两个TetR家族的转录因子介导耻垢分枝杆菌药物抗性调控的分子机制研究

发布时间：2018-04-27 10:42

本文选题：耻垢分枝杆菌 + TetR家族转录调控因子　；参考：《华中农业大学》2016年博士论文

【摘要】：结核病是由结核分枝杆菌(Mycobacterium tuberculosis)引起的一种重要人类传染病,其产生的抗药性问题已经引发了全球范围的广泛关注。然而,致病性的结核分枝杆菌生长非常缓慢,感染性极强,目前对其抗药调控机制的认识仍然十分有限。相比而言,耻垢分枝杆菌(M.smegmatis)生长较快,遗传操作方法成熟,而且很多基因与结核分枝杆菌高度同源,因此已经成为研究病原性分枝杆菌的基因调控和生理代谢特点的重要模式菌株。本研究以耻垢分枝杆菌为研究对象,发现了两个新的TetR家族的转录因子,分别正调控耻垢分枝杆菌对利福平和乙胺丁醇药物的抗性。具体结果如下:(1)Ms4022是耻垢分枝杆菌基因组编码的一个功能未知的转录因子,由199个氨基酸组成,其N端含有一个TetR家族的HTH结构域。通过构建ms4022敲除和超表达重组菌株并测定利福平药物对它们的最小抑菌浓度(MIC),结果发现:ms4022敲除菌株的MIC(3.13μg/ml)比野生型菌株的(6.25μg/ml)低2倍,而超表达菌株的MIC(25μg/ml)大约是野生型菌株的4倍。因此,ms4022基因的表达水平显著影响耻垢分枝杆菌对利福平的抗性。进一步,通过EMSA和DNA足迹实验发现,Ms4022在其自身启动子区域含有有两个结合位点:第一个命名为motif 1,其长度为19 bp,靠近翻译起始位点;第二个则包含了两个motif,分别命名为motif 2和motif 3,它们距离翻译起始位点都较远。序列比对分析显示,这三个motif的5’端9 bp序列的同源性很高,具有明显的保守性。利用Ms4022识别的3个motif在全基因组范围内搜索耻垢分枝杆菌的启动子区域,发现Ms4022可广泛调节约315个潜在靶基因的表达。有趣的是,这些潜在靶基因中包括有31个运输相关的基因(占9.84%)。随后,采用EMSA和Ch IP实验证实Ms4022在耻垢分枝杆菌体内体外能与这些运输相关基因的靶启动子序列特异性结合。进一步采用qRT-PCR分析发现,与野生型菌株相比,ms4022敲除菌株中绝大部分这些运输相关靶基因的表达量都明显下调;相反,ms4022超表达菌株中它们的表达量则显著上调。进一步的β-半乳糖苷酶活实验也清楚证明,Ms4022正调控这些基因的表达。最后,通过分别构建这些运输相关靶基因的超表达菌株并测定生长曲线,结果发现7个靶基因的超表达能够特异性增强耻垢分枝杆菌对利福平的抗药性。综合这些结果,Ms4022是一个广泛调控因子,发挥一个转录激活子的作用,它通过正调控与运输相关的靶基因的表达,增强耻垢分枝杆菌对利福平的抗药性。(2)LerR是耻垢分枝杆菌基因组编码的另一个转录因子,含有Lux R结构域,也属于TetR家族;它与激酶LerS组成一对双组分系统,共同调节耻垢分枝杆菌对抗结核药物乙胺丁醇的抗性。通过构建lerR超表达菌株并测定它在不同抗结核药物胁迫下的生长曲线,结果发现:与野生型菌株相比,超表达菌株对乙胺丁醇的抗性能力有显著提高。随后,EMSA和Ch IP实验证实LerR能够在体内和体外特异性地结合Ms6242和Ms6239(1,3-丙二醇脱氢酶PDH)的启动子序列。进一步的qRT-PCR分析发现,与野生型菌株相比,lerR敲除菌株中PDH以及操纵子Ms6242、Ms6241、Ms6240的表达量都显著下降;相反,lerR超表达菌株中这些靶基因的表达量则增加。比较转录组分析发现,与没有药物胁迫相比,乙胺丁醇处理下的耻垢分枝杆菌中lerR及其靶基因PDH、Ms6242、Ms6241、Ms6240的表达量增加了2-4.5倍,说明这些基因均能响应乙胺丁醇的刺激。最后,通过分别构建LerR靶基因的超表达菌株并测定生长曲线,发现只有超表达PDH的耻垢分枝杆菌对乙胺丁醇的抗药性增加。因此,LerR在耻垢分枝杆菌中也是一个正调控蛋白,通过激活包括PDH在内的多个靶基因的表达,调节耻垢分枝杆菌对乙胺丁醇的抗性。
[Abstract]:Tuberculosis is an important human infectious disease caused by Mycobacterium tuberculosis (Mycobacterium tuberculosis), and its resistance to drug resistance has attracted worldwide attention. However, the pathogenicity of Mycobacterium tuberculosis is very slow and highly infectious, and the understanding of its anti drug regulation mechanism is still limited. In comparison, Mycobacterium foul Mycobacterium (M.smegmatis) grows rapidly, the genetic manipulation is mature, and many genes are highly homologous with Mycobacterium tuberculosis. Therefore, it has become an important model for the study of gene regulation and physiological metabolism of Mycobacterium tuberculosis. This study was based on the study of Mycobacterium foul Mycobacterium and found two The transcriptional factor of the new TetR family regulates the resistance of Mycobacterium tumefaciens to rifampicin and ethambutol respectively. The specific results are as follows: (1) Ms4022 is a functional unknown transcription factor, composed of 199 amino acids, which contains a HTH domain of the TetR family in the N end of the Mycobacterium tumefaciens genome. Through the construction of ms4022 The recombinant strain was knocked out and overexpressed and the minimal inhibitory concentration (MIC) of rifampin was measured. The results showed that the MIC (3.13 mu g/ml) of the ms4022 knockout strain was 2 times lower than that of the wild type (6.25 mu g/ml), and the MIC (25 mu g/ml) of the overexpression strain was about 4 times that of the wild type. Therefore, the expression level of ms4022 gene significantly affected the disgrace scale. The resistance of Mycobacterium to rifampin. Further, through EMSA and DNA footprints experiments, it is found that Ms4022 has two binding sites in its own promoter region: the first is named motif 1, and its length is 19 BP, close to the translation initiation site, and the second contains two motif, named motif 2 and motif 3 respectively, which are from the beginning of translation. The sequence alignment analysis showed that the 5 'end 9 BP sequences of the three motif were highly homologous and conserved. Using 3 motif identified by Ms4022 to search the promoter region of Mycobacterium scale in the whole genome, it was found that Ms4022 could regulate the expression of about 315 potential target genes. 