Bacillus Anthracis Edema Toxin Research Articles

Imaging of anthrax intoxication in mice reveals shared and individual functions of surface receptors CMG-2 and TEM-8 in cellular toxin entry

Bacillus anthracis lethal toxin and edema toxin are binary toxins that consist of a common cell-binding moiety, protective antigen (PA), and the enzymatic moieties, lethal factor (LF) and edema factor (EF). PA binds to either of two receptors, capillary morphogenesis protein-2 (CMG-2) or tumor endothelial marker-8 (TEM-8), which triggers the binding and cytoplasmic translocation of LF and EF. However, the distribution of functional TEM-8 and CMG-2 receptors during anthrax toxin intoxication in animals has not been fully elucidated. Herein, we describe an assay to image anthrax toxin intoxication in animals, and we use it to visualize TEM-8- and CMG-2-dependent intoxication in mice. Specifically, we generated a chimeric protein consisting of the N-terminal domain of LF fused to a nuclear localization signal-tagged Cre recombinase (LFn-NLS-Cre). When PA and LFn-NLS-Cre were coadministered to transgenic mice expressing a red fluorescent protein in the absence of Cre and a green fluorescent protein in the presence of Cre, intoxication could be visualized at single-cell resolution by confocal microscopy or flow cytometry. Using this assay, we found that: (a) CMG-2 is critical for intoxication in the liver and heart, (b) TEM-8 is required for intoxication in the kidney and spleen, (c) CMG-2 and TEM-8 are redundant for intoxication of some organs, (d) combined loss of CMG-2 and TEM-8 completely abolishes intoxication, and (e) CMG-2 is the dominant receptor on leukocytes. The novel assay will be useful for basic and clinical/translational studies of Bacillus anthracis infection and for clinical development of reengineered toxin variants for cancer treatment.

Nociceptive Sensory Neurons Mediate Inflammation Induced by Bacillus Anthracis Edema Toxin.

Bacterial products are able to act on nociceptive neurons during pathogenic infection. Neurogenic inflammation is an active part of pain signaling and has recently been shown to impact host-pathogen defense. Bacillus anthracis Edema Toxin (ET) produces striking edema in peripheral tissues, but the cellular mechanisms involved in tissue swelling are not completely understood. Here, we find that nociceptive neurons play a role in ET-induced edema and inflammation in mice. Subcutaneous footpad infection of B. anthracis Sterne caused ET-dependent local mechanical allodynia, paw swelling and body weight gain. Subcutaneous administration of ET induced paw swelling and vascular leakage, the early phases of which were attenuated in the absence of Trpv1+ or Nav1.8+ nociceptive neurons. Nociceptive neurons express the anthrax toxin receptor ANTXR2, but this did not mediate ET-induced edema. ET induced local cytokine expression and neutrophil recruitment, which were dependent in part on Trpv1+ nociceptive neurons. Ablation of Trpv1+ or Nav1.8+ nociceptive neurons also attenuated early increases in paw swelling and body weight gain during live B. anthracis infection. Our findings indicate that nociceptive neurons play an active role in inflammation caused by B. anthracis and Edema Toxin to potentially influence bacterial pathogenesis.

Bacillus anthracis Edema Toxin Inhibits Efferocytosis in Human Macrophages and Alters Efferocytic Receptor Signaling.

The Bacillus anthracis Edema Toxin (ET), composed of a Protective Antigen (PA) and the Edema Factor (EF), is a cellular adenylate cyclase that alters host responses by elevating cyclic adenosine monophosphate (cAMP) to supraphysiologic levels. However, the role of ET in systemic anthrax is unclear. Efferocytosis is a cAMP-sensitive, anti-inflammatory process of apoptotic cell engulfment, the inhibition of which may promote sepsis in systemic anthrax. Here, we tested the hypothesis that ET inhibits efferocytosis by primary human macrophages and evaluated the mechanisms of altered efferocytic signaling. ET, but not PA or EF alone, inhibited the efferocytosis of early apoptotic neutrophils (PMN) by primary human M2 macrophages (polarized with IL-4, IL-10, and/or dexamethasone) at concentrations relevant to those encountered in systemic infection. ET inhibited Protein S- and MFGE8-dependent efferocytosis initiated by signaling through MerTK and αVβ5 receptors, respectively. ET inhibited Rac1 activation as well as the phosphorylation of Rac1 and key activating sites of calcium calmodulin-dependent kinases CamK1α, CamK4, and vasodilator-stimulated phosphoprotein, that were induced by the exposure of M2(Dex) macrophages to Protein S-opsonized apoptotic PMN. These results show that ET impairs macrophage efferocytosis and alters efferocytic receptor signaling.

