【作者】 商正玲；

【导师】 鲍朗；

【作者基本信息】 四川大学 ， 免疫学， 2007， 博士

【摘要】 结核病是由结核分枝杆菌(mycobacterium tuberculosis，MTB)引起的一种慢性传染病，已困扰人类数千年。尽管目前的正规联合治疗方案(DOTS)取得良好的效果，但与其他感染性疾病相比，该病的治疗存在病程迁延、易复发的问题。资料表明全球大约有1/3的人口具有结核分枝杆菌的潜伏感染，随着社会老龄化、HIV感染和耐药菌株的流行，这一问题日渐突出，成为人类防治结核病的重要“瓶颈问题”。结核分枝杆菌的持留状态或称持留菌(Mycobacterial tuberculosis persister)被认为是结核病病程迁延、复发的根本原因。因此，探索MTB长期持留的分子机制，寻找抗结核潜伏感染的有效途径将为人类战胜结核病带来新的希望。目前关于结核分枝杆菌持留状态的分子生物学机制众说纷纭。近年在原核生物染色体基因组上广泛存在被认为参与原核生物基因表达质量控制的TA系统(Toxin-antitoxin model)可能与细菌在压力因素下的长期存活密切相关。Gerdes等通过比较基因组学方法发现在结核杆菌H37Rv株中含有38个TA loci，其中包括3个relBE位点。但关于这些基因在结核分枝杆菌中的生物学意义国内外鲜见报道。因此对这些未知功能的假想蛋白基因进行研究不仅对结核分枝杆菌后基因组学的研究增添实验资料，而且可能对深入认识、理解结核杆菌持留状态的分子生物机制提供新的研究方向。本研究在文献资料和生物信息学分析的基础上以结核杆菌H37Rv株基因组DNA为模板，首次对结核分枝杆菌Rv1246c和Rv1247c基因进行克隆和融合表达，并构建了pMV-relBE(含Rv1246c-Rv1247c重叠基因)大肠杆菌—分枝杆菌重组表达穿梭质粒，用电穿孔方法将该质粒导入快速生长型非致病性耻垢分枝杆菌mc~2155株，并对该重组菌在体外压力环境和巨噬细胞内的存活情况及对巨噬细胞功能影响进行实验研究，初步探索目的基因在菌体存活、繁殖速率以及抵抗体内外环境压力等方面的功能；同时利用酵母双杂交系统，在酵母细胞体内观察Rv1246c-Rv1247c基因编码假想蛋白间的相互作用，为研究该对基因是否具有TA系统抗毒素—毒素通用模式提供实验依据。以重组耻垢分枝杆菌MCBE(负载Rv1246c-Rv1247c重叠基因)和MC261(负载pMV261空质粒)为实验对象，在体外营养丰富，培养条件适宜的环境下监测细菌的生长曲线，表明二者增殖特性相似，外源基因在该条件下不影响正常耻垢分枝杆菌的生长。在与上述相同培养条件下分别施加不同时间的热压力(53℃)和酸压力(pH 4.0)，通过对不同压力时间处理后细菌CFU(克隆形成单位)变化的统计分析，推测结核Rv1246c-Rv1247c基因具有在体外压力环境中使菌体增殖延缓、通过调整菌体生长代谢状态以对抗环境压力的分子功能。感染小鼠ANA-1巨噬细胞的实验表明：与对照组相比较，负载Rv1246c-Rv1247c重叠基因的重组耻垢分枝杆菌MCBE感染巨噬细胞后其胞内繁殖速度较缓慢，且表现为触发感染细胞凋亡的能力较弱及刺激细胞明显活化反应相对迟缓的现象。酵母双杂交实验表明结核Rv1246c和Rv1247c基因编码的假想蛋白在酵母细胞内可以相互作用，推测二者具有TA系统抗毒素—毒素蛋白共同的胞内天然复合体形式。综上所述，本研究从生物信息学角度推测结核分枝杆菌Rv1246c和Rv1247c基因编码的假想蛋白属于原核生物TA系统relBE家族；构建能够表达Rv1246c和Rv1247c基因编码假想保守蛋白的重组耻垢分枝杆菌能较对照组更易对抗体内外压力及抵御小鼠巨噬细胞活化、凋亡等作用，酵母双杂交系统明确二者具有胞内相互作用的功能，初步证实结核分枝杆菌Rv1246c-Rv1247c基因具有TA系统relBE家族压力反应元件的功能，在国内外尚属首次报道。本研究为研究结核分枝杆菌基因组TA系统的功能提供重要的实验资料，为探索结核杆菌菌体持留状态的分子机制提供了新的研究思路。更多还原

