結核病仍是全球疾病和死亡的主要原因。雖然人均新發結核病例自2003年以來一直下降，耐多藥（MDR）和廣泛耐藥（XDR）的結核病例的突然增加為全球疾病控制帶來了新的威脅。結核分枝杆菌（MTB）北京株在過去十年越来越受重視，皆因其席捲亞洲，前蘇聯，和包括美國在內的好幾個地方。北京株在動物實驗中也表現出高毒性和耐多藥的傾向。目前結核菌廣泛耐藥定義為至少對異煙肼和利福平耐藥，再加上任何氟喹諾酮類，和至少一個二線藥物。我們對這種病菌的生物知識仍然有限。在這研究，我們為來自香港和福建五株MTB北京株進行了基因組測序，其中兩株的耐藥性遠超XDR標準 - “全耐藥“（TDR）的表型。五個北京株的比較基因組學為我們提供了在北京株的毒力相關基因的啟示。一個約4 KB大小的片段被找出来了，此片段是所有已知MTB中都没有的。我們討論了此片段對MTB進化的含義。當我們研究在北京耐藥株的獨特基因變化時發現，DNA修復和香葉醇降解有關連。我們還觀察到大的缺失（D）和截斷（T）的事件，顯著高於框移位（F）的突變。此外，在TDR菌株出現的FDT事件更頻繁地涉及到最佳生長和麻風分枝杆菌的基因組中保留的基因。這方面的証據表明，MTB通過缩减進化發展極端耐藥性。適應度的顯著降低也許解釋了TDR菌株的稀缺 。 / Tuberculosis (TB) remains one of the major causes of illness and death globally. Although the number of new TB cases per capita has been falling since 2003, the emergence of multidrug resistant (MDR) and extensively drug resistant (XDR) cases of TB poses new threat to the successful worldwide control of the disease (WHO, 2008; Iseman, 2007). The Beijing lineage of Mycobacterium tuberculosis (MTB) has received much attention over the past decade due to its prevalence throughout Asia, parts of the former Soviet Union, and several other geographical locations including the United States. The strain also demonstrated hypervirulence in animal models and an increased likelihood to develop multidrug resistance. The current definition of XDR in TB is defined as resistance to at least isoniazid and rifampicin, any fluoroquinolone, and with at least one of the three second-line drugs. Here we show that our knowledge of the biology of this pathogen is still limited. We performed genome sequencing and reported the complete genomes of five Beijing isolates from Hong Kong and Fujian, of which two were shown to have drug resistance that is far beyond the current XDR standard - a "Totally Drug Resistance" (TDR) phenotype. Comparative genomics of the five Beijing isolates provided us insights into the virulence-related genes in the Beijing family. A distinct region of about 4 kb in size that are absent in all known complete genomes of MTB was also identified. The evolutionary implications of this region were discussed. When we investigated the unique genetic changes in drug resistant Beijing strains, a correlation to DNA repair and geraniol degradation was implicated. We have also observed that the big deletions (D) and truncations (T) events were significant higher when compare with frameshift (F) mutations. Moreover, the FDT events in TDR strains were more frequently found in genes that are involved in growth attenuation and retained in the genome of the Mycobacterium leprae. This evidence suggests that MTB develops its extreme drug resistance through the reductive evolution. The significant decrease in the fitness may explain the rareness of TDR strains. / Detailed summary in vernacular field only. / Leung, Ka Kit. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 93-108). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Epidemiology - a ubiquitous threat --- p.1 / Chapter 1.2 --- Surviving the Hell --- p.3 / Chapter 1.3 --- Relatives of M. tuberculosis --- p.4 / Chapter 1.4 --- The age of M. tuberculosis --- p.5 / Chapter 1.5 --- Characteristics of Beijing strains --- p.6 / Chapter 1.6 --- Drug resistance --- p.7 / Chapter 1.7 --- Genome sequencing --- p.9 / Chapter 1.7.1 --- Conventional sequencing --- p.9 / Chapter 1.7.2 --- High-throughput sequencing --- p.10 / Chapter 1.8 --- Sequence assembly --- p.11 / Chapter 1.8.1 --- De novo assembly --- p.11 / Chapter 1.8.2 --- Reference mapping --- p.12 / Chapter Chapter 2 --- Materials and Methods --- p.14 / Chapter 2.1 --- Sample preparation --- p.14 / Chapter 2.2 --- DNA extraction and genome sequencing --- p.18 / Chapter 2.3 --- Gap filling and finishing --- p.20 / Chapter 2.3.1 --- In silico gap verification --- p.20 / Chapter 2.3.2 --- Comparison among different reference mapped contigs --- p.24 / Chapter 2.3.3 --- Experimental work --- p.26 / Chapter 2.4 --- Bioinformatics analysis --- p.27 / Chapter 2.4.1 --- Genome