title: Trehalose-6-Phosphate-Mediated Toxicity Determines Essentiality of OtsB2 in Mycobacterium tuberculosis In Vitro and in Mice.
abstract: Trehalose biosynthesis is considered an attractive target for the development of antimicrobials against fungal, helminthic and bacterial pathogens including Mycobacterium tuberculosis. The most common biosynthetic route involves trehalose-6-phosphate (T6P) synthase OtsA and T6P phosphatase OtsB that generate trehalose from ADP/UDP-glucose and glucose-6-phosphate. In order to assess the drug target potential of T6P phosphatase, we generated a conditional mutant of M. tuberculosis allowing the regulated gene silencing of the T6P phosphatase gene otsB2. We found that otsB2 is essential for growth of M. tuberculosis in vitro as well as for the acute infection phase in mice following aerosol infection. By contrast, otsB2 is not essential for the chronic infection phase in mice, highlighting the substantial remodelling of trehalose metabolism during infection by M. tuberculosis. Blocking OtsB2 resulted in the accumulation of its substrate T6P, which appears to be toxic, leading to the self-poisoning of cells. Accordingly, blocking T6P production in a ΔotsA mutant abrogated otsB2 essentiality. T6P accumulation elicited a global upregulation of more than 800 genes, which might result from an increase in RNA stability implied by the enhanced neutralization of toxins exhibiting ribonuclease activity. Surprisingly, overlap with the stress response caused by the accumulation of another toxic sugar phosphate molecule, maltose-1-phosphate, was minimal. A genome-wide screen for synthetic lethal interactions with otsA identified numerous genes, revealing additional potential drug targets synergistic with OtsB2 suitable for combination therapies that would minimize the emergence of resistance to OtsB2 inhibitors.
authors.affiliation: Institute for Pharmaceutical Biology and Biotechnology, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany.|Institute for Medical Microbiology and Hospital Hygiene, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Korte
authors.affiliation: Institute for Medical Microbiology and Hospital Hygiene, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany.
authors.forename: Marina
authors.identifier: http://orcid.org/0000-0003-1377-0998
authors.initials: M
authors.lastname: Alber
authors.affiliation: Department of Microbiology and Immunology, Weill Cornell Medical College, New York, New York, United States of America.
authors.forename: Carolina M
authors.identifier: http://orcid.org/0000-0002-9601-820X
authors.initials: CM
authors.lastname: Trujillo
authors.affiliation: Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich, Norfolk, United Kingdom.
authors.forename: Karl
authors.identifier: http://orcid.org/0000-0002-9624-7221
authors.initials: K
authors.lastname: Syson
authors.affiliation: Institute for Medical Microbiology and Hospital Hygiene, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany.
authors.forename: Hendrik
authors.initials: H
authors.lastname: Koliwer-Brandl
authors.affiliation: Biological and Medical Research Center (BMFZ), Cluster of Excellence on Plant Sciences (CEPLAS), Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany.
authors.forename: René
authors.identifier: http://orcid.org/0000-0003-4520-3023
authors.initials: R
authors.lastname: Deenen
authors.affiliation: Biological and Medical Research Center (BMFZ), Cluster of Excellence on Plant Sciences (CEPLAS), Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany.
authors.forename: Karl
authors.identifier: http://orcid.org/0000-0003-3644-2022
authors.initials: K
authors.lastname: Köhrer
authors.affiliation: Department of Computer Science, Texas A&M University, College Station, Texas, United States of America.
authors.forename: Michael A
authors.identifier: http://orcid.org/0000-0003-3867-0299
authors.initials: MA
authors.lastname: DeJesus
authors.affiliation: Department of Microbiology and Immunology, Howard Hughes Medical Institute, Albert Einstein College of Medicine, Bronx, New York, United States of America.
authors.forename: Travis
authors.initials: T
authors.lastname: Hartman
authors.affiliation: Department of Microbiology and Immunology, Howard Hughes Medical Institute, Albert Einstein College of Medicine, Bronx, New York, United States of America.
authors.forename: William R
authors.initials: WR
authors.lastname: Jacobs
authors.affiliation: Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich, Norfolk, United Kingdom.
authors.forename: Stephen
authors.identifier: http://orcid.org/0000-0001-5930-9473
authors.initials: S
authors.lastname: Bornemann
authors.affiliation: Department of Computer Science, Texas A&M University, College Station, Texas, United States of America.
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Ioerger
authors.affiliation: Department of Microbiology and Immunology, Weill Cornell Medical College, New York, New York, United States of America.
authors.forename: Sabine
authors.initials: S
authors.lastname: Ehrt
authors.affiliation: Institute for Pharmaceutical Biology and Biotechnology, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany.|Institute for Medical Microbiology and Hospital Hygiene, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany.
authors.forename: Rainer
authors.identifier: http://orcid.org/0000-0002-3378-2067
authors.initials: R
authors.lastname: Kalscheuer
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D002855:Chromatography, Thin Layer; D004195:Disease Models, Animal; D005260:Female; D020869:Gene Expression Profiling; D055785:Gene Knockdown Techniques; D005964:Glucosyltransferases / Q000378:metabolism; D066298:In Vitro Techniques; D051379:Mice; D008810:Mice, Inbred C57BL; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D013403:Sugar Phosphates / Q000378:metabolism*; D014199:Trehalose / Q000031:analogs & derivatives* / Q000378:metabolism; D014376:Tuberculosis / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D013403:Sugar Phosphates; C082722:trehalose-6-phosphate; D014199:Trehalose; D005964:Glucosyltransferases; C023180:trehalose-6-phosphate synthase; C023178:trehalose-phosphatase; D010744:Phosphoric Monoester Hydrolases