A Novel Supplementation Approach to Enhance Host Response to Sublingual Vaccination

Sublingual immunization is emerging as an alternative to nasal immunization and induction of mucosal IgA responses. Using Bacillus anthracis edema toxin (EdTx) as an adjuvant, we previously showed that innate responses triggered after sublingual immunization could limit generation of IgA responses. We tested whether co-administration of a neutrophil elastase inhibitor (NEI) could rescue the ability of EdTx to induce broad antibody responses, including mucosal IgA. NEI supplementation of sublingual vaccines containing EdTx promoted antigen-specific serum IgA responses but also enhanced serum IgG1, and IgG2b responses. This enhancing effect of NEI did not extend to all antibody isotypes and IgG sublclasses, since NEI reduced serum IgE responses and did not affect IgG2a/c and IgG3 responses. NEI supplementation also promoted anti-Bacillus anthracis protective antigen (PA) neutralizing antibodies and enhanced high affinity IgG1 and IgA antibodies. In addition to serum IgA, NEI supplementation stimulated antigen-specific mucosal IgA responses in the GI tract, and enhanced antigen-specific IgG responses in vaginal washes. Analysis of CD4+ T helper cell responses revealed that co-administration of NEI broadened the profile of cytokine responses, by stimulating Th1, Th2, Th17, and Tfh cytokines. We also noted that NEI had a higher stimulatory effect on IL-5, IL-10, IL-17 responses.

Unique, Intersecting, and Overlapping Roles of C/EBP \u03b2 and CREB in Cells of the Innate Immune System

CREB and C/EBP β signaling pathways are modulated during inflammation and also targeted by Bacillus anthracis edema toxin (ET), but how these factors individually and jointly contribute to changes in immune cell function is poorly understood. Using CRISPR/Cas9 gene editing, macrophage cell lines lacking CREB and isoforms of C/EBP β were generated and analyzed for changes in responses to LPS, ET, and IL-4. Macrophages lacking C/EBP β suppressed induction of IL-10 and Arg1, while IL-6 was increased in these cells following exposure to LPS. Examination of C/EBP β isoforms indicated the 38 kDa isoform was necessary for the expression of IL-10 and Arg1. ChIP-Seq analysis of CREB and C/EBP β binding to targets on the chromosome of human PBMC identified several regions where both factors overlapped in their binding, suggesting similar gene targeting or cooperative effects. Based on the ChIP-Seq data, a panel of previously unknown targets of CREB and C/EBP β was identified and includes genes such as VNN2, GINS4, CTNNBL1, and SULF2. Isoforms of a transcriptional corepressor, transducin-like enhancer of Split (TLE), were also found to have CREB and C/EBP β binding their promoter and were up regulated by ET. Finally, we explore a possible layer of C/EBP β regulation by a protein complex consisting of adenomatous polyposis coli (APC) and PKA. Collectively, these data provide new insights into the role of CREB and C/EBP β as immunosignaling regulators and targets of an important bacterial virulence factor.

Compartmentalized Cyclic AMP Production by the Bordetella pertussis and Bacillus anthracis Adenylate Cyclase Toxins Differentially Affects the Immune Synapse in T Lymphocytes.

A central feature of the immune synapse (IS) is the tight compartmentalization of membrane receptors and signaling mediators that is functional for its ability to coordinate T cell activation. Second messengers centrally implicated in this process, such as Ca2+ and diacyl glycerol, also undergo compartmentalization at the IS. Current evidence suggests a more complex scenario for cyclic AMP (cAMP), which acts both as positive and as negative regulator of T-cell antigen receptor (TCR) signaling and which, as such, must be subjected to a tight spatiotemporal control to allow for signaling at the IS. Here, we have used two bacterial adenylate cyclase toxins that produce cAMP at different subcellular localizations as the result of their distinct routes of cell invasion, namely Bordetella pertussis CyaA and Bacillus anthracis edema toxin (ET), to address the ability of the T cell to confine cAMP to the site of production and to address the impact of compartmentalized cAMP production on IS assembly and function. We show that CyaA, which produces cAMP close to the synaptic membrane, affects IS stability by modulating not only the distribution of LFA-1 and its partners talin and L-plastin, as previously partly reported but also by promoting the sustained synaptic accumulation of the A-kinase adaptor protein ezrin and protein kinase A while suppressing the β-arrestin-mediated recruitment of phosphodiesterase 4B. These effects are dependent on the catalytic activity of the toxin and can be reproduced by treatment with a non-hydrolyzable cAMP analog. Remarkably, none of these effects are elicited by ET, which produces cAMP at a perinuclear localization, despite its ability to suppress TCR signaling and T cell activation through its cAMP-elevating activity. These results show that the IS responds solely to local elevations of cAMP and provide evidence that potent compartmentalization mechanisms are operational in T cells to contain cAMP close to the site of production, even when produced at supraphysiological levels.