annotation --- p.27 / Chapter 2.4.2 --- Phylogeny analysis --- p.27 / Chapter 2.4.3 --- Variation analysis --- p.28 / Chapter 2.4.4 --- In silico functionality analyses --- p.29 / Chapter Chapter 3 --- Results --- p.30 / Chapter 3.1 --- Genome features of M. tuberculosis Beijing genotype strains --- p.30 / Chapter 3.2 --- Phylogeny of M. tuberculosis Beijing genotype strains --- p.36 / Chapter 3.3 --- Evolutionary implications of a 4kb-insertion in Beijing strains --- p.40 / Chapter 3.4 --- Beijing family specific gene variations --- p.48 / Chapter 3.5 --- Drug resistance --- p.52 / Chapter Chapter 4 --- Discussions --- p.75 / Chapter 4.1 --- 4kb insertion, a potential bridge to our knowledge gap --- p.75 / Chapter 4.2 --- Beijing common and Beijing drug resistant specific variations --- p.77 / Chapter 4.3 --- Regions of deletion --- p.79 / Chapter Chapter 5 --- Conclusions --- p.82 / Chapter Chapter 6 --- Future Work --- p.84 / Chapter 6.1 --- Compensatory mutation study --- p.84 / Chapter 6.1.1 --- Database construction for drug resistance compensatory mutations --- p.85 / Chapter 6.2 --- Non-protein coding region study --- p.92

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

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009. / Härtill 4 uppsatser.

Conventional drug susceptibility testing techniques, the ‘gold standard’ for M. tuberculosis are slow, requiring about 3-6 weeks from a positive culture. This diagnostic delay, before initiation of appropriate treatment, contributes to increased transmission rates. Molecular techniques provide rapid results and therefore present an alternative to conventional tests. The aim of this project was to develop an inhouse reverse line blot hybridization assay (RIFO assay) that could detect mutations associated with Rifampicin resistance directly in clinical specimens of patients in KwaZulu Natal. A 437 bp region of the rpoB gene was sequenced to ascertain the most frequently occurring mutations conferring resistance to rifampicin in isolates in KwaZulu-Natal. Wildtype and mutant probes designed to target these mutations, were immobilized on a Biodyne C membrane. Hybridization conditions were optimized using biotin labeled PCR products from culture. Detection was performed with peroxidase labeled streptavidin using enhanced chemiluminescence. Four DNA extraction methods were evaluated on sputum specimens to determine the one with the least inhibitory effect on amplification. A total of 11 mutations were found in 236 clinical isolates: 531TTG (109, 58.3%), 516GTC (26, 13%), 533CCG/516GGC (20, 10%), 533CCG (18, 9.6%), other mutations < 5% each. The chelex extraction method was found to be optimal for removing inhibitors in sputum specimens. Sputum specimens of 404 patients hospitalized at King George V Hospital between 2005 and 2006 were rifoligotyped. The RIFO assay was optimised on clinical isolates and then applied to sputum specimens. The RIFO assay on culture and sputum correlated well with the DST (sensitivity 92% and 94% respectively). However, the specificity was very low in both culture and sputum specimens compared to DST (38% and 35% respectively). This could be attributed to the presence of silent mutations, mixed infections, mixed populations of bacteria or the small number of susceptible strains used in this study. The in-house RIFO assay can be used directly on sputum specimens to predict Rifampicin resistance and therefore MDR-TB in less than a week compared to the gold standards. A total of 43 samples can be tested simultaneously at low cost and the membrane is reusable compared to commercial kits such as the Hains test that is expensive and strips are not reusable. A similar assay can be designed to target mutations for the detection of XDR-TB. Future studies should be conducted in a clinical setting on patients with sensitive strains to increase the specificity. / Thesis (M.Med.)-University of KwaZulu-Natal, Durban, 2011.

