Conferences

C1 - ^{ANTIGENIC VARIATION IN AFRICAN TRYPANOSOMES AND OTHER PATHOGENIC MICRO-ORGANISMS}

C2 - ^{IMMUNOLOGICAL MARKERS OF CLINICAL EVOLUTION IN HUMAN LEISHMANIASIS}

C3 - ^{BLOODFEEDING IN MOSQUITOES AND THE TRANSMISSION OF DISEASES}

C4 - ^{LIFE AND DEATH IN TB.RHODESIENSE}

C5 - ^{O-GLYCOSYLATION OF TRYPANOSOMA CRUZI PROTEINS: STRUCTURAL DIVERSITY AND CHARACTERISATION OF A NOVEL URIDINE-DIPHOSPHO-N-ACETYL-GLUCOSAMINE: POLYPEPTIDE N-ACETYLGLUCOSAMINYLTRANSFERASE}

C6 - ^{TOXOPLASMA GONDII — A GENETIC MODEL FOR THE INVESTIGATION OF DRUG RESISTANCE MECHANISMS, PARASITE DIFFERENTIATION, AND THE EVOLUTION OF EUKARYOTIC ORGANELLES}

C7 - ^{INFLUENCE OF PLASMODIUM FALCIPARUM INFECTION MEDIATED IMMUNE STIMULATION ON HIV-1 REPLICATION}

C8 - ^{THE CLONAL-HISTOTROPIC MODEL FOR THE PATHOGENESIS OF CHAGAS DISEASE}

C9 - ^{GENETIC ANALYSIS OF THE PROTOZOAN PARASITE TOXOPLASMA GONDII HAS UNDERGONE A RAPID EXPLOSION IN RECENT YEARS.}

C10 - ^{THE MINI-EXON GENE IS A DISTINCTIVE NUCLEAR MARKER FOR GROUPING PROTISTIS OF THE KINETOPLASTID/EUGLENOID LINEAGE}

C11 - ^{PRESENT SITUATION AND FUTURE OF HUMAN CHAGAS DISEASE IN BRAZIL}

C12 - ^{DEVELOPMENT OF A HETEROLOGOUS TRANSPOSON SYSTEM FOR LEISHMANIA}

C1 - ^{ANTIGENIC VARIATION IN AFRICAN TRYPANOSOMES AND OTHER PATHOGENIC MICRO-ORGANISMS}

P. Borst

The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam

Antigenic variation looks like a simple and effective method for a parasite to elude the immune system of its mammalian host. By regularly changing the surface coat of a small sub-fraction of the parasite population, the parasite can remain in the mammalian bloodstream to meet its insect vector taking a blood meal. In practice this strategy is more complex than one would think, as our analysis of antigenic variation in African trypanosomes has shown. There are at least five basic requirements for making antigenic variation work:

1. A large repertoire of surface antigens.

2. A mechanism for switching the surface antigen expressed in a sub-fraction of the trypanosome population, before the host has made antibodies against this antigen.

3. The ability to express surface antigens in a defined order to avoid population heterogeneity.

4. The ability to combine antigenic variation with substrate uptake (requiring invariant receptors and translocators).

5. The ability to survive in multiple hosts, offering different (macromolecular) substrates.

I shall delineate how Trypanosoma brucei meets these requirements as also discussed in recent reviews (1-3).

In addition, I shall discuss our recent experiments on J and on the transferrin receptor of T. brucei. J is a novel base, þ-glucosylhydroxymethyluracil, discovered in bloodstream form T. brucei (4) and recently shown to be also present in other kinetoplastida, such as T. cruzi, Leishmania and Crithidia (5). In T. brucei, J replaces about 1% of T and this replacement is not only DNA sequence specific but also DNA context specific. J is present in repeated sequences and especially in both strands of the telomeric (GGGTTA)n repeats of T. brucei (6).

Indirect evidence suggests that the conversion of T into J occurs in two steps: first, a sequence specific oxygenase converts T into hydroxymethylU; then a relatively non-specific glucosyl transferase adds the glucosyl group (7). The function of J is still a matter of speculation. If it would be essential, it would be an interesting target for drugdevelopment.

A transferrin receptor (Tf-R) is present in the flagellar pocket of T. brucei and has unusual properties. It is heterodimeric, attached via a GPI anchor in the membrane, and is encoded by a telomeric gene family, present in the expression sites for Variant Surface Glycoprotein (VSG) genes (see 2). There are some twenty VSG gene expression sites per trypanosome nucleus; usually only one is active at a time. We have shown that different expression sites encode Tf-Rs that are similar, but not identical. These small differences can have profound effects on the binding affinity for Tfs from different mammals and on the ability of trypanosomes to grow in the sera of these mammals. Our results suggest that the ability to switch between different Tf-R genes allows T. brucei to cope with the large sequence diversity in the Tfs of its hosts (2,8).

References

1. Borst P, Rudenko G, Taylor MC, Blundell PA, Van Leeuwen F, Bitter W, Cross M, McCulloch R. Antigenic variation in trypanosomes. Archives of Medical Research 1996; 27: 379-388.

2. Borst P, Bitter W, Blundell P, Cross M, McCulloch R, Rudenko G, Taylor MC, Van Leeuwen F. The expression sites for variant surface glycoproteins of Trypanosoma brucei. In: Hide G, Mottram JC, Coombs GH and Holmes PH (eds.), Trypanosomiasis and Leishmaniasis: biology and control. British Society for Parasitology/CAB International, Oxford, 1997, chapter 7: pp. 109-131.

3. Borst P, Rudenko G, Blundell PA, Van Leeuwen F, Cross MA, McCulloch R, Gerrits H, Chaves IMF. Mechanisms of antigenic variation in African trypanosomes. Behring Inst. Mitt. 1997; 99: 1-15.

4. Gommers-Ampt JH, Van Leeuwen F, De Beer ALJ, Vliegenthart FG, Dizdaroglu M, Kowalak JA, Crain PF, Borst P. Beta-D-glucosyl-hydroxymethyluracil: a novel modified base present in the DNA of the parasitic protozoan Trypanosoma brucei. Cell 1993; 75: 1129-1136.

5. Van Leeuwen F, Borst P, unpublished.

6. Van Leeuwen F, Wijsman ER, Kuyl-Yeheskiely E, Van der Marel GA, Van Boom JH, Borst P. The telomeric GGGTTA repeats of Trypanosoma brucei contain the hypermodifiedbase J in both strands. Nucl. Acid Res. 1996; 24: 2476-2482.

7. Borst P, Gommers-Ampt JH, Ligtenberg MJL, Rudenko G, Kieft R, Taylor MC, Blundell PA, Van Leeuwen F. Control of antigenic variation in African trypanosomes. Cold SpringHarbor Symposia on Quantitative Biology, DNA and Chromosomes, volume 58, 1993: 105-115.

8. Bitter W, Gerrits H, Borst P, unpublished.

C2 - ^{IMMUNOLOGICAL MARKERS OF CLINICAL EVOLUTION IN HUMAN LEISHMANIASIS}

Edgar de Carvalho

Universidade Federal da Bahia, Salvador, Ba/Brasil

Abstract not received.

C3 - ^{BLOODFEEDING IN MOSQUITOES AND THE TRANSMISSION OF DISEASES}

Anthony A. James, Ph.D.

Department of Molecular Biology and Biochemistry, 3205 Bio Sci II, University of California, Irvine, CA 92697-3900 USA [phone] [phone] FAX [e-mail]

The application of molecular biological approaches to research on insect vectors of disease and vector-parasite interactions has broadened the interest in developing disease control strategies that rely on attacking the vector. A number of different strategies have been proposed, and one receiving substantial attention is the effort to genetically modulate vector competence in vector-species (James and Collins, 1996). The hypothesis to be tested is that increasing antiparasitic allele or gene frequencies in populations of vector insects should lead to a decrease in transmission of disease. A specific gene or allele that interferes with parasite development or propagation is to be spread through a target vector species and this will result in less transmission and less disease. Following implementation of this strategy, there should be a concrete and measurable effect. As such, this is a testable hypothesis.

This hypothesis is the subject of lively debate (Collins, 1994: Spielman, Kitron, and Pollack, 1993) and for specific diseases such as malaria it has been suggested that reducing transmission without eradicating disease may actually be worse than no intervention at all. However, it is unreasonable to expect that any one strategy alone will be successful for the control of parasitic diseases. It behooves us to study all possible approaches, and that includes modest technology strategies such as the use of insecticide impregnated bednets (Curtin, 1996) and the high-profile efforts to develop vaccines (Jones and Hoffman, 1994). Most likely, control or elimination of disease will require a manifold approach using the best tools for each specific situation. Furthermore, a broad-based approach to disease control is likely to reveal new perspectives and insights that may provide additional and at this time, unpredicted, means of controlling disease.

