SciELO - Scientific Electronic Library Online

 
vol.92 suppl.1 author indexsubject indexarticles search
Home Pagealphabetic serial listing  

Memórias do Instituto Oswaldo Cruz

Print version ISSN 0074-0276

Mem. Inst. Oswaldo Cruz vol.92  s.1 Rio de Janeiro Nov. 1997

http://dx.doi.org/10.1590/S0074-02761997000700005 

Biochemistry and Molecular Biology
129 - 138
139 - 148
149 - 158
159 - 168
169 - 178
179 - 188
189 - 198
199 - 208
209 - 218
219 - 228
229 - 238
239 - 248
249 - 258
259 - 268
269 - 276

129

FLOW CYTOFLUORIMETRIC ANALYSIS OF CHITIN ON THE SURFACE OF TRICHOMONADS.

Ferreira, L. N.1, Soares, R.M.A.1, De Souza, W.2, Angluster, J.1& Alviano, C.S.1

1Instituto de Microbiologia Prof. Paulo de Góes, CCS, UFRJ, Cidade Universitária, Rio de Janeiro, 21941-590, RJ, Brasil. E-mail: immgceu@microbio.ufrj.br2 Instituto de Biofísica Carlos Chagas Filho, CCS, UFRJ.

Trichomonas vaginalis and Tritrichomonas foetus are the causative agents of human and bovine trichomoniasis, respectively. The mechanism used by these parasites to exert their pathogenic effect is not fully understood. In this work, the expression of polysaccharide chitin as an exposed component on trichomonads surface was studied by using flow cytometry analysis. Fixed cells were incubated with recombinant (rec-) chitinase, N-acetyl-b-D-glucosaminidase (NAGase) or N,N',N"triacetylchitotriose (GlcNAc)3. Parasites either untreated or treated with the glycosidases or (GlcNAc)3 were incubated with fluorescein isothiocyanate (FITC) labeled Lycopersicon esculentum (TOL) and -Solanum tuberosum (STL) lectins. Rec-chitinase treatment markedly decreased the interaction of FITC-TOL and -STL lectins with the trichomonads surface, whereas NAGase which cleaves terminal b-linked D-GlcNAc, had no effect in lectins binding. These results show that the lectins receptors on the parasites surface are b-linked D-GlcNAc polymers and not terminal residues. We could also conclude, that the lectin binding to the trichomonads surface was specific, since it could be completely inhibited by the specific sugar (GlcNAc)3. These data support our previous findings which showed the presence of chitin on the parasitic surface by others techniques. The presence of chitinous structural components in trichomonads raises the possibility that chitin synthesis inhibitors may have a role in prevention and control of these parasitic infections.

Supported by CNPq, FINEP and PRONEX.


130

CALCIUM STORE COMPARTMENTS OF TRITRICHOMONAS FOETUS: A BIOCHEMICAL AND ULTRASTRUCTURAL STUDY

Aquino-Almeida, J.C.1,4, Okorokov, L.A.2, Benchimol, M.3 & De Souza, W.1,4

Laboratório de Biologia Celular e Tecidual1, Laboratório de Fisiologia e Bioquímica de Microorganismos2, CBB, UENF, Av. Alberto Lamego 2000, Campos, 28015-620, RJ, Brasil; Universidade Santa Úrsula3, Rua Jornalista Orlando Dantas 59, 90 and., Rio de Janeiro, 22231-010 , RJ, Brazil; Laboratório de Ultraestrutura Celular Hertha Meyer4, IBCCFo, UFRJ, Ilha do Fundão, Rio de Janeiro, 21949-900, RJ, Brasil.

Control of cellular Ca2+ plays an essential role in the physiology of eukaryotic cells, affecting many critical processes like signal transduction, secretion and cell-cycle control. There are few studies about Ca2+ homeostasis in parasitic protozoa. In this work we analyze the calcium store compartments of Tritrichomonas foetus, a parasitic protozoan from the urogenital tract of cattle. T. foetus is the causative agent of trichomoniasis, a sexually transmitted disease that causes abortion and sterility. Ca2+ uptake was measured after incubation of membranes with 1mM 45Ca2+ in the presence of ATP by routine millipore filtration technique. Radioactivity retained was measured by scintillation spectrophotometry. We found that the Ca2+ uptake by total membranes of T. foetus is not inhibited by even 10mM of the protonophore FCCP. However, it is blocked by vanadate (inhibitor of P-type ATPases) with I50 160mM. To identify organelles involved in Ca2+ uptake total membranes were submitted to fractionation in a sucrose density gradient. The fractions were characterized by routine transmission electron microscopy, by determination of Ca2+uptake and activity of marker enzymes of hydrogenosomes, Golgi and endoplasmic reticulum. Membranes of T. foetus present a weak Ca2+ uptake in several peaks, including the hydrogenosome one. Noteworthy that a huge peak of Ca2+ uptake activity corresponds to a peak of GDPase activity. Electron microscopy observation shows many typical Golgi stacks in this peak. Our data support the hypothesis that: 1)The Ca2+ is stored in several compartments, with the Golgi as an absolutely predominant one; 2)The Ca2+ transport is due to a Ca2+-ATPase(s) with small or no participation of Ca2+/H+ antiporter.

Financial Support: PRONEX, CNPq, FENORTE and FINEP .


131

EVIDENCE OF THE Na+/H+ ANTIPORTER ACTIVITY IN HERPETOMONAS SAMUELPESSOAI

Vieira, C.M.J.; Moreira, E.S.A & Pinto, A.S.

Departamento de Microbiologia, Instituto de Ciências Biológicas, CP 486, Universidade Federal de Minas Gerais.

The Na+/H+ antiporter is a transport system widely distributed in eukaryotic cells, which catalyses the exchange of one extracelullar sodium (or lithium ion) for one intracelluar proton, and is implicated in many cellular functions, such as control of pH, volume and proliferation of cells. To investigate the presence of a Na+/H+ antiporter in Herpetomonas samuelpessoai, a lithium-tolerant cell line was obtained by exposins wild type cells to 200 mM LiCl, at 37oC (one step protocol). Growth of the wild type and Li-tol lines was inhibited by lithium and amiloride, at pH 6.5 and 7.5, but a high degree of inhibition was observed for the wild type. Both drugs inhibited the cellular differentiation of wild type cells, but no significant effect was observed with the Li-tol cells. The Li-tol line was more tolerant to NaCl than the wild type. Changes in the intracellular pH (pHi) and Na+ concentration [Na+]i were determined fluorimetrically with probes BCECF-AM and SBFI, respectively. Addition of NaCl to the cells during NH4Cl pulse results in recovery towards initial pHi and an increase in [Na+]i in both wild-type an Li-tol cells. In the presence of amiloride, an Na+/H+ antiporter inhibitor, [Na+]i was not altered. Together, these results suggest that in Herpetomonas samuelpessoai, a Na+/H+ antiporter may be a mechanism for the regulation of cytoplamic homeostasis. RFLP and PFGE analyses were used in order to detect genomic differences between wild type and Li-tol cells lines. PFGE showed the presence of one band of approximate 1000 Kb in DNA of cells of wild type, that was absent in lithium tolerant cells. This difference may be associated with lithium tolerance, perhaps by amplification or delection mechanisms. Future work involving DNA hybridization with probes specific for the gene coding for a protein of the Na+/H+ antiporter will help clarify this question. Supported by CNPq.


132

ASSAY OF QUINOLINATE PHOSPHORIBOSYL TRANSFERASE IN HERPETOMONAS SAMUELPESSOAI AND CRITHIDIA FASCICULATA.

Zamboni, D. S. & Roitman, I.*

Laboratório de Microbiologia, Departamento de Biologia Celular, Universidade de Brasília, *Universidade de Mogi das Cruzes-SP.

All trypanosomatids studied so far have shown an absolute requirement for nicotinamide or nicotinic acid. It was demonstrated previously that quinolinic acid can replace nicotinamide in Herpetomonas samuelpessoai, H. anglusteri, Crithidia desouzai, C. deanei, Leptomonas seymouri and Phytomonas serpens, while in C. fasciculata, C. acanthocephali, C. guilhermei and in C. luciliae, quinolinic acid could not replace nicotinamide. Quinolinate phosphoribosyltransferase is one of the NAD biosynthesis enzyme, converting quinolinate in nicotinate mononucleotide. Here we try to demonstrate the presence of quinolinate phosphoribosyltransferase in Herpetomonas samuelpessoai, and in Crithidia fasciculata extracts.

H. samuelpessoai, and C. fasciculata was grown for 48 hours in defined medium for trypanosomatids. The cells were transfered, for one hour, to a defined medium without nicotinamide and nicotinic acid supplied with 5mg% of quinolinic acid to induce quinolinate phosphoribosyltransferase. Homogenates of the two flagellates were obtained with alumina followed by centrifugation. The supernatant was used for testing the enzyme activity after protein determination. The enzyme test assay was performed according to Packman and Jakoby (1970). Rat liver extract was used as positive control while boiled C. fasciculata extract was used as negative control.

Quinolinate phosphoribosyltransferase activity was verified in both cell extracts suggesting that the inability of quinolinic acid in replacing nicotinamide in Crithidia fasciculata is not caused by the absence of the enzyme. Difference in cell permeability of quinolinic acid in both cells can explain the behaviour previously reported. The diversity of species in terms of cell permeability of metabolites of NAD synthesis could be considered for phylogenetic studies of Trypanosomatidae.

Supported by PIBIC-UnB, CNPq.


133

GLYCOPROTEINS OF 200 kDa FROM RING AND TROPHOZOITE STAGES OF PLASMODIUM FALCIPARUM CONTAIN N-LINKED OLIGOSACCHARIDES.

M. L. Uhrig, E.A. Kimura*, V. Peres*, A. Katzin* and A. Couto.

Departamento de Quimica Organica, Facultad de Cs. Exactas y Naturales, Universidad de Buenos Aires,Buenos Aires, Argentina. *Departamento de Parasitologia, ICB, USP, Brazil.

We have already reported the presence of N-linked glycoproteins in the ring stage and in young trophozoites. The aim of this study is the structural analysis of the N-linked oligosaccharides obtained from purified glycoproteins of approx. 200 kDa which have been related to the differentiation of the intraerythrocytic stages in P. falciparum (Kimura et al, J. Biol Chem. 271:14452, 1996).

Parasites were metabolically labeled with L-[35S]-methionine. Each stage was purified by Percoll gradient, lysed and subjected to 8% polyacrylamide gel electrophoresis (SDS-PAGE). A glycoprotein of >200 kDa present in the ring forms was excised from the gel and digested with N-glycanase. The hydrolysate was reduced with NaB3H4 and the released radioactive oligosaccharides were analysed by high performance anion exchange chromatography (HPAE-PAD). The sample showed a major peak corresponding to Man3GlcNAc2 (1,59 glucose units, GU). This structure was confirmed by sequencial treatment with a-mannosidase, b-mannosidase. Two other peaks eluting approx. 3 GU and 4 GU corresponding to N-linked chains that appear later than Man9GlcNAc2 were also detected. A similar analysis performed on the 200 kDa glycoprotein obtained from young trophozoites showed the presence of labeled oligosaccharides eluting in the same range of glucose units. The same treatment performed on glycoproteins metabolically labeled with [14C]-glucose confirmed the presence of high molecular weight oligosaccharides.

Supported: CONICET (Argentina) FAPESP (Brasil) Fundación Antorchas (Projeto bi-nacional)


134

STRUCTURAL CHARACTERIZATION OF NOVEL SIALOGLYCOLIPIDS ISOLATED FROM ENDOTRYPANUM SCHAUDINNI LV58 STRAIN

Xavier da Silveira E. 1, Jones C. 2, Wait R. 3, Previato J.O. 1 & Mendonça-Previato L. 1

1 Instituto de Microbiologia, UFRJ, RJ, Brasil; 2 Laboratory for Molecular Structures, NIBSC, Herts, UK; 3 Center for Applied Microbiology and Research, Wilts, UK

Several studies characterizing the nature and structure of the sialic acid-containing glycoprotein in T. cruzi and T. brucei have been published. In T. cruzi strains Y and G the sialic acid is expressed in O-linked oligosaccharides from mucin-like glycoproteins (1,2) whereas in T. brucei, the GPI-glycan of procyclin is the main sialic acid acceptor (3). In both parasites, the sialic acid was incorporated on the surface molecules by trans-sialidase, a glycosyltransferase which catalyzes the transfer of a2-3-linked sialic acid from exogenous sialoglycoconjugates to acceptor molecules, containing terminal b-D-galactopyranosyl residues. Trans-sialidase is, however, not restricted to trypanosomes as it also occurs in Endotrypanum, a genus of sloth parasites, which uniquely among trypanosomatids, invade host erythrocytes (4). Nevertheless, the presence of sialic acid containing glycoconjugates in Endotrypanum is controversial, and no natural sialic acid acceptor has yet been identified. In the present work, we described sialic acid-containing glycophosphosphingolipids synthesized by strain LV58 of E. schaudinni. Their structures were determined by a combination of chemical analysis, FAB-mass spectrometry and NMR spectroscopy and shown to be:

NeuAca(2-3)Galpb(1-3)Galpb(1-3)Man a(1-3)Mana(1-4)GlcNa(1-6)Ins1-PO4-Cer (Structure I)

NeuAca(2-3)Galpb(1-3)Galpb(1-3)Gal pb(1-3)Mana(1-3)Mana(1-4)GlcNa(1-6)Ins1-PO 4-Cer (Structure II)

Although the biological significance of the sialoglycophosphosphingolipids in Endotrypanum is unknown, in T. cruzi, sialoglycoproteins have been implicated in the recognition or invasion of host cells (5 ).

(1) Previato et al., J. Biol. Chem. 270: 7242, 1995 (2) Previato et al., Biochem. J. 301: 151, 1994 (3) Ferguson et al., Biochem. J. 291: 51, 1993 (4) Medina-Acosta et al., Mol. Biochem. Parasitol. 64: 273, 1994 (5) Andrews and Burleigh, Annu. Rev. Microbiol. 49: 175, 1995

Supported by: CNPq (RHAE, PADCT), FINEP, CEPG/UFRJ, PRONEX


135

GLYCOSYLPHOSPHATIDYLINOSITOL ANCHORED MUCIN-LIKE GLYCOPROTEINS (GPI-mucins) PURIFIED FROM TRYPANOSOMA CRUZI INTRACELLULAR AMASTIGOTES ARE POTENT INDUCERS OF PRO-INFLAMMATORY CYTOKINES AND NITRIC OXIDE BY MACROPHAGES.

Gazzinelli, R.T.1,2; Camargo, M.M.1,2; Travassos, L.R.4; Ferguson, M.A J.3; and Almeida, I.C.3

1 Departmento de Bioquímica e Imunologia, UFMG; 2 Laboratório de Chagas, CPqRR-FIOCRUZ, Belo Horizonte, MG, Brazil; 3 Department of Biochemistry, Dundee University, Dundee, Scotland; and 4Disciplina de Biologia Celular, DPMI-UNIFESP, São Paulo, SP, Brazil.

In recent studies we have demonstrated that live amastigote and trypomastigote stages of Trypanosoma cruzi are potent activators of pro-inflammatory cytokines and nitric oxide synthesis by macrophages. In contrast, live epimastigote or metacyclic forms were unable to trigger any of the tested macrophage functions. In these studies, we have also demonstrated that GPI-mucins isolated from trypomastigote (tGPI-mucins), but not from epimastigote or metacyclic forms of T. cruzi, are potent inducers of cytokine synthesis and microbicidal activity by macrophages (Camargo et al. 1997, J. Immunol. 158:5890 and Camargo et al. 1997, submitted). In the present study, we isolated and partially characterized GPI-mucins from intracellular amastigote forms (aGPI-mucins) and tested their activity on macrophages. An identical protocol to that used to purify tGPI-mucins (Almeida et al. 1994, Biochem. J. 304:793) was employed to obtain aGPI-mucins. Briefly, a pellet of amastigotes obtained from disrupted LLCMK2 cells was submitted to extraction with chloroform:methanol:water followed by butanol:water partition and then fractionation in a hydrophobic interaction column. The different fractions eluted from octyl-Sepharose using a propan-1-ol gradient had their myo-inositol content as well as IL-12, TNF-a and nitric oxide inducing activity assayed. As control we used fractions obtained from octyl-Sepharose loaded with aqueous phase extracted from either trypomastigote or epimastigote extracts and eluted with propan-1-ol gradient. Consistent with our experiments with live parasites, aGPI-mucins were also able to trigger cytokine and nitric oxide synthesis by macrophages. The maximum peak of activity of the fractions from the octyl-Sepharose, loaded with either amastigote or trypomastigote extracts, corresponded to those eluting with 22-27% and 20-28% propan-1-ol, respectively. An overlap between cytokine inducing activity and myo-inositol peaks was observed in the different fractions eluted from octyl-Sepharose, further suggesting the role of GPI-anchors in cytokine inducing activity of GPI-mucins. It is noteworthy that aGPI-mucins were three to four fold less active than tGPI-mucins on the basis of myo-inositol concentration. Nevertheless, aGPI-mucins were at least 100 fold more active than epimastigote GPI-mucins (eGPI-mucins), which were eluted at 20-40% propan-1-ol and showed only a residual activity on macrophages. A third peak was released at fractions eluting with 30-42% propan-1-ol from the octyl-Sepharose loaded with epimastigote extracts. This peak corresponded to LPPG and GIPLs from epimastigotes and have no macrophage-inducing activity even at high concentrations. Our previous studies suggested a possible role for unsaturated fatty acids from GPI-anchors as important components of cytokine-inducing activity of tGPI-mucins. In fact, most of the phosphatidylinositol (PI) species isolated from the GPI-anchor of tGPI-mucins contain unsaturated fatty acid chains (mainly C18:1 and C18:2). In contrast, as previously observed (Acosta-Serrano et al., 1995, J. Biol. Chem. 270:27244), GPI anchors obtained from eGPI-mucins have only saturated fatty acid chains in their PI moiety. Furthermore, preliminary structural analysis indicates that aGPI-mucins are composed PI species containing either saturated or unsaturated fatty acid chains. Thus, the data presented here further suggest the role of unsaturated fatty acids in the GPI anchor of T. cruzi mucins as a marker for stage-specific induction of pro-inflammatory cytokines.

Supported by CNPq and FAPESP (Fellowship No. 96/4260)


136

ISOLATION OF NEUTRAL GLYCOLIPIDS FROM METACYCLIC FORMS OF TRYPANOSOMA CRUZI

Martins, R M ; Soares, R M A ; Bonaldo, M C ; Lima, A C ; Branquinha, M H & Vermelho, A B.

Dept. Microbiologia Geral, Instituto de Microbiologia Prof. Paulo de Góes, UFRJ.

Glycolipids, as components of the cellular surface, are believed to be involved in cell-cell interactions, differentiation, immunogenicity and oncogenesis ( Curatolo, W. 1987. Biochem. Biophys. Acta 906, 137-160 ). In this work, we have investigated Trypanosoma cruzi clone Dm28c neutral glycolipids during the conversion of epimastigotes into metacyclic trypomastigotes under chemically defined conditions in TAUAAG medium (Contreras et al, 1985. Mol. Biochem. Parasitol. 16, 315-327). Neutral glycolipids were extracted from metacyclic forms successively with chloroform / methanol 2:1 and 1:2 (v / v) and partitioned according to Folch (J. Biol. Chem. , 1957, 226: 497 - 509). The lower phase, after drying, was dissolved in chloroform/methanol 90:10 (v/v) and chromatographed on a silica gel column. This column was eluted sequentially with chloroform / methanol 90:10, 80:20, 70:30 and 60:40 ( v / v ). Final purification was obtained on a small Iatrobeads column by step - wise elution with chloroform/methanol 97:3, 95:5, 90:10, 80:20, 75:25 and 50:50 (v/v). Neutral glycolipids were detected with orcinol reagent on high performance thin layer chromatography ( HPTLC ).

Two glycolipids were eluted from silica gel and Iatrobeads columns with chloroform / methanol 90:10 e 80:20. The chromatographic mobility of these glycolipids was similar to the galactosyl ceramide used as standard. Studies are in progress to determine the primary structure of these molecules.

Supported by : FINEP-(PRONEX-CARBOHYDRATES), FINEP-BID, FAPERJ, CNPq, FUJB, CEPG-UFRJ.


137

PURIFICATION OF G PROTEINS IN TRYPANOSOMA CRUZI. DIFFERENTIAL EXPRESSION OF Ga SUBUNITS IN EACH FORM OF THE PARASITE.

J. Paba1, J. Santana2, P.A. Millner3., M. V. Sousa1 and C. A. O. Ricart1

1-Laboratório de Bioquímica e Química de Proteinas, Dep. Biologia Celular, Universidade de Brasília, Brasília 70910-900 - DF 2-Laboratorio Multidisciplinar de Pesquisa em Doença de Chagas, Universidade de Brasilia, Brasília 70910-900 - DF. 3-Department of Biochemistry and Molecular Biology, University of Leeds, UK

Heterotrimeric G proteins are involved in transmembrane signal transduction leading to the modulation of different cellular events as differentiation, cell cycle, protein vesicle trafficking and mating. This effects are a consequence of the direct or indirect action of its three subunits (a, b, g) on different effectors, such as adenylyl ciclase, phospholipase and ion channels.

T. cruzi epimastigote extracts containing immunologically recognized putative Ga subunits Gao, Gai, Gacommon and Gas (Paba et al, 1996, Mem.Inst. Oswaldo Cruz Vol 91, Suppl.,p 294), were subjected to FPLC separation on a Resource Q column. The separated bands corresponding to a subunits were either transferred to PVDF membrane in order to obtain an aminoterminal sequence, or submitted to tryptic in situ gel digestion. The resulting peptides are now being separated on HPLC and sequenced to obtain several internal sequences. Other four proteins (55, 43, 30 and 43 Kda) purified by affinity chromatography on a GTP agarose resin but not recognized by any of the anti Ga antisera, were submitted to amino terminal and internal sequencing as described above. The amino terminal sequences showed homology with some GTP binding proteins and several other nucleotide binding proteins.

Whole extracts of the three different forms of the parasite were separated by SDS PAGE and immunoblotted using antisera against Gao, Gai, Gacommon and Gas subunits. The results revealed a differential expression of the Gao (tryp>epi>ama) and Gas (epi>tryp>ama) proteins, suggesting a possible role of the heterotrimeric G proteins in the Trypanosoma cell cycle. The Gai and Gacommon proteins were not analized due to the non-specific detection of other proteins running with the same molecular mass on the gel.

Supported by CNPq, FAPDF and UnB


138

FCaBP IS A MYRISTOYL/PALMITOYL SWITCH PROTEIN THAT ASSOCIATES WITH THE FLAGELLAR MEMBRANE IN A CALCIUM-DEPENDENT MANNER

Godsel, L.M., Olson, C.L. and Engman, D.M.

Departments of Pathology and Microbiology-Immunology
Northwestern University, Chicago IL 60611 USA

The flagellar calcium binding protein (FCaBP) of Trypanosoma cruzi is a 24-kDa EF-hand calcium binding protein of the calflagin family. In amastigotes and trypomastigotes the protein is found in both the flagellum and cell body, whereas in epimastigotes it is localized solely to the flagellum, where it is associated with the internal face of the flagellar plasma membrane. Myristoylation and palmitoylation of the N-terminus are necessary and sufficient for the flagellar localization of FCaBP. Calcium is required for localization as well. Chelation of calcium from permeabilized cells leads to dissociation of FCaBP from the flagellum. Green fluorescent protein containing the N-terminal FCaBP flagellar targeting sequence does not dissociate, indicating that the calcium-binding domains are critical for calcium-regulated localization. Thus, FCaBP is a novel member of the calcium-myristoyl switch protein family and is the first protein described in which amino acylation specifies flagellar localization. Partial FCaBP knock-out mutants display significantly slowed growth and delayed cell division, suggesting that FCaBP might play a role in the process of cell growth.


139

THE TRANSFERRIN RECEPTOR OF TRYPANOSOMA BRUCEI IS CONSTRUCTED WITH N-TERMINAL DOMAINS OF THE VARIANT SURFACE GLYCOPROTEIN (VSG) AND THE LIGAND BINDING SITE CORRESPONDS TO THE MOST EXPOSED SURFACE LOOPS OF THE VSG WHICH EXIBIT THE TRYPANOSOME VARIANT EPITOPES.

Salmon, D, Hanocq-Quertier, J., Paturiaux-Hanocq, F., Nolan, D., Pays, A., Tebabi, P., Michel, A1, and Pays, E.

Department of Molecular Biology, University of Brussels, 67 rue des chevaux, B1640 Rhode St Genèse, Belgium.
1 Laboratory of Biological Chemistry, University of Mons, Belgium.

The transferrin (Tf) receptor expressed in bloodstream forms of Trypanosoma brucei is a glycosylphosphatidylinositol (GPI)-anchored heterodimer encoded by ESAG 7 and ESAG 6, two related genes contained in the different polycistronic transcription units of the variant surface glycoprotein (VSG) gene.

The slight sequence variations between ESAG 7/6 from different units and the expression alternative of different units in the bloodstream forms during the variation antigenic of the parasite allow the successive expression of several variants of the Tf receptor with different affinity. Based on the homology between pESAG 7/6 and the N-terminal domain of VSGs, it can be predicted that four blocks containing the major differences between pESAG 7 and 6 form surface-exposed loops and generate the ligand binding site. In accordance with this prediction, we show that in this region, the exchange of a few amino acids between pESAG 6s encoded by different VSG expression sites greatly increases the affinity for bovine Tf. Similar changes in other regions are ineffective, and mutations that alter the VSG structure abolish the Tf binding. Moreover, chimeric proteins constructed with the dimerization domain of the VSG and the presomptive ligand binding region of pESAG 7 and pESAG 6 efficently associate to bind Tf. It is concluded that the Tf receptor is constructed with specilized VSG N-terminal domains, and that the different VSG expression sites (around 20) encode a collection of transferrin receptors with a wide range of affinities for the ligands.


140

A NOVEL RECEPTOR MEDIATED ATP TRANSPORT SYSTEM REGULATED BY TYROSINE AND SERINE/THREONINE PHOSPHOKINASES IN TRYPANOSOMA CRUZI TRYPOMASTIGOTES

Sadigursky, M., Santos-Buch, C. A.

Faculty of Medicine, Federal University of Bahia -Salvador-Bahia-Brazil and Cornell University Medical College - New York-NY-USA

Trypanosoma cruzi has a plasma membrane ATP transport system that may consist of an exterior receptor domain (ATP-R) and an interior domain regulated by tyrosine and serine/threonine kinases. The addition of exogenous ATP to freely swimming trypomastigotes resulted in a receptor-mediated inward movement of the nucleotide, and the system obeyed mass action law

( Km 9.42uM and Vmax 77.7nmol.min-1x106 trypomastigotes-1). Preloaded [H3]ADP was not exchanged for ATP following the addition of increasing concentrations of exogenous ATPo to swimming trypomastigotes. Trypomastigote[ATP]o«ATP-R®ATP] i transport was [ATP]o-dependent and saturable at100uM. [ATP]o«ATP-R®[ATP]i transport was abrogated by the tyrosine kinase inhibitors, genistein and lavendustin A. [ATP]o«ATP-R®[ATP]i transport was also inhibited by the serine/threonine/kinase inhibitor, staurosporin. Suramin, the antagonist of P2x and P2y purinergic receptors, was also a very effective competitive inhibitor of the trypomastigote ATP transport system. The action of exogenous [y32P]ATPo resulted in the initial and simultaneous phosphorylation of a 63-kDa polypeptide (p63) and of a 92.4-kDa polypeptide (92.4), which was followed by the abrupt phosphorylation of many other substrate proteins. The trypomastigote p63/p92.4 polypeptides may represent substrate proteins of a putative ATP-R-related tyrosine phosphokinase, and ATP receptors may transmit their signals by phosphorylation of specific substrate proteins.

Supported by CAPES/Fulbright program


141

DETECTION OF PROTEINASE ACTIVITIES IN TWO ISOLATES OF ACANTHAMOEBA POLYPHAGA

Correia, C E B, Varotti, F P, Motegi, S A, Firmani, C M & Alfieri, S C

Depto. de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, Av. Prof. Lineu Prestes 1374, CEP 05508-900, São Paulo, SP, Brasil

Among the small, free-living amoebas, species of the genus Acanthamoeba can be potentially pathogenic, and responsible for two types of human diseases: granulomatous amebic encephalitis, and keratites. The mechanisms underlying tissue damage and invasion are poorly understood, but the involvement of as yet uncharacterized trophozoites' proteinases has been suggested. Here we have employed gelatin-containing SDS-acrylamide gels and azocasein enzymatic assays to examine proteinase activities in two A. polyphaga isolates (ATCC 30461, originally from a case of keratitis, and 30872, a soil isolate). In freshly prepared lysates of both isolates, azocasein hydrolysing activity, although detectable over a broad pH range (4.0 - 8.0), was much higher at pH 4.0 and 5.0. The acidic proteinases were stimulated by DTT, suggesting the involvement of cysteine proteinases. In gelatin gels, total lysates of both isolates contained proteinases which were not totally inactivated by SDS, and responsible for extensive substrate degradation in gels fixed and stained immediately after electrophoresis. The SDS-resistant enzymes were potently inhibited by PMSF, but not by E-64, o-phenanthroline, TPCK or TLCK added prior to electrophoresis, thus indicating the involvement of serine proteinases. Blockage of serine proteinase activity prior to electrophoresis permitted the visualization of several bands of activity, of which most were stimulated by reducing agents and optimally detected at acidic pH. The enzymes of the two isolates examined were differently distributed in substrate gels: four bands (100, 74, 43, and 39 kDa) were detected in lysates of isolate 30872, and six (120, 100, 68, 59, 43, and 39 kDa) were depicted in isolate 30461. In both cases, the activity associated with the high Mr, 68-120 kDa proteinases was shown to predominate; these enzymes were partially inhibited by antipain and E-64, suggesting a relation to the cysteinyl class of peptidases.

Work supported by FAPESP, CAPES, and CNPq


142

OCCURRENCE OF CYSTEINE-PROTEINASES IN BAT TRYPANOSOMES CULTURE SUPERNATANT

Branquinha, MH, Petinate, SDG, Almeida, FVS & Vermelho, AB.

Dept. Microbiologia Geral, Inst. Microbiologia Prof. Paulo de Góes, UFRJ.

Epimastigote forms of bat trypanosomatids from the subgenus Schizotrypanum contain high cysteine-proteinase activity, possibly associated to cytoplasmic organelles called reservosomes (Soares et al., 1992, J. Cell Sci., 102:157-167).

In this study, a Schizotrypanum trypanosomatid isolated from the bat Phyllostomus hastatus (Teixeira et al., 1993, Parasitol. Res., 79:497-500) and Trypanosoma (S.) dionisii were grown in yeast extract-peptone-sucrose medium. After centrifugation, culture supernatant was concentrated and the extracellular proteolytic activity was determined by SDS-PAGE containing co-polymerized gelatin as substrate (Heussen & Dowdle, 1980, Anal. Biochem., 102:196-202).

This analysis in the Schizotrypanum isolate revealed the presence of at least four bands at 30 kDa, 40 kDa, 55 kDa and 70 kDa in cell-free culture supernatant. In T. dionisii, three bands were detected at 30 kDa, 50 kDa and 70 kDa. Addition of E-64 resulted in total inhibition of these proteolytic activities, suggesting that these enzymes are cysteine-proteinases. Secretion of these enzymes began on the fourth day of culture and reached a maximun on the sixth day, corresponding to the end of the log phase of culture. These similarities could point to the importance of cysteine-proteinase activity on bat trypanosomes cell cycle.

Supported by: FINEP-BID, CNPq, FUJB, CEPG-UFRJ


143

INFLUENCE OF TWO DIFFERENT SUBSTRATES ON HERPETOMONAS MEGASELIAE EXTRACELLULAR PROTEINASE PROFILES

Melo, A C N, D'ávila, C M, Branquinha, M H & Vermelho, A B

Departamento de Microbiologia Geral, Instituto de Microbiologia, UFRJ.

The genus Herpetomonas comprises monoxenic parasites of insects that present pro- and opisthomastigote forms in their life cycle. These trypanosomatids secrete proteinases that are important in host-parasite interactions, intracellular survival, escape and in the processing of host proteins for nutrition purposes.

In the present report the effect of two different substrates added to the growth medium (BHI) in the expression of extracellular proteinases of Herpetomonas megaseliae is related. The microorganism was grown on BHI supplemented with 1% BSA (bovine serum albumin) or 1% gelatin for four days at 28° C. The culture media were centrifugated, the cells were removed and the supernatants were concentrated against polyethyleneglycol. Proteolytic activities were detected on 7.5% SDS-PAGE containing gelatin as substrate (Heussen & Dowdle, Anal. Biochem., 102:196-202). The gels were incubated overnight at 37° C in 50 mM phosphate buffer, pH 5.5 and in 50 mM glycine-NaOH buffer, pH 10.0, and stained for 1 hour with 0.1% amido black.

When Herpetomonas megaseliae was cultivated in BHI a band of 60 kDa was observed (Melo et al., XXVI Reunião Anual da Sociedade Brasileira de Bioquímica e Biologia Molecular, 1997, p.95). The addition of BSA increased the extracellular proteinase production: two bands were detected in this medium migrating at 70 kDa and 60 kDa. No proteolytic activity was detected in BHI-gelatin, indicating a repression due to this substrate. In the media tested the proteolytic activity was the same either at pH 5.5 or at pH 10.0.

Supported by: FINEP-BID, FUJB, CNPq, CEPG/UFRJ.


144

BIOCHEMICAL CHARACTERIZATION AND SUBCELLULAR LOCALIZATION OF THE ENDOGENOUS T. cruzi INHIBITOR OF CYSTEINE PROTEASES (Tc-CPI).

Monteiro, A.C.S.a, Abrahamson, M.b, Vannier, M.A.S.c, Schelble, G. aand Scharfstein, J.a

aLaboratório de Imunologia Molecular, IBCCF,U.F.R.J.; bDepartment of Clinical Chemistry, University of Lund, Sweden,c Laboratório de Ultraestrutura Celular Hertha Meyer, IBCCF,U.F.R.J.

A superfamily of cysteine proteinases inhibitors designated as cystatins is widely distributed in mammalian tissues and body fluids, plants and lower organisms. Since uncontrolled proteolysis by endogenous proteases is a potential threat for the integrity of cells, the activities of these enzymes must be carefully regulated. T. cruzi cysteine proteases (CP) (cruzipains) play essential role in the life cycle of the parasite. By analogy with other organisms, endogenous T. cruzi inhibitors might exist to regulate the intracellular proteolytic activity. It has been previously reported that an 11 kDa inhibitory protein (Tc-CPI) was identified in lysates obtained from DM28c epimastigotes (EPI). The c-DNA gene encoding this protein was cloned and expressed. A recombinant protein was expressed in E. coli and showed potent activity against CP. A polyclonal antiserum raised against the active recombinant protein recognized a 11 kDa inhibitory protein in lysates of DM28c EPI. The rate of inactivation for n-cruzipain (natural form, that is, isolated from parasite extracts), r-cruzain (recombinant form, a gift from J.H. McKerrow) and cruzipain 2 (recombinant isoform, distinct from cruzain) was determined for natural and recombinant Tc-CPI. Both proteins bound tightly to all investigated enzymes with Ki (equilibrium constant for dissociation) values in the picomolar range . The Ki values were : rTc-CPI/n-cruzipain = 0.0024 nM and nTc-CPI/n-cruzipain = 0.0067 nM; rTc-CPI/r-cruzain = 0.0095 nM and Tc-CPI/r-cruzain= 0.0124 nM; rTc-CPI/r-cruzipain 2 = 0.039 nM and nTc-CPI/r-cruzipain 2 = 0.075 nM. Tc-CPI display broad specificity and high affinity binding to other cysteine proteases, including cathepsin B, papain and falcipain. The primary structure deduced from the sequence of the cDNA clone shows no homology with cystatins or with any other protein in the data bank, indicating that Tc-CPI is a novel class of tight reversible CP inhibitors. Northern blot analysis revealed that the Tc-CPI message has approximately 800 bp, and is expressed in all three developmental stages of the life cycle of T. cruzi. Our results shows that the level of expression is higher in trypomastigotes than in amastigotes or epimastigotes. Immunolocalization sstudies performed with affinity purified anti-Tc-CPI antibodies showed that the protein is present in the flagellar pocket, flagellum and secretory vesicles. Interestingly, Tc-CPI is also found at the surface of the amastigotes. The mechanism whereby Tc-CPI controls proteolytic activity of T. cruzi cysteine proteases is under investigation. Supported by CNPq and PADCT-CNPq.


145

MODIFICATION OF PLACENTAL ALKALINE PHOSPHATASE ACTIVITY BY TRYPANOSOMA CRUZI IN VITRO.

Lin S., Sartori M.J., Fretes R.E., Fabro S. P. de.

IIa Cátedra de Histología, Emb. y Genética e Instituto de Biología Celular. Facultad de Ciencias Médicas. Universidad Nacional de Córdoba. Agencia Postal N° 4. Ciudad Universitaria. (5000) Córdoba. Argentina.

Supported by CONICOR (Consejo de Investigación Científica de la Provincia de Córdoba) and SECyT (Secretaría de Ciencia y Técnica de la Universidad Nacional de Córdoba).

Previous works have demonstrated that placental alkaline phosphatase activity (PLAP) decreases in plasma of chagasic pregnant women in the third trimestre. In vitro, Trypanosoma cruzi induces changes on the protein pattern of human syncytiotrophoblast and alkaline phosphatase activity is modified by trypomastigotes in cultured human placental villi. Trypomastigotes present neuraminidases that are implied in the interiorization of T. cruzi into host cells (Pereira 1983 a and b, Schenkman 1993). This suggests that a possible mechanism of modification of the PLAP by T. cruzi could be through the action of neuraminidase. In the present work we cultured human placental villi with trypomastigotes forms of T. cruzi and compared these results induced by the parasite with those from placental villi cultured in presence of neuraminidase (Sigma). Central villi of placental cotyledones were co-cultured with 5.6 x105 bloodstream trypomastigotes (Tulahuen strain) of T. cruzi or 5x10-4 gr of neuraminidase at 37ºC in a final volume of 1.5 ml of M-199 culture medium and harvested after 24 and 72 hours. Controls were maintained at same conditions without T.cruzi. After the cultures, placental tissues were either processed for immunofluorescence or homogeneized for biochemical analysis. Electrophoresis in SDS-PAGE , PLAP activity (Messer 1975) and protein levels (Lowry et al 1951) were measured in placental homogenates and culture media. PLAP was detected in cultured tissue by monoclonal antibody (Biogenex Ab 228M, mouse source), labelled with fluorescened goat IgG anti-mouse. PLAP activity diminishes in culture media and homogenates from placental villi co-cultured with T. cruzi in 41% and 43 %. This decrease was also observed in cultures with neuraminidase in 59% and 39% respectively. Electrophoresis in both experimental conditions show differences in protein patterns with regard to controls. PLAP antibody marks more intensively some areas of control than the two experimental samples. The fact that trypomastigotes, as the neuraminidase, decreases PLAP activity both in placental homogenate and culture medium suggests that these parasites probably modifie PLAP activity through the action of neuraminidase.


146

DETECTION OF ALKALINE PHOSPHATASE ACTIVITY ON CELL SURFACE OF PROMASTIGOTE FORMS OF LEISHMANIA AMAZONENSIS

Mattos A1, Almeida Souza A1, Morgado Díaz JA1., and Giovanni-De-Simone S1,2.

1 Departamento de Bioquímica e Biologia molecular, Instituto Oswaldo Cruz, FIOCRUZ, RJ, Brasil. 2 Departamento de Biologia celular e Molecular, Instituto de Biolgia, Universidade Federal Fluminense, Niterói, RJ, Brasil.

