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Metabolic regulation of mycobacterial growth and antibiotic sensitivity.

Baek SH, Li AH, Sassetti CM - PLoS Biol. (2011)

Bottom Line: This pathway plays a causal role in reducing growth and antibiotic efficacy by redirecting cellular carbon fluxes away from the tricarboxylic acid cycle.Mutants in which this metabolic switch is disrupted are unable to arrest their growth in response to stress and remain sensitive to antibiotics during infection.Thus, this regulatory pathway contributes to antibiotic tolerance in vivo, and its modulation may represent a novel strategy for accelerating TB treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.

ABSTRACT
Treatment of chronic bacterial infections, such as tuberculosis (TB), requires a remarkably long course of therapy, despite the availability of drugs that are rapidly bacteriocidal in vitro. This observation has long been attributed to the presence of bacterial populations in the host that are "drug-tolerant" because of their slow replication and low rate of metabolism. However, both the physiologic state of these hypothetical drug-tolerant populations and the bacterial pathways that regulate growth and metabolism in vivo remain obscure. Here we demonstrate that diverse growth-limiting stresses trigger a common signal transduction pathway in Mycobacterium tuberculosis that leads to the induction of triglyceride synthesis. This pathway plays a causal role in reducing growth and antibiotic efficacy by redirecting cellular carbon fluxes away from the tricarboxylic acid cycle. Mutants in which this metabolic switch is disrupted are unable to arrest their growth in response to stress and remain sensitive to antibiotics during infection. Thus, this regulatory pathway contributes to antibiotic tolerance in vivo, and its modulation may represent a novel strategy for accelerating TB treatment.

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Modulating carbon fluxes reverses the antibiotic tolerance induced                            during infection.Mice were infected via the aerosol route with the indicated bacterial                            strains. Total bacterial burden in the spleens (A, C, E) and lungs (B,                            D, F) is shown. Mice were treated at the indicated times with isoniazid                            (“INH”, A, B), ethambutol (“EMB”, C, D), or                            isoniazid plus pyrazinamide (“INH+PZA”, E, F). Dotted                            line represents the detection limit of the experiment. “ND”                            indicates no colonies detected. ND* indicates two colonies were                            detected but neither retained the citA overexpression                            plasmid. Means ± SD from three to five mice are shown.
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pbio-1001065-g004: Modulating carbon fluxes reverses the antibiotic tolerance induced during infection.Mice were infected via the aerosol route with the indicated bacterial strains. Total bacterial burden in the spleens (A, C, E) and lungs (B, D, F) is shown. Mice were treated at the indicated times with isoniazid (“INH”, A, B), ethambutol (“EMB”, C, D), or isoniazid plus pyrazinamide (“INH+PZA”, E, F). Dotted line represents the detection limit of the experiment. “ND” indicates no colonies detected. ND* indicates two colonies were detected but neither retained the citA overexpression plasmid. Means ± SD from three to five mice are shown.

Mentions: Induction of the tgs1 gene and TAG accumulation occur during infection [20], and TCA activity appears to be limited in this environment [25]. Therefore, we next investigated whether TCA limitation by TAG synthesis was also required for antibiotic tolerance in vivo. The Δtgs1 mutation did not overtly disrupt the physiology of the bacterium in vivo, as only subtle defects in bacterial viability were observed in mice infected with the mutant (Figure S8). Despite this apparently normal behavior, the metabolic state of the mutant was clearly different from wild type, as the Δtgs1 strain remained significantly more sensitive to several antibiotic regimens targeting different cellular functions (Figure 4). Consistent with a central role for TCA activity in antibiotic tolerance in vivo, we found that overexpressing citrate synthase had a more pronounced effect. The citA* strain displayed a modest growth or survival defect in mice (Figures 4A,B and S8), indicating that increased TCA flux under these conditions decreased overall fitness. More importantly, this strain remained even more sensitive to antibiotics during infection than the Δtgs1 mutant, as we had previously observed under in vitro stress conditions. After 28 d of monotherapy, the number of viable wild bacteria had only decreased 20-fold, while the number of viable citA overexpressors was reduced below the limit of detection (Figure 4A,B).


