title: Stringent Response Factors PPX1 and PPK2 Play an Important Role in Mycobacterium tuberculosis Metabolism, Biofilm Formation, and Sensitivity to Isoniazid In Vivo.
abstract: 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.
authors.affiliation: Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Yu-Min
authors.initials: YM
authors.lastname: Chuang
authors.affiliation: Department of Medicine, Johns Hopkins University School of Medicine, Baltimore Maryland, USA.
authors.forename: Noton K
authors.initials: NK
authors.lastname: Dutta
authors.affiliation: Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.|Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Chien-Fu
authors.initials: CF
authors.lastname: Hung
authors.affiliation: Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.|Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.|Department of Obstetrics and Gynecology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.|Department of Molecular Microbiology and Immunology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: T-C
authors.initials: TC
authors.lastname: Wu
authors.affiliation: Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, USA.
authors.forename: Harvey
authors.initials: H
authors.lastname: Rubin
authors.affiliation: Department of Medicine, Johns Hopkins University School of Medicine, Baltimore Maryland, USA petros@jhmi.edu.|Department of International Health, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA.
authors.forename: Petros C
authors.initials: PC
authors.lastname: Karakousis
mesh_terms: D017766:Acid Anhydride Hydrolases / Q000172:deficiency / Q000235:genetics*; D000818:Animals; D000995:Antitubercular Agents / Q000494:pharmacology*; D018441:Biofilms / Q000187:drug effects / Q000254:growth & development; D002952:Citric Acid Cycle / Q000235:genetics; D002991:Clofazimine / Q000494:pharmacology; D004195:Disease Models, Animal; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D015870:Gene Expression; D005994:Glycerophosphates / Q000378:metabolism; D007527:Isoenzymes / Q000172:deficiency / Q000235:genetics; D007538:Isoniazid / Q000494:pharmacology*; D000077731:Meropenem; D051379:Mice; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D009285:Naphthoquinones / Q000494:pharmacology; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000172:deficiency / Q000235:genetics*; D011122:Polyphosphates / Q000378:metabolism; D013845:Thienamycins / Q000494:pharmacology; D032581:Tuberculosis Vaccines / Q000008:administration & dosage*; D018088:Tuberculosis, Multidrug-Resistant / Q000188:drug therapy* / Q000276:immunology / Q000382:microbiology / Q000517:prevention & control; D019444:Vaccines, DNA / Q000008:administration & dosage; D014994:Xylose / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D000995:Antitubercular Agents; D005994:Glycerophosphates; D007527:Isoenzymes; D009285:Naphthoquinones; D011122:Polyphosphates; D013845:Thienamycins; D032581:Tuberculosis Vaccines; D019444:Vaccines, DNA; C492551:deoxyxylulose phosphate; C029620:alpha-glycerophosphoric acid; D014994:Xylose; D002991:Clofazimine; D017856:Phosphotransferases (Phosphate Group Acceptor); C022424:polyphosphate kinase; D017766:Acid Anhydride Hydrolases; C020137:exopolyphosphatase; D000077731:Meropenem; D007538:Isoniazid; C014758:plumbagin