Several works have demonstrated the presence of an acid phosphatase in various parasitic protozoa of the Trypanosomatidae family. This enzyme has been localizated on the parasite surface or was found secreted in the surrounding medium. However, little or nothing is known on the existence of an alkaline phosphatase in these parasites. In this work, we showed that living promastigote forms of Leishmania amazonensis were able to hydrolyze r-nitrophenyl phosphate (5mM, pH 7.2 at 26° C in various time intervals), a substrate for phosphatases. This activity appears to be due to an alkaline phosphatase because: (a) it was enhanced at high pH values (9.0 - 9.5), (b) it was inhibited by the alkaline phosphatase inhibitor EDTA (20mM and 3mM Tetramisole), but not by the acid phosphatase inhibitor sodium fluoride (10 mM). Furthermore, studies at various temperatures (4 - 40°C), indicate that at least some of this alkaline phosphatase activity may be associated with the surface of the parasites, rather than with endocityc or intracellular systems. This was supported by subcellular fractionation of parasites which showed some cosedimentation of alkaline phosphatase (3-fold) with an enriched membrane fraction.

Supported by CNPq, FAPERJ/FIOCRUZ


147

INDUCTION OF IL-12(p40) BY MICROBIAL PRODUCTS REQUIRES ACTIVATION OF THE P38/RK MAP KINASE AND IS REGULATED BY CYCLIC AMP

Camargo,M.M.1,2; Clifton, A3; Almeida,I.C.3; Lee, J.4; Ferguson, M.A J.3; Cohen,P.3 and Gazzinelli, R.T.1,2

1Depart. de Bioquímica e Imunologia, UFMG; 2Laboratorio de Chagas, CPqRR-FIOCRUZ, Belo Horizonte, MG, Brazil; 3Depart. of Biochemistry, Dundee University, Dundee, Scotland; and 4SmithKline Beecham Pharmaceuticals, King of Prussia, PA ,USA.

IL-12 is a heterodimer composed of an inducible chain of 40 kDa (p40) produced by activated cells from the monocytic/macrophage lineage, and a 35kDa polypeptide (p35) which is expressed constitutively by a variety of cells. IL-12 has also been shown to be a key cytokine involved in initiation of cell-mediated-immunity (and INF-g synthesis) and resistance to different intracellular pathogens. Our previous studies (Gazzinelli et al. 1993, PNAS 90:6115 and Camargo et al. 1997, J. Immunol. 158:5890) show that live tachyzoite and trypomastigotes stages of the intracellular protozoa Toxoplasma gondii and Trypanosoma cruzi, respectively, are potent inducers of IL-12 synthesis by inflammatory macrophages. We have further demonstrated that glycolipids present in soluble tachyzoite antigens (STAg) or purified from trypomastigote extracts (tGPI-mucins) also display the same activity as live parasites in inducing IL-12 synthesis by macrophages. The ability to trigger IL-12 synthesis by different microbial products is highly potentiated by macrophage priming with INF-g. Despite of recent demonstrations that a NF-kB half-site (Murphy et al. 1996, J. Mol. Cell Biol. 15:5258) and a new ets element (MA et al. 1996, J. Exp. Med. 183:147) may be important transcription factors required for induction of the p40 chain of IL-12, the signal transduction pathway(s) involved in its induction is(are) poorly understood. The present study is our initial effort in trying to characterize the signaling pathway(s) involved in IL-12(p40) gene expression in inflammatory macrophages exposed to microbial products and INF-g. Among different inhibitors for protein kinases, we found that SB203580, a pyridinyl-imidazole compound and specific inhibitor for p38/RK MAP-kinase (Lee at al. Nature 372:741 and Cuenda et al. FEBS-Lett 364, 229-233) is a potent inhibitor of IL-12(p40) synthesis by macrophages exposed to either tGPI-mucins, STAg or Escherichia coli derived endotoxin (LPS) plus INF-g. SB 203580 inhibited IL-12 synthesis with an IC50 of 1.2 mM. We also measured the activation of MAPKAP-K2, a downstream target of p38/RK MAP kinase. Our data show that MAPKAP-K2 activity is induced by either LPS or tGPI-mucins, and that induction of this activity was blocked by SB 203580, but not by Cholera Toxin. Although significant, the induction of MAPKAP-K2 activity by tGPI-mucins was much weaker than the activation induced by LPS. IFN-? did not induce any MAPKAP-K2 activity on its own, but slightly increased the MAPKAP-K2 activity induced by tGPI-mucins. In contrast, PD 98059, a specific inhibitor of the activation of MAPKK-1, had no effect on IL-12 synthesis by macrophages exposed to different microbial products with or without INF-g. Cholera Toxin, an activator of adenylate cyclase, was also shown to inhibit the synthesis of IL-12 with an IC50 of 25 pg/ml. In order to confirm the cAMP dependency for IL-12 synthesis we used two cAMP analogues that are known to mimic the action of cAMP in vivo. 8-bromo cAMP (IC50:1 mM) or dibutyryl cAMP (IC50: 0.3 mM) were both found to be potent inhibitors of IL-12 synthesis by macrophages as well. However, cholera toxin and cAMP analogues did not prevent the activation of MAPKAP-K2 by LPS or tGPI mucins. We are currently investigating whether macrophage exposure to tGPI-mucins triggers phosphorylation of other components of MAPKAP-K2 cascade. Our data support the hypothesis that induction of IL-12 synthesis by microbial products combined with INF-g maybe regulated positively by RK/p38 MAP kinase and negatively by cAMP. Supported by CNPq and FAPESP (Fellowship No. 96/4260)


148

EFFECT OF PLATELET-ACTIVATING FACTOR ON ECTO-PHOSPHATASE AND ON SECRETED PHOSPHATASE ACTIVITIES OF TRYPANOSOMA CRUZI.

Rodrigues, C.O.1, Barros, F.S.1, Lopes, A.H.C.S.1, Souto-Padrón, T.2, Dutra, P.M.L.1, Grillo, L.A.M.1 and Meyer-Fernandes, J.R.3

1Instituto de Microbiologia Prof. Paulo de Góes, 2Instituto de Biofísica Carlos Chagas Filho, 3Departamento de Bioquímica Médica, I.C.B., Universidade Federal do Rio de Janeiro, Ilha do Fundão, 21941-590, Rio de Janeiro, R.J., Brasil.

Acid phosphatase activity has been characterized in some members of the family Trypanosomatidae, like Trypanosoma, Leishmania, Phytomonas and Herpetomonas. A few very important physiological roles have been attributed to this enzyme, as hydrolysis of phosphomonoesters, to provide the parasites with a source of inorganic phosphate, involvement in cell differentiation and adaptation of the microorganisms to their intracellular environment.

Platelet activating factor (PAF) is a potent phospholipid mediator which exerts a wide range of biological activities such as hemostasy, inflammation, allergy and cellular differentiation. PAF promotes its effects by activating specific cell surface receptors and signal transduction between these receptors and their effector systems, which involve G-protein(s) and the activation of phospholipase C. As a consequence, hydrolysis of polyphosphoinositides generats IP3, which promotes the release of Ca2+ from intracellular stores and DAG, which activates protein kinase C (Chao & Olson, 1993. Biochem. J. 292:617-629). We have recently shown that PAF triggers the process of cell differentiation of Trypanosoma cruzi (Rodrigues et al, 1996. Biochem. Biophys. Res. Comm. 223:735-740) and of Herpetomonas muscarum muscarum (Lopes et al, 1997. J. Euk. Microbiol. 44(4): 321-325).

In this work we used Trypanosoma cruzi clone Dm 28C grown in LIT (liver infusion tryptose) for 5 days at 28o C. The effects of 10-9 M PAF on both ecto-phosphatase and on secreted phosphatase activities were investigated. The control intact parasites shown a phosphatase activity of 5.7 + 0.42 and the PAF treated parasites shown an activity of 8.63 + 0.82 nmol Pi / mg. min. The phosphatase activity measured in the supernatant of the control cells was of 0.50 + 0.09, while in the supernatant of the PAF treated parasites the activity was of 0.98 + 0.09 nmol Pi / mg. min. These effects were also shown by cytochemical analysis. Both ecto-phosphatase and secreted phosphatase activities of the PAF treated parasites were reverted to the control levels by a PAF-receptor specific antagonist, WEB 2086, which suggests the presence of PAF receptors on the cell surface of Trypanosoma cruzi. We used the protein kinase C modulators sphyngosine (50 ng/ml) and TPA (20 ng/ml), which abrogated PAF-induced effects on ecto-phosphatase and secreted phosphatase activities, suggesting the involvement of PKC in these processes.

Supported by: FINEP, CNPq and PRONEX (41/96 0885.00)


149

ECTO-PHOSPHATASE ACTIVITY IN TRYPANOSOMA CRUZI : A FURTHER COMPARATIVE STUDY BETWEEN Y AND COLOMBIANA STRAINS

Couto, L.M.1, Dutra, P.M.L.1, Souto-Padrón, T.3, Teixeira, R.L.F.1, Leite-Lopes, F.1, Meyer-Fernandes, J.R.2, Saad-Nehme, J.2 & Lopes, A.H.C.S.1

1Instituto de Microbiologia Prof. Paulo de Góes, 2Departamento de Bioquímica Médica, I.C.B., 3 Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Ilha do Fundão, 21941-590, Rio de Janeiro, R.J., Brasil.

It has been reported that acid phosphatase is present in some members of the family Trypanosomatidae, such as Trypanosoma and Leishmania. In Trypanosoma cruzi acid phosphatase was found to be bound to the plasma membranes (Pereira et al., 1985. Exp. Parasitol. 46 : 225-234) and in Leishmania spp, the secreted acid phosphatase is supposed to be a virulence marker. So far, the role of this enzyme in these protozoa keeps unknown. However, these enzyme seems to be involved in important processes such as cell differentiation, nutrition, adaptation of the microorganisms to their intracellular enviroment, protection in the digestive tract of the insect vector and metacyclogenesis, among others. In this work, we have characterized a phosphatase activity bound to plasma membranes of intact cells of two strains of Trypanosoma cruzi: a myotropic strain (Colombiana) and a macrophagotropic one (Y). It has been shown that Colombiana strain is more infective to heart mouse muscle cells than Y strain (Meirelles, M.N.L. et al., 1986. Europ. J. Cell Biol. 41: 198-206) and to myoblasts (Araújo-Jorge et al., 1986. Z. Parasitenk. 72 : 577-584). The parasites were grown in LIT (liver infusion tryptose medium) for 7 days at 28°C. Y strain parasites presented two enzymes: one independent of Mg2+ and other Mg2+-stimulated, while the Colombiana strain parasites seem to have only one enzyme bound to the plasma membrane, which is not stimulated by Mg2+ as well as a secreted phosphatase. The two enzymes of Y strain shown a distinct pattern of response to pH variation. In the pH range from 6.0 to 8.0, the Mg2+-independent activity reached a maximum at pH 6.0, whereas the Mg 2+-stimulated activity reached a maximum at pH 8.0. Both Mg2+-independent activity of Y strain and the ecto-phosphatase activity of Colombiana strain decreased with the concomitant increase of pH. On the other hand, the Mg2+-dependent activity increased with the concomitant increase of pH. These three enzymes presented a distinct pattern of sensitivity to some classical phosphatase inhibitors, such as sodium orthovanadate, sodium tartrate, sodium fluoride and zinc chloride.

Supported by: FINEP, CNPq and PRONEX (41/96 0885.00)


150

PARTIAL CHARACTERIZATION OF SECRETED PHOSPHATASE OF TRYPANOSOMA CRUZI COLOMBIANA STRAIN

Couto, L.C.1, Meyer-Fernandes, J.R.2, Souto-Padrón, T.3, Santos, M.A.A.1, Lopes, A.H.C.S.1, Jesus, J.B.1 & Dutra, P.M.L.1

1Instituto de Microbiologia Prof. Paulo de Góes, 2Departamento de Bioquímica Médica, I.C.B., 3 Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Ilha do Fundão, 21941-590 Rio de Janeiro, R.J., Brasil.

Phosphatase activity has been characterized in some members of theTrypanosomatidae family such as Leishmania and Trypanosoma. It has been suggested the involvement of these enzymes in some important process to parasite survival, such as cell differentiation and adaptation of the microorganisms to their intracellular enviroment. In Leishmania spp, secreted acid phosphatase activity is supposed to be a marker of virulence and it is considered to be one of the mediators of Leishmania-macrophage interaction (Vannier-Santos et al 1995. Eur. J. Cell Biol. 67:112-119). Phosphatase activity has also been reported to be related to the metacyclogenesis process of Trypanosoma brucei and Trypanosoma cruzi. In this work, we have characterized the secreted phosphatase activity of Trypanosoma cruzi Colombiana strain. It has been shown that this strain of Trypanosoma cruzi is more infective to heart mouse muscle cells (Meirelles et al., 1986. Eur. J. Cell Biol. 41: 198-206) and to myoblasts (Araújo-Jorge et al., 1986. Z. Parasitenk. 72 : 577-584) than some others strains, such as Y and CL. The parasites of Colombiana strain were grown in LIT (liver infusion tryptose medium) for 7 days at 28 °C. The cells were incubated in Tris-HCl (100 mM)/sucrose (250 mM), pH 6.8 for 1 h. The supernatant was used to detect the phosphatase activity and it was able to hydrolyze p-nytrophenylphosphate (p-NPP) at a rate of 3.00 nmol Pi/mg . min. A pH curve was done and the optimum activity was at pH 5.5. The secreted phosphatase shown a hyperbolic dependence of substrate concentration (Km = 6.91 mM p-NPP) . Sodium tartrate, a known secreted phosphatase inhibitor was able to inhibit the phosphatase activity in 80% and the classical phosphatase inhibitors, such as zinc chloride, sodium fluoride, sodium orthovanadate and amonium molybdate, were also able to inhibit this phosphatase activity (58%, 70%, 60% and 90% inhibition, respectively).

Supported by: FINEP, CNPq and PRONEX (41/96 0885.00)


151

PURIFICATION, CHARACTERISATION AND SUB-CELLULAR LOCALISATION OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE FROM TRYPANOSOMA BRUCEI PROCYCLIC FORMS

Heise, N & Opperdoes, FR

Research Unit for Tropical Diseases, lnternational Institute of Cellular and Moiecular Pathology (ICP), and Laboratory of Biochemistry, Catholic University of Louvain, Avenue Hippocrate 74+ I, B- 1200, Brussels, Belgium

Several lines of evidence suggest the presence of an operational pentose-phosphate pathway (PPP) in the procyclic form of Trypanosoma brucei: these are: (i) liberation of l4C 02 from [I-l4C]glucose; (ii) the requirement of ribose 5phosphate for the synthesis of nucleotides; and (iii) the detection in procyclic forms of all enzymes of the classical PPP. ln general the PPP was considerei to be entirely cytosolic and the first enzyme of the pathway, glucose-6-phosphate dehydrogenase (G6PDH), was even chosen as a marker for the trypanosome's cytosol in earlier cell fractionation experiments.

Now, by using digitonin titration and cell-fractionation experiments, we demonstrate that 30-45% of the total NADP+dependent G6PDH activity is associated with glycosomes. ln the purified organelles the specific activity of G6PDIÁ was increased by 4-5 times as compared to the starting homogenates. ln purified glycosomes, similar to the glycosomal marker enzyme hexokinase, G6PDH showed a latency of 88-97% and in the absence of detergents this activity was totaliy resistant to the action of trypsin. The cytosolic counterpart of G6PDH showed neither latency nor trypsin resistance. During purification both cytosolic and glycosomal G6PDH activities showed identical behaviour on phenyl-, CM-, heparin- and affigel-blue-sepharose columns. Both isoenzymes had a subunit molecular mass of 62 ± 2 kDa and an isoeletric point of 6.85. ln addition, kinetic studies carried out on the partially purified G6PDH isoenzymes also did not reveal any differences. Both enzymes had an apparent Km for glucose 6-phosphate and NADP+ of 150 gM and 5gM, respectively. We conclude that although in T brucei procyclics G6PDH activity is present in two different cell compartments, i.e. the cytosol and the glycosomes, these activities most likely represent one and the same isoenzyme. Supported by the National Bank of Belgium and by an ICP fellowship to N.H.


152

PURIFICATION AND PARTIAL CHARACTERIZATION OF TRYPANOSOMA CRUZI TRIOSEPHOSPHATE ISOMERASE

Bourguignon SC *, Meirelles MN**, Pacheco R * and Giovanni-De-Simone S */+

*Departamento de Bioquímica e Biologia Molecular, §Departamento de Ultraestrutura Celular, Instituto Oswaldo Cruz, FIOCRUZ, Av. Brasil 4365, 21040-900, Rio de Janeiro, RJ, Brasil; *Departamento de Biologia Celular e Molecular, Instituto de Biologia, Universidade Federal Fluminense, Niterói, RJ, Brasil.

The T. cruzi and other kinetoplastids are distinguished from diverse eucaryotic cells by the presence of glycosomes, a microbody-like organelle containing glycolytic and glycerol metabolic enzymes. These proteins are encoded by different genes and it was observed that the inhibition of some glycosomal enzymes of T. brucei may result in the death of this parasite. In view of this and that some of these enzymes may be a target for drugs design, in this work we purified and partially characterized the enzyme triosephosphate isomerase (TPI, EC 5.3.1.1) from T. cruzi. The purification steps included: hydrophobic interaction chromatography on phenyl-Sepharose, CM-Sepharose, and high performance liquid gel filtration chromatography. The CM-Sepharose material contained two bands (27 and 25 kDa) with similar isoelectric points (pI 9.3-9.5) which could be separated by HPLC gel filtration. Polyclonal antibodies raised against the porcine TPI, detected one single polypeptide on western blot with a molecular weight (27 kDa) identical to that purified from T. cruzi. These antibodies also recognized only one band of identical molecular weight in western blots of several other trypanosomatids (B. culicis, P. serpens, L. major like, H. s. pessoai). The presence of only one enzymatic form of TPI in T. cruzi epimastigotes was confirmed by agarose gel activity assay and its localization was established by immunocytochemical analysis. The T. cruzi purified TPI (as well as other trypanosomatid's TPI) is a dimmeric protein, consisted of two identical subunits with an approximate mw of 27,000 and it is resolved on two dimensional gel electrophoresis with a pI of 9.3. Sequence analysis of the N-terminal portion of the 27 kDa protein revealed a high homology to the L. mexicana and T. brucei protein.

Supported in part by the CNPq, FINEP and FIOCRUZ


153

PRELIMINAR STUDIES ON THE SUBCELLULAR LOCALIZATION OF ACID PROTEASE FROM LEISHMANIA AMAZONENSIS PROMASTIGOTES

Morgado Díaz J.A1., Alves C.R1., Soares M.J2., and Giovanni De Simone S1,3.

1 Departamento de Bioquímica e Biologia Molecular, Instituto Oswaldo Cruz, FIOCRUZ, RJ, Brasil. 2 Departamento de Ultraestrutura e Biologia Celular, Instituto Oswaldo Cruz, FIOCRUZ, RJ, Brasil. 3 Departamento de Biologia Celular e Molecular, Instituto de Biologia, Universidade Federal Fluminense, Niteroi, RJ, Brasil

Parasite proteases are being extensively studied to elucidate their roles in parasite survival and pathogenicity, and also with a view to exploiting the enzymes as targets for novel anti-parasite agents. Previous studies from our laboratory (Mem. Inst. Oswaldo Cruz. Vol. 91, Suppl. 193) have reported the purification and partial characterization of an aspartic proteinase (AP) from Leishmania amazonensis. The purified enzyme was obtained using a differential extraction procedure followed by affinity chromatography and gel-filtration HPLC. In the present work, we describe data on the subcellular distribution of this enzyme from the same parasite. Four representative fractions were achieved using differential centrifugation, after cell rupture. The cell breakage method used a combination of osmotic stress followed by mechanical shear (Dounce homogenizer). This step aided the separation of different organelles which seem to maintain their morphological characteristics as assessed by electron microscopy. Enzymatic assays of this fractions were carried out using 2.5% hemoglobin as a substrate in 100 mM sodium citrate, pH 3.5, containing 0.1% Triton X-100, and at 37°C. Acid protease activities were observed in all fractions with a significant enrichment (5-fold) in the microsomal fraction, as compared to the whole homogenate. This activity was sensitive (range 70 - 100%) to an inhibitor specific for aspartic protease. Furthermore, when the different fractions were analyzed to proteinase activity by SDS-PAGE gels containing copolymerized gelatin, the same molecular weight band was associated with the four fractions, however, with noticeable differences in activities among they. This data suggest that the enzyme is associated mainly with the microsomal fraction, at least in this parasitic developmental stage.

Supported by FAPERJ/ FIOCRUZ, CNPq


154

PURIFICATION AND PARTIAL CHARACTERIZATION OF LEISHMANIA AMAZONENSIS SERINE PROTEASES

Lopez RES and Giovanni-De-Simone S.

Departamento de Bioquímica e Biologia Molecular, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, RJ, Brasil; Departamento de Biologia Celular e Molecular, Instituto de Biologia, Universidade Federal Fluminense, Niterói, RJ, Brasil

In recent years, groups of investigators have begun to characterize on a molecular level proteinases of infectious agents since the inhibition of their activities may be a new approach to anti-infectious therapy. So, many proteinases of flagellate protozoa have been purified and or cloned, and their principal functional properties revealed. In our study, we are investigating the biological importance and functional properties of the L. amazonensis serine proteinase. This enzyme appear to be widely distributed in eukariotic systems where it plays different but important functions such as intracellular protein degradation and, perhaps more importantly, in post-translational processing of some biologically important protein and cells of immune system. Thus in order to investigate the physiological function of this proteinase class it is necessary the purification of the protein in a native state. So, two L. amazonenses serine proteinases were identified, purified and partially characterized. The purification involved freeze-fraction, differential centrifugation, detergent extraction and a step of affinity chromatography followed by gel filtration HPLC.

The concentration of the water-soluble enzyme was 2.5 times higher than the detergent-soluble protein. While the first presented a higher affinity to lower substrate, the second was more active to larger ones, as gelatin and azocasein. Both were inhibited by specific serine protease inhibitors (PMSF, TPCK, TLCK and others) but differed in the kinetics parameters, optimum pH and substrate specificity. They were thermostable from 25 to 42oC and heat label above 50oC.

Supported by CNPq and FIOCRUZ.


155

MUTATIONS IN PLASMODIUM FALCIPARUM DHFR & DHPS OF ISOLATES FROM THE AMAZON REGION OF BRAZIL.

Vasconcelos, KF1, Marques, C2, Silveira, MS1, Pang. L2, Milhous, WK2, Wirth, DF4, Plowe, CV4 and Zalis, MZ4

1Instituto de Biofisica Carlos Chagas Filho, Federal University Brazil; 2Department of Experimental Therapeutics, WRAIR, Washington, DC; US-Medical Research Unit-Brazil, AmConGen-Rio APO 34030, 3University of Maryland School of Medicine and 4Harvard School of Public Health

Since the late 1970s Fansidar-TM (pyrimethamine + sulfadoxine, PS; Hoffman-LaRoche) has been used for the first line therapy of uncomplicated malaria in the Amazon basin. Unfortunately, resistance has developed over the last 10 years in many regions of the Amazon and PS now can no longer be used in Brazil. In vitro resistance to pyrimethamine and cycloguanil (the active metabolite of proguanil) is caused by specific point mutations in P. falciparum dihydrofolate reductase (DHFR), and in vitro resistance to sulfadoxine has been associated with mutations in dihydropteroate synthase (DHPS). In association with a proguanil / sulphametaxazol clinical trial in Brazil , we performed a nested mutation-specific PCR to measured the prevalence of DHFR mutations at amino acids positions 50, 51, 59, 108 and 164 and DHPS mutations at positions 436, 581 and 613. All the analyzed samples contained the mutants Asn-108, Arg-59, Ile-51 and the wild type Ile 164. These findings indicate a association between these mutations and a high level of in vivo PS resistance in Brazil. Although proguanil has not previously been used in Brazil and the Leu-164 mutation conferring cross resistance to pyrimethamine and cycloroguanil was not found, in vitro tests with these isolates showed some reduced susceptibility to cycloguanil. All parasites analyzed showed the wild type codons Ser-436, Ala-581, Ala-613.

This work was supported by CNPq and NIH/NIAID


156

INHIBITION OF BIOSYNTHESIS OF GLYCOPROTEINS IN PLASMODIUM FALCIPARUM TREATED WITH COMPACTIN.

Kimura E.A., Couto A.S.1 Peres V.J., and Katzin A.M.

Depto de Parasitologia ICB-USP, Brasil. 1 Depto de Quimica Orgânica FCEyN-UBA, Argentina.

In previous studies, we have demonstrated that compactin (3-hidroxy-3-methylglutaryl coenzyme-A reductase inhibitor) possess antiparasitic activity in vitro against intraerythrocityc P. falciparum stages. Differences in profile from ring, young and old trophozoite stages (between treated and untreated parasites) were detected by SDS/PAGE after labeling with D-[U14C] glucose.

In the present study we show that treatment with 120 µM compactin inhibits N-linked glycoproteins.

This fact was demonstrated by treating P. falciparum lysates corresponding a each stage with specific Glycanases (N- and O-Glycanase). An asynchronous culture of P. falciparum (25% parasitemia) was metabolically labeled for 18h with D-[U-14C]glucose and each stage was purified using Percoll gradient. The samples were analysed by SDS/PAGE side by side with samples treated with compactin (120 µM). The same bands that disappeared or decreased in intensity after treatment with N-Glycanase corresponded to bands that disappeared or decreased in intensity when parasites were treated with compactin. We are interested to know if this fact is correlated with lack of production of dolichol. Preliminary results, analysed by HPLC from samples obtained of parasites labeled with [1-14C]acetic acid show one peak corresponding to dolichol whereas in samples obtained from parasites treated with compactin this peak disappears. Further experiments are necessaries to confirm this data.

Supported by FAPESP, CNPq, Fund Antorchas, CONICET, UBA.


157

EFFECT OF STEROLS BIOSYNTHESIS INHIBITORS ON CARBOHIDRATES TRANSPORT OF TRYPANOSOMA CRUZI EPIMASTIGOTES

Vivas1, J,. y Perez-Kepp2 y Ruiz E1 ,3.

1 Instituto de Biología Experimental. Facultad de Ciencias. Universidad Central de Venezuela. Apartado 47860,

Los Chaguaramos, Caracas 1041 D.F. Venezuela. 2 Departamento de Biología. Facultad de Ciencias y Tecnología. Universidad de Carabobo. Valencia. Edo Carabobo. 3 Universidad Simón Bolivar

Previously we showed the inhibitory action of two sterols analogs, 22,26-azasterol (AZA) and 24(R,S)25-epiminolanosterol (EIL), and a derivate of imidazol, Ketoconazole (Keto), on proliferation of T.cruzi epimastigotes and amastigotes, as well as strong change of the neutral lipid profiles of epimastigotes treated. Both effect were very closed related (Urbina, Vivas y col. Chemotherapy. 42:294-307. 1996, Urbina, Vivas y col. Mol. Biochem. Parasitol. 73:199-210. 1995). Now we report the action of this compound on D-glucose transport in T.cruzi epimastigotes. The drugs were added in exponential growth phase, 96 hours after the parasites were collected and made the transport assays with [14C]-2-deoxiglucose, a non metabolized analog. We have obtained effect on transport with concentration from 1 to 3 µM of the drugs used. We did not observe effect on the initial rate of accumulation but we obtained 50% of maximum accumulation. The efflux in precharged cells was higher in treated cells when compared with control cells. We will discuss the synergetic effect of the combination of compounds.


158

N-LINKED OLIGOSACCHARIDE STRUCTURES OF THE FML ANTIGEN OF LEISHMANIA DONOVANI.

Bernardo, RR1, Palatnik de Sousa, CB2 & Parente, JP1.

Núcleo de Pesquisas de Produtos Naturais1 & Instituto de Microbiologia "Prof. Paulo de Góes", Universidade Federal do Rio de Janeiro, Caixa Postal 68040, Rio de Janeiro, 21941-590, RJ, Brasil.

Leishmania are digenetic parasites which are the causative agents in a number of serious diseases affecting human populations throughout the tropical and subtropical world. The Fucose Manose Ligand (FML) is a complex glycoprotein fraction present on the surface of pro and amastigotes of L. donovani, that strongly inhibits the in vitro macrophage infection by both forms of the parasite. Manose (47%), Galactose (12%), Glucose (30%) and Fucose (10%) were detected as its main sugar components. In this work, we describe the isolation of the N-linked oligosaccharides of the FML antigen of L. donovani, their separation by HPLC, gel permeation chromatography and their caracterization by chemical analysis. The FML fraction was prepared according to Palatnik et al (1989. Infection and Immunity. 57: 754). The oligosaccharides were released from FML by hydrazinolysis (Bayard & Fournet. 1975. Carbohyd. Res. 46: 75). These conditions separe the glicidic from the protein moieties of N-linked glycoproteins. The resulting oligosaccharides were N-reacetylated according to Reading (1978. J. Biol. Chem. 253: 5600) and further reduced with NaBH4. The oligosaccharides were subjected to HPLC on an 5mm RP-18 column using 500mM potassium dihydrogen phosphate (KH2PO4) as eluent giving 15 fractions. The major fraction (33.5 %) was purified by Bio-Gel P-2 column chromatography. GLC analysis disclosed the presence of Gal, Man, Fuc. Methylation analysis of this fraction gave residues of galactopyranose (2,3,4,6, Me4-Gal, 32.8%), manopyranose (2,3,6 Me3-Man 41.5%, 2,4,6 Me3-Man 22.4%), fucopyranose (3,4-Me2-Fuc 3.2%) and 3,4,6, Me3-Man in trace amounts, which were identified and quantitated by GLC-MS.

Support: PRONEX, FNS-PNUD, CNPQ, FINEP, RHAE-CNPQ, CEPG-UFRJ, FUJB-UFRJ.


159

OUABAIN-INSENSITIVE Na+-STIMULATED ATPase ACTIVITY IN TRYPANOSOMA CRUZI EPIMASTIGOTES

Einicker-Lamas, M , Caruso-Neves, C , Oliveira, MM and Lopes, AG

In eukaryotes the principal primary active transport involved in the translocation of Na+ through the plasma membrane is the ouabain sensitive (Na++K+) ATPase. Recently, we have demonstrated the presence of this enzyme in T. cruzi (Caruso-Neves, C et al. in press). In the present work, we studied the presence of the ouabain-insensitive Na+-ATPase in T. cruzi epimastigotes

We used epimastigotes from CL 14 clone and NIH NTY strain. The ATPase activity was measured as described (Grubmeyer, C and Penefsky, HS. J.Biol.Chem. 256:3718-3727, 1981). In the presence of 10mM Mg2* and 2mM ouabain, the ATPase activity was 77,1±12,0 and 66,9±6,8 nmol Pi x mg-1 x min-1 in the CL 14 clone and NIH NTY strain respectively. The addition of 100mM Na+ increased the ATPase activity to 103,7±9,0 and 96,1±9,2 nmol Pi x mg-1 x min-1 in the CL 14 clone, and NIH NTY strain respectively. The Na+ stimulated ATPase activity was completely abolished by the addition of 2mM furosemide, or in a Mg2+-free medium. We also observed that Na+ stimulated the ATPase activity in the presence of 2mM ouabain in a dose-dependent manner.

These results suggest that T. cruzi presents a Na+ stimulated ATPase activity, Mg2+-dependent, ouabain-insensitive and sensitive to furosemide. We are still working on this theme, to understand the complete role of this enzyme in the parasite homeostasis.

Supported by: PADCT, FINEP, CNPq and FAPERJ.


160

THE USE OF A TRIPEPTIDE COMBINATORIAL LIBRARY IN OBTAINING INHIBITORS OF A SERINE PROTEASE FROM T. CRUZI.

Vendeville, S.a, Grellier, P.b, Santana, J.M.c, Teixeira, A.R.L.c, Schrevel, J.b & Sergheraert, C.a

a Institut de Biologie et Institut Pasteur de Lille, URA 1309 CNRS, Faculté de Pharmacie, 1 rue du Pr. Calmette, 59021 Lille, France

b Laboratoire de Biologie Parasitaire et Chimiothérapie, ERS CNRS 156, Muséum National d'Histoire Naturelle, 61 rue Buffon, 75231 Paris Cédex 05

c Laboratorio Multidisciplinar de Pesquisa em Doença de Chagas, Departementos de Biologica Celular e de Patologia, Universidade de Brasilia, CP 04436, 70919-970, Brasilia

The combinatorial approach was selected to quickly identify a lead for Tc 80, a serine protease found in each of the three stages of T. Cruzi, the aetiological agent of Chagas disease. This protein, which could possess the ability to facilitate the infection of host cells by the degradation of collagens of the extracellular matrix, was obtained by four successive steps of column chromatography.

An inhibition test using the fluorogenic substrate N-suc-GPLGP-Amc was employed to screen a combinatorial library containing 15625 tripeptides prepared from 25 unnatural amino acids (23 D and 2 non-chiral), and distributed in 125 sub-libraries of 125 mixed trimers. From the most active sub-library on Tc 80, deconvolutions were carried out in order to isolate the trimer(s) responsible for inhibition. In this way, for each position (N-terminal, intermediate, C-terminal), the amino acid corresponding to the optimum inhibition was determined.

Synthesis of the selected tripeptide yielded an inhibitor in the low micromolar range (10 µm), which was identified as a Ipe-DTic-Glu p.thiocresol by-product formed in the peptide-resin cleavage step (p.thiocresol was employed as a carbocation scavenger). The inhibitor proved inactive toward other enzymes such as trypsin or chymotrypsin; was effective against the invasion of rat muscular cells (L6) by the parasite and was therefore chosen as a lead.

Several modifications to the lead were studied to establish structure-activity relationships.

- replacement of the reactive p.thiocresol moiety by more stable entities: p.cresol, p.toluidine in a or g position of the glutamic residue.

- replacement of the N-terminal residue, which according to deconvolution in this position, proved less important for recognition.

Funding sources : CNRS and Région Nord - Pas de Calais – Picardie


161

MULTIPLE MECHANIMS DETERMINE DRUG-RESISTANCE AGAINST PEPTIDYL-BASED INHIBITORS OF T. CRUZI CYSTEINE-PROTEASES

Yong, V., Schmitz, V., Sampaio, T.C., Lima, A.P.L. and Scharfstein, J.

Instituto de Biofisica Carlos Chagas Filho-UFRJ, Dept. Bioquímica Médica, ICB, Rio de Janeiro.

In the past few years, the systematic biochemical characterization of T. cruzi has uncovered new potential targets for anti-parasitic drugs. Notable among these are a group of cathepsin L-like cysteine proteinases (cruzipain or cruzain) that are highly sensitive to peptidyl diazomethane inhibitors such as Z-(SBz)Cys-Phe-CHN2. We have previously shown that these membrane-permeable inhibitors can effectively block the intracellular multiplication of amastigotes. Recently, a mutant cell line (R-Dm28) displaying phenotypic resistance to this cathepsin L-like peptidyl inhibitor (LD50 20 mM vs 1.5 mM) was obtained by applying seletive pressure on Dm28 epimastigotes. R-Dm28 had a stable resistant-phenotype in the absence of the seletive drug. Drug-resistance appears to be seletive for the peptidyl CP inhibitor because the cell-line remained as sensitive to unrelated trypanocydal drugs (eg. benznidazol) as wildt-type cells. Importantly, R-Dm28 cells transformed into metacyclics and were infective in vitro. We now report that multiple mechanims contribute to the drug-resistance phenotype. Using monoclonal antibodies and biotinylated peptidyl diazomethane inhibitors to analyse the intracellular contents of cruzipain on Wt-Dm28 vs R-Dm28, we observed that this protease is drastically reduced in the mutant cell lines; consistent with these findings, northern blotting indicated that the cruzipain message was 1.8 fold lower in R-Dm28 epi. This abnormality has led to a drastic reduction in polypeptide catabolism, this being accompanied by a remarkable increase in intracellular concentrations of soluble proteins. Interestingly, the decreased accumulation of the cathepsin L-like cruzipain target was compensated by a relative increase in the contents of a different cysteine-protease (30 KDa). This protease was less sensitive to the cathepsin L-like inhibitor, being thus possibly involved in the resistant-phenotype. We then labelled the N-terminus of NH2-(SBz)cys-Phe-O-Me with the fluorochrome BODIPY, and used this probe to measure rates of cellular uptake by FACS. Our data showed that R-Dm28 had 36% lower uptake of BODIPY-(SBz)Cys-Phe-O-Me as compared to wild-type epimastigotes. There was no evidence for enhanced extrusion rates in these cells. In conclusion, the adaptive changes of R-Dm28 seem to depend on at least two independent but converging mechanisms: decreased drug internalization and reduced target availability. Supported by PADCT, CNPQ, CAPES.


162

PURIFICATION AND CHARACTERIZATION OF AN 73-KDA SIALIDASE SECRETED BY TRYPANOSOMA RANGELI EPIMASTIGOTE.

1,2Saldaña, A.**, 1Harris, R., 1Orn, A. & 2Souza, O.E.

1Microbiology and Tumorbiology Center, Karolinska Institute, BOX 280, S-17177, Stockholm - SWEDEN and 2Center for Research and Diagnosis of Parasitic Diseases, Faculty of Medicine, University of Panama - PANAMA.

The epimastigote stage of Trypanosoma rangeli release a sialidase with a high sialic acid hydrolysis capacity. We demonstrated that the sialidase secretion is an active process that is reduced at low temperatures and in the presence of sodium azide. The enzyme is continuously released until certain maximally active concentrations are attained in the BHI culture medium when the parasite density reach 20 - 30 million/ml. When introduced into culture medium already containing such enzyme levels, freshly harvested parasites do not secrete addicional sialidase. These findings suggest a self regulating mechanisms and a biological role for the secreted T. rangeli sialidase. The secreted enzyme was purified to homogeneity by fractionation with ammonium sulphate and mucin affinity chromatography. No change of molecular weight (73 KDa) or additional bands were observed after DTT treatment, suggesting that the enzyme is not composed by different polypetide chains hold together. Also DTT at 20 mM did not affect the enzymatic activity indicating that thiol groups are not essential for catalytic activity. Antibodies raised against the purified molecule recognized antigens of similar molecular weight in the immunoblotting analysis of T. rangeli and T. cruzi whole cell lysates. No antigenic recognition were recorded against T. cruzi concentrated tissue culture supernatant which express high sialidase/trans-sialidase activity or comercial bacterial sialidases. These observations may indicate a poor antigenic cross-reaction between the T. rangeli sialidase and the active sialidase/trans-sialidase molecules (120-220 KDa) of T.cruzi.

Supported by Sida/SAREC and CIDEP - University of Panama.


163

TRYPANOTHIONE-DEPENDENT PEROXIDASE ACTIVITY IN TRYPANOSOMA CRUZI

López, J.A1., Nogoceke, E.2, Montemartini, M2., Kalisz, H2., Flohé, L2., Carvalho, T. U3,4., de Souza, W3,4. Colli, W.1 & Alves, M.J.M.1

1Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, C.P. 26077, 05599-970 SP, Brazil

2Gesellschaft für Biotechnologische Forschung mbH (GBF), Braunschweig-Stöckheim, Germany

3Universidade Estadual do Norte Fluminense (UENF) and 4Universidade Federal do Rio de Janeiro (UFRJ)

Protozoan parasites are exposed to a variety of oxygen concentrations depending on whether they are intracellular parasites or remain free in circulation. In either case, the cells need to be protected against the products of the partial reduction of oxygen. Trypanosomes are particularly weak in antioxidant defenses. Although they possess superoxide dismutase activity, they lack glutathione peroxidase and catalase which are necessary for the removal of hydrogen peroxide. Moreover, glutathione, the major antioxidant sulphydryl compound in mammalian cells, is present in low concentrations in the trypanosomes. Instead they contain a unique cofactor known as trypanothione (N1,N8-bis(glutathionyl)spermidine) which together with its ancillary enzymes, trypanothione reductase and trypanothione peroxidase, appear to play a central role in the antioxidant defense mechanisms of the trypanosomatids and would represent particularly important targets for a direct chemotherapeutic attack.

A low but measurable activity of trypanothione peroxidase was found in crude, liophylised extracts of epimastigotes and trypomastigotes of T. cruzi using t-butyl hydroperoxide as oxidant. The reaction was measured by following spectrophotometrically the transformation of NADPH to NADP in the presence of externally added trypanothione reductase purified from C. fasciculata. A polyclonal antibody raised against a bona fide trypanothione peroxidase from C. fasciculata was employed to identify, by immunoprecipitation and Western blot, a T. cruzi protein with an apparent molecular mass of 21 kDa. This antibody recognized internal structures in epimastigotes and trypomastigotes as detected by indirect immunofluorescence and immunoelectronmicroscopy.

