ETD Collection

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    The fitness costs of drug resistance mutations in Mycobacteria
    (2012-01-17) Koch, Anastasia Sideris
    The 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.
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    The construction and phenotypic characterization of mycobacterial mutants deficient in DNA glycosylases
    (2009-04-09T08:53:06Z) Goosens, Vivianne Jacoba
    Mycobacterium tuberculosis is an exquisitely adapted intracellular pathogen that encounters hostile, host-derived reactive nitrogen and oxygen intermediates during the course of infection of its human host. These radicals cause DNA damage, which is repaired through various pathways to allow for the continued survival of the organism. Base excision repair (BER) is one such pathway, which depends on DNA glycosylases to identify and excise damaged DNA bases. Formamidopyrimidine DNA glycosylase (Fpg/ MutM/ FAPY) and Endonuclease VIII (Nei) are such enzymes, which both target oxidatively damaged DNA and together, form the Fpg family of DNA glycosylases. Bioinformatic analyses identified two copies each of Fpg and Nei-encoding genes in M. tuberculosis as well as in its non-pathogenic relative, Mycobacterium smegmatis. To understand the role of these multiple glycosylases in the maintenance of genomic integrity and survival of mycobacteria, the genes encoding the four Fpg/Nei glycosylases were individually deleted in M. smegmatis strain mc2155 by homologous recombination. In addition to the four single mutants, double and triple Fpg and Nei glycosylase knockout mutants were generated by sequential gene knockout. When compared to the parental strain, the single and double mutants showed no variation in growth kinetics, no increased sensitivity to hydrogen peroxide and no increase in spontaneous mutation rates. However, a slight increase in frequency of spontaneous C T transition mutations was observed in double knockout mutants compared to the wild type and single mutant strains. These results suggest that these enzymes may be part of an extensive network of enzymes which collectively work to enhance the overall survival of M. smegmatis through the repair of oxidatively damaged DNA.