title: A new dehydratase conferring innate resistance to thiacetazone and intra-amoebal survival of Mycobacterium smegmatis.
abstract: Nontuberculous mycobacteria are innately resistant to most antibiotics, although the mechanisms responsible for their drug resistance remain poorly understood. They are particularly refractory to thiacetazone (TAC), a second-line antitubercular drug. Herein, we identified MSMEG_6754 as essential for the innate resistance of Mycobacterium smegmatis to TAC. Transposon-mediated and targeted disruption of MSMEG_6754 resulted in hypersusceptibility to TAC. Conversely, introduction of MSMEG_6754 into Mycobacterium tuberculosis increased resistance 100-fold. Resolution of the crystal structure of MSMEG_6754 revealed a homodimer in which each monomer comprises two hot-dog domains characteristic of dehydratase-like proteins and very similar to the HadAB complex involved in mycolic acid biosynthesis. Gene inactivation of the essential hadB dehydratase could be achieved in M. smegmatis and M. tuberculosis only when the strains carried an integrated copy of MSMEG_6754, supporting the idea that MSMEG_6754 and HadB share redundant dehydratase activity. Using M. smegmatis-Acanthamoeba co-cultures, we found that intra-amoebal growth of the MSMEG_6754 deleted strain was significantly reduced compared with the parental strain. This in vivo growth defect was fully restored upon complementation with catalytically active MSMEG_6754 or HadABC, indicating that MSMEG_6754 plays a critical role in the survival of M. smegmatis within the environmental host.
authors.affiliation: Laboratoire de Dynamique des Interactions Membranaires Normales et Pathologiques, CNRS UMR 5235, Université de Montpellier, case 107, Place Eugène Bataillon, 34095 Montpellier Cedex 05, France.|INSERM U1058, Université de Montpellier and Department of Bacteriology-Virology, CHU de Montpellier, Montpellier, 34095, France.
authors.forename: Séverine
authors.initials: S
authors.lastname: Carrère-Kremer
authors.affiliation: CARB Centre, Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10c, DK-8000 Aarhus C, Denmark.
authors.forename: Mickael
authors.initials: M
authors.lastname: Blaise
authors.affiliation: Laboratoire de Dynamique des Interactions Membranaires Normales et Pathologiques, CNRS UMR 5235, Université de Montpellier, case 107, Place Eugène Bataillon, 34095 Montpellier Cedex 05, France.
authors.forename: Vipul K
authors.initials: VK
authors.lastname: Singh
authors.affiliation: Laboratoire de Dynamique des Interactions Membranaires Normales et Pathologiques, CNRS UMR 5235, Université de Montpellier, case 107, Place Eugène Bataillon, 34095 Montpellier Cedex 05, France.
authors.forename: Laeticia
authors.initials: L
authors.lastname: Alibaud
authors.affiliation: INSERM U1058, Université de Montpellier and Department of Bacteriology-Virology, CHU de Montpellier, Montpellier, 34095, France.
authors.forename: Edouard
authors.initials: E
authors.lastname: Tuaillon
authors.affiliation: Laboratoire de Dynamique des Interactions Membranaires Normales et Pathologiques, CNRS UMR 5235, Université de Montpellier, case 107, Place Eugène Bataillon, 34095 Montpellier Cedex 05, France.
authors.forename: Iman
authors.initials: I
authors.lastname: Halloum
authors.affiliation: Department of Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam, 1081 BT, The Netherlands.
authors.forename: Robert
authors.initials: R
authors.lastname: van de Weerd
authors.affiliation: Unité de Glycobiologie Structurale et Fonctionnelle, CNRS UMR 8576, IFR 147, Université des Sciences et Technologies de Lille, 59655 Villeneuve d'Ascq cedex, France.
authors.forename: Yann
authors.initials: Y
authors.lastname: Guérardel
authors.affiliation: URMITE, UMR63, CNRS 7278, IRD 198, INSERM 1095, Université Aix-Marseille, Marseille, France.
authors.forename: Michel
authors.initials: M
authors.lastname: Drancourt
authors.affiliation: Laboratorio de Genética Molecular, CMBC, IVIC, Caracas, 1020A, Venezuela.
authors.forename: Howard
authors.initials: H
authors.lastname: Takiff
authors.affiliation: Department of Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam, 1081 BT, The Netherlands.
authors.forename: Jeroen
authors.initials: J
authors.lastname: Geurtsen
authors.affiliation: Laboratoire de Dynamique des Interactions Membranaires Normales et Pathologiques, CNRS UMR 5235, Université de Montpellier, case 107, Place Eugène Bataillon, 34095 Montpellier Cedex 05, France.|INSERM, DIMNP, Place Eugène Bataillon, 34095 Montpellier Cedex 05, France.
authors.forename: Laurent
authors.initials: L
authors.lastname: Kremer
mesh_terms: D048248:Acanthamoeba castellanii / Q000382:microbiology*; D000818:Animals; D000995:Antitubercular Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D004285:Dogs; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics; D020868:Gene Silencing; D005816:Genetic Complementation Test; D006836:Hydro-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D050296:Microbial Viability / Q000187:drug effects; D008968:Molecular Conformation; D020102:Mycobacterium smegmatis / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics / Q000502:physiology*; D009169:Mycobacterium tuberculosis / Q000235:genetics; D016415:Sequence Alignment; D017384:Sequence Deletion; D013828:Thioacetazone / Q000494:pharmacology*
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D006836:Hydro-Lyases; D013828:Thioacetazone