ETD Collection
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Item Antibodies to mycobacterium tuberculosis mycolic acids in patients with pulmonary tuberculosis(2001-09-11) Schleicher., Gunter, Klaus.Introduction and Aim: The waxy outer cell wall of mycobacteria consists mainly of mycolic acids (MA). The unique immuno-stimulatory properties of MA via the CD 1-restricted antigen presentation pathway have been demonstrated in humans. Purification and isolation of M.tuberculosis (MTB) MA has allowed them to be applied as an antigen in an ELISA-based sero-diagnostic assay to detect specific antibodies in the sera of humans. The aim of the study was to measure the levels of antibody to MA in the sera of patients with culture proven pulmonary tuberculosis (PTB), and in control subjects without evidence of tuberculosis.Item Characterization of DD-carboxypeptidase function in mycobacteria: genetic knockout and recombinant protein production(2016-10-12) Ismail, Zaahida SheikTuberculosis (TB), a disease caused by the pathogenic bacterium Mycobacterium tuberculosis (Mtb), is responsible for killing over one million people each year with an alarming number categorized as multidrug resistant (MDR) or extensively drug resistant (XDR) infections. These high numbers, coupled with other factors such as the ability of Mtb to adapt to its host, its synergistic relationship with Human Immunodeficiency Virus (HIV) and the protracted treatment regimen required to treat TB has resulted in the urgent need for new TB drugs. In this regard, the peptidoglycan (PG) layer of the mycobacterial cell wall, which requires an array of enzymes for synthesis of this mesh-like polymer, have been of particular interest. The PG consists of sugars cross-linked by stem peptides and is synthesized, cross-linked and remodeled by carefully regulated enzymes such as penicillin binding proteins (PBPs) which perform the final crosslinking step in PG biosynthesis. This study focuses on a specific group of low molecular weight PBPs, namely the DD-carboxypeptidases (DD-CPases), which are responsible for regulating the amount of cross-links found in the PG by cleaving the terminal D-Alanine (D-Ala) from the stem peptide of nascent PG units. To date, these proteins have remained largely uncharacterized in mycobacteria. To investigate the functions of these proteins in Mycobacterium smegmatis, two double knockout mutants lacking different combinations of the DD-CPase-encoding genes (MSMEG_1661, MSMEG_2432 and/or MSMEG_2433) were created using two-step allelic exchange and assessed using a range of phenotypic analyses. In addition recombinant protein production of these DD-CPases as well as MSMEG_6113 was attempted. We were unable to create a double knockout mutant lacking both MSMEG_2432 and MSMEG_2433 from an existing single mutant strain, suggesting that the operonic structure of these two genes may require a different approach. We were able to generate two double knockout mutants, ΔMSMEG_1661 ΔMSMEG_2432 and ΔMSMEG_2433 ΔMSMEG_1661, lacking two DDCPases. Southern blot and gene expression analyses confirmed loss of the respective genes from M. smegmatis. The ΔMSMEG_1661 ΔMSMEG_2432 mutant and ΔMSMEG_2433 ΔMSMEG_1661 mutant displayed no alterations in colony morphology, biofilm formation, sliding motility and sodium dodecyl sulphate (SDS) sensitivity. However, loss of two DD-CPases resulted in increased sensitivity of M. smegmatis to vancomycin and a range of β-lactams antibiotics. Analysis of cellular morphology using transmission electron microscopy (TEM) demonstrated that the septum was fully formed in all strains but in some cases was not degraded during daughter cell separation. Scanning electron microscopy (SEM) and spatial localization of new PG units using BODIPY-labeled vancomycin revealed that late division processes were hampered in these double knockout mutants with new PG inserted across the cell as well as cells with multiple unresolved septa. To create recombinant derivatives of MSMEG_6113, MSMEG_1661, MSMEG_2432 and MSMEG_2433, proteins were His-tagged and purified using affinity chromatography. However, under all conditions tested soluble protein could not be acquired. Collectively, these data provide the first evidence that the DD-CPases of M. smegmatis may play a direct role in the late cell