Mycobacterium abscessus HelR interacts with RNA Polymerase to confer intrinsic rifamycin resistance

Abstract

Rifampicin (RIF) constitutes the frontline therapy against M. tuberculosis as well as most slow-growing non-tuberculous mycobacteria (NTM). However, RIF is completely ineffective against M. abscessus despite the absence of mutations in the rifampicin resistance determining region of Mab_rpoB. This has been attributed to the presence of an ADP-ribosyltransferase (Arr) activity that inactivates RIF. Rifabutin (RBT), a close analogue of RIF, has recently been shown to be effective against M. abscessus in vitro and in a mouse model and comprises a promising therapeutic against M. abscessus infections. Using RNA sequencing we show that exposure of M. abscessus to sublethal doses of RIF and RBT results in [~]25-fold upregulation of Mab_helR in laboratory and clinical isolates; an isogenic deletion of Mab_helR is hypersensitive to RIF and RBT, and over-expression of Mab_helR confers RIF tolerance in M. tuberculosis implying that helR constitutes a significant determinant of inducible RIF and RBT resistance. We demonstrate a preferential association of MabHelR with RNA polymerase in vivo in bacteria exposed to RIF and showed that purified MabHelR can rescue transcription inhibition in the presence of RIF in in vitro transcription assays. Furthermore, MabHelR can dissociate RNAP from RIF-stalled initiation complexes in vitro, a species we envisage accumulates upon RIF exposure. Lastly, we show that the tip of the PCh-loop of Mab_helR, in particular residues E496 and D497 that are in proximity to RIF, is critical for conferring RIF resistance without being required for RNAP dissociation from stalled complexes. This suggests that HelR may be additionally involved in displacing RIF bound to RNAP and function as an RNAP protection protein. Significance StatementBacterial RNA polymerase is a target for the potent and broad-spectrum rifamycin group of antibiotics. Mutations within rpoB and inactivation by a diverse group of enzymes constitute the most widespread mechanisms of resistance. Herein we report an unprecedented mechanism of rifamycin resistance in M. abscessus mediated by MabHelR, a putative SF1 like helicase, that involves disassembly of RIF-stalled initiation complexes, likely followed by displacement of the antibiotic, leading to RNAP recycling. The mechanism is reminiscent of the role of HflX and ribosome protection proteins in resistance to ribosome targeting antibiotics and suggests that removal of stalled macromolecular complexes and their recycling comprises a widespread but underappreciated mechanism of antibiotic resistance. Rifampicin (RIF) is pivotal in the control of M. tuberculosis infections but ineffective against M. abscessus. Identification of inducible rifamycin resistance determinants in M. abscessus is therefore particularly crucial for informing treatment strategies and development of novel therapeutic approaches.