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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
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国家重点研发计划(2017YFA0505901,2014CB744402);国家自然科学基金(31670137,31600114,31700128)


Mechanisms of MSMEG_3312-mediated collective antibiotic tolerance to erythromycin in mycobacteria revealed by quantitative proteomic analysis
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    [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mc2 155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mc2 155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mc2 155,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。

    Abstract:

    [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 mc2 155 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 mc2 155 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 mc2 155, while growing in 7H9 medium. In contrast, 448 proteins were identified as being differentially expressed between mc2 155 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 mc2 155.[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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张玉娇,胡新玲,李晓静,邓海腾,米凯霞. 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. 微生物学报, 2018, 58(12): 2186-2203

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  • 收稿日期:2018-01-26
  • 最后修改日期:2018-03-30
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  • 在线发布日期: 2018-12-05
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