Regulatory responses to rifampicin exposure in Mycobacterium tuberculosis

[摘要] ENGLISH ABSTRACT : There is increasing evidence that the mechanisms of drug resistance in Mycobacterium tuberculosis are not limited to mutations in the known drug resistance causing genes. Classically a mutation in one gene confers resistance to a drug, for example a mutation in the rpoB gene confers rifampicin resistance. However varying levels of resistance and fitness observed, as well as the lack of mutations in some clinically resistant isolates has guided researchers to focus on alternate genetic and physiological factors influencing drug resistance in M. tuberculosis. Whole genome sequencing (WGS) has previously identified polymorphisms in the rpoC gene of M. tuberculosis which were later confirmed to compensate for the loss of fitness occurring with the acquisition of rpoB mutations. Similarly, numerous mutations have been identified to be ancillary to drug resistance. Additionally, WGS has been used to investigate the evolution of drug resistance in M. tuberculosis. In addition to genetic factors, the activity of energy dependent efflux pumps has been associated with drug resistance and has been demonstrated to provide low levels of drug resistance to anti-TB drugs to allow for the development of drug resistance causing mutations. This study aimed to further investigate both genetic and efflux based mechanisms of resistance in M. tuberculosis, as well the response of M. tuberculosis to rifampicin exposure. It was therefore hypothesised that M. tuberculosis acquires additional genomic mutations during the evolution of rifampicin mono-resistance to multidrug resistant (MDR). In addition this study hypothesised that efflux pump activity contributes to the level of rifampicin resistance in M. tuberculosis, and this activity is dependent on the presence of specific rpoB mutations. Lastly, this study hypothesised that rifampicin exposure induces the expression of energy metabolism genes and efflux pumps in M. tuberculosis.The Department of Biomedical Sciences strain bank was interrogated and a total of 6 clinical isolates originating from 3 patients representing evolution from rifampicin mono-resistance to MDR were selected for WGS analysis. WGS identified novel genetic variants occurring during the evolution of drug resistance. Numerous variants were only present in a proportion of the population and were observed to change (emerge or disappear) during the course of treatment. This heterogeneity suggests that M. tuberculosis populations are dynamic during the evolution of drug resistance, accumulating numerous genetic changes. Experiments investigating the role of efflux pump activity in rifampicin resistance demonstrated that efflux pump activity differs in M. tuberculosis isolates with an rpoB Ser531Leu mutation compared to those with an rpoB His526Leu mutation. In addition, the efficacy of verapamil as an efflux pump inhibitor differed between these isolates. This finding may have implications for the consideration of the inclusion of efflux pump inhibitors in treatment regimens.Lastly, this study is the first to identify mmpL5 expression in response to rifampicin exposure; however the consequence of this association remains unknown. An additional finding of the investigation into the response of M. tuberculosis to rifampicin exposure is the identification of a proposed transcriptional regulator responding to rifampicin exposure. Together these findings demonstrate the numerous genetic and physiological mechanisms contributing to drug resistance in M. tuberculosis.

[发布机构] Stellenbosch University