结核分枝杆菌ABC转运蛋白Rv1473的功能研究

发布时间：2018-06-05 01:09

本文选题：抗生素耐受结核分枝杆菌 + Rv1473　；参考：《西南大学》2017年硕士论文

【摘要】：细菌对抗生素的耐受已经成为全球面临的大问题,特别是多重耐药菌的出现,给临床上抗菌治疗带来极大的困难,一些原本具有良好效果的药物也失去了疗效,对耐药菌感染的治疗迫在眉睫。细菌对抗生素的耐药有多种途径,药物靶基因的突变和药物外排泵的活动是其中两个重要途径。药物外排泵可以把抗生素主动排出,降低胞内抗生素含量,实现药物耐受。外排泵的作用除了直接降低胞内抗生素浓度外,还会提高细菌耐药突变率,造成高水平、多种抗生素交叉耐药。因此,对药物外排泵的深入研究,对于临床上抗菌治疗具有重要意义。结核病是由结核分枝杆菌引起的,仅次于艾滋病的第二大传染病,极大地威胁全球人类的健康。临床上结核分枝杆菌的耐药同样严重,特别是近年来出现的多耐药、广泛耐药甚至是全耐药结核分枝杆菌的出现,再加上和HIV的共感染,结核病的治疗形势非常严峻,结核分枝杆菌耐药机理的研究对于结核病的控制和治疗十分重要。药物外排泵的活动也是结核分枝杆菌耐药的重要途径,根据分子结构及转运底物的不同,结核分枝杆菌外排泵可以分为5个亚家族,即ATP结合盒超家族(ABC)、主要易化超家族(MFS)、耐受小节分裂区家族(RND)、小耐多药性家族(SMR)及多药及毒性外排家族(MATE),其中ABC型外排泵需要ATP水解驱动,其他四种需要依靠质子动力势驱动。在结核分枝杆菌中有一些外排泵被发现参与抗结核药物的外排,包括异烟肼、利福平、氨基糖苷类、氟喹诺酮类等。本课题以结核分枝杆菌Rv1473为研究对象,Rv1473属于ABC亚家族,含有NBD结构域,可以结合和水解ATP,生物信息学预测其可能是大环内酯类外排泵。为了研究Rv1473蛋白的功能,我们首先在大肠杆菌中构建了Rv1473基因的过表达菌株,表达并纯化了Rv1473重组蛋白,通过ATPase检测试剂盒发现Rv1473重组蛋白可以和ATP结合,具有较高的ATPase活性,表明Rv1473可能水解ATP为物质跨膜转运提供能量。生长快、无毒的耻垢分枝杆菌是研究致病性分枝杆菌的理想模型,耻垢分枝杆菌MSMEG_3140和Rv1473是同源基因,其编码蛋白的氨基酸序列同源性在80%以上。我们通过同源重组的方法构建了耻垢分枝杆菌MEMEG_3140基因的敲除菌株(△MSMEG_3140),并构建了回补菌株(△MSMEG_3140::Rv1473)。我们发现相比较野生型,△MSMEG_3140缺失菌株对大环内酯类更敏感,而且缺失菌株在相同时间内积累的EB更多,回补菌株可以恢复到野生表型,这表明Rv1473可能和大环内酯类外排有关系。我们用RT-PCR检测了耻垢分枝杆菌MSMEG_3140在大环内酯类处理下转录水平,发现MEMEG_3140在大环内酯类处理下表达上调。根据转录组数据显示Rv1473在大环内酯类家族成员罗红霉素处理下表达上调,为了验证这个结果,我们测定了Rv1473的启动子在大环内酯类处理下的活性,发现其启动子活性增强,以上结果可以充分说明Rv1473是一个大环内酯类外排泵。另外,有文章报道外排泵蛋白可能参与生物膜的形成,我们测定了野生型、缺失型和回补型菌株的生物膜形成能力,并没有显著性差异,说明Rv1473可能不参与耻垢分枝杆菌生物膜的形成。Rv1473和ctpD、trxA、trxB、echA12和Rv1473A共转录,我们推测Rv1473可能和ctpD合作共同完成大环内酯类的跨膜转运。Rv1473可能参与到whiB7介导的结核分枝杆菌对大环内酯类的天然抗性调节过程中。本论文首次揭示了Rv1473是一个新的大环内酯类外排泵,所得的初步研究结果将为深入开展以外排泵为靶点相关抑制剂的开发提供参考。
[Abstract]:The tolerance of bacteria to antibiotics has become a major problem facing the world, especially the emergence of multidrug-resistant bacteria, which brings great difficulties to the clinical antibiotic treatment. Some drugs that have good effects have also lost the curative effect. The treatment of antibiotic resistant bacteria is imminent. The mutation and the activity of the drug efflux pump are two important ways. The drug efflux pump can discharge antibiotics voluntarily, reduce the intracellular antibiotic content and achieve drug tolerance. The efflux pump can increase the rate of bacterial resistance in addition to the intracellular antibiotic concentration directly, and cause a high level of antibiotic cross resistance. Therefore, the in-depth study of the drug efflux pump is of great significance for the clinical antibacterial treatment. Tuberculosis is caused by Mycobacterium tuberculosis, which is second only to the second major infectious diseases of AIDS, which greatly threatens the health of the world. Drug-resistant and even all drug-resistant Mycobacterium tuberculosis, combined with the co infection of HIV, is very severe in the treatment of tuberculosis. The study of the mechanism of Mycobacterium tuberculosis is important for the control and treatment of tuberculosis. The activity of the drug efflux pump is also an important way for the drug resistance of Mycobacterium tuberculosis, according to the molecular structure and transport. Different substrates, Mycobacterium tuberculosis efflux pump can be divided into 5 subfamilies, the ATP binding cassette superfamily (ABC), the main superfamily (MFS), the