division process that lead to daughter cell separation in mycobacteria.Item Construction and phenotypic characterization of Mycobacterium smegmatis mutants deficient in the MutY DNA glycosylase(2014-02-19) Hassim, FarzanahMycobacterium tuberculosis is a facultative pathogen that causes tuberculosis and it accounted for 1.4 million deaths worldwide in 2011. During infection reactive species are released by macrophages as part of the hosts‟ immune response, causing damage to the pathogen‟s DNA. Since mycobacterial genomes are G+C rich the guanine base is more susceptible to oxidative damage which results in the formation of 7,8-dihydro-8-oxoguanine (8-oxoG) lesions which are subsequently repaired by the Fpg/Nei family of DNA glycosylases of the base excision repair (BER) pathway. Mycobacteria possess four copies of the Fpg/Nei glycosylases with the Fpg homologue displaying an overlapping role with MutM in the GO system. Loss of the Fpg/Nei DNA glycosylases leads to mispaired bases during replication which are subsequently repaired by the MutY DNA glycosylase. Previously, in our laboratory we showed that a deficiency of two Fpg/Nei glycosylases displayed no differences in survival of M. smegmatis mutants under oxidative stress and a 2-3 log difference was observed only when three or all four of the DNA glycosylases were inactivated. Surprisingly, there was no corresponding increase in mutator phenotype for all the combinatorial Fpg/Nei deficient mutants compared to the parental strain. A recent study further showed that a deficiency in M. smegmatis MutY glycosylase also displayed no notable susceptibility to oxidative stress or increase in mutagenesis. Since in E.coli a double mutM and mutY mutant displayed an increased mutator phenotype compared to the individual mutants, it was plausible to investigate the role of MutY in combination with the Fpg/Nei family of DNA glycosylases in mycobacteria to understand whether these DNA glycosylases display overlapping and/or compensatory functions in dealing with oxidative damage. Using homologous recombination the mutY gene was deleted in the parental and in the Fpg/Nei deficient mutant strains. Deletion of mutY together with the Fpg/Nei family of DNA glycosylasesdisplayed similar in vitro growth kinetics as the parental strain under normal culture conditions. However, under in vitro oxidative stress conditions the mutY deficiency especially in the absence of all four Fpg/Nei DNA glycosylases results in a greater reduction in survival of the mutants with a general increase in mutation rates. Consistent with these data was the significant increase in C → A and A → C mutations with the progressive loss of the DNA glycosylases as assessed by the spectral analysis of rifampicin resistant mutants. Taken together these data indicate that the mycobacterial MutY DNA glycosylase has antimutator properties and possibly has a more significant role in mycobacterial genome maintenance compared to the Fpg/Nei family of DNA glycosylases.Item Analysis of peptidoglycan degrading amidases in Mycobacterium smegmatis(2014-02-18) Senzani, SibusisoTuberculosis (TB) is a worldwide pandemic, which claims approximately 2 million lives annually. Though treatable through chemotherapy, TB still causes 8-10 million new infections annually. The problem is further complicated by the emergence of multi-drug resistant strains of Mycobacterium tuberculosis (Mtb) the causative agent of TB. The unabated spread of this disease and associated high levels of mortality has prompted the search for new drugs with novel modes of action. The peptidoglycan (PG) component of the cell wall of Mtb is an incredibly complex structure and has been the focus of antimicrobial development in other organisms. In this study, we characterize PG remodelling N-acetylmuramyl-L-alanine amidases (cell wall amidases) in Mycobacterium smegmatis (Msm), a model organism for TB research by gene knockout/knockdown. Cell wall amidases cleave the bond between the stem peptide and the glycan backbone in PG and have been shown to play an essential role in cell growth, cell signalling and antibiotic resistance in other organisms. Our bioinformatics analysis revealed that M. smegmatis encodes three possible amidase homologues designated ami1, ami2 and ami3. Deletion mutagenesis in Msm resulted in