Metabolic regulation of mycobacterial growth and antibiotic sensitivity.

Baek SH, Li AH, Sassetti CM - PLoS Biol. (2011)

Modulating carbon fluxes reverses the antibiotic tolerance induced                            during infection.Mice were infected via the aerosol route with the indicated bacterial                            strains. Total bacterial burden in the spleens (A, C, E) and lungs (B,                            D, F) is shown. Mice were treated at the indicated times with isoniazid                            (“INH”, A, B), ethambutol (“EMB”, C, D), or                            isoniazid plus pyrazinamide (“INH+PZA”, E, F). Dotted                            line represents the detection limit of the experiment. “ND”                            indicates no colonies detected. ND* indicates two colonies were                            detected but neither retained the citA overexpression                            plasmid. Means ± SD from three to five mice are shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3101192&req=5

pbio-1001065-g004: Modulating carbon fluxes reverses the antibiotic tolerance induced during infection.Mice were infected via the aerosol route with the indicated bacterial strains. Total bacterial burden in the spleens (A, C, E) and lungs (B, D, F) is shown. Mice were treated at the indicated times with isoniazid (“INH”, A, B), ethambutol (“EMB”, C, D), or isoniazid plus pyrazinamide (“INH+PZA”, E, F). Dotted line represents the detection limit of the experiment. “ND” indicates no colonies detected. ND* indicates two colonies were detected but neither retained the citA overexpression plasmid. Means ± SD from three to five mice are shown.
Mentions: Induction of the tgs1 gene and TAG accumulation occur during infection [20], and TCA activity appears to be limited in this environment [25]. Therefore, we next investigated whether TCA limitation by TAG synthesis was also required for antibiotic tolerance in vivo. The Δtgs1 mutation did not overtly disrupt the physiology of the bacterium in vivo, as only subtle defects in bacterial viability were observed in mice infected with the mutant (Figure S8). Despite this apparently normal behavior, the metabolic state of the mutant was clearly different from wild type, as the Δtgs1 strain remained significantly more sensitive to several antibiotic regimens targeting different cellular functions (Figure 4). Consistent with a central role for TCA activity in antibiotic tolerance in vivo, we found that overexpressing citrate synthase had a more pronounced effect. The citA* strain displayed a modest growth or survival defect in mice (Figures 4A,B and S8), indicating that increased TCA flux under these conditions decreased overall fitness. More importantly, this strain remained even more sensitive to antibiotics during infection than the Δtgs1 mutant, as we had previously observed under in vitro stress conditions. After 28 d of monotherapy, the number of viable wild bacteria had only decreased 20-fold, while the number of viable citA overexpressors was reduced below the limit of detection (Figure 4A,B).

Bottom Line: This pathway plays a causal role in reducing growth and antibiotic efficacy by redirecting cellular carbon fluxes away from the tricarboxylic acid cycle.Mutants in which this metabolic switch is disrupted are unable to arrest their growth in response to stress and remain sensitive to antibiotics during infection.Thus, this regulatory pathway contributes to antibiotic tolerance in vivo, and its modulation may represent a novel strategy for accelerating TB treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.

ABSTRACT
Treatment of chronic bacterial infections, such as tuberculosis (TB), requires a remarkably long course of therapy, despite the availability of drugs that are rapidly bacteriocidal in vitro. This observation has long been attributed to the presence of bacterial populations in the host that are "drug-tolerant" because of their slow replication and low rate of metabolism. However, both the physiologic state of these hypothetical drug-tolerant populations and the bacterial pathways that regulate growth and metabolism in vivo remain obscure. Here we demonstrate that diverse growth-limiting stresses trigger a common signal transduction pathway in Mycobacterium tuberculosis that leads to the induction of triglyceride synthesis. This pathway plays a causal role in reducing growth and antibiotic efficacy by redirecting cellular carbon fluxes away from the tricarboxylic acid cycle. Mutants in which this metabolic switch is disrupted are unable to arrest their growth in response to stress and remain sensitive to antibiotics during infection. Thus, this regulatory pathway contributes to antibiotic tolerance in vivo, and its modulation may represent a novel strategy for accelerating TB treatment.

Show MeSH
Related in: MedlinePlus