菌丝特异性基因和Candidalysin对白念珠菌培养特性及致病性的影响  

Effect of hypha-specific gene and Candidalysin on the culture characteristics and pathogenicity of Candida albicans

作　　者：王迪 黄劲[3,4] 姚昌昊 王梅竹 程敏 郭鑫瑶 廖万清 康颖倩 WANG Di;HUANG Jing;YAO Changhao;WANG Meizhu;CHENG Min;GUO Xinyao;LIAO Wanqing;KANG Yingqian(Department of Microbiology,School of Basic Medical Sciences,Guizhou Medical University,Guiyang 550025,Guizhou,China;Guizhou Talent Base for Research on the Relationship between Microbiology and Human Health&Key Laboratory of PathogenBiology of Guizhou Higher Education Department,Guiyang 550025,Guizhou,China;Key Laboratory of Environmental Pollution Monitoring and Disease Control of Guizhou Medical University of Ministry of Education,Guiyang 550025,Guizhou,China;Department of Biochemistry and Molecular Biology,Basic Medical Sciences,Guizhou Medical University,Guiyang 550025,Guizhou,China;Department of Dermatology,Changzheng Hospital Affiliated to Navy Medical University,Shanghai 200003,China;Shanghai Key Laboratory of Molecular Medical Mycology,Shanghai 200003,China;Guizhou Academy of Tobacco Science,Guiyang 550009,Guizhou,China)

机构地区：[1]贵州医科大学基础医学院微生物学教研室,贵州贵阳550025 [2]贵州省微生物与人类健康关系研究人才基地&贵州省普通高校病原生物学特色重点实验室,贵州贵阳550025 [3]贵州医科大学环境污染与疾病监控教育部重点实验室,贵州贵阳550025 [4]贵州医科大学基础医学院生物化学与分子生物学教研室,贵州贵阳550025 [5]海军军医大学长征医院皮肤科,上海200003 [6]上海市医学真菌分子生物学重点实验室,上海200003 [7]贵州省烟草科学研究院,贵州贵阳550009 [8]贵州医科大学

出　　处：《贵州医科大学学报》2021年第10期1126-1132,共7页Journal of Guizhou Medical University

基　　金：国家自然科学基金(32060034);国家科技部及教育部“111计划”(D20009);贵州省第六批人才基地项目(RCJD2018-22);贵州省科技厅社会发展攻关支撑项目[黔科合支撑(2019)2873];贵州省科学技术厅国际科技合作基地[黔科合平台人才(2020)4101];贵州省科技计划项目[黔科合支撑(2020)4Y220]。

摘　　要：目的探讨菌丝特异性基因(ECE 1)和Candidalysin对白念珠菌培养特性及致病性的影响。方法选取ece 1Δ/Δ菌株(ECE 1基因敲除菌株)、ece 1Δ/Δ+ECE 1菌株(ECE 1基因敲除回补菌株)、BWP17菌株(CiP30质粒转化的亲本菌株)及M2174菌株(Candidalysin毒素敲除菌株),采用沙氏葡萄糖琼脂培养基(SDA)37℃培养48 h并观察其培养特征,使用牛血清白蛋白培养基、卵黄培养基及三丁酸甘油酯培养基检测4种菌株的胞外水解酶活力,采用菌丝诱导实验观察4种菌株的菌丝形成能力;取4~6周龄Balb/c雌性小鼠70只,均分为BWP17组、ece 1Δ/Δ组及ece 1Δ/Δ+ECE 1组、M2174组及生理盐水(Saline,阴性对照)组,各组小鼠分别侧尾静脉注射2×10^(8) CFU/L上述4种菌液及等量Saline,观察各组小鼠注射后30 d内的体质量变化和90 d内的死亡情况,同时取各组死亡小鼠心脏、肝脏、脾脏、肺脏及肾脏制作切片、采用苏木精-伊红染色(HE)后观察各组组织的病理学特征。结果白念珠菌的菌落特征均表现为菌落较大、光滑湿润、呈奶油状圆形分布,在CHROMagar Candida培养基上的菌落颜色显示为浅绿色到深绿色;胞外水解酶检测结果显示ece 1Δ/Δ+ECE 1组、BWP17组及ece 1Δ/Δ组的蛋白酶(SAP)及磷脂酶(PL)活力强,脂肪酶(Lip)活力阴性,但M2174组只有磷脂酶活力高表达;白念珠菌ece 1Δ/Δ+ECE 1菌株的菌丝形成能力最强,ece 1Δ/Δ菌株菌丝形成能力最弱;感染白念珠菌后小鼠第28天时死亡率为21.43%~57.14%,第90天时死亡率为78.57%~100.00%;各感染组死亡小鼠肾脏的病理学改变情况最为明显,心、肝、脾及肺等病变较轻,以慢性炎症为主。结论ECE 1基因的表达增强了白念珠菌胞外水解酶的活力及其菌丝形成能力,ECE 1基因编码的Candidalysin对感染小鼠发挥了重要的毒力作用。Objective To explore the effect of hypha-specific gene(ECE)1 and Candidalysin on the culture characteristics and pathogenicity of Candida albicans.Methods Ece1Δ/Δstrain(ECE 1 gene knockout strain),ece 1Δ/Δ+ECE 1 strain(ECE1 gene knockout and replenishment strain),BWP17 strain(parent strain transformed by plasmid CiP30),and M2174 strain(Candidalysin toxin knockout strain)were selected and cultured with SDA for 48 hours at 37℃to observe their characteristics.The extracellular hydrolase activities of the four strains were detected by bovine serum albumin culture medium,yolk culture medium,and tributyrin culture medium,and the mycelium formation ability of the four strains was observed by mycelium induction test.Seventy female Balb/c mice aged 4-6 weeks were evenly divided into BWP17 group,ece 1Δ/Δgroup,ece 1Δ/Δ+ECE 1 group,M2174 group,and Saline(negative control)group,and 2×10^(8) CFU/L of the above four bacterial liquids and the same amount of saline were injected into the mouse tail veins of each group.Body mass changes within 30 days after infection and death within 90 days were observed;the heart,liver,spleen,lung,and kidney tissues of dead mice in each group were collected and the pathological characteristics of each tissue were detected by hematoxylin-eosin staining(HE).Results The colony characteristics of C.albicans were characterized by large colonies,smooth and moist,and cream-like circular distribution.The colony color on CHROMagar Candida culture medium ranged from light green to dark green.The extracellular hydrolase test found that ece 1Δ/Δ+ECE 1 group,BWP17 group,and ece 1Δ/Δgroup had intense protease(SAP)and phospholipase(PL)activities,the lipase(Lip)activity was negative,and M2174 group only had high expression of phospholipase activity.ece1Δ/Δ+ECE 1 strain had the strongest mycelium formation ability,and ece 1Δ/Δstrain had the weakest mycelium formation ability.The mortality rate of mice ranged from 21.43%-57.14%on day 28 after being infected with C.albicans,and from 78.57%-100.0

关 键 词：念珠菌 白色 动物实验 Candidalysin 毒力因子 胞外水解酶 菌丝诱导 

分 类 号：R379.4[医药卫生—病原生物学]