3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item Characterization of resuscitation promoting factors in Mycobacterium smegmatis(2012-09-12) Mapela, Lusanda ThatoMycobacterium tuberculosis, the causative agent of tuberculosis (TB) has infected one third of the world’s population and continues to claim more lives annually than any other infectious disease agent. A significant proportion of individuals carry latent TB infection (LBTI) which is characterized by the absence of clinical symptoms and it has been postulated that the tubercle bacilli are in a dormant-like state during this type of infection. Resuscitation promoting factors (Rpfs) are cell wall hydrolases which cleave glycosidic bonds within the peptidoglycan (PG), a mechanism thought to result in reactivation of bacteria from the state of dormancy. M. tuberculosis encodes five rpf-like homologues which are collectively dispensable for growth but are required for reactivation from dormancy in vitro, and for virulence in the mouse model of infection. LTBI thus poses a huge threat to the global burden of active disease. The purpose of this study was to further investigate the biological roles of Rpfs by assessing the effects of rpf gene deletion in M. smegmatis, a model organism used for TB research. M. smegmatis encodes four rpf-like genes designated rpfA, rpfB, rpfC and rpfE, and deletion mutants that lack one or more of these genes were constructed by allelic exchange mutagenesis. M. smegmatis mutant strains that lack either rpfA or rpfB display no significant differences in growth both on solid and in liquid medium when compared to wild type. However, loss of rpfA resulted in bacterial clumping during stationary-phase growth in broth culture and changes in cell morphology. Moreover; the rpfA rpfB double mutant and its derivative strain lacking rpfC, rpfA rpfB rpfC displayed a ca. 2-4 log increase in susceptibility to erythromycin and vancomycin. Furthermore, unusual colonial morphologies with reduced serpentine cording and smooth peripheries were observed for these multiple mutants. Cell surface defects and cell distortions were evidenced as wrinkle-textured, bent cells and polar tip bulges for the abovementioned mutants. The multiple deletion strains also displayed a defect in biofilm formation revealing an inability for the mutants to form complex cell-cell interactions. Collectively, the data are suggestive of a loss of bacterial cell wall integrity due to rpf gene deletion and it is proposed that rpfA, in combination with other genes, is largely responsible for cell wall integrity maintenance. Our data indicate that these factors play an important role in cell growth and division and therefore represent an untapped source of novel targets for anti-tubercular drug discovery.