DNA damage repair mechanisms are critical to the adaptive evolution of Mycobacterium tuberculosis as obligate human pathogen, including the emergence of drug-resistance during anti-tuberculosis (TB) chemotherapy. In experimental models, DnaE2-dependent translesion synthesis (TLS) and UvrB-dependent nucleotide excision repair (NER) have been identified as major mediators of DNA damage tolerance and repair, respectively. Given the inferred dominance of these pathways, this thesis aimed to elucidate otherwise cryptic repair mechanisms which might buffer loss of DnaE2 and UvrB in bacilli exposed to genotoxic stress. Using dnaE2 and uvrB deletion mutants of the model mycobacterium, M. smegmatis (MSM) mc2 155, we applied genome-wide transposon (Tn) mutagenesis to identify conditionally essential repair pathways under treatment with genotoxins of different mechanistic classes. To this end, the DNA crosslinking agent, mitomycin C (MMC), and the gyrase inhibitor and clinically relevant TB drug, moxifloxacin (MOX), were used. The goal was to reveal potential targets for co-drugs that might shorten treatment duration and reduce the risk of drug resistance by severely limiting the intrinsic capacity of MTB to tolerate lethal drugs for extended periods. Among others, our analysis identified GlgB, which is involved in glycogen biosynthesis, and mycothiol biosynthesis proteins, MSMEG_0933 and MSMEG_5261, as compensating the absence of UvrB during MMC treatment. Under MOX treatment, the absence of UvrB was compensated by the RecC/Single-strand Annealing pathway. In contrast, DnaE2 deficiency revealed the conditional essentiality of the PadR family transcriptional regulator, MSMEG_2868, under MMC exposure. Importantly, in all cases, results from the Tn screen were validated using CRISPR interference targeting the identified genes. Of particular interest, we observed that UvrB was essential to compensate loss of DnaE2, whereas the reciprocal was less definitive: while DnaE2 appeared dispensable in MMC-treated uvrB, Tn analyses suggested that dnaE2 might be essential in the untreated ∆uvrB mutant. This result, which is consistent with very recent results suggesting the co-ordination of NER and DnaE2 functions in Caulobacter crescentus, is intriguing in potentially revealing a previously unappreciated role for DnaE2 in mycobacterial NER function. Taken together, these results support the utility of Tn-based whole-genome screens in revealing unexpected genegene interaction networks, and provide additional impetus to explore ancillary, non-essential metabolic functions as alternative targets for novel combination therapies designed to cripple intrinsic mechanisms of mycobacterial resistance.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:uct/oai:localhost:11427/35781 |
| Date | 15 February 2022 |
| Creators | Gobe, Irene |
| Contributors | Warner, Digby, Mizrahi, Valerie, Iorger, Thomas R |
| Publisher | Faculty of Health Sciences, Division of Medical Microbiology |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Doctoral Thesis, Doctoral, PhD |
| Format | application/pdf |
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