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Impairment of D-alanine biosynthesis in Mycobacterium smegmatis determines decreased intracellular survival in human macrophages

¹ Department of Veterinary and Biomedical Sciences, University of Nebraska, Lincoln, NE 68583, USA
² Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A & M University, College Station, TX 77843, USA
³ Sección de Bacteriología, Corporación para Investigaciones Biológicas (CIB), Carrera 72A No. 78B 141, A.A. 7378, Medellín, Colombia
⁴ Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, OR 97331, USA
⁵ Department of Statistics, University of Nebraska, Lincoln, NE 68583, USA

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.

Correspondence
Raúl G. Barletta
rbarletta{at}unl.edu
L. Garry Adams
gadams{at}cvm.tamu.edu

Three supplementary figures are available with the online version of this paper, showing growth curves for M. smegmatis parental, alr mutant and complemented strains grown in MADC-TW and minimal medium, with or without D-alanine supplementation, and the susceptibility of M. smegmatis to hydrogen peroxide and sodium nitrite.