31 transport related genes were included in the target gene (9.84%). Subsequently, EMSA and Ch IP experiments were used to confirm that Ms4022 can specifically bind to the target promoter sequences of these transport related genes in vitro and in vitro. Further qRT-PCR analysis was used to find that most of the ms4022 knockout strains were compared with the wild type strains. The expression of these transport related target genes was obviously down; on the contrary, the expression of ms4022 overexpressed strains was significantly up-regulated. Further beta galactosidase experiment also clearly demonstrated that Ms4022 was regulating the expression of these genes. Finally, the overexpressed strains of these transport related target genes were constructed and the growth was determined. The results show that the overexpression of 7 target genes can specifically enhance the resistance of Mycobacterium tumefaciens to rifampicin. These results suggest that Ms4022 is a wide regulator and plays a role in a transcriptional activator. It enhances the resistance of Mycobacterium tumefaciens to rifampicin by regulating the expression of target genes associated with transport. (2) (2) LerR is another transcription factor that encodes the genome of Mycobacterium tumefaciens, which contains the Lux R domain and also belongs to the TetR family; it forms a pair of two component systems with kinase LerS to regulate the resistance of Mycobacterium tumefaciens to the anti tuberculosis drug ethambutol. By constructing a lerR superexpression strain and determining it in different anti tuberculosis drug coerced strains. The results showed that the resistance ability of the overexpression strain to ethambutol was significantly improved compared with the wild strain. Subsequently, EMSA and Ch IP experiments confirmed that LerR could specifically combine the promoter sequence of Ms6242 and Ms6239 (1,3- propanediol dehydrogenase PDH) in vivo and in vitro. The expression of PDH, operon Ms6242, Ms6241 and Ms6240 in the lerR knockout strains decreased significantly compared with the strain of the strains; on the contrary, the expression of these target genes increased in the lerR overexpression strain. The comparison of the transcriptional analysis found that the lerR and its target gene PDH, Ms624 in ethambutol under the absence of drug stress, lerR and its target gene PDH, Ms624 2, the expression of Ms6241 and Ms6240 increased by 2-4.5 times, indicating that all the genes could respond to the stimulation of ethambutol. Finally, the growth curve was determined by constructing the overexpressed strains of the LerR target gene and the growth curve was measured. It was found that only the overexpressed PDH was anti drug of ethambutol. Therefore, LerR was also one of the mycobacteria in Mycobacterium foul. A positive regulatory protein regulates the resistance of Mycobacterium tuberculosis to ethambutol by activating multiple target genes including PDH.

【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R378.91

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