Phenotypic characterization of a Mycobacterium smegmatis strain lacking MSMEG_0858
No Thumbnail Available
Date
2021
Authors
Branti, Vuyani
Journal Title
Journal ISSN
Volume Title
Publisher
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
Description
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