結核病是由結核桿菌感染而引起的慢性傳染病，它是危害人類健康的主要殺手。根據世界衛生組織的報導，目前在全球範圍內有三分之一的人口感染了結核桿菌，每年約有915 萬人口被確診患有結核病。耐藥結核病尤其是對最有效的一線抗結核藥物異煙阱和利福平產生抗藥的耐多藥結核病的出現，令有效的控制結核病更加棘手。 / 在本研究中，我們首先用利福平誘導得到五株伴有明顯生長緩慢的高水平利褔卒耐藥的恥垢分支桿菌。通過比較基因組學研究發現，在編碼區有四個突變，其中兩個位於中rpoB 基因(N484T and 1488F) ，一個位於MSMEG_0436 (V49M) ,一個位於MSMEG_6872 (V181L)。rpoB 基因突變是該恥垢分支桿菌利福平耐藥的主要原因。而生長緩慢主要源於MSMEG_6872基因的影響。更為有趣的是，我們發現一個與MSMEG_6872具有相同的蛋白模序的結核分支桿菌蛋白質Rv1367 在不間的結核分支桿菌菌株之間存在I193V 多態性。193V 主要存在于北京株或者在耐藥的非北京株上。進一步的研究發現，過量表達MSMEG_6872或者Rv1367c 的恥垢分支桿菌形態上呈現為細長棒狀，而他們的親代則為短棒狀。 / 為獲得耐藥性，以及在高濃度的抗生素環境下生存，細菌必須付出一定的生物學代價。本研究中，恥垢分支桿菌以生長缺陷為代價獲得了對利褔平的耐藥，而這個代價可能是由於MSMEG_6872 基因的突變或者過量表達打破了細胞壁延長和分裂的平衡引起。 / Mycobacterium tuberculosis (MTB), which is the pathogen of tuberculosis (TB), remains a major human public health problem throughout the world. According to the report from the World Health Organization, currently about one third of the world's population was infected by MTB and there is globally 9.15 million recorded cases of TB annually. The occurrence of resistance to drugs used to combat TB, particularly multi-drug resistant TB (MDR-TB), defined as resistance to at least isoniazid and rifampin (RIF), has become a significant public health problem in a number of countries and an obstacle to effective global TB control. / In this project, we firstly obtained high level RIF resistant Mycobacterium smegmatis (MSM) strains with obviously growth retardation by repeated drug selection. Comparative analysis of genomic sequences revealed 4 mutations in coding sequences, including two in rpoB (N484T and I488F), one in MSMEG 0436 (y 49M), and one in MSMEG 6872 (y181L). Characterization of these four mutations showed that the two mutations in rpoB were correlated to RIF resistance. The one in MSMEG_6872 can render obviously growth retardation when MSMEG_6872 is over-expressed. Interestingly, we found an MTB protein, Rv1367c, which has the same motif with MSMEG_6872, had an I193V polymorphism in different MTB strains. The 193V variant was mainly found in Beijing/W or drug resistant non-Beijing/W family strains. The transformants, no matter MSMEG_6872 or Rv 1367 c, all exhibited slim and long rod shape compared to stocky and short rod appearance of the parental strain. / Mycobacterial cells must pay biological cost in order to obtain RIF resistance and survive in the high concentration of RIF. In our case, the growth arrest may be due to the mutation of MSMEG_6872 which disrupts the balance of cell wall elongation and cell division. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Guan, Bing. / "November 2011." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 139-143). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Acknowledgements --- p.I / Abstract --- p.II / Abstract in Chinese --- p.IV / List of Abbreviations --- p.V / List of Tables --- p.VI / List of Figures --- p.VII / Contents --- p.IX / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- Overview of Tuberculosis --- p.1 / Chapter 1.1.1 --- Pathogens --- p.2 / Chapter 1.1.2 --- Syndromes --- p.2 / Chapter 1.1.3 --- Transmission --- p.3 / Chapter 1.1.4 --- Diagnosis --- p.4 / Chapter 1.1.5 --- Epidemiology --- p.6 / Chapter 1.1.6 --- Mortality --- p.8 / Chapter 1.2 --- The Anti-TB Strategies --- p.8 / Chapter 1.2.1 --- Chemotherapy Treatment for MTB --- p.8 / Chapter 1.2.2 --- Vaccine Development for MTB --- p.9 / Chapter 1.3 --- Genome Sequencing of