Bacillus anthracis Edema Toxin Increases Fractional Free Water and Sodium Reabsorption in an Isolated Perfused Rat Kidney Model.

Bacillus anthracis edema toxin (ET) consists of protective antigen (PA), necessary for host cell toxin uptake, and edema factor (EF), the toxic moiety which increases host cell cyclic AMP (cAMP). Since vasopressin stimulates renal water and sodium reabsorption via increased tubular cell cAMP levels, we hypothesized the ET would also do so. To test this hypothesis, we employed an isolated perfused rat kidney model. Kidneys were isolated and perfused with modified Krebs-Henseleit buffer. Perfusate and urine samples were obtained at baseline and every 10 min over 150 min following the addition of challenges with or without treatments to the perfusate. In kidneys perfused under constant flow or constant pressure, compared to PA challenge (n = 14 or 15 kidneys, respectively), ET (13 or 15 kidneys, respectively) progressively increased urine cAMP levels, water and sodium reabsorption, and urine osmolality and decreased urine output (P ≤ 0.04, except for sodium reabsorption under constant pressure [P = 0.17]). In ET-challenged kidneys, compared to placebo treatment, adefovir, an EF inhibitor, decreased urine cAMP levels, water and sodium reabsorption, and urine osmolality and increased urine output, while raxibacumab, a PA-directed monoclonal antibody (MAb), decreased urine cAMP levels, free water reabsorption, and urine osmolality and increased urine output (P ≤ 0.03 except for urine output with raxibacumab [P = 0.17]). Upon immunohistochemistry, aquaporin 2 was concentrated along the apical membrane of tubular cells with ET but not PA, and urine aquaporin 2 levels were higher with ET (5.52 ± 1.06 ng/ml versus 1.51 ± 0.44 ng/ml [means ± standard errors of the means {SEM}; P = 0.0001). Edema toxin has renal effects that could contribute to extravascular fluid collection characterizing anthrax infection clinically.

A Simple Luminescent Adenylate-Cyclase Functional Assay for Evaluation of Bacillus anthracis Edema Factor Activity

Edema Factor (EF), the toxic sub-unit of the Bacillus anthracis Edema Toxin (ET) is a calmodulin-dependent adenylate cyclase whose detrimental activity in the infected host results in severe edema. EF is therefore a major virulence factor of B. anthracis. We describe a simple, rapid and reliable functional adenylate-cyclase assay based on inhibition of a luciferase-mediated luminescence reaction. The assay exploits the efficient adenylate cyclase-mediated depletion of adenosine tri-phosphate (ATP), and the strict dependence on ATP of the light-emitting luciferase-catalyzed luciferin-conversion to oxyluciferin, which can be easily visualized. The assay exhibits a robust EF-dose response decrease in luminescence, which may be specifically reverted by anti-EF antibodies. The application of the assay is exemplified in: (a) determining the presence of EF in B. anthracis cultures, or its absence in cultures of EF-defective strains; (b) evaluating the anti-EF humoral response in experimental animals infected/vaccinated with B. anthracis; and (c) rapid discrimination between EF producing and non-producing bacterial colonies. Furthermore, the assay may be amenable with high-throughput screening for EF inhibitory molecules.

Nitric oxide production contributes to Bacillus anthracis edema toxin-associated arterial hypotension and lethality: ex vivo and in vivo studies in the rat.

