结核分枝杆菌致病基因簇的全基因组偶联定位及功能分析

发布时间：2018-03-16 07:33

本文选题：肺结核　切入点：网络meta分析　出处：《吉林大学》2015年博士论文　论文类型：学位论文

【摘要】：肺结核（pulmonary tuberculosis）是由结核分枝杆菌(Mycobacteriumtuberculosis, MTB)感染引起的以侵袭肺部为主的慢性传染病。目前，肺结核已经成为危害人类公共健康的全球性问题。据世界卫生组织（world healthorganization，WHO）发布的统计结果，2010年全球肺结核新发病例880万，约有145万人死于肺结核。随着抗生素的广泛使用，结核分枝杆菌的耐药问题日益严重，随之产生的多重耐药肺结核(multidrug resistance tuberculosis, MDR-TB)，甚至广泛耐药肺结核(extensively drug tuberculosis resistant, XDR-TB)更成为了全球结核病防控的主要威胁。普通的肺结核服用一线抗结核药物，半年左右即可治愈，而耐药肺结核的治疗周期一般不少于18个月，且所服药物的副作用较之普通药物更大，但治疗效果却十分有限。因此，在肺结核的治疗过程中，选择有效性及安全性俱佳的药物是耐药肺结核治疗的关键因素。同时，避开结核分枝杆菌传统药物产生的细菌耐药性，开发针对结核分枝杆菌的有效作用靶点的新型药物迫在眉睫。结核分枝杆菌作为结核病的病原菌，其致病基因成分复杂，在病原菌的进化过程中，基因水平转移(horizontal gene transfer，HGT)是目前结核分枝杆菌致病的主要推动因素。毒力岛(pathogenicity island，PAI)正是来源于大片段DNA的水平转移，位于基因组的特殊区域，可以将若干功能基因进行有效偶联，实现基因功能的互补及整合，是病原菌毒力进化的新方式。近年来随着高通量生物计算技术的快速发展，使病原菌毒力岛的高通量筛选成为可能。对结核分枝杆菌毒力岛的泛基因组扫描及功能解析将为我们全面注释该病原菌的致病机制奠定分子基础，对于结核分枝杆菌的诊断，疫苗及药物研发具有至关重要的作用。多重耐药肺结核较之药物敏感性肺结核疗程长、费用大且死亡率高。尽管目前有很多对于抗MDR-TB药物的报道，但还没有公认的有效药物用于MDR-TB的治疗。本研究通过网络meta分析的方法旨在对现有6种抗MDR-TB药物的有效性及安全性加以评估，寻找有效性和安全性俱佳的抗结核药物用于耐药肺结核的临床用药。本研究使用贝叶斯网络meta分析模型，通过6种抗MDR-TB药物的随机临床实验来评估抗结核药物贝宁喹啉、delamanid、利奈唑胺、左氧氟沙星、甲硝唑、莫西沙星的有效性和安全性。本研究系统分析了13项随机临床实验（12篇文献及ClinicalTrials.gov中的8项随机临床实验），1549例样本，使用比值比（oddsratios，OR）作为效应量。结果发现，6种抗耐药结核药物间的有效性和安全性具有统计学差异，利奈唑胺可能是最为有效的药物，随之依次为贝宁喹啉、delamanid、左氧氟沙星、莫西沙星和甲硝唑。毒力岛是一组紧密连锁且功能上密切相关的大分子DNA水平转移片段，，它编码重要的毒力调控成分，毒力岛在病原菌的进化过程中通过基因重组的方式进入宿主菌，从而赋予了宿主菌新的毒力特征，也为病原菌的致病机制提供分子基础。本研究通过对结核分枝杆菌基因组任意1000bp片段4-mer核苷酸字符串频率谱的计算实现结核分枝杆菌的基因组条形码可视化注释，通过异常条形码区域欧式距离的计算及筛选，实现对结核分枝杆菌毒力岛的泛基因组扫描，随后应用Pfam_Scan和Blast2GO功能注释工具对已筛选得到的毒力岛进行功能分析。通过上述分析，我们共筛选得到三个结核分枝杆菌毒力岛区域，其中即包括了功能已知的毒力岛MPI-2和MPI-3，也包括功能未知的毒力岛MPI-1。MPI-2和MPI-3这2个毒力岛均携带编码PE/PPE家族蛋白的基因，MPI-2编码so-called Type VIIsecretion systems，功能分析显示其可作为结核分枝杆菌早期感染的诊断标志；MPI-3主要参与细胞壁中霉菌酸的合成及与宿主细胞的相互作用，功能分析前世其可作为抗结核药物的重要分子靶点；MPI-1的功能目前尚不清楚，但是通过蛋白结构域分析显示，此毒力岛上的基因可能编码integral membrane nitriteextrusion protein，促进结核分枝杆菌从有氧生长转变为非复制持留状态(nonreplicating persistence, NPR)，与结核分枝杆菌的持续性感染密切相关。基因组条形码技术整合了基因组更多的遗传信息，具有更高的分辨率。通过对结核分枝杆菌毒力岛的泛基因组扫描及功能解析将为我们全面注释该病原菌的致病机制奠定分子基础，也对结核分枝杆菌的诊断及疫苗和药物的研发起到至关重要的作用。
[Abstract]:Pulmonary tuberculosis (pulmonary tuberculosis) by Mycobacterium tuberculosis (Mycobacteriumtuberculosis, MTB) infection caused by the invasion of lung infection and chronic diseases. Currently, tuberculosis has become a global problem endangering human public health. According to the WHO (World HealthOrganization WHO) statistics released in 2010, 8 million 800 thousand new cases of pulmonary tuberculosis in the world. About 1 million 450 thousand people died of tuberculosis. With the widespread use of antibiotics, drug resistance of Mycobacterium tuberculosis is becoming increasingly serious, resulting in multiple drug resistant pulmonary tuberculosis (multidrug resistance, tuberculosis, MDR-TB) and extensively drug-resistant tuberculosis (extensively drug tuberculosis resistant, XDR-TB) has become a major threat to the global TB prevention and control of tuberculosis in general. The first-line anti tuberculosis drugs can be cured for about half a year, and a cycle of treatment of drug resistant tuberculosis Like not less than 18 months, and the service is better than the ordinary drug side effects of drugs are more, but the treatment effect is very limited. Therefore, in the treatment of pulmonary tuberculosis, drug efficacy and safety and is a key factor in drug resistant tuberculosis treatment. At the same time, avoid the bacterial resistance of Mycobacterium tuberculosis the traditional drugs, new drug targets for the effective development of Mycobacterium tuberculosis is imminent. Mycobacterium tuberculosis as a pathogen of tuberculosis, the causative gene of complex components, in the process of evolution of pathogenic bacteria, horizontal gene transfer (horizontal gene transfer, HGT) is currently the main push of Mycobacterium tuberculosis pathogenic factors. Pathogenicity island (pathogenicity island, PAI) is derived from the large fragment of DNA horizontal transfer, special area is located in the genome can be effectively coupled several functional genes, gene function The complementary and integration, is a new way of evolution of virulent pathogens. In recent years, with the rapid development of high-throughput biological computing technology, the high-throughput pathogenicity island pathogens to be screened. Analytical scanning and function the pan genome of Mycobacterium tuberculosis virulence island will lay the molecular basis for the pathogenesis of the pathogen was us face annotation, for the diagnosis of Mycobacterium tuberculosis, vaccine and drug development has a crucial role.