MTB Isolates --- p.9 / Chapter 1.4 --- Drug Resistance of MTB --- p.13 / Chapter 1.4.1 --- MDR-TB and XDR-TB --- p.15 / Chapter 1.4.2 --- Mechanism of Drug Resistance --- p.18 / Chapter 1.4.2.1 --- Intrinsic Resistance of Mycobacterium Species --- p.20 / Chapter 1.4.2.2 --- Acquired Resistance of Mycobacterium Species --- p.22 / Chapter 1.4.3 --- RIF Resistant MTB --- p.24 / Chapter 1.5 --- Useful tool for MTB Research --- p.26 / Chapter 1.6 --- The Biological Cost of Antibiotic Resistance in MTB --- p.27 / Chapter 1.6.1 --- The meaning of Biological Cost --- p.27 / Chapter 1.6.2 --- Factors Involved in Biological Cost of Mycobacterium Species --- p.29 / Chapter 1.17 --- Objectives of the Project and Experimental Strategies --- p.30 / Chapter CHAPTER 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Selection of RIF Resistant MSM mc²155 Strains --- p.31 / Chapter 2.1.1 --- Bacterial Strains, Media, and Growth Conditions --- p.31 / Chapter 2.1.2 --- Selection of RIF Resistant Strain --- p.31 / Chapter 2.2 --- Minimum-Inhibitory-Concentration (MIC) Assay --- p.34 / Chapter 2.3 --- Detection of Mutations in the rpoB Gene of RIF Resistance Strains --- p.36 / Chapter 2.3.1 --- Primers Design --- p.36 / Chapter 2.3.2 --- PCR and Direct Sequencing --- p.36 / Chapter 2.4 --- Characterization of the RpoB Gene --- p.38 / Chapter 2.4.1 --- Construction of Recombinant Clones --- p.38 / Chapter 2.4.2 --- Preparation of MSM competent cell. --- p.38 / Chapter 2.4.3 --- Electroporation of plasmid into MSM competent cells --- p.39 / Chapter 2.4.4 --- Site-directed Mutagenesis of the RpoB Clone --- p.39 / Chapter 2.5 --- Whole Genome Sequencing of Parental and Drug --- p.43 / Chapter 2.5.1 --- MSM Genomic DNA Extraction --- p.43 / Chapter 2.5.2 --- Genomic Sequencing --- p.44 / Chapter 2.5.3 --- Data Analysis and SNPs Identification --- p.45 / Chapter 2.6 --- Validation of Mutations by PCR and Direct Sequencing --- p.46 / Chapter 2.6.1 --- PCR Primers Design --- p.46 / Chapter 2.6.2 --- PCR and Direct Sequencing --- p.46 / Chapter 2.7 --- Characterization of MSMEG 0436 and MSMEG 6872 --- p.47 / Chapter 2.7.1 --- Construction of the recombinant clones --- p.47 / Chapter 2.8 --- Assay of Ethidium Bromide in Intact Cells --- p.48 / Chapter 2.9 --- Quantitative Real-time PCR to Expression of the Measure the ATP-binding Cassette (ABC) Superfamily Efflux Pumps --- p.49 / Chapter 2.9.1 --- RNA Extraction --- p.49 / Chapter 2.9.2 --- Synthesis of Double-stranded cDNA from Total RNA --- p.49 / Chapter 2.9.3 --- Real-time PCR to Quantify the Efflux Pump Gene Expression Level --- p.49 / Chapter 2.10 --- The construction of the Growth Curve --- p.53 / Chapter 2.11 --- Generation of ΔMSMEG_6872 Mutant Strain --- p.54 / Chapter 2.11.1 --- Preparation of Recombination Strain Stocks --- p.54 / Chapter 2.11.2 --- Preparation of the Electrocompetent Cells of the Recombination Strain --- p.54 / Chapter 2.11.3 --- Preparation of Allelic Exchange Substrate (AES) for Generating Gene Replacement Mutants --- p.55 / Chapter 2.12 --- Validation of Rv1367c (MT1414) in MTB --- p.60 / Chapter 2.12.1 --- Primer Design --- p.60 / Chapter 2.12.2 --- Strain Selection --- p.60 / Chapter 2.12.3 --- PCR and Direct Sequencing --- p.60 / Chapter 2.12.4 --- Alignment the Gene Sequence of Rv1367c of Different MTB Strains --- p.61 / Chapter 2.13 --- Model building of the RpoB protein --- p.62 / Chapter 2.14 --- MSM staining method --- p.63 / Chapter CHAPTER 3 --- RESULTS / Chapter 3.1 --- dentification of RIF Resistant Related-genes Using Induced RIF Resistant MSM Model --- p.64 / Chapter 3.1.1 --- Emergence ofRIF Resistant Strains after the Prolonged Drug Exposure --- p.64 / Chapter 3.1.2 --- Induced RIF Resistance Were Stable In the Absence of Selection --- p.66 / Chapter 3.1.3 --- The Growth State of 5 RIF Resistance MSM mc²155 Strain --- p.68 / Chapter 3.1.4 --- Involvement of RpoB in the Mechanisms of the Emergence of RIF Resistance in MSM --- p.71 / Chapter 3.1.4.1 --- Mutations in the RpoB Gene --- p.71 / Chapter 3.1.4.2 --- Identical Mutations of RpoB Gene in Different RIF Resistance Isolates from Different Generation --- p.74 / Chapter 3.1.4.3 --- Characterization of RpoB in MSM strains --- p.76 / Chapter 3.1.4.4 --- Rifampin-Binding Pockets of RpoB Protein Model --- p.80 / Chapter 3.1.5 --- Other Genetic Alternations possibly Involved in RIF Resistance --- p.83 / Chapter 3.1.5.1 --- Whole Genome Sequencing of the Patental and P5 MSM mc²155 Strains --- p.83 / Chapter 3.1.5.2 --- Validation of the 32 Selected Alterations --- p.88 / Chapter 3.1.5.3 --- Characterization of MSMEG_0436 and MSMEG_6872 in RIF Resistance --- p.91 / Chapter 3.1.5.4 --- Characterization of MSMEG_0436 in the Growth Rate of MSM --- p.93 / Chapter 3.1.5.5 --- Characterization of MSMEG_6872 in the Growth Rate of MSM --- p.95 / Chapter 3.1.5.6 --- MSMEG_6872 Knock-out Strain Exhibited Normal Phenotype as its Parent --- p.98 / Chapter 3.1.5.7 --- Identification of Mutations in the Beta-Iactamase Gene of Mycobacterium Tuberculosis (MTB) --- p.101 / Chapter 3.1.5.8 --- Characterization of Rv 1367 c in Mycobacterium Growth Rate --- p.108 / Chapter 3.1.5.9 --- Morphology Changes of the Rv1367c and MSMEG_6872 Transformants --- p.110 / Chapter 3.2 --- Genetic Alterations in Non-coding Sequence --- p.112 / Chapter 3.2.1 --- ATP-binding Cassette (ABC) Superfamily Efflux Pumps Up-regulated in Drug Resistant M Smegmatis Strain --- p.112 / Chapter 3.2.2 --- RIF Resistant M smegmatis mc²155 Strain exhibited Low Level Cross-drug Resistance to INH --- p.115 / Chapter 3.2.3 --- RIF Resistant M smegmatis mc²155 Strain Showed Low level Accumulation of Ethidium Bromide --- p.117 / Chapter CHAPTER 4 --- DISCUSSION / Chapter 4.1 --- The Protocol for the Preparation RIF Resistant Strains --- p.121 / Chapter 4.2 --- RIF Induced Stable Chromosomal Mutations in RIF Resistant MSM Strains --- p.123 / Chapter 4.3 --- MIC Levels of the RIF Resistant Strains --- p.125 / Chapter 4.4 --- Factors May involved in RIF Resistant MSM Strains --- p.128 / Chapter 4.5 --- Cell Shape and Growth Regulation --- p.129 / Chapter 4.6 --- MSMEG _6872 and Twin-Arginine Translocase (TAT) Secretion System --- p.135 / Chapter 4.7 --- Conclusion --- p.137 / Chapter 4.8 --- Future Perspectives --- p.138 / REFERENCES --- p.139
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_327983 |
| Date | January 2012 |
| Contributors | Guan, Bing., Chinese University of Hong Kong Graduate School. Division of Biomedical Sciences. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | electronic resource, electronic resource, remote, 1 online resource (xiii, 143 leaves) : ill. (some col.), col. maps |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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