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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 under                            growth-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 Site                            Hybridization to result in overrepresentation after hypoxia. OAA,                            oxaloacetate; MAG, monoacylglycerol; DAG, diacylglycerol. (B)                                Δtgs1 bacteria grow to a higher cell density in                            hypoxic cultures. (C) Δtgs1 mutants continue to                            replicate in hypoxic culture. The replication dynamics of the indicated                            strains were assessed by quantifying the rate at which unstable plasmid                            pBP10 was lost (right axis, open symbols). The “cumulative                            bacterial number” (left axis, closed symbols) represents the total                            number of organisms that would have been present if cell death was                            negated. Arrows in (B) and (C) indicate the initiation of hypoxia based                            on methylene blue decolorization. (D and E) Growth of M.                                tuberculosis strains at an initial pH of 5.5 (D) and in low                            iron medium (E). Optical density measurements are shown (similar data                            were obtained by quantifying CFU). Means ± SD of two independent                            experiments each performed in duplicate or triplicate are shown. Insets                            demonstrate the lack of TAG accumulation (upper species) in                                Δtgs1 bacteria, as assessed by thin layer                            chromatography. Each TLC was developed independently. In inset,                            “a,” H37Rv; “b,” Δtgs1; and                            “c,” complemented strain                                Δtgs1+pTGS1.
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pbio-1001065-g001: Triglyceride synthesis mutants continue to replicate under growth-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 Site Hybridization to result in overrepresentation after hypoxia. OAA, oxaloacetate; MAG, monoacylglycerol; DAG, diacylglycerol. (B) Δtgs1 bacteria grow to a higher cell density in hypoxic cultures. (C) Δtgs1 mutants continue to replicate in hypoxic culture. The replication dynamics of the indicated strains were assessed by quantifying the rate at which unstable plasmid pBP10 was lost (right axis, open symbols). The “cumulative bacterial number” (left axis, closed symbols) represents the total number of organisms that would have been present if cell death was negated. Arrows in (B) and (C) indicate the initiation of hypoxia based on methylene blue decolorization. (D and E) Growth of M. tuberculosis strains at an initial pH of 5.5 (D) and in low iron medium (E). Optical density measurements are shown (similar data were obtained by quantifying CFU). Means ± SD of two independent experiments each performed in duplicate or triplicate are shown. Insets demonstrate the lack of TAG accumulation (upper species) in Δtgs1 bacteria, as assessed by thin layer chromatography. Each TLC was developed independently. In inset, “a,” H37Rv; “b,” Δtgs1; and “c,” complemented strain Δtgs1+pTGS1.

Mentions: Prominent among the 34 identified genes (Table S1) were several predicted to encode the enzymes necessary to produce triacylglycerol (TAG) from glycerol and acyl-CoA (Figure 1A). The gene that appeared to play the most important role, tgs1, encodes a well-characterized TAG synthase that represents the dominant triglyceride synthetic activity under hypoxia [17],[18]. The importance of the tgs1 gene under this condition is likely due to its transcriptional induction via the DosR regulator, which controls the earliest response to hypoxia [19]. Consistent with the known regulatory relationship between tgs1 and DosR, our genetic screen also indicated that dosR mutants were overrepresented in the library exposed to hypoxia. The similar phenotypes of mutants lacking virtually every step in this pathway indicated that DosR-triggered TAG accumulation was critical for hypoxia-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 under                            growth-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 Site                            Hybridization to result in overrepresentation after hypoxia. OAA,                            oxaloacetate; MAG, monoacylglycerol; DAG, diacylglycerol. (B)                                Δtgs1 bacteria grow to a higher cell density in                            hypoxic cultures. (C) Δtgs1 mutants continue to                            replicate in hypoxic culture. The replication dynamics of the indicated                            strains were assessed by quantifying the rate at which unstable plasmid                            pBP10 was lost (right axis, open symbols). The “cumulative                            bacterial number” (left axis, closed symbols) represents the total                            number of organisms that would have been present if cell death was                            negated. Arrows in (B) and (C) indicate the initiation of hypoxia based                            on methylene blue decolorization. (D and E) Growth of M.                                tuberculosis strains at an initial pH of 5.5 (D) and in low                            iron medium (E). Optical density measurements are shown (similar data                            were obtained by quantifying CFU). Means ± SD of two independent                            experiments each performed in duplicate or triplicate are shown. Insets                            demonstrate the lack of TAG accumulation (upper species) in                                Δtgs1 bacteria, as assessed by thin layer                            chromatography. Each TLC was developed independently. In inset,                            “a,” H37Rv; “b,” Δtgs1; and                            “c,” complemented strain                                Δtgs1+pTGS1.
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pbio-1001065-g001: Triglyceride synthesis mutants continue to replicate under growth-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 Site Hybridization to result in overrepresentation after hypoxia. OAA, oxaloacetate; MAG, monoacylglycerol; DAG, diacylglycerol. (B) Δtgs1 bacteria grow to a higher cell density in hypoxic cultures. (C) Δtgs1 mutants continue to replicate in hypoxic culture. The replication dynamics of the indicated strains were assessed by quantifying the rate at which unstable plasmid pBP10 was lost (right axis, open symbols). The “cumulative bacterial number” (left axis, closed symbols) represents the total number of organisms that would have been present if cell death was negated. Arrows in (B) and (C) indicate the initiation of hypoxia based on methylene blue decolorization. (D and E) Growth of M. tuberculosis strains at an initial pH of 5.5 (D) and in low iron medium (E). Optical density measurements are shown (similar data were obtained by quantifying CFU). Means ± SD of two independent experiments each performed in duplicate or triplicate are shown. Insets demonstrate the lack of TAG accumulation (upper species) in Δtgs1 bacteria, as assessed by thin layer chromatography. Each TLC was developed independently. In inset, “a,” H37Rv; “b,” Δtgs1; and “c,” complemented strain Δtgs1+pTGS1.
Mentions: Prominent among the 34 identified genes (Table S1) were several predicted to encode the enzymes necessary to produce triacylglycerol (TAG) from glycerol and acyl-CoA (Figure 1A). The gene that appeared to play the most important role, tgs1, encodes a well-characterized TAG synthase that represents the dominant triglyceride synthetic activity under hypoxia [17],[18]. The importance of the tgs1 gene under this condition is likely due to its transcriptional induction via the DosR regulator, which controls the earliest response to hypoxia [19]. Consistent with the known regulatory relationship between tgs1 and DosR, our genetic screen also indicated that dosR mutants were overrepresented in the library exposed to hypoxia. The similar phenotypes of mutants lacking virtually every step in this pathway indicated that DosR-triggered TAG accumulation was critical for hypoxia-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