REACTIVITY OF ANTI-GP43 ANTIBODIES FROM PARACOCCIDIOIDES BRASILIENSIS ANTISERUM WITH EXTRACTS FROM CUTANEOUS LESIONS OF LOBO'S DISEASE: PRELIMINARY NOTE

REACTIVITY OF ANTI-GP43 ANTIBODIES FROM PARACOCCIDIOIDES BRASILIENSIS ANTISERUM WITH EXTRACTS FROM CUTANEOUS LESIONS OF LOBO’S DISEASE. PRELIMINARY NOTE

Mônica Scarpelli Martinelli VIDAL ^{( 1 )} , Selma Aliotti PALACIOS ^{( 2 )} , Natalina TAKAHASHI DE MELO ^{( 1 )} & Carlos da Silva LACAZ ^{( 1 , 2 )}

VIDAL, M.S.M.; PALACIOS, S.A.; MELO, N.T. de & LACAZ, C. da S. – Reactivity of anti-GP43 antibodies from Paracoccidioides brasiliensis antiserum with extarcts from cutaneous lesions of lobo’s disease. Preliminary note. Rev. Inst. Med. trop. S. Paulo, 39(1):000-000, 1997.

SUMMARY

We demonstrated through several immunochemical tests the presence of GP-43 from P. brasiliensis in extracts of cutaneous lesions from Jorge Lobo’s disease. This glicoprotein is one of the immunodominant antigens in this species, and is used to identify it.

The demonstration of GP-43 tissues infected by the agent of Jorge Lobo’s disease is an additional evidence for classifying it in the genera Paracoccidioides, species loboi.

KEYWORDS:Paracoccidioides brasiliensis; Anti-GP-43 Antibodies, Lobo’s disease.

The 43 kDa glycoprotein is the major antigen of Paracoccidioides brasiliensis (Splendore, 1912) Almeida, 1930. Its detection guarantees the identification of this species using different immunochemical tests⁵. It has also been demonstrated that antibodies to this glycoprotein are present in the sera of patients with paracoccidioidomycosis, histoplasmosis and Lobo’s disease and can be determined by ELISA⁶. Using anti-GP-43 and anti-P. brasiliensis antisera, SANDOVAL et al.⁷ reported that P. brasiliensis antigens are present in biopsies from lesions of paracoccidioidomycosis and Lobo’s disease. Other data from the literature have demonstrated that the cell walls of the agents of paracoccidioidomycosis and Lobo’s disease may express similar constituents (common antigens), the only difference being that P. loboi has not yet been cultivated. Recently the GP-43 gene from P. brasiliensis has been cloned and sequenced by CISALPINO et al.².

The present note demonstrates the recognition of a 43 kDa antigen in the extracts of cutaneous lesions from a patient with Lobo’s disease, by anti-GP-43 antibodies from P. brasiliensis antiserum. This reactivity was demonstrated by the following immunochemical tests: a) SDS PAGE³ on linear 12.5% gel. The antigen was precipitated by trichloroacetic acid (TCA), the precipitate diluted in the sample buffer and 50 µL of this solution applied in each slot of the gel. The staining method used was silver nitrate¹. b) Immunoblotting¹⁰ carried out with P. brasiliensis anti-GP-43 polyclonal antiserum produced in rabbit at 1:50 dilution. c) Immunoelectrophoresis⁹ with the same reference antiserum. These tests were carried out with the antigen obtained by trypsin treatment (2%) and grinding of biopsies from lesions of patients with Lobo’s disease (JL3 somatic antigen). The protein yield, measured by Lowry’s method⁴ was 1.64 mg/mL. The carbohydrate concentration, measured by Scott & Melvin’s method⁸ was 0,58 mg/mL. This experiment was made in duplicate.

As control, a cutaneous biopsy from a healthy donor was obtained and similarly processed. All procedures indicated above for detection of GP-43 were repeated with this specimen and gave negative results.

Figures 1, 2 and 3 demonstrate the presence of GP-43 in the biopsies from patients with Lobo’s disease. By immunoelectrophoresis, the cathodal migration¹¹ arch has a different shape from that obtained with the P. brasiliensis antigen, but the precipitation reaction with specific antiserum is clear.

 

Fig. 1 – SDS-PAGE of biopsy extract (Silver nitrate stain). A) Molecular weight standards; B) Somatic antigen JL3 from P. loboi.

 

Fig. 2 – Immunoblotting of biopsy extract with anti-GP-43 from P. brasiliensis antiserum. A) Molecular weight standards; B) Somatic antigen JL3 from P. loboi; C) Extract from biopsy of normal skin.

