Hurst-Hess, K. R. | bioRxiv Science

bioRxiv2023

Hierarchy and networks in the transcriptional response of Mycobacterium abscessus to antibiotics

Mycobacterium abscessus causes acute and chronic pulmonary infection in patients with chronic lung damage. It is intrinsically resistance to antibiotics effective against other pathogenic mycobacteria largely due to the drug-induced expression of genes that confer resistance. Induction of genes upon exposure to ribosome targeting antibiotics proceeds via WhiB7-dependent and -independent pathways. WhiB7 controls the expression of >100 genes, a few of which are known determinants of drug resistance. The function of the vast majority of genes within the regulon is unknown, but some conceivably encode additional mechanisms of resistance. Furthermore, the hierarchy of gene expression within the regulon, if any, is poorly understood. In the present work we have identified 56 WhiB7 binding sites using chromatin immunoprecipitation sequencing (CHIP-Seq) which accounts for the WhiB7-dependent upregulation of 70 genes, and find that M. abscessus WhiB7 functions exclusively as a transcriptional activator at promoters recognized by {sigma}A/{sigma}B We have investigated the role of 18 WhiB7 regulated genes in drug resistance and demonstrated the role of MAB_1409c and MAB_4324c in aminoglycoside resistance. Further, we identify a {sigma}H-dependent pathway in aminoglycoside and tigecycline resistance which is induced upon drug exposure and is further activated by WhiB7 demonstrating the existence of a crosstalk between components of the WhiB7-dependent and -independent circuits. Abstract ImportanceThe induction of multiple genes that confer resistance to structurally diverse ribosome-targeting antibiotics is funneled through the induction of a single transcriptional activator, WhiB7, by antibiotic-stalled ribosomes. This poses a severe restriction in M. abscessus therapy as treatment with one ribosome-targeting antibiotic confers resistance to all other ribosome-targeting antibiotics. Here we uncover the intricacies of the WhiB7 regulatory circuit, identify three previously unknown determinants of aminoglycoside resistance and unveil a communication between WhiB7 dependent and independent components. This not only expands our understanding of the antibiotic resistance potential of M. abscessus but can also inform the development of much needed therapeutic options.

microbiology↗

bioRxiv2021

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

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.

microbiology↗