Inhibitors And Detection Of Phospholipases Secreted By Cryptococcus Neoformans

Objective:To evaluate the phospholipases secreted by Cryptococcus neoformans from different sources, serotypes and incubation conditions; To confirm the medicine as macrocylin, sodium deoxycholic and chlorpromazine has the characters to inhibit the activity of phospholipases secreted by Cryptococcus neoformans; To detect the effect of macrocylin and chlorpromazine on inhibiting the virulence of Cryptococcus neoformans in vivo.Methods:Forty Cryptococcus neoformans isolates were detected for extracelluar phospholpases activity after being incubated onto egg yolk agar. Phospholipase activity of every isolate was measured and then compared by the ratio of the diameter of the colony to the total diameter of the colony plus precipitation zone (PZ). The medicines, including macrocylin, sodium deoxycholic and chlorpromazine, which have different concentration, were put into the cultures and the bacterium solutions of the isolate which has greatest phospholpase activity. By comparing the PZ of the medicines in deferent concentration, the effects of the medicines on isolate phospholpase activity were measured. The macrocylin and chlorpromazine in deferent concentrations were injected via abdomen to the mouse infected with Cryptococcus neoformans. The survival rate and CFU in brain of different group mouse were observed.Results :Thirty-nine of forty isolates produced distinct precipitation zone implicating the extracellular phospholipase activity. PZs were 0.561±0.123 at the incubation temperature of 30℃ and 0.563±0.130 at 37℃. Incubated at 37℃,PZs of serotype A, B and AD with D were 0.576±0.104, 0.589±0.079 and 0.472±0.051, respectively; PZs of clinical and enviromental isolates were 0.621±0.133 and 0.527±0.104, respectively; PZs of 22 from cerebrospinal fluid of 26 clinical isolates1. and the remainings from lung and skin were 0.508±0.103 and 0.593±0.095 ,respectively.2. On experiment with the medicine inhibiting the phospholipase activity, the PZs of control group and chlorpromazine groups in different concentration were 0.386±0.023, 0.524±0.065, 0.790±0.089, 0.802±0.085 and 0.892±0.059,respectively; the PZs of control group and denmacrocylin groups in different concentration were 0.387±0.039, 0.479±0.086, 0.702±0.029, 0.891±0.072 and 0.955±0.021,respectively; the PZs of control group and sodium deoxycholic groups in different concentration were 0.390±0.050, 0.782±0.038, 0.875±0.074, 0.914±0.048 and 0.885±0.091,respectively.3. When detected the effect of macrocylin and chlorpromazine on inhibiting the virulence of Cryptococcus neoformans in vivo, the mean survival time of control group, macrocylin and chlorpromazine groups were 18.90d, 19.92d, 19.20d, 26.39d, 25.94d, respectively; the CFU in brain of different groups were 518.67±203.86, 423.53±241.54, 469.07±243.13, 261.73±225.82 and 226.47±196.13, respectively. Conclusions:1. The data showed that no significant difference was found in phospholipase activity between the isolates incubated at 37℃ and 30℃(P＝0.191), between serotype A, B and C with AD(P＝0.225). There was significant difference between the clinical and enviromental isolates(P＝0.031). Among clinical isolates, isolates from cerebrospinal fluid were significant difference from that from lung and skin(P＝0.047). The phospholipase activity of isolates from cerebrospinal fluid was most active.In vitro, there was significant difference between control group and and chlorpromazine groups in different concentration (p<0.001),2. which proved the inhibition character of chlorpromazine to phospholipase activity. The inhibition concentration was 0.3% and there was not difference between the concentration of chlorpromazine above 0.6% ( P＞0.05). There was significant difference between control group and and macrocylin groups in different concentration (p<0.001). The inhibition concentration was 0.36% and there was not difference between the concentration of macrocylin above 0.36% (P＞0.05). The inhibition concentration of sodiu...