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. ...

In the current study, we examined the regulatory interactions of a serine/threonine phosphatase (BA-Stp1), serine/threonine kinase (BA-Stk1) pair in Bacillus anthracis. B. anthracis STPK101, a null mutant lacking BA-Stp1 and BA-Stk1, was impaired in its ability to survive within macrophages, and this correlated with an observed reduction in virulence in a mouse model of pulmonary anthrax. Biochemical analyses confirmed that BA-Stp1 is a PP2C phosphatase and dephosphorylates phosphoserine and phosphothreonine residues. Treatment of BA-Stk1 with BA-Stp1 altered BA-Stk1 kinase activity, indicating that the enzymatic function of BA-Stk1 can be influenced by BA-Stp1 dephosphorylation. Using a combination of mass spectrometry and mutagenesis approaches, three phosphorylated residues, T165, S173, and S214, in BA-Stk1 were identified as putative regulatory targets of BA-Stp1. Further analysis found that T165 and S173 were necessary for optimal substrate phosphorylation, while S214 was necessary for complete ATP hydrolysis, autophosphorylation, and substrate phosphorylation. These findings provide insight into a previously undescribed Stp/Stk pair in B. anthracis.A profile of the intracellular signaling proteins that regulate transition of Bacillus anthracis from dormancy to expression of virulence factors is emerging. Like many prokaryotes, B. anthracis utilizes two-component histidine kinase systems to regulate physiological changes and the expression of virulence factors. These systems include the Spo0 histidine kinase-based phosphorelay pathway (32, 37) and the Bacillus respiratory response A and B system involved in regulating toxin expression (36). Unlike for histidine kinase systems, little is known about reversible serine/threonine phosphorylation events in B. anthracis. These systems are common to eukaryotic cells (3,14,25,40) but were only recently found in prokaryotes to modulate a variety of metabolic and physiological processes (1,2,7,11,12,15,17,24,28,35,38). Whether reversible serine/threonine phosphorylation contributes to similar events in B. anthracis is not known.The current paradigm for prokaryotic serine/threonine kinases (STK) is based in part on the structure of PknB, a serine/threonine kinase from Mycobacterium tuberculosis that is structurally related to eukaryotic Hanks-type kinases (39). PknB autophosphorylates and is dephosphorylated by an M. tuberculosis phosphatase, PstP, in order to alter kinase activity (4). Similar to the findings for PnkB, Madec et al. identified critical autophosphorylated residues and autophosphorylated domains of PrkC, an STK from Bacillus subtilis (22), which suggested that the phosphorylation state of these residues impacts the activation of PrkC (22). These studies suggested that prokaryotic STKs exhibited activities similar to those of their eukaryotic homologs and were regulated by cognate phosphatases. Hence, studies of serine/threonine phosphatase (STP)/STK pairs may help define a core regulatory module in bacterial physiology and virulence, wherein the kinase ...

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Resistance to antibiotics currently used empirically to treat newborns is present in bacteremia-producing E. coli. Clonal spread among newborns of multidrug-resistant E. coli is possible; therefore, continued surveillance is needed. Identification of additional virulence factors associated with increased invasion in neonatal E. coli strains is important and further studies are warranted.

The production of cAMP from Bacillus anthracis edema toxin (ET) activates gene expression in macrophages through a complex array of signaling pathways, most of which remain poorly defined. In this study, the tumor suppressor protein adenomatous polyposis coli (APC) was found to be important for the up-regulation of previously defined ET-stimulated genes (Vegfa, Ptgs2, Arg2, Cxcl2, Sdc1, and Cebpb). A reduction in the expression of these genes after ET exposure was observed when APC was disrupted in macrophages using siRNA or in bone marrow-derived macrophages obtained from C57BL/6J-Apc Min mice, which are heterozygous for a truncated form of APC. In line with this observation, ET increased the expression of APC at the transcriptional level, leading to increased amounts of APC in the nucleus. The mechanism utilized by APC to increase ETinduced gene expression was determined to depend on the ability of APC to interact with C/EBP ␤, which is a transcription factor activated by cAMP. Coimmunoprecipitation experiments found that APC associated with C/EBP ␤ and that levels of this complex increase after ET exposure. A further connection was uncovered when silencing APC was determined to reduce the ET-induced phosphorylation of C/EBP ␤ at Thr-188. This ET-mediated phosphorylation of C/EBP ␤ was blocked by glycogen synthase kinase 3 (GSK-3) inhibitors, suggesting that GSK-3 is involved in the activation of C/EBP ␤ and supporting the idea of APC helping direct interactions between GSK-3 and C/EBP ␤. These results indicate that ET stimulates gene expression by promoting the formation of an inducible protein complex consisting of APC and C/EBP ␤.