Investigating RNase HI as a synergistic target for Mycobacterium tuberculosis antibiotics

Abstract

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), infected 10 million people and accounted for 1.5 million deaths globally in 2019. Global infection rates for M. tuberculosis remain high, with an estimation that latent infections affect one in every three people. Treatment of tuberculosis is severely challenged by rising resistance against front-line anti-tubercular drugs such as rifampicin. Synergy testing in Mycolicibacterium smegmatis, the laboratory model of M. tuberculosis, has revealed that the genetic depletion of the endonuclease, - ribonuclease HI (RNase HI) which resolves RNA:DNA hybrids produces a 100-fold enhancement of bacterial cell killing by rifampicin highlighting RNase HI as a valuable new drug target. Validation of RNase HI as a candidate drug target requires insight into the cellular consequences of its inhibition, specifically: its ability to generate mutants towards its synergistic partner. The effect RNase HI inhibition has on preventing mutagenesis to rifampicin resistance, or re-sensitizing rifampicin resistant mutants to treatment is currently unknown. In this thesis the complex relationship between length of antibiotic exposure, concentration of the antibiotic pressure applied and the generation of genotypic or phenotypic resistors was investigated in a context which closely mimicked the dynamic antibiotic exposure cells experience within a host’s internal environment. A diffusion-based agar plate assay capable of producing a gradient of antibiotic concentrations was created which could be modified to produce different ratios of phenotypic to genotypic rifampicin resistors. Colonies isolated from this assay, displayed identifiable mutations to rifampicin, validating this approach. This work confirmed that the way in which an antimicrobial pressure is applied can itself generate antimicrobial resistance. It also provides a facile way to screen compounds that can disrupt phenotypic adaptation. The developed assay was validated to both generate mutants towards rifampicin treatment and to monitor the effects of varying rifampicin concentration on the generation of phenotypic versus genotypic resistance. This assay is facile, quick and inexpensive, suggesting that it will find broad utility for assaying other antibiotics and other organisms.