As discussed in Collins and James, (1996) research in three areas needs to be done to test the hypothesis of genetic control of vectors and disease. In the laboratory, we must be able to develop insects that are resistant or refractory to parasites. Along this line, the production of a Dengue-resistant resistant yellow fever mosquito, Aedes aegypti (Olson et al. 1996 ) provides proof of principle. Recent development of Trypanosome cruzi resistant bugs (Darvasulla, et al) also shows that this part of the approach can be done. The second arena involves moving genes developed in the laboratory into wild target populations. It is clear that an approach utilizing Mendelian transmission of vectors will be too protracted in time to sustain support and interest. However, concepts of genes spreading in an infection-like wave have been developed, and there is enthusiasm for using transposable elements or other mobile nucleic acid vectors to spread genes through population (Kidwell and Ribeiro, 1992).

Finally, we must have sufficient information about the target vector population so that we can model and then predict how the individual gene will behave in the population. This is important for both the introduction of the gene and establishing parameters by which the success of the introduction will be measured. The genetic structure, population migration indices, gene flow, and other population genetic factors will have a major impact on the outcome of a genetic strategy and their effects must be anticipated and accounted for.

Our research group has focused on the laboratory component of this strategy. In order to make a parasite-resistant vector insect, in this case, a mosquito, we have identified three areas of specific research. The first goal was to identify a tissue in which specific interactions between the parasite and host take place and which would be the target of intervention. We chose the salivary glands and have characterized a number of genes expressed specifically in the salivary glands (James, 1994, Champagne et al., 1995, Smart et al. 1995). It is our intention to use the promoter regions of these genes to express antiparasitic coding regions. We need a system for stable introduction of genes into the mosquito. Transgenesis techniques are absolutely essential for us to test the hypothesis. Recently, we have shown that the transposable elements, Hermes and mariner, are capable of mobility in embryos and of integration into the genome of Ae. aegypti (Sarkar et al., 1997, Jasinskiene et al., 1997). These elements provide powerful tools for the molecular analysis of vector-parasite interactions using transgenic technology.

Finally, we need to identify the specific interactions between parasite and the target tissue. We have been investigating the development of the avian malaria parasite, Plasmodium gallinaceum, as it transitions from an oocyst sporozoite to a salivary gland sporozoite.. This development is characterized by the presence, and subsequent to invasion, the loss of the ability to penetrate salivary glands (Touray et al, 1992), and the acquisition of resistance to vertebrate host complement that allows it to infect the chicken (Touray, Seely and Miller, 1994). We have hypothesized that these changes in the pathogenic properties of the parasite result from gene expression, and we have ongoing efforts to identify the genes responsible. It is our goal to use the transformation system to introduce an antiparasitic hybrid gene consisting of a salivary gland promoter controlling the expression of a molecule that will interfere with the oocyst-to-salivary gland development of the sporozoite.

References

Champagne, D.E., Smartt, C.T., Ribeiro, J.M.C. and James, A.A. (1995). The salivary gland-specific apyrase of the mosquito, Aedes aegypti, is a member of the 5'-nucleotidase family. Proc. Natl. Acad. Sci. USA. V92, 694-698.

Coates, C.J., Jasinskiene, N., Miyashiro, L., and James, A.A. (1997) Mariner transposition and transformation of the yellow fever mosquito, Aedes aegypti. Submitted.

Collins F.H. (1994) Prospects for malaria control through the genetic manipulation of its vectors. Parasitology Today, V10 N10: 370-371.

Collins, F.H. and James, A.A. (1996) Genetic modification of mosquitoes. Science and Medicine V3: 52-61.

Curtis C.F. (1996) Impregnated Bednets, Malaria Control And Child Mortality in Africa. Tropical Medicine & International Health, V1 N2: 137-138.

Durvasula, RV, Gumbs, A, Panackal, A, Kruglov, O, Aksoy, S., Merrifield, B.R., Richards, F.R. and Beard, C.B.(1997). Prevention of insect-borne disease: An approach using transgenic symbioticbacteria. Proc. Natl. Acad. Sci. USA, 94, 3274-3278.

James, A.A. (1994) Molecular and biochemical analyses of the salivary glands of vector mosquitoes. Bulletin de l'Institut Pasteur V92, 113-150.

James, A.A. (1996) Dengue hemorrhagic fever. Science, V272, 829.

Jasinskiene, N., Coates, C.J., Benedict, M.Q., Cornel, A.J., Salazar-Rafferty, C., James, A.A., and Collins, F.H. (1997) Stable, transposon-mediated transformation of the yellow fever mosquito, Aedes aegypti, using the Hermes element from the housefly. Submitted.

Jones T.R.; Hoffman S.L. (1994) Malaria vaccine development. Clinical Microbiology Reviews, V7 N3: 303-310.

Kidwell M.G.; Ribeiro J.M.C. (1992) Can transposable elements be used to drive disease refractoriness genes into vector populations. Parasitology Today, V8 N10: 325-329.

Olson K.E., Higgs, S. Gaines P.J., Powers A.M. et al., (1996) Genetically engineered resistance to dengue-2 virus transmission in mosquitos. Science, V272 N5263: 884-886.

Sarkar, A., Yardley, K., Atkinson, P.W., James, A.A and O'Brochta (1997) Transposition of the Hermes element in embryos of the vector mosquito, Aedes aegypti. Insect Biochem. and Molec. Biol., 27: 359-363.

Smartt, C.T., Kim, A.P., Grossman, G.L. and James, A.A. (1995) The Apyrase gene of the vector mosquito, Aedes aegypti, is expressed specifically in the adult female salivary glands. Experimental Parasitology V 81, 239-248.

Spielman A; Kitron U; Pollack RJ. (1993) Time limitation and the role of research in the worldwide attempt to eradicate malaria. Journal of Medical Entomology, V30 N1: 6-19.

Touray MG; Seeley DC; Miller LH. (1994) Plasmodium gallinaceum - differential lysis of two developmental stages of malaria sporozoites by the alternative pathway of complement. Experimental Parasitology, V78 N3: 294-301.

Touray MG; Warburg A; Laughinghouse A; Krettli A.U. (1992) and others.developmentally regulated infectivity of malaria sporozoites for mosquito salivary glands and the vertebrate host. Journal of Experimental Medicine, V175 N6:1607-1612.

Warburg A; Touray M; Krettli AU; Miller LH. (1992) Pasmodium-gallinaceum - antibodies to circumsporozoite protein prevent sporozoites from invading the salivary glands of aedes-aegypti. Experimental Parasitology, V75 N3: 303-307.

C4 - ^{LIFE AND DEATH IN TB.RHODESIENSE}

lan Maudlin and Susan C.Welburn

Division of Molecular Genetics, Institute of Biomedical and Life Sciences, University of Glasgow, Anderson College, 56 Dumbarton Road, Glasgow Gll 6NU

Trypanosoma brucei ssp undergo complex morphological and biochemical changes during cyclical transrnission between vertebrate host and insect vector. On entering the fly midgut parasites transfortn from bloodstream to insect form (procyclic) and either die in the midguts of refractory tsetse or survive to form persistent procyclic infections in the ectoperitrophic space in susceptible flies. ln susceptible tsetse, parasites either remain as a 'core' population of procyclics or a proportion of them may migrate to the salivary glands to establish a population of mamnialian infective (metacyclic) trypanosomes. Trypanosomes therefore have two hurdles to overcome in tsetse to complete their life cycle i) establishment and ii) maturation and surprisingly few parasites overcome both hurdles.

To establish, incoming parasites transform and move to the ectoperitrophic space of the gut to fonn an actively dividing population of procyclic forms. Most tsetse are refractory to establishment through the action of a tsetse midgut lectin which induces death in procyclics. Lectin inhibition in vivo through addition of inhibitory oligosaccharides greatly enhances susceptibility to infection in tsetse. Electron micrographs of procyclics dying in the midguts of refractory flies show several morphological features characteristic of apoptosis. Apoptosis can be induced in vitro in procyclic T b. rhodesiense using Con A, the treated parasites showing the characteristics of apoptotic cells including surface membrane vesiculation and fragmentation of nuclear DNA. Trypanosomes which do establish midgut infections do so by circumventing the natural defences of the vector. ln susceptible flies this is due to the presence of endosymbiotic bacteria (RLO) in the midgut cells which produce chitinases which degrade chitin during pupatíon, resulting in a build up of oligosaccharide specifically inhibitory to the lectin.

