Mutation of Rv2887, a marR-like gene, confers Mycobacterium tuberculosis resistance to an imidazopyridine-based agent

Abstract

Drug resistance is a major problem in Mycobacterium tuberculosis control, and it is critical to identify novel drug targets and new antimycobacterial compounds. We have previously identified an imidazo[1,2-a]pyridine-4-carbonitrile-based agent, MP-III-71, with strong activity against M. tuberculosis. In this study, we evaluated mechanisms of resistance to MP-III-71. We derived three independent M. tuberculosis mutants resistant to MP-III-71 and conducted whole-genome sequencing of these mutants. Loss-of-function mutations in Rv2887 were common to all three MP-III-71-resistant mutants, and we confirmed the role of Rv2887 as a gene required for MP-III-71 susceptibility using complementation. The Rv2887 protein was previously unannotated, but domain and homology analyses suggested it to be a transcriptional regulator in the MarR (multiple antibiotic resistance repressor) family, a group of proteins first identified in Escherichia coli to negatively regulate efflux pumps and other mechanisms of multidrug resistance. We found that two efflux pump inhibitors, verapamil and chlorpromazine, potentiate the action of MP-III-71 and that mutation of Rv2887 abrogates their activity. We also used transcriptome sequencing (RNA-seq) to identify genes which are differentially expressed in the presence and absence of a functional Rv2887 protein. We found that genes involved in benzoquinone and menaquinone biosynthesis were repressed by functional Rv2887. Thus, inactivating mutations of Rv2887, encoding a putative MarR-like transcriptional regulator, confer resistance to MP-III-71, an effective antimycobacterial compound that shows no cross-resistance to existing antituberculosis drugs. The mechanism of resistance of M. tuberculosis Rv2887 mutants may involve efflux pump upregulation and also drug methylation.

FIG 1

The 3,183-bp deletion in mutant 1, which includes Rv2887. (A) Coverage analysis of sequencing results for M. tuberculosis H37Rv and mutant 1, showing a lack of reads in this region for the mutant but not the parent H37Rv strain. (B) Confirmation of the deletion mutation. Lane 0, Invitrogen 1-kb Plus DNA ladder. Numbers over the bars indicate the size in base pairs of the ladder. Lane 1, PCR of genomic DNA from the parent H37Rv strain with primers outside the region of the deletion. Lane 2, PCR of genomic DNA from mutant 1 with the same primers as for lane 1. Lane 3, PCR of genomic DNA from H37Rv with primers inside the deletion (in Rv2887). Lane 4, PCR of mutant 1 genomic DNA with the same primers as for lane 3.

FIG 2

Rv2887 is in the MarR family. (A) Alignment of Rv2887 to the consensus sequence for Pfam01047 (MarR) and four other MarR family proteins (selected as the five most diverse members in the conserved domain database, including the consensus sequence) (14). The bottom row is the consistency, as estimated by PRALINE (17). (B) Secondary structure prediction from Phyre2 for Rv2887 (16). (C) Secondary structure prediction for the C terminus of mutant 3. The black boxes in panels A and B indicate V61, which is mutated to alanine in mutant 2.