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Enhanced respiration prevents drug tolerance and drug resistance in Mycobacterium tuberculosis.

ABSTRACT: Persistence, manifested as drug tolerance, represents a significant obstacle to global tuberculosis control. The bactericidal drugs isoniazid and rifampicin kill greater than 99% of exponentially growing Mycobacterium tuberculosis (Mtb) cells, but the remaining cells are persisters, cells with decreased metabolic rate, refractory to killing by these drugs, and able to generate drug-resistant mutants. We discovered that the combination of cysteine or other small thiols with either isoniazid or rifampicin prevents the formation of drug-tolerant and drug-resistant cells in Mtb cultures. This effect was concentration- and time-dependent, relying on increased oxygen consumption that triggered enhanced production of reactive oxygen species. In infected murine macrophages, the addition of N-acetylcysteine to isoniazid treatment potentiated the killing of Mtb Furthermore, we demonstrate that the addition of small thiols to Mtb drug treatment shifted the menaquinol/menaquinone balance toward a reduced state that stimulates Mtb respiration and converts persister cells to metabolically active cells. This prevention of both persister cell formation and drug resistance leads ultimately to mycobacterial cell death. Strategies to enhance respiration and initiate oxidative damage should improve tuberculosis chemotherapies.

SUBMITTER: Vilcheze C

PROVIDER: S-EPMC5410800 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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Enhanced respiration prevents drug tolerance and drug resistance in Mycobacterium tuberculosis.

Vilchèze Catherine C Hartman Travis T Weinrick Brian B Jain Paras P Weisbrod Torin R TR Leung Lawrence W LW Freundlich Joel S JS Jacobs William R WR

Proceedings of the National Academy of Sciences of the United States of America 20170410 17

Persistence, manifested as drug tolerance, represents a significant obstacle to global tuberculosis control. The bactericidal drugs isoniazid and rifampicin kill greater than 99% of exponentially growing Mycobacterium tuberculosis (Mtb) cells, but the remaining cells are persisters, cells with decreased metabolic rate, refractory to killing by these drugs, and able to generate drug-resistant mutants. We discovered that the combination of cysteine or other small thiols with either i ...[more]

PMID: 28396391

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Project description:Initial responses to tuberculosis treatment are poor predictors of final therapeutic outcomes in drug-susceptible disease, suggesting that treatment success depends on features that are hidden within a small minority of the overall infecting Mycobacterium tuberculosis population. We developed a multitranswell robotic system to perform numerous parallel cultures of genetically barcoded M. tuberculosis exposed to steady-state concentrations of rifampicin to uncover these difficult-to-eliminate minority populations. We found that tolerance emerged repeatedly from at least two subpopulations of barcoded cells, namely, one that could not grow on solid agar media and a second that could form colonies, but whose kill curves diverged from the general bacterial population within 4 and 16 days of drug exposure, respectively. These tolerant subpopulations reproducibly passed through a phase characterized by multiple unfixed resistance mutations followed by emergent drug resistance in some cultures. Barcodes associated with drug resistance identified an especially privileged subpopulation that was rarely eliminated despite 20 days of drug treatment even in cultures that did not contain any drug-resistant mutants. The association of this evolutionary scenario with a defined subset of barcodes across multiple independent cultures suggested a transiently heritable phenotype, and indeed, glpK phase variation mutants were associated with up to 16% of the resistant cultures. Drug tolerance and resistance were eliminated in a ΔruvA mutant, consistent with the importance of bacterial stress responses. This work provides a window into the origin and dynamics of bacterial drug-tolerant subpopulations whose elimination may be critical for developing rapid and resistance-free cures. IMPORTANCE Tuberculosis is unusual among bacterial diseases in that treatments which can rapidly resolve symptoms do not predictably lead to a durable cure unless treatment is continued for months after all clinical and microbiological signs of disease have been eradicated. Using a novel steady-state antibiotic exposure system combined with chromosomal barcoding, we identified small hidden Mycobacterium tuberculosis subpopulations that repeatedly enter a state of drug tolerance with a predisposition to develop fixed drug resistance after first developing a cloud of unfixed resistance mutations. The existence of these difficult-to-eradicate subpopulations may explain the need for extended treatment regimen for tuberculosis. Their identification provides opportunities to test genetic and therapeutic approaches that may result in shorter and more effective TB treatments.

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Enhanced respiration prevents drug tolerance and drug resistance in Mycobacterium tuberculosis.

Publications

Enhanced respiration prevents drug tolerance and drug resistance in <i>Mycobacterium tuberculosis</i>.

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