| Summary: | A dissertation submitted to the Faculty of Health Science, University of Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science in Medicine.
2016 === Tuberculosis (TB), a disease caused by the pathogenic bacterium Mycobacterium tuberculosis
(Mtb), is responsible for killing over one million people each year with an alarming number
categorized as multidrug resistant (MDR) or extensively drug resistant (XDR) infections. These
high numbers, coupled with other factors such as the ability of Mtb to adapt to its host, its
synergistic relationship with Human Immunodeficiency Virus (HIV) and the protracted treatment
regimen required to treat TB has resulted in the urgent need for new TB drugs. In this regard, the
peptidoglycan (PG) layer of the mycobacterial cell wall, which requires an array of enzymes for
synthesis of this mesh-like polymer, have been of particular interest. The PG consists of sugars
cross-linked by stem peptides and is synthesized, cross-linked and remodeled by carefully
regulated enzymes such as penicillin binding proteins (PBPs) which perform the final crosslinking
step in PG biosynthesis. This study focuses on a specific group of low molecular weight
PBPs, namely the DD-carboxypeptidases (DD-CPases), which are responsible for regulating the
amount of cross-links found in the PG by cleaving the terminal D-Alanine (D-Ala) from the stem
peptide of nascent PG units. To date, these proteins have remained largely uncharacterized in
mycobacteria. To investigate the functions of these proteins in Mycobacterium smegmatis, two
double knockout mutants lacking different combinations of the DD-CPase-encoding genes
(MSMEG_1661, MSMEG_2432 and/or MSMEG_2433) were created using two-step allelic
exchange and assessed using a range of phenotypic analyses. In addition recombinant protein
production of these DD-CPases as well as MSMEG_6113 was attempted. We were unable to
create a double knockout mutant lacking both MSMEG_2432 and MSMEG_2433 from an
existing single mutant strain, suggesting that the operonic structure of these two genes may
require a different approach. We were able to generate two double knockout mutants,
ΔMSMEG_1661 ΔMSMEG_2432 and ΔMSMEG_2433 ΔMSMEG_1661, lacking two DDCPases.
Southern blot and gene expression analyses confirmed loss of the respective genes from
M. smegmatis. The ΔMSMEG_1661 ΔMSMEG_2432 mutant and ΔMSMEG_2433
ΔMSMEG_1661 mutant displayed no alterations in colony morphology, biofilm formation,
sliding motility and sodium dodecyl sulphate (SDS) sensitivity. However, loss of two DD-CPases
resulted in increased sensitivity of M. smegmatis to vancomycin and a range of β-lactams
antibiotics. Analysis of cellular morphology using transmission electron microscopy (TEM)
demonstrated that the septum was fully formed in all strains but in some cases was not degraded
during daughter cell separation. Scanning electron microscopy (SEM) and spatial localization of
new PG units using BODIPY-labeled vancomycin revealed that late division processes were
hampered in these double knockout mutants with new PG inserted across the cell as well as cells
with multiple unresolved septa. To create recombinant derivatives of MSMEG_6113,
MSMEG_1661, MSMEG_2432 and MSMEG_2433, proteins were His-tagged and purified
using affinity chromatography. However, under all conditions tested soluble protein could not be
acquired. Collectively, these data provide the first evidence that the DD-CPases of M. smegmatis
may play a direct role in the late cell division process that lead to daughter cell separation in
mycobacteria. === MT2016
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