Oligonucleotides constructed with sequences based on the DNA coding sequence of trypanothione peroxidase from C. fasciculata were synthesized. Using these primers in PCR assays with total T. cruzi DNA, a product with approximately 600 bp was amplified. Southern and Northern blot analyses allowed the detection of the T. cruzi gene for the peroxidase and its corresponding mRNA. The available evidence confirms the existence of trypanothione peroxidase in T. cruzi, as opposed to previous claims.

J. A. López is a doctoral student from CNPq . Work supported by FAPESP and CNPq/PADCT.


164

THE REPETITIVE DOMAIN OF TRYPANOSOMA CRUZI TRANS-SIALIDASE MODULATES THE ENZYMATIC ACTIVITY IN BLOOD

Buscaglia, C.A.*, Campetella, O.E.*, Leguizamón, M.S.‡ & Frasch A.C.C.*

*Instituto de Investigaciones Biotecnológicas, Universidad Nacional de General San Martín; (1650) San Martín, Casilla de Correo 30, and ‡Departamento de Microbiología, Facultad de Medicina, Universidad de Buenos Aires, Argentina.

Supported by the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR), the Department for Research Cooperation (SAREC) from the Swedish International Development Cooperation Agency (SIDA) and the Howard Hughes Medical Institute.

The trans-sialidase, a specific virulence factor from Trypanosoma cruzi, seems to be a naturally chimeric protein containing a catalytic region responsible for the enzymatic activity and a C-terminal extension composed essentially of immunodominant repetitive sequences (termed SAPA-repeats). The presence of repetitive sequences is a common feature among antigens from several protozoan parasites, nevertheless their possible roles are still poorly understood. Due to their immunogenicity, they are suggested to act as immune distractors, preventing and/or delaying the humoral immune response against relevant molecules. To investigate the in vivo functions of SAPA-repeats, we have intravenously administered recombinant trans-sialidases either containing or lacking the C-terminal domain in mice. Since trans-sialidase is shed into the bloodstream by infective trypomastigotes, this immunization route is expected to mimic the effect of enzyme released during the course of infection. Here we show that the solely presence of SAPA-repeats in cis accounts for two unexpected functions. On the one hand they enhanced the persistence of the trans-sialidase activity in blood by an yet undefined mechanism. On the other hand, the early and strong antibody response against the immunodominant SAPA-repeats did not prevent but instead promoted the production of antibodies directed to the enzymatic domain that inhibited its activity. These latter antibodies were only generated when the immunization was performed with a native trans-sialidase, suggesting that an active conformation of the molecule is required. Altogether, these results strongly suggest that SAPA-repeats, albeit not necessary for the enzymatic activity, modulate the host immune response against the catalytic domain through a dual mechanism, initially favoring the infection process and, on a later phase, helping to control parasite dissemination and ensuring both, host and parasite survival.


165

STUDY OF A CDC2- RELATED PROTEIN KINASES (CRK) IN TRYPANOSOMA CRUZI

Gómez, E. B., Santori, M. I. & Tellez-Iñón, M. T. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular (INGEBI-CONICET) and Facultad Ciencias Exactas y Naturales (UBA), Buenos Aires, Argentina

In eukaryotic organisms G1/S and G2/M cell cycle transitions are controlled by the activity of cyclin-dependent protein kinases (CDK). A cdc2-related protein kinase, TCRK2, was cloned from the protozoan parasite Trypanosoma cruzi.. TCRK2 encodes a 33 kDa protein sharing 52.7 % identity with human cdc2 and a high degree of identity (> 78 %) with T. brucei CRK1, Leishmania mexicana CRK1 and T. congolense CRK1.

TCRK2 was expressed in E.coli as a fusion protein coupled to gluthation-S transferase. The purified recombinant protein was able to phosphorylate histone HI and retinoblastome protein and was used as antigen to raise a polyclonal antibody. Western blot analysis with this antiserum showed a single 33 kDa protein in whole cell extracts of the three life cycle stages of the parasite. No differences in protein expression can be observed between the three forms. In many species a specific antiserum (anti-PSTAIRE) able to recognize protein kinases of the CDC2 family is used to analyze CRK proteins. The PSTAIRE antiserum recognized, at comparable concentrations, a 33 kDa protein in the different stages of the parasite; this polypeptide is likely to be TCRK2. The antiserum also revealed, in all three stages, a 32 kDa protein with higher expression in epimastigote forms. A third 35 kDa protein also detected by the PSTAIRE antiserum, was not present in amastigotes, slightly expressed in trypomastigotes and highly in epimastigote forms. This antiserum cross-reacted with the recombinant GST-TCRK2 protein, in which the serine of the PSTAIRE box is substituted by a cysteine.

Western blot analysis with the TCRK2 antiserum showed that this protein is localized mainly in the nuclear and cytosolic fractions of the parasite. TCRK2 antiserum was used to immunoprecipitate the crk protein from the cytosolic fraction (S100) and was tested for kinase activity; the IPs were able to phosphorylate histone H1 and the retinoblastome protein.

To determine whether tcrk2 could complement the cdc28 gene function, tcrk2 was used to transform a S. cerevisiae cdc28 temperature-sensitive (ts) mutant. Under these conditions the T. cruzi protein could not rescue the cdc28-4 ts mutation.

The identification of a role for each of the CRKs proteins should help to resolve how trypanosomes control their cell cycle during differentiation.


166

DETRIMENTAL EFFECT OF NITRIC OXIDE ON TRYPANOSOMA CRUZI AND LEISHMANIA MAJOR- LIKE CELLS

Bourguignon S. C., Alves C.R. and Giovanni-De-Simone S.

Departamento de Bioquímica e Biologia Molecular, Instituto Oswaldo Cruz, FIOCRUZ, Av. Brasil 4365, 21045-900, Rio de Janeiro, RJ, Brasil; Departamento de Biologia Celular e Molecular, Instituto de Biologia, Universidade Federal Fluminense, Niterói 24210, RJ, Brasil.

Intracellular parasites of mammalians as Leishmania and Trypanosoma cruzi undergo multiplication almost exclusively in macrophages. Although, several assumptions have been aimed concerning mechanisms whereby these parasites inhibit or resist toxic processes generated in this cell the reason of its survive within the potentially severe environment of the phagolysosomal compartment remains an intrigating and unsolved question. The production of nitric oxide (NO) by activated macrophages has been reported to be a non-specific immune-effect mechanism against several parasites. In this work we investigate if the NO might have a detrimental effect on the intracellular Leishmania and T. cruzi parasites.. This was assessed by cocultivating infective (promastigotes of Leishmania and trypomastigotes of T. cruzi) and non-infective (epimastigotes) forms of the parasite with the NO releasing molecule, S-nitroso-N-acetyl-DL-penicillamine (SNAP). It was demonstrated that the NO inhibits the growth of either parasite stage in a concentration dependant manner. In addition, it was demonstrated that the NO may regulate the GAPDH activity of both parasites. This was analysed both in purified protein and cell homogenates of L. major (promastigotes) and T. cruzi (epimastigotes and trypomastigotes). As this enzyme appears to be a multifunctional protein it may be regulated, via its S-nitrosilation (ADP-ribosylation), through intracellular concentration of NO. Thus, NO mediated cytotoxicity towards T. cruzi trypomastigotes and Leishmania promastigote forms may be correlated with glycolysis inhibition. Altered energy supply and cytotoxic NO effects are possible correlated. Accordingly, these observations may be useful to the understanding of the pathogenesis of these intracellular parasites and its relationship with macrophage cells.

Supported by CNPq, FINEP, FIOCRUZ


167

ACTIVATION OF CYTOLYTIC ACTIVITY IN LEISHMANIA GUYANENSIS MEMBRANE EXTRACTS BY LEISHMANIA AMAZONENSIS CYTOSOLIC FRACTION

Almeida-Campos, FR and Horta, MF.

Departamento de Bioquímica-Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, C.P. 486, Belo Horizonte, 30161-970, MG, Brasil

Previous studies from our laboratory have shown that extracts of L. amazonensis, but not of L. guyanensis, are lytic to erythrocytes and nucleated cells, including the macrophage. L. amazonensis-mediated lysis is caused by a membrane-associated pore-forming protein (leishporin), optimally active at acid pH (Horta, MF. 1997. Trends in Microbiology 5 (9), in press). Although the cytosolic fraction of the parasites is hemolytically inactive, it contains serine-proteases that can activate the membrane-associated cytolysin. In the present work, we show that membrane extracts of L. guyanensis promastigotes become hemolytic after incubation for 24 hours at 37ºC at pH 7.0 with the cytosolic fraction of L. amazonensis. The cytosolic fraction of L. guyanensis is unable to generate hemolytic activity in both L. amazonensis and L. guyanensis membrane extracts. These results suggest that L. guyanensis membrane fraction has a potential lytic activity, but the parasite does not have the protease(s) for the activation of the cytolysin. We have also investigated whether exogenous proteases could be effective in the generation of L. guyanensis cytolytic activity. The membrane fraction of promastigotes extracts was incubated for 24 h at 37ºC in the presence of the following proteases: trypsin, chymotrypsin, collagenase, proteinase K and pronase at the concentration of 1, 10 or 100 mg/ml. While effective to generate hemolytic activity in the membrane fraction of L. amazonensis, these enzymes were unable to generate cytolytic activity in the membrane fraction of L. guyanensis. This suggests that the cytolysins of the two species may be different in structure and/or are activated by different mechanisms.


168

EVALUATING OF THE MICRO-CALORIMETRIC TECHNIQUE FOR SCREENING OF DRUGS AGAINST LEISHMANIA (L.) AMAZONENSIS PROTOZOA.

* Silva-Alves, J.M.; * Santoro, M.M.; ** Melo, M.N.; * Mares-Guia, M.L.

* Dep. de Bioquímica e Imunologia, ICB-UFMG, Caixa Postal 486, Belo Horizonte, MG, Brasil.
** Dep. de Parasitologia, ICB-UFMG, Belo Horizonte, MG, Brasil.

Micro-calorimetry is a highly sensitive, non agressive technique for attaining measurements of thermodynamic parameters. It can be used to directly evaluate the metabolic activity of a given biological system by determining the rates of heat production. The micro-calorimetric signal (dq/dt versus time) results in a continuous register of the metabolic processes. Any biological substance capable of interfering with metabolism may lead to changes in the power/time curve, making possible the study of its biological activity. Considering that the basal metabolic rates of in vitro cultivated cells directly relates to heat production, we decided to apply conduction micro-calorimetry to establish a fast and useful methodology for the screening of anti-metabolic agents and drugs used in therapy of Leishmania (L.) amazonensis promastigotes. The results show that the ammount of heat released after addition of fructose (2.5 mM) is about 60% higher than the amount observed in basal metabolism during a 900 s incubation. Addition of Iodacetamide caused inhibition of released heat with concentrations higher than 2.5 mM, and this effect is more pronounced with concentrations over 7.5 mM. Glucantime (58.27 mM) did not cause any modification in heat release rates, indicating that under our experimental conditions the strain used for experiments is not sensitive to the drug, confirming reported data. Amphotericin B caused a rapid inhibition of heat releasing with concentrations of 0.45 to 4.51 mM. Increasing the concentration to the range of 45.09 to 450.89 mM cell death was observed. Summarizing, our results demonstrate that conduction calorimetry is a fast, sensitive and reproducible methodology to evaluate the metabolic conditions, to determine the time of cell death and to screen anti-metabolic agents and drugs used against Leishmania protozoa.

Supported by: CAPES, bioBRÁS and WHO.


169

ENERGY-DEPENDENT EFFLUX FROM LEISHMANIA PROMASTIGOTES OF SUBTRATES OF THE MAMMALIAN MULTIDRUG RESITANCE PUMPS

1Essodaigui, M., 2Frézard, F., 3Moreira, E.S.A., 1Garnier-Suillerot, A.

1Laboratoire de Physicochimie Biomoléculaire et Cellulaire, (CNRS - 2056), UFR Biomédicale, Université Paris Nord, 74 rue Marcel Cachin, Bobigny cedex 93017, France. 2Depart. de Fisiologia e Biofísica, 3Depart. de Microbiologia, ICB, UFMG, C.P.486, Belo Horizonte, 30161-910, MG, Brasil.

The underlying mechanisms that contribute to drug resistance in Leishmania (L.) parasites are still poorly understood. Recent works suggest that ATP-dependent efflux pump(s), similar to the mammalian P-glycoprotein, may be involved. Members of the P-glycoprotein gene (pgp) family have been identified in Leishmania, and variation in the pgp copy number have been implicated as a basis for drug resistance. On the other hand, an ATP-dependent pump, capable of transporting arsenite-glutathione complex, have been identified in everted plasma-membrane-enriched vesicles prepared from L.promastigotes. The aim of the present study was to investigate in intact promastigotes the presence of energy-dependent pump(s), using two fluorescent probes of the mammalian multidrug resistance (MDR) transporters, calcein acetoxy-methyl ester (Calc-AM) and the doxorubicin derivative pirarubicin (Thp-DOX). We observed that energy depletion of L. promastigotes enhanced intracellular accumulation of both Calc-AM and Thp-DOX. A L. braziliensis strain was found to be more efficient in reducing intracellular accumulation of Calc-AM than a L. (V.) guyanensis strain. An energy-dependent efflux of Thp-DOX was clearly demonstrated in the case of the L. braziliensis strain. Calc-AM, once incorporated in cells, is rapidly transformed into calcein by intracellular esterases. In desenergized promastigotes, we showed that calcein remained in the cytoplasm. However, when promastigotes were maintained in presence of glucose, calcein was extruded from the cytoplasm to the external medium. In conclusion, we identified, in intact L. promastigotes, energy-dependent efflux pump(s), through their ability to transport two substrates of the mammalian MDR transporters. These fluorescent dyes should be useful for further exploring the function of these transporters. (supported by Université Paris-Nord and FAPEMIG).


170

PURIFICATION OF LEISHPORIN, A CYTOLYSIN OF LEISHMANIA AMAZONENSIS

Noronha, FSM., Pontes, LA # and Horta, MF. #

Departamento de Bioquímica-Imunologia#, Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, C.P. 486, Belo Horizonte, 30161-970, MG, Brasil

We have recently shown that Leishmania amazonensis promastigotes extract are lytic to erythrocytes and nucleateds cells, including macrophages, the parasite's host cell. Lysis is colloid-osmotic and is mediated by a protein associated to the parasite membrane fraction (Noronha et al. 1996. Infec. Immun. 64: 3975). Using the patch clamp technique we have conclusively shown that parasite extracts induce the formation of non-selective pores on the macrophage membrane, indicating the presence of a pore forming cytolysin in L. amazonensis, which we called leishporin. Here, we describe the purification of leisporin using proteolytic treatment followed by liquid chromatography in FPLC. Previous results from our laboratory have shown that the treatment of the parasite membrane fraction with proteases, especially proteinase K, increases its specific hemolytic activity. This is possibly due to the activation of leishporin either by limited proteolysis of an inactive precursor or by degradation of a protein inhibitor. While hemolytic activity increases in the membrane extracts, there is a marked decrease in the total protein concentration. We took advantage of this fact as an approach to purify leishporin. Crude parasite extracts, obtained by freeze-and-thaw were centrifuged at 100,000 x g and the membrane-rich pellet was solubilized with 0.4% CHAPS. The solubilized membrane-associated molecules (sExt) were then treated with 10 mg/ml proteinase K. This treatment produces a 2- to 5-fold increase in the hemolytic activity of the extract and approximately 80% reduction in the protein content. Proteinase K-treated sExt was then submitted to a 3-step chromatographic purification, using Resource Q, pH 8.5, Phenyl Superose and again Resource Q pH 7.5, in this order. The hemolytic activity was monitored in each fraction and, after the last step, the hemolytic activity was co-purified with one single silver-stained band in the range of 55-60 kDa as detected by SDS-PAGE.

Supported by: CAPES, CNPq, FAPEMIG and FINEP


171

A ROLE FOR PI 3-KINASE IN THE INTERACTION OF T. CRUZI WITH MACROPHAGES

Einicker-Lamas, Ma,c, Malaquias, ATa, De Castro, SLc, Guilherme, ALb & Oliveira, Mecia M. a

aInstituto de Biofísica Carlos Chagas Filho e bDepartamento de Bioquímica Médica, Universidade Federal do Rio de Janeiro, 21949-900 Rio de Janeiro; cDepartamento de Ultraestrutura e Biologia Celular, FIOCRUZ, 21045.900 Rio de Janeiro, Brazil.

It is already well established that the T. cruzi invasion in non-phagocytic cells occurs through calcium (1-2) and TGF-b (3) signalling pathways in the host cells. As for the parasite interaction with phagocytic cells the above mentioned pathways were not observed (3), and its mechanism is not completely understood (4). We now propose, following experimental results, that one possible modulation for the interaction T. cruzi - macrophage implicates in the activation of the phosphoinositide-3 kinase (PI 3-K), recently recognised important metabolic pathway modulating many cellular processes (5).

Pre-treatment of macrophages with the PI 3-K inhibitor, wortmannin (1-20nM), blocked T. cruzi infection in a dose-dependent manner, reaching the maximum of 96% inhibition. PI 3-K activation during infection was observed by the appearance of a phospholipid among the lipids from [32P]-Pi pre-labelled macrophages with the same chromatographic mobility as authentic phosphatidylinositol-3-phosphate. Also in the in vitro assay of PI 3-K co-preciptated with anti P-Tyr antibodies6, the phosphorylation of exogenous substrate was enhanced in infected cells. Our results suggest a possible role for the PI 3-K pathway in the interaction of the parasite with that host cell, and further experiments are under way to clarify this point.

1Tardieux, I, et al. J. Exp. Med., 179: 1017, 1994. 2Moreno et al., J. Exp. Med., 180: 1535, 1994. 3Ming et al., Cell, 82: 287, 1995. 4Burleigh, BA & Andrews, NW Annu. Rev. Microbiol., 49: 175, 1995. 5Toker, A & Cantley LC. Nature 387:673, 1997. 6Guilherme, AL et al. J. Biol. Chem. 271:29533, 1996.


172

ESSENTIAL ROLE OF PHOSPHOLIPASE C IN TRYPANOSOMA CRUZI PROLIFERATION.

Malaquias, AT; Silva, LCF; Soares, MS & Oliveira, Mecia M.

Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 21949-900 Rio de Janeiro, Brazil.

During our studies of phosphoinositides participation in T. cruzi growth control, we have observed that a functionally intact phospholipase C (PLC) is essential for the mitogenic stimuli from FCS proceed to the final stages of cell division. Experiments were done with PLC inhibitors pre-incubated with cells before the external stimuli was given, this impaired the early events of PI hydrolysis (Oliveira, M.M., et al. Mol. Cell. Biochem., 124: 2, 1993) as well as DNA replication, accessed by [3H]-thymidine incorporation. The eventual participation of another enzyme of the phospholipid (PL) metabolism, namely phospholipase A2 (PLA2) was investigated to its metabolite lyso-phosphatidic acid (LPA). LPA was reported as the active substance in FCS-induced cellular alterations (2Tigy, G. et al., Proc. Natl. Acad. Sci. USA 87:1521-1525, 1990), therefore it was very important for the understanding of our results. The addition of LPA to T. cruzi cultures (24nM-200µM) did not alter cell growth. Parallel experiments to check eventual effect of LPA were done, such as dialyze and methanolic extraction of FCS, and all this experiments did not alter the FCS-induced effect, thus eliminating LPA as the active factor in FCS-induced T. cruzi growth.

Supported by grants from CNPq, FAPERJ and FINEP.


173

PRELIMINARY CHARACTERIZATION OF TRYPANOSOMA CRUZI CELL LINES TRANSFECTED WITH THE CYSTEINE PROTEASE ISOFORM CRUZIPAIN 2.

Lima, A.P.C. A., Schmitz, V., Reis, F. C. G., Colonese, A. P. and Scharfstein, J.

Instituto de Biofísica Carlos Chagas Filho - U.F.R.J., Rio de Janeiro, RJ, Brasil.

Cysteine proteases have been implicated in the survival of Trypanosoma cruzi in mammalian cells. Its importance for parasite replication and survival has validated these enzymes as potencial targets for the development of new anti-parasitic drugs. The major cysteine proteases of T. cruzi, cruzipains, are members of a polymorphic multi-gene family and share over 80% of amino acid identity. Recently, we have described the existence of cruzipain isoforms that diverge more significantly from the family prototype, the cathepsin-L like enzyme designated as cruzain. The amino acid substitutions found in the isoform cruzipain 2 are concentrated in the catalytic domain, resulting in a protease with kinetic properties different from those of cruzain. Indeed, the characterization of recombinant cruzipain 2, produced in S. cerevisiae, demonstrated that this isoform differs in substrate specificity and pH stability when compared to recombinant cruzain. In attempt to investigate the functional role of this enzyme, we have transfected T. cruzi epimastigotes with a plasmid (pTEX) containing the cruzipain 2 gene. Parasites selected for resistance against 800 mg/ml of neomycin were analysed in respect of their overexpression of cruzipain 2, growth rates and infectivity in VERO cells. Two independently transfected cell lines, DM28c and X10.6 epimastigotes grow normally in liquid medium and displayed no obvious abnormalities. Trypomastigotes from both transfected strains, however, showed an increase in infectivity when compared to wild type or parasites transfected with the plasmid alone. The mechanisms whereby cruzipain 2 participates in the invasion of mammalian cells by T. cruzi is under investigation.

Supported by PADCT, CNPq and CEPG.


174

PROTEIN TYROSINE PHOSPHORYLATION AND Ca2+ MOBILIZATION IN TRYPANOSOMA CRUZI ARE ASSOCIATED EVENTS REQUIRED FOR MAMMALIAN CELL INVASION

Silvio Favoreto Jr.*, Alice T. Ferreira#, Miriam L. Dorta* and Nobuko Yoshida*

* Departo. de Microbiologia, Imunologia e Parasitologia, # Departo de Biofísica, Escola Paulista de Medicina, Universidade Federal de São Paulo, R. Botucatu, 862, São Paulo, S.P., Brasil

We investigated in this study whether tyrosine phosphorylation of Trypanosoma cruzi proteins is essential for parasite entry into mammalian cells, and whether that event is associated with Ca2+ mobilization. Treatment of metacyclic or tissue culture trypomastigotes with genistein, an inhibitor of protein tyrosine kinase activity, markedly decreased invasion of HeLa cells. Genistein inhibited tyrosine phosphorylation of p175 in trypomastigotes, a 175 kDa protein undetectable in non invasive epimastigotes. A soluble factor, contained in HeLa cell extract and absent in the extract ot T. cruzi-resistant K562 cells, enhanced phosphorylation levels of p175. The phosphorylation-inducing activity of Hela cell extract was abrogated when it was adsorbed with metacyclic trypomastigotes but not with epimastigotes, or when it was mixed with recombinant protein J18, which contains the entire peptide sequence of gp82, a metacyclic stage-specific surface glycoprotein implicated in target cell invasion. HeLa cell extract, but not K562 cell extract, triggered Ca2+ mobilization in metacyclic trypomastigotes. Ca2+ response was not induced by Hela cell extract in genistein-treated metacyclic forms or in epimastigotes. When mixed with recombinant protein J18, Hela cell extract failed to display Ca2+ signaling activity towards metacyclic forms. All these data suggest that Ca2+ signaling, which is required for T. cruzi entry into host cells, is associated in metacyclic trypomastigotes with upstream tyrosine phosphorylation events that are mediated by surface molecule gp82.

Supported by FAPESP, CNPq/PADCT.


175

FUNCTIONAL CHARACTERISATION OF A STAGE-REGULATED GENE IN LEISHMANIA

L. M. Cesila1, D. F. Smith2 and S. R. B. Uliana1

1Departamento de Parasitologia-ICB/USP, São Paulo, Brasil – 2Imperial College of Science, Techcnology and Medicine, London, UK

The expression of the meta 1 gene in Leishmania is highly upregulated in metacyclic promastigotes. The gene encodes an 11.5 kDa protein present in metacyclic forms, barely detectable in procyclic promastigotes and not detectable in amastigotes. Attempts to knock out the meta 1 gene in L. major were unsuccesfull, suggesting that this might be an essential gene. As an alternative to investigate the function of this gene in L. major and L. amazonensis we have chosen to use antissense RNA to block its expression. The open reading frame of the meta1 gene (ORF) was cloned in pET17 upstream to the T7 gene 10 Tag. The fragment ORF/TAG in the inverted orientation was then cloned into vectors suitable for expression in Leishmania. The constructs obtained will be used in transfection experiments. Transfected parasites will be analysed for transcription of antisense RNA and expression of the native protein. Blocking meta 1 protein translation will enable us to verify if this gene is indeed essential for promastigote survival.

Supported by FAPESP and PIBIC/CNPq.


176

PRESENCE OF MINIEXON EXTRA COPIES INCREASE LEISHMANIA MAJOR VIRULENCE IN VIVO.

Antoniazi, S.A.1, Lima, J.H.C.2 & Cruz, A.K.1

1 Depto de BioquRmica - FMRP/USP, Av. Bandeirantes, 3900, CEP14049-900 - Ribeirno Preto, SP, Brasil. 2 Depto de Micro, Imuno e Parasitologia, UFSC, Florian\polis, SC, Brasil.

As part of a general project to develop the physical mapping of L.major parasite, we mapped a region of chromosome two (LV-39 clone, Mem. Inst. Oswaldo Cruz, vol. 91, suppl. 1996, p.190) which contains the miniexon array, tandemly repeated. It was possible to start some functional studies with this gene. Gene amplification within the miniexon array is, at least partially responsible for increasing the chromosome size (350 Kb chromosome) and it is suggested that this might be related to Leishmania virulence (Iovannisci and Beverley, 1989. Mol. Bioch. Parasitol., 34: 177-188). To test this assumption, one clone from this mapped region bearing 40 Kb of miniexon repeats, was transfected into an avirulent clonal line of L.major (MHMO/IR/83/LT 252). In order to verify whether this manipulation alters the virulence capacity of transfected clones, we have evaluated the growth rate and the metacyclogenesis in vitro. It was shown that the transfection did not affect the growth rate neither the metacyclogenesis of any of the transfectants (Mem. Inst. Oswaldo Cruz, vol. 91, suppl. 1996, p. 188). To evaluate the pattern of virulence, three of the transfectants (D2, D6 e C5) were injected subcutaneously on susceptible animals (BALB/c). LV-39 virulent line, CC1 avirulent line and a transfectant containing only the cLHYG vector were inoculated as control lines. They were studied under minimal (16mg/mL) and high (40mg/mL) levels of hygromicin B to verify possible alterations in the pattern of virulence in vivo. Under these conditions only one transfectant (D2) showed significant increase of virulence as compared to parental avirulent line. RNA levels were studied and an increased expression of the miniexon was evident under 40mg/mL hygromicin B pressure. To test whether this effect is related to the presence of miniexon extra copies we "cured" transfectants D2 and D6. The clones were maintained in culture (D2 e D6) under no drug pressure and the "cured" clones were selected in a dilution assay. The absence of the extrachromosomal molecule was confirmed by Southern blot hybridization of the "cured" transfectants (D2C-1 e D6C-1). In vivo experiments with these transfectants are on dealing.

Supported by UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR); FAPESP, CNPq and CAPES.


177

TARGETED DISRUPTION OF THE TRANSCRIPTION FACTOR NFAT1 RESULTS IN A SUSCEPTIBILITY TO LEISHMANIA MAJOR INFECTION

Viola, J.P.B.1,2; Kiani, A.1 and Rao, A.1

1The Center for Blood Research and Department of Pathology, Harvard Medical School, Boston, MA, USA, 02115; 2Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, RJ, Brasil, 21949-900.

The newly-identified NFAT (Nuclear Factor of Activated T Cells) family of transcription factors is thought to play a critical role in immune response. NFAT DNA-binding activity has been detected in nuclear extracts of antigen-stimulated T cells, B cells, mast cells and NK cells, and NFAT binding sites have been identified in the promoter/enhancer regions of many genes encoding immunoregulatory cytokines such as IL-2, TNF-a, IL-3, GM-CSF, IL-4 and IL-5. The NFAT family comprises several structurally related proteins that are encoded by at least four distinct genes, named NFAT1, NFATc, NFAT3 and NFAT4. The differential expression of NFAT proteins in tissues, and the differences in their binding preferences for recognition sites in cytokine genes, suggest that each NFAT protein may control the expression of a distinct set of genes in vivo. To study the unique functions of NFAT1 in vivo, we generated mutant mice carrying a disrupted NFAT1 allele by gene targeting. The targeting vector was designed to delete most of an exon encoding amino acids near the amino-terminus of the DNA-binding domain. Protein immunoblot and gel mobility shift assay demonstrated that the homozygous mutant mice possessed a null phenotype for NFAT1. Here we show that NFAT1 selectively downregulates the late phase of IL-4 gene transcription, thus potentially inhibiting the development of the Th2 responses in normal mice. Whereas T cells from wild-type littermates show a transient increase and then a rapid decline in the steady-state levels of mRNA, in an in vitro stimulation with anti-CD3, the levels of IL-4 gene transcripts in NFAT1-deficient T cells are maintained at high levels under the same conditions. Consistent with this in vitro phenotype, NFAT1-/- mice are more susceptible to Leishmania major infection than are their littermates, despite a C57BL/6 genetic background. Furthermore, when NFAT1-/- mice were treated with anti-IL4 neutralizing antibody there was a reduction of the lesion size that was comparable with the wild-type and anti-IL4 treated-Balb/c mice. We conclude that NFAT1 uniquely regulates IL-4 expression and T helper subset differentiation by facilitating a late feedback inhibitory mechanism that downregulates IL-4 gene transcription in normal T cells.

This work was supported by NIH grants.


178

INCREASING IN THE EXPRESSION OF PROTEIN KINASE C IS ASSOCIATED WITH THE INFECTIVITY OF LEISHMANIA AMAZONENSIS PROMASTIGOTES

Aguiar-Alves, F.*, Gomes-Cardoso, L.*, Cysne-Finkelstein, L.** and Leon, L.L.*

* Departamento de Imunologia; ** Departamento de Protozoologia - IOC - FIOCRUZ.

There are few data in the literature concerning to signaling proccess in protozoa and most of them are not associated with biological activity of the parasite. In this work we demonstrated that the activity of the phosphorilative enzyme protein kinase C (PKC) is directly associated with metacyclogenesis, as well as infectivity of Leishmania amazonensis promastigotes. Infective and non-infective forms of L. amazonensis were grown in Schneider's medium in presence of a phorbol ester (phorbol-12-myristate-13-acetate, PMA), an agonist of PKC activity and quercetin as antagonist of these activity. PKC was purified from soluble and enriched membrane fractions obtained from the stationary phase of both parasite forms, through anion exchange chromatography. The PKC activity was assayed in three different conditions: in the presence of Ca2+, EGTA and sphingosine, using [32 P]g-ATP and histone as substrate. The results showed that the purified PKC is Ca2+ independent, suggesting that this isoform belongs to the new protein kinase C classe (nPKC). The highest PKC activity was observed in the enriched membrane fraction of the infective form, which contained about 80% percent of metacyclic promastigotes. This fact clearly demonstrated that the infectivity of the parasite was directly associated with the PKC activity in Leishmania amazonensis promastigotes.

Supported by CNPq/FIOCRUZ.


179

CHARACTERIZATION OF AN ANTIGEN OF LEISHMANIA (L.) CHAGASI AMASTIGOTES INVOLVED IN PROTECTIVE RESPONSES IN BALB/c MICE.

Lopes, R.A.M., Beyrodt, C.G.P., Gris, S.K., Freymüller, E.H. & Barbiéri, C.L.

Disciplina de Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, Caixa Postal 20.342, São Paulo, 04023-062, SP, Brasil

Cysteine proteinases with a low molecular weight pattern have been described in megasomes of amastigotes from species belonging to L. (L.) mexicana complex (Pupkis and Coombs, 1984, J. Gen. Microbiol. 130, 2375; Alfieri et al., 1995, Parasitol. Res., 81, 240; Traub-Czeko et al., 1993, Mol. Biochem. Parasitol., 57, 101), whereas other species of Leishmania, as L. (L.) donovani and L. (L.) major, exhibit very low cysteine proteinase activity and few structures equivalent to megasomes (Pupkis et al., 1986, Exp. Parasitol., 62, 29).

We have previously identified an antigen of 30 kDa in L. (L.) amazonensis amastigotes which induces lymphoproliferative reponses in vitro and significant protection against homologous infection in BALB/c mice. Characterization L. (L.) amazonensis p30 was performed by use of a monoclonal antibody specific for this antigen. Immunoaffinity purified p30 showed cysteine proteinase activity and immunoelectron microscopy studies revealed that it is predominantly distributed in megasomes of L. (L.) amazonensis amastigotes (Beyrodt et al., 1997, Infect. Immun., 65, 2052).

We have recently identified antigens from L. (L.) chagasi amastigotes implicated in cellular immune responses among which a 30 kDa one is able to elicit lymphoproliferative responses in vitro mediated by CD4+ Th1 and significant protection against challenge with L. (L.) chagasi in BALB/c mice.

In contrast to the high cysteine proteinase activity observed in the band pattern of L. (L.) amazonensis amastigotes, no activity was found in the 30 kDa region of L. (L.) chagasi amastigote extracts. Since the monoclonal antibody directed to L. (L.) amazonensis p30 also recognizes the p30 from L. (L.) chagasi amastigotes, we are now involved in the purification of the antigen by immunoaffinity chromatography and in immunoelectron microscopy studies in order to define the subcellular localization of p30 in L. (L.) chagasi amastigotes.

Supported by FAPESP and CNPq/PADCT


180

PARTIAL MOLECULAR CHARACTERIZATION OF THE SMALL GTPase Rac IN TRYPANOSOMA CRUZI

Nepomuceno-Silva J.L., de Sá F. G. Paixão J. C. & Lopes U.G.

IBCCF, Laboratório de Parasitologia Molecular, UFRJ
Bloco G, CCS, Cidade Universitária, Av. Brigadeiro Trompovski s/no
Rio de Janeiro, Brasil, CEP 21949-900, tel (021) 260-9705

In order to characterize genes of the Ras superfamily GTPases in Trypanosoma cruzi, we have previously cloned a RT-PCR fragment which shares around 30% of homology and 52% of similarity with Rac, a member of the Rho family of GTPases. In other organisms studied so far, this gene family is involved in signal transduction pathways that mainly leads to rearrangements of the actin cytoskeleton modifying cellular morphology. This fragment, named Tcrac, spans three of the five conserved functional domains found in Ras superfamily GTPases and was used as a homologous probe in the screening of a Dm28clEMBL3 genomic library. A genomic clone of about 10 Kb obtained from the library had its Southern blot patterns compared to a genomic Southern blot. It seemed to contain all of the coding and intergenic regions. From this genomic clone we subcloned both an EcoRI fragment of 3.8Kb containing the 5'P intergenic and coding region and a SalI fragment of 2Kb containing the remaining of the 3'OH region.

The sequencing and analysis of coding and flanking regions are in progress as well as Northern blot analysis and control of genic expression experiments. We also intend to perform functional experiments concerning the role of the Rac protein on T. cruzi physiology.

This work is supported by PRONEX, FUJB and FINEP.


181

PARTIAL CHARACTERIZATION OF AN ARF GTPase GENE FROM TRYPANOSOMA CRUZI.

Freire, A.S., Paixão, J.P. & Lopes, U.G.

Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro
Av. Brigadeiro Trompovski s/no, Ilha do Fundão CEP 21949-900 Rio de Janeiro-RJ

The RAS-related small GTPase ARF has been characterized in several eukaryotic cells. Many important cellular functions have been attributed to ARF proteins such as: assembling of COPs (Coat proteins), initiation of the budding vesicles from Golgi complex, acting as a cofactor for cholera toxin and recently, as an activator of phospholipase D (PLD). We have cloned by Similar Display Technique (ref.) a RT-PCR product, designated pTcARF1, highly homologous to several eukaryotic ARF genes. We have screened a T. cruzi DM28 genomic library and we were able to isolate a lambda clone carrying the full length gene. The restriction map analysis of genomic sub-clones has been carried out and sequencing analysis of such clones is in progress. To get insight on the function of ARF in T cruzi we aim to subclone it in T. cruzi transfection vector to perform function assays . Moreover specifics antibodies will be raise against ARF to immunolocalization.

This work was supported by PRONEX and FINEP.

Reference: Mem. Inst. Oswaldo Cruz, Rio de Janeiro, vol. 91, Suppl., November, 1996/Page 304


182

TRYPANOSOMA CRUZI rab27: PRELIMINAR MOLECULAR CHARACTERIZATION.

Mendonça, S.M.; Nepomuceno, J.L.; Gonçalves, N.S.; Paixão, J.C.;Habib Jr., J.L.and Lopes, U.G.

IBCCF, Laboratório de Parasitologia Molecular, UFRJ
Bloco G, CCS, Cidade Universitária, Av. Brigadeiro Trompovski s/n°
Rio de Janeiro, Brasil, CEP 21949-900, Tel (021) 260-9705

Rab proteins are members of Ras superfamily of small GTP-binding proteins , and are involved in both endocytics and exocytics steps of vesicular traffic. In a previous work we showed a novel RT-PCR based assay that we developed in order to clone ras related sequences in the protozoa parasite Trypanosoma cruzi using a set of degenerated oligonucleotides designed based on the G3 domain aminoacid sequence, and the anchor primer which exhibits part of the mini-exon sequence. One of the obtained clone has 393 bps and showed a predicted aminoacid sequence that share 52,2% de identity with human rab27 in a overlap of 46 aa, this level of identity is consistent with the different levels of homology found among rab subfamilies. The rab27 clone, named Tcrab27, spans three of the five conserved domains presents in Ras superfamily GTPases and was used as molecular probe in Southern analysis. The discrete pattern of bands displayed in this experiment suggested the presence of at least two copies of Tcrab27 in the parasite genome.

Moreover, a T.cruzi Dm28c genomic library has been screened against this fragment and the characterization of corresponding clone are in progress.

This work is supported by PRONEX, FUJB and FINEP.


183

RAB7 GENE IN TRYPANOSOMA CRUZI : OBTENTION OF A PARTIAL KNOCK OUT

Araripe, J.R. ; Leal, S.T. ; Urmenyi, T.P. & Rondinelli, E.

RAB7 GENE IN Trypanosoma cruzi: OBTENTION OF A PARTIAL KNOCK OUT.

Araripe, J.R.; Leal, S.T.;Ürniényi, T.P. & Rond'melli, E.

Lab. Metabolismo Macromolecular Firniino Torres de Castro, Instituto de Biofisica Carlos Chagas Filho, UFRJ, 21949-900, Rio de Janeiro, RJ74

T'he small monoimric GTP-binding proteins that belong to the Rab subfamily are key regulators of membrane úaffic in eukaryotic cells. These proteins can act as a catalyst, accelerating the fusion reaction between donor and acceptor vesicles promoted after vlt-SNARE complex assembly. The Rab7 protein is associated with late endosomes in mammalian cells and is involved in the anchoring step of these vesicles duáng endocytosis. The rab7 gene of Trypanosoma cruzi has already been sequenced and characterized in our laboratory. lt is present in the genome of this trypanosomatid as a single copy gene enabling us to try to obtain null mutants. Plasmids were constructed in which the hygromycin phosphotransferase gene (HYG) and the phleomycin ressarce gene (BLE) were each flanked by 5' upstream and 3' downstream sequenccs of the rab7 codlng region (prab7HYGrab7 and prab7BLErab7 constmcts, respectively). Both constructs were digested with restriction enzymes and the linearized fragrnents were transfected mto T cruzí epimastigotes, clone CL Brener. Characterization of double transformants showed that, although the drug selection markers were integrated in the genome and sometimes amplified, both alleles of rab 7 were still present. We therefore repeated the transfonnation assay, starting with the prab7BLErab7 construct. We selected the phleomycin resistant cells and characterization of the heterogeneous population showed that BLE was integrated in the parasite genome. These parasites were cloned by limiting dilution and the stably transforined cells were grown without the drug. We used three different probes to characterize the integration events: I I- the rab 7 coding region; 21- a fragrnent containing part of the 5'upstream region of the rab7 locus and aproximately 70 base palrs of the rab7 codlng region; 31- the BLE coding rcgion. Southern blot analysis of genomic DNAs from cloned transformants showed that one out of the 5 clones analyzed had the BLE gene integrated at the expected position in the rab7 locus. The clone with the corrw integration event was transfected with linearized prab7HYGrab7 and the drug selection and cloning are currently being performecl The pafflal rab7 knock out transformant shows morphological alterations at the anterior region of the parasite when observed at electron núcroscopy. Further characterization of this transformant is in progress.