Multi drug resistant pulmonary tuberculosis with drug sensitive TB treatment course is long, high cost and high mortality. Although there are many reports for anti MDR-TB drugs, but there is no effective drug accepted for the treatment of MDR-TB. Methods this study through the network meta analysis to the efficacy and safety of the existing 6 kinds of anti MDR-TB drugs to assess, for the safety and efficacy of good anti tuberculosis drugs for drug resistant tuberculosis clinical medication. This study uses Bayesian network meta analysis model, through 6 randomized clinical trials of anti MDR-TB drugs to evaluate the anti TB drugs Benin quinoline, delamanid, linezolid, levofloxacin, metronidazole, effectiveness and safety of moxifloxacin. This study analyzed 13 randomized clinical trials (8 randomized clinical trials of 12 articles in ClinicalTrials.gov), 1549 cases of samples, the odds ratio (o Ddsratios (OR) was used as an effective dose. It was found that the effectiveness and safety of 6 kinds of drug-resistant TB drugs were statistically different. Linezolid is probably the most effective drug, followed by quinoline, delamanid, levofloxacin, moxifloxacin and metronidazole in Benin.
The island is a group of closely linked and function is closely related to the molecular level of DNA fragment encoding it transfer, an important component of the regulation of virulence, pathogenicity island in the evolutionary process of pathogenic bacteria by gene recombination into host bacteria, thus giving the virulence of the new characteristics of the host bacteria, but also provide a molecular basis for the pathogenic mechanism of pathogens bacteria. Based on the calculation of the genome of Mycobacterium tuberculosis 1000bp 4-mer nucleotide fragment of arbitrary string frequency spectrum to achieve the genomic barcode visualization annotation of Mycobacterium tuberculosis, and abnormal screening by calculating the Euclidean distance of the barcode area, realize the pan genome scan of Mycobacterium tuberculosis virulence Island, then analyze the function of the island has been selected the application of Pfam_Scan and Blast2GO functional annotation tools. Through the above analysis, we screened three virulence of Mycobacterium tuberculosis The island area, which include the function of known pathogenicity island MPI-2 and MPI-3, including the unknown function virulent MPI-1.MPI-2 and MPI-3 2 were carrying PE/PPE pathogenicity island encoding protein family genes encoding MPI-2 so-called Type VIIsecretion systems, a functional analysis shows that it can be used as early diagnosis of Mycobacterium tuberculosis infection marker; MPI-3 involved in cell wall synthesis of mycolic acids and host cell interaction, functional analysis of past life which can be used as an important molecular target of anti tuberculosis drugs; the function of MPI-1 is unclear, but through the protein domain analysis showed that this gene may be virulence on the island of integral membrane nitriteextrusion protein encoding, promotion of Mycobacterium tuberculosis coli from aerobic growth into non replicating retention state (nonreplicating persistence, NPR), and the sustainability of Mycobacterium tuberculosis infection. Cut. Genomic barcode technology integrates more genetic information of the genome, with higher resolution. By analyzing the function of scanning and the pan genome of Mycobacterium tuberculosis virulence island for the pathogenic mechanism of the bacteria we fully annotated lay the molecular basis, also research on diagnosis of Mycobacterium tuberculosis and the vaccine and drug to the vital role.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R378.911

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