title: Impairment of D-alanine biosynthesis in Mycobacterium smegmatis determines decreased intracellular survival in human macrophages.
abstract: d-Alanine is a structural component of mycobacterial peptidoglycan. The primary route of d-alanine biosynthesis in eubacteria is the enantiomeric conversion from l-alanine, a reaction catalysed by d-alanine racemase (Alr). Mycobacterium smegmatis alr insertion mutants are not dependent on d-alanine for growth and display a metabolic pattern consistent with an alternative pathway for d-alanine biosynthesis. In this study, we demonstrate that the M. smegmatis alr insertion mutant TAM23 can synthesize d-alanine at lower levels than the parental strain. The insertional inactivation of the alr gene also decreases the intracellular survival of mutant strains within primary human monocyte-derived macrophages. By complementation studies, we confirmed that the impairment of alr gene function is responsible for this reduced survival. Inhibition of superoxide anion and nitric oxide formation in macrophages suppresses the differential survival. In contrast, for bacteria grown in broth, both strains had approximately the same susceptibility to hydrogen peroxide, acidified sodium nitrite, low pH and polymyxin B. In contrast, TAM23 exhibited increased resistance to lysozyme. d-Alanine supplementation considerably increased TAM23 viability in nutritionally deficient media and within macrophages. These results suggest that nutrient deprivation in phagocytic cells combined with killing mediated by reactive intermediates underlies the decreased survival of alr mutants. This knowledge may be valuable in the construction of mycobacterial auxotrophic vaccine candidates.
authors.affiliation: Sección de Bacteriología, Corporación para Investigaciones Biológicas (CIB), Carrera 72A No. 78B 141, A.A. 7378, Medellín, Colombia.|Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A & M University, College Station, TX 77843, USA.|Department of Veterinary and Biomedical Sciences, University of Nebraska, Lincoln, NE 68583, USA.
authors.forename: Ofelia
authors.initials: O
authors.lastname: Chacon
authors.affiliation: Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, OR 97331, USA.
authors.forename: Luiz E
authors.initials: LE
authors.lastname: Bermudez
authors.affiliation: Department of Veterinary and Biomedical Sciences, University of Nebraska, Lincoln, NE 68583, USA.
authors.forename: Denise K
authors.initials: DK
authors.lastname: Zinniel
authors.affiliation: Department of Veterinary and Biomedical Sciences, University of Nebraska, Lincoln, NE 68583, USA.
authors.forename: Harpreet K
authors.initials: HK
authors.lastname: Chahal
authors.affiliation: Department of Veterinary and Biomedical Sciences, University of Nebraska, Lincoln, NE 68583, USA.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Fenton
authors.affiliation: Department of Veterinary and Biomedical Sciences, University of Nebraska, Lincoln, NE 68583, USA.
authors.forename: Zhengyu
authors.initials: Z
authors.lastname: Feng
authors.affiliation: Department of Statistics, University of Nebraska, Lincoln, NE 68583, USA.
authors.forename: Kathy
authors.initials: K
authors.lastname: Hanford
authors.affiliation: Sección de Bacteriología, Corporación para Investigaciones Biológicas (CIB), Carrera 72A No. 78B 141, A.A. 7378, Medellín, Colombia.
authors.forename: L Garry
authors.initials: LG
authors.lastname: Adams
authors.affiliation: Department of Veterinary and Biomedical Sciences, University of Nebraska, Lincoln, NE 68583, USA.
authors.forename: Raúl G
authors.initials: RG
authors.lastname: Barletta
mesh_terms: D000409:Alanine / Q000096:biosynthesis*; D002478:Cells, Cultured; D006801:Humans; D008264:Macrophages / Q000382:microbiology*; D016254:Mutagenesis, Insertional; D009165:Mycobacterium Infections, Nontuberculous / Q000382:microbiology*; D020102:Mycobacterium smegmatis / Q000235:genetics / Q000502:physiology*
chemical_list: D000409:Alanine