Once established, procyclic trypanosomes will reside in the ectoperitrophic space for the life of the fly. During this period the parasite population density remains remarkably constant and is not accounted for by loss of trypanosomes through migration to the salivary glands. Such a state of equilibrium would be advantageous to both parasite and fiy which are in competition for proline as an energy source and it is likely that this is effected by cell division being balanced by parasite controlled cell death.

The second hurdle restricting trypanosome development in tsetse flies is for the established procyclic population to mature through epimasitigote to mammalian infective fonns (metacyclics) in the salivary glands. Several factors are involved in the regulation of maturation; the most important being the positive effect of the GlcNAc/glucosamine-specific midgut lectin, trypanosome genotype and fly sex. While inhibition of the activity of the midgut lectin is essential for trypanosome survival in the midgut, activity of the same molecule is required for parasite maturation, indicating that maturation may be an altemative pathway to cell death. Maturation of midgut infections in can be blocked by continuous feeding of lectin inhibitory olligosaccaride. The process of maturation is triggered during a specific time period following establishment in the midgut. Higher rates of maturation are achieved in stocks which mature earlier in tsetse flies. This implies that a 'window of opportunity' exists for T brucei s.l. in the midgut to receive stimulation to mature, which is stock/strain specific. For most T brucei s.l. studied, this lies between 8-11 days following infection (shortly after/ at the point of establishment).

Transmissibility is also markedly affected by the sex of the vector - male flies maturing significantly more midgut infections that females. The underlying mechanism of these sex differences appears to be the operation of a product(s) of an X-Iinked gene which kills or prevents migrating parasites from maturing.

To dissect the relationship between death and differentiation at the parasite vector interface we are now using RADES pcr to identify genes in Tb.rhodesiense which are upregulated during programmed cell death in vitro and in tsetse and which are also involved in parasite maturation in vivo.

C5 - ^{O-GLYCOSYLATION OF TRYPANOSOMA CRUZI PROTEINS: STRUCTURAL DIVERSITY AND CHARACTERISATION OF A NOVEL URIDINE-DIPHOSPHO-N-ACETYL-GLUCOSAMINE: POLYPEPTIDE N-ACETYLGLUCOSAMINYLTRANSFERASE}

¹Mendonça-Previato, L, ²Sola-Penna, M, ³Jones, C, ⁴Wait, R, ¹Agrellos, OA, ¹Todeschine, AR & ¹Previato, JO.

¹Instituto de Microbiologia, and ²Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil; ³National Institute for Biological and Control, Potters Bar, UK; ⁴Centre for Applied Microbiology and Research, Salisbury, UK

The two most diverse glycoprotein classes, related to protein-bound oligosaccharide linkages, are the N- and O-glycans. All N-glycans share the common GlcNAcb-Asp complex linked to a core structure containing Mana1-6(Mana1-3)Manb1-4GlcNAcb1-4®. There is, however, an enormous variety in the oligosaccharides attached to this core. O-glycans containing linkage between HexNAc and hydroxylated aminoacids, are found as glycoproteins containing one to several hundred carbohydrate chains attached to the peptide by a-O-glycosidic linkages between GalNAc and Ser and/or Thr (mucin-type). These structures have been assigned to a diverse array of functions, such as in cell-cell recognition, host-pathogen interactions, and protection from proteolytic degradation [1]. O-glycans also include a protein modification in which single residues of GlcNAc are O-linked to Ser and/or Thr. This type of linkage was found on a wide variety of cytoplasmic and nucleoplasmic proteins. Evidence suggests that this form of protein modification may play a regulatory role analogous to protein phosphorylation [2]. More recently, GlcNAc preferentially O-linked to Thr residues was found in glycoproteins present on T. cruzi cell surface. This core can be elongated to form oligosaccharide regions by addition of Galp in b1-2, b1-3, b1-4 and b1-6 linkages and Galf in b1-4 linkage. Occasionally, the termini are formed by further addition of sialic acid. Some reports indicate that the sialic acid containing molecules from T. cruzi perform an important role in the recognition or invasion of host cells [3].

Evidences for protein O-glycosylation in T. cruzi were first reported following b-elimination of a pure GP-25 kDa glycoprotein preparation [4]. This glycoprotein, which corresponds to T. cruzi cruzipain C-terminal domain [5], contains, at least to some extend, di- and tetrasaccharide O-glycosidically linked to Ser and/or Thr. Later on, the presence of a sialylated oligosaccharide of about 10 glucose units in size, attached via O-linkages to the 35/50-kDa antigens of T. cruzi metacyclic forms was described [6]. O-linked oligosaccharide containing glycoproteins were also identified in cell-cultured trypomastigote forms [7].

The complete structure of T. cruzi O-linked oligosaccharides was characterized in glycoproteins from epimastigote forms of strains G [8], Dm-28c [9], Y [10], CL [11], and Tulahuen [12]. Mild alkaline reductive degradation of glycoproteins resulted in b-elimination of glycosylated Thr, and the liberation of N-acetylglucosaminitol (GlcNAc-ol), galactobiosyl, galactotriosyl, galactotetraosyl and galactopentaosyl-N-acetylglucosaminitol. The oligosaccharide primary structure, resolved by methylation analysis, Fab-mass spectrometry and high field nmr spectroscopy established the presence of three different groups of molecules:

I - Galfb1-4(Galp1-6)GlcNAc
Galpb1-3Galpb1-6(Galfb1-4)GlcNAc
[(Galpb1-3)(Galpb1-2)Galpb1-6](Gal fb1-4)GlcNAc
[(Galpb1-3)(Galpb1-2)Galpb1-6](Gal pb1-2Galfb1-4)GlcNAc II - Galpb1-4GlcNAc
Galpb1-6(Galpb1-4)GlcNAc
Galpb1-2Galpb1-6(Galpb1-4)GlcNAc III - Galpb1-4GlcNAc
Galfb1-4GlcNAc
Glcpb1-4GlcNAc
Galpb1-2Galfb1-4GlcNAc
Galfb1-2Galfb1-4GlcNAc
Galpb1-3Galpb1-6(Galfb1-4)GlcNAc
[(Galpb1-3)(Galpb1-2)Gal pb1-6](Galfb1-4)GlcNAc
[(Galpb1-3)(Galpb1-2)Galpb1-6](Gal fb1-2Galfb1-4)GlcNAc

The T. cruzi strains G and Dm-28c; Y and CL; and Tulahuen synthesize O-linked oligosaccharides containing structures I, II and III respectively. However the most abundante glycan in all strains analysed is unsubstituted GlcNAc (approximately 30 %). Strains Y, CL and Tulahuen express O-glycan linked to the peptide through GalNAc [12]. Glycoproteins containing O-linked oligosaccharides are the major acceptor for sialic acid transferred by trans-sialidase of T. cruzi [13]. Compounds NeuNAca2-3Galpb1-4GlcNAc and NeuNAca2-3Galpb1-6Galpb1-4GlcNAc were purified from strains CL, Dm-28c and Tulahuen, showing that sialic acid residues are preferentially transferred to small O-linked oligosaccharides. Regulated sialylation in vitro was observed using O-glycans from Y-strain. The incorporation of one sialyl unit into an O-linked oligosaccaride containing two potential sites of sialylation strongly hinders entry of a second sialic acid residue [10].

These observations provide the first description of O-GlcNAc protein modification for a surface glycoprotein, and prompted us to assay the enzyme responsible for the addition of O-GlcNAc to T. cruzi surface proteins. A synthetic peptide was used as substrate for the enzyme, based on the partial peptide sequence of a deglycosylated O-GlcNAc containing 40/45 kDa glycoprotein from Y-strain, and from MUC gene family products from T. cruzi [14]. UDP-GlcNAc was used as the sugar donor. Characterization of the in vitro glycosylated peptide product, after mild base catalyzed b-elimination, gel-filtration chromatography and HPLC, demonstrated the presence of glycosylation site containing GlcNAc covalently attached to the peptide. This unique transferase is pivotal to the biosynthesis of the sialic acid acceptor molecules in T. cruzi. Studies to analyse the regulation of this novel UDP-glucosaminyltransferase are justify, due to the possibility of designing chemical inhibitors of its enzymatic activity.

