Bacterial Genome-Wide Association Identifies Novel Factors That Contribute to Ethionamide and Prothionamide Susceptibility in Mycobacterium tuberculosis.

Nathan D Hicks,Jian Yang,Yanlin Zhao,Sarah M Fortune,Christina L Stallings,Allison F Carey

doi:10.1128/mbio.00616-19

Abstract

In Mycobacterium tuberculosis, recent genome-wide association studies have identified a novel constellation of mutations that are correlated with high-level drug resistances. Interpreting the functional importance of the new resistance-associated mutations has been complicated, however, by a lack of experimental validation and a poor understanding of the epistatic factors influencing these correlations, including strain background and programmatic variation in treatment regimens. Here we perform a genome-wide association analysis in a panel of Mycobacterium tuberculosis strains from China to identify variants correlated with resistance to the second-line prodrug ethionamide (ETH). Mutations in a bacterial monooxygenase, Rv0565c, are significantly associated with ETH resistance. We demonstrate that Rv0565c is a novel activator of ETH, independent of the two known activators, EthA and MymA. Clinically prevalent mutations abrogate Rv0565c function, and deletion of Rv0565c confers a consistent fitness benefit on M. tuberculosis in the presence of partially inhibitory doses of ETH. Interestingly, Rv0565c activity affects susceptibility to prothionamide (PTH), the ETH analog used in China, to a greater degree. Further, clinical isolates vary in their susceptibility to both ETH and PTH, to an extent that correlates with the total expression of ETH/PTH activators (EthA, MymA, and Rv0565c). These results suggest that clinical strains considered susceptible to ETH/PTH are not equally fit during treatment due to both Rv0565c mutations and more global variation in the expression of the prodrug activators.IMPORTANCE Phenotypic antibiotic susceptibility testing in Mycobacterium tuberculosis is slow and cumbersome. Rapid molecular diagnostics promise to help guide therapy, but such assays rely on complete knowledge of the molecular determinants of altered antibiotic susceptibility. Recent genomic studies of antibiotic-resistant M. tuberculosis have identified several candidate loci beyond those already known to contribute to antibiotic resistance; however, efforts to provide experimental validation have lagged. Our study identifies a gene (Rv0565c) that is associated with resistance to the second-line antibiotic ethionamide at a population level. We then use bacterial genetics to show that the variants found in clinical strains of M. tuberculosis improve bacterial survival after ethionamide exposure.

Highlights

In Mycobacterium tuberculosis, recent genome-wide association studies have identified a novel constellation of mutations that are correlated with high-level drug resistances
Even as the global incidence and mortality of tuberculosis disease (TB) have steadily decreased over the last several years, the incidence of multidrug-resistant TB (MDR-TB) is projected to rise over the decade [1]. The discrepancy in these trends is in part due to large differences in the efficacy of antibiotic treatment for drugsusceptible tuberculosis (DS-TB) versus MDR-TB; DS-TB is treated for 6 months with a combination of four potent first-line antibiotics capable of achieving a durable cure in ϳ95% of patients [2, 3], while MDR-TB is treated using more complex and longer regimens that have significantly lower success rates [4]
To identify genes and intergenic regions in which mutations are associated with ETH resistance in clinical isolates, we used phyOverlap [9] to perform genomewide association testing on a set of sequenced M. tuberculosis strains from China for which drug susceptibility phenotypes were available [11]

Summary

Introduction

In Mycobacterium tuberculosis, recent genome-wide association studies have identified a novel constellation of mutations that are correlated with high-level drug resistances. Genome-wide association (GWA) approaches have been applied to identify genetic determinants of drug resistance in several bacterial pathogens [8] These studies benefit from directly examining mutations that have evolved in the host environment with physiological levels and timing of antibiotic exposure. Recurrent loss-of-function mutants in genes encoding two additional bacterial monooxygenases, mymA (Rv3083) and Rv0565c, were identified in a large clinical data set from Russia [19], and a recent study implicated mymA in ETH susceptibility during in vitro growth, where mutation of either ethA or mymA conferred intermediate resistance to ETH, whereas the double mutant conferred a much higher level of resistance [20]. Another study suggested that Rv0565c mutations were correlated with ethA mutations in only one bacterial lineage [13] and proposed that Rv0565c mutations may be compensatory mutations reflecting the complexity of assessing genetic contributions to drug susceptibility

Methods

Results

Conclusion