A dissertation submitted to the Faculty of Health Science, University of the Witwatersrand,
Johannesburg, in fulfillment of the requirements for the degree of Master of Science in
Medicine.
2017 / Mycobacterium tuberculosis assembles a complex cell wall consisting of mycolic acids, arabinogalactan and peptidoglycan layers. The peptidoglycan is important for structural maintenance and osmotic protection of the cell. Beta-lactam antibiotics, such as penicillin, perturb biogenesis of cross-linked peptidoglycan by inhibition of penicillin-binding proteins and cause cell death. As a result, penicillin-binding proteins have been extensively used in antimicrobial development. However, penicillin insensitive enzymes involved in peptidoglycan biogenesis such as amidases, transglycosylases and endopeptidases remain to be exploited for anti-TB drug development, a field that urgently requires new drugs in light of the rapid emergence of drug resistant strains. In this study, we functionally characterize a novel class of LytM domain containing peptidoglycan endopeptidases (also known as M23 peptidases) in mycobacteria. Bio-informatics tools were used to identify LytM domain-containing homologues in Mycobacterium smegmatis, designated MepB1-MepB4. These were deleted using standard allelic exchange mutagenesis and recombination techniques and the resulting mutants were assessed for cell wall related defects. We found that mycobacterial LytM endopeptidases have important roles in bacterial growth as demonstrated by delayed cell growth kinetics in a ΔmepB1 deletion mutant. We noted no growth defects in ΔmepB2 and ΔmepB3 single deletion mutants but observed defective cell division in a ΔmepB2 ΔmepB3 double deletion mutant. In this double mutant, spatial localization of new cell wall biosynthesis revealed the inability to degrade the septal bridge joining two daughter cells, pointing to a critical role for these enzymes in cell separation. MepB1 is sequestered from the peptidoglycan by cytosolic localization and its absence causes a septal and polar buldging phenotype. To further investigate the biological roles of these putative peptidoglycan endopeptidases, protein interaction studies were conducted using the bacterial two-hybrid mycobacterial protein fragment complementation assay. This analysis identified FtsX, a key cell division protein, as an interacting partner for both MepB2 and MepB3, thus identifying these proteins as novel components of the mycobacterial divisome. Collectively, these observations provide the first insight into a new group of potential drug targets for tuberculosis disease and notably enhance the overall understanding of peptidoglycan turnover, which is of general revelevance in bacterial pathogens. / MT2017
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/23164 |
| Date | January 2017 |
| Creators | Shaku, Tube Moagi |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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