Supported by CNPQ, FAPERJ, CAPES and FINEP


184

CHARACTERIZATION OF POR NULL MUTANTS OF TRYPANOSOMA CRUZI.

Cortés A., Swindle J.* and González A.

Instituto de Parasitología y Biomedicina. CSIC. Calle Ventanilla 11. E-18001 Granada, Spain.
(*) Seattle Biomedical Research Institute. 4 Nickerson St. Seattle, WA 98109, USA.

We have previously reported the cloning of a T. cruzi gene, POR, coding for a protein, TcPOR, immunologically related to the complement cascade component C9 and to TcTOX, the acid active porin putatively responsible for the parasite escape from the phagolysosomic vacuole. Anti-TcPOR antibodies react in Western blots of acid amastigote-conditioned media with a protein of the same size than TcTOX, inhibit the hemolytic activity of acid amastigote supernatants and reduce the infective capacity of trypomastigotes when present in the culture. Since POR is single-copied per haploid genome, we have generated POR null mutants to further investigate the role of its encoded protein in the infection process and determine if TcTOX and TcPOR are in fact the same protein. Upon cotransformation with constructions containing antibiotic resistance genes flanked by the sequences bounding the protein coding sequence, transformants were selected in medium supplemented with hygromycin B and geneticin and clonal lines selected by limit dilution. Southern blot analysis demonstrated the replacement of the targeted POR sequences with the antibiotic resistance genes. Further, in Western blots of conditioned medium derived from the parasite carrying the null mutation, no bands were observed which reacted with anti-TcPOR antibodies strongly suggesting that indeed TcTOX and TcPOR are the same protein. Additionally, the residual hemolytic activity, which can be measured after incubating for 6 hours at pH 5.5 wild-type epimastigotes with erythrocytes, is no longer detected with null mutant epimastigotes. Lastly, although metacyclic trypomastigotes carrying the POR null mutation invade LLC-MK2 epithelial cells as efficiently as wild type parasites, unlike the wild type parasites they fail to multiply once internalized suggesting the null mutant lacks the porin activity needed to enter into the host cell cytoplasm.


185

T-DAF GENE CLONING

Mayer, M.G.1, Floeter-Winter, L.M.2, Cortez, A. P.1, Kipnis, T.L.3

Laboratório de Genética do Instituto Butantan Depto. de Parassitologia do ICB
3 Laboratório de Biologia do Reconhecer CBB-UENF.

Trypanosoma cruz! cycles between insect vectors and mammalian hosts. The epimastígote noninfective insect form is lysed by complement. Metacyclics and trypomastigotes, which are infective for the mammalian hosts, bear surface regulatory molecules that inhibit the assembiy of C3 convertases, and resist complement lysis in the absence of immune sera from infected animals.A 87-93kDa protein named T-DAF (Trypomastigote Decay Accelerating Factor) is present on the surface of infective forms and inhibits complement activation in a manner functionally similar to the mammalian complement regulator Decay Accelerating Factor(DAF).Screening of a Miranda 88 CDNA library with specific polyclonal sera raised against T-DAF protein rendered a 285bp fragment wich showed sequence and functional similarities to human DAF (infection and lmmunity 61:3656-3663, 1993).

Southern blots of Y strain Tcruzí genomic DNA digested with BamHl showed only one 3kb band wich hybridizes with the 285bp fragment used as a probe. Fragments affound this size (24kb) were then generated and fractionated in I % agarose gel for the construction of a partial genomic líbrary ín pUC 18 vector. Using the 285bp fragment as a probe we isolated four recombinant clones. Southern blot of plasmid DNA digested with BamHl showed that one of this cíones (4.1 clone), whose inseri was 3kb in length, hybridized with the probe. ln order to determine the complete nucleotide sequence of the 3kb fragment we generated a nested set of deletion mutants. Partial sequences of this 3kb fragment are being presented as weii as simíiarities with sequences found in Genebank data bank.

Supported by CAPES, FAPESP, CNPQ and FENORTE.


186

TCTOP2 GENE ENCODES AN EXCLUSIVELY NUCLEAR TOPOISOMERASE II IN TRYPANOSOMA CRUZI.

Fragoso, S.P.; Carreira, M.A.C.; Salles, J.M. & Goldenberg, S.

Fiocruz, Inst. Oswaldo Cruz, Av. Brasil 4365, Rio de Janeiro, RJ, 21045-900

Topoisomerases are enzymes that participate in many cellular functions involving manipulation of the DNA strands. There are two types of topoisomerases in the cell: a) type I topoisomerases and b) type II topoisomerases (topo II). Many studies have showed topo II as a target for many drugs against cancer tumors and it may be a way to be followed in the search for a chemotherapy treatment to diseases caused by parasites, such as Chagas' disease, where the high rate of T.cruzi proliferation and the unique aspects of the biology of the parasite, such as the replication of kDNA, may imply the involvement of a specific topo II. In the search for such activity, the TcTOP2 gene (3.7Kb) was expressed in a system based on the original pGEx kit (Invitrogen Inc.) and the resulting recombinant protein (140 KDa) was used to raise an antiserum in rabbit. Confocal microscopy showed that polyclonal antiserum against TcTOPO2 recognized only a nuclear topo II but not the mitocondrial counterpart, indicating that TcTOP2 encodes an exclusively nuclear enzyme. The TcTOP2 gene was also cloned in the pET system (Novagen Inc.) that expresses the recombinant protein fused with a histidine tag. TcTOPO2 was produced in a soluble form and we have started the procedures to purify the enzyme in nickel columns and the assays to analyze the different activities and sensibility of the enzyme to topo II inhibitors. Other important aspect we are interested in, refers to the import signals that directs the TcTOPO2 to the nucleus. We have identified an eukaryotic bipartite nuclear signal (NLS) at the carboxy domain of the enzyme, and we are presently testing its function using the jellyfish gene (that encodes a green fluorescent protein, GFP) as reporter gene under the control of the T.cruzi ribosomal promoter.

Support: Fiocruz-PAPES, CNPq, EEC


187

POLYMORPHISMS OF THE TOPOISOMERASE GENE LOCUS AMONG DIFFERENT TRYPANOSOMA CRUZI STRAINS

Dos Santos, W. G.* & Buck, G. A.**

*Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, Caixa Postal 486, Belo Horizonte, MG, 30161-970, Brasil; Centro de Pesquisas René Rachou-FIOCRUZ, Av. Augusto de Lima, 1715, Belo Horizonte, MG, 30190-002.

**Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia, 23298-0678.U.S.A.

We have analysed the genomic location of the topoisomerase II gene (topo II) in different Trypanosoma cruzi strains by restriction digestion, pulse field gel electrophoresis (PFGE) and sequencing. The molecular karyotype obtained by PFGE showed a marked heterogeneity in the size of the chromosomes of the strains studied. Hybridization of southern blots of these gels with a probe corresponding to the 5'end of the topo II gene from Dm28c strain also showed differences in number and size of chromosomes containing the gene which varied from 800 kb to 1950 kb depending on the strain analysed. It is not clear if these different sized hybridizing bands represent homologous or heterologous chromosomes. However, this data suggests that chromosomes presumably homologous can vary widely in size and possibly gene composition in different T. cruzi strains. The analysis of DNA restriction fragment lenght polymorphisms (RFLPs) using different restriction endonucleases and comparison of the 5'coding region sequences of the gene among different T. cruzi strains divided them into two major groups. These two groups reflect those previously observed using other molecular markers and reinforce the hypothesis of an ancient divergence among these protozoan parasites. Moreover, taken together these results confirm the plasticity of T. cruzi genome and extend evidences for diploidy in these organisms.

Partially supported by CNPq


188

Abstract not received.


189

EXPRESSION OF A TRYPANOSOMA CRUZI SURFACE GLYCOPROTEIN (GP82) BY MAMMALIAN CELLS INDICATES THE EXISTENCE OF DIFFERENT SIGNAL SEQUENCE REQUIREMENTS AND PROCESSING

Ramirez, M.I.1, Boscardin, S.B.1, Araya, J.3, Sang, W.H.2, Yoshida, N.1, Mortara, R.A.1 & Franco da Silveira, J.1

Deptos de Microbiologia, Imunologia e Parasitologia1 e Biofísica2 UNIFESP, Escola Paulista de Medicina, Rua Botucatu ,862, CEP: 04023-062, SP - Brasil; Universidade de Antofagasta3, Chile.

Metacyclic trypomastigotes of Trypanosoma cruzi express a developmentally regulated 82 kDa surface protein which has been implicated in the invasion of the mammalian host cells by the parasite. Analysis of cDNA clones encoding gp82 revealed the existence of several post-translational modifications such as n-glycosylation and addition of a GPI anchor. We had reported previously the expression of gp82 protein in mammalian cells transfected with the plasmid pcDNA3 carrying the complete open reading frame of gp82 gene. The gp82 cDNA was subcloned in plasmid pcDNA3 (pc-gp82) maintaining putative sequences for addition of GPI anchor and the signal peptide (NH2-terminal cleavable sequence). Transfected cells expressed 4 insoluble polypeptides under 60 kDa present suggesting that the signal peptide found in gp82 is not recognized by the mammalian cell system responsible for the sorting of membrane proteins.

To test this hypothesis, the signal peptide sequence from the the hemagglutinin virus protein was cloned cloning in frame with the gp82 gene generating a new construction named pc-gp82+PS. We have compared the expression of both pc-gp82 and pc-gp82+PS by immunoblotting, immunofluorescence and N-glycosylation inhibition through tunicamycin. Transfected cells with pc-gp82+PS expressed a 75 kDa protein detected with Mab3F6 (specific for gp82). When the transfection was made in presence of tunicamycin a faster migrating form of gp82 (55 kDa) was detected. It that had a mobility on gel matching that of deglycosylated gp82. Transfected cells with pc-gp82 expressed the same 4 polypeptides (under 60 kDa) in the presence or absence of tunicamycin.

Intracellular localization was defined by immunochemistry and confocal microscopy. Transfected cells containing pc-gp82 accumulated immunoreactive gp82 primarily near to the nucleus (possibly at endoplasmic reticulum as a non processed protein) and those with pc-gp82+PS showed distribution through the cytoplasm as a processed protein. These results suggest that the peptide signal from hemagglutinin protein gene in frame with gp82 gene allowed endoplasmic membrane targeting and glycosylation of gp82 protein in transfected cells. Most likely indicating that the signal sequence processing in higher eucaryotic cells is distinct from that of trypanosomatids.

Supported by FAPESP, CNPq/PADCT, FINEP/BID, International Atomic Energy Agency (Vienna, Austria).


190

CLONING AND EXPRESSION OF THE ACID a-MANNOSIDASE OF TRYPANOSOMA CRUZI

Oeltmann, T N, Vandersall-Nairn, A, O'Brien, K, Merkle, R K & Moremen, K W,

Department of Biochemistry and Molecular Biology and The Complex Carbohydrate Research Center, University of Georgia, Athens, G A, USA, and Department of Medicine and Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA.

We have isolated a full-length genomic clone of the acid a-mannosidase from an epimastigote genomic library of Trypanosoma cruzi (1). Degenerate oligonucleotide primers were generated from areas of sequence similarity between the Dictyostelium discoideum a-lysosomal mannosidase and the murine Golgi a-mannosidase II (2). These primers were used to generate a 643 bp PCR product from T. cruzi genomic DNA which was used as a probe to screen the epimastigote genomic library of a Tulahuen strain of T. cruzi which was constructed in the lFLX vector. Twelve positive clones were identified that hybridized to the amplimer probe. One clone that contained a full-length mannosidase gene was chosen for further characterization. A 5 kb Spei-Xhol fragment that hybridized to the amplimer was subcloned into the pZErOÔ1 vector and used for subsequent sequencing and restriction analyses. Preliminary sequence analysis revealed a single open reading frame of approximately 3 kb encoding the a-mannosidase gene. The size of the open reading frame is consistent with other lysosomal mannosidases including murine (2), Dictyostelium discoideum (3), and human (4), Translation of the open reading frame sequence predicts an amino acid sequence which contains regions of similarity with other lysosomal mannosidases, as well as, with short peptide sequences isolated and sequenced from the purified T. cruzi acid a-mannosidase. Expression studies in Pichia pastoris has resulted in high levels of acid a-mannosidase activity.

Supported by NIH Research Grants GM47533 and RR05351

1Swanson, P.M., Carter, C.E., Hager, C., Kim, W.J., Obermeier, S. and Oeltman, T.N. (1992) Glycobiology, 2:563-569.

2Merkle, R.K. and Moremen, K.W. (1993) Glycoconjugate J., 10: 240.

3Schatzle, J., Bush, J. and Cardelli, J. (1992) J. Biol. Chem., 267, 4000-4007.

4Liao, Y.F., Lal, A. and Moremen, K.W. (1997) J. Biol. Chem., in press.


191

EXPRESSION OF GREEN FLUORESCENT PROTEIN IN TRYPANOSOMA CRUZI

Yamauchi, L.M. Ullu, E. and Schenkman, S.

Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo - Escola Paulista de Medicina. R. Botucatu, 862 8o and, 04023-062, São Paulo - SP, Brasil, Department of Int. Medicine, Yale School of Medicine, New Haven, C.T., USA.

To study protein localization and expression in T. cruzi, we have transfected epimastigote forms with an expression vector containing the amino-terminal domain of histone H2 gene fused with green fluorescent protein (GFP) of Aequorea victoria. The expression vector was derived from the plasmid pTEX (Kelly et al. Nuc.Acid.Res. 20:3963-3969, 1992), which contained the promotor for the 18S ribosomal RNA. Twelve hours after eletroporation parasites with fluorescent nucleus were detected. The number of fluorescent parasites decreased after long incubations, suggesting that the plasmid was eliminated. The presence of the promotor sequence in the right orientation was required for expression. The number of fluorescent parasites was dependent on the electroporation conditions, and was higher when we used exponentially growing parasites at optimal culturing conditions. Recently differentiated epimastigotes were poorly transformed. To obtain stable transfectants cultures were incubated in at least 0.2 mg/ml of geneticin G418, and three to four weeks latter parasites were recovered when fresh human blood was added. Expression of GFP containing proteins should therefore be useful to establish best conditions for eletroporation, transformation and selection aiming studies of cellular localization and expression of proteins in T. cruzi.

Supported by: FAPESP, CNPq, CAPES


192

EXPRESSION OF GREEN FLUORESCENT PROTEIN (GFP) AND NEOMYCIN PHOSPHOTRANSFERASE (NPTII) BY TRYPANOSOMA CRUZI

Dos Santos, W. G.* & Buck, G. A.**

*Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, Caixa Postal 486, Belo Horizonte, MG, 30161-970, Brasil; Centro de Pesquisas René Rachou-FIOCRUZ, Av. Augusto de Lima, 1715, Belo Horizonte, MG, 30190-002.

**Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia, 23298-0678.U.S.A.

We have constructed a plasmid termed pPAGFPAN that contains the ribosomal RNA promoter cloned upstream the green fluorescent protein gene from the jellyfish Aequorea victoria followed by the bacterial neomycin phosphotransferase gene. In order to be properly processed by trans-splicing, a splicing site acceptor was inserted upstrean of each gene. The plasmid was transfected into Trypanosoma cruzi epimastigotes by electroporation using a Gene Pulser Bio-Rad apparattus adjusted to 2,25 V/cm and 500 mF. After selection of transformants in LIT medium supplemented with G418 an neomycin stable resistant (neor) population was obtained which also expressed GFP as seen by immunofluorescence microscopy. A bright green fluorescence was distributed throughout the transfected parasites, readily distinguishable from control parasites. The neor population was cloned in solid medium supplemented with 500 mg/ml of G418. Colony PCR of single colonies using appropriate set of primers showed the presence of the correct sized fragments corresponding to GFP and NPTII genes. Molecular analysis by pulse field gel electrophoresis and by restriction endonucleases demonstrated that the plasmid integrated into T. cruzi genome in chromossomes of different sizes including the 1.5 kb chromosome which contains the ribosomal RNA promoter cistron. These data suggest the use of this plasmid as a vector to express foreing genes in T. cruzi and introduce the use of GFP as a marker for monitoring gene expression in this parasite.

Partially supported by CNPq


193

GREEN FLUORESCENT PROTEIN AS A REPORTER FOR THE STUDY OF CYSTEINE PROTEINASE TARGETING IN LEISHMANIA

Costa-Pinto, D., McMahon-Pratt, D.* & Traub-Cseko, Y.M.

Fundação Oswaldo Cruz, IOC, DBBM, C.P.926, Rio de Janeiro, RJ, 21045-900; *Yale School of Medicine, EPH, 60 College St., New Haven, Ct. 06520-8034

We have previously identified two distinct cysteine proteinase genes of Leishmania pifanoi axenic amastigotes, lpcys1 and lpcys2, using the polymerase chain reation (PCR). Cysteine proteinases have been found in a variety of organisms and have been implicated in many processes related to pathogenesis in parasites. We have been studying the cellular targeting and the effects of overproduction of cysteine proteinases on infectivity, virulence and biochemical and morphological aspects in various species of Leishmania. Cysteine proteinases are synthesized as precursor molecules containing a pre and a pro region, the mature proteinase and, in some instances, a C-terminal extension. The pro region has been implicated in cellular trafficking in other organisms, and, in the case or trypanosomatids, targeting to the lysosome was considered a potencial function for the long C-terminal extension. We are investigating the possible role in trafficking for these regions by cloning them into Leishmania transfection vectors coding for a reporter protein, the green fluorencent protein (GFP) of Aequorea victoria. We have amplified the pre-pro regions of lpcys1 and lpcys2 genes, and the C-terminal extension of lpcys2 using PCR and cloned these into pXG-GFP+2' and pXG-'GFP+. The fusion proteins containing these putative signal sequences in the N-terminus or C-terminus of GFP, respectivelly, will allow us to study the role of these sequence in the trafficking of cysteine proteinases of Leishmania.


194

CLONING AND CHARACTERIZATION OF A GENE ENCODING A CYSTEINE PROTEINASE OF L. (L.) AMAZONENSIS AMASTIGOTES.

Beyrodt, C.G.P., Silveira, J.F. & Barbiéri, C.L.

Disciplina de Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, Caixa Postal 20.342, São Paulo, 04023-062, SP, Brasil

An antigen of apparent molecular mass 30 kDa (p30) was identified in L. (L.) amazonensis amastigotes and shown to be implicated in lymphoproliferative responses in BALB/c mice mediated by CD4+ Th1. Characterization of p30 was performed by use of a monoclonal antibody directed to the antigen. Immunoaffinity purification of p30 revealed that it presents cysteine proteinase activity and immunoelectron microscopy studies showed that the antigen is localized predominantly in megasomes of L. (L.) amazonensis amastigotes. Immunization of BALB/c mice with p30 was able to induce a significant protection against L. (L.) amazonensis infection (Beyrodt et al., 1997, Infect. Immun. 65, 2.052).

In order to identify a p30 gene, a cDNA library of L. (L.) amazonensis amastigotes was constructed in lgt11 and screened with the monoclonal antibody specific for p30 antigen. Positive clones were further screened by hybridization with a 500 bp fragment. This fragment was obtained by amplification by PCR using genomic DNA of L. (L.) amazonensis amastigotes and a pair of primers derived from active sites cys25 and asp145 of Dictyostelium discoideum cysteine proteinase. Nucleotide sequence analysis of the amplified fragment showed a high degree of homology to sequences of cysteine proteinase genes from Leishmania (Traub-Cseko et al., 1993, Mol. Biochem. Parasitol., 57, 101). DNA from two positive clones was digested with EcoRI endonuclease restriction enzyme and revealed a 4 kb fragment which was purified and subcloned in pUC18/EcoRI-BAP vector for subsequent restriction and nucleotide sequence analysis.

Supported by CNPq/PADCT and FAPESP


195

SITE-DIRECTED MUTAGENESIS OF TWO POTENTIAL N-GLYCOSYLATION SITES OF CYSTEINE PROTEINASE GENE FROM LEISHMANIA PIFANOI (LPCYS2) BY POLYMERASE CHAIN REACTION.

Boukai, L. K., Pacheco, L. L.P., McMahon-Pratt, D*. & Traub-Cseko Y.M.

Fundação Oswaldo Cruz, I.O.C., Departamento de Bioquímica e Biologia Molecular, C.P. 926, R.J. CEP21045-900, Brazil

*Yale Medical School , Departament of Epidemiology and Public Health, 60college St., New Haven, C.T. 06510, USA.

Amastigote forms of leishmanias from the mexicana complex have been shown to contain high levels of proteinase activity, mostly due to cysteine proteinases, in large lysosomal organelles called megasomes. The abundance of cysteine proteinases in amastigotes make them particularly attractive targets for chemotherapeutic attack. In L. pifanoi we have previously isolated two distinct cysteine proteinase genes (lpcys1 and lpcys2) through PCR, using active sites as degenerate primers and c-DNA. Analysis of restriction patterns of partial digestions from genomic DNA revealed that lpcys1 is a single copy while lpcys2 has approximately 8-20 copies in the cluster. The translated product of lpcys2 shares many features in common with cysteine proteinases from other trypanosomatids. The N-terminus has an hydrophobic signal sequence and there are four well defined domains including a long C-terminal extension. Cysteine proteinases of all trypanosomatids described so far have also putative N-glycosylation sites and mannnose residues have been found. In mammals mannose-6-phosphate is an important signal to target lysosomal enzymes. In this study we are investigating the possible involvement of glycosylation in targeting Lpcys2 to megasomes, through site-directed mutagenesis. In Lpcys2 there are two potential N-glycosylation sites: one at the central domain and the other in the C-terminal extension. The mutations were introduced by PCR in both N-glycosylated sites using oligonucleotides with substitution in the asparagine residue of a lpcys2 gene containing sequence coding for epitope tag cloned at the C-terminal domain. The PCR was performed in two different steps. The mutated products were cloned into transfection vectors pX63Neo and pXG1 and transfected into leishmanias in and out of the mexicana complex. Positive transfectants will be analysed through immunofluorescence and eletron microscopy, using monoclonal antibodies against the epitope tag.


196

MOLECULAR MODELLING OF CYSTEINE PROTEASE INHIBITION

Carvalho M. L, Dardenne L. E, Bisch P. M

Laboratório de Física Biológica Instituto de Biofísica- UFRJ

Based on the structure of the catalytic site of the Cruzipain - a T. cruzi cysteine protease, we are investigating an enzyme inhibitor, MW16, to develop a new drug against Chagas Disease. In order to understand the iteration inhibitor-catalytic site, we performed several computer analysis:1.aligment of the aminoacid sequence of papain and cruzipain by the FASTA software to identify the essential aminoacids in the catalytic site; 2. characterize a putative inhibitor-catalytic interaction; 3. geometrical optimization of the inhibitor into catalytic site; 4.quantum and classic calculus of molecular dynamics. The mechanics and molecular dynamics simulations were done using the program THOR and the quantum calculus was done by semi-empirical methods, AM1 and PM3. The inhibitor conformation in the water presented planar structure due to a intramolecular hydrogen bond. This planar conformation facilitates the prototype to enter into the enzyme active site. In addition, the conformational preferences adopted by the complex enzyme-inhibitor in the course of the simulations were also analyzed. In summary, we believe that MW16 is a good candidate to elaboration of a specific drug for Chagas disease.

Supported by: CNPq, FUJB


197

CHARACTERIZATION OF THE ACTIVE SITE OF ARGINASE FROM LEISHMANIA (L.) AMAZONENSIS BASED ON THE ENZYME STRUCTURE DEFINED FOR RATTUS NORVEGICUS.

da Silva, E.R. & Floeter-Winter, L.M.

Departamento de Parasitologia - Instituto de Ciências Biomédicas - Universidade de São Paulo Caixa Postal 66208, São Paulo, SP - 05389-970

Arginase is an enzyme involved in the urea cycle that catalyzes the conversion of arginine to urea and ornithine. Using oligonucleotides based on the conserved regions of arginase genes of yeast, Xenopus, rat and human, we obtained a PCR fragment corresponding to the central part of the arginase gene of L. (L) amazonensis. The fragment was cloned into pMOS-blue vector, generating the pLarg-9 clone. The base composition of the cloned DNA was determined and compared to the published sequences (da Silva et al., Mem. Inst. Oswaldo Cruz 89: 110,1994). This clone was used as a probe in the screening of a L. (L.) amazonensis genomic cosmid library. Nine clones were isolated and suitable subcloning was performed to determine the nucleotide sequence of the arginase gene.

The bioinorganic chemistry of arginase is particularly rich because this enzyme is one of the very few that specifically requires a spin-coupled Mn2+-Mn2+ cluster for catalytic activity in vitro and in vivo (Kanyo et al, Nature 383, 554 - 1996).

The analysis of the primary structure of L. amazonensis arginase showed that some aminoacids responsible for the coordination of the binuclear center formed by the spin-coupled Mn2+-Mn2+ were conserved. Importantly, in the same region, other aminoacids presented a low level of conservation. Moreover, two deletions were detected in L. (L.) amazonensis between His 141 and Asp 232 (both involved in the catalysis) in positions 160 and 161. These differences may prove relevant in the analysis of the parasite arginase activity and its potential as a target for therapeutic drugs.

Financial support: FAPESP and CNPq


198

IS THE ARGINASE GENE SEQUENCE CONSERVED IN TRYPANOSOMATIDS?

Pioker, F.C., da Silva, E.R. & Floeter-Winter, L.M.

Departamento de Parasitologia – Instituto de Ciências Biomédicas – Universidade de São Paulo Caixa Postal 66208, São Paulo, SP – 05389-970

The activity of arginase, an enzyme of the urea cycle which catalyses the conversion of arginine to urea and ornithine, varies according to a genus pattern within the organisms of Trypanosomatid family and is a useful character for identification of these organims (Camargo, 1979).

Genomic DNA of eight genera of that family was probed with the DNA of a clone that comprises the 3' region of the gene of Leishmania (L.) amazonensis. The results showed the tested organisms presented sequences similar to the probe (da Silva & Floeter-Winter, 1996), indicating the presence of the nucleotide coding sequence of the enzyme. Then, the absence of enzyme activity could be due to an inativation of the gene or the sequence could be so altered that originated an inactive protein.

The degenerated oligonucleotides, originally used to clone the central region of arginase gene of L. (L.) amazonesis, were then used to PCR Trypanosoma cruzi genomic DNA. The resulting fragment, with the expected length if we consider a conservative position of the oligos inside the sequence, was cloned into pMOS or pUC19 vectors and the sequence of the amplified product was determined. The comparison of this sequence to those of L.(L.) amazonensis showed a low level of similarity, however some residues, showed some conservation. The natural implications in the evolution of this sequence within the genome of the trypanosomatid family will be discussed.

Financial support: FAPESP and CNPq


199

CLONING OF THE HISTONE H1 GENE FROM LEISHMANIA AMAZONENSIS.

Mendonça, SCF, Ransijn, A, Noll, T, Da Cruz, AM & Fasel, N.

Instituto Oswaldo Cruz, FIOCRUZ, Caixa Postal 926, Rio de Janeiro, 21045-900, RJ, Brasil and Institut de Biochimie, Université de Lausanne, 1066, Epalinges, Switzerland.

The double substraction principle was used to identify Leishmania major early infection genes. This strategy has led to the isolation of a gene coding for a histone H1. The expression of this gene, termed SW3, was found to be increased during metacyclogenesis. The high expression level is maintained in amastigotes. Recent studies in mice challenged with L. major have indicated that the histone H1 may be a potential candidate vaccine subunit. Lymphocytes from patients with Old World and New World cutaneous leishmaniasis proliferate in vitro in response to synthetic peptides designed according to the published L. major histone H1 sequence (Fasel et al, Mol. Biochem. Parasitol. 62: 321-323, 1993). These results prompted us to isolate the homologous genes from New World Leishmania, particularly from L. amazonensis IFLA/BR/67/PH8, the strain currently used in the preparation of both the vaccine against leishmaniasis and the Montenegro skin test antigen industrially produced by Biobras (Montes Claros, MG, Brasil). Cytoplasmic RNA was first extracted from stationary phase promastigotes and used in RT-PCR with primers designed according to the L. major sequence. DNA fragments with the expected size were obtained, purified by QIAEX extraction (QIAGEN AG, Switzerland) and inserted into the pZErOTM- vector (Invitrogen, USA). The L. amazonensis histone H1 gene was sequenced using the dioxide chain termination procedure. L. major and L. amazonensis sequences are rich in lysine and proline and appear to have also an anti-sense open reading frame (ORF). Unpublished data indicate that, at least in L. major, the anti-sense ORF is also translated into protein. Comparing the. L. major and L. amazonensis sequences there is a 79,9% similarity at the nucleotide level, 67,3% at the sense ORF and 61,4% at the anti-sense ORF. We hope that the cloning and sequencing of this gene will lead to the production of the corresponding recombinant protein and synthetic peptides with potential use in humans as diagnostic or immunoprophylactic tools.

Financial support: CNPq and Swiss National Fund.


200

GENE ORGANIZATION AND EXPRESSION OF HEAT SHOCK PROTEIN 60 (HSP60) IN LEISHMANIA BRAZILIENSIS

Lees, R.A. and Rondinelli, E.

Lab. Metabolismo Macromolecular Firmino Torres de Castro, Inst. Biofísica Carlos Chagas Filho, UFRJ,21949-900 CCS, bloco G, Ilha do Fundão,0Rio de Janeiro - RJ

One important feature of Leishmania species life cycles is the obligatory temperature shift upon passage from the insect host to the mammal host: the parasites are required to adapt to temperatures of 37oC for visceral leshmaniasis species or 34oC for cutaneous leshmaniasis species, such as L. braziliensis. This temperature shift can suggest a role for HSPs in survival and adaptation to the mammalian environment.

Our laboratory has been studying heat shock proteins (HSPs) in the trypanosomatids T. cruzi and Crithidia fasciculata , with emphasis on HSP60. We showed that the hsp60 gene in T.cruzi clone CL14 is arranged in a tandem repeat containing at least three copies (Giambiag-deMarval et al, 1993 - MBP 59:25-31). Engam end collaborators showed that in the PBOL strain hsp60 is arranged in two clusters containing at least ten copies each (Sullivan et al, 1994 - MBP 68:197-208).

We isolated the L. braziliensis hsp60 gene from a genomic library constructed in flEMBL3 (kindly provided by Dr. U.G. Lopes - IBCCF - UFRJ) by using our T. cruzi hsp60 sequence as a probe. Southern blot analysis of total DNA partia|ly digested with Eag I suggests a tandem organization for this gene. When L. brazilensis cultures where submited to heat shock, northern blot analysis did not reveal any change in expression of hsp60 RNA, although we observe induction of the protein at 34oC. Thies suggests a postranscriptional regulation for hsp60, a commonplace in trypanosomatids.

Supported by CNPq, FAPERJ and FINEP


201

CLONING AND FUNCTIONAL EXPRESSION OF A NUCLEOSIDE TRANSPORTER GENE FROM LEISHMANIA DONOVANI

Vasudevan*, G., Carter, N.S.**, Drew, M.E.*, Beverley, S.M.***, Ullman, B.** & Landfear, S.M.*

Departments of *Molecular Microbiology and Immunology and **Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland, OR, 97201,U.S.A. and ***Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110, U.S.A.

All parasitic protozoa that have been studied to date are purine auxotrophs that must salvage purines from their hosts. The first step in this important

salvage pathway is the transport of purine nucleobases or nucleosides across the parasite plasma membrane. There is considerable interest in nucleoside transporters among these protozoa, since their central role in the biochemistry of these parasites suggests that they could be exploited for development of novel anti-parasite chemotherapies. Potential pharmacological strategies vary from inhibition of these transporters to prevent uptake of their essential substrates to use of these transporters to deliver to the parasite selectively toxic nucleoside analogs. We have used genetic complementation to clone a gene encoding a nucleoside transporter from the parasitic protozoan Leishmania donovani. The TUBA5 mutant of L. donovani (1) is deficient in the adenosine/pyrimidine transporter, and as a result this mutant is highly resistant to the cytotoxic adenosine analog tubercidin. To clone the gene for this adenosine/pyrimidine transporter (the Ldnt1 gene which encodes the LdNT1 transporter), we transfected the TUBA5 line with a cosmid expression library (2) containing large fragments of L. donovani genomic DNA. We then screened approximately 5,000 transformants for acquiredsensitivity to nanomolar concentrations of tubercidin. From this screen we obtained 5 independent transformants that contained distinct but overlapping cosmid inserts. One of these cosmid clones has been analyzed in detail. Restriction digestion and subcloning of this cosmid revealed that it contained two adjacent restriction fragments that could confer sensitivity to tubercidin when transfected into TUBA5 parasites. Hence, there is a locus which contains at least two Ldnt1 genes. We have sequenced the smaller restriction fragment and demonstrated that it contains an open reading frame encoding 492 aminoacids. This open reading frame encodes a very hydrophobic protein containing 11predicted trans-membrane segments and displaying marked sequence identity to the human facilitative nucleoside transporter hENT1 (3). Transfection of TUBA5 parasites with this open reading frame restored sensitivity to nanomolar concentrations of tubercidin and concurrently restored the ability of the parasites to take up tritiated adenosine in transport assays. These results confirm that the open reading frame encodes a functional nucleoside transporter. Furthermore, expression of this Ldnt1 gene in Xenopus oocytes significantly stimulated their ability to take up radiolabeled adenosine, establishing that the LdNT1 protein alone is sufficient to mediate adenosine transport. The isolation of the Ldnt1 gene opens the way to a thorough structural and functional analysis of this important parasite transporter.

This work was supported by NIH grants to S.M.L., B.U. and S.M.B. S.M.L. and B.U. are currently and S.M.B. was formerly a Burroughs Wellcome Scholar in Molecular Parasitology.

REFERENCES: 1. Iovannisci, D.M. et al. (1984) Mol. Cell. Biol. 4, 1013-1019. 2. Ryan, K.A. et al. (1993) Gene 131, 145-150. 3. Griffiths, M. et al. (1997) Nature Medicine 3, 89-93.


202

STRUCTURAL ANALYSIS OF GENE AMPLIFICATION RELATED WITH MULTIDRUG RESISTANCE IN GLUCANTIME-RESISTANT LEISHMANIA (V.) GUYANENSIS CELL LINE

Anacleto, C.; Abdo, M. C. B.; Fernandes, A. P. S. M.; Petrillo-Peixoto, M. L.; Moreira, E. S. A.

Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Caixa Postal 486, Belo Horizonte, 31 . 270 - 901, MG, Brasil

We have demonstrated that a Leishmania (V.) guyanensis Glucantime-resistant cell line, obtained by one-step protocol (Line R.21), is also resistant to other drugs. Genetic analysis demonstrated the presence of an amplified circular extrachromosomal element. Results from restriction digestions with different enzymes suggest that this element is monomeric with a complexity of about 30 Kb. Hybridization analysis showed that this element contains a homologue of the ltpgpA gene which is related with multidrug resistant phenotype in Leishmania tarentolae. Partial sequence of the amplified region of R.21 line showed a sequence similarity to ltpgpA. Physical map of the fragment containing the sequences homologous to the ltpgpA indicates a peculiar profile when compared with other genes family of the multidrug resistance in Leishmania (ltpgpA, B, C and D). Pulse field gel electrophoresis (CHEF) of total DNA of wild type and R.21 cell lines followed by hybridization indicated that the pgpA gene was present in a chromosomal band of approximately 850 Kb in both cell lines and was also present as an extrachromosomal element only in line R.21. The presence of the pgpA gene was checked in other species of Leishmania and it was shown to be contained in a small chromosome ranging from 900 - 750 Kb depending on the species analyzed. Different intensity of the hybridization signal among samples suggests that the copy number of the pgpA gene may vary among strains. The presence of other genes related to drug resistance in R.21 cell line will be investigated.

Supported by CAPES and CNPq.


203

INTERCONNECTION BETWEEN EXPRESSION OF SAG-1 (p30), SAG-2 (p22) OF TOXOPLASMA GONDII AND 70 kDa HEAT SHOCK PROTEIN DURING IN VIVO CONVERSION OF BRADYZOITES INTO TACHYZOITES

Silva, N.M.1, Gazzinelli, R.T.2, Silva, D.A.O.1, Ferro, E.A.V.1, Kasper, L.H.3 and Mineo, J.R.1

1Laboratory of Immunology, Department of Pathology, Universidade Federal de Uberlândia, Uberlândia, MG, Brasil, 2Centro de Pesquisas Renée Rachou, Belo Horizonte, MG, Brasil and 3Department of Medicine, Dartmouth Medical School, Hanover, NH, USA.

Stage conversion between bradyzoites and tachyzoites of T. gondii was investigated in vivo. Experiments were carried out in nervous system from C57BL/6 chronically infected mice with ME-49 strain of T. gondii. In order to promote bradyzoite/tachyzoite interconversion, mice were deleted in vivo of CD4+ and/or CD8+ T lymphocytes, or treated with neutralizing doses of anti-IFNg or anti-TNF-a antibodies. Expression of tachyzoite specific genes SAG-1 and SAG-2 was visualized in the cerebral tissue by immunocitochemistry and measured by photometric assay. The depletion of either CD8+ or the combination of CD4+ plus CD8+ lymphocytes increased the SAG-1 and SAG-2 expression inside cysts, showing a crescent stage interconversion. The immunossupressive effect of anti-INF-g or anti-TNF-a treatments was immediate and more potent, when compared with T lymphocytes depletion, leading to greatest parasite stage conversion and replication. Expression of 70 kDa heat shock protein (hsp70) was also studied in brain cysts of these mice. We observed that the parasites express in vivo detectable levels of hsp70 only during a short period of T. gondii of stage conversion. In addition, fully differentiated tachyzoites did not express detectable amounts of hsp70. Taken together, our results indicate that INF-g or TNF-a are important mediators of parasite dormancy during chronic infection with T. gondii and that their decreasing favors bradyzoite/tachyzoite stage conversion. Our data also suggest that hsp70 may take an important role on parasite adaptation during developmental stage interconversion.


204

PARTIAL CLONING AND CHARACTERIZATION OF A cDNA SPECIFIC FOR AMASTIGOTE OF TRYPANOSOMA CRUZI .

Olivas-Rubio, M. & Rosales-Encina J.L. Departamento de Patología Experimental, Centro de Investigación y Estudios Avanzados del IPN. Apartado Postal 14-740, México D.F. , 07300, México.

Trypanosoma cruzi the causative agent of Chagas disease, undergoes morphologic and physiologic changes during its life cycle. The parasite's cycle is initiated when metacyclic trypomastigotes are eliminated in the feces of the triatomine vector and enter the mamalian cells. Inside the cell, trypomastigotes transform into amastigotes in a process that is characterizated by changes in the major suface glycoprteins. Then the amastigotes multiply and transform again into trypomastigotes, lysate the cell and are released into the circulation. Amastigotes are found both inside the host cells and in circulation, and as trypomastigotes they infect mammalian cells. It has been reported the expression of a stage specific glycoprotein (Ssp-4) on the surface of the parasite during differentiation of trypomastygotes into amastigotes. Ssp-4 is a 70-84kDa glycoprotein recognized by the monoclonal antibody 2C2, and is expressed on the recently transformed amastigotes both intra or extracellulary. To clone the gene for this glycoprotein, a genomic expression library was screened with a sera against deglycosylated Ssp-4, and the DNA insert of one positive clone was used to isolate a clone from a cDNA library. Using the 2.3 kb DNA insert of this last clone (TcA230) as a probe, it was found by dot blot analysis of mRNA, that the corresponding gene was expressed mainly in the amastigote form, and by Southern blot experiments that the gene is comprised in a family or related genes. A fusion protein (A230) of 125kDa was produced and affinity purified when the DNA insert was subcloned in the expression vector pMAL-C2. Antigenic specificity to the fusion protein was demonstrated in Western blots using antibodies against deglycosilated Ssp-4. Anti-A230 antibodies reacted strongly with a 66kDa polypeptide and weakly with a 80kDa polypeptide only in amastigotes. Indirect immunofluorescence studies showed that these antibodies bind on the surface of both intra- and extracellularly developed amastigotes. The above results indicates that the TcA230 insert codifies for an amastigote specific surface protein.