Mycobacterium tuberculosis (MTB) is an airborne pathogen that is easily transmitted from person to person. An intact immune system prevents the organism from causing disease in most individuals. In South Africa, the prevalence of human immunodeficiency virus (HIV) has reached astronomical levels and is now fuelling the tuberculosis (TB) epidemic. Drug resistant MTB strains combined with a weakened host immune system is a lethal combination. Multi-drug resistant (MDR) including extensively drug resistant (XDR) tuberculosis is on the increase, with Tugela Ferry in KwaZulu-Natal South Africa, reporting the largest cluster of XDR cases in the world. It is unknown whether a single clone of the drug resistant strain is circulating in this area or whether there are multiple strains at play. Using 2 complementary genotyping methods, we showed that the MDR strains present are the result of clonal spread associated with the F28 family, as well as de novo resistance which manifests as unique patterns. The XDR epidemic in Tugela Ferry is the result of clonal spread of a strain belonging to the F15/LAM4/KZN family. / Thesis (M.Med.)-University of KwaZulu-Natal, Durban, 2011.

The World Health Organization has identified antimicrobial resistance as one of the top three threats to human health. Gram-negative bacteria such as Escherichia coli are intrinsically more resistant to antimicrobials. There are very few drugs either on the market or in the pharmaceutical pipeline targeting Gram-negative pathogens. Two mechanisms, the protection of the outer membrane and the active efflux by the multidrug transporters, play important roles in conferring multidrug resistance to Gram-negative bacteria. My work focuses on two main directions, each aligning with one of the known multidrug resistance mechanisms. The first direction of my research is in the area of the biogenesis of the bacterial outer membrane. The outer membrane serves as a permeability barrier in Gram-negative bacteria. Antibiotics cross the membrane barrier mainly via diffusion into the lipid bilayer or channels formed by outer membrane proteins. Therefore, bacterial drug resistance is closely correlated with the integrity of the outer membrane, which depends on the correct folding of the outer membrane proteins. The folding of the outer membrane proteins has been studied extensively in dilute buffer solution. However, the cell periplasm, where the folding actually occurs, is a crowded environment. In Chapter 2, effects of the macromolecular crowding on the folding mechanisms of two bacterial outer membrane proteins (OmpA and OmpT) were examined. Our results suggested that the periplasmic domain of OmpA improved the efficiency of the OmpA maturation under the crowding condition, while refolding of OmpT was barely affected by the crowding. The second direction of my research focuses on the major multidrug efflux transporter in Gram-negative bacteria, AcrB. AcrB is an obligate trimer, which exists and functions exclusively in a trimeric state. In Chapter 3, the unfolding of the AcrB trimer was investigated. Our results revealed that sodium dodecyl sulfate induced unfolding of the trimeric AcrB started with a local structural rearrangement. While the refolding of secondary structure in individual monomers could be achieved, the re-association of the trimer might be the limiting factor to obtain folded wild type AcrB. In Chapter 4, the correlation between the AcrB trimer stability and the transporter activity was studied. A non-linear correlation was observed, in which the threshold trimer stability was required to maintain the efflux activity. Finally, in Chapter 5, the stability of another inner membrane protein, AqpZ, was studied. AqpZ was remarkably stable. Several molecular engineering approaches were tested to improve the thermal stability of the protein.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 6 uppsatser.

Multidrug efflux pumps are membrane proteins that actively transport foreign objects out of cells. The active efflux of these pumps is a critical self-defense mechanism that enables the survival of bacteria under hostile environments. Efflux pump AcrB is a member of the Resistance-Nodulation-Division (RND) super family. In E. coli, it associates with periplasmic protein AcrA and outer membrane channel TolC to extrude a variety of noxious compounds out of cell from both the cytoplasm and the periplasm. My dissertation research focused on two aspects of this multidrug efflux pump: the oligomerization process during the biogenesis of AcrB and its degradation. Oligomerization is an important aspect of the structure and function for many proteins and has been the subject of many studies. However, most of such studies focused on soluble proteins. The oligomerization process of membrane proteins, including AcrB, is rarely explored. In chapter 2, the co-assembly of AcrB variants co-expressed in the same cell was used as a tool to investigate the assembly of AcrB trimers during its bio-production. By monitoring the portion of pure trimers containing only one type of subunit and hybrid trimers containing a mixture of the two kinds of subunits, it was found that the oligomerization of membrane proteins is not a random process as the formation of pure trimer is favored. In chapter 3, the GALLEX system was used to monitor AcrB oligomerization in cells under the native condition. Previously GALLEX has only been used to monitor the oligomerization of small transmembrane proteins. By constructing a series of fusion proteins with different linker length between LexA and AcrB, and optimizing inducer concentration, we finally developed a system that could be used to differentiate AcrB trimers of different oligomerization affinities. While chapters 2 and 3 focus on the trimerization of AcrB, a critical step of its biogenesis, chapters 4 and 5 focus on its life time and degradation. In chapter 4, the life time of AcrB was measured by incorporating non-natural amino acid azidohomoalanine (AHA) into protein translation. Using this method, it was determined that that the half-life of both AcrA and AcrB in E. coli were six days. The surprisingly long lifetime of these detoxification proteins might represent a strategy by the bacteria to conserve energy and maximize their competition niche for survival in a hostile environment. In chapter 5, the degradation process of ssra tagged AcrB was investigated. In-vivo degradation test showed that properly inserted AcrB can be digested after addition of ssra-tag to its C-terminus. It was found that cytoplasmic unfoldase-protease complex ClpXP and chaperone SspB are involved in the degradation. In vitro assay is still being optimized to quantitatively analyze the activity of ClpXP in the degradation of AcrB.

