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 ...

Herpes simplex virus type 1 (HSV-1) infection of the cornea induces VEGF-A-dependent lymphangiogenesis that continues to develop well beyond the resolution of infection. Inflammatory leukocytes infiltrate the cornea and have been implicated to be essential for corneal neovascularization, an important clinically relevant manifestation of stromal keratitis. Here, we report that cornea infiltrating leukocytes including neutrophils and T cells do not have a significant role in corneal neovascularization past virus clearance. Antibody mediated depletion of these cells did not impact lymphatic or blood vessel genesis. Multiple pro-angiogenic factors including IL-6, angiopoietin-2, HGF, FGF-2, VEGF-A, and MMP-9 were expressed within the cornea following virus clearance. A single bolus of dexamethasone (DEX) at day 10 pi resulted in suppression of blood vessel genesis and regression of lymphatic vessels at day 21 pi compared to control-treated mice. Whereas IL-6 neutralization had a modest impact on hemangiogenesis (day 14–21 pi) and lymphangiogenesis (day 21 pi) in a time-dependent fashion, neutralization of FGF-2 had a more pronounced effect on the suppression of neovascularization (blood and lymphatic vessels) in a time-dependent, leukocyte-independent manner. Furthermore, FGF-2 neutralization suppressed the expression of all pro-angiogenic factors measured and preserved visual acuity.

In an effort to better understand the mechanisms by which Bacillus anthracis establishes disease, experiments were undertaken to identify the genes essential for intracellular germination. Eighteen diverse genetic loci were identified via an enrichment protocol using a transposon-mutated library of B. anthracis spores, which was screened for mutants delayed in intracellular germination. Fourteen transposon mutants were identified in genes not previously associated with B. anthracis germination and included disruption of factors involved in membrane transport, transcriptional regulation, and intracellular signaling. Four mutants contained transposon insertions in gerHA, gerHB, gerHC, and pagA, respectively, each of which has been previously associated with germination or survival of B. anthracis within macrophages. Strain MIGD101 (named for macrophage intracellular germination defective 101) was of particular interest, since this mutant contained a transposon insertion in an intergenic region between BAs2807 and BAs2808, and was the most highly represented mutant in the enrichment. Analysis of B. anthracis MIGD101 by confocal microscopy and differential heat sensitivity following macrophage infection revealed ungerminated spores within the cell. Moreover, B. anthracis MIGD101 was attenuated in cell killing relative to the parent strain. Further experimental analysis found that B. anthracis MIGD101 was defective in five known B. anthracis germination pathways, supporting a mechanism wherein the intergenic region between BAs2807 and BAs2808 has a global affect on germination of this pathogen. Collectively, these findings provide insight into the mechanisms supporting B. anthracis germination within host cells.Bacillus anthracis spores germinate during early stages of inhalational anthrax, and this is considered to be a critical step in the progression of anthrax disease (4, 5, 7). Indeed, transition from dormant spore to vegetative bacilli is essential for growth, bacteremia, toxin production, and synthesis of a poly-D-glutamic acid capsule, each of which is important to the virulence of B. anthracis.Results from a series of studies indicate B. anthracis spores are engaged by, and can germinate within, alveolar macrophages (13, 15). Sanz et al. recently described the detection of B. anthracis spores in alveolar macrophages under in vivo infection conditions using a mouse model of inhalational anthrax (15). The molecular basis of spore tropism for alveolar macrophages may be due to the collagenlike exosporium protein, BclA, which interacts with the macrophage receptor, Mac-1 (13), while at the same time preventing the interaction of spores with other types of cells. Spores are taken up by Mac-1-dependent phagocytosis into LAMP-1 positive vesicles, where germination is triggered, and the germinated spores become susceptible to killing by the macrophage (10).Very little is known about the bacterial factors that promote, repress, or otherwise regulate germination of B. anthracis spores within macrophages. To date, expe...