【Abstract】 Tuberculosis(TB) is one of the chronic and infectious diseases, caused by a causative bacterial agent, Mycobaterium tuberculosis(MTB). Although the directly observed treatment short-course (DOTS) has good effct on treatment of TB, the long term of the disease and its relapse,however, are the main barrier of the control of TB. It is estimated that one third of the world population harbor persistent M.tuberculosis infection. With the human immunodeficiency virus (HIV) epidemic unabated, population aging and the emergency of multidrug resistant strains of tubercle bacillus wordwide, those latently infected individuals make the control of TB more difficult. As the persitent states have been realized a major obstacle to eradicating M.tuberculosis infection, undoubtedly, to understand the molecular mechanisms operative in persistence will lead to new and more effective strategies to control or eradicate this phathogen.It is still unclear about the molecular mechanisms of how M.tuberculosis enter the persistent stage. Recent findings showed that prokaryotic chromosomes code for toxin-antitoxin (TA) loci are stress-response elements that help cells survive unfavorable growth conditions, which offer the quality control of gene expression. Gerdes, K et al surprisingly identified that M.tuberculosis H37Rv and CDC1551 have 38 and 36 TA loci, respectively, including 3 relBE family. Researchers also found that free-living slowly growing prokaryotes had particularly many TA loci and put forward the hypothesis that the number of TA loci may be beneficial for organisms characterized by slow growth i.e. the chromosome of M.smegmatis, a fast-growing close relative of M.tuberculosis , encodes two TA loci only. Despite the large amount of TA loci in M. tuberculosis genome, little is known about its functions in the pathogensis of the organism. Futher study on the functions of these hypothetical proteins encoded by M.tuberculosis chromosomes TA loci will not only be accelerated the biological knowledge of M.tuberculosis in the post-genome era but also provide a new ideal to clarify the persistent mechanisms of the causative pathogen.According to the bioinformation analysis, there exist homologies, to some extent, between hypothetical proteins of Rv1246c and Rv1247c gene and that of relBE family, one of E. coli chromosomal TA loci. We amplified the gene Rv1246c and Rv1247c from the genome of H37Rv for the first time to make an attempt to investigate the functions of them. The recombinant fused expression plasmid pET-relB and pET-relE were constructed respectively. The recombinant plasmids could express the fusion protein stably, thus provided the basis for the further study of the gene Rv1246c and Rv1247c. After being carefully analyzed the overlap gene Rv1246c-Rv1247c was proved to be a unique sequence in the M.tuberculosis Complex, a recombinant plasmid bearing gene Rv1246c-Rv1247c was constructed using the E.coil-Mycobacterium shuttle vector pMV261. Then it was electroporated into avirulent Mycobacterium smegmatis mc~2155 which is lack of the sequence. The transformants were induced to express the predicted proteins of Rv1246c and Rv1247c. Under the heat stress and acid stress condition respectively in vitro, the viability of the Recombinant Mycobacterium smegrnatis (MCBE) was evaluated. According to the stress assays, we suggested that the functions of gene Rv1246c-Rv1247c could inhibit cell growth and reduce the number of colony-forming units, which offer advantages to bacteria when the organisms lived under limiting growth conditions in vitro. Furthermore, we employed the MCBE recombinant strian to infect mouse bone marrow macrophage line ANA-1. The viability of the infected ANA-1 cells, the intracellular survival assay of the mycobacteria, ANA-1 cells apoptosis and the expression levels of iNOS mRNA and NO in ANA-1 cells were detected at different times postinfection. The transformant containing Rv1246c-Rv1247c showed more advantages to survive and a lower speed to be eliminated by macrophages than the control group. Although the infected ANA-1 cells underwent obvious apoptosis compared with normal cells as control, but the recombinant M.smegmatis only containing the pMV261 plasmid could significantly induce more ANA-1 apoptosis than the recombinant ones bearing the gene Rv1246c-Rv1247c. Worthy of note, the results of the activity response of macrophages indicated that the MCBE strain also could inhibit the excessively activity response of the mice macrophage ANA-1 cells.To investigate the protein-protein interaction between Rv1246c and Rv1247c in vivo, the genes were amplified from M. tuberculosis H37Rv genome DNA. The Rv1247c gene and Rv1246c gene were subcloned into pGBKT7 and pGADT7-Rec respectively. The recombinant vectors were transformed into yeast cell AH109. Rv1246c and Rv1247c protein could interact with each other in yeast cells as shown from the results. It also showed that the protein pairs encoded by Rv1246c and Rv1247c have the basic proterties that are required to be the TA module.It is the first time to study the biological functions of Rv12461-Rv1247c gene of M. tuberculosis by constructing recombinant M. smegmatis. In summary, Rv1246c-Rv1247c may be the stress-response element and belong to the relBE family of TA system. It also is a new way and important information to provide insights into the mechanism that M.tuberculosis has evolved into adopt its successful highly model of persistence.更多还原