Thesis (MScMedSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Drug resistance in Mycobacterium tuberculosis is an increasing global problem. Drug resistance is mostly caused by single nucleotide polymorphisms (SNPs) within the bacterial genome. This observed increase in global incidence of drug resistant tuberculosis (TB) has sparked the search for new anti-TB drugs and the repurposing of drugs that are currently used against other organisms or species of mycobacteria. One such repurposed drug, clofazimine (CFZ), is currently used for the treatment of leprosy, caused by Mycobacterium leprae. The mechanism of action of CFZ is not clear, but it is hypothesized that CFZ is reduced by a mycobacterial type II NADH oxidoreductase (NDH-2). The reduction of CFZ drives the production of reactive oxygen species (ROS) which is toxic to the pathogen. The aim of this study was to elucidate the mechanism of CFZ resistance. Towards this aim, spontaneous in vitro CFZ resistant mutants were selected, characterized and whole genome was used identify SNPs which may cause CFZ resistance. Mutations were identified in a transcriptional regulator encoded by Rv0678, fatty-acid-AMP ligase, or FadD28 (Rv2941) and glycerol kinase or GlpK (Rv3696c). Mutations in Rv0678 have previously been shown to play a role in both CFZ resistance and bedaquiline (BDQ) cross-resistance, while no link has been found between CFZ resistance and mutations in fadD28 and glpK. The novel, non-synonymous SNP identified in Rv0678 resulted in the replacement of an alanine residue with threonine at codon 84, which is located in the DNA binding domain. Virtual modelling of the mutated Rv0678 protein showed that the A84T mutation may influence DNA binding, possibly due to its proximity to the DNA binding domain. This mutation caused a change in hydrophobicity, which may influence binding to DNA. Previous studies showed that mutations in Rv0678 resulted in the upregulation of mmpL5, a putative efflux pump. However, the mechanism whereby CFZ resistance occurs via increased abundance of this efflux pump in the cell wall is not clear and needs further investigation. The cross-resistance between CFZ and BDQ, caused by mutations in Rv0678, is of concern and may influence the planning of anti-TB drug regimens for the future. The roles of the other two mutations identified in this study in CFZ resistance is also not clear and requires further investigation. Finally, the findings of this study support the role of Rv0678 in CFZ resistance thereby suggesting that this gene could be useful as a diagnostic marker to test for CFZ resistance in clinical isolates. / AFRIKAANSE OPSOMMING: Middelweerstandigheid in Mycobacterium tuberculosis is 'n wêreldwye toenemende probleem. Middelweerstandigheid word meestal veroorsaak deur enkel nukleotied polimorfismes (SNPs) in die bakteriële genoom. Hierdie toename in middelweerstandige tuberkulose (TB) het gelei tot die soektog na nuwe anti-TB-middels en die alternatiewe aanwending van middels wat tans teen ander organismes of spesies van mikobakterieë gebruik word. Een so 'n alternatiewe middel, clofazimine (CFZ), word tans gebruik vir die behandeling van melaatsheid wat veroorsaak word deur Mycobacterium leprae. CFZ se meganisme van werking is nie duidelik nie, maar dit word vermoed dat CFZ gereduseer word deur 'n mikobakteriële tipe II NADH oksidoreduktase (NDH-2). Die reduksie van CFZ dryf die produksie van reaktiewe suurstof spesies wat giftig is vir die patogeen. Die doel van hierdie studie was om die meganisme van CFZ weerstandigheid te ondersoek. Om hierdie doel te bereik was spontane in vitro CFZ weerstandige mutante gekies, gekarakteriseer en heel genoom volgorde bepaling is gebruik om SNPs te identifiseer wat CFZ weerstandigheid veroorsaak. Mutasies in Rv0678, 'n transkripsie reguleerder, vetsuur-AMP ligase, of FadD28 (Rv2941) en gliserol