CORRESPONDENCE
Proteolytic activity of the 43,000 molecular weight antigen secreted by Paracoccidioides brasiliensis
Paracoccidioidomycosis is a infection caused by the dimorphic fungus Paracoccidioides brasiliensis. The disease is usually initiated by inhalation of airbone conidia or mycelial fragments8. Most individuals exposed to the fungus develop asymptomatic infection. However, clinical manifestations are observed in some cases. The disease may affect different organs and systems, with multiple clinical features. Relatively little is known of the biochemical mechanisms involved in the pathogenicity of this fungus, including its first contact and subsequent stages of paracoccidioidomycosis development.
Filamentous fungi which sucessfully colonize substrates in nature typically do so by the action of specific, digestive exoenzymes2.
Proteinases produced by certain fungi pathogenic in humans have been recognized as potentially important virulence factors3. It has been suggested that extracellular proteinases of such fungi may play a role in adherence and survival of the pathogen on host mucosal surfaces1, invasion of host tissues9,11, and digestion of immunoglobulins10,14.
Mycelial and yeast forms of P. brasiliensis synthesize soluble antigens which comprise up to 60 components, including glycoproteins and proteins12. One of these, the 43 kDa glycoprotein was found as of diagnostic value6,13. This fraction is easily found in culture supernatants and in sera of patients presenting the active disease7.
Here we report preliminary studies related to the proteolytic activity of the 43 kDa glycoprotein.
Enzymatic characteristics as inhibitors and optimal pH were determined by SDS-polyacrylamide gel electrophoresis with substrates incorporated into the gel4. The specificity of the proteolytic activity toward casein 5 and elastin at different pH and temperatures was spectrophotometrically determined.
Extensive proteolysis (destaining) was observed when the 43 kDa fraction was submitted to electrophoresis in gelatin, gelatin-albumin or casein containing SDS-polyacrylamide gels and then incubated overnight (18h) at pH 6.0. Complete elastin solubilization and casein hydrolisis were observed at pH 6.0 and 35ºC (optimal pH and temperature). Caseinolytic and collagenolytic activities were not affected by pepstatin, hydroxymercuribenzoic acid, iodoacetamide, PMSF (phenyl methyl sulfonyl fluoride) but were inhibited by EDTA. This indicates the proteolytic activity of 43 kDa fraction as metal-dependent. The digestion of tissue structural proteins eg. collagen, elastin, suggests that this fraction may play a role in the virulence of P. brasiliensis.
REFERENCES
1. BORG, M. & RUCHEL, R. Expression of extracellular acid proteinase by proteolytic Candida spp during experimental infection of oral mucosa. Infect. Immun., 53: 626-631, 1988.
2. GRIFFIN, D.H. Fungal physiology. New York, John Willey & Sons, 1981.
3. KWON-CHUNG, K.J.; LEHMAN, D.; GOOD, C. & MAGEE, P.T. Genetic evidence for role of extracellular proteinase in virulence of Candida albicans. Infect. Immun., 49: 571-575, 1985.
4. LACKS, S.A. & SPRINGHORN, S.S. Renaturation of enzymes after polyacrylamide gel electrophoresis. J. biol. Chem., 255: 7467-7473, 1980.
5. LALUCE, C. & MOLINARI, R. Fractionation of the proteolytic and amylolytic complex enzyme system of Streptomyces aureofaciens and some properties of fractions. Biotechnol. Bioeng., 21: 915-938, 1979.
6. MENDES-GIANNINI, M.J.S.; BUENO, J.P.; SHIKANAI-YASUDA, M.A.; STOLF, A.M.; MASUDA, A.; AMATO NETO, V. & FERREIRA, A.W. Immunoglobulin G antibody response to 43 KD glycoprotein of Paracoccidioides brasiliensis as marker for the evaluation of patients under treatment. Rev. ibér. Micol, 5(suppl. 1): 72, 1988.
7. MENDES-GIANNINI, M.J.S.; BUENO, J.P.; SHIKANAI-YASUDA, M.A.; FERREIRA, A W. & MASUDA, A. Detection of the 43.000 molecular weight glycoprotein in sera of patients with paracoccidioidomycosis. J. clin. Microbiol., 27: 2842-2845, 1989.
8. RESTREPO, A.M. The ecology of Paracoccidioides brasiliensis: a puzzle still unsolved. Sabouraudia, 23: 323-333, 1985.
9. RUCHEL, R. On the role of proteinases from Candida albicans in the pathogenesis of acronecrosis. Zbl. Bakteriol. (A), 255: 368-379, 1983.
10. RUCHEL, R. Cleavage of immunoglobulin by pathogenic yeast of the genus Candida. Microbiol. Sci., 3: 316-319, 1986.
11. RUCHEL, R.; BONING, B. & BORG, M. Characterization of a secretory proteinase of Candida parapsilosis and evidence for the absence of the enzyme during infection in vitro. Infect. Immun., 53: 411-419, 1986.
12. YARZÁBAL, L. Composición antigénica de Paracoccidioides brasiliensis. In: DEL NEGRO, G.; LACAZ, C.S. & FIORILLO, A.M., ed. Paracoccidioidomicose. São Paulo, Sarvier, EDUSP, 1982. p. 59-67.
13. YARZÁBAL, L.A.; BOUT, D.; NAQUIRA, F.; FRUÍT, J. & ANDRIEU, S. Identification and purification of the specific antigen of Paracoccidioides brasiliensis responsible for immunoelectrophoretic band E. Sabouraudia, 15: 79-85, 1977.
14. YUAN, L. & COLE, G.T. Isolation and characterization of an extracellular proteinase of Coccidioides immitis. Infect. Immun., 55: 1970-1978, 1987.
Faculdade de Ciências Farmacêuticas
UNESP
Rua Expedicionários do Brasil, 1.621
Caixa Postal 502
14800 ARARAQUARA, SP., BRASIL
Rosangela Aparecida MORAES
Terezinha Aparecida RICCI
Recebido para publicação em 22/08/90
Research supported by CNPq (Proc. 402041/89-5/CL)
Publication Dates
-
Publication in this collection
12 Sept 2006 -
Date of issue
Oct 1990
