Mycobacterium amidases: biological function and putative role in cell wall remodeling

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2018

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Sikhosana, Nombeko

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Abstract

Mycobacterium tuberculosis, the causative agent of tuberculosis, uses macrophages to cross host mucosal barriers and initiate infection at sterile sites in the alveoli, where it establishes infection and evades immune detection. Despite significant effort, the mechanism through which mycobacteria replicate and persist within host tissues remains enigmatic. Bacterial replication requires substantive rearrangement of the cell surface and in this context, the bacterial cell wall and the enzymes that remodel the various polymers present in this structure are of particular interest. Within the cell wall, the bacterial peptidoglycan (PG) layer has been the subject of intense research, given the therapeutic benefit of targeting this macromolecule in various diseases. PG hydrolases specifically, N-acetylmuramyl-L-alanine amidases (amidases) in other bacterial species have been shown to contribute to essential cell division processes and pathogenesis. In this study, the role of mycobacterial amidases in cell growth and division was investigated to assess their function in Mycobacterium smegmatis, a model organism routinely used for tuberculosis research. For this, putative amidase-encoding genes ami3, ami4 and ami5 were individually deleted using homologous recombination. The resulting mutant strains were then phenotypically characterized by investigating nascent cell wall incorporation, cell elongation and division mechanisms. Deletion of ami3 resulted in severe cell division defects, in many cases these were associated with mislocalization of the cell elongation and division apparatus. In addition to this, cell wall permeability was severely affected, with the strain displaying increased susceptibility to cell wall targeting antibiotics. Deletion of ami4 resulted in the formation of twisted and morphologically atypical cells, some of which died after the first cell division event. In contrast, deletion of ami5 resulted in the formation of short cells, suggesting that this enzyme plays a role in cell growth/elongation. Collectively, these data describe novel and non-redundant roles for amidases in mycobacterial cell growth and division, thus validating them as potential new drug targets for tuberculosis disease

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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. March 2018.

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