 

Fig. 3 – Immunoelectrophoresis of antigenic preparation containing GP-43 epitopes. 1) Rabbit anti-GP-43 antiserum; 2) Somatic antigen JL3 from P. loboi; 3) P. brasiliensis strain 113 antigen.

The demonstration of GP-43 in human tissue infected by the agent of Lobo’s disease represents an additional argument in favor of assigning it to the genus Paracoccidioides species loboi.

RESUMO

Reatividade do soro anti-Gp-43 do P. brasiliensis com extratos de lesões cutâneas da doença de Jorge Lobo

Através de várias provas imunoquímicas foi demonstrada a presença da GP-43 em extratos de lesões cutâneas da doença de Jorge Lobo. A glicoproteína de 43 kDa é um dos antígenos dominantes do Paracoccidioides brasiliensis, permitindo a identificação desta espécie fúngica.

A demonstração da GP-43 em tecidos infectados com o agente da doença de Jorge Lobo, constitui mais um argumento para colocá-lo no gênero Paracoccidioides, espécie loboi.

ACKNOWLEDGEMENT

The authors thank Dr. Zoilo P. de Camargo for kindly supplying the polyclonal anti-GP-43 antiserum.

REFERENCES

1. ANSORGE, W. – Fast visualization of protein bands by impregnation in potassium permanganate and silver nitrate. In: STATHAKOS, D., ed. Eletrophoresis 82. Berlim, Walter de Gruyter, 1983. p. 235-242.

2. CISALPINO, P.S.; PUCCIA, R.; YAMAUCHI, L.M. et al. – Cloning characterization and epitope expression of the major diagnostic antigen of Paracoccidioides brasiliensis. J. biol. Chem., 271:4553-4560, 1996.

3. LAEMMLI, V.K. – Clivage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227:680-685, 1970.

4. LOWRY, O.H.; ROSEBROUGH, N.J.; FARR, A.L. & RANADAL, R.J. – Protein measurement with the Folin-phenol reagent. J. biol. Chem., 193:265-275, 1951.

5. PUCCIA, R.; SCHENKMAN, S.; GORIN, P.A.J. & TRAVASSOS, L.R. – Exocellular components of Paracoccidoides brasiliensis: identification of a specific antigen. Infect. Immun., 53:199-206, 1986.

6. PUCCIA, R. & TRAVASSOS, L.R. – 43 Kilodalton glycoprotein from Paracoccidioides brasiliensis. Immunochemical reactions with sera from patients with paracoccidioidomycosis, histoplasmosis or Jorge Lobo’s diseases. J. clin. Microbiol., 29:1610-1615, 1991.

7. SANDOVAL, M.; DE BRITO, T.; SOTTO, M.N.; SANTOS, R.T. & FRANCO, M.F. – Antigen distribution in mucocutaneous biopsies of human paracoccidioidomycosis. Int. J. surg. Path., 3:181-188, 1996.

8. SCOTT, T.A. & MELVIN, E.H. – Determination of dextran with antrone. Analyt. Chem., 25:1656-1661, 1953.

9. SIQUEIRA, A.M. de – Avaliação da sensibilidade e especificidade de algumas provas sorológicas no diagnóstico, prognóstico e controle de cura da paracoccidioidomicose. Caracterização imunoquímica do antígeno E₂ do Paracoccidioides brasiliensis. São Paulo, 1982. (Tese de Doutoramento – Instituto de Ciências Biomédicas da Universidade de São Paulo).

10. TOWBIN, H. & GORDON, J. – Immunoblotting and dot blotting – current status and outloor. J. immunol. Meth., 72:313-340, 1984.

11. YARZÁBAL, L.A.; BOUT, D.; NAQUIRA, F.; FRUIT, J. & ANDRIEWS, S. – Identification and purification of the specific antigen of Paracoccidioides brasiliensis responsible for immunoeletrophoretic band E. Sabouraudia, 15:79-85, 1977.

Recebido para publicação em 20/09/1996

Aceito para publicação em 14/02/1997

(

¹) Instituto de Medicina Tropical de São Paulo.

(2

) Laboratório de Investigação Médica 53 do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo.

Correspondence to: Monica S.M. Vidal. Instituto de Medicina Tropical. Av. Dr. Enéas C. Aguiar, 470, 05403-000 São Paulo, SP, Brasil.

Publication Dates

• Publication in this collection
  14 Oct 1998
• Date of issue
  Jan 1997

History

• Received
  20 Sept 1996
• Accepted
  14 Feb 1997