205

USE OF REPRESENTATION OF DIFFERENTIAL EXPRESSION TO CLONE GENES EXPRESSED DURING THE ONSET OF TRYPANOSOMA CRUZI METACYCLOGENESIS.

Plazanet-Menut C., d'Escoffier L.N., Avila A., Krieger M.A., Goldenberg S.

FIOCRUZ, Instituto Oswaldo Cruz, Departamento de Bioquímica e Biologia Molecular, Avenida Brasil 4365, CEP 21045-900, Rio de Janeiro-RJ, Brasil.

The differentiation of T. cruzi epimastigotes into metacyclic trypomastigotes is preceded by the adhesion of parasites to a substrate either inside the triatomine invertebrate host or in a in-vitro model using the chemically defined TAU3AAG medium. This substrate adhesion occurs within the first six hours of differentiation and is accompanied by the expression of stage specific genes. Therefore, it can be considered as an early event during the metacyclogenesis process.

A new molecular method has been developed recently for cloning the differences between two genomes, called Representational Differential Analysis (RDA), based on the principles of subtractive hybridization followed by PCR amplification (1). An adaptation of this method, that we call Representation of Differential Expression (RDE), was developed in our laboratory and allows the cloning of trypanosomatid stage specific genes (2). This method was applied for studying genes expressed within the first six hours of T. cruzi metacyclogenesis. We have generated a cDNA population representative of six hours adhered epimastigotes by reverse transcription of polysomal RNA extracted from parasites at this differentiation stage, followed by a Polymerase Chain Reaction using oligonucleotides corresponding to the conserved spliced leadder (or mini-exon) sequence and oligo(dT) as primers. This population of molecules was then hybridized to an excess of equivalent cDNA molecules representative of LIT medium exponentially growing epimastigotes. The non-hybridized molecules, specific from six hours adhered epimastigotes, were selectively amplified by PCR. This subtraction-amplification procedure was repeated three times, using an increasing excess of exponentially growing epimastigotes molecules. As a result, we obtained 8 distinct DNA bands with respective sizes varying from 0.6 to 1.1 kb, which were subsequently cloned into pBSKII+. Each clone separately was used as a probe and hybridized against six hours adhered epimastigotes RNA and against exponential epimastigotes RNA to check their specificity. 5 out of the 8 clones appeared to be specific from six hours adhered epimastigotes. We are presently characterizing the genes corresponding to these clones in terms of their nucleotide sequence, expression and organization within T.cruzi.

Support PAPES-Fiocruz, CNPq

References:

(1) N. Lisitsyn ,N. Lisitsyn and M. Wigler, Science 259, 946(1993).

(2) M. A. Krieger and S. Goldenberg, Parasitology Today, in press


206

CLONING AND CHARACTERIZATION OF TWO GENES EARLY EXPRESSED DURING TRYPANOSOMA CRUZI METACYCLOGENESIS.

Ávila A. R.; Krieger M. A.; Plazanet-Menut, C.; Linss, J. & Goldenberg S.

FIOCRUZ, Instituto Oswaldo Cruz, Dept. Bioquímica e Biologia Molecular, Av. Brasil 4365, Rio de Janeiro - RJ - Brasil, 21045-900.

Previous work in our laboratory resulted in the development of chemically defined conditions that mimic the metacyclogenesis process of T. cruzi (Contreras et al., Mol.Biochem.Parasitol. 16:315,1985). Under in vitro conditions, differentiating epimastigotes adhere to the culture flask and are released to the culture medium upon transformation into metacyclic trypomastigotes. We have recently developed a method for the amplification and cloning of T. cruzi genes specifically expressed at different times of the metacyclogenesis process (Krieger & Goldenberg, Parasitol. Today, in press). The method, named Representation of Differential Expression (RDE), is based on the PCR amplification of DNA sequences unique to a given cell population (tester), after hybridization with an excess of DNA sequences of a related cell population (driver) to remove sequences common to tester and driver populations.

In order to characterize genes expressed at different times of the metacyclogenesis process, we have used cDNAs obtained from polysomal mRNAs of 24h-adhered epimastigotes and exponentially growing epimastigotes as tester and driver populations, respectively, in a RDE procedure. Two genes (clones 18 and 21) were fully sequenced and characterized in terms of their genomic organization. Search of GenBank sequences showed that none of them display homology to available gene sequences. Southern blot analysis and pulsed-field gel electrophoresis indicate that these genes exist as low copy number genes in T. cruzi Dm28c. We are presently raising antisera against recombinant proteins derived from these genes in order to determine their precise location within the parasite.

Support: CNPq and Fiocruz-PAPES.


207

CLONING OF A TRYPANOSOMA CRUZI TRYPOMASTIGOTE STAGE SPECIFIC cDNA BELONGING TO THE Tc-85 MULTIGENE FAMILY

`Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, C.P. 26077, 05599-970 SP, Brazil

· Department of Biochemistry and Molecular Biology, University of California, lrvine, USA

We have previously described a partial CDNA insert (Tc85-1) coding for a polypeptide recognized by the HlAlO monocional antibody. ln order to obtain the complete codon sequences of a Tc-85 gene, a new CDNA library constructed in XZAP vector was screened with the Tc85-1 insert. Positive ciones were selected and subcloned into pTrcHis expression vector. Bacterial colonies were grown, induced with IPTG and the lysates analysed by reaction with monocional antibody HlAlO in Western biots. One cione, named Tc85-11, was seiected by positive reaction with the monocional antibody and subcioned into pUCl9 and pBS/KS vectors for sequencing. Several clones were generated by Exo 111 partial digestion and the whole coding region of the gene was sequenced. The open reading frame has 2,361 bp and codes for a protein with an expected molecular weight of 84,549 Da and a predicted pi of 5.00. The Tc85-11 gene has a high sequence identity with severai members of the trans-sialidase multigene famiiy, for instance, 82% identity with SA85-1 and 62% identity with TSA-1. ln addition, the gene possesses two non-degenerated neuraminidase motifs (SKDNGSTW and SCDMGKTW corresponding to nucleotides 835-858 and 970-993, respectively) and the common consensus sequence found in ali members of the family, namely, VTVTNVFLYNR. Presently, the whole gene is being subcioned into pET expression vector and the fusion protein will be further studied.

R. Giordano ís a post-doctoral feilow from FAPESP. Work supported by FAPESP and CNPq/PADCT.


208

CLONING OF DIFFERENTIALLY EXPRESSED GENE FRAGMENTS FROM MACROPHAGES ACTIVATED BY TRYPANOSOMA CRUZI DERIVED GPI-MUCINS AND/OR INF-g USING MRNA DIFFERENTIAL DISPLAY.

Ferreira, L.R.P. 1, 4; Silva, A M.2, 3; Reis, L.F.L.2, 3; Gazzinelli, R.T.1,4

1IMPAR, Departmento de Bioquímica e Imunologia, UFMG, Belo Horizonte, MG; 2Departamento de Microbiologia , UFMG, Belo Horizonte, 3Instituto Ludwig de Pesquisa sobre o Cancer, São Paulo, SP; 4Laboratório de Chagas, CPqRR-FIOCRUZ, Belo Horizonte, MG, BRAZIL

Macrophages (Mfs) play multiple roles in resistance to infection as well as pathogenesis of experimental Chagas'disease. The Mf activation by T. cruzi result in initiation of cytokine synthesis and inflammation, establishment of adaptive immune-response, as well as induction of microbicidal effector function. We have recently demonstrated that tGPI-mucins (trypomastigote derived glycosylphosphatidylinositol anchored mucin-like glycoproteins) are potent inducers of cytokine synthesis and macrobicidal activity by macrophages (Camargo et al. 1997, J. Immunol. 158:5890). In the studies presented here, we performed a comparative analysis of mRNAs isolated from Mfs cultured in the presence of medium alone (control), activated with tGPI-mucins and/or IFNg by using the Differential Display (DD) technique (Liang and Pardee 1992 , Science 257: 967 ). Initially the level of activation of macrophages was evaluated by RT-PCR specific for the cytokines, IL-10, MIP-1a, IL-12(p40), IP-10 and HPRT as a house-keeping gene. Our data showed that tGPI-mucins induced expression IL-10, MIP-1a, IL-12(p40) and IP-10 genes. In contrast, INF-g induced IP-10, potentiated the tGPI-mucin on IL-12(p40), but inhibited the expression of IL-10. INF-g had no effect on MIP-1a expression. For the DD, mRNA extracted from the different Mf populations were used to prepare cDNA fragments employing the following primers: ctgatccatg; ctgctctcaa; cttgattgcc and a T11XC (anchored oligo 5'primer). The cDNA fragments were then fractionated in a sequencing gel. Although most of the cDNA fragments were identical in all four cDNA preparations, we were able to find some fragments which were present on cDNAs prepared from activated Mf that were absent in the cDNA preparation from resting Mf. In order to confirm that such fragments were not artefact, we isolated each one of these fragments and performed a re-amplification using the same set of primers employed in DD. From a total of 15 fragments, 10 yield a PCR product of the expected size ( GPI-3, GPI-4., GPI-6, GPI-7, GPI-8, GPI-9, GPI-10, IFNg-1, Cont-2, GPIg-3). The PCR products were then cloned using the PUC18 plasmide inserted in SmaI site. The specificity of each cloned fragment was tested by both PCR and digestion with the restriction enzymes BamHI and Kpn I, that should yield a nucleotide with similar size of the original fragment detected in the DD. Finally the cloned fragments were sequenced and used as probe to hybridized in Northern Blot analysis, in order to confirm that they were being expressed by macrophages activated with tGPI-mucins and/or IFNg.

Supported by CNPq and CAPES


209

SEQUENCE ANALYSIS OF T. CRUZI AMASTIGOTE CDNA CLONES WITH HOMOLOGY TO MEMBERS OF GP 85/SIALIDASE SUPERFAMILY

Verbisck, N.V.#, Franco da Silveira, J. & Mortara, R.A.

Disciplina de Parasitologia, Escola Paulista de Medicina-Universidade Federal de São Paulo Rua Botucatu, 862, 6o andar, São Paulo 04023-062, SP, Brasil #E-mail: newtonviskdmip@epm.br

We have previously isolated and partialy characterized four amastigote cDNA clones after screening a Sylvio X-10/4 T. cruzi amastigote cDNA library (kindly provided by Dr. David Engman) using as a probe Tt34c1 gene, which was described as a member of the multigenic gp85/sialidase superfamily (Takle & Cross, Mol. Biochem. Parasitol., 48: 185-198, 1991; Cross & Takle, Annu. Rev. Microbiol., 47: 385-411, 1993). Initial analysis of such clones revealed that they are members of gp85/sialidase family dispersed through the genome in multiple copies. Within those clones, sx38 (insert size ~ 2 kb) was chosen for further sequence analysis, because its amastigote-specific expression was detected after Northern-blot experiments. Preliminary results indicate that sx38 has homology to 3' untranslated region of Tt34c1 gene. In addition, sx38 is probably a portion of a gene distinct from MTS-gp82 and MTS-gp90 genes previously described. We are now subcloning portions of sx38 in order to identify the open-reading frame.

Financial support: FAPESP, CNPq-PADCT, CAPES.


210

DIFFERENTIAL EXPRESSION OF MEMBERS OF TRANS-SIALIDASE GENE FAMILY OF TRYPANOSOMA CRUZI IS REGULATED BY DISTINCT FACTORS

Abuin, G., & Schenkman, S.

Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina Universidade Federal de São Paulo, R. Botucatu 862 /8o, 04023-062 São Paulo, S.P. Brasil.

Trypomastigote forms of T. cruzi invade mammalian cells, escape to the cytoplasm, transform into amastigotes and then proliferate. A few hours before breaking the infected cells amastigotes transform into intermediate, and then into trypomastigote forms that are released from the cell. Here, we have studied the expression of the trans-sialidase gene family during these transformations. The steady state levels of trans-sialidase mRNA increased about 100 fold in the intermediate forms and decreased in trypomastigotes. In contrast, mRNA levels of 85 kDa glycoproteins was high only in trypomastigotes. Incorporation of [32P]-labeled UTP in lysolecithin permeable-parasites showed that different members of this gene family were transcribed at similar rates in all parasite stages. Therefore, the steady-state levels of mRNA must be post-transcriptionally regulated. By inhibiting transcription with actinomycin D, we found that mRNA for trans-sialidase was quite stable in the intermediate forms, but was rapidly degraded in trypomastigotes, explaining why trans-sialidase expression decreased in trypomastigotes. In the presence of cycloheximide, which inhibit protein synthesis, the expression of TS mRNA was not affected in intermediate and trypomastigote forms. However, a 4 to 6 fold increase in the transcripts for the 85 kDa glycoprotein was detected in intermediate forms, suggesting that a labile negative regulator might target these transcripts for degradation or prevent their processing. Decrease in protein synthesis in trypomastigote forms might increase expression of 85 kDa glycoproteins. Therefore these genes are regulated by different factors, which in the case of trans-sialidase seem to promote stabilization of mRNA. In the case of 85 kDa glycoproteins these factors have a short half life, promoting either mRNA degradation, or preventing RNA processing.

Financial support: CNPq, CAPES, PADCT, FAPESP.


211

HIGHLY DIVERSE MUCIN-LIKE GENES ARE EXPRESSED DIFFERENTIALLY IN TRYPANOSOMA CRUZI

Freitas-Junior, L. & Schenkman, S.

Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, UNIFESP, R. Botucatu 862 8o A, São Paulo, S.P. 04023-062, Brasil

Sialic acid acceptors of Trypanosoma cruzi are characterized as mucin-like glycoproteins probably encoded by a multigene family, which shows a high degree of similarity with mammalian mucins (Di Noia et al., J. Biol. Chem, 1996, 271:32078). To study the expression of these genes in T. cruzi, we have isolated 120 mucin-like genes by RT-PCR from cDNA obtained from epimastigote and trypomastigote forms. The clones revealed a high heterogeneity in size, hybridization pattern and sequence. At least 21 different groups were found. They contained a conserved 5'and 3' that included respectively the signal sequence and the sequence for GPI addition. The internal portions of the genes, which code for threonine rich stretches, were highly variable in size and sequence. By RT-PCR and Northern blot analysis we found that these genes were expressed in all developmental stages of the parasite. We found however, that some genes were more expressed in some particular stage than others, by using probes derived from individual genes. Therefore, the expression of different mucin-like genes are developmentally regulated.

Financial support: FAPESP, CNPq, FINEP and PADCT.


212

SMALL NUCLEOLAR RNAS FROM THE SPLICED LEADER-ASSOCIATED RNA LOCUS IN THE KINETOPLASTID PROTOZO

aRoberts, T.G.1, Sturm, N.R.1, Yee, B.1, Yu, M.C.1, Hartshorne, T.3, Agabian, N.2, and Campbell, D.A.11Department of Microbiology and Immunology, University of California, Los Angeles, CA. 90095 & 2Program in Molecular Pathogenesis, University of California, San Francisco, CA. 94143 & 3Biochemistry & Molecular Biology, Albany Medical College, Albany NY 12208, USA

Small RNAs are involved in a variety of functions in the processing and maturation of RNA substrates. Small nucleolar RNAs provide direction for methylation and cleavage of ribosomal RNAs (rRNAs) and a well-defined set of small RNAs are necessary for the messenger RNA splicing reaction in a diverse range of eukaryotes. In kinetoplastid protozoa another small RNA is linked to the trans-splicing process; the spliced leader associated (SLA) RNA. First characterized in T. brucei, the SLA RNA gene locus has now been isolated from the distantly-related kinetoplastids Leishmania tarentolae and Trypanosoma cruzi. In addition to the SLA RNA, both L. tarentolae and T. cruzi SLA RNA repeat units yield three additional RNAs of 75-76 nt, 92-94 nt and a species of about 250 nt which migrates aberrantly in L. tarentolae. These RNAs have significant sequence identity to the previously described transcripts from the Trypanosoma brucei SLA RNA repeat. The three transcripts meet the criteria for small nucleolar RNAs: they possess box C/D elements, localize to the nucleolar compartment, and show potential basepairing with rRNAs. In addition, the target site for the 75-76-nt RNA is a highly conserved portion of the SSU rRNA that is methylated in Human and Xenopus; a corresponding snoRNA, U25, has been described in these systems and shows conservation with the kinetoplastid 75-76-nt RNAs. In vivo UV/psoralen treatment in L. tarentolae generates a cross-link between the spliced leader RNA and the SLA RNA, mirroring the interaction seen in T. brucei. The level of sequence similarity amongst these RNAs in three kinetoplastid species supports their conserved functions in parasite RNA processing.


213

MUTATION OF THE SPLICED LEADER RNA GENE AFFECTS TRANSCRIPT TERMINATION, PROCESSING AND TRANS-SPLICING IN LEISHMANIA TARENTOLAE

Sturm, N.R.1, Fleischmann, J., and Campbell, D.A.1

1Department of Microbiology and Immunology, UCLA School of Medicine, University of California, Los Angeles, CA 90095

The process of trans-splicing is basic to the expression of nuclear genes among the trypanosomatids. A remarkable level of sequence conservation has been noted in the exon of the mini-exon or spliced leader (SL) that is present on every messenger RNA examined to date. Conservation of the intron sequences correlates with genus and species boundaries. Using the stable transfection system that was used successfully to define SL RNA promoter elements in our laboratory, we have extended our studies to examine the exon and intron components required for the trans-splicing process. Exon mutagenesis has demonstrated that internal sequences are not required for transcription, in contrast to Ascaris. However, as in Ascaris these mutagenized exon sequences are substrates for trans-splicing. Linker scanning mutagenesis was performed through the intron region of the SL RNA gene in Leishmania tarentolae, and the ability of the resulting transcripts to trans-splice was followed with an exon-localized tag sequence by total RNA analyses and an RT-PCR assay. Most of the linker-scan intron mutants are not efficiently trans-spliced, nor do they possess a complete cap4 structure at their 5' ends. Formation of the 3' end was affected by mutations in and downstream of the Sm-binding site, and transcription termination was elimated when the downstream T-track was disrupted. Specific stem-loop structures and sequence elements within the intron were challenged to more precisely determine the nature of their roles in the maturation of the SL RNA. We will show the results of these mutations on trans-splicing, 5'-end modification, 3'-end formation and transcriptional termination.


214

IN VITRO TRANS-SPLICING IN NUCLEAR EXTRACTS FROM TRYPANOSOMA CRUZI

Bach-Elias, M., Trigueros, S. Guil, S., Codony, C. & Cicarelli, R.M.B.*

IBCMCB Centro Mixto CSIC-IMIM, Barcelona, España. *Faculdade de Ciências Farmacêuticas-UNESP, Caixa Postal 502, Araraquara, 14801-902, SP, Brasil.

An in vitro trans-splicing system by using nuclear extracts from T. cruzi has been developed with some modifications of the Dignam et al. protocol (Nucl. Acids. Res. (1989) 11, 1475-89). These modifications allow to break the parasite cells in a soft manner and to recover the nuclei. Extracts from two different strains rendered active extracts. Nuclear extracts can be done with table-centrifuges which is very convenient for laboratories with few equipment. The in vitro reaction has been performed with two acceptor pre-mRNA: the previous acceptor used in the nematode system and one African trypanosome acceptor obtained from the tubulin gene. Both acceptors suffer the trans-splicing reaction with endogenous T. cruzi SL RNAs. Some characteristics of the reaction has been studied: a) the system requires less amount of ATP than cis-splicing; b) ionic strength and Mg2+ better conditions are similar to the ones used with the nematode system; c) trans-spliceosomes can be detected on native gels; d) so far it has not been possible to use A. lumbricoides SL RNA as donor in the protozoa trans-splicing reaction, that could indicate a major dependence on the T. cruzi in vitro reaction to the proper SL RNA. Higher concentrations of A. lumbricoides SL RNA could even inhibit the trans-splicing reaction; e) U5snRNA from HeLa origin did not affect the trans-splicing reaction of T. cruzi nuclear extracts. Experiments, which will be showed, are ongoing to demonstrate that the trans-splicing reaction is accurate, to study whether human U1snRNP could affect the trypanosome trans-splicing reaction and to map the presence of T. cruzi SL RNAs and UsnRNAs inside the trans-spliceosomes complexes.

RMBC was supported by CAPES (1471/94).


215

MAPPING OF THE 5' AND 3' ENDS OF TRYPANOSOMA CRUZI POLY-A BINDING PROTEIN (PABP) mRNA REVEALS UNUSUALLY LONG UNTRANSLATED REGIONS.

João A. N. Batista and Cezar Martins de Sá

Departamento de Biologia Celular, Universidade de Brasília, Brasília-DF, 70910-900, Brasil.

We have previously isolated cDNAs encoding T. cruzi PABP and shown they are incomplete due to the difference in size between the 2 kb cDNAs and the single 5.5 kb mRNA detected. In this work we mapped the 5' and 3' ends of T. cruzi PABP mRNA, confirming they are unusually long, and analyzed flanking regions of the gene. The 5' end was mapped by RT-PCR, which identified two spliced leader addition sites located 476 and 525 bp upstream from the start codon. In accordance, primer extension analysis detected two bands with corresponding sizes. Similar to other PABP mRNAs the 5' untranslated region (UTR) of T. cruzi PABP mRNA contains extensive A-rich sequences, which in higher eukaryotes autoregulate PABP synthesis, suggesting the same mechanism operates in T. cruzi. At the 3' end, RACE experiments identified two polyadenylation sites, located 2844 and 2849 bp downstream from the stop codon. Sequence analysis of the entire region, from a genomic clone, identified several A-rich sequences, which account for the cDNAs incomplete 3' ends and rendered the 3' RACE analysis particularly troublesome. Since 5' and 3' UTRs play an important role in differential gene expression, the reason for this long 3' UTR in a constitutively expressed gene is unclear. Sequence analysis and probing on Northern blots of sequences 1.8 kb upstream and 0.9 kb downstream from the 5' and 3' ends, respectively, revealed no additional open reading frames or detectable transcripts. At the 5' end, about 750 bp upstream from the spliced leader addition sites, is a region with high identity (76% in 225 bp overlap) to the reverse and complementary strand of the 5' Ribosomal Mobile Element (RIME) of the L1Tc non-LTR Retrotransposon. This sequence is also found upstream of other T. cruzi genes, but its functional meaning is unknown.

Supported by CNPq/PADCT.


216

STRUCTURE AND FUNCTION OF THE rRNA GENE PROMOTER REGIONS OF TRYPANOSOMA CRUZI CL BRENER AND DM28 STRAINS

Stolf, B.S.1, Souto, R.P.1, Floeter-Winter, L.M.2 and Zingales, B.1

1 Departamento de Bioquímica, IQ-USP, C. Postal 26077, CEP 05599-970, São Paulo, Brasil

2 Departamento de Parasitologia, ICB-USP, CEP 05508-900, São Paulo, Brasil

Our group has defined two major phylogenetic lineages in Trypanosoma cruzi based on rRNA and mini-exon gene sequences and RAPD analysis (Souto et al., 1996 Mol. Biochem. Parasitol. 83, 141-152). Functional differences in the rRNA promoter regions were observed between the two lineages. In fact, a plasmid construct bearing the promoter sequence from CL strain (lineage 1) was shown to efficiently drive orientation-dependent expression of bacterial chloramphenicol acetyltransferase (CAT) when transfected into lineage 1 isolates, but not when transfected into lineage 2 strains (Floeter-Winter et al., 1997 Exp. Parasitol. in press). We have cloned the rRNA promoter region from Dm28 strain, typed as T. cruzi lineage 2. Sequence alignment of the rRNA promoter regions of Dm28 and CL strains showed only 82% identity, reinforcings lineage division based on sequence divergency. Plasmids bearing the promoter from the two lineages were electroporated into CL Brener and Dm28 epimastigotes. It was observed that Dm28 epimastigotes express CAT only from the homologous rRNA promoter. On the other hand, CL Brener showed CAT expression directed by both lineage 1 and lineage 2 promoters. Surprisingly, the activity in CL Brener was up to six-fold higher when directed from the heterologous promoter. The structure and function of the rRNA promoter regions of the two lineages is under analysis. The transcriptional start points from Dm28 and CL Brener were mapped by primer extension at approximately 1800bp from the 5' end of the 18S rRNA gene. In order to elucidate the structural characteristics of the Dm 28 strain promoter region that determine its activity in both T. cruzi lineages, this region has been subdivided in three portions. The resulting fragments have been combined in different plasmid constructs, which will be employed to assess CAT activity in CL Brener and Dm28 strains.

Supported by FAPESP and CNPq


217

STRUCTURAL AND FUNCTIONAL COMPARISON OF rDNA PROMOTER REGIONS OF SOME LEISHMANIA SPECIES.

Stempliuk,V. A.; Uliana, S.R.B. & Floeter-Winter, L.M.

Departamento de Parasitologia, Instituto de Ciências Biomédicas, USP – CP 66208 CEP 05389-970 São Paulo SP, Brasil

The rDNA promoter region of L. amazonensis has been structurally and functionally characterized (Uliana et al, Mol. Biochem. Parasitol.,76: 245, 1996). Constructs containing this rDNA promoter upstream to the chloramphenicol acethyltransferase (CAT) gene proved to be functional when transfected into L.major and L.mexicana. Interestingly, the levels of CAT expression attributable to activity of the L. amazonensis promoter in these species were higher than in the homologous species (Stempliuk et al, Mem. Inst. Oswaldo Cruz, 90:134, 1995).

To determine the reasons for the differential levels of expression, the regions mapped as being the minimal functional domains in L.amazonensis were characterized in the other species. Two regions, encompassing the UBF plus SL1 binding sites (nucleotides –196 to + 170) and the SL1 binding site alone ( –77 to 170) of the L. amazonensis, L. major and L. mexicana promoter sequences were obtained by PCR, cloned and sequenced. Preliminary results indicate a high degree of homology for these sequences.

Constructs bearing these minimal promoter regions derived from the three different Leishmania species, upstream to the CAT reporter gene, will be used in functional cross-species activity studies in an attempt to explain the differential levels of expression previously observed.

Supported by FAPESP and CNPq.


218

FUNCTIONAL ANALYSIS OF THE RRNA PROMOTER OF LEISHMANIA TARENTOLAE

Orlando, T.C., Fujikawa, G.Y., Campbell, D.A* & Floeter-Winter, L.M.

Departamento de Parasitologia - Instituto de Ciências Biomédicas - Universidade de São Paulo - Brasil

*Department of Microbiology and Immunology, University of California, Los Angeles, CA90095, USA

Transcription of the rRNA genes is said to be highly species-selective since there is no consensus sequence for the RNA polymerase I recognition. This recognition used to occur only among closely related organisms (Sommerville, J. Nature 310: 189-190,1984).

A construct containing the rRNA promoter sequence of Leishmania amazonensis upstream the bacterial chloramphenical acethyl transferase reporter gene (CAT) was transfected into L. tarentolae promastigotes resulting in no CAT activity (Orlando e cols. Mem. Inst. Osw. Cruz, 91(Suppl): 189, 1996.)

Another construct encompassing the IGS/ETS region and the rRNA promoter of L. tarentolae cloned upstream the CAT gene was transfected into promastigotes of the homologous species, L. amazonensis, L. major and L. hoogstraali (another lizard infecting parasite). Surprisingly, CAT activity was detected in all organisms, but the level of activity obtained for L. hoogstraali was of one order of magnitude higher in relation to the homologous species. The amount of transfected DNA was quantified and CAT expression normalized to assure that the observed high level of expression was not due to a variation of transfection experiments.

In view of these results the relationship between L. tarentolae and L. hoogstraali was examined using the rRNA SSU sequence as a molecular marker. The L. hoogstraali SSU was obtained by PCR, using conserved 5' and 3'oligonucleotides (Uliana e cols. J. Euk. Microbiol., 41(4): 324-330, 1994) and the 700nt of its 3' end were determined, showing 100% of similarity with the same region of L. tarentolae. This region shows three differences when compared to L. donovani and thirteen to Crithidia fasciculata.

These results open an interesting approach to understand the species-seletivity mechanisms of rDNA expression, mainly between lizard-infecting parasites and pathogenic species.

Supported by FAPESP, CNPq and NIH.


219

LOCALIZATION AND CHARACTERIZATION OF TELOMERIC AND CENTROMERIC SEQUENCES OF LEISHMANIA MAJOR.

1Pedrosa, A L, 1Casagrande, L ., 3Schwarz, J., 2Beverley,S.M. & 1Cruz, A K

Departamento de Bioquímica, FMRP/USP, Av Bandeirantes, 3900, Ribeirão Preto, SP, Brasil.

Dept of Molecular Microbiology, WUSM, 660, S Euclid Ave., St Louis, MO, USA, 3Depto of Molecular Microbiology and Immunology, University of Missouri, Columbia, MO, USA.

One of the major achievements of Genome Projects is the generation of tools for further studies to understand organization and dynamics of genomes, localizing not only genes but also structural sequences such as telomeres and centromeres.

In our laboratory, we have used a chromosome specific strategy to build up a physical map of Leishmania chromosomes. In the process of mapping L.major (LV39) DHFR-TS chromosome, a 300 KB contig was built. Map extension indicated a peculiar feature of sequence organization in subtelomeric regions where chromosome-specificity of mapping is lost. End-probes generated from clones mapping to the extremes of a 300 Kb "contig" identified a high percentage of 2 types of clones from the genomic library, one of which showed positive hybridization to the hexameric telomere repeat(Van der Ploeg, Cell 36: 459, 1984). It is called clone T.

Clone T is currently under characterization. We have previously determined that its sequences are at the ends of the chromosomes, and that long stretches of the clone are present in virtually all chromosomes. Our previous results also indicate the presence of polymorphism at the chromosome ends (Tosi et al, Parasitology 114: 521, 1997).

Telomeric and subtelomeric sequences have been implicated in the genome plasticity of protozoan parasites. Thus, to understand the Leishmania telomeric regions we are sequencing some subcloned fragments from clone T, which has been assigned to a specific chromosomal band using an internal region (distant 7.4 Kb from the hexameric repeat). We have also identified three other clones carrying telomeric repeats (named U, V and X) from the library. Each one of them had been assigned to different chromosomal bands. Chromosomal ends represented by cosmids T, U, V and X are being mapped to identify unique, repetitive and/or polymorphic regions.

The representation of DHFR-TS chromosome turned to be an interesting tool for the search of possible discrete centromeric regions in Leishmania. In order to localize and characterize the presence of such sequences we are using linear vectors to subclone large fragments of DHFR-TS chromosome recombinants. Such linear recombinants are transfected into Leishmania and tested for maintenance under no drug pressure. Besides, a genomic library constructed in a linear vector has been transfected in L. major. The maintenance of the exogenous molecule is periodically analyzed.

Supported by UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR); FAPESP, CNPq and CAPES


220

CLONING OF TOXOPLASMA GONDII REPETITIVE DNA ELEMENT USING A TELOMERIC-LIKE PROBE

Echeverria, P, Matrajt, M, Guarnera, E, Garberi, JC & Angel, SO.

Dpto. Parasitología, ANLIS-Carlos G. Malbran, Av. Velez Sarsfield 563, 1281-Buenos Aires, Argentina.

The genome of T. gondii is haploid and has an estimated mass of 7.7 108 bp per nucleus. It is believed that there are 11 chromosomes in the nuclear genome. In order to study some aspects of T. gondii genomic organization , an oligonucleotide based on Eimeria tenella telomeric-like sequence was designed and used as probe. Southern blot analysis of T. gondii chromosomes separated by pulse field gel electrophoresis (PFGE) showed that telomeric-like probe hybridized with all parasite chromosomes. This telomeric-like probe was used to screen a T. gondii cosmid library (Dr. D. Roos, University of Pennsylvania, USA). A positive clone was isolated containing an 1.9 kb EcoRI fragment (Tg 1.9) that hybridized with the probe. Southern blot analysis using probe Tg 1.9 showed a characteristic pattern of repetitive DNA elements. Thus, probe Tg 1.9 hybridized with all T. gondii chromosomes separated by PFGE. Partial sequence of Tg 1.9 was searched in GenBank and EMBL without finding any significative homology. Complete sequencing of the fragment is in progress. The data presented here indicates that the 1.9 kb Eco RI fragment contains a repetitive DNA element located in all T. gondii chromosomes, which hybridized with a telomeric-like DNA sequence and has not yet been described for T. gondii.


221

ANTIGENICITY OF RECOMBINANT ROP 2 PROTEIN OF TOXOPLASMA GONDII EXPRESSED IN ESCHERICHIA COLI

Martin, V, Cespedes, G, Santillan, G, Pszenny, V, Guarnera, E, Garberi, JC & Angel SO.

Dpto. Parasitología, ANLIS Carlos G. Malbran, Av. Velez Sarsfield 563, 1281-Buenos Aires, Argentina.

Rop 2 (P54) is a T. gondii rhoptry protein which was cloned by immunoscreening of a T. gondii lgt 11 library with a pool of sera from chronically infected donors. Rop 2 was found to be one of the parasite antigens, against which human Toxoplasma-specific T cell reactivity is directed, producing interleukin-2 and interferon gamma. In order to evaluate the humoral antigenic value of Rop 2, it was expressed in E. coli as a fusion protein containing 44-kDa of the 55 k-Da of mature Rop 2, supplied with six histidyl residues at the N-terminal end. With this purpose, a Bam HI-Hind III DNA fragment from the digestion of a Rop 2 containing plasmid (Dr. K. A. Joiner, University of Yale School of Medicine, USA) was subcloned into pQE plasmid. The recombinant protein was purified on a Ni-chelate column. Purification was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by Western blot. The antigenic value of recombinant Rop 2 was analyzed by ELISA with sera from individuals with chronic T. gondii infection according to serological data. Antibody reactivity was observed in 88% of the 25 sera with anti-T. gondii IgGs indirect immunofluoresce (G-IIF) titers between 256 to 4,096. Analyzing 5 sera with anti-T. gondii G-IIF titer value of 64, none gave positive. On the other hand, 25 sera from Toxoplasma seronegative individuals did not react against the fusion protein. Therefore, we suggest that recombinant Rop 2 would be suitable for use in diagnostic systems in combination with other T. gondii antigens.


222

DIFFERENT CHROMOSOMAL LOCALIZATION OF HISTONE H1 AND H3 GENES IN TRYPANOSOMA CRUZI. DIVERGENCY WITH OTHER GENERA.

Triana, O.; Galindo, M.; Toro, G.C. and Galanti, N.

Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile.

Trypanosomatids are parasitic protozoa which characteristically do not condense their chromatin to chromosomes during cellular division. This may be related to a short histone H1 and a high divergency in the sequence of the core nucleosome histones, as compared to higher eukaryotes.

The aim of this work was to determine the chromosomal localization of histone H3 and H1 genes in three genera of the Trypanosomatidae family.

Chromosomes of T. cruzi (two strains and one clone), T. rangeli (three strains), L. mexicana (one strain) and C. fasciculata (one strain) were separated by PFGE and hybridized to H3 and H1 probes from T. cruzi. Hybridization was specific for T. cruzi, either using a high stringent condition or a low stringency. Similar results were obtained using a multi-probe of 35 Kbp from T. cruzi. Under high stringency, the H1 gen showed a strong hybridization signal in a chromosome of approximately 2200 Kbp, with different intensity in the three T. cruzi strains. Using low stringent conditions other bands were observed at 1000 Kbp, probably related with genic variants of this protein. Histone H3 showed one hybridization signal in the zone of 700 to 1000 Kpb, for each of the three T. cruzi strains.

Our results suggest an important divergency in the sequences of the genes coding for histones H1 and H3, in the three genera of the Trypanosomatidae family. Differences in the intensity of the hybridization signals among T. cruzi strains may be related to differences in the number of genes. Moreover, contrary to higher eukaryotes, Trypanosomatid histone genes are localized in different chromosomes.

Supported by Project SIDA/SAREC


223

STABILITY OF THE MOLECULAR KARYOTYPE DURING CELL PROLIFERATION AND DIFFERENTIATION IN TRYPANOSOMA CRUZI.

Galindo, M.; Sabaj, V. and Galanti, N.

Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile.

Conventional cytogenetics is based on the observation of mitotic chromosomes at a particular stage of the cell cycle: metaphase. On the other hand, PFGE is used to separate intact chromosomes from unicellular parasites and lower eukaryotes, in asynchronic cultures. This approach does not consider variations in the replicative status of the chromosomal DNA during the cell cycle or possible lost of DNA during cell differentiation.

PFGE molecular karyotypes were obtained from asynchronic cultures of T. cruzi epimastigotes, from hydroxyurea-synchronized epimastigotes and from trypomastigotes. The chromosomal pattern of epimastigotes did not change along the growth curve. No important differences between the molecular karyotypes of synchronic and asynchronic cultures were observed, except in the intensity of the bands. Similarly, the karyotypes obtained from the replicative epimastigotes and the Go non replicative trypomastigotes were identical. Finally, the molecular karyotype of 52 days old cultures, in which only 15% of the cells are viable, showed a pattern similar to the other conditions tested.

Our results suggest a high stability of the T.cruzi karyotype during cell proliferation, cell quiescence and cell differentiation of this parasite.

Supported by Project SIDA/SAREC


224

RESTRICTION FRAGMENT LENGTH POLYMORPHISM OF PCR AMPLIFIED PRODUCTS CORRESPONDING TO THE VARIABLE REGIONS OF KDNA MINICIRCLE OF LEISHMANIA (VIANNIA)

Derré, R.1; Degrave, W2.; Saravia, N.3 & Fernandes, O.1,4

1. Departamento de Patologia, Universidade do Estado do Rio de Janeiro; 2. Departamento de Bioquímica e Biologia Molecular, Instituto Oswaldo Cruz, Fiocruz; 3. Fundación CIDEIM, Colombia; 4. Departamento de Medicina Tropical, IOC, Fiocruz.

Restriction Fragment Lenght Polymorphism of the kinetoplast DNA (kDNA) has been widely used as a typing tool for several trypanosomatid species. This technique, which was named schizodeme analysis, has proven to be very specific and has the potential of typing different isolates from the same genus. In Leishmania Viannia, schizodeme analysis has demonstrated that L. braziliensis, L. panamensis and L. guyanensis show distinct profiles. These differences are due to sequence heterogeneity in the variable region of the minicircle molecule, as the conserved region is almost identical among these species. Our group has previously demonstrated that the Polymerase Chain Reaction can amplify solely the variable region pool of (L. Viannia) isolates, using a set of primers directed outwardly from the minicircle conserved region (5'-TTCG(G/C)AGAACGCCCCT(A/C)CCC & 5'-GGGTTGGTGTAA(A/T)ATAG(G/T)(G/C)(G/C)C - Fernandes et al., Mem. Inst. Oswaldo Cruz, 90 (Suppl. I): 138, 1995). Such PCR products were used in hybridization assays showing a high degree of specificity in the hybridization patterns. In order to further investigate this same phenomenon we performed a RFLP analysis, using several restriction endonucleases to digest PCR products corresponding to the variable region of the (Viannia) minicircle molecule. The restriction fragments were analyzed by poliacrylamide gradient gel electrophoresis and silver staining. A highly heterogeneous profile can be found with some enzymes confirming the different hybridization patterns that were evidenced when the variable regions were used as hybridization probes. The RFLP analysis of the PCR products corresponding to the pool of variable region sequences of the minicircle molecules offers several advantages when compared to traditional schizodeme experiments, as it is not necessary to submit the protozoa to in vitro culture, a procedure that could select sub-populations, nor even extract the DNA from the kinetoplast, a fastidious and cumbersome task.

This research is sponsored by the Latin America Linkage Grant - W.H.O. - U.N.D.P. Special program for Tropical Disease Research.


225

ORIGIN OF TRYPANOSOMA CRUZI GENETIC DIVERSITY BY CLONAL EVOLUTION AND OCCASIONAL RECOMBINATION

Brisse S.; Barbanabe C.; Souchon A. and Tibayrenc M.

