Isolation and characterization of Mycobacterium smegmatis D-alinine racemase mutants /
Mycobacterium bovis and Mycobacterium tuberculosis are agents of serious infections of animals and human beings. Mycobacterial infections have re-emerged with the appearance of multiple drug resistant strains. This situation is difficult to control by current vaccines and anti-mycobacterial agents...
| Main Author: | |
|---|---|
| Format: | Thesis Book |
| Language: | English |
| Published: |
[Place of publication not identified] :
[publisher not identified] ;
2002.
|
| Subjects: | |
| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=765069481&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | Mycobacterium bovis and Mycobacterium tuberculosis are agents of serious infections of animals and human beings. Mycobacterial infections have re-emerged with the appearance of multiple drug resistant strains. This situation is difficult to control by current vaccines and anti-mycobacterial agents. Mycobacterium smegmatis is a fast-growing non-pathogenic Mycobacterium, widely used to study cellular processes in search of targets for the development of new drugs and vaccines. This study focused on the D-alanine racemase gene (alrA), involved in the synthesis of D-alanine, a basic component of peptidoglycan that forms the backbone of the mycobacterial cell wall. M. smegmatis alrA null mutants were generated by homologous recombination and characterized. The viability of the mutants and independence of D-alanine for growth indicate that the gene is not essential. In a medium without D-alanine, mutant cells formed colonies with a flaky appearance and irregular borders, and grew with a phenotypic lag of at least ten generation times. These mutants displayed reduced survival in human macrophages and hypersusceptibility to the anti-mycobacterial agent D-cycloserine. Mutant cells underwent a 500-fold reduction in the number of intracellular bacilli at 72 hours post-infection, which contrasted with a 5-fold reduction for wild type cells. The minimal inhibitory concentration of the mutant strain for D-cycloserine was 2.56 []g ml⁻¹, 30-fold less than the wild type strain. In contrast, mutants displayed the same acid-fastness, ultrastructure, and susceptibility to hydrogen peroxide and lysozyme, as the wild type strain. In summary, the properties of M. smegmatis alrA mutants suggest the existence of a new pathway of D-alanine or peptidoglycan biosynthesis. The reduced survival in macrophages identifies the alrA gene as a candidate target for the attenuation of pathogenic mycobacteria and eventual design of novel vaccines. The hypersusceptibility of the mutant cells to D-cycloserine is consistent with the identification of D-alanine racemase as a target, but underlies the existence of a more fundamental one. This outcome is of importance for the design of novel anti-tuberculosis drugs targeting the mycobacterial peptidoglycan. |
|---|---|
| Item Description: | Vita. "Major Subject: Veterinary Microbiology". |
| Physical Description: | xiii, 134 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilm Inc. |
| Bibliography: | Includes bibliographical references (leaves 101, 133). |