We showed previously that Bacillus anthracis edema toxin (ET), comprised of protective antigen (PA) and edema factor (EF), inhibits phenylephrine (PE)-induced contraction in rat aortic rings and these effects are diminished in endothelial-denuded rings. Therefore, employing rat aortic ring and in vivo models, we tested the hypothesis that nitric oxide (NO) contributes to ET's arterial effects. Compared with rings challenged with PA alone, ET (PA + EF) reduced PE-stimulated maximal contractile force (MCF) and increased the PE concentration producing 50% MCF (EC50) (P < 0.0001). Compared with placebo, l-nitro-arginine methyl-ester (l-NAME), an NO synthase (NOS) inhibitor, reduced ET's effects on MCF and EC50 in patterns that approached or were significant (P = 0.06 and 0.03, respectively). In animals challenged with 24-h ET infusions, l-NAME (0.5 or 1.0 mg·kg(-1)·h(-1)) coadministration increased survival to 17 of 28 animals (60.7%) compared with 4 of 27 (14.8%) given placebo (P = 0.01). Animals receiving l-NAME but no ET all survived. Compared with PBS challenge, ET increased NO levels at 24 h and l-NAME decreased these increases (P < 0.0001). ET infusion decreased mean arterial blood pressure (MAP) in placebo and l-NAME-treated animals (P < 0.0001) but l-NAME reduced decreases in MAP with ET from 9 to 24 h (P = 0.03 for the time interaction). S-methyl-l-thiocitrulline, a selective neuronal NOS inhibitor, had effects in rings and, at a high dose in vivo models, comparable to l-NAME, whereas N'-[3-(aminomethyl)benzyl]-acetimidamide, a selective inducible NOS inhibitor, did not. NO production contributes to ET's arterial relaxant, hypotensive, and lethal effects in the rat.

Micropatterned Macrophage Analysis Reveals Global Cytoskeleton Constraints Induced by Bacillus anthracis Edema Toxin

Bacillus anthracis secretes the edema toxin (ET) that disrupts the cellular physiology of endothelial and immune cells, ultimately affecting the adherens junction integrity of blood vessels that in turn leads to edema. The effects of ET on the cytoskeleton, which is critical in cell physiology, have not been described thus far on macrophages. In this study, we have developed different adhesive micropatterned surfaces (L and crossbow) to control the shape of bone marrow-derived macrophages (BMDMs) and primary peritoneal macrophages. We found that macrophage F-actin cytoskeleton adopts a specific polar organization slightly different from classical human HeLa cells on the micropatterns. Moreover, ET induced a major quantitative reorganization of F-actin within 16 h with a collapse at the nonadhesive side of BMDMs along the nucleus. There was an increase in size and deformation into a kidney-like shape, followed by a decrease in size that correlates with a global cellular collapse. The collapse of F-actin was correlated with a release of focal adhesion on the patterns and decreased cell size. Finally, the cell nucleus was affected by actin reorganization. By using this technology, we could describe many previously unknown macrophage cellular dysfunctions induced by ET. This novel tool could be used to analyze more broadly the effects of toxins and other virulence factors that target the cytoskeleton.

Neutrophils negatively regulate induction of mucosal IgA antibody responses to sublingual immunization (MUC4P.835)

Abstract Secretory IgA antibody plays an important role in the protection of mucosal surfaces against microbial pathogens. Induction of the IgA antibody has been known to be largely dependent upon the cytokine milieu. However, the importance of innate immune cell subsets for IgA responses has not been fully appreciated yet. We investigated the role of myeloid cell-subsets on IgA responses to sublingual immunization. In contrast with cholera toxin as an adjuvant which promotes IgA responses, sublingual application of the Bacillus anthracis edema toxin (EdTx) to C57BL/6 mice transiently increased the frequencies of neutrophils in sublingual tissue and cervical lymph nodes (CLNs), and promoted antigen-specific serum IgG, but not serum or mucosal IgA responses. However, EdTx as an adjuvant failed to increase frequencies of neutrophils in sublingual tissue and CLNs of mice lacking IKKβ in myeloid cells (IKKβΔMye), and these mice developed antigen-specific mucosal IgA responses. Interestingly, depletion of neutrophils in C57BL/6 mice promoted antigen-specific fecal and serum IgA responses, similar to those of IKKβΔMye mice, and this effect was associated with increased frequencies of surface IgA expressing plasma cells, α4β7+CCR9+ B cells in lamina propria and number of Peyer’s patch. Our results show that the presence of neutrophils at mucosal inductive sites negatively influences induction of IgA responses and underlying mechanisms are under investigations.

