Mce4C蛋白参与结核分枝杆菌摄取利用胆固醇的实验研究  

Experimental study on the role of Mce4C in the uptake and utilization of cholesterol by Mycobacterium tuberculosis

作　　者：胡一凡 杜博平 吴亚东 朱传智[1] 张蓝月 贾红彦[1] 孙琦[1] 潘丽萍[1] 张宗德[1] 李自慧[1] Hu Yifan;Du Boping;Wu Yadong;Zhu Chuanzhi;Zhang Lanyue;Jia Hongyan;Sun Qi;Pan Liping;Zhang Zongde;Li Zihui(Beijing Tuberculosis and Thoracic Tumor Research Institute,Beijing Chest Hospital,Capital Medical University,Beijing Key Laboratory for Drug Resistant Tuberculosis Research,Beijing 101149,China)

机构地区：[1]北京市结核病胸部肿瘤研究所/首都医科大学附属北京胸科医院/耐药结核病研究北京市重点实验室,北京101149

出　　处：《中国防痨杂志》2025年第4期444-453,共10页Chinese Journal of Antituberculosis

基　　金：国家自然科学基金(82070012,81702097);北京市自然科学基金(7212012);通州区两高人才工程运河计划项目(YH201908);北京市优秀人才培养资助项目(20081D0301300095)。

摘　　要：目的:探讨Mce4C蛋白是否参与结核分枝杆菌(Mycobacterium tuberculosis,MTB)摄取利用胆固醇。方法:利用定量PCR技术分析不同浓度胆固醇(无胆固醇组、0.01%胆固醇组和0.1%胆固醇组)对MTB mce4C基因表达的影响。通过紫外-可见分光光度计测定培养菌液A 600值绘制生长曲线和扫描电镜分析,了解MTB H37Rv野生株(WT)、mce4C基因敲除株(Δmce4C)和敲除回补株(Δmce4C+mce4C)的生长速率和形态差异。通过试剂盒定量测定或NBD-胆固醇荧光值测定菌体本身、培养基及感染细胞裂解液中胆固醇含量变化,明确Mce4C蛋白是否参与MTB摄取利用胆固醇。通过MTB不同组分分离结合免疫印迹实验明确Mce4C蛋白亚细胞定位。结果:mce4C基因表达量随苏通培养基中胆固醇浓度的升高(无胆固醇组、0.01%胆固醇组和0.1%胆固醇组)而升高,培养1周时mce4C相对表达量分别为1.000±0.588、1.390±0.162和3.622±1.031,差异有统计学意义(F=28.200,P=0.001)。随着胆固醇苏通培养基培养时间(20、40、60、70、80 d)的延长,Δmce4C A 600值相比WT和Δmce4C+mce4C降低逐渐明显,至第80天时,Δmce4C的A 600值(0.913±0.017)明显低于WT(1.245±0.011)和Δmce4C+mce4C(1.246±0.029),差异均有统计学意义(t=28.182,P<0.001;t=17.140,P<0.001)。用胆固醇苏通培养基培养3种菌株时,Δmce4C菌体胆固醇含量相比WT和Δmce4C+mce4C降低,而其培养上清中胆固醇含量升高,至21 d时,Δmce4C菌体总胆固醇含量[(1.058±0.012)μg/ml]明显低于WT[(1.347±0.087)μg/ml]和Δmce4C+mce4C[(1.505±0.021)μg/ml],而培养上清中Δmce4C胆固醇含量[(16.371±0.753)μg/ml]明显高于WT[(7.740±0.422)μg/ml]和Δmce4C+mce4C[(7.274±0.131)μg/ml],差异均有统计学意义(t=4.621,P=0.044;t=25.679,P=0.002;t=-14.135,P=0.005;t=-16.827,P=0.004)。在感染THP-1细胞不同时间点(4、24、48、72 h),Δmce4C感染细胞裂解液中胆固醇含量均明显高于WT和Δmce4C+mce4C,即使在感染4 h最低点时,Δmce4C感染细胞裂�Objective:To investigate whether Mce4C protein is involved in the uptake and utilization of cholesterol by Mycobacterium tuberculosis(MTB).Methods:The impact of different cholesterol concentrations(cholesterol-free group,0.01%cholesterol group,and 0.1%cholesterol group)on the expression of MTB mce4C gene was analyzed by quantitative polymerase chain reaction.Growth curves plotted by measuring A 600 value of cultured bacterial solutions with an ultraviolet-visible spectrophotometer and supplemented by scanning electron microscopy,were used to analyze the differences in growth rate and morphology among the wild-type MTB H37Rv reference strain(WT),mce4C knockout strain(Δmce4C),and its complemented strain(Δmce4C+mce4C).To determine whether Mce4C was involved in the uptake of cholesterol by MTB,the changes of cholesterol levels in bacteria,bacterial medium and lysates of infected cells were measured either by using testing kits to quantitatively measure cholesterol content or by determining the fluorescence value of NBD-cholesterol.The subcellular localization of the Mce4C protein was determined through immunoblotting experiments on separation and combination of different MTB components.Results:The expression level of mce4C gene increased with elevation of cholesterol concentration in Sauton medium.After one week’s culture,the mce4C relative expression levels were 1.000±0.588,1.390±0.162,and 3.622±1.031 respectively for cholesterol-free group,0.01%cholesterol group,and 0.1%cholesterol group,with statistically significant difference(F=28.200,P=0.001).As the culture time in cholesterol-containing Sauton medium extended(20,40,60,70,80 days),the decrease of A 600 value ofΔmce4C became increasingly more pronounced compared to WT andΔmce4C+mce4C.By the 80th day,the A 600 value ofΔmce4C(0.913±0.017)was significantly lower than that of WT(1.245±0.011)andΔmce4C+mce4C(1.246±0.029),with all differences being statistically significant(t=28.182,P<0.001;t=17.140,P<0.001).When the three strains were cultured in cholest

关 键 词：分枝杆菌 结核 胆固醇 营养需要 能量摄取 免疫蛋白质类 

分 类 号：R52[医药卫生—内科学] R-33[医药卫生—临床医学]