Centre d'Etudes sur le Polymorphisme des Microorganismes, UMR CNRS/ORSTOM 9926, ORSTOM, 911avenue Agropolis, 34032 Montpellier Cedex IOne hundred stocks representative of the major phylogenetic lineages of T cruzi were studied by Multilocus Enzyme Electrophoresis (MLEE), multiprimer RAPD fingerprinting, Restriction Fragments Length Polymorphism (RFLP), specific PCR, and sequencing of mitochondrial genes. Related bat trypanosomes were included in the study as extemal outgroups. The multilocus approaches (MLEE and RAPD) revealed the partition of the clones into two major phylogenetic lineages, the second being further subdivided into five additional smaller clades. PCR primers were designed to generate clade-specific PCR amplifications. The sequencing of PCR fragments showed that at least two clades were generated by past recombination events between clones belonging to other clades, as was also suggested by MLEE, RAPD and RFLP data. The two recombinant clades contained stocks previously described as zymodeme 39 and zymodeme 43, respectively, while the parental clones were related to zymodemes 32 and 36 (Tibayrenc et al., 1986). The sequences of variable maxicircle genes were in accordance with the recombination hypothesis, and further showed that zymodeme 32 had a very divergent maxicircle compared to its nuclear genes, which is suggestive of a horizontal transfer from a less-related donor. However, the maxicircle sequences of three different bat trypanosome species did not reveal any relationships with zymodeme 32, which was more related to the other T. cruzi lineages than to the bat trypanosomes. Altogether, these results strongly suggest that the genetic diversity of T. cruzi was generated by long-term clonal evolution and occasional phenomena of hybridization between clonal lines.


226

POLYMORPHIC MICROSATELLITES REVEAL DETAILS OF THE GENETIC POPULATION STRUCTURE OF TRYPANOSOMA CRUZI

Oliveira, R.P., Melo, A.I.R., Macedo, A.M. & Pena, S.D.J.

Depto. de Bioquimica e Imunologia, UFMG, Av. Antônio Carlos 6627, Caixa Postal 486, CEP 31.270-010, Belo Horizonte, Brazil

Trypanosoma cruzi presents great heterogeneity in its biological, biochemical and genetic characteristics. Indeed, a complex clonal population structure has been suggested on the basis of isozyme studies. We have now, for the first time, performed analyses of T. cruzi populations using microsatellite polymorphisms, which have the advantages of being hypervariable. We cloned and sequenced eight polymorphic (CA)n repeat loci from the T. cruzi genome and used them to study 26 different T. cruzi strains and clones, isolated from sylvatic mammals, vectors and chagasic patients. Primers were fluorescently labeled and the PCR products were run in the ALF automatic sequencer to determinate the allele sizes. Many clones exhibited heterozygosity at several microsatellite loci, demonstrating diploidy for the T .cruzi genome. Six strains, isolated from opossum, triatomid bugs and acute chagasic patients, showed presence of more than 2 alleles, strongly suggesting that they were composed of more than one clone. The observed heterozigosity levels for the loci varied from 0.20 to 0.80. The observed genotypes were not distributed according to Hardy-Weinberg proportions and there was strong linkage disequilibrium between the loci, indicating that T. cruzi has a clonal population structure with absent or rare sexual reproduction. No single multilocus genotype was observed more than once, in agreement with our previous suggestion, based on DNA fingerprint studies, that there is absolute genetic individuality of clonal lineages in T. cruzi. The CA-repeat genotypes were also used to investigate phylogenetic relationships among the strains based on parsimony method. Assuming a stepwise mutation model, the genetic distance used to construct the Wagner tree was based on the minimum number of mutational steps separating any two strains. The Wagner tree showed considerable genetic distance among the T. cruzi strains, with a minimum number of 17 mutational steps between any pair of strains. The Wagner tree topology was in excellent agreement with the rRNA 24Sa gene dimorphic strain classification.

Supported by PRONEX (FINEP), CNPq and FAPEMIG


227

TRYPANOSOMA CRUZI GENOME PROJECT: CONSTRUCTION OF CDNA LIBRARIES AND FACILITATION OF GENE DISCOVERY

Fernandes, M.1, Soares, M.B.2, Bonaldo, M.F.2, Rondinelli, E.1 & Ürményi, T.P.1

1Instituto de Biofísica Carlos Chagas Filho, CCS, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21949-900, Brazil. 2The University of Iowa, Iowa City, IA, USA.

A joint effort of several laboratories is in progress to map and sequence the entire Trypanosoma cruzi genome. Complete genomic nucleotide sequences allow whole genome studies, which include large scale genome organization, sistematic knockout to determine gene function, characterization of the complete set of expressed sequences of a given cell type (sometimes called a transcriptome) and evolutionary comparisons between organisms. Genome projects are greatly aided by the sistematic, large scale sequencing of randomly picked clones of cDNA libraries, which generates expressed sequence tags (ESTs) for the mapping effort and facilitates gene discovery. We have constructed four directionally cloned, oligo(dT)-primed cDNA libraries of poly(A)+ RNA from clone CL Brener epimastigotes: two standard, non-normalized libraries, a library enriched with abundant clones and a normalized cDNA library. The frequency of individual cDNA clones in a normalized library are brought within a narrow range, thereby greatly increasing the cost-effectiveness of large scale sequencing. The T. cruzi library was normalized by a recently described method based on reassociation kinetics (Soares et al., PNAS USA 91:9228-9232, 1994; Bonaldo et al., Genome Res. 6:791-806, 1996). The normalized library has been distributed to several groups and large scale sequencing is currently in progress. More than 1700 clones have been sequenced so far, 30% of which a match has been found in the public sequence databases. Less than 4% of the total number of clones sequenced were sequenced twice or more than twice, clearly showing the advantages in cost-effectiveness of a normalized cDNA library for this approach. We are currently obtaining metacycnic trypomatigotes for poly(A)+ RNA isolation and construction of a cDNA library.

Supported by CNPq, FINEP, CEPG-UGRJ, INGEBI and UNDP/World Bank/WHO.


228

CONSTRUCTION OF A Tc-85 LIBRARY AND EXPRESSION OF Tc-85 IN EPIMASTIGOTES OF TRYPANOSOMA CRUZI

Giordano, R., Alves, L.C.C., Pereira, V.T., Colli, W. & Alves, M.J.M.

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, C.P. 26077, 05599-970 SP, Brazil

Using synthetic nucleotides a Tc-85 library into pTEX vector was generated by PCR. In brief, the oligonucleotides were used to subject T. cruzi genomic DNA to PCR and the product was ligated to the pTEX vector and digested with EcoRV/CIAP. The ligation reaction was used to transform E. coli DH5a and approximately 3,500 clones have been obtained. These clones were pooled, amplified and stored as plasmids which have been purified in large scale preparations using CsCl gradients. Twelve clones were selected for sequencing analyses for the 5' and 3' ends of the genes. Ten clones had high identity with previously described members of the trans-sialidase multigene family (56-93% sequence identity with SA85-1, SA85-2 and Tc85-11). One of the clones showed an identity of 56% with TSA-1. Two clones did not show any significant identity with previously described members of the trans-sialidase family. Since the gene cloning into pTEX was a blunt-ended ligation, the genes could ligate in two different orientations in relation to the pTEX promoter region. The sequence analysis revealed 5 clones (50%) that subcloned into the correct orientation and 5 (50%) in the opposite orientation, as expected. The clones in the correct orientation were selected for further studies. The library was also used to transfect epimastigotes which, after transfection, were grown for 3 days, fixed and reacted with an anti-Tc-85 polyclonal serum and H1A10 monoclonal antibody. Most of the parasites (about 50%) reacted with the anti-Tc-85 serum and a smaller percentage with the monoclonal antibody, a result which strongly suggests that the Tc-85 genes may have been expressed by the epimastigotes.

R. Giordano is a post-doctoral fellow from FAPESP. Work supported by FAPESP and CNPq/PADCT.


229

VIPER, A NEW TRYPANOSOMA CRUZI DISPERSED ELEMENT IS COMPOSED WITH A HALF OF SIRE ELEMENT AT EITHER END

Martin Vazquez, Claudia Ben-Dov and Mariano J. Levin.

Institute of Genetic Engineering and Molecular Biology (INGEBI-CONICET-FCEyN), University of Buenos Aires, Argentina.

Approximately 2000-3000 copies of the 433 bp short interspersed repetitive element (SIRE) occur in the nuclear genome of Trypanosoma cruzi, accounting for 1-2 % of total genomic DNA (Vazquez, M. et al, Mol. Biochem. Parasitol. 64, 327-336, 1994). Screening of genomic libraries with SIRE probe led us to the identification of another larger element (2359 bp) named VIPER that is composed with a half of SIRE at either end. Each half (5' of 182 bp and 3' of 226 bp) is separated by a 1923 bp connecting segment. This particular structure is found also in the non-LTR retrotransposon ingi of T.brucei, composed with halves of RIME at either end. VIPER encodes a gene for a putative protein of 230 aminoacids that is transcribed and its mRNA includes the 3' half of SIRE. This protein showed restricted homologies with a region of a DNA polymerase of HBV and with ribonuclease H domains. In contrast, VIPER is much less abundant than SIRE (around 10 times) and its distribution is restricted to only 4 chromosomes ranging in size from 0.9 to 1.6 Mbp in CL-Brener strain. The question remains open if SIRE originates from a deletion in VIPER or if VIPER is originated by insertion in SIRE of a genomic fragment with coding sequences.

Supported by CEPH-INGEBI cooperation program-CyTED-WHO/TDR-UBACyT.


230

EXPRESSION OF SIRE ELEMENTS IN TRYPANOSOMA CRUZI

Martin Vazquez, Claudia Ben-Dov and Mariano J. Levin.

Institute of Genetic Engineering and Molecular Biology (INGEBI-CONICET-FCEyN), University of Buenos Aires, Argentina.

The 433 bp Short Interspersed Repetitive Element (SIRE) is present in about 2000 copies in the genome of T.cruzi and is distributed through out all chromosomes. We first described the element associated to a tandem of two TcP2b genes, where the insertion of SIRE upstream of each gene provides a novel functional trans-splicing acceptor site and polypyrimidine region (Vazquez, M. et al, Mol. Biochem. Parasitol. 64, 327-336, 1994). In order to study if SIRE is transcribed elsewhere in the genome, we performed northern blot analysis on total RNA and found that several bands from 0.8 to 6 kb reacted with SIRE probe. Further analysis using RT-PCR technique showed that complete SIRE sequences are represented in T.cruzi poli A+ RNA. We used two cDNA CL-Brener libraries, normalized and non-normalized (kindly provided by Dr. Edson Rondinelli), to analyze these SIRE transcribed sequences and found that SIRE is present in the 3'UTR of several different parasite mRNAs (at least 20), including histone H2A. Most of these mRNAs correspond to single copy genes or tandemly arranged genes. Interestingly, we found that SIRE is transcribed always in the same orientation and in many cases polyadenylation occurs within the element. At present, we are performing transfection experiments to address the influence of SIRE in mRNA stability.

Supported by CEPH-INGEBI cooperation program-CyTED-WHO/TDR-UBACyT.


231

TOWARD A YAC-BASED PHYSICAL MAP OF TRYPANOSOMA CRUZI CHROMOSOMES XIX AND XX: GENERATION OF SPECIFIC STSs AND ESTs

Hernán Lorenzi; Cecilia Medrano; Alejandro Schijman; Mariana Catalani; Mariano J. Levin.

Laboratorio de biología Molecular de la Enfermedad de Chagas, INGEBI-CONICET, FCEN-UBA, Buenos Aires, Argentina. E-mail: hlorenzi@proteus.dna.uba.ar

The physical map of the Trypanosoma cruzi genome is an essential tool for localizing the complete inventory of T.cruzi genes, understanding its genome organization and allowing to device sequencing strategies for the T.cruzi genome project. The aim of our work is the construction of YAC-based physical maps of the two largest chromosomes of this parasite named chromosomes XIX and XX. Given their large size (3.3 Mb and 3.5Mpb respectively), physical mapping requires using clones with very large inserts, of the order of 100 kb or more. Since YACs are able to propagate such large DNA fragments, they are very useful for covering large regions such as the cases of chromosomes XIX and XX.. In addition, YAC-based physical maps are important intermediates to provide "ready to sequence" tool consisting of smaller clones like BACs or cosmids.

Our strategy can be divided in three main parts: 1) generation of subsets of YACs enriched in chromosome-specific clones, 2) isolation of ESTs and STSs specific for chromosomes XIX and XX and 3) construction of YAC-contigs and physical maps assembly.

The first screening of 1250 YACs of the T.cruzi genomic YAC library with total DNA of chromosome XX as a probe allowed the isolation of about 60 positive clones, which represent 17% of the analyzed library. Similar results were obtained when the library was screened with chromosome XIX total DNA. The first step towards the characterization of the isolated YACs was the construction of vectorette libraries to obtain: 1) YAC-derived STSs useful as probes for contig assembly and 2) "ready to sequence" YAC extremities, applying the bubble-vector PCR methodology (Munroe, D.J.; Haas, M.; Bric, E.; Whitton, T.; Aburatani, H.; Hunter, K.; Ward, D.; Housman, D.E. Genomics 19: 506-514). Since some YACs presented the repetitive element SIRE we further applied a SIRE-bubble PCR approach to generate additional STSs named SAS (SIRE-associated STS).

Finally, in order to generate chromosome specific ESTs we screened a pT3T718D cDNA library with chromosome XIX total DNA. Up to now 3 out of 500 cDNA clones have been isolated and they are under characterization. Additional cDNA clones will be isolated by this method and those specifics for chromosomes XIX and XX will be used in the screening of YAC subsets.


232

TRYPANOSOMA CRUZI GENOME PROJECT: CONSTRUCTION OF YAC CONTIG AND INTEGRATED GENETIC MAP OF A REGION OF 880 KB ON CHROMOSOME XVI (2.3 MBP)

Santos, M.R.M1, Lorenzi, H2, Brandariz, S2, Schijman, A2 ,Ferrari, I2., A., Carmo, M.S1, Gomes, H.B.M1, Brandão, A3, Degrave, W3, Levin, M.J2 and Franco da Silveira, J1.

1. Depto Micro, Imuno e Parasitologia, Escola Paulista de Medicina, UNIFESP, Rua Botucatu, 862-CEP 04023-062, SP, Brasil (email: mrmachados.dmip@epm.br); 2. Ingebi, Buenos Aires, Argentina; 3. DBBM, Fiocruz, RJ, Brasil.

The objective of this work is to construct an ordered telomere to telomere contig map of chromosome XVI by isolating overlapping YAC and cosmid clones from whole genomic libraries. A T. cruzi YAC library, containing about 3,000 recombinant clones with a mean insert size of 365 kb and representing more than 10 genome equivalents, was used to isolate the recombinant clones. Contig assembly has begun around well characterized loci, such as H49 (also named JL7, Ag1 and FRA) and JL8 (also named Ag30, CRA, TCR27) which encode two large immunodominant repetitive antigens located on the flagellum and cytoplasm of the parasite. H49 and JL8 genes were mapped on two chromosomal bands of 2.3 and 2.6 Mbp (chromosomes XVI and XVII, respectively).

Several markers were mapped on the chromosomal band XVI: 6 polymorphic repetitive elements (C6, SIRE, 196 bp-minisatellite, SRE, B11), 9 genes encoding proteins and structural RNAs (gp85, gp82, gp90, H49, JL8, cDNA40, cDNA78, cDNA68, spliced leader sequence) and 2 anonymous markers (SZ23, SZ14).

We have constructed a YAC contig and integrated genetic map covering a region of 880 kb of chromosome XVI (2.3 Mb). The contig comprises 11 overlapping YAC clones (inserts: 150 to 600 kb), 2 l phages and 1 cosmid that were aligned based on the presence/absence of 17 DNA markers (6 STS polymorphic markers, 7 known genes encoding protein or RNA, 2 EST, 2 anonymous markers). The alignment was also confirmed by comparison of the enzyme restriction site patterns of the recombinant clones with the genomic DNA. Average genetic markers spacing in the contig was estimated to be 64 kb/marker.

In order to investigate whether H49 and JL8 loci are located near at telomeric region, agarose blocks containing whole chromosomal T. cruzi DNA were digested with increasing amounts of exonuclease Bal31 and digested with SfI restriction enzyme. The resulting fragments were hybridized with H49 and JL8 probes. H49 probe hybridized with SfI fragments of about 50 kb. These fragments were derived from a ~ 400 kb Sf I fragment that shown to be sensitive to the treatment with Bal31. On the other hand, JL8 probe reacted with two SfI fragments (50 and 23 kb) that were not affected by the treatment with Bal31. These results suggest that the gene order is: telomer ®....® H49 ® Jl8 ®....®centromer.

Supported by FAPESP, CNPq, FINEP/BID, CYTED and WHO/TDR.


233

PHYSICAL MAPPING ACROSS LEISHMANIA MAJOR GENOME: EST AS A MARKER AND AS A TOOL FOR GENE DISCOVERY.

Ruiz, J.C, Iribar, M. P. , Cruz, A..K..

Departamento de Bioquímica, FMRP, Universidade de São Paulo, Av. Bandeirantes, 3900, Ribeirão Preto, 14049-900, SP, Brasil.

Physical mapping constitutes an important step in genome projects and allows the organization of the subsequent stages in genome mapping and sequencing. It consists of ordering genomic DNA fragments reproducing their chromosome location, in a process called contig generation. In our effort to physically map the Leishmania major genome we have employed a random mapping strategy. This approach uses expressed sequences to build up contigs across L. major genome (LV39, Rho/SU/59/P). Here, we show that the chosen strategy proved to be efficient. We have used 300 ESTs (Expressed sequence tags) for the construction of "contigs" in several chromosomal bands of the parasite genome. The ESTs are used to rescue recombinants from the L. major genomic library, by hybridization to filters containing a high density array of this library. The same probe is also hybridized to PFGE (Pulse Field Gel Electrophoresis) separated chromosomes, allowing the assignment of markers and contigs to a specific chromosomal band. We used ESTs generated from a lambda UNIZAP LV39 cDNA library at J. Blackwell (Cambridge, UK ) and H. Schneider (UFPA, Belém, Brazil) laboratories. All data have been stored as lists of positive clones and corresponding ESTs in an Excel File. We developed a program under FoxPro to prepare and format data for SAM (a contig assembler software developed at Sanger Centre, UK). We will present a number of possible contigs assembled by SAM. Various solutions across different chromosomal bands have been confirmed through restriction profile and hybridization analysis, indicating the efficiency of the designed strategy. As part of our strategy for the construction of a physical/transcriptional map the LV39, cDNA has been pooled to be used as a tool to increase markers density onto growing contigs.

The ESTs are also an important source for gene discovery. 70% of the Leishmania sequenced tags have no similarity to other transcribed sequences available on databases. Thus, as a branch of the EST mapping project, we are carrying studies for the characterization of a gene with unknown function. Preliminary results on the genomic organization of such gene and its flanking sequences will be presented.

Supported by UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR); FAPESP; CNPq, CAPES.


234

ACANTHAMOEBA PHYLOGENETIC RELATIONSHIPS INFERRED FROM SSU RDNA FINGERPRINTING

Alves, J.M.P., Teixeira, M.M.G., Foronda, A.S. & Affonso, M.H.T.

Depto. de Parasitologia, Instituto de Ciências Biomédicas - USP, 05508-900, São Paulo – SP.

Two severe pathologies of humans are caused by some species of free-living amoebae of the Acanthamoeba genus: a painful sight-threatening keratitis and granulomatous amebic encephalitis (GAE). Rapid identification of their ethiological agents are very important in order to improve efficient diagnosis and treatment of the patients (in case of keratitis, because GAE leads to death). Despite the intensive use of Acanthamoeba species in classical molecular and cellular studies, the identification, taxonomy and phylogeny of the genus remain confuse and not well established. Different molecular methods are currently been used in an attempt to solve taxonomic problems that traditional morphological criteria is unable to do.

This study involved 8 ATCC strains and 11 cornea isolates from Brazil. All keratitis isolates and ATCC reference strains were analyzed by total DNA restriction fragment length polymorphism - RFLP (see Alves, J.M.P. et al, 1996 - Mem. Inst. Oswaldo Cruz, 91, Suppl.:75). Polymerase chain reaction (PCR) was then performed in order to isolate the sequence that encodes the ribosomal small subunit DNA (SSU rDNA). The oligonucleotides for PCR were obtained by aligning GenBank-available Acanthamoeba SSU rDNA sequences. Highly conserved SSU flanking 18-mers oligonucleotides were chosen and used as primers in the reactions. Digestion of amplified SSU rDNAs was performed with some restriction enzymes (MspI, HhaI, RsaI and others) in order to generate patterns that can be used in computer aided cluster analyses, as previously done with total DNA RFLPs. Total DNA analyses revealed high intra and interspecific genetic diversities among the ATCC reference species and the human cornea isolates. Preliminary analyses of the SSU rDNA RFLPs indicated a still high diversity, although not so prominent as detected before using total DNA. This fact is probably due to the more conserved, and biologically significant nature of the rDNA sequences, which is advantageous in making more reliable groupings.

Data matrices, based on molecular weights of the bands presented in RFLP gels, will be constructed and genetic distances and phenograms will be determined. It will make possible to group the Acanthamoeba strains and isolates into distinct subgroups according to their degree of similarity. These results will be compared with those previously obtained with total DNA RFLPs.

Supported by FAPESP and CNPq


235

ANALYSIS OF GENETIC DIVERSITY OF PLASMODIUM FALCIPARUM FIELD ISOLATES FROM THE BRAZILIAN AMAZON USING THE PCR-SSCP METHOD.

Sallenave-Sales, S1; Ferreira da Cruz, MF1; Durlacher, R2; Pang, L2; Daniel-Ribeiro, CT1 & Zalis, MG3. Department of Immunology, IOC, Fiocruz, RJ-Brazil1; USA Medical Research Unit, WRAIR, RJ-Brazil2 & IBCCF, Federal University, RJ-Brazil3

Analysis through PCR of the genetic polymorphism of P. falciparum populations has been an important tool for epidemiological studies and for evaluation of antimalarial treatment follow-up studies in malaria endemic areas. In the present work, we assess the degree of clonal diversity of 42 P. falciparum isolates from Peixoto de Azevedo (MT-Brazil), by amplifying the repeated variable region of MSA-2 gene. The PCR fragments size variation were first analyzed in a 2% agarose gel and then by the Single Strand Conformational Polymorphism (SSCP) which allows the discrimination of sequence microheterogeneities in the amplified products. In our preliminary analysis, we succeed to amplify the MSA-2 fragment in all samples. In an agarose gel, a low frequency of isolates (12%) presenting mixed infections were found, while 37(88%) presented only one fragment. Four different size allelic variants of MSA-2 gene were observed. Size allelic variant of 520 bp were presented in 27 out of 42 isolates (64%) and of 620bp in 9 isolates (21.4%), while fragments of 600bp or 400bp was present in a single isolate each one (2%). In order to verify sequence variations we performed a SSPC technique. Each allelic amplified product was digested with RsaI and analyzed in a polyacrilamide gel under denaturing conditions.This approach confirmed the results observed in agarose gels, showing that there is no sequence difference between the fragments with the same size.

Supported by CNPq


236

ALLELIC DIVERSITY AT THE MEROZOITE SURFACE PROTEIN-1 LOCUS OF PLASMODIUM FALCIPARUM IN CLINICAL ISOLATES FROM THE SOUTHWESTERN BRAZILIAN AMAZON.

Ferreira MU,1,2 Liu Q,2 Kaneko O,3 Kimura M,4 Tanabe K,5 Kimura EAS,1 Katzin AM,1 Isomura S2 & Kawamoto F2

1Department of Parasitology, Institute for Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazil; 2Department of Medical Zoology, Nagoya University School of Medicine, Nagoya, Japan; 3Department of Medical Zoology and 4Laboratory of Biophysics, Osaka City University Medical School, Osaka, Japan; 5Laboratory of Biology, Osaka Institute of Technology, Osaka, Japan.

Nucleotide sequences of each variable block in the Plasmodium falciparum merozoite surface protein-1 gene (PfMSP-1) may be grouped into one of two or three possible allelic types, named after the reference isolates MAD20, K1 and RO33. Allelic diversity at this locus basically results from different combinations of allelic types in variable blocks. Here we used a polymerase chain reaction (PCR)-based strategy to type the variable blocks 2, 4a, 4b and 10 of the PfMSP-1 gene of P. falciparum isolates from 54 symptomatic malaria patients living in Rondonia, a hypoendemic area in the southwestern Brazilian Amazon. Ten different PfMSP-1 gene types, defined as unique combinations of allelic types in variable blocks, were identified among the 54 isolates. Twenty-one isolates (39%) harbored more than one gene type and two had at least three genetically distinct clones. Hybrid sequences, with a MAD20-type sequence in the 5' segment (4a) and a K1-type sequence in the 3' segment (4b), were quite common in block 4. Direct sequencing of block 4 PCR products revealed a new putative recombination site in four isolates. In contrast with previous studies, the observed distribution of gene types does not deviate significantly from that expected under the null hypothesis of random association between allelic types detected in each variable block. These contradictory data are discussed with reference to the immuno-epidemiological features prevailing in distinct malaria-endemic areas.


237

MALARIA SURVEY IN MONKEYS CAPTURED IN HYDROELECTRIC FLOODING AREA AT SERRA DA MESA, GOIAS, BRAZIL.

Duarte, A.M.R.C. 1 , Hoffmann E.E.H.1, Malafronte R. S.1, Oliveira, S.2, Brígido, M.C.O.3 , Kloetzel, J.K.1,4

1-Inst. Med. Tropical de S.Paulo. Av. Enéas Carvalho Aguiar, 470, São Paulo, 05403-000, Brasil 2-Inst.Evandro Chagas, Belém PA. 3-Centro Nacional de Primatas, FNS, Ananindeua, Pa. 4-Dept. Parasitologia, ICB-USP.

Literature on Brazilian monkey malaria infection, based mainly on morphological data, described P.brasilianum as being present in the Northern part, P.brasilianum and P.simium in the Southern part of Brazil.. The study of monkey malaria is important to clarify the relationship between human and monkey malaria, since homologies have been described, both for morphology, biology and CS protein repeats. Tandem repeats of the circumsporozoite protein (CS) are homologous for P.brasilianum/P.malariae [(NAAG)4]4, P.simium I/ P.vivax I (GDRADGQPA)2(GDRAAAGQPA)2(GDRADGQPA), P.simium II/P.vivaxVK247 II (ANGAGNQPG)4 and P.simiovale/human P.vivax-like (APGANQEGGAA)3. In the present paper we describe results of the serological and parasitological study of 51 monkeys from Serra da Mesa, Goias. Non-human primates were captured for salvation, in an area that is being flooded for the construction of a hydroelectric plant. The specimens were 42 Alouatta, 4 Cebus, 5 Callithrix. Animals were anesthetized, blood drawn .Thin and thick smears were prepared for each specimen. Both plasma and rbc were frozen, at -20oC, and shipped on dry ice. Indirect immunofluorescence assay for both IgG and IgM anti P.vivax and P.malariae asexual antibodies, as well as peptide ELISA for the above repeats were performed. Cut-off points for the reactions were standardized, based on mean OD plus 3 s.d. of 37 monkey sera of known origin born or kept in captivity. Among the 51 samples, there were 12 positive reactions for Pb/Pm, 14 for PsI/PvI, 7 PsII/PvII, 15 for Pso/Pvlike, 9 of these were positive for more than one peptide, while 21 tested negative. IFA was negative for IgG antibodies, 7 tested positive for IgM anti-P.malariae and 2 P.vivax, titers varying 1:40-1:160. None of the smears had malaria parasites. DNA was extracted from rbc and PCR performed with specific primers for the terminal region of the CS gene of P.brasilianum/P.malariae, P.vivax/P.simium as well as a genus-specific 18S rRNA primer, all with negative results.This is the first survey of monkey malaria in West-Central Brazil.Financed by CNPq-FAPESP-LIM49-HC


238

PLASMODIUM PARASITES WITH QUARTAN PERIODICITIES ARE NOT CLOSELY RELATED TO EACH OTHER EVOLUTIONARILY.

Jessica C. Kissinger1,3, William E. Collins2, Thomas McCutchan3

1 Centro de Pesquisas René Rachou, FIOCRUZ, Minas Gerais, 2 Centers for Disease Control and Prevention, PHS, Atlanta, Georgia, U.S.A. 3 National Institutes of Health, Laboratory for Parasitic Diseases, Bethesda, Maryland U.S.A.

Three different species of Plasmodium (P. malariae, P. brasilianum and P. inui) have quartan periodicities (72 hour asexual cycle) in their respective vertebrate hosts. P. malariae infects humans and has a patchy distribution in both the Old and New Worlds, P. brasilianum infects monkeys in South and Central America, and P. inui infects Old World monkeys in isolated pockets throughout Indonesia, Malaysia, Philippines and the Celebes. Despite differences in their respective geographical distributions these three parasites have traditionally been considered to be closely related to each other based on their 72 hour periodicities, their extended sporogonic cycle and the time needed for development of pre-erythrocytic stages in the liver. We have amplified portions of the small subunit ribosomal RNA (SSUrRNA) Type A, from 16 different isolates of P. inui collected over a 20 years from different geographical locations. Nine of 16 RT-PCR products were cloned and sequenced and two PCR products were cloned and sequenced. A molecular phylogenetic analysis of the P. inui SSUrRNA Type A gene in combination with other Plasmodium sequences reveals that P. inui SSUrRNA is more closely related to SSUrRNA genes of Plasmodium "vivax-type" species than to either P. malariae or P. brasilianum. Moreover, antigenically-distinct P. inui isolates from different geographical locations proved to have nearly identical SSUrRNA sequences.

This work was supported by: Sloan/National Science Foundation and CNPq


239

SCREENING FOR ACTIVE TOXOPLASMOSIS IN PATIENTS BY DNA HYBRIDIZATION WITH THE ABGTg7 PROBE IN BLOOD SAMPLES

Garberi, J. C. 1 ; Angel, S. O.1 ; Matrajt, M.1 ; Margarit, J.1; Nigro, M. 1 ; Illescas, E.1; Pszenny, V.1; Alves, A. S.2; Marzochi, M. C. A.3; Guarnera, E.1 & Amendoeira, M. R. R. 2

1-Centro National de Toxoplasmosis, Department Parasitologia, Institute National de Microbiology Dr. Carlos G. Malbrán, CP 1281, Buenos Aires, Argentina; 2- Institute Oswaldo Cruz-FIOCRUZ ; 3- ENSP-FIOCRUZ.

We report the potential use of a specific Toxoplasma gondii DNA probe (ABGTg7). We applied a dot blot hybridization assay to blood samples for the diagnosis of cerebral toxoplasmosis (CT), acute toxoplasmic lymphadenopathy (ATL), and disseminated toxoplasmosis in transplant recipients (TRs). We studied a total of 84 individuals: 38 patients and 46 controls. We found positive hybridization signals for 12 (66,7%) of 18 patients with confirmed CT, 9 (52,9%) of 17 patients with ATL, and 2 (66,7%) of 3 TRs. PCR assays were performed in parallel for patients with ATL, resulting in T. gondii DNA detection for 10 patients (58,8%). A comparative study between dot blot and PCR assays performed with the blood of mice that had been experimentally infected with tachyzoites gave similar results: 60 and 70% positive results, respectively. Finally, the sum of positive values obtained by both DNA tests (dot blot assay plus PCR) increased the rate of positivity for ATL patients to 76,4%. These results demonstrate that the T. gondii ABGTg7 repetitive DNA element is an additional useful resource for diagnosing Toxoplasma parasitemia in patients with CT and ATL and in TRs. Thus, our ABGTg7-based dot blot test may lead to an improvement in T. gondii detection methods in patients with acute toxoplasmosis.

Supported by CABBIO and CNPq


240

NUCLEAR AND MITOCHONDRIAL DNA ANALYSES DEFINE GROUPS OF SPECIES IN THE GENUS CRITHIDIA

Pires, M.Q*.; Fernandes, O**.;Sousa, M..A*** & Pacheco, R.. S*

* Dept. Bioquimica e Biologia Molecular, **Dept. Medicina Tropical, ***Dept. Protozoologia Instituto Oswaldo Cruz - FIOCRUZ.

We have previously shown that the genusCrithidia comprises a group of heterogeneous species ( Pacheco et al. J. Protozool. Res. 4: 71- 82, 1994; Pires et al. Mem. Inst. Oswaldo Cruz 90( I ): 280, 1995; Fernandes et al. J. Eukariotic Microbiol. 1996 in press)

In the present study we extend our first observations analysing the nuclear and mitochondrial genomes. Thirteen different Crithidia species have been investigated using different molecular approaches. By RsaI restriction fragment length polymorphism of kDNA analysis it was possible to separate 7 distinct groups (group kI to kVII). Interestingly, two species of Proteomonas (P. breviculae and P. inconstans) were placed within the group kIV. Seven distinct groups were also formed using PstI restriction analysis of nuclear DNA. C. fasciculata, C. luciliae and C. guilhermei belong to group nI. Group nII is composed by C. luciliae thermophila, C.hutneri and Crithidia sp (from Zelus leucogramus). To group nIII belong the species C. acanthocephali, C. harmosa and C. flexonema. C. mellificae is placed within the group nIV. C. oncopelti belongs to the group nV. C.desouzai and C.deanei to the groups nVI and nVII respectively. The phylogenetic relationship detected among theses group of species by mitochondrial markers was corroborated by nuclear DNA analyses. Although, minor variation had been detected at nuclear and mitochondrial levels (kII ¹ nII and kIII ¹ nIII), the species C. acanthocephali and C. harmosa from group kII are included in the group nIII together with C. flexonema. The species C. lucliae thermophila and Crithidia sp from kII are included in group nII. Further investigation using mini-exon gene sequence and total kDNA as probes have shown, in Southern and dot blot experiment that total kDNA from C. fasciculata shares high copy number of homologous sequence with kDNAs from group kI. No cross hybridisation was detected with other groups of species. Confirming their close phylogenetic relationship.The same was observed when C. fasciculata mini-exon gene was probed against genomic fragments of parasites from different groups, confirming the phylogenetic relationship in group nI. In addition, homologous sequences to C. acanthocephali mini-exon gene were found predominantly with the homologous nDNA. However, a faint hybridisation signal could be detected with C. harmosa nDNA. No cross-homology was found in Proteomonas with the probes used. Our results indicate that these integrated approaches are useful in typing and defining groups of trypanosomatids.

This work was supported by PAPES/ FIOCRUZ and CNPq


241

DETERMINATION OF PHYLOGENETIC RELATIONSHIPS AMONG PHYTOMONAS SPP. FROM PLANTS AND PHYTOPHAGOUS INSECTS USING RANDOMLY AMPLIFIED POLYMORPHIC DNA.

Serrano, M. G.; Campaner, M.; Camargo, E. P. & Teixeira, M. M. G. Departamento de Parasitologia. USP, São Paulo, Brasil.

Phytomonas spp. has been classified according to their morphology, developmental forms, and host and geographical origins, although these taxonomic criteria proved to be insufficient for both, classification in genera and species identification. The recognition of the importance of Phytomonas as plant pathogens have generated a search for efficient criteria for identifying this genus and its species, either from plants and vector insects. Recently, taxonomic markers specific to this genus were described. Although these parameters permit classification of trypanosomatids as Phytomonas they do not inform about the genetic heterogeneity of flagellates and thus, are not suitable for species identification. Species of Phytomonas from plants have been distinguished through zymodemes, kDNA fingerprinting, and sequences of rRNA and SL genes which are complex and time consuming methods. Therefore, genetic diversity of a great number of isolates from plants and insects has not yet been accomplished. Aiming an easy, quickly and sensitive assessment of genetic heterogeneity within Phytomonas, we have examined the genomic structure of this genus by RAPD analysis of 22 isolates from plants and 11 from phytophagous insects, classified as Phytomonas by morphological, biochemical and molecular markers previously defined for this taxon. We obtained RAPD profiles (rapdemes) characteristics for a given isolate as well as very similar rapdemes shared by two or more isolates. Thus, we identified isolate-specific fingerprints that could be helpful for species identification as well as group-specific markers. In addition, polymorphism were detected between isolates from one only species of plants or insects. These findings once again argues against defining species on the basis of host origin. Moreover, a high degree of similarity could be observed within groups of flagellates by comparison of rapdemes generated by primer 684, distributing most isolates in 5 groups by visual analysis. Aiming to assess the genetic relatedness of members of genus Phytomonas rapdemes were used to construct dendograms based on similarity matrices. The topology of dendogram also indicated the segregation of most isolates into 5 groups, similar to generated by visual assessment of rapdemes. Therefore, our results from RAPD studies of Phytomonas showed, either trough rapdemes or cladogram analysis, high genetic variability within this genus and revealed the existence of subgroups of closely related isolates, indicating that this is a complex taxon comprising several clusters composed by distinct species. To better evaluate the complexity of this genus it is necessary to determine the stability of these clusters by adding more isolates and by comparison with phylogenetic relationship based on other DNA markers. However, parity between groups defined by RAPD and ribosomal/spliced leader gene markers suggests that RAPD can be used as a reliable method to detect genetic markers for clade membership of Phytomonas. Considerable amounts of information must be accumulated and evaluated before giving taxonomic status for clusters or to create new species of Phytomonas.

Supported by FAPESP and CNPq.


242

TRYPANOSOMES ISOLATED FROM BATS IN FLORIANÓPOLIS, SANTA CATARINA STATE - BRAZIL MAY BE MEMBERS OF A DISTINCT BAT TRYPANOSOME SPECIES

Grisard, E.C.1, 2, Cordeiro, F.D.2; Steindel, M1; carvalho Pinto, C.J.1; Ribeiro-Rodrigues, R.2 & Romanha, A.J.2

1- Departamento de Microbiologia e Parasitologia - CCB - Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil - 88040-900; e-mail grisard@ccb.ufsc.br and 2- Laboratório de Parasitologia Celular e Molecular, Centro de Pesquisas René Rachou - FIOCRUZ, Belo Horizonte, MG, Brazil - 30190-002, e-mail: romanha@netra.cpqrr.fiocruz.br

Seven Trypanosoma (Schyzotrypanum) sp strains isolated from Eptesicus sp (Chiroptera: Vespertilionidae) were characterized using experimental infection in mice, triatomines and culicines; complement lysis; indirect fluorescence assay, isoenzymatic and random amplified polymorphic DNA profiles (RAPD). The Trypanosoma sp isolates were compared with T. cruzi, T. rangeli and two other bat trypanosomes species, T. vespertilionis and T. hastatus. Trypanosoma sp isolates were different from the other species in all experiments, except in complement lysis, were about 95% of the epimastigotes from the 7 Trypanosoma sp. isolates and from the T. cruzi strains were lysed in the presence of both human and guinea pig sera, whereas the T. rangeli strains were not lysed by both complement sources. Experimental infection of triatomines and culicines with Trypanosoma sp proved to be transitory, being these parasites non infective for both normal and immunosupressed mice. Isoenzymatic and RAPD profiles obtained for Trypanosoma sp were quite distinct from T. cruzi and T. rangeli and closely related to T. vespertilionis and T. hastatus. No cross-reaction was observed between sera from mice infected with Trypanosoma sp and the other trypanosomatids and vice-versa. Trypanosoma sp induced no protection against T. cruzi infection in mice. The very low or non-similarity between Trypanosoma sp isolates and the other species used in this study, suggests that they might be members of a distinct bat trypanosome species.

Supported by CNPq / CAPES-PICD and FIOCRUZ.


243

MINI-EXON GENE VARIABILITY AMONG TRYPANOSOMA RANGELI STRAINS ISOLATED FROM DIFFERENT GEOGRAPHIC REGIONS DETECTED BY LOW STRINGENCY SINGLE SPECIFIC PRIMER - PCR (LSSP-PCR)

Grisard, E. C.1, 2 & Romanha, A. J.2

1- Departamento de Microbiologia e Parasitologia - CCB - Universidade Federal de Santa Catarina, Florianópolis - SC - Brazil, e-mail: grisard@ccb.ufsc.br,, and 2- Laboratório de Parasitologia Celular e Molecular - Centro de Pesquisas René Rachou - FIOCRUZ, Belo Horizonte - MG - Brazil, e-mail: romanha@netra.cpqrr.fiocruz.br.

