EXOCELLULAR PROTEOLYTIC ACTIVITY OF PARACOCCIDIOIDES-BRASILIENSIS - CLEAVAGE OF COMPONENTS ASSOCIATED WITH THE BASEMENT-MEMBRANE

We have previously characterized an exocellular serine-thiol proteinas e activity in Paracoccidioides brasiliensis, using as substrates pepti des analogous of the internally quenched fluorogenic peptide Abz-MKWLT L-EDDnp. In this communication, detection of maximal proteinase activi ty in the culture supernatant fluids followed the abrupt increase in t he medium pH, owing to the accumulation of ammonia generated by urease activity. Culture supernatant fluids collected at the peak of protein ase activity against Abz-MRKLTL-EDDnp were able to cleave components o f the basal membrane of the extracellular matrix (EM), including lamin in, fibronectin, collagen type IV and proteoglycans, and the proteolyt ic activity was selectively inhibited both by PMSF and p-HMB (sodium 7 -hydroxymercuribenzoate), which are also specific inhibitors of the se rine-thiol proteinase. Human collagen I, bovine fibrinogen, human immu noglobulin G, BSA or P. brasiliensis gp43 were resistant to proteolysi s. The kinetics of appearance of the proteinase activity against EM su bstrates coincided with that of proteolysis of Abz-MKRLTL-EDDnp. Moreo ver, chromatographic fractions of culture supernatants containing the serine-thiol proteinase at high specific activity were also active aga inst EM substrates. These data suggest the involvement of this enzyme activity in the degradation of the basement membrane, which is the fir st step for fungal tissue invasion.