[1] Roussel,P. & Lamblin,G. 1996. In Glycoproteins and Diseases. Montreuil, Vliegenhart and Schachter Eds. Elsevier Science B.V., pp. 351-393

[2] Hart,G.W., Kreppel,L.K., Comer,F.I., Arnold,C.S., Snow,D.M., Ye,Z., Xiaogang,C., DellaManna,D., Caine,D.S., Earles,B.J., Akimoto,Y-, Cole,R.N. & Hayes,B.K. 1996. Glycobiology 6, 711-716

[3] Burleigh,B. & Andrews,N.W. 1995. Annu.Rev.Microbiol. 49, 175-200

[4] Mendonça-Previato,L., Gorin,P.A.J., Braga,A.F., Scharfstein,J. & Previato,J.O. 1983. Biochemistry 22, 4980-4987

[5] Cazzulo,J.J., Stoka,V. & Turk,V. 1997. Biol. Chem. 378, 1-10

[6] Schenkman,S., Ferguson,M.A.J., Heise,N., Cardoso de Almeida,M.L., Mortara,R.A. & Yoshida,N. 1993. Mol. Biochem. Parasitol. 59, 293-304

[7] Almeida,I.C., Ferguson,M.A.J., Schenkman,S. & Travassos,L.R. 1994. Biochem. J. 304, 793-802

[8] Previato,J.O., Jones,C., Gonçalves,L.P.B., Wait,R., Travassos,L.R. & Mendonça-Previato,L. 1994. Biochem. J. 301, 151-159

[9] Agrellos,O.A., Santos,P.T., Bilate,A.M.B., Jones,C., Previato,J.O. & Mendonça-previato,L. 1997. Resumos da XXVI Reunião Anual da SBBq, pp. 100

[10] Previato,J.O., Jones,C., Xavier,M.T., Wait,R., Travassos,L.R., Parodi, A.J. & Mendonça-Previato,L. 1995. J. Biol. Chem. 270, 7241-7250

[11] Todeschini,A.R., Jones,C., Wait,R., Agrellos,O.A., Previato,J.O. & Mendonça-Previato,L. 1996. Mem. Inst. Oswaldo Cruz 91, 282

[12] Jones,C., Previato,J.O., Todeschini,A.R., Wait,R. & Mendonça-Previato,L. 1997. Abstract for EuroCarb 9 (Utrech), B36

[13] Previato,J.O., Andrade,A.F.B., Pessolani,M.C.V. & Mendonça-Previato,L. 1985. Mol. Biochem. Parasitol. 16, 85-96

[14] DiNoia,J.M., Pollevick,G.D., Xavier,M.T., Previato,J.O., Mendonça-Previato,L., Sánchez,D.O. & Frasch,C.C. 1996. J. Biol. Chem. 271, 32078-32083

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

C6 - ^{TOXOPLASMA GONDII — A GENETIC MODEL FOR THE INVESTIGATION OF DRUG RESISTANCE MECHANISMS, PARASITE DIFFERENTIATION, AND THE EVOLUTION OF EUKARYOTIC ORGANELLES}

David S. Roos, Dep't Biology, Univ. Pennsylvania, Philadelphia PA 19104-6018 USA

Toxoplasma gondii is a protozoan parasite that replicates within specialized vacuoles inside the cells of infected animals. Although normally harmless to healthy individuals, this ubiquitous pathogen is a major source of congenital birth defects, and has recently achieved considerable notoriety as a leading cause of death in AIDS patients. T. gondii also provides an appealing model system for parasites less amenable to study in the lab, such as Plasmodium (the causative agent of malaria) — an organism closely related in structure, lifestyle, and evolutionary history. Beyond its direct medical importance, as an ancient eukaryote, Toxoplasma also offers unique insights into the origins of molecular and cell biological processes.

T. gondii parasites are convenient and safe to grow in culture, and their unusually wide host range allows experiments conceptually analogous to somatic cell genetic studies, taking advantage of mammalian cell mutants to ask what the parasites can do for themselves and what they require from the host. Toxoplasma also undergoes sexual replication (in cats; analogous to the mosquito vector for malaria), making classical genetic crosses feasible. Micromanipulation permits the isolation of individual meiotic progeny (analogous to tetrad analysis in yeast). Most importantly, we and others have developed transformation vectors and protocols suitable for transient and stable transformation of T. gondii parasites, using both integrating and episomal vectors. The molecular genetics of T. gondii is distinguished by two remarkable features:

»Frequencies of stable molecular transformation are sufficiently high to permit complementation cloning (>10^-2).

»Genomic integration can be accomplished by either homologous or non-homologous recombination (depending on the length of contiguous genomic DNA introduced).

Homologous recombination allows targeted gene knock-outs and allelic replacements, while high-frequency nonhomologous recombination permits insertional mutagenesis, analogous to transposon tagging in bacteria, yeast, Drosophila, etc. The entire T. gondii genome can be tagged in a single experiment, and tagged loci are readily cloned by plasmid-rescue techniques.

Toxoplasma is unusually accessible in cell biological terms as well, with superb ultrastructural resolution that facilitates a wide variety of studies on the structure, function, and evolution of eukaryotic cells and organelles. The small size and highly polarized organization of T. gondii tachyzoites provides an example of a minimal eukaryote, which nevertheless remains instantly recognizable to the mainstream cell biologist. Taking advantage of the unique accessibility of the T. gondii system, investigations in this laboratory include:

•Studies on metabolic pathways, and the identification of drug targets and antibiotic resistance mechanisms.

•Genetic dissection of the temporal and developmental controls which regulate differentiation through the complex parasite life cycle.

•Examination of protein secretory pathways in this 'minimal eukaryote'.

•Characterization of endosymbiotic organelles, and studies on extrachromosomal genome evolution (including the 'apicoplast' — a novel organelle specific to the apicomplexa, which was acquired by secondary endosymbiosis of a green algal plastid).

C7 - ^{INFLUENCE OF PLASMODIUM FALCIPARUM INFECTION MEDIATED IMMUNE STIMULATION ON HIV-1 REPLICATION}

Altaf A. Lal

Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta GA, USA

HIV/AIDS is a major global public health problem with 93% of infections occuring in developing countries. Malaria is another major public health problem; nearly 500 million malaria cases occur annually, with 2-3 million infants dying every year of malaria. Despite earlier cross-sectional studies that failed to demonstrate an association HIV/AIDS and malaria, recent epidemiological studies suggest a strong interaction between these two diseases. CDC sponsored epidemiological studies have revealed that infant mortality is higher in babies born to mothers co-infected with placental malaria and HIV, and HIV infection impairs a pregnant woman's ability to limit P. falciparum infection. Because malaria infection-stimulated cytokine production may have deleterious effects on HIV-1 replication, we studied effects of P. falciparum antigen on HIV-1 replication. Stimulation with malarial antigens significantly enhanced HIV-1 replication of both LAV as well as primary HIV-1 isolates (subtype A) in CD8-depleted PBMC from naive donors. The malaria antigen induced activation of HIV-1 was due to cellular activation as judges by the expression of cell activation markers (CD69 and HLA-DR expression) and proliferative responses (mean SI= 5 to 20). While malarial antigen stimulation increased expression of TNF-alpha and IL-6, only mABs to TNF-alpha inhibited HIV-1 replication . Malarial antigen preparations increased HIV-1 replication both by increasing viral mRNA expression as well as by activation LTR-directed viral transcription. Taken together, these data suggest that malaria infection can modulate HIV-1 pathogenesis both by promoting the infection through the activation of lymphocytes, and by increasing viral replication through the production of cytokines. These studies establish na urgent need for further studies so that a complete picture of the interaction between malaria and HIV/AIDS can be obtained. The outcome of these studies could be utilized in drawing definite public health-related conclusions.

C8 - ^{THE CLONAL-HISTOTROPIC MODEL FOR THE PATHOGENESIS OF CHAGAS DISEASE}

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

Departamento de (1) Bioquímica e Imunologia, (2) Parasitologia , (3) Morfologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Caixa Postal 486, 31270-910 - Belo Horizonte , MG, Brasil

The lack of microscopic visualization of parasites in affected tissue lesions in the chronic phase of Chagas disease and also the findings of intense T-cell-rich inflammatory mononuclear cell infiltrates and circulating anti-T. cruzi antibodies that crossreacted with target organ epitopes, have led to doubts about a major role of the parasite in the pathogenesis of the disease and to the genesis of the autoimmune hypothesis^1,2. More recently, however, a direct pathogenic role of the parasite has gained strong support from very sensitive new immunohistochemical techniques³ and applications of the polymerase chain reaction (PCR)^4-6, both of which have shown a strict correlation between the presence of the parasite and the tissue lesions. This has rescued the notion, already implicit in the work of Carlos Chagas, that genetic variation of T. cruzi might influence the course of the disease.

