Mycobacterium 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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/11937 |
| Date | 12 September 2012 |
| Creators | Mapela, Lusanda Thato |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf, application/pdf |
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