Dissertation submitted to the Faculty of Health Science, University of the Witwatersrand,
Johannesburg, in fulfilment of the requirements for the degree of Master of Science in
Medicine.
June 2016Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is responsible for the
largest number of deaths due to a single bacterial pathogen. The persistence of TB in society
is exacerbated by multiple factors, including the emergence of drug resistant forms of M.
tuberculosis such as multi-, extensively- and totally-drug strains, which have fuelled a race
for the discovery of new drugs with novel modes of action. However, the metabolic
flexibility inherent in the tubercle bacillus has made the identification of vulnerable drug
targets difficult. The peptidoglycan (PG) layer in the mycobacterial cell wall plays an
essential role in bacterial growth, survival and antibiotic resistance but has not been
exploited for TB drug development. In this study, we characterized the physiological
function of mycobacterial DD-Carboxypeptidases (DD-CPases), a subgroup of Low
Molecular Weight penicillin binding proteins (LMW PBPs), implicated in modulating the
amount of PG cross-linking, thereby regulating the growth and remodelling of the PG
polymer. Given that DD-CPases have not been characterized in actinobacteria, this study
comprised the identification of M. smegmatis DD-CPases interacting partners, using the
mycobacterial Protein Fragment complementation assay (mPFC), and interrogation of the
functionality of the M. tuberculosis DD-CPase homologues through heterologous
complementation in M. smegmatis. The Two DD-CPases, encoded by the MSMEG_2433 and
MSMEG_6113 loci in M. smegmatis, were chosen for the identification of putative
interacting partners. Screening of a genomic library using the mPFC assay identified 30 and
10 possible interacting partners for MSMEG_2433 and MSMEG_6113 respectively.
Notably, some of these interacting partners included key regulators of cell elongation
(PonA1) or division (PbpA and PbpB) and point to an important function for DD-CPases
during mycobacterial growth. To further assess if genetic multiplicity for DD-CPases in M.
tuberculosis is reflective of an underlying functional redundancy between these enzymes, the
M. tuberculosis DD-CPase homologues (Rv2911, Rv3627c and Rv3330) were assessed for
functionality through ectopic expression in M. smegmatis. The resulting heterologous M.
smegmatis strains displayed altered colony morphology, with smooth colonies that displayed
a mucoid surface and cording defects. Ectopic expression of the M. tuberculosis DD-CPases
in M. smegmatis also resulted in aberrant biofilm formation and affected bacterial sliding on
semi-solid agar, indicative of an alteration in the three-dimensional packing of cells in a
community or changes in membrane properties. Over-expression of Rv2911 and Rv3627c
resulted in reduced bacterial cell length accompanied by division defects. Transmission
electron microscopy revealed that the M. tuberculosis DD-CPase homologues are involved in
maintaining normal bacterial cell wall architecture. Over-expression of these homologues in
native wild type M. tuberculosis H37RvS revealed a role for Rv3627c in regulating bacterial
colony morphology. Cellular localization studies with rseGFP C-terminally tagged
derivatives of these proteins revealed that they localize at the poles, mid-cell or and quartercell
positions, further suggestive of a role in temporal and spatial co-ordination of cell
growth and division. Collectively, the data point to important, possibly redundant roles for
the multiple DD-CPases-encoding genes in M. tuberculosis and highlights these proteins as
possible new drug targets for TB.MB201