Trypanosoma rangeli is the second trypanosome that infect humans, as well as sylvatic and domestic animals, in Central and South America. It is extremely important in the epidemiology of Chagas' disease, since it has been found in the same reservoirs and vectors of T. cruzi. The variability of the intergenic region of the nuclear and repetitive mini-exon gene of T. rangeli was evaluated through LSSP-PCR. T. rangeli strains from Honduras (H8GS), Venezuela (Macias, Palma-2), Colombia (Choachi, San Agostin) and Brazil (SC-58) were used. Additionally, two clones of the SC-58 strain (Cl-26 and Cl-32) were used. The LSSP-PCR described by Pena et alli (PNAS, 91:1946-1949, 1994), is the fingerprinting of a DNA target segment. It is obtained by the reamplification of a specific PCR product with one of the specific primers at low stringency conditions. Variabilities on LSSP-PCR patterns are correlated with variabilities in nucleotide sequence of the DNA segment. T. rangeli DNA amplification products obtained with primers TrINT 1/2 and 3/2, homologous to the intergenic region of the gene, were submitted to electrophoresis in 1% ethidium bromide stained agarose gel. The specific bands were excised, eluted from agarose and used as template for amplification by LSSP-PCR using primers TrINT-1, 2 or 3 independently. Primer TrINT-1 produced the most informative patterns, whereas TrINT-2 and 3 produced the least informative with almost no bands. SC-58 strain and its clones presented identical patterns but quite distinct from the other strains, that revealed similar patterns among themselves. The intraspecific sequence polymorphisms found among these strains in the intergenic region of T. rangeli mini-exon gene through LSSP-PCR, confirm our previous findings with isoenzyme and RAPD analyses that the T. rangeli strain SC-58 isolated in Santa Catarina, South Brazil, is genetically distinct from the strains isolated in north of South America and Central America.

Supported by CAPES-PICD and FIOCRUZ


244

EVALUATION OF A RANDOMLY AMPLIFIED POLYMORPHIC DNA FRAGMENT AS A DIAGNOSTIC MARKER FOR TRYPANOSOMA EVANSI.

Ventura, R.1; Silva, R.A2; Nunes V.L.B.3; Takeda, G.K F.1 & Teixeira, M.M.G.1.

1Depto de Parasitologia, ICB/USP, São Paulo, S.P.; 2EMBRAPA-Pantanal; 3Depto de Parasitologia, UFMS.

T. evansi, the agent of a wasting disease named "Surra", is the only member of the subgenus Trypanozoon, which also comprises T. brucei and T. equiperdum, infecting domestic and wild animals in Brazil. This parasite is restricted to the Pantanal of Mato Grosso in Brazil, while widespread in Africa and Asia. T. evansi is characterized by the absence of kDNA maxicircle and consequently is mechanically transmitted through blood-sucking insects. Animals infected with T. evansi can be infected with several other trypanosomes and identification of this species is still based on morphological and biometrical studies of blood parasites and mouse inoculation. Detection of trypanosome by these methods is impractical for epidemiological studies. We have characterized 14 T. evansi isolates from horses, dogs, capybaras, coatis and rats from Pantanal by morphological, biological and molecular methods. Mouse infection and morphology confirmed diagnosis of these isolates. Despite very distinct behavior in mice, analysis of ITS and SSU of rRNA and spliced leader gene as well as RAPD profiles did not reveal significant heterogeneity among T. evansi isolates. Aiming detection of T. evansi directly on their natural hosts we had evaluated diagnostic methods described by others. However, our results demonstrated that these methods are not helpful for Brazilian isolates because: a) Probe and PCR-based on repetitive DNA sequence of T. evansi (Wuyts et al., 1994) do not distinguished T. evansi from other trypanosomes; b) Probes based on kDNA are not useful for Brazilian stocks since all stocks analyzed by showed to be totally dyskinetoplastic, either by DAPI staining or by probing with conserved replication origin of kDNA minicircle. Although ribosomal and spliced leader gene markers did not distinguish T. evansi from other species of Trypanozoon, all patterns allowed discrimination of this subgenus. However, besides subgenus differentiation, RAPD analysis generated T. evansi-specific profiles. Moreover, it was also observed that one primer generated a single DNA amplicon (Te664) that showed to be monomorphic for all T. evansi stocks and was different from profiles observed for all other trypanosomes, even the closely related species T. equiperdum and T. brucei. The homogeneity of this RAPD fragment among T. evansi stocks was determined by restriction analysis and confirmed using cloned fragments as probes. Results demonstrated that Te664 DNA sequence is species-specific and suggests that can be useful through PCR or DNA probing to detect T. evansi. Recently, we standardized this RAPD assay using crude preparations of infected mice blood as DNA templates for PCR, either collected in microfuge tubes or in glass slides. Thus, preliminary results suggest that Te664 fragment may be suitable as a sensitive diagnostic tool for T. evansi, distinguishing this species from all other trypanosomes and detecting even diskinetoplastic strains. Presently, we are sequencing Te664 in order to improve this diagnostic method constructing primers for PCR amplification of T. evansi directly on naturally infected blood horse.

Supported by FAPESP and CNPq.


245

FROM MORPHOLOGICAL TO MOLECULAR MARKERS AS TAXONOMIC CRITERIA FOR TRYPANOSOMA (MEGATRYPANUM) SPP.

Rodrigues, A.C.; Dell' Porto; Campaner, M.; Takata, C.S.A.; Takeda, G. F. & Teixeira, M.M.G.

Departamento de Parasitologia, ICB/USP, São Paulo, S.P.

The subgenus Megatrypanum (Stercoraria) are constituted by mammalian trypanosomes with widespread host range and wide geographical distribution transmitted by hematophagous insects. Mammalian hosts of Megatrypanum spp. (bovids, primates, rodents, etc) could also be infected by trypanosomes of other subgenus, according to their geographic origin. Thus, in Brazil, domestic and wild animals can be infected by the subgenus Schizotrypanum (T. cruzi), Herpetosoma (T. rangeli), Dutonella (T. vivax) and Trypanozoon (T. evansi). While T. vivax and T. evansi, that are morphologically quite distinct from Megatrypanum, could be cultured "in vitro" only in very special conditions, species of other subgenus can be isolated and cultured. Mammals trypanosomes have been classified as Megatrypanum considering exclusively morphological features of flagellates on blood of infected animals. Moreover, besides the need of experts to distinguish all subgenus, Megatrypanum is rarely observed in blood and thus, is detectable, in most cases, only by hemocultures. Thus, in order to evaluated the value, as taxonomic criteria, of culture forms we compared distinct subgenus using different media and culture conditions. Large tripo- and epimastigotes, characteristics of Megatrypanum were substituted, after 10-15 days of culture, by smaller epimastigotes, indistinguishable from those presented by T. cruzi and T. rangeli. On the other hand, when Megatrypanum were co-cultured with a monolayer of LLCMK2 cells large extra-cellular trypomastigotes, with morphological characteristics compatible with those described for Megatrypanum were observed. However, this is a fastidious and very time consuming classification method. Moreover, isolates from distinct hosts, although morphologically indistinguishable, have been identified at specific level only considering the host of origin. So, isolates from cattle and buffaloes have been classified as T. theileri. Aiming to develop an easy and quick method for classification and species identification of Megatrypanum spp. we are investigating ribosomal, spliced leader and RAPD markers of several trypanosomes of this subgenus isolated from cow, buffalo, rat, opossum, anteater and sloth. Analysis by PCR amplification of ITS rRNA showed length and restriction patterns conserved between cattle and buffaloes isolates. However, differences were detected among Megatrypanum spp. from bovids and wild animals as well as from species of other subgenus. Thus, results suggested that cattle and buffalo isolates are more closely related to each other than to other Megatrypanum spp. However, RAPD analysis disclosed genetic variability that permitted partition of buffalo and cattle isolates. Isolates from other animals showed very heterogeneous patterns. Moreover, while polymorphism could be detected among cattle isolates by RAPD profiles, this analyzes did not show significant variability among buffaloes isolates. Thus, our results demonstrated the existence of genetic variability between Megatrypanum isolates from cattle and buffaloes, indicating that these animals could be infected by different species, in disagreement with previous classification using host origin as taxonomic criteria.

Supported by FAPESP and CNPq.


246

IDENTIFICATION OF TRYPANOSOMA RANGELI GENES RELATED TO TRYPANOSOMA CRUZI METACYCLIC GLYCOPROTEIN SURFACE ANTIGENS

Renata C. P.Baida1, Jorge Araya2, Nancy E. V. Bonilla3, Bianca Zingales3 and José Franco da Silveira1

1. Depto Micro, Imuno e Parasitologia, Escola Paulista de Medicina, UNIFESP, Rua Botucatu, 862-CEP 04023-062, SP, Brasil (email: franco.dmip@epm.br); 2. Universidade de Antofagasta, Antofagasta, Chile; 3. Instituto de Química, USP, SP, Brasil.

Trypanosoma cruzi metacyclic trypomastigotes express two major surface glycoproteins (gp90 and gp82) which are involved in cell invasion and induction of protective immune response in mammalian hosts. Gp82 and gp90 belong to a superfamily of surface molecules whose members are characterized by the presence of two partially conserved amino acid motifs: subterminal repeat Fn3 (VTVxNVxLYNR) and Asp box (SxDxGxTW). The non pathogenic protozoan Trypanosoma rangeli and T. cruzi share several characteristics, including the minicircle structure and several antigenic determinants, suggesting that both parasites are closely related. A comparative analysis of the T. cruzi gp82/gp90 and T. rangeli related sequences may aid in the determination of the features of gp82 and gp90 proteins that contribute to its function in host cell interactions.

The presence of multiple copies of genes in T. rangeli encoding products related to T. cruzi gp82 and gp90 was revealed when genomic T. rangeli DNA was hybridized at moderate and high stringencies with gp82 and gp90 genes. Even at high stringency conditions, both probes hybridized with several genomic fragments suggesting that gp82 and gp90 related sequences are interspersed in the genome rather than arranged in tandem repeats.

We have also used PCR amplification to identify gp90 and gp82 related sequences in T. rangeli genomic DNA. A 1600-bp fragment, carrying the complete open reading frame (ORF) of T. rangeli gp82 gene, was amplified using a pair of primers derived from T. cruzi gp82. The size of the amplified fragment is slightly lower than the ORF found in T. cruzi indicating the existence of some differences between them. The amplified fragment was cloned in pMOS vector and the analysis of its nucleotide sequence is underway.

Supported by FAPESP, FINEP/BID, CNPq/PADCT.


247

CHARACTERIZATION OF REPETITIVE DNA SEQUENCES IN TRYPANOSOMA RANGELI

Vargas, N & Zingales, B.

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Caixa Postal 26.077, CEP 05599-970 São Paulo, SP, Brasil

Trypanosoma rangeli is a mammalian trypanosome whose geographical distribution coincides with Trypanosoma cruzi in Central America and in the Northern part of South America. Both parasite species are transmitted by triatomine vectors where simultaneous infections are observed. In addition, both trypanosomes can infect humans and produce mixed infections. In contrast to T. cruzi, the causative agent of Chagas disease, T. rangeli is apparently non-pathogenic to humans. However, both parasites induce cross-reacting antibodies causing frequent diagnostic errors. The coincidence observed between T. rangeli and T. cruzi in geographical and host distribution, as well as the immunological cross-reactivity recommend the search of molecular markers for differentiation of these parasites aiming clinical and epidemiological studies. In order to characterize repetitive DNA sequences of T. rangeli, DNA from the San Agustin strain was isolated and digested with TaqI, which does not present restriction sites in the parasite kDNA. Analysis in agarose gels showed the expected DNA smear along with two bands of 0.78 kb and 1.25 kb intensely stained with ethidium bromide. These bands, which could correspond to highly repetitive DNA sequences, were isolated and ligated to the vector pBluescript digested with AccI. Several recombinants were obtained. One of these recombinants, presenting an insert of 0.78 kb (clone I-31), hybridizes to T. rangeli chromosomal bands of 1.25 Mbp; 0.78 Mbp and 0.58 Mbp. No hybridization was detected with T. cruzi chromosomes. Three recombinants presenting inserts of 1.25 kb were isolated and partially sequenced. Alignment of the sequences of these clones shows 40-50% similarity. The four recombinants already obtained, as well as other clones, will be further characterized to assess T. rangeli specificity and the representativeness in the parasite genome.

Supported by FAPESP, CNPq.


248

MOLECULAR CHARACTERIZATION OF THE 21SF STRAIN OF TRYPANOSOMA CRUZI: A STABLE AND LABORATORY PRESERVED STRAIN OF TRYPANOSOMA CRUZI: A STABLE AND LABORATORY PRESERVED STRAIN, ITS CLONES AND SUBCLONES.

Campos,R.F; Gonçalves, M.S; Reis,E.A.G; Reis,M.G. and Andrade, S.G.

Centro de Pesquisas Gonçalo Moniz, FIOCRUZ, Rua Waldemar Falcão, 121,

40295-001,Salvador,Bahia - Brazil

Trypanosoma cruzi , the ethiologic agent of Chagas disease, has been found in heterogeneous populations with different biological, biochemical and genetic characteristics.Those different characters has been used for the classification of T. cruzi strains into biological Types or biodemes, different zymodemes and schizodemes. The clonal structure proposed for T.cruzi strains suggests the presence of natural clones that may influence the biological behaviour of the strains and their pathogenicity. Considering the stability of the biological characters of some laboratory strains it is interesting to investigate the genetic characters of their clonal populations and identify its possible homogeneity. A stable strain of Trypanosoma cruzi (21SF) from the endemic area of São Felipe, Bahia-Brazil, maintained by serial passages in mice for 15 years, with preserved biological and biochemical characteristics, has been submitted to a molecular analysis to clarify whether this stability is dependent on its clonal composition. The 21SF strain has been cloned; its clones and subclones were classified as Type II (biodeme) and Z2 (zymodeme). Kinetoplast DNA (kDNA) was isolated from culture forms of the parental strain, five clones and fourteen subclones.Schizodeme analysis was performed by polymerase chain rection (PCR), using a set of primers from the variable regions of the minicircle DNA molecule. Fragments were digested with restriction endonucleases Rsa I and Hinf I. Comparison of digestion patterns was performed in silver-stained polyacrylamide gel. Results demonstrated high degree of homology between the restriction fragment lenght polymorphism (RFLP) for the parental strain and its clones (67 to 100%), between individual clones (70 to 97%, Rsa I and 70 to 100%, Hinf I) and between clones and their subclones (79 to 100%). Present findings suggest the existence of a high degree of association between biologic, isoenzymic and genomic characteristics of the 21 SF strain . Since previous studies have demonstrated that all strains isolated from the area of São Felipe - Ba belonged to the same biologic and isoenzymic types, it is possible that there is a predominance of principal T.cruzi clones or an homogeneity of the clonal populations of the strains circulating in this endemic area.


249

MOLECULAR TYPING OF RECENTLY ISOLATED TRYPANOSOMA CRUZI STOCKS FROM THE SYLVATIC ENVIRONMENT (RIO DE JANEIRO, BRAZIL) BASED ON MINI-EXON GENE NON-TRANSCRIBED SPACER

Fernandes, O.1, 2; Mangia, R.H.3; Lisbôa, C.V.4; Pinho, A.P.4; Morel, C.M.3; Zingales, B.5; Campbell, D.A.6; Jansen, A.M.4

1.Departamento de Medicina Tropical, Instituto Oswaldo Cruz, Fiocruz, Av. Brasil, 4365, Rio de Janeiro, Brasil, CEP 21045-900, 2. Departamento de Patologia, Universidade do Estado do Rio de Janeiro, 3. Departamento de Bioquímica e Biologia Molecular, IOC; 4. Departamento de Protozoologia, IOC; 5. Departamento de Bioquímica, Instituto de Química, Unversidade de São Paulo, Brasil; 6. Department of Microbiology and Immunology, UCLA School of Medicine, Los Angeles, California, USA.

American trypanosomiasis occurs in nature as a sylvatic cycle, where Trypanosoma cruzi interacts with wild triatomines and mammalian reservoirs, such as marsupials, rodents, armadillos and other animals. Due to difficulties in trying to isolate T. cruzi stocks from the sylvatic cycle, very few studies have been performed in order to understand the parasite infection on natural environments. Traditionally T. cruzi has been considered to be composed of a highly heterogeneous population of parasites. In contrast, we have shown that T. cruzi stocks can be clustered into two major phylogenetic groups: lineage1 and lineage 2, when the mini-exon and the 24Sa rRNA gene loci are analyzed.

In this report, we have typed sixty-one recently isolated T. cruzi samples from the sylvatic cycle belonging to different geographical origins in Rio de Janeiro State (Teresópolis, Jaguanum Island, Miguel Pereira, Biological Reserve of Poço das Antas and Itaguaí), based on a variable spot in the non-transcribed spacer of the mini-exon gene. Eight isolates were from triatomines, twenty-six stocks were from golden-lion tamarins (an endangered species of primates endemic to the Atlantic Coast Rainforest - Biological Reserve of Poço das Antas), twenty-three from opossum, three from rodents and one from a three-toed sloth. Twenty-seven (44% - 27/61) isolates were typed as lineage 1, while thirty-two (52% - 32/61) isolates were typed as lineage 2. Two opossums presented mixed infection. Therefore, three percent (2/61) of the isolates were typed as lineage 1 + lineage 2.Using these regions as models of sylvatic environments, it was observed that all opossums were infected by lineage 2 isolates, while all twenty-six golden-lion tamarins were infected by lineage 1. Our group has shown in previous epidemiological studies that there is a preferential association of lineage 1 to the domestic cycle, and consequently to the human host. The results presented in this abstract, strongly suggest that the preferential distribution of lineage 1 among humans could be explained by an eventual pre-adaptation of this given sub-population to primates and the possibility for lineage 1 to evade from the sylvatic to the domestic cycle. Further studies on the T. cruzi-primate interaction is still necessary to address the question of the parasitism phenomena regarding American tripanosomiasis.

This research is sponsored by FIOCRUZ/PAPES, CNPq and International Atomic Energy Agency.


250

TWO MAJOR SUB-POPULATIONS OF TRYPANOSOMA RANGELI IN COLOMBIA DEFINED BY SUB-SPECIFIC kDNA PROBES

Vallejo, G.A.1; Silva, J.C.1; Castañeda, N.1; Jaramillo, J.C.1; Carranza, J.C.2; Sánchez, J.L.2 y Guhl F.2

1Departamento de Biología, Instituto de Ciencias, Universidad del Tolima, A.A. 546 Ibagué-Colombia.

2Centro de Investigaciones en Microbiología y Parasitología Tropical (CIMPAT), Universidad de los Andes, A.A. 4976 Santafé de Bogotá-Colombia.

In previous works, DNA fingerprinting with the multilocal probe 33.15 showed a great divergence between the T. rangeli strains form Santa Catarina (southern Brazil) and the others isolated from Honduras, Colombia and Venezuela suggesting the existence of two groups of T. rangeli (Macedo, et al., 1993. In: DNA fingerprinting: State of the Science. PENA, S.D.J., CHAKRABORTY, R., EPPLEN, J.T. & JEFFREYS, A.J. Eds., Birkhauser Verlag Basel/Switzerland, p. 321-329). Further isoenzyme and RAPD analysis revealed two distinct groups of T. rangeli strains, one formed by strains from Santa Catarina (Brazil) and the other , by the strains from Central America and the northern part of South America (Steindel et al., 1994. J. Euk. Microbiol., 41:261-267).

T. rangeli contains two distinct classes of minicircles of kDNA with different size and molecular organization called KP1 and KP2 (Vallejo et al., 1994. Molecular and Biochemical Parasitology. 67:245-253). Polymerase chain reaction and hybridization studies suggested that the sequence of KP1 is conserved in several T. rangeli strains from Honduras, Colombia and Venezuela but absent in strains from southern Brazil.

In this study, we analyzed strains of T. rangeli isolated from sylvatic Rhodnius robustus, Didelphis marsupialis and domestic Rhodnius prolixus in Tolima State (Central Colombia). All strains of T. rangeli isolated from domestic cycle showed positive hybridization with KP1 probe. Primers designed for KP1 minicircle, amplified, by Polymerase chain reaction, a 165 bp band that were obtained from all T. rangeli strains derived from domestic cycle but absent from all strains from sylvatic cycle. These and other results together suggest the existence of the same two groups of T. rangeli in the same geographic area.

Financial Support by Instituto Colombiano para el Desarrollo de la Ciencia y la Tecnología Francisco José de Caldas ( COLCIENCIAS). Proyecto 1105-04-181-95.


251

POLYMERASE CHAIN REACTION CROSS-REACTIVITY BETWEEN TRYPANOSOMA CRUZI AND TRYPANOSOMA RANGELI.

Vidigal, P.G.1,3; Grisard, E.C.2,3 & Romanha, A.J.3

1- Depto. de Propedêutica Complementar - FM/UFMG - Belo Horizonte - MG - Brazil; 2- Depto. de Microbiologia e Parasitologia - CCB/UFSC - Florianópolis - SC - Brazil and 3- Centro de Pesquisas René Rachou - FIOCRUZ, Belo Horizonte - MG - Brazil, 30190-002, e-mail: grisard@ccb.ufsc.br.

The Polymerase Chain Reaction (PCR) has been used as a new alternative confirmatory method for human Chagas' disease diagnosis, specially in it's chronic phase. The main advantages of the PCR when compared to the classic serological and parasitological methods are its high sensitivity, specificity and quickness. Many PCR primers have been recently developed and used for Chagas'disease diagnosis as well as for differentiation between T. cruzi and T. rangeli infections. The primers S-35 and S-36 were designed to amplify T. cruzi kinetoplast DNA minicircle sequences (kDNA) (Sturm et al., 1989, Mol. Biochem. Parasitol. 33: 205-214). However, since T. rangeli and T. cruzi kDNA have similar sequences, we decided to evaluate the ability of these primers to detect T. rangeli DNA, and their capacity to differentiate these two parasites. Using serial dilutions from 100 pg to 0,001 pg of total DNA extracted from CL strain of T. cruzi and SC-58 strain of T. rangeli as template, we observed the previously described specific amplification products of approximately 330 to 450 bp for T. cruzi and the 760 bp for T. rangeli. These primers can detect until 0,01 pg of T. rangeli DNA and 0,0001 pg of T. cruzi DNA. Using DNA obtained from another three strains of T. rangeli from Honduras, Colombia and Venezuela, the same amplifications products and sensivity values were observed. Despite of this sensitivity, these primers were not able to differentiate DNA from each parasite species, since T. rangeli DNA revealed products around 330 to 450 bp and T. cruzi near 760bp. In order to confirm these results, DNA from T. rangeli and T. cruzi were mixed and used as PCR template. The multi band patterns obtained were not able to differentiate the parasites. Since human, domestic and sylvatic animal infections with T. rangeli have been recently reported in Central and South America, the use of these primers for human Chagas' disease diagnosis and for differentiation between T. cruzi and T. rangeli should be used with caution, specially in areas were these parasites coexist.

Supported by CNPq, CAPES and PAPES/FIOCRUZ.


252

ISOENZYME AND RANDOMLY AMPLIFIED POLYMORPHIC DNA (RAPD) CHARACTERIZATION OF TRYPANOSOMA CRUZI STRAINS ISOLATED FROM TRIATOMA SORDIDA AND CHRONIC CHAGASIC PATIENTS FROM THE COUNTY OF PORTEIRINHA, MG, BRAZIL

Fernandes, A. J.1, Diotaiuti, L.1, Murta, S. M. F.1,2, Meira, W. S. F.2, Duarte A. P. M. 1, Chiari, E.3 & Romanha, A. J.1

1-Centro de Pesquisas René Rachou/FIOCRUZ, 2- Depto Bioquímica e Imunologia, 3- Depto Parasitologia, ICB/UFMG, Brasil

After the control of the domiciliar Triatoma infestans by insecticide, T. sordida appears as one of the species of greater epidemiologic importance due to its wide dispersion and high peridomiciliary infestation. In 12 localities of the county of Porteirinha, MG, 24 T. cruzi strains were isolated from chronic chagasic patients and 19 from T. sordida, 15 captured in the peridomicile and 4 in the intradomicile. These strains were characterized using isoenzymatic profiles of 6 different enzymes (ALAT, ASAT, GPI, PGM, ME and G6PD) and randomly amplified polymorphic DNA (RAPD) profiles obtained with 2 different primers (M13F and LS15996). The isoenzymatic characterization of 19 T. cruzi strains isolated from T. sordida showed basically Z1 pattern for all enzymes (Z1-1), except four strains that presented a ZB variant for ALAT (Z1-2). Twenty three T. cruzi strains isolated from chronic chagasic patients showed Z2 pattern, and one presented ZB pattern. Although complex, the RAPD profiles divided the T. cruzi strains into two groups, one formed by the strains from T. sordida and the other group formed by the strains from patients. Therefore RAPD and isoenzyme profiles divided the T. cruzi strains into the same 2 groups. The parity observed between RAPD and isoenzyme profiles is consistent with data previously obtained for genetic characterization of T. cruzi strains. The results show that T. sordida is the main vector of T. cruzi Z1 in the peridomicile but not in the domicile. T. cruzi Z2 predominate in intradomicile (patients), showing the very low or null participation of the T. sordida in vectorial transmission of human Chagas' disease in that region. T. infestans seems to be the vector responsible for transmission of Chagas' disease to man in Porteirinha in a recent past. Fortunately, the natural transmission was successfully interrupted by the Chagas Disease Control Program (CDCP) started in 1979 by Fundação Nacional de Saúde. These data emphasize the importance of continuing the CDCP to avoid the intradomiciliary infestation by those triatomines that represent a real threaten to domiciliar transmission in that region.

Supported by CNPq, FIOCRUZ and FNS


253

EXPERIMENTAL TRYPANOSOMA CRUZI CLONAL GENOTYPES MIXED INFECTION IN TRIATOMA INFESTANS: DIRECT IDENTIFICATION BY HYBRIDIZATION WITH PCR PRODUCTS USING MOLECULAR PROBES

Pinto, A.S.*; Lana, M.**; Brito, C.F.P.C.***; Bastrenta, B.****& Tibayrenc, M.****

*Departamento de Microbiologia, Instituto de Ciências Biológicas, CP486, Universidade Federal de Minas Gerais, 31270-010, Belo Horizonte, MG, Brasil. **Departamento de Análises Clínicas, Escola de Farmácia, Rua Costa Sena, Universidade Federal de Ouro Preto, MG, Brasil.***Departamento de Bioquímica e Biologia Molecular, Instituto Oswaldo Cruz, Av. Brasil, 4365, 21045-900, Rio de Janeiro, Brasil.****Centre d'Études sur le Polymorphysm des Micrroganisms, Unité Mixte de Recherche 9926, CNRS/ORSTOM, BP 50045, 32032, Montpellier, Cedex 1, France.

Fifteen mixtures involving 9 different stocks attributed to 19/20, 32 and 39 clonal genotypes of Trypanosome cruzi were used to infect with monitored inoculum third instars nymphs of Triatoma infestans by use of an artificial feeding device. Direct detection of distincts T. cuzi genotypes in the insects was performed by combination of polymerase chain reaction (PCR) and genotype-specific k-DNA hybridization. Eighty-five out of 90 insects examined, 85 (94,4%) were positive by PCR amplification. Mixed infection between two clonal genotypes was still present after completion of the cycle in the vector as follow: 19/20 + 32 = 16.6; 19/20 + 39 = 53.3%; 32 + 39 = 83.3%. Cases in which one genotype was detectable were as follows: 19/20 + 32, 19/20 = 43.3% and 32 = 36.6%; 19/20 + 39, 19/20 = 20% and 39 = 20%; 32 + 39, 32 = 0% and 39 = 10%. These results indicate that both selection or independent transmission of clonal genotypes may occur in the vector insect.


254

COMPARISON OF SEROLOGICAL AND PARASITOLOGICAL METHODS WITH POLYMERASE CHAIN REACTION (PCR) FOR DIAGNOSIS OF HUMAN CHAGAS' DISEASE

Fernandes, A.J.1,2; Grisard, E.C. 2,3; Vidigal, P.G.2,5; Chaves, A.C.L.2; Busek, S.1; Chiari, E.4 & Romanha, A.J.2

1- Laboratório de Biologia de Triatomíneos e Epidemiologia da Doença de Chagas and 2- Laboratório de Parasitologia Celular e Molecular - Centro de Pesquisas René Rachou - FIOCRUZ, Belo Horizonte - MG - Brazil, e-mail: romanha@netra.cpqrr.fiocruz.br; 3- Departamento de Microbiologia e Parasitologia - CCB - UFSC, Florianópolis - SC - Brazil; 4- Departamento de Parasitologia - ICB, and 5- Departamento de Propedêutica Complementar - FM - UFMG, Belo Horizonte - MG - Brazil.

After a previous serological survey, 103 blood samples were collected from inhabitants of the Chagas'disease endemic area of Porteirinha, 650 km far from Belo Horizonte, north of Minas Gerais State. The serum was assayed by indirect immunofluorescence (IFI), indirect hemaglutination (IH) and complement fixation test (CF) and the whole blood, stored in guanidine-EDTA solution, was assayed by polymerase chain reaction (PCR). The serological cut-offs were IFI (1:80), IH (1:32) and CF (reactive/non reactive). Eighty nine (86.4%) individuals were seroreactive (chagasic) and 14 (13,6%) were non reactive (non chagasic). The seroreactive patients were submitted to hemoculture (n=88) in LIT medium and xenodiagnosis (n=73) with forty Triatoma infestans 3-4th instar nymphs. Double-blind PCR assays were carried out with all samples. The PCR was made using the primers described by Diaz et alli (Am. J. Trop. Med. Hyg., 46:616-623, 1992) with the reaction conditions stablished in our laboratory. In order to discard the possibility of inhibition of the reaction, the constitutive human gliceraldehyde phosphate dehydrogenase gene was amplified through PCR in all negative samples. DNA extraction and PCR amplification were made at different places to avoid or disminish contamination. A non chagasic blood was introduced in each set of blood samples from the DNA extraction up to PCR reaction. Furthermore, controls with no DNA and 1 and 100 fg of T. cruzi DNA were added. Compared to the serological tests, PCR sensitivity was 82.0% (73/89), presented 2 false-positive in 14 reactions (14.3%) and a concordance of 82.5% (85/103). From 88 seroreactive patients, 28 (32.0%) were hemoculture positive and 71 (80.7%) PCR positive. From 73 seroreactive patients, 11 were xenodiagnosis positive (15.0%) and 57 (78.1%) PCR positive. Six out of 28 patients with positive hemoculture and 1 out of 11 with positive xenodiagnosis were PCR negative. Considering both parasitological methods, 31 out of 73 (42.5%) patients were positive, and 56 (76.7%) were PCR positive. As previously described by other authors, PCR was far more sensitive than both parasitological tests together and less sensitive than the serological methods. Supported by CAPES-PICD/CNPq/PAPES-FIOCRUZ/FNS.


255

CHAGAS DISEASE DIAGNOSIS: COMPARATIVE ANALYSIS AMONG PARASITOLOGIC, MOLECULAR AND SEROLOGIC METHODS

Gomes, M.L.1,3, Galvão, L.M.C.1, Macedo, A.M.2, Pena, S.D.J.2, & Chiari, E.1

Departamentos de 1Parasitologia, e 2Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Caixa Postal 486, 31270-901 Belo Horizonte, MG, Brasil e-mail: chiari@icb.ufmg.br; 3Departamento de Análises Clínicas, Universidade Estadual de Maringá, 87020-900, Maringá, PR, Brasil.

During the course of chronic chagasic infection, low parasitaemia levels inhibit parasite detection by current techniques such as haemoculture and xenodiagnosis. Diagnosis mainly relies on the detection of antibodies against T. cruzi antigens and not the presence of the parasite itself. Since serologic tests have sensitivity but lack specificity, molecular assays based on polymerase chain reaction (PCR) have been proposed as an alternative tool for parasite detection in individuals with chronic Chagas disease. A variable degrees o PCR efficiency has been reported in the literature and illustrates the need for further evaluation of PCR among large numbers of chagasic patients. In this study we compared an optimized PCR technique with haemoculture and complement-mediated lysis (CoML) in 126 individual from different endemic areas of Brazil who had conventional serologic results that were either positive, negative or inconclusive. PCR amplification yielded positive results in 83,5% (66/79) of individuals with positive serology, 29,4% (10/34) with negative serology and 46,2% (6/13) with inconclusive serology. CoML and PCR results were agreed across the three groups studied. When compared to haemoculture, PCR was positive for all individuals who had positive haemoculture, for 37 individuals with negative haemoculture and for two of six individuals with inconclusive serology and negative haemoculture. Our results show that among the same individuals, PCR was consistently more sensitive than haemoculture in detecting the presence of the parasite. PCR and CoML results were similar independent of the groups studied which suggest that our protocol may be effective in the evaluation of cure in patients that received anti-parasite treatment. Further, our findings suggest that conventional serologic tests may be unable to detect infection in some individuals.

Supported by CAPES-PICD/CNPq/FAPEMIG/PRONEX


256

PCR DETECTION OF TRYPANNOSOMA CRUZI kDNA IN ELUATES OF BLOOD COLLECTED ON FILTER PAPER.

Mercado, R.1, Fernández, J.2 and Diaz, W1.

1 Departamento de Parasitología. Facultad de Medicina. Universidad de Chile. Casilla 9183 Santiago Chile. 2 Unidad de Desarrollo. Instituto de Salud Pública de Chile.

As the natural and transfusional ways of transmission of Chagas disease is controlled health entities must shift attention to congenital form of transmission (Bittencourt, 1992). Mercado et al (1996) proposed the use of a sistematical diagnostic test to detect congenital cases of Chagas disease, because most newborns infected by T. cruzi are asymptomatics (Schenone et. al.,1989).

Scarce quantities of blood samples for diagnostic proposals are provided by children probably infected. Collection of blood onto filter papers was described, both for serodiagnosis of Chagas disease in children and for epidemiological.

PCR is a new tool for detection of T. cruzi DNA in blood samples. Here, we evaluate the yield of PCR to detect T. cruzi DNA in eluates of blood collected on filter paper blood samples with T. cruzi (Tulahuen strain) was obtained from mice periodically infected. Microscopical observation of blood confirmed the presence of abundant trypomastigotes. Blood was gathered onto filter paper (Whatman N° 1) and air dried at room temperature. PCR was run according with Kain & Lanar (1991) with modifications. Eluates were obtained cutting filter paper and soaked in destilled water for two hours at room temperature. Eluates were seen in a 2% agarose gel. Amplified.330 bp fragments in eluates and and whole blood were detected. No difference in sensitivity of the PCR in eluates and whole blood was observed.

Kain & Lanar (1991) described the detection of DNA of Plasmodium falciparum from filter paper impregnated with whole blood by using enzimatically amplified DNA technique. We demostrate the feasibility of PCR amplification of T. cruzi kDNA in eluates of blood collected on filter paper. Studies with blood samples obtained from infected patients are underway.

Financial support by DP/FM/U. de Chile and the Instituto de Salud Pública de Chile.


257

TRYPANNOSOMA CRUZI: OPTIMIZATION OF POLYMERASE CHAIN REACTION FOR DETECTION IN HUMAN BLOOD

Gomes, M.L.1,3, Macedo, A.M.2, Vago, A.R.2, Pena, S.D.J.2, Galvão, L.M.C.1 & Chiari, E.1

Departamentos de 1Parasitologia, e 2Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Caixa Postal 486, 31270-901 Belo Horizonte, MG, Brasil e-mail: chiari@icb.ufmg.br; ,3Departamento de Análises Clínicas, Universidade Estadual de Maringá, 87020-900, Maringá, PR, Brasil.

The procedure often used for detection of Trypanosoma cruzi by polymerase chain reaction (PCR) in blood from chronic chagasic patients is the amplification of 330 bp kDNA fragment (Ávila et al. 1991, 1993). A high sensitivity (96%-100%) of PCR was described prior (Wincker et al., 1994; Ávila et al., 1993), but a low sensitivity (45%-60%) was also reported (Britto et al., 1995; Junqueira et al., 1996). Because this discrepancy the actual potential usefulness of the PCR for chronic Chagas disease diagnosis need further investigation. In this report we have optimized the conditions for DNA extraction and PCR amplification to diagnose the presence of Trypanosoma cruzi DNA in blood and serum of patients with chronic Chagas disease. The » 330 bp fragment of the kinetoplast minicircles was used as a target for amplification. Our protocol has shown a sensitivity of 10 fg T. cruzi DNA by silver staining and below this concentration the PCR products were detected after hybridization with a specific alkaline phosphatase conjugated oligonucleotide probe increasing the sensitivity to 0.1 fg of T. cruzi kDNA. An additional product of aproximately 200 bp inadvertently amplified from the human genome was observed in human blood frm T. cruzi negative and positive samples and served as an internal control of the amplification. Samples from other mammalian hosts were also assayed using the PCR protocol. The higher sensitivity of our PCR method observed in both acute and chronic phases of T. cruzi infections in mice and dog respectively, could be useful in monitoring the course of infection during esperimental drug tests in laboratory animals. Since this procedure showed a higher sensitivity than other protocols in the literature, it may be a suitable routine test to diagnose Chagas disease, especially for patients presenting very low parasitemia levels.

Supported by CAPES-PICD/CNPq/ FAPEMIG/PRONEX.


258

STUDY OF TISSUE TROPISM OF TRYPANOSOMA CRUZI POPULATIONS AND ITS INFLUENCE IN THE PATHOGENESIS OF CHAGAS DISEASE

Andrade, L.O1; Chiari, E.2; Machado, C.R.S.3; Pena, S.D.J.1; Macedo, A.M.1

1. Depto Bioquímca e Imunologia / 2. Depto de Parasitogia / 3. Depto de Morfologia - ICB - UFMG, Belo Horizonte , Caixa Postal 486, Minas Gerais, 31270-010

Chagas' disease is characterized by a variable clinical course, ranging from the absence of symptoms to severe cardiac or gastrointestinal involvement. The factors influencing this clinical variability are not completely understood, but most likely involve genetic variability of both human hosts and parasites. In this work we tested the hypothesis that the genetic variability of parasites might influence the pathogenesis of the disease, through differential tissue tropism of T.cruzi clonal lineages. Two distinct T.cruzi populations, the Col1.7G2clone and the apparently monoclonal JG strain, isolated or in mixtures, were used to infect BALB/c mice. In the acute (20 days) or chronic (3and 6 months) phases of infection we collected blood and fragments from brain, heart, esophagus and rectum. The detection and identification of the specific clone(s) present in each of the different tissues was performed by PCR amplification of kDNA minicircles and LSSP-PCR analysis. Tissue fragments were also sent for microscopic examination. In the acute phase of infection no clear tissue tropism was found, except for a preference of Col1.7G2 clone for the rectum of the mice. However, after three months, half of the mice infected exclusively with the JG strain showed absence of parasites in the rectum, in contrast with 100% presence in mice infected with Col1.7G2 or the mixture of clones (Col1.7G2 + JG). LSSP-PCR showed that in mice infected with mixtures, all parasites present in the rectum belonged to theCol1.7G2 clone. Moreover, the esophagus, diaphragm, blood and, after six months, the brain, were also parasitized exclusively by the Col 1.7G2clone, whereas conversely the heart presented only parasites of the JG strain. Histological analysis from the heart and rectum of these mice revealed a perfect correlation between the presence of parasites and tissue injury. Our data demonstrate clear tissue tropism of T.cruzi in the mouse, providing strong support to the hypothesis that genetic characteristics of T.cruzi clones are relevant to the pathogenesis of Chagas' disease (clonal-histotropic model).

Supported by PRONEX (FINEP), CNPq and FAPEMIG


259

TRYPANOSOMA CRUZI STRAIN-, ZYMODEME-, AND SPECIES-SPECIFIC MOLECULAR MARKER GENERATED IN A SINGLE PCR REACTION: SIGNIFICANCE FOR THE STUDY OF THE EPIDEMIOLOGY OF CHAGAS DISEASE.

Carrasco, H.J.1, Valente, S.A.2 and Miles, M.A.3.

1-Dpto. de Ciencias Básicas, Esc. Nutrición y Dietética, Fac. Medicina, UCV, Apartado postal 47176, Z.P. 1041-A, Caracas, Venezuela; 2-Instituto Evandro Chagas, Belém-Pará, Brasil; 3-Department of Medical Parasitology, London School of Hygiene and Tropical Medicine, UK.

