Tuberculosis (TB) is a worldwide pandemic, which claims approximately 2 million lives annually. Though treatable through chemotherapy, TB still causes 8-10 million new infections annually. The problem is further complicated by the emergence of multi-drug resistant strains of Mycobacterium tuberculosis (Mtb) the causative agent of TB. The unabated spread of this disease and associated high levels of mortality has prompted the search for new drugs with novel modes of action. The peptidoglycan (PG) component of the cell wall of Mtb is an incredibly complex structure and has been the focus of antimicrobial development in other organisms. In this study, we characterize PG remodelling N-acetylmuramyl-L-alanine amidases (cell wall amidases) in Mycobacterium smegmatis (Msm), a model organism for TB research by gene knockout/knockdown. Cell wall amidases cleave the bond between the stem peptide and the glycan backbone in PG and have been shown to play an essential role in cell growth, cell signalling and antibiotic resistance in other organisms. Our bioinformatics analysis revealed that M. smegmatis encodes three possible amidase homologues designated ami1, ami2 and ami3. Deletion mutagenesis in Msm resulted in successful deletion of ami1 whilst repeated attempts to delete ami2 did not yield a knockout mutant, suggesting that ami2 is essential for growth. Deletion of ami1 results in the formation of long filaments consisting of 3 to 8 cells attached to each other due to incomplete resolution of the septum. In these filaments, lateral growth at both the internal septation sites and extreme poles resulted in irregular cell width, strongly implicating Ami1 in bacterial cell division, the maintenance of bacterial cell shape and possibly balancing the growth at the cell pole. Since deletion of ami2 was not possible, a knockdown strain allowing for anhydrotetracycline (ATC)-regulated conditional gene expression of ami2 was generated. In this system, withdrawal of ATC results in repression of expression. Expression analysis of ami2 in this strain revealed that
whilst significant gene knockdown was achieved, ami2 expression was not completely abolished in the absence of the inducer ATC, suggesting the presence of basal level ami2 expression. The repression of ami2 expression results in retarded growth, diminished motility, unusual colony morphology consisting of miniature colonies lacking any form of serpentine cording and the formation of miniature cells consisting of globular bulges. These data implicate Ami2 in bacterial growth and maintenance of bacterial cell shape. Collectively, our data comprise the first demonstration of an important role for peptidoglycan degrading amidases in mycobacterial growth and cell division. Furthermore, the phenotypic defects in colony formation due to deletion or depletion of amidases suggest that these enzymes play an important role in cell-cell communication during colony formation. These data validate this class of enzymes as an untapped, legitimate 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:10539/13852 |
| Date | 18 February 2014 |
| Creators | Senzani, Sibusiso |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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