Clonorchis sinensis cysteine protease participated in metacercarial excystation
Objective To understand the role of cysteine protease in promoting excystation in Clonorchis sinensis.Methods The metacercariae were collected by artificial digestion and underwent in vitro excystation,with 100 metacercariae in each group.The metacercariae in experimental group was treated with L-trans-3-carboxyoxiran-2-carbonyl-L-leucylagmatine trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane N-(trans-epoxysuccinyl)-L-leucine 4-guanidinobutylamide(E-64)(20,40 μmol/L),iodoacetic acid(IAA)(50,100 μmol/L),and maturation domain of cysteine protease in Clonorchis sinensis(CsCPL-m),and the control group only added buffer,at 37 ℃ for 30 min;the liquid was discarded,and then 10 μg/mL trypsin-PBS solution(pH 7.5)was added;the number of excystation was counted every 5 minutes,6 times in total;the excystation rate was calculated;under stereomicroscope,the empty cysts in each group after excystation were divided into three categories to calculate the composition ratio.Results The excystation rates of the rCsCPL-m treatment group and the control group were similar at 5 minutes and 10 minutes,and the difference was not statistically significant(x2=0.070,0.163;all P＞0.05).By 15 minutes,the excystation rates of both groups reached 99％;the excystation rates of E-64 and IAA treatment groups were lower than the control group at 5,10,15,and 20 min,and the difference was statistically significant(x2=73.895,286.003,203.959,41.451;all P＜0.001);at 25 min,the excystation rate of the E-64 and IAA(100 μmol/L)group was still lower than that of the control group,and the difference was statistically significant(x2=10.318,11.428,16.807;all P＜0.001);the excystation rates of the IAA treatment group(50μmol/L)at 6 statistical time points were higher than those of the 100 μmol/L treatment group,the difference was statistically significant(x2=10.285,22.623,14.654,12.934,7.789,4.308;all P＜0.05);the composition ratio of the three types of empty cyst morphology in the rCsCPL-m group showed no statistically significant difference compared to the control group(x2=2.993,P＞0.05);the E-64 and IAA treatment groups both showed a decreased proportion of type A and an increased proportion of type C when compared to the control group.The difference was statistically significant(x2=21.495,17.324;all P＜0.01).Conclusions The cysteine protease of Clonorchis sinensis might promote excystation by enzymatically softening the inner layer of the cyst wall.Whether CsCPL is involved in excystation still needs further confirmation.
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