title: Glutamate Dehydrogenase Is Required by Mycobacterium bovis BCG for Resistance to Cellular Stress.
abstract: We recently reported on our success to generate deletion mutants of the genes encoding glutamate dehydrogenase (GDH) and glutamine oxoglutarate aminotransferase (GOGAT) in M. bovis BCG, despite their in vitro essentiality in M. tuberculosis. We could use these mutants to delineate the roles of GDH and GOGAT in mycobacterial nitrogen metabolism by using M. bovis BCG as a model for M. tuberculosis specifically. Here, we extended our investigation towards the involvement of GDH and GOGAT in other aspects of M. bovis BCG physiology, including the use of glutamate as a carbon source and resistance to known phagosomal stresses, as well as in survival inside macrophages. We find that gdh is indispensable for the utilization of glutamate as a major carbon source, in low pH environments and when challenged with nitric oxide. On the other hand, the gltBD mutant had increased viability under low pH conditions and was unaffected by a challenge with nitric oxide. Strikingly, GDH was required to sustain M. bovis BCG during infection of both murine RAW 264.7 and bone-marrow derived and macrophages, while GOGAT was not. We conclude that the catabolism of glutamate in slow growing mycobacteria may be a crucial function during infection of macrophage cells and demonstrate a novel requirement for M. bovis BCG GDH in the protection against acidic and nitrosative stress. These results provide strong clues on the role of GDH in intracellular survival of M. tuberculosis, in which the essentiality of the gdh gene complicates knock out studies making the study of the role of this enzyme in pathogenesis difficult.
authors.affiliation: DST-NRF Centre of Excellence for Biomedical Tuberculosis Research, SA MRC Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, Cape Town, South Africa.
authors.forename: James L
authors.initials: JL
authors.lastname: Gallant
authors.affiliation: Centre National de la Recherche Scientifique FRE 3689, Centre d'études d'agents Pathogènes et Biotechnologies pour la Santé, Université de Montpellier, 1919 route de Mende, Montpellier, France.
authors.forename: Albertus J
authors.initials: AJ
authors.lastname: Viljoen
authors.affiliation: DST-NRF Centre of Excellence for Biomedical Tuberculosis Research, SA MRC Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, Cape Town, South Africa.
authors.forename: Paul D
authors.initials: PD
authors.lastname: van Helden
authors.affiliation: DST-NRF Centre of Excellence for Biomedical Tuberculosis Research, SA MRC Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, Cape Town, South Africa.
authors.forename: Ian J F
authors.initials: IJ
authors.lastname: Wiid
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D015870:Gene Expression; D005816:Genetic Complementation Test; D005969:Glutamate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism; D008264:Macrophages / Q000382:microbiology; D051379:Mice; D009154:Mutation; D009163:Mycobacterium bovis / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D009169:Mycobacterium tuberculosis / Q000201:enzymology / Q000235:genetics; D009569:Nitric Oxide / Q000494:pharmacology; D020030:Nitric Oxide Donors / Q000737:chemistry / Q000494:pharmacology; D061251:Primary Cell Culture; D013312:Stress, Physiological; D000637:Transaminases / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D007656:Ketoglutaric Acids; D020030:Nitric Oxide Donors; D005973:Glutamine; D009569:Nitric Oxide; D018698:Glutamic Acid; D005969:Glutamate Dehydrogenase; D000637:Transaminases; C020656:glutamine-pyruvate aminotransferase
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