Modulation of Notch signaling by intracellular bacterial toxins (610.3)

The Notch signaling pathway has recently been recognized as a critical signaling pathway in macrophages. This study examines how Notch signaling functions in the context of infectious disease as well as how contrasting intracellular bacterial toxins modify this pathway. In this work, we investigate how the Notch signaling pathway is altered in macrophages by Bacillus anthracis edema toxin (ET) and Clostridium difficile toxin B (TcdB). In preliminary studies, ET was found to activate and augment Notch signaling in macrophages through a mechanism involving transducin‐like enhancer of split (TLE) and recombinant recognition sequence binding protein at the Jκ site (RBP‐J). Because ET, an immunosuppressive toxin, activates Notch signaling in macrophages, we hypothesized that TcdB, an inflammation stimulating toxin, may have an alternative effect on this signaling pathway. To address this possibility, the Notch signaling pathway was examined in primary macrophages exposed to TcdB, and results from these studies found that Notch signaling was suppressed by TcdB. Further analyses have suggested that the TcdB induced suppression of Notch signaling is through a mechanism involving the stimulation of interferon signaling. These results provide insight into mechanisms utilized by disparate bacterial toxins to modify Notch signaling in macrophages.

Imaging Bacillus anthracis Edema Toxin Effects on Normalized Murine Dendritic Cells and Macrophages

Event Abstract Back to Event Imaging Bacillus anthracis Edema Toxin Effects on Normalized Murine Dendritic Cells and Macrophages Yannick Trescos1, 2*, Danel Fiole1, Marion Mulier1 and Jean-Nicolas Tournier1, 2* 1 Institut de Recherche Biomédicale des Armées (IRBA), Department of microbiology, France 2 Ecole du Val-de-Grâce (EVDG), France Virulence of Bacillus anthracis, the agent of anthrax is caused by two main factors, the capsule and two toxins. One of these toxins, edema toxin (ET) is an adenylate cyclase, producing a rise in intracellular cAMP concentrations. ET deregulates the immune system but little is known on its effects on immune cell cytoskeleton. Using adhesive micropattern array, initially designed for cells capable of stretching on micropatterns and forming stress fibers, we have developed a method of depositing immune cells to normalize their morphology. First, we present an innovative, robust and sensitive model to study murine bone-marrow-dendritic cells (BMDCs) and macrophages (BMDMs) in vitro, based on their adhesion to micro-patterns to standardize their morphology. This model reduces the variability of shape inherent in our cell cultures and gain sensitivity and reproducibility. Thus, the quantification of sub-cellular phenomena has become possible for studying many functions of immune cells. Second, our micro-patterning model has been used to identify and quantify the effects of ET on cytoskeleton of BMDCs and BMDMs. We observed that ET caused a time-dependent retraction of the actin cytoskeleton on both cells. This retraction begins with the formation of actin-rich cortical protrusions, appearing like membranes ruffles. This reorganization is accompanied by a perinuclear accumulation of actin, responsible of a deformation of the nucleus. We thus infer that our imaging model on immune cells opens new perspectives for high throughput analysis of morphology and functions of BMDCs and BMDMs in vitro. Acknowledgements This presentation was made possible by Direction Générale de l’Armement funding and ANR ANABIO. Keywords: Anthrax, Dendritic Cells, Macrophages, micropatterning, edema toxin, Cytoskeleton Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Host-pathogen interactions Citation: Trescos Y, Fiole D, Mulier M and Tournier J (2013). Imaging Bacillus anthracis Edema Toxin Effects on Normalized Murine Dendritic Cells and Macrophages. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.01089 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 29 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Yannick Trescos, Institut de Recherche Biomédicale des Armées (IRBA), Department of microbiology, Brétigny-sur-Orge, 91220, France, yannick.trescos@yahoo.fr Prof. Jean-Nicolas Tournier, Institut de Recherche Biomédicale des Armées (IRBA), Department of microbiology, Brétigny-sur-Orge, 91220, France, jntournier@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Yannick Trescos Danel Fiole Marion Mulier Jean-Nicolas Tournier Google Yannick Trescos Danel Fiole Marion Mulier Jean-Nicolas Tournier Google Scholar Yannick Trescos Danel Fiole Marion Mulier Jean-Nicolas Tournier PubMed Yannick Trescos Danel Fiole Marion Mulier Jean-Nicolas Tournier Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