successful deletion of ami1 whilst repeated attempts to delete ami2 did not yield a knockout mutant, suggesting that ami2 is essential for growth. Deletion of ami1 results in the formation of long filaments consisting of 3 to 8 cells attached to each other due to incomplete resolution of the septum. In these filaments, lateral growth at both the internal septation sites and extreme poles resulted in irregular cell width, strongly implicating Ami1 in bacterial cell division, the maintenance of bacterial cell shape and possibly balancing the growth at the cell pole. Since deletion of ami2 was not possible, a knockdown strain allowing for anhydrotetracycline (ATC)-regulated conditional gene expression of ami2 was generated. In this system, withdrawal of ATC results in repression of expression. Expression analysis of ami2 in this strain revealed that whilst significant gene knockdown was achieved, ami2 expression was not completely abolished in the absence of the inducer ATC, suggesting the presence of basal level ami2 expression. The repression of ami2 expression results in retarded growth, diminished motility, unusual colony morphology consisting of miniature colonies lacking any form of serpentine cording and the formation of miniature cells consisting of globular bulges. These data implicate Ami2 in bacterial growth and maintenance of bacterial cell shape. Collectively, our data comprise the first demonstration of an important role for peptidoglycan degrading amidases in mycobacterial growth and cell division. Furthermore, the phenotypic defects in colony formation due to deletion or depletion of amidases suggest that these enzymes play an important role in cell-cell communication during colony formation. These data validate this class of enzymes as an untapped, legitimate source of novel targets for anti-tubercular drug discovery.Item The role of resuscitation promoting factors in peptidoglycan hydrolysis and reactivation from dormancy in Mycobacterium smegmatis(2014-02-18) Beukes, Germar MatthewThe global burden of tuberculosis (TB) has reached an alarming status with numerous countries reporting an incidence of greater than 300 cases per 100 000 population per capita. By far, the most concerning statistic regarding TB is that estimates indicate 2 billion people worldwide are infected with Mycobacterium tuberculosis, the causative agent of TB. Only a small proportion (10%) of these individuals progress to active disease whilst the majority control the infection in an asymptomatic state that is termed latent TB infection (LTBI). These individuals carry a lifetime risk, which is significantly enhanced in the presence of HIV co-infection, of progressing to active disease. It has been hypothesized that during LTBI, the infecting bacterial population adopts a non-growing, dormant state and that progression to active disease is a result of reactivation of these organisms from dormancy. M. tuberculosis encodes for 5 homologues for the resuscitation promoting factor (Rpf), designated rpfA-E which have been shown to be important for reactivation from dormancy and critical for virulence during TB infection. As such, these factors may play an important role in bacterial reactivation during LTBI. In this study, we further characterize Rpf function in Mycobacterium smegmatis, a non-pathogenic relative of M. tuberculosis. M. smegmatis encodes four rpf–like homologues and using sequential deletion mutagenesis, we constructed a panel of deletion mutants that were defective in one, two, three or all four rpf-like genes in M. smegmatis. The successful construction of a quadruple mutant lacking all four rpf-like genes demonstrated that, as observed in M. tuberculosis, the rpf-like genes are collectively dispensable for growth of M. smegmatis. A select group of rpf deficient mutant strains were then characterized further. Our analysis indicates that the double ΔrpfA ΔrpfB, triple ΔrpfA ΔrpfB ΔrpfC, and quadruple ΔrpfA ΔrpfB ΔrpfC ΔrpfE, mutants displayed no growth defects in vitro but were impaired for biofilm formation and formed abnormal colonies that lacked the surface cording which is characteristic of mycobacterial colonies. Genetic complementation reversed these defects. Furthermore, the triple ΔrpfA ΔrpfB ΔrpfC, and quadruple ΔrpfA ΔrpfB ΔrpfC ΔrpfE, mutants displayed increased