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The role of the novel exopolyphosphatase MT0516 in Mycobacterium tuberculosis drug tolerance and persistence.

ABSTRACT: Inorganic polyphosphate (poly P) has been postulated to play a regulatory role in the transition to bacterial persistence. In bacteria, poly P balance in the cell is maintained by the hydrolysis activity of the exopolyphosphatase PPX. However, the Mycobacterium tuberculosis PPX has not been characterized previously. Here we show that recombinant MT0516 hydrolyzes poly P, and an MT0516-deficient M. tuberculosis mutant exhibits elevated intracellular levels of poly P and increased expression of the genes mprB, sigE, and rel relative to the isogenic wild-type strain, indicating poly P-mediated signaling. Deficiency of MT0516 resulted in decelerated growth during logarithmic-phase in axenic cultures, and tolerance to the cell wall-active drug isoniazid. The MT0516-deficient mutant showed a significant survival defect in activated human macrophages and reduced persistence in the lungs of guinea pigs. We conclude that exopolyphosphatase is required for long-term survival of M. tuberculosis in necrotic lung lesions.

SUBMITTER: Thayil SM

PROVIDER: S-EPMC3221697 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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The role of the novel exopolyphosphatase MT0516 in Mycobacterium tuberculosis drug tolerance and persistence.

Thayil Seema M SM Morrison Norman N Schechter Norman N Rubin Harvey H Karakousis Petros C PC

PloS one 20111121 11

Inorganic polyphosphate (poly P) has been postulated to play a regulatory role in the transition to bacterial persistence. In bacteria, poly P balance in the cell is maintained by the hydrolysis activity of the exopolyphosphatase PPX. However, the Mycobacterium tuberculosis PPX has not been characterized previously. Here we show that recombinant MT0516 hydrolyzes poly P, and an MT0516-deficient M. tuberculosis mutant exhibits elevated intracellular levels of poly P and increased expression of th ...[more]

PMID: 22132215

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Project description:UNLABELLED:Mycobacterium tuberculosis can persist for decades in the human host. Stringent response pathways involving inorganic polyphosphate [poly(P)], which is synthesized and hydrolyzed by polyphosphate kinase (PPK) and exopolyphosphatase (PPX), respectively, are believed to play a key regulatory role in bacterial persistence. We show here that M. tuberculosis poly(P) accumulation is temporally linked to bacillary growth restriction. We also identify M. tuberculosis Rv1026 as a novel exopolyphosphatase with hydrolytic activity against long-chain poly(P). Using a tetracycline-inducible expression system to knock down expression of Rv1026 (ppx2), we found that M. tuberculosis poly(P) accumulation leads to slowed growth and reduced susceptibility to isoniazid, increased resistance to heat and acid pH, and enhanced intracellular survival during macrophage infection. By transmission electron microscopy, the ppx2 knockdown strain exhibited increased cell wall thickness, which was associated with reduced cell wall permeability to hydrophilic drugs rather than induction of drug efflux pumps or altered biofilm formation relative to the empty vector control. Transcriptomic and metabolomic analysis revealed a metabolic downshift of the ppx2 knockdown characterized by reduced transcription and translation and a downshift of glycerol-3-phosphate levels. In summary, poly(P) plays an important role in M. tuberculosis growth restriction and metabolic downshift and contributes to antibiotic tolerance through altered cell wall permeability. IMPORTANCE:The stringent response, involving the regulatory molecules inorganic polyphosphate [poly(P)] and (p)ppGpp, is believed to mediate Mycobacterium tuberculosis persistence. In this study, we identified a novel enzyme (Rv1026, PPX2) responsible for hydrolyzing long-chain poly(P). A genetically engineered M. tuberculosis strain deficient in the ppx2 gene showed increased poly(P) levels, which were associated with early bacterial growth arrest and reduced susceptibility to the first-line drug isoniazid, as well as increased bacterial survival during exposure to stress conditions and within macrophages. Relative to the control strain, the mutant showed increased thickness of the cell wall and reduced drug permeability. Global gene expression and metabolite analysis revealed reduced expression of the transcriptional and translational machinery and a shift in carbon source utilization. In summary, regulation of the poly(P) balance is critical for persister formation in M. tuberculosis.

Project description:ABSTRACT The chronic nature of tuberculosis (TB), its requirement of long duration of treatment, its ability to evade immune intervention, and its propensity to relapse after drug treatment is discontinued are reminiscent of other chronic, biofilm-associated bacterial diseases. Historically, Mycobacterium tuberculosis was grown as a pellicle, a biofilm-like structure, at the liquid-air interface in a variety of synthetic media. Notably, the most widely administered human vaccine, BCG, is grown as a pellicle for vaccine production. However, the molecular requirements for this growth remain ill defined. Here, we demonstrate that keto-mycolic acids (keto-MA) are essential for pellicle growth, and mutants lacking in or depleted of this MA species are unable to form a pellicle. We investigated the role of the pellicle biofilm in the reduction of antibiotic sensitivity known as drug tolerance using the pellicle-defective ?mmaA4 mutant strain. We discovered that the ?mmaA4 mutant, which is both pellicle defective and highly sensitive to rifampicin (RIF) under planktonic growth, when incorporated within the wild-type pellicle biofilm, was protected from the bactericidal activity of RIF. The observation that growth within the M. tuberculosis pellicle biofilm can confer drug tolerance to a drug-hypersensitive strain suggests that identifying molecular requirements for pellicle growth could lead to development of novel interventions against mycobacterial infections. Our findings also suggest that a class of drugs that can disrupt M. tuberculosis biofilm formation, when used in conjunction with conventional antibiotics, has the potential to overcome drug tolerance. IMPORTANCE Two of the most important questions in tuberculosis (TB) research are (i) how does Mycobacterium tuberculosis persist in the human host for decades in the face of an active immune response and (ii) why does it take six months and four drugs to treat uncomplicated TB. Both these aspects of M. tuberculosis biology are reminiscent of infections caused by organisms capable of forming biofilms. M. tuberculosis is capable of growing as a biofilm-like structure called the pellicle. In this study, we demonstrate that a specific cell wall component, keto-mycolic acid, is essential for pellicle growth. We also demonstrate that a strain of M. tuberculosis that is both drug sensitive and pellicle defective exhibits commensal behavior and becomes drug tolerant by becoming part of a heterogeneous pellicle, a characteristic of multispecies biofilms. These observations could have important implications for identifying novel pathways for M. tuberculosis drug tolerance and the design of new modalities to rapidly treat TB.

Dataset Information

The role of the novel exopolyphosphatase MT0516 in Mycobacterium tuberculosis drug tolerance and persistence.

Publications

The role of the novel exopolyphosphatase MT0516 in Mycobacterium tuberculosis drug tolerance and persistence.

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