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 fact that inflammation-sensitive proteins were identified as increased in pancreatic cancer sera supports the hypothesis that inflammatory-driven processes are involved in pancreatic carcinogenesis. Liquid ESI-MS analyses of sera hold promise for future pancreatic cancer blood tests as well as for understanding mechanisms of pancreatic carcinogenesis. The variability observed between the low-mass regions of normal versus pancreatic cancer spectra may aid in diagnosis and therapy.
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 ...
Zinc finger motifs are present in a wide variety of regulatory proteins and generally function as interaction modules between macromolecules. These functional interactions are controlled by mechanisms of zinc (Zn2+)-binding and release. Besides Zn2+ certain electrophilic metals can potentially react with zinc finger domains and lead to changes in the structure and function of those domains. In these studies, the Cys2His2 zinc finger was chosen as a model for understanding how the gold (I) (Au1+) drug, aurothiomalate (AuTM), interacts mechanistically with the Zn2+ coordination sphere. DNA binding assays were used to analyze functional interactions between AuTM and two model Cys2His2 zinc finger transcription factors, TFIIIA and Sp1; inhibition in the micromolar range of AuTM was observed in both cases. Electrospray ionization mass spectrometry (ESI-MS) was utilized to examine molecular interactions between AuTM and a zinc finger peptide modeled after the third finger of Sp1 (Sp1-3). These experiments demonstrated Au1+ ions can bind the zinc finger structure and trigger the release of the Zn2+ ion. Quantifying the ESI-MS data allowed for a relative affinity value between Zn2+ and Au1+ ions to be calculated and shows Au1+ has a 4-fold higher affinity for Sp1-3 than Zn2+. Mechanistic differences between Zn2+ and Au1+ binding to the model Sp1-3 zinc finger were analyzed at isotopic resolution, and the metal-coordination spheres were probed with small molecules (H+, hydrogen peroxide, glutathione disulfide, and iodoacetamide). Natural isotope cluster analysis suggested the presence of a metal-thiol bond in the Cys2His2 zinc finger structure. Metal exchange reactions between zinc fingers demonstrated Zn2+ ions exchanged more rapidly than Au1+ ions. Circular dichroism (CD) exhibited differences in the secondary structure of the Sp1-3 model peptide when binding Zn2+ or Au1+ ions.
Exogenous CD1d-binding glycolipid (α-Galactosylceramide, α-GC) stimulates TCR signaling and activation of type-1 natural killer–like T (NKT) cells. Activated NKT cells play a central role in the regulation of adaptive and protective immune responses against pathogens and tumors. In the present study, we tested the effect of Bacillus anthracis lethal toxin (LT) on NKT cells both in vivo and in vitro. LT is a binary toxin known to suppress host immune responses during anthrax disease and intoxicates cells by protective antigen (PA)-mediated intracellular delivery of lethal factor (LF), a potent metalloprotease. We observed that NKT cells expressed anthrax toxin receptors (CMG-2 and TEM-8) and bound more PA than other immune cell types. A sub-lethal dose of LT administered in vivo in C57BL/6 mice decreased expression of the activation receptor NKG2D by NKT cells but not by NK cells. The in vivo administration of LT led to decreased TCR-induced cytokine secretion but did not affect TCR expression. Further analysis revealed LT-dependent inhibition of TCR-stimulated MAP kinase signaling in NKT cells attributable to LT cleavage of the MAP kinase kinase MEK-2. We propose that Bacillus anthracis–derived LT causes a novel form of functional anergy in NKT cells and therefore has potential for contributing to immune evasion by the pathogen.
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