Endogenous D-alanine Synthesis and Auto-inhibition of Bacillus anthracis Germination: Effects on in vitro and in vivo Infections

Department of Microbiology and Immunology, The University of Oklahoma Health Sciences Center, Oklahoma City, OK, 73014; Department of Microbiology and the Institute for Genomic Biology, University of Illinois, Urbana, IL, 68101; Department of Internal Medicine, University of New Mexico Health Sciences Center, Albuquerque, NM, 87131

^* To whom correspondence should be addressed. Email: jimmy-ballard{at}ouhsc.edu.

	Abstract

Bacillus anthracis transitions from a dormant spore to a vegetative bacillus through a series of structural and biochemical changes collectively referred to as germination. The timing of germination is important during early steps in infection, and may determine if B. anthracis survives or succumbs to responsive macrophages. In the current study experiments determined the contribution of endogenous D-alanine production to the efficiency and timing of B. anthracis spore germination under in vitro and in vivo conditions. Racemase-mediated production of endogenous D-alanine by B. anthracis altered the kinetics for initiation of germination over a range of spore-densities, and exhibited a threshold effect wherein small changes in spore number resulted in major changes in germination efficiency. This threshold effect correlated with D-alanine production, was prevented by an alanine racemase inhibitor, and required L-alanine. Interestingly, endogenous production of inhibitory levels of D-alanine was detected under experimental conditions that did not support germination, and in a germination deficient mutant of B. anthracis. Racemase-dependent production of D-alanine enhanced survival of B. anthracis during interaction with murine macrophages, suggesting a role for inhibition of germination during interaction with these cells. Finally, in vivo experiments revealed an approximately 2-fold decrease in the LD₅₀ of B. anthracis spores administered in the presence of D-alanine, indicating rates of germination may be directly influenced by the levels of this amino-acid during early stages of disease.