Several different biological, biochemical and molecular based techniques (including zymodemes, schizodemes, RAPD, DNA fingerprinting, karyotyping, etc.) have been used to study the genetic variability of T. cruzi. With none of them has any correlation been found with the clinical aspects of the disease. We believe that the reason for this is that essentially all the approaches used to profile variability have the drawback of being applicable only to the study of cultured parasites. In this respect, an important confounding factor is that patients in endemic areas probably are infected by multiple contacts with different triatomids and these, in turn, may feed on different infected individuals. This promiscuity certainly propitiates the formation of multiclonal populations in hosts and vectors and in fact we have strong evidences that many T. cruzi population are multiclonal. Since culture involves adaptation to new life conditions and remarkable population expansion, there is ample opportunity for clonal selection. Consequently the parasite population obtained from a patient could be very different from that circulating in the blood. On the other hand, the circulating parasite population is not necessary identical to the set of clones that actually infected the heart or any other specific patient tissue. Among the "constellation" of clones infecting an individual we may have clones with specific tropism to different tissues, whose distribution may determine the clinical course of that patient. This scenario constitutes what we call of clonal-histotropic model for the pathogenesis of Chagas disease and explain why none correlation between the parasite genetic aspects and the patient clinical manifestations could be found until now.

In the last couple of years we have developed new methodology for the study of genetic variability of T. cruzi. The first was the isolation and sequencing of eight hypervariable CA-repeat microsatellites from the T. cruzi genome. Study of natural parasite populations with these markers have permitted detailed characterization of the genetic structure of T. cruzi and yielded results along the lines predicted from our model. More important was the utilization of a technique called LSSP-PCR for the genetic typing of T. cruzi directly in the tissue lesion. LSSP-PCR investigations in both chronic Chagasic patients and experimentally infected mice have provided strong support in favor of the clonal-histotropic model.

(Supported by PRONEX-FINEP and CNPq)

References

1. Levin, M. J. et al. (1989) Identification of major Trypanosoma cruzi antigenic determinants in chronic Chagas heart disease. Am. J. Trop. Med. Hyg. 41, 530-539.

2. Levin, M. J. et al. (1990) Autoantibodies in Chagas heart disease: possible markers of severe Chagas heart complaint. 85 (4), 539-543.

3. Higushi, M.L. et al. (1993) Correlation between Trypanosoma cruzi parasitism and miocardial inflamatory infiltrate in human chronic chagasic myocarditis: light microscopy and immunohistochemical findings. Cardiovasc. Pathol. 2, 101-105

4. Jones, E. M. et al. (1993) Amplification of Trypanosoma cruzi DNA sequence from inflammatory lesions in human chagasic cardiomyopathy. Am. J. Trop. Med. Hyg. 48, 348-357.

5. Brandariz, S. et al. (1996) Detection of parasite DNA in Chagas heart disease. Lancet 347, 914.

6. Vago, A. R. et al.(1996a) PCR detection of Trypanosoma cruzi DNA in esophageal tissues of patients with chronic digestive. Lancet 348, 891-892

C9 – ^{GENETIC ANALYSIS OF THE PROTOZOAN PARASITE TOXOPLASMA GONDII HAS UNDERGONE A RAPID EXPLOSION IN RECENT YEARS.}

John Boothroyd, Stanford University, San Francisco, CA, USA

This is due to effort in a number of labs that have worked on the development of molecular genetic techniques. It is also due, however, to the natural biology of this system (including a well-described sexual cycle) that makes possible genetic mapping of the F1 progeny from a cross. In this talk, I will present recent work from our group on how to study invasion and development using molecular genetic means. This will focus on using mutants to study developmentally regulated gene expression and invasion. Candidate genes that are involved in or affected by these processes have been identified and will be discussed.

C10 - ^{THE MINI-EXON GENE IS A DISTINCTIVE NUCLEAR MARKER FOR GROUPING PROTISTIS OF THE KINETOPLASTID/EUGLENOID LINEAGE}

David A. Campbell, Octavio Fernandes and Nancy R. Sturm

Department of Microbiology and Immunology, UCLA School of Medicine, University of California, Los Angeles, CA 90095-1747, U.S.A.

The kinetoplastid protozoa use the trans-splicing pathway to add a common 39-nucleotide exon to the 5'-end of all nuclear messenger RNAs. Trans-splicing is mechanistically similar to cis-splicing (the removal of introns from pre-mRNA), however it refers to the joining of two independent RNA molecules. The conserved 39-nt 5'-exon, the mini-exon or spliced leader, is synthesized as a precursor molecule, the mini-exon-donor RNA (medRNA) or spliced leader RNA, whose size varies between 95-nt and 140-nt depending on the genus/species. Two major goals of our research are to understand how the mini-exon gene is transcribed and how the medRNA functions in trans-splicing. These objectives are being achieved in Leishmania tarentolae using stable transfection of pX-based plasmids that contain mutated copies of the mini-exon gene. The functional features of the upstream promoter of the mini-exon gene have been mapped [1]. Structural features of the medRNA that determine transcription termination, 3'-end processing, 5'-end 'cap 4' formation and trans-splicing have also been mapped.

During the early stages of these structure/function studies, DNA sequence alignments of mini-exon gene repeats were performed to identify conserved domains that may be phylogenetically (and hence functionally) conserved. To obtain such information as rapidly as possible, mini-exon gene repeats were amplified from a variety of kinetoplastids using a universal Polymerase Chain Reaction assay that was successful due to the high sequence conservation of the mini-exon and the multicopy tandemly-repeated nature of the genes. From these studies and DNA sequences reported in the literature, it was apparent that both sequence conservation and sequence variation in the mini-exon gene reflected taxonomic patterns. The original mini-exon PCR assay has been developed subsequently to A) evaluate and challenge existing taxonomic definitions, and B) determine the potential of the mini-exon gene as a diagnostic marker.

A study of isolates representing 14 different species of Leishmania isolated from humans indicated that the exon and intron (transcribed) sequences were almost identical to each other [2]. The mini-exon gene transcribed regions were also very similar in Leishmania isolated from lizard (L. tarentolae) and gerbil (L. enriettii). Thus in Leishmania, the intron sequence conservation correlates with the genus boundary. Moreover, comparison of the non-transcribed spacer regions indicated that the most common species isolated from humans could be placed into one of four groups that correlate with geographic and pathologic criteria: Old World dermotropic, New World dermotropic, New World Viannia subspecies and Viscerotropic [2]. The non-transcribed spacer sequences have been used as hybridisation targets for oligonucleotides probes, which may also distinguish among New World dermotropic species: L. aethiopica, L. major and L. tropica [3]. Non-transcribed spacer sequences can also be used to identify Endotrypanum schaudinni [4], which may be present with Leishmania in sandfly intestines.

In the genus Trypanosoma, the intron sequences are conserved only among subspecies (e.g. the T. brucei complex). Thus in Trypanosoma, both intron and non-transcribed spacer sequences correlate with species boundaries. Variation in the mini-exon gene repeats allowed discrimination between closely related organisms such as A) T. cruzi and T. rangeli [5], and B) T. (Nannomonas) simiae and other species of the subgenus Nannomonas, (T. (N.) congolense subgroups and T. (N.) godfreyi). This heterogeneity of mini-exon gene sequences is consistent with evolutionary studies that indicate the genus Trypanosoma is more diverse than the genus Leishmania.

Surprisingly, we detected _30% variation in the mini-exon genes from multiple isolates of T. cruzi, which suggested that the species consisted of two groups [6]. This dimorphism was mirrored by the clustering of the same isolates using a ribosomal RNA gene marker and the appearance of two corresponding branches created by RAPD analysis [6]. The combined data have a bearing on the issue of subspeciation within the taxon.

True plant trypanosomes of the genus Phytomonas show great conservation of intron sequences, however isolates from phloem, latex and fruit show substantial variation in non-transcribed spacer sequences [7]. Because the phloem-restricted Phytomonas may cause diseases such as wilts of economically important palms and other plants we are using of mini-exon genes from cells associated with disease to assess the relatedness of pathogenic isolates.

Like Trypanosoma, the genus Crithidia showed substantial heterogeneity. We have defined six groups within Crithidia based on mini-exon gene repeat sequence and hybridisation studies [8]. The same approach is being applied to isolates of Blastocrithidia and Herpetomonas.

The mini-exon gene has also been used to explore Bodonids and more distantly related protozoa in the kinetoplastid/euglenoid lineage. Both Bodo caudatus and Trypanoplasma borreli possess trypanosomatid-like mini-exon genes [9,10]. The repeat in each organism contains the 5S ribosomal RNA gene, an association also found in T. rangeli, T. vivax and Herpetomonas.

