Phenotypic characterization of a Mycobacterium smegmatis strain lacking MSMEG_0858

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2021

Authors

Branti, Vuyani

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Abstract

The genus Mycobacterium represents a complex family containing a number of clinically significant pathogens such as Mycobacterium tuberculosis (Mtb), Mycobacterium ulcerans, and Mycobacterium leprae. Tuberculosis remains a global health issue, claiming approximately two million lives annually, with much of this burden occurring in developing countries such as India and South Africa. Due to the emergence of drug-resistance, there is an urgent need to identify drugs with novel modes of action or different drug targets. In this regard, mycobacterial proteins that are essential for various cellular processes may serve as a starting point for the identification of potential targets. This study focuses on the protein product of MSMEG_0858, Cdc48, which is a member of the AAA+ protein family involved with protein homeostasis via direct interaction with the 20S proteasome. A previous study suggested that Cdc48 interacted with an essential peptidoglycan associated enzyme (MSMEG_6113, DacB). This study aimed to explore this further. A Mycobacterium smegmatis (Msm) strain lacking MSMEG_0858 was phenotypically characterized using several assays but prior to this, the strain was genotypically confirmed using PCR, Southern hybridization and mRNA transcript analysis. The mutant strain was shown to be dispensable for growth in 7H9 (nutrient rich) media, but when cultured in carbon-limited media lacking glycerol, growth rate was reduced, an observation that was reversed following genetic complementation. The ∆cdc48 strain was more permeable to EtBr uptake but despite this, no differences in sensitivity to several antibiotics (including amoxicillin, vancomycin and D cycloserine), cell damaging agents (lysozyme and formaldehyde), heat-shock or exposure to detergents (SDS), were observed. Collectively, our observations suggest a non-essential role for Cdc48 under nutrient-rich conditions. Under carbon-limiting conditions, the protein may play a more central role but more work is required to describe the physiological mechanism and to target this protein for drug development against Mtb

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A dissertation submitted in fulfilment of the requirements for the degree of Master of Science in Medicine to the Faculty of Health Sciences, School of Pathology, University of the Witwatersrand, Johannesburg, 2021

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