定量蛋白质组分析蚯蚓血红蛋白样蛋白MSMEG_3312介导的分枝杆菌耐红霉素机制（英文）  

Mechanisms of MSMEG_3312-mediated collective antibiotic tolerance to erythromycin in mycobacteria revealed by quantitative proteomic analysis

作　　者：张玉娇[1,2] 胡新玲 李晓静[1] 邓海腾[3] 米凯霞[1,4] Yujiao Zhang;Xinling Hu;Xiaojing Li;Haiteng Deng;Kaixia Mi(CAS Key Laboratory of Pathogenic Microbiology and Immunology,Institute of Microbiology,Chinese Academy of Science,Beijing 100101,China;College of Life Sciences,University of Chinese Academy of Sciences,Beijing 101408,China;School of Life Sciences,Tsinghua University,Beijing 100084,China;Savaid Medical School,University of Chinese Academy of Sciences,Beijing 101408,China)

机构地区：[1]中国科学院微生物研究所,中国科学院病原微生物与免疫学重点实验室,北京100101 [2]中国科学院大学生命科学学院,北京101408 [3]清华大学生命科学学院,北京100084 [4]中国科学院大学存济医学院,北京101408

出　　处：《微生物学报》2018年第12期2186-2203,共18页Acta Microbiologica Sinica

基　　金：国家重点研发计划(2017YFA0505901);国家自然科学基金(31670137,31600114,31700128).

摘　　要：【目的】细菌耐药机制是个复杂的机制,系统生物学是系统性揭示耐药机制的有力研究手段。我们课题组前期研究结果显示,蚯蚓血红蛋白样蛋白msmeg_3312基因敲除后能够增加耻垢分枝杆菌对红霉素的耐药性,本文系统研究MSMEG_3312参与红霉素耐药性形成的机制。【方法】首先纯化MSMEG_3312蛋白,利用光谱及圆二色谱描述MSMEG-3312蛋白。利用定量蛋白质组学的方法比较分析敲除菌株Δmsmeg_3312与野生型菌株mc^2155蛋白表达的差异,并通过qRT-PCR进行验证。利用红霉素ELASA试剂盒测定Δmsmeg_3312与mc^2155的胞内药物浓度。【结果】光谱及圆二色谱分析确定MSMEG_3312是蚯蚓血红蛋白样蛋白。定量蛋白质组学分析发现,红霉素未处理的条件下,相比于野生型菌株mc^2155,敲除菌株Δmsmeg_3312有包括3种转运蛋白在内的8种蛋白表达水平上调,14种蛋白表达下调;而红霉素处理后,Δmsmeg_3312中有448种蛋白差异表达,其中有11种转运蛋白表达上调,26种蛋白与氨基酸合成通路相关。胞内药物浓度检测显示敲除菌株Δmsmeg_3312的胞内红霉素浓度显著低于野生型菌株。【结论】蚯蚓血红蛋白样蛋白MSMEG_3312调控改变了细菌对红霉素药物处理的反应网络,其介导的红霉素耐药是一种集合抗生素耐受机制。[Objective]The effects of antibiotics on bacteria are complex,and bacterial response to antibiotics is just beginning to be understood using systems biology.We previously showed that a hemerythrin-like protein, MSMEG_3312,is involved in erythromycin susceptibility.In this study,we explore the mechanisms of collective antibiotic tolerance to erythromycin in mycobacteria through the hemerythrin-like protein MSMEG_3312. [Methods]We analyzed MSMEG_3312secondary structure using spectrophotometric and circular dichroism (CD) methods.Tandem mass tag(TMT)-labeled quantitative proteomics was used to compare protein level changes between the wild type strain mc^2 155and the knockout strain △msmeg_3312,following bioinformatics analysis. Differentially expressed proteins were also verified by qPCR.To confirm our analyses'conclusions that transporters are involved in MSMEG_3312-related erythromycin susceptibility,we also measured the concentration of mycobacterial erythromycin in vivo in the wild type strain mc2155and △msmeg_3312using an erythromycin ELISA kit.[Results]Initially,we confirmed that MSMEG_3312is a redox-related hemerythrin-like protein using spectrophotometric and CD analysis.Quantitative proteomic analysis revealed that △msmeg_3312has eight up-regulated proteins,including three transporters,and 14down-regulated proteins,compared with the wild type strain mc^2 155,while growing in 7H9 medium.In contrast,448proteins were identified as being differentially expressed between mc^2 155 and △msmeg_3312,when treated with erythromycin,of which 11were identified as up-regulated transporter proteins,and 26were associated with amino acid synthetic pathways.The intracellular erythromycin concentration in △msmeg3312was also lower than in mc^2 155.[Conclusion]We show that MSMEG_3312mediates erythromycin resistance due to collective antibiotic tolerance arising from antibiotic titration and high-density populations.

关 键 词：定量蛋白质组 蚯蚓血红蛋白样蛋白 MSMEG_3312 分枝杆菌 红霉素 耐药性 

分 类 号：R378.91[医药卫生—病原生物学]