The marine biflagellates Diplonema and Isonema are of uncertain position in the kinetoplastid/euglenoid lineage. By PCR, we have shown that both possess a trypanosomatid-like mini-exon gene repeat. Thus by this criterion they appear more closely related to the kinetoplastids than the euglenoids, which also process mRNA by trans-splicing but have a distinctively different 22-nt common 5'-exon.

Summary. We have studied the mini-exon repeats from over 37 different species of kinetoplastid, or related, protozoa. We have found that the mini-exon gene is a very useful marker for distinguishing among existing taxonomic groupings. The sequence of the mini-exon is characteristic of the kinetoplastid lineage and distinct from the homologous exon in the euglenoid lineage. The sequence of the intron is discriminative at the boundaries of either genus (e.g. Leishmania) or species (e.g. Trypanosoma and Crithidia). Typically, the non-transcribed spacer sequences between the tandemly-repeated mini-exon genes correlate with existing species, or species group, designations.

Acknowledgements. Work in this laboratory has been supported by NIH grant AI34536, Rockefeller Foundation grant RF87049, and a Burroughs Wellcome New Investigator Award. We thank our many colleagues who have provided willingly cells and DNA samples from their tryypanosomatid collections for this study.

References

1.Saito, R.M., Elgort, M.G. and Campbell, D.A. (1994) A conserved upstream element is essential for transcription of the Leishmania tarentolae mini-exon gene. The EMBO Journal 13: 5460-5469.

2.Fernandes, O., Murthy, V.K., Kurath, U., Degrave, W.M. and Campbell, D.A. (1994) Mini-exon gene variation in human pathogenic Leishmania species. Molecular and Biochemical Parasitology 66: 261-271.

3. Ramos, A., Maslov, D.A., Fernandes, O., Campbell, D.A. and Simpson, L. (1996) Detection and identification of human pathogenic Leishmania and Trypanosoma species by hybridization of PCR-amplified mini-exon repeats. Experimental Parasitology 82: 242-250.

4. Fernandes, O., Degrave, W.M. and Campbell, D.A. (1993) The mini-exon gene: A molecular marker for Endotrypanum schaudinni. Parasitology 107: 219-224.

5. Murthy, V.K., Dibbern, K.M. and Campbell, D.A. (1992) PCR amplification of mini-exon genes differentiates Trypanosoma cruzi from Trypanosoma rangeli. Molecular and Cellular Probes 6: 237-243.

6. Souto, R.P., Fernandes, O., Macedo, A.M., Campbell, D.A. and Zingales, B. (1996) DNA markers define two major phylogenetic lineages of Trypanosoma cruzi. Molecular and Biochemical Parasitology 83: 141-152.

7. Sturm, N.R., Fernandes, O. and Campbell, D.A. (1995) The mini-exon genes of three Phytomonas isolates that differ in plant tissue tropism. FEMS Microbiology Letters 130: 177-182.

8. Fernandes, O., Teixeira, M.M.G., Sturm, N.R., Sousa, M.A., Camargo, E.P., Degrave, W.M. and Campbell, D.A. (1997) Mini-exon gene sequences define six groups within the genus Crithidia. Journal of Eukaryotic Microbiology : In Press

9. Campbell, D.A. (1992) Bodo caudatus medRNA and 5S genes: Tandem arrangement and phylogenetic analysis. Biochemical and Biophysical Research Communications 182: 1053-1058.

10. Maslov, D.A., Elgort, M.G., Wong, S., Peckova, H., Lom, J., Simpson, L. and Campbell, D.A. (1993) Organization of the mini-exon and 5S rRNA genes in the kinetoplastid Trypanoplasma borreli. Molecular and Biochemical Parasitology 61: 127-136.

C11 - ^{PRESENT SITUATION AND FUTURE OF HUMAN CHAGAS DISEASE IN BRAZIL}

João Carlos Pinto Dias - National Health Foundation, Federal University of Minas Gerais and Oswaldo Cruz

Foundation. E mail.- [e-mail]

Human Chagas disease (HCD) has been one of the most important problems of Brazilian Public Health in this century, being responsible at least for 16,000 deaths/year in the last two decades (Akhavan, 1997). According to Schmunis and WHO (l997), HCD is the most impacting parasitic disease in terms of social & economic disability in Latin America, only being surpassed by diarrheal and respiratory diseases and AIDS. ln Brazil, at least 3,5 million of individuais are presently infected by Trypanosoma cruzi, with 16 - 18 million in Latin America (Dias, 1997, Schmunis, 1997). ln Brazil, it can be estimated a minimum rate of 20% of chagasic individuais presenting a chronic myocardiopathy, the most important clinical form of HCD, from the epidemiological standpoint. Other important clinical pictures of HCD correspond to digestive dysperistalsis, chiefly of esophagus and terminal colon, with a general prevalence of I O to 20% among infected individuais (Brazil, 1996, Dias & Coura, 1997). The transmission of T cruzi to men was very intensive in Brazil before 1980, when was estimate an incidence of IOO,OOO new cases/year. The classic geographical distribution of HCD in Brazil corresponds to the natural distribution of the domestic vector species involved on the transmission of the parasite (Triatomines: Hemiptera, Reduviidae). HCD was classically an endemic rural disease depending on the contact susceptible human beings/ infected triatomines, which progressively, became a new urban disease in Brazil: today it is estimate that at least 60% of infected people are living in urban spaces, in consequence of a very intensive rural=>urban migration in the last 40 years (Dias & Coura, 1997). The second more important mechanism of transmission is by blood transfusion, corresponding, in 1979, to 15 - 20% of HCD in the country (Dias, 1979). The great changes occurred in the epidemiological trame of HCD in Brazil during the past twenty years depended both of the social evolution ("urbanization", industrialization) and technical intervention (governmental program). ln the beginning of the years 1980, after an important pressure from the scientifical community, a political decision was assumed by the Brazilian Govern and the Chagas disease program was priorised. After two national surveys (entomological and serologic), the complete coverage of the endemic area was carried out during many years, mainly throughout a rational and vertical insecticide spraying program which reduced drastically the infestation of domestic triatomines. The principal target of the campaign was Triatoma infestans, the most domestic vector of HCD in the country and certainly the main responsible for the prevalence and morbidity of the disease in Brazil. ln the former 711 municipality infested by T infestans in 1978, the program reached the eradication of the species in more than 86%, with a so drastic reduction of the insect densities in the few remaining foci of two or three states, in the 199Os (Brazil, 1996, WHO, 1997). By the way, the same program reached a very low dwelling infestation by the other more important species (PanstronáWlus megistus, Triatoma brasiliensis, T sordida and T pseudomaculata), which present rates of intra-domestic infestation are bellow 2% in more than 97% of the former infested municipalities (Brazil, 1996, Dias & Coura, 1997). As the most convincing result of this continuous governmental program, it was observed that the prevalence of HCD in the age group 7-14 years in 10 Brazilian states decreased from 4.2% in 1980 to 0. 15% in 1994, that means a reduction of 96.5% (Brazil, 1996, Silveira & Rezende, 1994). On the other hand, as a practical product of the AIDS campaign started in the past decade, the blood transfusion transmitted Chagas disease also presented a significant reduction in Brazil. From a mean average of 6.5% at the beginning of the 198O's for the whole country, the prevalence of HCD among blood donors decreased to I% in 1991 and O.7% in 1994 (Brazil, 1996, WHO, 1997). This reduction was obtained not only through the control performed in blood banks (where the coverage of screening changed from 68% in 1988 to more than 90% in 1995), but also as a natural result of the vector controí carried out in the Country. By the same reason, the proportion of about 5% of fertile women infected by Tcruzi twenty years ago decreased to I% or less at present time, so reducing substantially the risk of congenital Chagas disease, the third more important mechanism of transmission of HCD (Dias & Coura, 1997). At the practical standpoint, it can be assumed that the transmission of HCD has been virtually eliminated in Brazil (WHO, 1997). On the other hand, morbidity and mortality due to HCD in Brazil are also being reduced in the last years, according to official data. Hospital internment by Chagas disease is decreasing in all the country and the doctors assume that the pictures of chagasic heart failure and severe digestive "megas" are becoming more and more rare in the daily practice On the mortality side, not only the total proportion of deaths due to HCD is decreasing after 1980, but also the mean age of death is being displaced to elder individuais: from the classical mortality of HCD in the years 1950 (deaths around 30-40 years), the present available data show the majority of deaths of chagasic people occurring after the 60 years of tife ( Akhavan, 1997, Dias & Coura, 1997, Silveira & Rezende, 1994). The main reasons to explain this reduction of morbidity and mortality of HCD in Brazil are not yet well established, but at least a coincidence exists between these facts and the improvement of the national Chagas control program. Perhaps a relationship could exist with the decreasing of new antigen stimuli in the infected individuais by the reduction of domestic infected triatomines (role of reinfection), but also it must be considerei the improvement of medical care in endemic areas, with a precocious attention to infected people (Dias & Coura, 1997).

