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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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Triglyceride synthesis mutants continue to replicate undergrowth-limiting conditions.(A) The predicted TAG biosynthetic pathway of M.tuberculosis and its relationship to the TCA cycle.Mutations in the underlined genes were predicted by Transposon SiteHybridization to result in overrepresentation after hypoxia. OAA,oxaloacetate; MAG, monoacylglycerol; DAG, diacylglycerol. (B)Δtgs1 bacteria grow to a higher cell density inhypoxic cultures. (C) Δtgs1 mutants continue toreplicate in hypoxic culture. The replication dynamics of the indicatedstrains were assessed by quantifying the rate at which unstable plasmidpBP10 was lost (right axis, open symbols). The “cumulativebacterial number” (left axis, closed symbols) represents the totalnumber of organisms that would have been present if cell death wasnegated. Arrows in (B) and (C) indicate the initiation of hypoxia basedon methylene blue decolorization. (D and E) Growth of M.tuberculosis strains at an initial pH of 5.5 (D) and in lowiron medium (E). Optical density measurements are shown (similar datawere obtained by quantifying CFU). Means ± SD of two independentexperiments each performed in duplicate or triplicate are shown. Insetsdemonstrate the lack of TAG accumulation (upper species) inΔtgs1 bacteria, as assessed by thin layerchromatography. Each TLC was developed independently. In inset,“a,” H37Rv; “b,” Δtgs1; and“c,” complemented strainΔtgs1+pTGS1.
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pbio-1001065-g001: Triglyceride synthesis mutants continue to replicate undergrowth-limiting conditions.(A) The predicted TAG biosynthetic pathway of M.tuberculosis and its relationship to the TCA cycle.Mutations in the underlined genes were predicted by Transposon SiteHybridization to result in overrepresentation after hypoxia. OAA,oxaloacetate; MAG, monoacylglycerol; DAG, diacylglycerol. (B)Δtgs1 bacteria grow to a higher cell density inhypoxic cultures. (C) Δtgs1 mutants continue toreplicate in hypoxic culture. The replication dynamics of the indicatedstrains were assessed by quantifying the rate at which unstable plasmidpBP10 was lost (right axis, open symbols). The “cumulativebacterial number” (left axis, closed symbols) represents the totalnumber of organisms that would have been present if cell death wasnegated. Arrows in (B) and (C) indicate the initiation of hypoxia basedon methylene blue decolorization. (D and E) Growth of M.tuberculosis strains at an initial pH of 5.5 (D) and in lowiron medium (E). Optical density measurements are shown (similar datawere obtained by quantifying CFU). Means ± SD of two independentexperiments each performed in duplicate or triplicate are shown. Insetsdemonstrate the lack of TAG accumulation (upper species) inΔtgs1 bacteria, as assessed by thin layerchromatography. Each TLC was developed independently. In inset,“a,” H37Rv; “b,” Δtgs1; and“c,” complemented strainΔtgs1+pTGS1.

Mentions: Prominent among the 34 identified genes (Table S1) were several predicted to encodethe enzymes necessary to produce triacylglycerol (TAG) from glycerol andacyl-CoA (Figure 1A). Thegene that appeared to play the most important role, tgs1,encodes a well-characterized TAG synthase that represents the dominanttriglyceride synthetic activity under hypoxia [17],[18]. The importance of thetgs1 gene under this condition is likely due to itstranscriptional induction via the DosR regulator, which controls the earliestresponse to hypoxia [19]. Consistent with the known regulatory relationshipbetween tgs1 and DosR, our genetic screen also indicated thatdosR mutants were overrepresented in the library exposed tohypoxia. The similar phenotypes of mutants lacking virtually every step in thispathway indicated that DosR-triggered TAG accumulation was critical forhypoxia-induced growth arrest.


Metabolic regulation of mycobacterial growth and antibiotic sensitivity.

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

Triglyceride synthesis mutants continue to replicate undergrowth-limiting conditions.(A) The predicted TAG biosynthetic pathway of M.tuberculosis and its relationship to the TCA cycle.Mutations in the underlined genes were predicted by Transposon SiteHybridization to result in overrepresentation after hypoxia. OAA,oxaloacetate; MAG, monoacylglycerol; DAG, diacylglycerol. (B)Δtgs1 bacteria grow to a higher cell density inhypoxic cultures. (C) Δtgs1 mutants continue toreplicate in hypoxic culture. The replication dynamics of the indicatedstrains were assessed by quantifying the rate at which unstable plasmidpBP10 was lost (right axis, open symbols). The “cumulativebacterial number” (left axis, closed symbols) represents the totalnumber of organisms that would have been present if cell death wasnegated. Arrows in (B) and (C) indicate the initiation of hypoxia basedon methylene blue decolorization. (D and E) Growth of M.tuberculosis strains at an initial pH of 5.5 (D) and in lowiron medium (E). Optical density measurements are shown (similar datawere obtained by quantifying CFU). Means ± SD of two independentexperiments each performed in duplicate or triplicate are shown. Insetsdemonstrate the lack of TAG accumulation (upper species) inΔtgs1 bacteria, as assessed by thin layerchromatography. Each TLC was developed independently. In inset,“a,” H37Rv; “b,” Δtgs1; and“c,” complemented strainΔtgs1+pTGS1.
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pbio-1001065-g001: Triglyceride synthesis mutants continue to replicate undergrowth-limiting conditions.(A) The predicted TAG biosynthetic pathway of M.tuberculosis and its relationship to the TCA cycle.Mutations in the underlined genes were predicted by Transposon SiteHybridization to result in overrepresentation after hypoxia. OAA,oxaloacetate; MAG, monoacylglycerol; DAG, diacylglycerol. (B)Δtgs1 bacteria grow to a higher cell density inhypoxic cultures. (C) Δtgs1 mutants continue toreplicate in hypoxic culture. The replication dynamics of the indicatedstrains were assessed by quantifying the rate at which unstable plasmidpBP10 was lost (right axis, open symbols). The “cumulativebacterial number” (left axis, closed symbols) represents the totalnumber of organisms that would have been present if cell death wasnegated. Arrows in (B) and (C) indicate the initiation of hypoxia basedon methylene blue decolorization. (D and E) Growth of M.tuberculosis strains at an initial pH of 5.5 (D) and in lowiron medium (E). Optical density measurements are shown (similar datawere obtained by quantifying CFU). Means ± SD of two independentexperiments each performed in duplicate or triplicate are shown. Insetsdemonstrate the lack of TAG accumulation (upper species) inΔtgs1 bacteria, as assessed by thin layerchromatography. Each TLC was developed independently. In inset,“a,” H37Rv; “b,” Δtgs1; and“c,” complemented strainΔtgs1+pTGS1.
Mentions: Prominent among the 34 identified genes (Table S1) were several predicted to encodethe enzymes necessary to produce triacylglycerol (TAG) from glycerol andacyl-CoA (Figure 1A). Thegene that appeared to play the most important role, tgs1,encodes a well-characterized TAG synthase that represents the dominanttriglyceride synthetic activity under hypoxia [17],[18]. The importance of thetgs1 gene under this condition is likely due to itstranscriptional induction via the DosR regulator, which controls the earliestresponse to hypoxia [19]. Consistent with the known regulatory relationshipbetween tgs1 and DosR, our genetic screen also indicated thatdosR mutants were overrepresented in the library exposed tohypoxia. The similar phenotypes of mutants lacking virtually every step in thispathway indicated that DosR-triggered TAG accumulation was critical forhypoxia-induced growth arrest.

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