Isocitrate lyase mediates broad antibiotic tolerance in Mycobacterium tuberculosis

Nandakumar, Madhumitha; Nathan, Carl; Rhee, Kyu Y.

doi:10.1038/ncomms5306

Cited by 249 publications

(259 citation statements)

References 36 publications

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“…Characterization of the role of other enzymes (e.g., MetK) in the SAM biosynthesis pathway will help to dissect this mechanism further. Some aspects of the metabolic trauma described here are similar to the oxidative stress elicited by many front-line TB drugs (28). The induction of glyoxylate and methylcitrate pathways and of oxidative stress response genes is consistent with a recently proposed role for anaplerosis in antioxidant defense in Mtb (28).…”

Section: Discussionsupporting

confidence: 57%

Essential roles of methionine and S -adenosylmethionine in the autarkic lifestyle of Mycobacterium tuberculosis

Berney

Berney-Meyer

Wong

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

121

163

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Multidrug resistance, strong side effects, and compliance problems in TB chemotherapy mandate new ways to kill Mycobacterium tuberculosis (Mtb). Here we show that deletion of the gene encoding homoserine transacetylase (metA) inactivates methionine and S-adenosylmethionine (SAM) biosynthesis in Mtb and renders this pathogen exquisitely sensitive to killing in immunocompetent or immunocompromised mice, leading to rapid clearance from host tissues. Mtb ΔmetA is unable to proliferate in primary human macrophages, and in vitro starvation leads to extraordinarily rapid killing with no appearance of suppressor mutants. Cell death of Mtb ΔmetA is faster than that of other auxotrophic mutants (i.e., tryptophan, pantothenate, leucine, biotin), suggesting a particularly potent mechanism of killing. Time-course metabolomics showed complete depletion of intracellular methionine and SAM. SAM depletion was consistent with a significant decrease in methylation at the DNA level (measured by single-molecule real-time sequencing) and with the induction of several essential methyltransferases involved in biotin and menaquinone biosynthesis, both of which are vital biological processes and validated targets of antimycobacterial drugs. Mtb ΔmetA could be partially rescued by biotin supplementation, confirming a multitarget cell death mechanism. The work presented here uncovers a previously unidentified vulnerability of Mtb-the incapacity to scavenge intermediates of SAM and methionine biosynthesis from the host. This vulnerability unveils an entirely new drug target space with the promise of rapid killing of the tubercle bacillus by a new mechanism of action.host-pathogen interaction | bactericidal auxotrophy | amino acid biosynthesis | metabolism U nderstanding the metabolic interactions between an invading microbe and its host is becoming a new cornerstone of host-pathogen research (1-3). Many intracellular pathogens modulate the host response to satisfy their nutritional needs and as a result have become auxotrophic for several essential amino acids and cofactors (4-6). Mycobacterium tuberculosis (Mtb), arguably the most deadly bacterial pathogen in the world (7), adopted a different strategy. This ultra-slow-growing bacterium is prototrophic for all essential cofactors and amino acids, suggesting that it either dwells in host compartments where such metabolites are unavailable or actively chooses this autarkic lifestyle to retain metabolic flexibility and remain invisible to the host. Indeed, much of Mtb's long-term success as a human pathogen is ascribed to its extraordinary stealth in the face of host immunity (8, 9); Mtb's ability to evade detection by the host might explain why devising an efficient vaccine has failed thus far and why drug therapy is difficult. Therefore, understanding Mtb's in vivo metabolic requirements could help in the development of much-needed new strategies for antimycobacterial therapy.Methionine and S-adenosylmethionine (SAM) are essential metabolites that have gained considerable scientific attenti...

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Section: Discussionsupporting

confidence: 57%

Essential roles of methionine and S -adenosylmethionine in the autarkic lifestyle of Mycobacterium tuberculosis

Berney

Berney-Meyer

Wong

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

121

163

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“…Our data raise the possibility that poly(P) accumulation leads to upregulation of the arginine deiminase pathway, perhaps reflecting a switch to alternative energy sources. Remodeling of the citric acid cycle, including increased production of succinate and decreased production of ␣-ketoglutarate, has been observed during M. tuberculosis adaptation to hypoxia and following bacillary exposure to antibiotics (56,57). In E. coli, citrate and succinate were found to accumulate during exposure to bactericidal antibiotics (58).…”

Section: Discussionmentioning

confidence: 94%

Stringent Response Factors PPX1 and PPK2 Play an Important Role in Mycobacterium tuberculosis Metabolism, Biofilm Formation, and Sensitivity to Isoniazid In Vivo