susceptibility to vancomycin, erythromycin and cephalosporins, suggesting some change in peptidoglycan structure or composition. Microscopic analysis of rpf deletion mutants revealed that the double ΔrpfA ΔrpfB, triple ΔrpfA ΔrpfB ΔrpfC, and quadruple ΔrpfA ΔrpfB ΔrpfC ΔrpfE, mutants displayed cells that were smaller than the wild type. In an interesting development, analysis of growth and survival in an in vitro model of mycobacterial dormancy indicated that the triple ΔrpfA ΔrpfB ΔrpfC, and quadruple ΔrpfA ΔrpfB ΔrpfC ΔrpfE mutants were unable to enter into a viable but non-culturable state. These results point to a novel role for Rpfs in the establishment of the dormant state in mycobacteria. Transmission Electron Microscopy on cells isolated from the dormancy model confirmed the progressive accumulation of inclusion bodies in the double ΔrpfA ΔrpfB, triple ΔrpfA ΔrpfB ΔrpfC, and quadruple ΔrpfA ΔrpfB ΔrpfC ΔrpfE mutants as these stains were passaged through our model of dormancy. Further analysis with Nile Red staining identified these inclusion bodies as lipid bodies. Collectively, our data reveal an important role for Rpfs in regulating essential growth processes and reveal a new role for Rpfs in the ordered shutdown of bacterial growth and establishment of dormancy in M. smegmatis.Item The fitness costs of drug resistance mutations in Mycobacteria(2012-01-17) Koch, Anastasia SiderisThe increasing emergence of drug-resistant pathogens poses a major threat to public health. Although influenced by multiple factors, resistance is often associated with mutations in drug target-encoding or associated genes. The potential fitness cost of such resistance mutations is, in turn, a key determinant of the spread of drug-resistant strains. Rifampicin (RIF) is a frontline anti-tuberculosis agent that targets the rpoB-encoded β-subunit of the DNA-dependent RNA polymerase (RNAP). RIF resistance (RIFR) maps primarily to mutations in rpoB that might be expected to affect transcription and so the ability of the organism to cause disease. Accordingly, numerous studies have assessed the impact of RIFR on key fitness indicators in pathogens including Mycobacterium tuberculosis (MTB). In contrast, the specific consequences of RIFR for bacterial physiology remain poorly understood. Notably, previous studies of the effects of RIFR-associated rpoB mutations on mycobacterial physiology have been conducted using strains generated by RIF exposure, without accounting for the potential impact of second-site mutations that may compensate for fitness costs or contribute to drug resistance. In this study, site-directed mutagenesis and allelic exchange were employed to generate a panel of M. smegmatis (MSM) strains containing clinically-relevant RIFR-associated point mutations. Importantly, this methodology enables the introduction of rpoB mutations into defined strain backgrounds in the complete absence of RIF. Using this approach, we constructed “RIF naive” MSM rpoB mutant strains carrying either an S531L or H526Y mutation. The resulting mutants were 100-fold less susceptible to RIF than the isogenic, parental strain. Notably, the inclusion of selected efflux inhibitors in susceptibility assays had little impact on mutant susceptibility to RIF. In contrast, restoration of the wild-type allele returned the observed susceptibility to parental levels, thereby providing strong evidence of the sufficiency of a single rpoB mutation for clinical RIFR in mycobacteria. Competitive growth assays utilizing the S531L mutant and the parental strain exposed a growth defect for the S531L mutant. However, discriminating between wild-type and mutant rpoB strains proved a significant technical challenge, again highlighting the difficulties associated with inferring in vivo fitness from in vitro assays conducted under a limited number of different conditions. In summary, our results suggest the benefit of a deeper exploration of the physiological and fitness implications of RIFR-associated mutations. In addition, in coupling a system which enables an evaluation of the physiological consequences of drug resistance-associated mutations with evolutionary analyses, we provide preliminary evidence of the benefits of a multipronged approach to elucidating the physiological implications of drug resistance in MTB.