Activity detection of extracellular proteinase and serine protease secreted by Cryptococcus neo formans / 第二军医大学学报

ABSTRACT

Objective: To evaluate the activities of extracellular proteinase and serine protease secreted by Cryptococcus neoformans (C. neoformans) of different sources and serotypes and incubated under different conditions. Methods: Thirty-six C. neoformans strains of different sources and serotypes were incubated in protein agar medium at 30°C and 37°C separately. Activities of extracellular proteinase and serine protease were analyzed by determining Clearance Halos(CHs) produced by C. neoformans. Activity changes of the 2 proteases were analyzed after treated with serine protease inhibitors. The activity and the molecular weight of extracellular proteinase in the concentrated supernatant were measured using zymography assay. Results: Thirty-three of 36 C. neoformans strains produced specific Clearance Halos around the colonies, and their mean CHs at 30°C and 37°C were 0.558±0.170 and 0.575±0.169, respectively (P>0.05). The mean CHs of serotype A(n=13), B(n=13), and D/AD (n=6) strains were 0.564±0.144, 0.515±0.078 and 0.482±0.072, respectively (P>0.05); the mean CHs of clinical(n=23), environmental(n=9),and uncapsuled strains(n=4) were 0.570±0.177, 0.513±0.069, and 0.942±0.075, respectively (P<0.05). The mean CHs of control group (without protease inhibitor) and groups treated with protease inhibitor (1.2 mU and 1.6 mU) were 0.459±0.188, 0.975±0.287 and 0.733±0.252, respectively(P<0.01). Rich extracellular protease was found in the concentrated supernatant. Conclusion: The incubation temperature (30°C and 37°C) has no effect on activities of extracellular proteinase and serine protease, and the activities of clinical strains are significantly stronger than those of other 2 strains; the activities in different serotypes groups have no significant difference and can be inhibited by serine protease inhibitors.