Chuang

Dutta

Hung

et al. 2016

Antimicrob Agents Chemother

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g Mycobacterium tuberculosis remains a global health threat largely due to the lengthy duration of curative antibiotic treatment, contributing to medical nonadherence and the emergence of drug resistance. This prolonged therapy is likely due to the presence of M. tuberculosis persisters, which exhibit antibiotic tolerance. Inorganic polyphosphate [poly(P)] is a key regulatory molecule in the M. tuberculosis stringent response mediating antibiotic tolerance. The polyphosphate kinase PPK1 is responsible for poly(P) synthesis in M. tuberculosis, while the exopolyphosphatases PPX1 and PPX2 and the GTP synthase PPK2 are responsible for poly(P) hydrolysis. In the present study, we show by liquid chromatography-tandem mass spectrometry that poly(P)-accumulating M. tuberculosis mutant strains deficient in ppx1 or ppk2 had significantly lower intracellular levels of glycerol-3-phosphate (G3P) and 1-deoxy-xylulose-5-phosphate. Real-time PCR revealed decreased expression of genes in the G3P synthesis pathway in each mutant. The ppx1-deficient mutant also showed a significant accumulation of metabolites in the tricarboxylic acid cycle, as well as altered arginine and NADH metabolism. Each poly(P)-accumulating strain showed defective biofilm formation, while deficiency of ppk2 was associated with increased sensitivity to plumbagin and meropenem and deficiency of ppx1 led to enhanced susceptibility to clofazimine. A DNA vaccine expressing ppx1 and ppk2, together with two other members of the M. tuberculosis stringent response, M. tuberculosis rel and sigE, did not show protective activity against aerosol challenge with M. tuberculosis, but vaccine-induced immunity enhanced the killing activity of isoniazid in a murine model of chronic tuberculosis. In summary, poly(P)-regulating factors of the M. tuberculosis stringent response play an important role in M. tuberculosis metabolism, biofilm formation, and antibiotic sensitivity in vivo.

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“…Hydroxyl radical-mediated oxidative stress can be toxic by damaging proteins, membranes, and DNA (11). Mycobacterium tuberculosis was found to up-regulate its glyoxylate shunt when exposed to three different kinds of clinically used tuberculosis antibiotics: isoniazid, rifampicin, and streptomycin (12). E. coli experiencing superoxide stress also increased metabolic fluxes (48%) through the glyoxylate shunt (13).…”

mentioning

confidence: 99%

Role of Glyoxylate Shunt in Oxidative Stress Response

Ahn

Jung

Jang

et al. 2016

Journal of Biological Chemistry

206

161

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The glyoxylate shunt (GS) is a two-step metabolic pathway (isocitrate lyase, aceA; and malate synthase, glcB) that serves as an alternative to the tricarboxylic acid cycle. The GS bypasses the carbon dioxide-producing steps of the tricarboxylic acid cycle and is essential for acetate and fatty acid metabolism in bacteria. GS can be up-regulated under conditions of oxidative stress, antibiotic stress, and host infection, which implies that it plays important but poorly explored roles in stress defense and pathogenesis. In many bacterial species, including Pseudomonas aeruginosa, aceA and glcB are not in an operon, unlike in Escherichia coli. In P. aeruginosa, we explored relationships between GS genes and growth, transcription profiles, and biofilm formation. Contrary to our expectations, deletion of aceA in P. aeruginosa improved cell growth under conditions of oxidative and antibiotic stress. Transcriptome data suggested that aceA mutants underwent a metabolic shift toward aerobic denitrification; this was supported by additional evidence, including up-regulation of denitrification-related genes, decreased oxygen consumption without lowering ATP yield, increased production of denitrification intermediates (NO and N 2 O), and increased cyanide resistance. The aceA mutants also produced a thicker exopolysaccharide layer; that is, a phenotype consistent with aerobic denitrification. A bioinformatic survey across known bacterial genomes showed that only microorganisms capable of aerobic metabolism possess the glyoxylate shunt. This trend is consistent with the hypothesis that the GS plays a previously unrecognized role in allowing bacteria to tolerate oxidative stress.

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Isocitrate lyase mediates broad antibiotic tolerance in Mycobacterium tuberculosis

Cited by 249 publications

References 36 publications

Essential roles of methionine and S -adenosylmethionine in the autarkic lifestyle of Mycobacterium tuberculosis

Essential roles of methionine and S -adenosylmethionine in the autarkic lifestyle of Mycobacterium tuberculosis

Stringent Response Factors PPX1 and PPK2 Play an Important Role in Mycobacterium tuberculosis Metabolism, Biofilm Formation, and Sensitivity to Isoniazid In Vivo

Role of Glyoxylate Shunt in Oxidative Stress Response

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