光滑念珠菌临床分离株胞外酶活性与蜡螟幼虫体内致病性的相关性研究

Correlation of extracellular enzymes activity of Candida glabrata clinical isolates with in vivo pathogenicity in Galleria mellonella larvae

目的基于蜡螟幼虫感染模型探讨光滑念珠菌临床分离株胞外酶活性与致病性之间的关系。方法采用实验性研究方法,收集青海省人民医院2021年6月至2022年1月临床分离的非重复光滑念珠菌71株。采用牛血清蛋白琼脂培养基、卵黄琼脂培养基、羊血琼脂培养基、吐温-80琼脂培养基和三丁酸甘油酯琼脂培养基分别检测光滑念珠菌的天冬氨酰蛋白酶、磷脂酶、溶血素、酯酶和脂肪酶的活性;采用光滑念珠菌ATCC2001不同浓度(1.25×108 CFU/ml、2.50×108 CFU/ml、3.75×108 CFU/ml、5.00×108 CFU/ml)的菌悬液感染蜡螟幼虫并计算半数致死浓度(LC50);采用蜡螟幼虫组织病理学及病原学分析,确定感染模型构建是否成功;将光滑念珠菌临床分离株配制成LC50浓度,通过注射感染蜡螟幼虫以检测对蜡螟幼虫致病性;采用Spearman检验或Pearson检验分析光滑念珠菌临床分离株胞外酶活性与蜡螟幼虫体内致病性之间的相关性。结果 71株临床分离的光滑念珠菌,培养2 d后均表现为低溶血活性;培养4 d后均可检测到天冬氨酰蛋白酶,其中有7株(9.86%)、19株(26.76%)、45株(63.38%)分别表现出低、中、高天冬氨酰蛋白酶活性;培养7 d后71株均未检测到磷脂酶、酯酶和脂肪酶活性。光滑念珠菌对蜡螟幼虫LC50= 2.50×108 CFU/ml;实验组蜡螟幼虫,肠道组织病理切片检见真菌孢子,培养后经微生物质谱鉴定为光滑念珠菌,而对照组病理切片及培养均未发现真菌。Spearman检验显示,天冬氨酰蛋白酶活性指数与蜡螟幼虫生存率呈线性正相关(r= 0.73,P<0.01),差异有统计学意义;Pearson检验显示,溶血活性指数与蜡螟幼虫生存率之间没有明显的线性关系(r=0.16,P>0.05),差异无统计学意义。结论本研究中光滑念珠菌临床分离株均有天冬氨酰蛋白酶活性和低溶血活性,无磷脂酶、酯酶和脂肪酶活性。光滑念珠菌天冬氨酰蛋白酶活性与蜡螟幼虫体内致病性呈正相关性。

Objective To explore the relationship between extracellular enzymes activity and virulence of Candida glabrata clinical isolates based on the infection model of Galleria mellonella larvae.Methods Using experimental research methods,71 strains of non-repetitive Candida glabrata were collected from Qinghai Provincial People′s Hospital from June 2021 to January 2022.Bovine serum protein agar medium,egg yolk agar medium,sheep blood agar medium,Tween-80 agar medium and triglyceride agar medium were used to detect the aspartyl protease activity,phospholipase activity,hemolysis activity,esterase activity and lipase activity of Candida glabrata.Median lethal concentration(LC50)was calculated by using 1.25×108 CFU/ml,2.50×108 CFU/ml,3.75×108 CFU/ml,5.00×108 CFU/ml suspension of Candida glabrata ATCC2001 to infect Galleria mellonella larvae.Histopathological and etiological analysis was performed to determine whether the infection model was successfully established.The clinical isolates of Candida glabrata were configured to infect Galleria mellonella larvae with LC50 concentration to detect the pathogenicity of Galleria mellonella larvae.Spearman test or Pearson test were used to analyze the correlation between the extracellular enzyme activity of Candida glabrata clinical isolates and the pathogenicity of Galleria mellonella larvae.Results 71 strains of Candida glabrata isolated clinically were detected to have low hemolytic activity after 2 days of culture.Aspartyl protease was detected after 4 days of culture,among which 7 strains(9.86%),19 strains(26.76%)and 45 strains(63.38%)showed low,medium and high aspartyl protease activity.After 7 days of culture,71 strains did not detect phospholipase,esterase and lipase activities.Candida glabrata on Galleria mellonella larvae of LC50=2.5×108 CFU/ml Fungal spore were found in the intestinal tissue pathological section of Galleria mellonella larvae in the experimental group,and Candida glabrata was identified by the microbial Mass Spectrometry after culture,while no fungi were found in the pathological section and culture of the control group.Spearman test shows that,there was a linear positive correlation between aspartyl protease activity and the survival rate of Galleria mellonella larvae(r=0.73,P<0.01),the difference was statistically significant.Pearson test shows that,there was no significant linear relationship between hemolytic activity and survival rate of Galleria mellonella larvae(r=0.16,P=0.34),the difference was not statistically significant.Conclusion The clinical isolates of Candida glabrata in this study had aspartyl protease activity and low hemolytic activity,but no phospholipase,esterase and lipase activity.The activity of aspartyl aspartyl protease of Candida glabrata was positively correlated with the pathogenicity of Galleria mellonella larvae.