As a general summary, the present situation of HCD in Brazii can be considerei comfortable and optimistic, in terms of control perspectives. At the political and operational side, the maintenance of the program involves a general cost of about 15 million US$/year and the progressive decentralization of the actions in view of an effective and continuous epidemiological surveillance (Dias, 1987, Schmunis, 1997). The economic trends of the program are also optimistic, showing a very good cost-benefit relation at long and medium terms and the progressive reduction of the costs according as the the surveillance phase is being improved (Akhavan, 1997, Schofield & Dias, 1991). At the blood transfusion side, the perspectives are also very good, since an official policy is being progressively improved in all the Country, and the coverage of blood selection by previous serology represents today at least 80% of the transfusions. The basic challenges, now, are the sustainability of the current program and the improvement of the medical care to those yet infected individuais, whose social and economic situation is still very precarious. A very practical problem, at the program side, is to assure the minimum quality and continuity in the vector control activities in the new decentralized and horizontal model which is being installed in Brazil ("municipalization"): the old and effeetive vertical program delivered from the classical malaria campaign is slowiy being replaced by local managed activities based on the primary health care activities, several times without the necessary know-how. The solution to this problem has been the maintenance of continuous supervision of the remain federal teams (FNS=National Health Foundation) in each State, as well as the maintenance of the whole work by the FNS agents in those poorer and isolated municipalities without the minimum conditions to carry on the program. The question of human resources is really the main present constraint of the program, since it is clearly foreseeable that in few years more it will be completely impossible to maintain a vertical program in Brazil: from a task force of 7,000 field agents in the beginning of the years 1980 (revising about 5 million and spraying between 500,000 and 750,000 houses/year), FNS has today no more than 2,500 men involved in the Chagas program; since 1989 no more contracts were permitted and the proportion of retirements is about 5%/year, at the present time. This transient situation is stimulating the search of partnerships between federal and municipal governments in order to transfer technology and improve the minimum required epidemiological surveillance (Dias, 1991). The available data are clearly showing that the consolidation of vector control, will constitute the main element in the control of HCD in Brazil. Eliminated the transmission of HCD, the remaining infected individuais will still require continuous medical attention: parasitological cure is highly desirable in the chronic phase of the infection, since is being demonstrable the great importance of the alive parasite in the progression of chronic lesions. At the entomological side, the new challenges and research needs can be summarized in 6 points (Dias, 1991): a) How to maintain continuous surveillance in areas with progressive decrease of endemicity? b) How to achieve good control of triatomines in peridomestic foci? c) How to improve local and regional data gathering in order to answer more quickly the program requirements? d) How to maintain program quality and continuity at the peripheral levels ? e) How to integrate vector control with other Chagas disease control activities, and f) How to prevent the spread of Chagas disease to new areas ?

Finally, it must be remembered that research must be a continuous feature of the surveillance phase, because the program involves such a close relation between local communities, technical support groups and the rapidly changing environment. The maintenance of continuity (persistence) and the quality of the program constitute the two main keys of success in HCD control. The recent episode shown by the television in the Northeast of the country, where the population was perplexed with thousands of triatomines being captured in human dwellings, is a good example of the consequences of a neglected program. As it was established by Carlos Chagas and Emmanuel Dias long ago, more than technical innovations, the final overcoming of HCD involves mainly political will and social responsibility.

References

1. Akhavan, D, 1997. Relatório sobre o custo e o beneficio do programa de controle da doença de Chagas no Brasil. Brasília. FNS, Ministério da Saúde, 26 p.

2. Brasil, 1996. Informe Técnico do Programa de Controle da doença de Chagas. FNS/MS, Brasília, 6 p.

3. Dias JCP, 1979. Mecanismos de transmissão. ln Trypanosoma cruzi e doença de Chagas. Brener, Z & Andrade, Z (organs), Rio de Janeiro, Guanabara Koogan Ed., p. 152-174.

4. Dias JCP, 1991. Chagas disease control in Brazil: which strategy after the attack phase? Annales de Ia Societé Bélgique de Médicine Tropicale, 71 (Suppl. 1): 75-86.

5. Dias JCP, 1987. Control of Chagas disease in Brazil. Parasitology Today, 3: 336-341.

6. Dias JCP & Coura JR. 1997. Epidemiologia. In Clínica e Terapêutica da Doença de Chagas: uma abordagem prática para o clínico Geral. Dias, JCP & Coura, JR (organs). Rio de Janeiro. FIOCRUZ Ed., p. 33-66.

7. Schofield, C.J. & Dias, J.C.P., 1991. A cost-benefit analysis of Chagas disease control. Memórias do Instituto Oswaldo Cruz, 86: 285-295.

8. Silveira AC & Rezende DF, 1994. Epidemiologia e controle da transmissão vetorial da doença de Chagas no Brasil. Revista da Sociedade Brasileira de Medicina Tropical, 27 (Suplemento III): 11-22.

9. Schmunis GA. 1997. Trypanossomíase americana: seu impacto nas Américas e perspectivas de eliminação. In Clínica e Terapêutica da Doença de Chagas: uma abordagem prática para o clínico Geral. Dias, JCP & Coura, JR (organs). Rio de Janeiro. FIOCRUZ Ed., p. 11-24.

10. WHO, 1997. Chagas disease. lnterruption of transmission. Weekly Epidem iological Record, 72: I - 5.

C12 - ^{DEVELOPMENT OF A HETEROLOGOUS TRANSPOSON SYSTEM FOR LEISHMANIA}

Frederico J. Gueiros-Filho and Stephen M. Beverley

Dept. of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston MA 02115, U.S.A. and Dept. of Molecular Microbiology, Washington University Medical School, St. Louis MO 63105 USA

Transposable elements are powerful tools for genetic analysis and have been successfully used as insertional mutagens, to characterize sets of coordinately expressed genes and their regulatory elements, to engineer the genome and as vectors for genetic transformation (1). These applications motivated us to pursue the development of a transposon system for Leishmania. Because there are no endogenous Leishmania transposons available, we chose to work with the mariner element of Drosophila mauritiana (2). Mariner belongs to a superfamily of DNA-based transposons that includes the C. elegans Tc1 element and has several attractive features. First, it is small (1.3 kb) and simple, encoding one protein (the transposase) flanked by 28 bp inverted repeats. Second, an active, well characterized element exists (Mos 1; [3]). Third, mariner's target sequence is the TA dinucleotide, abundant in any genome. Fourth, evolutionary and biochemical evidence suggested that mariner/Tc1 elements had little requirement for host specific factors to function (4). We thus anticipated that it could transpose when introduced in heterologous organisms.

We tested whether mariner could transpose in Leishmania by using a two-plasmid strategy (5): one plasmid (pX63TKNEO-TPASE) was designed to allow the expression of the mariner transposase in Leishmania, whereas a second donor plasmid (pX63PAC-Mos1) carried an intact copy of the transposon. Transfection of L. major with both pX63TKNEO-TPASE and pX63PAC-Mos1 caused the mobilization of the transposon from the donor plasmid into the Leishmania chromosomes in 20% of random transfectants, as assessed by Southern blots. Transposition was dependent on the expression of transposase and seemed to occur by the same mechanism as in Drosophila, with insertion into and duplication of a TA dinucleotide target site.

The high frequency of transposition in our initial experiment suggested that one could find insertions in virtually any gene. To test that we started with an L. major strain hemizygote for DHFR-TS (DHFR-TS/D - +/D1 strain) containing active transposons and subjected it to a selection for cells in which the remaining copy of this gene had been inactivated (6). We found that 1 out of 48 dhfr-ts^-lines recovered in this experiment had mariner inserted into DHFR-TS. These results demonstrate that mariner can be used as an insertional mutagen, and provided an estimate for the frequency of inactivation of a specific gene by transposition of ~2.5 x 10^-6.

