Identification of conditionally essential genes in Mycobacterium tuberculosis DD- carboxypeptidase mutant strains exposed to Augmentin

Jacobs, Olivia
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Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB), one of the most successful human pathogens to date. TB is the top ten leading cause of death worldwide despite the fact that it is curable. This is partially due to the emergence of multi-drug and extensively drug resistant TB. There is thus a need for new drugs and/or targets. The mycobacterial cell wall is a complex structure composed of various inner and outer layers providing a rich environment for potential drug targets. Of particular interest to this study is the peptidoglycan (PG) layer. PG is composed of glycan chains linked to short peptides. The PG layer is essential for cell stability, ensuring rigidity and resistance to internal turgor pressure. PG is consistently remodelled to allow for cell growth and division. One class of proteins involved in the biosynthetic and remodelling processes are penicillin binding proteins (PBPs). PBPs are divided into two categories, namely high molecular weight (HMW) and low molecular weight (LMW). The focus of this study was on LMW PBPs, specifically DD-carboxypeptidases (DDCPases) which cleave the terminal D-amino acid from stem peptides. There are three identified DD-CPase homologues in Mtb (Rv3330 (dacB1), Rv2911 (dacB2) and Rv3627c). Only Rv3627c is annotated as essential for in vitro growth. In Mycobacterium smegmatis (Msmeg), of the four homologues (MSMEG_1661, MSMEG_2432, MSMEG_2433 and MSMEG_6113 (dacB)), only dacB is essential. The aim of this project was to determine if LMW PBPs with DD-Cpase activity plays a role in response to antibiotic exposure either via direct peptidoglycan remodelling or as antibiotic sensors to recruit other proteins. Experiments were conducted in two mycobacterial species, namely Mtb and Msmeg. Mutant strains lacking DD-CPase homologues were assessed for changes in drug susceptibility, morphology, and PG biosynthesis. We observed an increased susceptibility of Msmeg mutant strains to RIF, the presence of bulging in Msmeg mutant cells when probed with fluorescent vancomycin as well as a shortening of the double mutant strain in Mtb relative to wild type. No other major phenotypes were observed suggesting that these proteins may only play minor roles in response to antibiotics, if any. We also sought to identify conditionally essential genes in an Mtb DDCpase mutant background exposed to Augmentin (β-lactam antibiotic plus β-lactamase inhibitor). We constructed MycoMarT7 transposon mutagenesis libraries in the Mtb strains but consistently noted that high mycobacteriophage titres (PFU/ml >1012) were associated with low library/genome coverage (maximum of 1x104 CFU/ml). As the project initially aimed to identify conditionally essential genes in Mtb, the low coverage was deemed inadequate to proceed further. In order to remedy this and improve library coverage, we sought to identify a novel mycobacteriophage and identified one from soil, subsequently named øIPJ3107. Phenotypic and genomic analysis of this mycobacteriophage suggested that is part of the Siphoviridae family and is classified under sub-cluster A1. Further modifications are required to make this a novel molecular tool for transposon mutagenesis libraries.
A dissertation submitted in fulfilment of the requirements for the degree of Master of Science in Molecular Medicine and Haematology to the Faculty of Health Sciences, School of Pathology, University of the Witwatersrand, Johannesburg, 2022