Possible role of a proteinase in endosporulation of Coccidioides immitis.

AUTOR(ES)

Yuan, L

RESUMO

We previously reported isolation of a serine proteinase from the soluble conidial wall fraction of Coccidioides immitis. The purified proteinase was identified as a polypeptide band of 36,000 Mr by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In this study, we raised monospecific antiserum in rabbits against the purified proteinase for use in immunoelectron microscopy. We showed that immunolabel was localized in the cell wall of both the saprobic and parasitic phases but was most concentrated in the wall of the segmentation apparatus of spherules just prior to endospore differentiation. The total wall fractions of the mycelial phase, as well as those of presegmented and endosporulating spherules, were isolated from in vitro grown cells and then treated with a proteinase inhibitor (phenylmethylsulfonyl fluoride [PMSF]) which irreversibly binds to the residual proteolytic enzyme in the wall isolates. Each fraction was dialyzed, lyophilized, and separately incubated with the active, purified 36,000-Mr proteinase. The reaction mixtures were examined spectrophotometrically (A280) for decomposition of the substrates. Only the PMSF-treated wall isolated from endosporulating spherules was significantly digested. Active, 36,000-Mr proteinase was isolated from intact and viable, endosporulating spherules by brief extraction of the cells with 1% octyl-beta-D-thioglucoside, a nonionic detergent. The serine proteinase may be partly responsible for autolysis of the segmentation apparatus of mature spherules, a morphogenetic process which is pivotal for release of endospores and subsequent proliferation of the pathogen.

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