Effects of Endogenous d -Alanine Synthesis and Autoinhibition of Bacillus anthracis Germination on In Vitro and In Vivo Infections

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 twofold decrease in the 50% lethal dose of B. anthracis spores administered in the presence of D-alanine, indicating that rates of germination may be directly influenced by the levels of this amino acid during early stages of disease.Bacillus anthracis spores transit from the lungs to the bloodstream during early stages of inhalational anthrax (7), and current dogma suggests that spores are engaged by resident macrophages during this step in infection (16). However, unlike vegetative B. anthracis, spores are resistant to killing by the macrophage (10, 14). Moreover, Hu et al. demonstrated that preventing spore germination using an antigerminant protects B. anthracis from macrophage-mediated destruction (10). These findings indicate that delayed germination could work to the advantage of B. anthracis by preventing macrophage-specific killing of the organism during the transition from localized to systemic infection. Hence, factors that influence the efficiency of germination may determine if B. anthracis is destroyed early in infection or survives to cause systemic disease.The influence of extrinsic and intrinsic factors on B. anthracis spore germination has been the focus of several studies (3,5,6,11,12,26,27). Yet the mechanisms by which these factors impact the timing and the location of B. anthracis germination during infection are poorly understood. More importantly, the factors that could delay germination of B. anthracis during early stages of inhalational anthrax have not been defined. At the most fundamental level, the efficiency of B. anthracis germination is influenced by the available concentrations of germinants and antigerminants. ...

The Exosporium Layer of Bacterial Spores: a Connection to the Environment and the Infected Host

“…The presence of Alr in the spore may function to inhibit premature germination of the spore under suboptimal growth conditions, such as in the intracellular macrophage environment. It may also prevent premature germination during sporulation (69,70). The distribution of Alr in the exosporium of B. anthracis is not uniform.…”

“…The timing of germination is thought to be critical to the outcome of the spore-macrophage interaction (70). The exosporium is involved in the initial spore interactions with the intracellular compartment of phagocytes following spore uptake.…”

“…Enzymes are present in the outer spore layers, apparently to prevent premature germination of the internalized spores and to provide protection to the emerging vegetative cell following germination of the spore (34). These spore-associated enzymes include alanine racemase to convert L-alanine (a germinant) to D-alanine (an inhibitor of L-alanine-induced germination) and at least two, and possibly more, inosine-preferring nucleoside hydrolases that may function to inactivate inosine (another germinant species) (35,69,70,101,142).…”

Opening moves: early events in anthrax pathogenesis and their consequences for dissemination

“…The dominant protein on the exosporium is BclA, a collagen-like protein that covers the exosporium with a hair-like nap [55]. However, other important proteins are present on the exosporium, such as the superoxide dismutase and an alanine racimase, which have important roles in pathogenesis and germination [56][57][58].…”

“…While L-amino acids are important in triggering germination, D-amino acids inhibit germination [65,66]. The alanine racemase enzyme on the exosporium takes advantage of this activity to prevent germination at inopportune times in the host [58]. Alanine racemase converts L-alanine to D-alanine when there is a low concentration in the environment, thereby acting like a germination buffer.…”