Kinetic Characterization of Rifamycin-Resistant M. tuberculosis RNA Polymerases and Novel Therapeutic Approach for Targeting Transcription

Abstract

Tuberculosis (TB) remains a critical threat to global human health. In 2016, 1.7 million people died from the disease. Rifampin (RMP) remains a key component of the front-line treatment for TB, though resistance has presented challenges for its efficacy. Resistance to RMP (RMPR) primarily occurs through point mutations of its target, RNA polymerase, within the rifamycin resistance determining region (RRDR) in the β-subunit. Three mutations constitute the bulk of RMPR, βD435V, βH445Y, and βS450L, with the latter being most prevalent in clinically resistant isolates. The molecular mechanisms which yield the observed distribution of RMPR mutations in MTB have been speculated upon; however, detailed in vitro studies of Mycobacterium tuberculosis (MTB) RNAP to elucidate those mechanisms have been lacking. This has likely been due, in part, to difficulty in acquiring pure MTB RNAP. To surmount this, an optimized methodology for the expression and purification of highly pure and active MTB RNAP is described. Co-expression of multiple vectors harboring all subunits of the RNAP holoenzyme allows for in vivo assembly of the holo RNAP complex. An optimized purification method was developed to acquire stoichiometric holo RNAP with high activity. In vivo fitness defects have been observed in RMPR mutants of MTB RNAP. These defects have been found to be ameliorated by the presence of secondary mutations in double-psi β-barrel (DPBB) of the RNAP β’-subunit. To identify factors contributing to this fitness defect, several in vitro transcription assays were utilized to probe initiation, elongation, termination and RNA primer hydrolysis with the wild-type and RMPR RNAPs. Secondary, compensatory mutations are predominantly associated with the βS450L mutant, therefore this mutant was also studied in the presence of secondary mutations. We found that the RMPR mutants exhibit significantly poorer termination efficiency relative to wild-type, an important factor for proper gene expression. This may contribute to the relative prevalence of the RMPR mutants observed in MTB clinical isolates. We also found that several mechanistic aspects of transcription of the RifR mutant RNAPs are impacted relative to wild-type, particularly the stability of the open-promoter complex and elongation rate. For the βS450L mutant, these defects are mitigated in the presence of secondary mutations in the DPBB of the β’-subunit, making the intrinsic properties of this mutant similar to those of the wild-type. These data provide insight into the cost of antibiotic resistance to the fitness of the organism and a mechanistic basis for how MTB alleviates fitness defects associated with drug resistance. Drug resistant TB has become pervasive in large part due to a lack of novel therapeutics which act by new mechanisms of action. CarD is a global transcription regulator which acts by stabilizing the open-promoter complex of MTB RNAP and has been shown to be required for MTB viability. This suggests that CarD may be an effective and novel target for therapeutic discovery for the treatment of tuberculosis. A fluorescence polarization assay which monitors the association of MTB RNAP, native rRNA promoter DNA and Bodipy-CarD has been developed, optimized and validated. A high throughput screen has been conducted to identify and characterize small molecule inhibitors which block the CarD•RNAP•DNA interaction. Several preliminary hits have been identified from this screen and initial secondary characterizations have been performed. This project will be the foundation for further investigation of CarD’s potential as a therapeutic target.