We also constructed a modified mariner element (MosHYG) carrying the hygromycin resistance gene and tested its ability to generate selectable gene fusions and therefore "trap" expressed regions of the Leishmania genome. This strategy is based on the fact that the drug resistance gene in MosHYG lacks a splice acceptor site and therefore is "silent" in the context of the donor plasmid. Once MosHYG transposes into a gene, however, it will capture its regulatory sequences, become activated and generate hygromycin resistant cells. Using this methodology we were able to "trap" and identify several putative 5' untranslated regions of previously unknown leishmanial genes. With the right combination of selectable marker/reporter and screening procedure, the same type of approach may lead to the identification of developmentally regulated genes, including genes expressed specifically upon host infection.

In summary, our results demonstrate that mariner can transpose efficiently in Leishmania. This establishes this transposon as a powerful tool for the dissection of the genetic basis of important aspects of Leishmania biology, such as virulence and pathogenesis. Furthermore, the demonstration that mariner can transpose in a heterologous host as evolutionarily distant as Leishmania suggests that this transposable element may conceivably become a general transformation/mutagenesis tool for a variety of organisms.

References

1. Berg, C.M., Berg, D.E. and Groisman, E.A. (1989) In Mobile DNA, D.E. Berg and M.M. Howe, eds., ASM Press, Washington DC.

2. Jacobson, J.W., Medhora, M.M. and Hartl, D.L. (1986) Proc. Natl. Acad. Sci. U.S.A. 83:8684.

3. Medhora, M.M., Maruyama, K. and Hartl, D.L. (1991) Genetics 128:311.

4. Plasterk, R.H. (1996) Curr. Topics Microbiol. Immunol. 204:125.

5. Gueiros-Filho, F.J. and Beverley, S.M. (1997) Science 276:1716.

6. Gueiros-Filho, F.J. and Beverley, S.M. (1996) Mol. Cell. Biol. 16:5655.

Marcello A. Barcinski

Departamento de Parasitologia, ICB/USP, Av. Prof. Lineu Prestes 1374, 05508-900 São Paulo, SP

Programmed Cell Death (PCD) or apoptosis, an active process of genetically controlled cell death, has been described in examples ranging from fungi to man. PCD is responsible for the physiological elimination of unnecessary, superfluous or potentially harmful cells during embriogenesis and as such it plays a central role in the normal development, maturation and organogenesis in a wide range of animal species. Cell death under the control of PCD is also pivotal for the proper function of continuous cell renewal systems in adult organisms. Aberrant, misplaced or extemporaneous induction or inhibition of PCD can lead to different diseases. The possibility of manipulating, under controlled conditions, the occurrence of PCD has enormous therapeutic potentialities. All these reasons have raised great excitement about PCD among the biomedical community. The signalization system and the selection process for the activation of life or death pathways, can be operated by a wide range of different cell bound or free ligands as well as by extracellular environmental inducers such as irradiation or heat-shock. In spite of the wide range of inducers and of cell types on which PCD occurs, the morphological and biochemical changes characterizing the process are very similar, strongly suggesting that the same basic biological mechanism underlies its occurrence. Also, a series of evolutionary conserved genes regulate the final common death pathway. They have been characterized by genetic studies in the nematode Caenorhabditis elegans and have been named ced genes (for cell death genes). Ced-3, ced-4 and ced-9 have been shown to affect PCD. Ced-3 and ced-4 are required for the death program while the ced-9 product counteracts the cell-inducing genes, protecting cells from PCD. Mammalian homologues of ced-3 and ced-9 gene products have been described as respectivelly, the bcl-2 family of cell death regulators and Interleukin-1b converting enzyme (ICE) family of aspartate-specific cell death executor proteases. The belief that unicellular organisms cannot benefit from an autoinflicting death program has led some authors to consider PCD as a prerogative of multicellular organisms and thus with an evolutionary origin necessarily upstream of the onset of multicellularity. This a priori view has been changed by the description of apoptosis in three examples of mammalian pathogenic trypanosomatids, with biochemical and morphological hallmarks very similar to the ones described in multicellular organisms. In Trypanosoma cruzi epimastigotes, apoptosis was described as regulated by culture conditions and changes in cell density, implying that extracellular signals define parasite's fate. Epimastigotes also undergo complement-mediated apoptosis (Ameisen et al. 1995). In the procyclic form of Trypanosoma brucei rhodesiense apoptosis was triggered by Con A as a study model of the effect of a lectin present in tsetse midgut and its putative role in parasite multiplication and differentiation (Welburn et al.1996). Finally, in promastigotes of Leishmania (Leishmania) amazonensis our group has described apoptotic death induced by heat-shock as a phenomenon happening at the site of infection in a mammalian host (Moreira et al. 1996). Heat-induced death in promastigotes is clearly Ca²⁺ dependent and the cells are protected from this type of death by GM-CSF (Barcinski et al.1992) an haemopoietic growth-factor that protects mammalian bone-marrow stem cells from apoptotic death. We will show that a non-lethal heat shock reduces the parasite's mitochondrial membrane potential and impairs ⁴⁵Ca²⁺ uptake. Ca²⁺ uptake is preserved by GM-CSF. The analysis, with the use of different Ca²⁺ indicators, of the specific uptake by the different calcium-buffering organelles have still not provided a definitive answer concerning the mechanism by which GM-CSF exerts its protective effect. However the reduction of the parasite's mitochondrial membrane potential by the use of a proton ionophore (FCCP) sensitizes the parasite to the effect of a non-lethal heat shock as well as to a non-lethal concentration of H₂O₂ .In this situation the promastigotes are exquisitelly sensitive to the protective effect of calcium chelators as well as to GM-CSF, suggesting that the mitochondria might be a responsive organelle for the action of the haematopoietic cytokine.

The description of apoptosis in unicellular eukaryotes raises questions regarding the evolutionary origin of PCD as well as its role in the emergence and maintenance of parasitism, the structure and function of the genes involved in the process and the constraints of the multicellular organization for the proper operation of a gene regulated cell death program. Our proposal is that PCD can occur in any situation where living cells display features of an organized network which operates throught interactions within themselves and/or with elements of their environment and division of labour among its members (Barcinski & Moreira, 1994; Welburn et al. 1997). This definition means that multicellularity is indeed a prerequisite for the operation of PCD but not necessarily organized as a single multicellular organism. The definition includes e.g. protozoan parasites growing and differentiating in culture, in their insect vectors, and/or in their mammalian host. Up to the moment, the description of apoptosis in kinetoplastids is restricted to mammalian pathogens with an heteroxenic life-style. Therefore the search for PCD in monoxenic and plant trypanosomatids can aid in the answer of some of the above-mentioned questions. The search for gene products involved in the regulation and execution of apoptosis in promastigotes of L. amazonensis suggest the presence of homologues of ICE/ced-3 and bcl-2/ced-9 in the parasite. Western blotting with polyclonal a-ICE and a-bcl-2 antibodies revealed bands with the expected sizes. Furthermore, incubation of the parasite with a specific biotinylated irreversible ICE inhibitor as well as with a rabbit anti-ICE antibody, developed respectively with avidin-fluorescein conjugate and anti-rabbit IgG conjugated to rhodamine and analysed by laser scanning confocal microscopy displayed ICE epitopes distributed mainly in the cell cytoplasmic ground substance. The search for bcl-2 epitopes is also currently under investigation.

References

1. Ameisen, JC., Idziorek, T., Billaut-Mulot, O., Loyens, M., Tissier, J., Potentier, A., and Ouassi, A. 1995. Apoptosis in a unicellular eukaryote (Trypanosoma cruzi): implications for the evolutionary origin and role of programmed cell death in the control of cell proliferation, differentiation and survival. Cell Death. Differ.2:9-22

2. Barcinski, MA.,and Moreira, MEC., 1994. Cellular response of protozoan parasites to host-derived cytokines. Parasitol. Today, 10: 352-355

3. Moreira, MEC., Del Portillo, HA., Milder, RV., Balanco, JMF., and Barcinski, MA. 1996. Heat shock induction of apoptosis in promastigotes of the unicellular organism Leishmania (L) amazonensis. J. Cell. Physiol. 167: 305-313.

4. Welburn, SC., Dale, C., Ellis, D., Beecroft, R. and Pearson, TW. 1996. Apoptosis in procyclic Trypanosoma brucei rhodesiense in vitro. Cell Death. Differ. 3: 229-236.

5. Welburn, SC., Barcinski, MA., and Williams, GT. 1997. Programmed cell death in trypanosomatids. Parasitol. Today. 13: 22-26

APOPTOSIS IN TRYPANOSOMATIDS:FACTS AND HYPOTHESES

APOPTOSIS IN TRYPANOSOMATIDS:FACTS AND HYPOTHESES

Publication Dates

• Publication in this collection
  30 Nov 2000
• Date of issue
  Nov 1997