[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mc2155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mc2155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mc2155,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_3312 secondary 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 mc2155 and the knockout strain Δmsmeg_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 mc2155 and Δmsmeg_3312 using an erythromycin ELISA kit.[Results] Initially,we confirmed that MSMEG_3312 is a redox-related hemerythrin-like protein using spectrophotometric and CD analysis.Quantitative proteomic analysis revealed that Δmsmeg_3312 has eight up-regulated proteins,including three transporters,and 14 down-regulated proteins,compared with the wild type strain mc2155,while growing in 7H9 medium.In contrast,448 proteins were identified as being differentially expressed between mc2155 and Δmsmeg_3312,when treated with erythromycin,of which 11 were identified as up-regulated transporter proteins,and 26 were associated with amino acid synthetic pathways.The intracellular erythromycin concentration in Δmsmeg_3312 was also lower than in mc2155.[Conclusion] We show that MSMEG_3312 mediates erythromycin resistance due to collective antibiotic tolerance arising from antibiotic titration and high-density populations.
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