CAMP Signaling by Anthrax Edema Toxin Induces Transendothelial Cell Tunnels, which Are Resealed by MIM via Arp2/3-Driven Actin Polymerization

RhoA-inhibitory bacterial toxins, such as Staphylococcus aureus EDIN toxin, induce large transendothelial cell macroaperture (TEM) tunnels that rupture the host endothelium barrier and promote bacterial dissemination. Host cells repair these tunnels by extending actin-rich membrane waves from the TEM edges. We reveal that cyclic-AMP signaling produced by Bacillus anthracis edema toxin (ET) also induces TEM formation, which correlates with increased vascular permeability. We show that ET-induced TEM formation resembles liquid dewetting, a physical process of nucleation and growth of holes within a thin liquid film. We also identify the cellular mechanisms of tunnel closure and reveal that the I-BAR domain protein Missing in Metastasis (MIM) senses de novo membrane curvature generated by the TEM, accumulates at the TEM edge, and triggers Arp2/3-dependent actin polymerization, which induces actin-rich membrane waves that close the TEM. Thus, the balance between ET-induced TEM formation and resealing likely determines the integrity of the host endothelium barrier.

The Adenylate Cyclase Toxins of Bacillus anthracis and Bordetella pertussis Promote Th2 Cell Development by Shaping T Cell Antigen Receptor Signaling

The adjuvanticity of bacterial adenylate cyclase toxins has been ascribed to their capacity, largely mediated by cAMP, to modulate APC activation, resulting in the expression of Th2–driving cytokines. On the other hand, cAMP has been demonstrated to induce a Th2 bias when present during T cell priming, suggesting that bacterial cAMP elevating toxins may directly affect the Th1/Th2 balance. Here we have investigated the effects on human CD4+ T cell differentiation of two adenylate cyclase toxins, Bacillus anthracis edema toxin (ET) and Bordetella pertussis CyaA, which differ in structure, mode of cell entry, and subcellular localization. We show that low concentrations of ET and CyaA, but not of their genetically detoxified adenylate cyclase defective counterparts, potently promote Th2 cell differentiation by inducing expression of the master Th2 transcription factors, c-maf and GATA-3. We also present evidence that the Th2–polarizing concentrations of ET and CyaA selectively inhibit TCR–dependent activation of Akt1, which is required for Th1 cell differentiation, while enhancing the activation of two TCR–signaling mediators, Vav1 and p38, implicated in Th2 cell differentiation. This is at variance from the immunosuppressive toxin concentrations, which interfere with the earliest step in TCR signaling, activation of the tyrosine kinase Lck, resulting in impaired CD3ζ phosphorylation and inhibition of TCR coupling to ZAP-70 and Erk activation. These results demonstrate that, notwithstanding their differences in their intracellular localization, which result in focalized cAMP production, both toxins directly affect the Th1/Th2 balance by interfering with the same steps in TCR signaling, and suggest that their adjuvanticity is likely to result from their combined effects on APC and CD4+ T cells. Furthermore, our results strongly support the key role of cAMP in the adjuvanticity of these toxins.

Bacillus anthracis Edema Toxin Causes Extensive Tissue Lesions and Rapid Lethality in Mice

Bacillus anthracis edema toxin (ET), an adenylyl cyclase, is an important virulence factor that contributes to anthrax disease. The role of ET in anthrax pathogenesis is, however, poorly understood. Previous studies using crude toxin preparations associated ET with subcutaneous edema, and ET-deficient strains of B. anthracis showed a reduction in virulence. We report the first comprehensive study of ET-induced pathology in an animal model. Highly purified ET caused death in BALB/cJ mice at lower doses and more rapidly than previously seen with the other major B. anthracis virulence factor, lethal toxin. Observations of gross pathology showed intestinal intralumenal fluid accumulation followed by focal hemorrhaging of the ileum and adrenal glands. Histopathological analyses of timed tissue harvests revealed lesions in several tissues including adrenal glands, lymphoid organs, bone, bone marrow, gastrointestinal mucosa, heart, and kidneys. Concomitant blood chemistry analyses supported the induction of tissue damage. Several cytokines increased after ET administration, including granulocyte colony-stimulating factor, eotaxin, keratinocyte-derived cytokine, MCP-1/JE, interleukin-6, interleukin-10, and interleukin-1beta. Physiological measurements also revealed a concurrent hypotension and bradycardia. These studies detail the extensive pathological lesions caused by ET and suggest that it causes death due to multiorgan failure.