The present study is based on the molecular characterisation of 115 different Trypanosoma cruzi isolates and reference strains, obtained from some widely separated geographical regions in Latin America (most from Brazil), from different mammals and triatomine bugs. Thirty six isolates were obtained from undisturbed and 28 from disturbed sylvatic cycles in the Amazon basin of Brazil. The remaining isolates were mainly from peridomestic and domestic cycles in different localities in Brazil, as well as some stocks from Chile, Mexico and Nicaragua. All the isolates were characterised by starch gel electrophoresis. Field isolates belonging to principal Z1 showed an important level of genomic variation as revealed by isoenzyme and RAPD analysis, and was the principal zymodeme in most of the natural cycles. RAPD generated molecular markers which wrere found to be specific at the species, zymodeme and strain level. Furthermore, RAPD also allowed the detection of mixed zymodeme populations in a human case of Chagas disease, and identification of T.cruzi at species and principal zymodeme levels when T.cruzi DNA was mixed with DNA from the closely related trypanosomatids, T.conorhini and T.rangeli, which share some mammal reservoir hosts and triatomine vectors with T.cruzi. Twenty five T.cruzi stocks characterised as principal zymodeme 1, obtained from individual triatomine bugs and 1 Didelphis marsupialis, circulating simpatrically in a disturbed sylvatic cycle in Barcarena, Pará State-Brazil, included some putative homozygous and heterozygous isoenzyme phenotypes with some enzymes. In a comparative study, 36 T.cruzi stocks, isolated from wild mammals (32 Didelphis marsupialis and 1 Philander opossum) and triatomine bugs (2 Rhodnius robustus and 1 unidentified bug) from an undisturbed sylvatic cycle in the Amazonian forest of Carajás, Pará State-Brazil, were characterised by isoenzyme and RAPD analysis as belonging to principal zymodeme 1. Isolates from Carajás, displayed two different homozygous and the corresponding heterozygous profiles for the enzyme phosphoglucomutase (PGM). Similarly, sharing of parental and hybrid characters was found with RAPD analysis. The frequency of PGM phenotypes within this population was as predicted by the theoretical Hardy-Weinberg equilibrium. Biological clones derived from hybrid stocks, retained the putative heterozygous characters. Further isoenzyme, RFLP and karyotype analysis of cloned stocks, indicated the presence of parental markers in the hybrid subpopulation. Overall results suggested that genetic exchange is a mechanism involved in generating genomic diversity during the sylvatic transmission of T.cruzi. This feature would confer the capacity to transfer to progeny drug resistance, changes in pathogenicity, and resistance to host defences.


260

GENETIC RELATIONSHIPS BETWEEN TRYPANOSOMA CRUZI STRAINS ISOLATED FROM CHRONIC CHAGASIC PATIENTS IN SOUTHERN BRAZIL AS REVEALED BY RAPD AND SSR-PCR

Gomes, M.L.1,3, Macedo, A.M.2, Pena, S.D.J.2 & Chiari, E.1

Departamentos de 1Parasitologia, e 2Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Caixa Postal 486, 31270-901 Belo Horizonte, MG, Brasil e-mail: chiari@icb.ufmg.br; 3Departamento de Análises Clínicas, Universidade Estadual de Maringá, 87020-900, Maringá, PR, Brasil.

Thirty Trypanosoma cruzi strains isolated from chronic chagasic patients were studied at the genotype level by RAPD (Random Amplified Polymorphic DNA) with arbitrary primers and SSR-PCR (Simple Sequence Repeat-Anchored PCR amplification) analysis. The genetic distance of strains was measured by the percentage of shared bands. The results showed that the strains isolated from chronic patients from different Chagas disease endemic areas of Brazil constituted a broad group significantly more correlated than reported in previous studies, presenting RAPD profiles with an average of 71% of shared bands and SSR-PCR patterns with a mean of 59% of shared bands. Different from other findings in the literature, these strains did not group on the basis of their geographic origin. These results suggest that a special adaptation of a parasite population in human hosts from mixed infective T. cruzi populations circulating in nature may have occurred.

Supported by CAPES-PICD/CNPq/FAPEMIG/PRONEX


261

POLYMERASE CHAIN REACTION AMPLIFICATION OF THREE DIFFERENT TRYPANOSOMA CRUZI DNA SEQUENCES FROM HUMAN CHAGASIC CARDIAC TISSUE

1Olivares-Villagómez, D., 2McCurley, T.L., 2Vnencak-Jones, C.L., 3Correa-Oliveira, R., 4Colley, D.G. & 1Carter, C.E.

1Department of Biology, Vanderbilt University, Nashville, Tennessee; 2Department Pathology, Vanderbilt University Medical Center, Nashville, Tennessee; 3Centro de Pesquisas René Rachou, FIOCRUZ, Belo Horizonte, Brasil; 4Division of Parasitic Diseases, National Center for Infectious Disease, Center for Disease Control and Prevention, Public Health Service, U.S. Department of Health and Human Services, Atlanta, Georgia.

The inability to consistently demonstrate T. cruzi by histologic techniques in inflammatory cardiac lesions has suggested that persistence may not be required for the pathology of the chronic phase. We have analyzed the persistence and localization of T. cruzi DNA in the hearts of seven patients with chronic chagasic cardiomyopathy, along with four indeterminate patients and seven control patients seronegative for T. cruzi infection. We have extracted DNA from selected inflammatory foci-positive (IFP) and inflammatory foci-negative (IFN) areas of hematoxylin and eosin stained cardiac tissue. We then used polymerase chain reaction methodology to amplify three different T. cruzi specific sequences (a minicircle sequence, MCS; a satellite repetitive sequence, RS; and a low copy number sequence within the gene coding for a flagellar proteins, FPS). MCS was detected in 100% of both the IFP and IFN areas analyzed. RS was detected in 37.5% and 23% of IFP and IFN areas respectively. FPS was rarely detected (2%) and was only present in DNA from IFP areas. MCS was also detected in most indeterminate cases although with a markedly diminished amplification signal relative to cardiomyopathy cases. MCS was not amplified from the cardiac tissues from seronegative controls.


262

DETECTION OF GENETIC POLYMORPHISM BY RAPD BETWEEN SIX STOCKS OF TRYPANOSOMA CRUZI ISOLATED FROM CHAGASIC PATIENTS.

Castro A.M.(1), Araújo T.C.C.(2), Ferreira K.S.(2), Oliveira E.C.(1), Rassi A.(1), Luquetti A.O.(1) and Soares C.M.A.(2)

1)Instituto de Patologia Tropical e Saúde Pública, 2)Instituto de Ciências Biológicas, Universidade Federal de Goiás, Goiânia.

Chagasic patients at the chronic phase present usually with different clinical manifestations, ranging from the indetermined form (no detectable disease) to severe cardiopathy and megaviscera. These differences have been attributed to both the genetic make up of the host and the strain involved. The purpose of this work was to explore the second alternative by the amplification of polymorphic segments of DNA of the parasites by Randon Aplified Plymorphic DNA (RAPD). Six stocks recently isolated from chagasic patients with different clinical forms of the disease were studied as well as the Y strain. Two patients were assymptomatic , one have slight megaesophagus(group I), one with a slight cardiopathy and one was an associated form (megadisease plus cardiopathy). All isolates(identification GOCH/357, 391, 412, 466, 491 and 519) were from xenodiagnosis and parasites were cultured in LIT medium. DNA was obtained as described by Borges (1990). Oligonucleotides were selected according to a better definition of DNA fragments on agar gels. The profile of amplification by 9 oligonucleotides was analised statistically. The algorhytm of groupment was UPGMA, program NTSYS-pc1.7, 1992. Indexes obtained through a similarity matrix varied from 0.8437 for stocks 519 and 412 to 0.3548 for stocks 466 and 357. The majority of stocks have had similarity higher than 50%.We concluded that the profile of stocks studied was rather similar.

Supported by: CNPq, FUNAPE-PRPPG UFGo.


263

THE USE OF THE RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) TECHNIQUE FOR THE DETECTION OF POLYMORPHISM IN POPULATIONS OF LEISHMANIA (VIANNIA) SHAWI.

Ishikawa, E.A.Y.1, Magalhães, A.L.P.1, Silveira, F.T.1, Shaw, J.J2.

1 Serviço de Parasitologia, Instituto Evandro Chagas, FNS, Belém, Pará; 2 Belém Research Projects, Instituto Evandro Chagas & Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo.

Eleven strains of L.(V.)shawi from three different regions of Pará State were examined for polymorphism by the random amplified polymorphic DNA (RAPD) technique using the oligonucleotide M13 (Forward 40). The isolates were from Paragominas (5), Serra dos Carajás (4) and Benevides (2) and all were provisionally identified as being L.(V.)shawi using a panel of 23 leishmanial monoclonal antibodies.

The data were analysed using the POP and MEGA programs and a phenogram was prepared using the simple cluster method UPGMA (Unweighted Pair-Group Method of Arithmetic averages ) which in other studies has given the best association of polymorphism and geographical origin for RAPD data. Except for two strains from Paragominas the isolates formed a homogenous group in spite of their different geographical origins. This is in contrast to populations of L.(V.)braziliensis from the same regions and may reflect different ecoepidemiologies. The grouping of the two strains from Paragominas was very distinctive and suggestive of a different taxon.

Supported bu CNPq; FNS.


264

DIAGNOSIS OF MUCOSAL LEISHMANIASIS BY POLYMERASE CHAIN REACTION

Gouvea CAB, Oliveira-Neto MP, Moreira J, Conceição-Silva F, Fernandes O & Pirmez, C.

Hospital Evandro Chagas, Dept. Tropical Medicine, Biochemistry & Molecular Biology and Protozoology Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brasil.

Mucosal leishmaniasis (ML) is most commonly caused by Leishmania (V.) braziliensis species. Diagnosis of ML is usually cumbersome because this particular species is difficult to grow in vitro and cultures are often lost by bacterial/fungal contamination. In addition, parasites are scarce in the lesions. Due to the high sensitivity of the polymerase chain reaction (PCR), we tried to amplify the conserved region of Leishmania in samples fixed in formalin and embedded in paraffin from 21 mucosal biopsies kept in our histopathological archives. Thirty sections of 5mm thickness were cut with a microtome using a disposable blade for each case. DNA was isolated by using the QIAmp tissue kit (QIAGEN, California, USA), ethanol precipited and resuspended in TE. Oligonucleotides that anneal to the origin of replication of both strands of the minicircle molecules were used in a hot start PCR, amplifying the conserved region of the minicircle kDNA. The amplified products were analysed by agarose gel electrophoresis, ethidium bromide staining and visualized under UV light. All products were applied to a nylon membrane using a dot-blot apparatus and hybridized with a preamplified product of L. braziliensis M2903 strain as a probe. Histopathological examination showed an intense inflammatory mononuclear cellular infiltrate, occasionally associated with an unorganized granuloma. Parasites were absent in all 21 cases, and could be isolated by culture in only 4/11 cases. In contrast, PCR was able to detect parasite DNA in 15 samples (71%).

Mucosal leishmaniasis is classically described as being a hyperegic form of the disease. Montenegro skin tests tend to be more intense and in vitro response against Leishamnia antigens is higher in ML than cutaneous disease. Moreover, the frequency of antigen-specific cells is 100 times higher in mucosal as compared to cutaneous lesions (Clin Exp Immunol 79:221, 1990). The higher negativity of mucosal lesions by standard methods such as culture, H&E and/or touch preparation could be explained by the intense inflammatory process within the lesions that would result in parasite destruction, and thus would make their visualization or isolation more difficult. The PCR would then be advantageous since it detects parasite DNA, even in the absence of entire parasites.

Supported by FAPERJ


265

BIOLOGICAL AND MOLECULAR CHARACTERIZATION OF LEISHMANIA SP ISOLATED FROM CUTANEOUS LEISHMANIASIS

Figueiredo, E. M.,* Gontijo, C. M. F.,* Brazil, R. P.*Pacheco, R.S. ***& Melo, M. N.**

Laboratório de Leishmanioses, Centro de Pesquisas René Rachou, FIOCRUZ, C. P. 1743, Belo Horizonte, 30190-002 MG, Brasil. * Departamento de Parasitologia, ICB/UFMG.**

Five strains of Leishmania sp isolated from human cases of Cutaneous Leishmaniasis from the casuistic of an outpatient clinic at Centro de Pesquisas René Rachou - FIOCRUZ, Reference Ambulatory for Cutaneous and Visceral Leishmaniasis of the Metropolitan Region of Belo Horizonte (MRBH), were studied using biological and biochemistry characteristics. Studies have been made on growth rates of promastigotes "in vitro", course of infection in experimental laboratory animals (Hamsters - Mesocricetus auratus), morphometric parameters of amastigotes, amplification of specific sequences of KDNA from L. braziliensis and L. mexicana groups, by polymerase chain reaction and electrophoretic mobility patterns of isoenzyme, in comparasion with WHO reference markers. The results of biological studies showed that four strains (RR052, RR021, RR028, RR051) were grouped together and characterized belonged to the subgenus Viannia and the remain strain (RR012) belonged to the subgenus Leishmania. The polymerase chain reaction confirmed the biological results of the strains included in braziliensis group, but strain RR012 did not showed any amplification with both set of primers from braziliensis and mexicana groups. Electrophoresis mobility patterns of 12 enzimatic systems: G6PDH (E. C. 1.1.1.49), ME (E.C.1.1.1.40), IDH (E.C. 1.1.1.42), MDH (E.C. 1.1.1.37), ACON (E.C. 4.2.1.3), PGM (E.C. 1.4.1.9), NH (E.C. 3.2.2.1), PEP D (L. prolina) (E.C. 3.4.13.9), 6PGDH (E.C. 1.1.1.43), GPI (E.C. 5.3.1.9), PEP 2 (L. L. alanina) (E.C. 3.4.11.1) and PEP 3 (L. alanina) (E.C. 3.4.11.1) showed that strains (RR052, RR021, RR028, RR051) are L. (V.) braziliensis and the strain RR012 are similar to L. (L.) amazonensis marker - PH8, but is probably a variant of this species. All the results taken together indicate that L. (V.) braziliensis and L. (Leishmania) of the amazonensis group are prevalent in the endemic areas of Metropolitan Region of Belo Horizonte, MG, Brazil, as showed by previous studies (PASSOS et al., Mem. Inst. Oswaldo Cruz, 88: 103-110, 1993; GONTIJO et al., Mem. Inst. Oswaldo Cruz, Rio de Janeiro, 90, Suppl. I: 143, 1995).

Supported by CAPES/FIOCRUZ


266

PRELIMINARY RESULTS OF BIOLOGICAL AND MOLECULAR CHARACTERIZATION OF LEISHMANIA MAJOR-LIKE PARASITES

Silva, S. O. ; Gomes, R. F.; Oliveira, M. R.; Macedo, A. M.; Melo, M. N.

Departamento de Parasitologia e Departamento de Bioquímica e Imunologia da Universidade Federal de Minas Gerais, Belo Horizonte - Minas Gerais.

Two strains (MHOM/BR/71/BH49 and MHOM/BR/71/BH121) of Leishmania, isolated in Brazil from two humans cases of cutaneous leishmaniasis, were characterized by their in "vitro" behaviour, including metacyclogenesis, assessed for aglutination by peanut agglutin (PNA) and their "in vivo" behaviour in Balb/c mice. The parasites were cultivated in Grace's medium supplemented with 20% bovine fetal serum, at pH 5,5 and 6,5. Growth was assessed by comparison with World Healt reference strains of L. major FN (MHOM/IL/80/Friedlin) and 5askh (MHOM/ SU/70/5ASKH). There were no significant differences in the growth of the strains at pH 6,5 but growth of FN was slower at pH 5,5. In separation with PNA, strain BH49 did not show a significant number of metacyclic forms (PNA-) and none were seen for strain BH121. BALB/c mice were inoculated with PNA+ forms (presumably non metacyclics). Preliminary results showed that strain BH49 caused a lesion at the site of inoculation 90 dias after inoculation where as strain FN gave rise to a lesion with 30 days. Groups of BALB/c mice inoculated with stationary phase forms of BH49, grown at pH 5,5 and pH 6,5 are still being studied. Molecular characterization was carried out by Random Amplification of Polymorphic DNA (RAPD) using nine different primers: QG1, L15966, M13 (-40), M13R, P53 (1), A07. B07, 3308 and SSR-PCR (Simple Sequence Repeat anchoring-PCR) with (CA)8 RY. Two New World reference strains - MHOM/BR/75/M2903 (L. braziliensis) and IFLA/BR/67/PH8 (L. amazonensis) were use as standards. An analysis of RAPD and SSR-PCR profiles showed a mean of 16,23 ± 3,05 bands between the pairs, varying between 15 and 38 bands depending on the primer used. Strains of BH49 and BH121 showed 98% of shared bands. No significant differences were found between strains BH49 and BH121 when compared with the reference strains of L. major with a mean of 89,4 ± 12,9 bands, with a variation of 69-100% depending on the primer used . Because of the great similarity with L. major strains BH49 and BH121 are designated as L. major like.

Supported by CNPq


267

VISCERAL LEISHMANIASIS IN BELO HORIZONTE CITY, STATE OF MINAS GERAIS, BRAZIL: MOLECULAR CHARACTERIZATION OF CANINE AND HUMAN SAMPLES.

Passos, VMA1, Volpini AC1, Fernandes O2,3.

1-Laboratório de Epidemiologia e Antropologia Médica, Centro de Pesquisas René Rachou-FIOCRUZ, Av. Augusto de Lima 1715, Belo Horizonte,CEP 30190-002, Minas Gerais, 2-Departamento de Medicina Tropical-IOC-FIOCRUZ, 3-Departamento de Patologia-Universidade do Estado do Rio de Janeiro.

Visceral Leishmaniasis (VL), although predominant in rural areas of Brazil, affects also people living in periurban regions of big cities such as Rio de Janeiro (Marzochi et al, Mem. Inst. Oswaldo Cruz, 80(3):349-357, 1985), Terezina (Costa et al., Rev. Saúde Púb. 24:361-372, 1990) and Belo Horizonte (Genaro et al., Rev. Soc. Bras. Med.Trop. 23 (2):121, 1990). From 1993 to 1996, 111 human cases were notified in almost all districts of Belo Horizonte and the canine incidence varied from 3,7% to 5,8% (Oliveira et al., XXXIII Cong. Soc. Bras. Med. Trop., Resumos:81, 1997). In 1995, peripheral blood and bone marrow samples were collected from one dog and from five patients (outpatient unit- Centro de Pesquisas René Rachou). All five human cases (one woman and four men), aging from 4 to 21 years-old (x=9,4+6,7) , had the Imunofluorescense Assay (IFA) positive (> 1:40) and all cases have been submitted to parasitological diagnosis, showing the presense of amastigotes on the bone marrow aspirate after Giemsa staining. In order to characterize the parasite, DNA was extracted from bone marrow by phenol:chloroform and the conserved region of the minicircle from Leishmania kDNA network was amplified by the Polymerase Chain Reaction (PCR) using a set of defined oligonucleotides. Reaction mixtures contained 200mM of dNTPs, 200ng of each primer, 2.5U of Taq DNA polymerase in the buffer recommended by the manufacturers, with a total concentration of 1.5 mM MgCl2. The reactions were performed in a total volume of 20ml after the addition of 2ml of the DNA sample. Positive controls with L. chagasi and negative controls with no DNA were included in each reaction set. PCR products were analyzed by ethidium bromide stained and visualized under UV light. As the amplification target is the conserved region of the minicircle molecule, the PCR detected the presence of Leishmania DNA in the sample, but did not distinguish among species. Therefore, the subgenus identification was made possible by hybridization with specific probe. The PCR products were denatured in 0.4N NaOH, applied to a nylon membrane using a dot-blot apparatus and hybridized with a cloned L. chagasi minicircle molecule radiolabeled. Membranes were then hybridized in Bovine Lacto Transfer Technique Optimizer at 600C, washed in 0.5X SSC at the same temperature and exposed to X-ray films overnight with an intensifying screen at -700C. All six samples were positive on dot hybridization with the L. chagasi probe, confirming this species of the parasite as more prevalent in human and canine cases of VL in the country as well in Belo Horizonte epidemy.

Supported by PAPES- FIOCRUZ, CAPES & PIBIC-CNPq.


268

AMERICAN TEGUMENTARY LEISHMANIASIS IN RIO DOCE VALLEY, STATE OF MINAS GERAIS, BRAZIL: MOLECULAR CHARACTERIZATION OF LEISHMANIA PARASITES.

Passos VMA1,2, Fernandes O3,4, Gontijo BA2, Lacerda PAF1, Volpini AC1,7 , Catanho M5, Mayrink W6 , Romanha A7.

1-Laboratório de Epidemiologia e Antropologia Médica, Centro de Pesquisas René Rachou-FIOCRUZ, Av. Augusto de Lima 1715, Belo Horizonte, 30190-002, Minas Gerais, 2-Dept. de Clínica Médica, Faculdade de Medicina-UFMG, 3-Dept. de Medicina Tropical-FIOCRUZ, 4-Dept. de Patologia-UERJ, 5-Dept. de Bioquímica e Biologia Molecular-FIOCRUZ, 6-Dept. de Parasitologia, ICB-UFMG, 7-Lab. de Parasitologia Celular e Molecular-CPqRR, romanha@netra.cpqrr.fiocruz.br.

Since 1965 many cases of American Tegumentary Leishmaniasis (ATL) were diagnosed and treated in an outpatient clinic at the city of Caratinga, Rio Doce valley, State of Minas Gerais. In order to characterize molecularly the parasites, 56 tissue biopsies were taken under sterile conditions from the border of one ulcer in each patient and were frozen at -200C. All biopsies have been submitted to conventional impriting preparation that demonstrated the presence of amastigotes. A tissue lysate was obtained by adding 25ml of TE , proteinase K (100mg/ml) and by incubating at 56oC for 2 h and subsequently at 370C overnight. The digested lysate was finally boiled for 15 min to inactivate the enzyme, spun-down and the supernatant used as DNA source. Primers designed to amplify the conserved region of the minicircle molecule from the Leishmania kDNA were used in Polymerase Chain Reaction (PCR). Reaction mixtures contained 200mM of dNTPs, 200ng of each primer, 2.5U of Taq DNA polymerase and 1.5mM MgCl2. The reactions were performed in a total volume of 20ml after the addition of 2ml of the tissue lysate. Positive controls with L. braziliensis and L. amazonensis reference strains and a negative control with no DNA were included in each reaction set. PCR products were analyzed in silver stained 8% polyacrylamide gel. As the amplification target is the conserved region of the minicircle molecule, the PCR detected the presence of Leishmania DNA in the sample, but did not distinguish among species. Therefore, the subgenus identification was made by hybridization with specific probes. The PCR products were denatured in 0.4N NaOH applied to a nylon membrane using a dot-blot apparatus and hybridized with cloned minicircles of L. (V.) panamensis and L (L.) mexicana labeled with 32P by random priming. Membranes were then hybridized in BLOTTO at 600C, washed in 0.5X SSC at the same temperature and exposed to X-ray film overnight with an intensifying screen at -700C. It was possible to perform the molecular typing of the Leishmania sp. infecting 56 patients (36 men and 20 women), aging from 5 to 54 years-old (x=27,6+14,7). Fifty two patients presented the cutaneous and 4 the mucocutaneous form of ATL. The number of lesions/patient varied from one to three (x=1,4+ 0,6). Fifty-three out of 56 (94,6%) of the characterized parasites belonged to the subgenus Viannia and the remaining 3 (5,4%) parasites belonged to the subgenus Leishmania. These results reveal that L. braziliensis is the main parasite of that area, as already observed in other regions of the State by Passos et al. Mem. Inst. Oswaldo Cruz 91(suppl): 183 (1996).

Supported by CAPES, CNPq, FIOCRUZ and FNS-UFMG- Secretaria de Saúde Municipal de Caratinga.


269

VISCERAL LEISHMANIASIS IN THE METROPOLITAN REGION OF BELO HORIZONTE, MINAS GERAIS - MOLECULAR CHARACTERZATION OF CANINE AND HUMAN ISOLATES.

*Silva, E.S., *Gontijo, C.M.F., **Pacheco,R.S. & *Brazil,R.P.

*Lab. Leishmanioses CPqRR/FIOCRUZ, Caixa Postal 1743 , Belo Horizonte, 30190-002 MG, Brasil - **Departamento de Bioquímica e Biologia Molecular, IOC-FIOCRUZ

Since 1994 when the first autochthonous case of visceral leishmaniasis was notified in Belo Horizonte (Minas Gerais State, Brazil) the number of cases increased notably. Having the dog as the more important domestic reservoir it's essential to detect canine infection in order to stablish adequate control measures. To diagnose infection and obtain isolated parasites from the metropolitan area of the city for characterization of strains we worked with specimens of blood and bone narrow colected from dogs from veterinarian clinics. Parasites collected from human especimens examined in our lab were also used to be characterized and compared with isolates from dogs. Sorology was performed in dogs and human cases using RIFI and ELISA techniques. Parasites were searched at the bone narrow from few cases, and positives had the parasites isolated and cultivated in NNN LIT medium. Parasites originated from nine canine and four human cases were analyzed trough the following techniques: kinetoplast DNA enzyme restriction analysis using Msp I and Mbo I endonucleases; agarosis gel eletrophoresis using nine enzymes; molecular hibridization. All the isolates presented a complex genotypic pattern consistent with the Leishmania chagasi reference strain in the fragments of the kDNA minicircle. Through the enzyme eletrophoresis we were able to divide the isolates from dogs in two zymodemes. Two isolates (normal skin and bone narrow) from a same dog revealed identical pattern. Heterogenity was present within the human isolates and between human and canine isolates. Southern Blot experiments showed homology in the kDNA sequence of the parasites isolated from dogs and men for the cloned minicircle of L. chagasi what confirms the anterior results. For the first time the Leishmania strains isolated in the metropolitan region of Belo Horizonte have been characterized by molecular and biochemical methods and the results obtained reveal that besides the genetic polimorphism the same strain of parasites is circulating between canine and human cases in the studied area. The present study reassures the importance of the dog as the domestic reservoir of the disease and the necessity of controlling the spread of disease among these animals.

Supported: CNPq, PAPES/FIOCRUZ


270

GENETIC VARIABILITY OF LEISHMANIA (V.) BRAZILIENSIS ISOLATES FROM AN ENDEMIC AREA OF CUTANEOUS LEISHMANIASIS IN THE ESPIRITO SANTO STATE, BRAZIL.

Lúcia Regina Brahim; Aloízio Falqueto1; Luíza O.R. Pereira; Antônio Teva; Miriam S. Pereira; Gabriel Grimaldi & Elisa Cupolillo

Laboratório de Leishmaniose, Dept. de Imunologia, Instituto Oswaldo Cruz, RJ. 1Núcleo de Medicina Tropical, Centro Biomédico, UFES.

Cutaneous leishmaniasis due to L. braziliensis has been known in Espírito Santo since the early 20th Century. The parasite is widespread in the State circulating in both (a) mountainous region, deforested, with reduced number of wild species, presenting Lutzomyia whitmani as the principal vector, and (b) valleys region of the bottom of the coastal mountain, with secondary forest, presenting Lutzomyia intermedia as the principal vector. To date, there is no evidence of a wild reservoir for this species. The existence of an urban cycle for L. braziliensis reflects the ability of these parasites and their vectors to adapt to changes in their original forest habitats (Falqueto et al. 1993. Research and Control of Leishmaniasis in Brazil: 59).

An extensive epidemiological study is taking place in Espirito Santo. Leishmania isolates from different vertebrate and invertebrate hosts, representing different endemic foci in this region, have been isolated and characterized. The enzymatic pattern for several enzymes has indicated that there is no enzymatic variation among the isolates.

We are looking for genetic variability in the parasite population for establishing a possible correlation with epidemiological features, such as the nature of the transmission cycle or geographic distribution of each Leishmania species. The ITS region of the rRNA gene has been amplified by PCR and the product digested with restriction enzymes. The profiles were analyzed by phenetic methods. The level of heterogeneity was not high. However, we could define some populations that are not restricted to a host or geographic area. This is an indication that the same population of the parasite can circulate all over the State and natural pressures are selecting different population.

Supported by CNPq, FIOCRUZ.


271

MOLECULAR EPIDEMIOLOGY OF PERUVIAN AND BOLIVIAN LEISHMANIAIS

Banuls A.L. I, Dujardin J.C.2, Guerrini F. I, Arevalo J.3, Solano M.4, Bermudez H.4, DeDoncker S.2, Jacquet D.2, Le Ray D.2 and Tibayrenc M. IICEPM, centre ORSTOM Montpellier, France; 2Laboratory of Protozoology, Prince Leopold

Institute of Tropical Medicine, Antwerpen, Belgium; 3Laboratory for Trypanosomatidae

Biochemistry, Instituto de Medicina Tropical Alexander Von Humbolt, Lima IOO, Peru

4Centro Universitario de Medicina, Universidad Mayor de San Simon, Casilla 3119,

Cochabamba, Bolivia.

ln this study, we analyzed by Multilocus Enzyme Electrophoresis (l6 loci) and Random Primer Amplified Polymorphic DNA (IO primers) a Peruvian and Bolivian sample of 188 Leishmania natural isolates.

Two main lines of results were reached:

(I) certain species were recorded for the first time in given countries: Leishmania lainsoni in Bolivia and L. guyanensis in the Peruvian Andean Valleys. (2) drastically different species can coexist in the same focus, and even, in the same host. These results emphasize the need for a sharp molecular identification of Leishmania species, the more so since that different species tend to be associated with distinct clinical forms. Both MLEE and RAPD provide markers specific of: (i) given species; (ii) given geographical areas; (iii) possibly given clinical forms, although this last result definitely has to be confirmei on larger sample.


272

IMPROVEMENTS OF THE RECOMBINANT CRA+FRA ELISA TEST FOR CHAGAS DISEASE DIAGNOSIS.

1d'Escoffier, L.N. ; 2Silva, E. D. ; 2Ferreira, A.G.P.; 1Krieger, M.A. & 1Goldenberg, S.

Fiocruz, Dept. de Bioquímica e Biologia Molecular (1) e Biomanguinhos (2). Avenida Brasil 4365, Rio de Janeiro, RJ, 21045-900 Brasil.

Previous work resulted in the development of an ELISA test for Chagas' disease diagnosis using recombinant antigens CRA and FRA (Krieger et al., Am.J.Trop. Med.Hyg. 46: 427, 1992). These antigens display a structure comprised by repetitions of 14 (CRA) and 68 (FRA) amino acids and are recognized by all chagasic sera tested so far. These antigens are normally produced fused to b-galactosidase (b-gal), and are purified by affinity chromatography on anti-b-gal-antibody bound to agarose, followed by digestion with factor Xa. Hence, the production of the recombinant antigens requires two fermentation steps.

In order to ameliorate the production of the ELISA test, we have constructed a CRA/FRA chimera and expressed it in E.coli fused to glutathione-S-transferase. The level of expression of the CRA/FRA recombinant protein was excellent resulting in yields at least as good as those obtained for the individual recombinant antigens. Western blot analysis using rabbit antisera against CRA or FRA and human chagasic sera showed that all of them recognized CRA/FRA. Furthermore, we have compared by ELISA the reactivity of the CRA/FRA fusion and the CRA and FRA mixture against chagasic and non-chagasic human sera. The results showed full-agreement between the results obtained using either ELISA. In conclusion, the use of a chimera CRA/FRA is suitable for the diagnosis of Chagas' disease and should reduce the time and cost of production of the ELISA diagnosis test.

Support: PAPES-Fiocruz; CNPq.


273

COMPARATIVE STUDIES OF DIFFERENT CERAMIDE MONOHEXOSIDE FROM TRYPANOSOMA CRUZI STRAINS BY ELISA

Villas Bôas, M.H.S.*, Silva, R. B.*, Wait, R.** & Barreto-Bergter, E.*

*Departamento de Microbiologia Geral, Instituto de Microbiologia Prof. Paulo de Góes, UFRJ, Rio de Janeiro, RJ, BR - E mail: immgbel@microbio.ufrj.br

**Centre for Applied Microbiology and Research, Salisbury, UK

Chaga's disease, caused by the trypanosomatid flagellate Trypanosoma cruzi, was first described by Carlos Chagas in 1909. This disease is endemic througout Central and South America, very complex, with several different clinical manifestations that range from a nearly complete lack symptoms to severe and often lethal cardiomyopathy, megacolon or megaesophagus. The etiology of the clinical variability of Chaga's disease is not understood, but one of possible determining factors is the infecting strain of T. cruzi.

Neutral glycosphingolipids are located primarily in the outer leaflet of plasma membranes. Glycosphingolipids are effective antigens and immunogens, and have shown to function as cell type-specific and developmental stage-specific antigens and isogeneic or heterophile antigens (i.e. histo-blood group antigens). Our previous immunological studies have revealed the antigenicity of neutral glycosphingolipids derived from the epimastigote T. cruzi Y strain (1). In this present study we have compared immunological and structurally glycosphingolipids from T. cruzi Dm 28c to those of the Y strain by a improvement ELISA (Enzyme-Linked Immunoadsorvent assay) technique. Rabbit antiserum against epimastigote T. cruzi Y strain was used. Crude lipid extract and ceramide dihexoside showed similar reactivity for both strains, whereas ceramide monohexoside from Y strain showed higher reactivity than Dm 28c strain. The different reactivity pattern presented for monohexosylceramide from two strains , probably is a consequence of lipid moiety heterogeneity.

(1)Villas Bôas, M.H., Silva, M.C.F., Oliveira, T.G., Travassos, L.R. & Barreto-Bergter, E. (1994) J. Clinical Laboratory Analysis, 8, 113-123.

Supported by: CNPq, FINEP, CEPG/UFRJ and PRONEX.


274

CLONING OF TWO IMMUNODOMINANT ANTIGEN GENES OF POTENTIAL VALUE FOR CHAGAS' DISEASE DIAGNOSIS

Tokiko K. Matsumoto2, Eufrosina S. Umezawa1, José Franco da Silveira3, Jorge C. Araya3 , Norival Kesper Jr1 & Paulo C. Cotrim1.

1- Instituto de Medicina Tropical de São Paulo-FMUSP, Av. Dr Enéas de Carvalho Aguiar 470,
CEP 15403-000, 2- Instituto Adolfo Lutz - São Paulo, 3- Escola Paulista de Medicina - UNIFESP.

E-mail: pccotrim@usp.br

Conventional serology for Chagas'disease using epimastigote antigens is highly sensitive to detect chronic infections, but has failed to detect acute and congenital infections. Moreover, it presents low specificity. Alternatively, trypomastigote-specific antigens could be exploited in the serodiagnosis of Chagas' disease. We thus decided to isolate two T. cruzi genes predominantly expressed in this evolutive form: (a) antigens called SAPA/TS (shed acute phase antigen/Transialidase), a target of acute-phase antibodies, and (b) the 160-kDa protein family, a target of chronic-phase antibodies. SAPA/TS and 160-kDa antigens are excreted/secreted from T. cruzi trypomastigotes (Y strain) into the supernatant of infected LLC-MK2 cells (Umezawa et al., J.Clin. Microbiol.34:2143-47, 1996). After separation by 7% SDS-PAGE, these excreted/secreted antigens were transferred to nitrocellulose membranes and incubated with a chronic chagasic serum. Specific antibodies against these two antigens were eluted from nitrocellulose membranes and used for immunological screening of a lgt11 expression library of genomic DNA from metacyclic T. cruzi. Among the selected recombinant clones, four expressed SAPA/TS and one expressed the 160-kDa antigen, as confirmed by dot blot analysis using specific rabbit antisera. These recombinant clones were PCR-amplified for further nucleotide sequence analysis and also subcloned into the expression vector pGEX for large-scale antigen production. Our preliminary data suggest that the use of these recombinant antigens can result in a substantial improvement in the serodiagnosis of Chagas' disease.

Financial support: FAPESP, LIM-48 and 49/FMUSP.


275

ISOLATION AND CHARACTERIZATION OF TRYPANOSOMA CRUZI RECOMBINANT ANTIGENS WITH ANTIBODIES FROM CHILEAN CHAGASIC PATIENTS

Araya, J.E.1, Manque, P.1, Franco da Silveira, J.2, Cano, M.I.2, Sagua, H.1 and Neira, I.1

1 Unidad de Parasitologia, Departamento de Tecnologia Medica, Universidad de Antofagasta, CHILE; 2 Depto., Micro, Imuno e Parasitologia, Escola Paulista de Medicina-UNIFESP, São Paulo, BRASIL.

In order to isolated T. cruzi antigenic determinants recognized by sera from Chilean chagasic patients, intact epimastigotes cells previously fixed with formaldehyde were incubated with the sera from non symptomatic chagasic patients and the bound antibodies were eluted by mild acid treatment of the cells. After neutralization, the selected antibodies were used to screen a T. cruzi (strain SPA-14, Antofagasta, Chile) epimastigote cDNA expression library constructed in l gt11.

Several recombinant reactive clones were isolated and one of them, named C9, was chosen for further analysis. C9 encodes a b-galactosidase fusion protein of 140 kDa that strongly reacted with a panel of Chilean chronic chagasic sera. The 573 bp insert of C9 was isolated and used to probe genomic Southern blots and chromoblots. DNA from different strains (SPA-14, Y, G, clone CL Brener) were digested with several restriction enzymes and hybrized with C9. The probe hybrized with a doublet of about 20 kbp in SPA-14, Y and G strains, and two genomic fragments of 20 and 9 kbp in clone CL Brener. C9 gene was mapped on a single chromosomal band of ~2.3 Mbp of strains SP-14, Y and G, and on two chromosomal bands of 2.3 and 2.6 Mbp of clone CL Brener. These data suggest that C9 gene is present in a small number copies in the genome.

Nucleotide sequence analysis showed that the insert of clone C9 display significant homology at nucleotide level with sequences present in genes enconding a high molecular weight flagellar repetitive antigen of T. cruzi (H49, FRA, Ag1, JL7). C9 encodes a polypeptide of ~21 kDa which carries the C-terminal domain of the flagellar repetitive antigen. C9 polypeptide contains one repeat (LAREKKLADRAFLDQKPE), previously described in the proteins cited above, followed by a short non-repetitive segment of the C-terminal domain. The C9 recombinant protein was subcloned in plasmid pGEX in fusion with glutathione S-transferase (GST) gene. The C9/GST fusion protein will be used to evaluate the potential of this protein in serodiagnosis of Chagas disease.

Supported by DNIV, Universidad de Antofagasta, Chile-Project PEI 9620, CNPq, International Atomic Agency (Vienna, Austria).


276

THE IMPACT OF HIV INFECTION ON T.CRUZI GENETIC POPULATION STRUCTURE

Perez-Ramirez L., Barnabé C. and Tibayrenc M.

ln Latin America with the spreading of the Human Inmiunodeficiency virus epidemic, reactivation cases of Chagas chronic disease have been described since 1990. These cases raise some questions about the parasite responsibility in the exacerbation of Chagas' disease. An inter-institutional study was performed in Brazil from June 1994 to May 1995 and from March 1996 to May 1996.

The aim of this study was to evaluate the specific impact of HIV immunodeficiency on the population structure of Tcruzi i) to compare the genetic diversity between T cruzi stocks from HIV positive and HIV negative patients ii) to test the clonal hypothesis in T cruzi stocks isolated from HIV positive patients, and (iii) to evaluate a possibie correlation between clinical forms and particular genotypes.

Ninety stocks of T cruzi isolated from 28 HIV positive patients and 16 HIV negative patients were analysed by multilocus enzyme electrophoresis (MLEE).

On one hand clinical forms of reactivation seem to be associated with very high parasitemia levels and suggest an important role of the parasite, the other hand, the genetic analysis showed that whatever the patients immune status, the stocks are related to previously described clonal genotypes 30, 32, 39 and 43 (]) . So it appears that the reactivation observed in HIV positive patients is not correlated with particular genotypes that would be only encountered in immunocompromised patients. Although if we do not observed new genotypes among the stocks isolated from HIV positive patients, these stocks showed a slightly higher degree of polymorphism in comparison with those isolated from HIV negative patients.

A strong linkage disequilibrium, which is considerei as a classical circumstantial evidence for clonal population structure, was evidenced within the stocks from all the patients whatever their immune status. This clonal population structure was present in both groups of patients, either VIH(+) or VIH(-).

No correlation between clinical fonns of Chagas disease and parasite genotypes was observed.

I) Tibayrenc M and Ayala FJ (l988). lsozyme variability in Trypanosoma cruzi, the agent of Chagas' disease : genetical, taxonomical, and epidemiological significance. Evolution 42, 277-292.