kinase of GlpK (Rv3696c) geïdentifiseer. Dit is al voorheen gevind dat mutasies in Rv0678 ‘n rol speel in beide CFZ weerstandigheid en bedaquiline (BDQ) kruis-weerstandigheid, terwyl geen verband gevind is tussen CFZ weerstandigheid en mutasies in fadD28 en glpK nie. Die nuwe, nie-sinonieme SNP, geïdentifiseer in Rv0678 het gelei to die vervanging van 'n alanien aminosuur met treonien by kodon 84, wat geleë is in die DNS bindings domein. Virtuele modellering van die gemuteerde Rv0678 proteïen het getoon dat die A84T mutasie DNS binding moontlik kan beïnvloed, as gevolg van sy nabyheid aan die DNS bindings domein. Hierdie mutasie veroorsaak 'n verandering in die hidrofobiese natuur, wat DNS binding kan beïnvloed. Vorige studies het getoon dat mutasies in Rv0678 lei tot die opregulering van mmpL5, 'n waarskynlike uitvloei pomp. Die meganisme waardeur CFZ weerstandigheid veroorsaak, deur ‘n groot aantal van hierdie uitvloei pompe in die selwand, is nie duidelik nie en moet verder ondersoek word. Die kruis-weerstandigheid tussen CFZ en BDQ, wat veroorsaak word deur mutasies in Rv0678, is van belang en kan die beplanning van anti-TB middel behandeling vir die toekoms beïnvloed. Die rolle van die ander twee mutasies, wat in hierdie studie geïdentifiseer is, in CFZ weerstandigheid is ook nie duidelik nie en vereis verdere ondersoek. Ten slotte, die bevindinge van hierdie studie steun die rol van Rv0678 in CFZ weerstandigheid en dit dui daarop dat hierdie geen gebruik kan word as 'n diagnostiese merker om vir CFZ weerstandigheid te toets in kliniese isolate.

Chemotherapeutic treatment of Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen, is substantially challenged by several membrane-spanning, multidrug-efflux pumps of the three-component RND family. Of these pumps, MexXY-OprM contributes to the intrinsic resistance of this organism by exporting clinically relevant antibiotics, most notably the ribosome-targeting aminoglycosides. Overproduction of MexXY-OprM is the most common mechanism providing pan-aminoglycoside resistance to P. aeruginosa cystic fibrosis clinical isolates. The mexXY genes are located in an operon, the expression of which is induced by ribosome-targeting antimicrobials. The mexXY operon is negatively regulated by MexZ, a repressor protein encoded by the divergently-transcribed gene mexZ. A second gene, PA5471, is also induced by ribosome-targeting antibiotics and is required for antibiotic induction of mexXY expression. One possibility is that PA5471 interacts with MexZ to alleviate repression of mexXY, thereby providing a mechanism for PA5471-dependent drug inducibility of mexXY. PA5471 interaction with MexZ was confirmed using a bacterial two-hybrid assay. To identify residues/regions of PA5471 important for interaction with MexZ, random chemical mutagenesis of the mexZ and PA5471 genes was carried out and the effects of these mutations on interaction of their protein products was assessed using the bacterial two-hybrid assay. Mutations of PA5471 that compromised interaction with MexZ included P68S, G76C, R216C, R221W, R221Q, G231D, and G252S, which occur within or in close proximity to a predicted surface-exposed α-helix of a PA5471 structural model that may contribute to the MexZ-interaction domain. Representative mutations P68S, G76C, R216C and R221W were placed into the chromosome of P. aeruginosa to assess their impact on drug-inducible mexXY expression. All of these mutations significantly reduced mexX upregulation in the presence of spectinomycin, where mutations R216C and R221W resulted in the near complete ablation of this antibiotic induction. These data suggest that PA5471 acts as a direct antirepressor of MexZ and that this interaction is key to mexXY upregulation in response to ribosome-targeting induction signals. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2013-02-26 13:32:39.307