Bacillus anthracis edema toxin (ET) generates high levels of cyclic AMP and impacts a complex network of signaling pathways in targeted cells. In the current study, we sought to identify kinase signaling pathways modulated by ET to better understand how this toxin alters cell physiology. Using a panel of small-molecule inhibitors of mammalian kinases, we found that inhibitors of glycogen synthase kinase 3 beta (GSK-3␤) protected cells from ET-induced changes in the cell cycle. GSK-3␤ inhibitors prevented declines in cellular levels of cyclin D1 and c-Jun following treatment of macrophages with ET. Strikingly, cell fractionation experiments and confocal immunofluorescence microscopy revealed that ET activates a compartmentalized pool of GSK-3␤ residing in the nuclei, but not in the cytoplasm, of macrophages. To investigate the outcome of this event, we examined the cellular location and activation state of ␤-catenin, a critical substrate of GSK-3␤, and found that the protein was inactivated within the nucleus following intoxication with ET. To determine if ET could overcome the effects of stimuli that inactivate GSK-3␤, we examined the impact of the toxin on the Wnt signaling pathway. The results of these experiments revealed that by targeting GSK-3␤ residing in the nucleus, ET circumvents the upstream cytoplasmic inactivation of GSK-3␤, which occurs following exposure to Wnt-3A. These findings suggest ET arrests the cell cycle by a mechanism involving activation of GSK-3␤ residing in the nucleus, and by using this novel mechanism of intoxication, ET avoids cellular systems that would otherwise reverse the effects of the toxin.Bacillus anthracis edema factor (EF) is a calcium-and calmodulin-dependent adenylate cyclase that generates high levels of cyclic AMP (cAMP) after delivery into the cell by protective antigen (PA) (20). The three-dimensional structure of EF has been resolved, and its catalytic mechanism is well understood (10,15,16,32). Edema toxin (ET), the combination of PA and EF, suppresses immune responses during the development and progression of anthrax disease (3,26,36). ET has also been found to sensitize mice to anthrax lethal toxin (12), increase the expression of anthrax toxin receptors in monocytic cells (22), and reduce cell viability (11, 39). Thus, while EF generates a common second messenger, cAMP, the toxin does so in a way that disrupts normal cellular activities.ET generates supraphysiological levels of cAMP, which is thought to accumulate in the perinuclear region of the cell (7,20). In the original description of EF as an adenylate cyclase, Leppla found that treatment of CHO K1A cells with ET increased levels of cAMP by approximately 200-fold (20). In a more recent study by Dal Molin et al., EF was shown to be delivered into the cell via a late endosome pathway, and by remaining associated with this compartment, the toxin generates cAMP in the perinuclear region, with decreasing gradients of cAMP radiating to the periphery of the cell (7). These observations support a toxicity model in...

Anthrax edema toxin (ET) is one of two binary toxins produced by Bacillus anthracis that contributes to the virulence of this pathogen. ET is an adenylate cyclase that generates high levels of cyclic AMP (cAMP), causing alterations in multiple host cell signaling pathways. We previously demonstrated that ET increases cell surface expression of the anthrax toxin receptors (ANTXR) in monocyte-derived cells and promotes dendritic cell (DC) migration toward the lymph node-homing chemokine MIP-3␤. In this work, we sought to determine if glycogen synthase kinase 3 (GSK-3) is important for ET-induced modulation of macrophage and DC function. We demonstrate that inhibition of GSK-3 dampens ET-induced maturation and migration processes of monocytederived dendritic cells (MDDCs). Additional studies reveal that the ET-induced expression of ANTXR in macrophages was decreased when GSK-3 activity was disrupted with chemical inhibitors or with small interfering RNA (siRNA) targeting GSK-3. Further examination of the ET induction of ANTXR revealed that a dominant negative form of CREB could block the ET induction of ANTXR, suggesting that CREB or a related family member was involved in the upregulation of ANTXR. Because CREB and GSK-3 activity appeared to be important for ET-induced ANTXR expression, the impact of GSK-3 on ET-induced CREB activity was examined in RAW 264.7 cells possessing a CRE-luciferase reporter. As with ANTXR expression, the ET induction of the CRE reporter was decreased by reducing GSK-3 activity. These studies not only provide insight into host pathways targeted by ET but also shed light on interactions between GSK-3 and CREB pathways in host immune cells.

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...