ATP1通过调节氧化应激促进白念珠菌逃逸巨噬细胞杀伤的研究  

ATP1 promotes Candida albicans to escape from macrophage killing through regulating oxidative stress

作　　者：吕妍 张艳丽 张展鹏 赵亚婧 张艺山[1] 李水秀[1] 张宏[1] Lyu Yan;Zhang Yanli;Zhang Zhanpeng;Zhao Yajing;Zhang Yishan;Li Shuixiu;Zhang Hong(Department of Dermatology,Institute of Mycology,The First Affiliated Hospital of Jinan University,Jinan 510632,Guangdong,China)

机构地区：[1]暨南大学附属第一医院皮肤科,暨南大学附属第一医院真菌病研究所,广州510632

出　　处：《中华皮肤科杂志》2020年第7期519-524,共6页Chinese Journal of Dermatology

基　　金：国家自然科学基金(81971913、81471995、81903675);中国博士后科学基金(2019M653291)。

摘　　要：目的通过反向遗传学方法,探索白念珠菌F1Fo-ATP合酶α亚基编码基因(ATP1)通过清除细胞内活性氧以逃逸巨噬细胞杀伤的生理作用。方法以白念珠菌ATP1缺失株及其亲本株为研究对象,接种平板培养后计算菌落数评估其体外细胞活性,通过尾静脉接种小鼠后计算肾脏组织形成菌落数评估体内细胞活性。将培养过夜的白念珠菌菌液与巨噬细胞共培养后,接种平板,计算菌落数,测定菌的存活率,通过乳酸脱氢酶释放法测定巨噬细胞乳酸脱氢酶水平;以过氧化氢建立模拟巨噬细胞内氧化应激模型,过氧化氢作用白念珠菌后,通过计算菌落数比较各菌株细胞活性,采用DCFH-DA染色测定细胞内活性氧水平,实时荧光定量PCR检测过氧化氢酶1(CAT1)基因、超氧化物歧化酶4(SOD4)基因和超氧化物歧化酶5(SOD5)基因mRNA水平。采用双因素方差分析或Studentt检验对数据进行统计学分析。结果在体外,亲本株组和ATP1缺失组菌落数随培养时间逐渐增多,24 h后ATP1缺失组菌落数仅为亲本株组的10%,差异有统计学意义(F=481.84,P<0.001)。在小鼠体内,亲本株组肾脏组织形成菌落数随时间逐渐增多,但是ATP1缺失组逐渐减少,两组差异有统计学意义(F=78.27,P=0.001)。与体内实验结果一致,体外白念珠菌菌体与巨噬细胞共培养后,ATP1缺失组白念珠菌存活率(62.67±3.51)%比亲本株组(82.33±2.52)%显著降低(t=7.88,P=0.001),与ATP1缺失组共培养的巨噬细胞乳酸脱氢酶释放百分比(27.80±3.54)%比与亲本株组共培养的巨噬细胞(87.78±0.17)%显著降低(t=33.89,P<0.001)。在模仿的巨噬细胞内氧化应激模型中,ATP1缺失组细胞活性比亲本株组显著降低,差异有统计学意义(F=3440.65,P<0.001)。在与巨噬细胞共培养以及模仿的巨噬细胞内氧化应激模型中,ATP1缺失组细胞内活性氧水平均比亲本株组显著增高,差异有统计学意义(均P<0.001)。ATP1缺失组CAT1、SOD4、SOD5 mObjective To investigate the physiological role of F1Fo-ATP synthaseα-subunit encoding gene(ATP1)in promoting Candida albicans(C.albicans)to escape from macrophage killing through eliminating intracellular reactive oxygen species(ROS)by using a reverse genetics approach.Methods An ATP1 deletion strain and a parental strain of C.albicans were cultured on the YPD media,and the number of formed colonies on the plates was counted to evaluate in vitro viability of C.albicans.To evaluate their in vivo viability,the ATP1 deletion strain and parental strain of C.albicans were inoculated into mice through the caudal vein,kidney tissues were taken out from the mice 1-7 days after the infection,and inoculated onto the YPD medium followed by numeration of colonies after 48 hours of culture.After co-culture of overnight-cultured C.albicans suspensions with macrophages,some of the C.albicans suspensions were inoculated onto the YPD solid medium followed by numeration of colonies and determination of survival rate,and some culture supernatants were inoculated into the 96-well plate for detection of the level of lactate dehydrogenase(LDH)released by macrophages by LDH release assay.A model mimicking oxidative stress in macrophages was established by using hydrogen peroxide.After treatment with hydrogen peroxide,the number of colonies was counted to compare the viability of the C.albicans strains.DCFH-DA staining was conducted to detect the intracellular ROS level in C.albicans after co-culture with macrophages or treatment with hydrogen peroxide,and real-time fluorescence-based quantitative PCR to measure mRNA expression of catalase 1(CAT1),superoxide dismutase 4(SOD4)and SOD5 genes in C.albicans after treatment with hydrogen peroxide.Statistical analysis was carried out by using two-way analysis of variance or Student t test.Results In vitro,the colony number in both the parental strain group and ATP1 deletion strain group gradually increased over time;after 24 hours,the colony number of the ATP1 deletion strain group was only

关 键 词：白念珠菌 氧化性应激 巨噬细胞 ATP1基因 

分 类 号：R519.3[医药卫生—内科学]