small division family (RND), the small resistant multidrug-resistant family (SMR) and the multidrug and toxic efflux family (MATE), in which the ABC type pump needs ATP hydrolysis drive and the other four need to rely on proton movement. Force potential drive. In Mycobacterium tuberculosis, some of the outer row pumps are found to participate in anti tuberculosis drugs, including isoniazid, rifampicin, aminoglycosides, fluoroquinolones, etc. this topic is based on Mycobacterium tuberculosis Rv1473. Rv1473 belongs to the ABC subfamily and contains the NBD domain, which can combine and hydrolyze ATP and predict bioinformatics. It may be a macrolide efflux pump. In order to study the function of Rv1473 protein, we first constructed the overexpressed strain of Rv1473 gene in Escherichia coli, expressed and purified the Rv1473 recombinant protein. The ATPase detection kit found that the recombinant protein of Rv1473 can be combined with ATP and has a high ATPase activity, indicating that Rv1473 may be water. The solution of ATP provides energy for the transmembrane transport of material. Fast growing and non-toxic Mycobacterium tumefaciens are ideal models for the study of pathogenic mycobacteria. MSMEG_3140 and Rv1473 are homologous genes, and the amino acid sequences of the encoded proteins are more than 80%. We constructed a Mycobacterium foul Mycobacterium MEMEG_31 by homologous recombination. The 40 gene knockout strain (delta MSMEG_3140) and the remedial strain (delta MSMEG_3140:: Rv1473) were constructed. We found that the wild type, the delta MSMEG_3140 deletion strain was more sensitive to the macrolide, and the missing strain accumulated more EB in the same time, and the remedial strain could be restored to the wild phenotype, which indicates that Rv1473 may be in the large ring. We used RT-PCR to detect the transcriptional level of MSMEG_3140 in macrolide treatment, and we found that MEMEG_3140 was up regulated under macrolide treatment. According to the transcriptional data, the expression of Rv1473 was up regulated by roxithromycin, a member of the macrolide family, in order to verify this result. The activity of Rv1473 promoter under the macrolide treatment was measured, and the promoter activity was found to be enhanced. The above results showed that Rv1473 was a macrolide efflux pump. In addition, it was reported that the outer row pump protein may be involved in the formation of biofilm. We have measured the biofilm of the wild type, the deletion type and the supplement type. There is no significant difference in formation ability, indicating that Rv1473 may not participate in the formation of.Rv1473 and ctpD, trxA, trxB, echA12 and Rv1473A in Mycobacterium tumefaciens biofilm. We speculate that Rv1473 may cooperate with ctpD to complete the transmembrane trans membrane transport of macrolides, which can be involved in whiB7 mediated Mycobacterium tuberculosis in the large ring. During the natural resistance regulation of esters, this paper first revealed that Rv1473 is a new macrolide efflux pump. The results of the preliminary study will provide a reference for the development of the target related inhibitors for the external exhaust pump.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R378.911

【相似文献】