Anthrax toxin: a tripartite lethal combination

Anthrax is a severe bacterial infection that occurs when Bacillus anthracis spores gain access into the body and germinate in macrophages, causing septicemia and toxemia. Anthrax toxin is a binary A-B toxin composed of protective antigen (PA), lethal factor (LF), and edema factor (EF). PA mediates the entry of either LF or EF into the cytosol of host cells. LF is a zinc metalloprotease that inactivates mitogen-activated protein kinase kinase inducing cell death, and EF is an adenylyl cyclase impairing host defences. Inhibitors targeting different steps of toxin activity have recently been developed. Anthrax toxin has also been exploited as a therapeutic agent against cancer.     

Anthrax toxin receptor 2-dependent lethal toxin killing in vivo

Anthrax toxin receptors 1 and 2 (ANTXR1 and ANTXR2) have a related integrin-like inserted (I) domain which interacts with a metal cation that is coordinated by residue D683 of the protective antigen (PA) subunit of anthrax toxin. The receptor-bound metal ion and PA residue D683 are critical for ANTXR1-PA binding. Since PA can bind to ANTXR2 with reduced affinity in the absence of metal ions, we reasoned that D683 mutant forms of PA might specifically interact with ANTXR2. We show here that this is the case. The differential ability of ANTXR1 and ANTXR2 to bind D683 mutant PA proteins was mapped to nonconserved receptor residues at the binding interface with PA domain 2. Moreover, a D683K mutant form of PA that bound specifically to human and rat ANTXR2 mediated killing of rats by anthrax lethal toxin, providing strong evidence for the physiological importance of ANTXR2 in anthrax disease pathogenesis.     

Anthrax toxin lethal factor contains a zinc metalloprotease consensus sequence which is required for lethal toxin activity

Comparison of the anthrax toxin lethal factor (LF) amino acid sequence with sequences in the Swiss protein database revealed short regions of similarity with the consensus zinc-binding site, HEXXH, that is characteristic of metalloproteases. Several protease inhibitors, including bestatin and captopril, prevented intoxication of macrophages by lethal toxin. LF was fully inactivated by site-directed mutagenesis that substituted Ala for either of the residues (H-686 and H-690) implicated in zinc binding. Similarly, LF was inactivated by substitution of Cys for E-687, which is thought to be an essential part of the catalytic site. In contrast, replacement of E-720 and E-721 with Ala had no effect on LF activity. LF bound ⁶⁵Zn both in solution and on protein blots. The ⁶⁵Zn binding was reduced for several of the LF mutants. These data suggest that anthrax toxin LF is a zinc metallopeptidase, the catalytic function of which is responsible for the lethal activity observed in cultured cells and in animals.     

Anthrax lethal toxin activates the inflammasome in sensitive rat macrophages

Anthrax lethal toxin (LT) is an important virulence factor for Bacillus anthracis. In mice, LT lyses macrophages from certain inbred strains in less than 2 h by activating the Nlrp1b inflammasome and caspase-1, while macrophages from other strains remain resistant to the toxin’s effects. We analyzed LT effects in toxin-sensitive and resistant rat macrophages to test if a similar pathway was involved in rat macrophage death. LT activates caspase-1 in rat macrophages from strains harboring LT-sensitive macrophages in a manner similar to that in toxin-sensitive murine macrophages. This activation of caspase-1 is dependent on proteasome activity, and sensitive macrophages are protected from LT’s lytic effects by lactacystin. Proteasome inhibition also delayed the death of rats in response to LT, confirming our previous data implicating the rat Nlrp1 inflammasome in animal death. Quinidine, caspase-1 inhibitors, the cathepsin B inhibitor CA-074Me, and heat shock also protected rat macrophages from LT toxicity. These data support the existence of an active functioning LT-responsive Nlrp1 inflammasome in rat macrophages. The activation of the rat Nlrp1 inflammasome is required for LT-mediated rat macrophage lysis and contributes to animal death.     

Anthrax lethal toxin paralyzes actin-based motility by blocking Hsp27 phosphorylation

Inhalation of anthrax causes fatal bacteremia, indicating a meager host immune response. We previously showed that anthrax lethal toxin (LT) paralyzes neutrophils, a major component of innate immunity. Here, we have found that LT also inhibits actin-based motility of the intracellular pathogen Listeria monocytogenes. LT inhibition of actin assembly is mediated by blockade of Hsp27 phosphorylation, and can be reproduced by treating cells with the p38 mitogen-activated protein (MAP) kinase inhibitor SB203580. Nonphosphorylated Hsp27 inhibits Listeria actin-based motility in cell extracts, and binds to and sequesters purified actin monomers. Phosphorylation of Hsp27 reverses these effects. RNA interference knockdown of Hsp27 blocks LT inhibition of Listeria actin-based motility. Rescue with wild-type Hsp27 accelerates Listeria speed in knockdown cells, whereas introduction of Hsp27 mutants incapable of phosphorylation or dephosphorylation causes slowing down. We propose that Hsp27 facilitates actin-based motility through a phosphorylation cycle that shuttles actin monomers to regions of new actin filament assembly. Our findings provide a previously unappreciated mechanism for LT virulence, and emphasize a central role for p38 MAP kinase-mediated phosphorylation of Hsp27 in actin-based motility and innate immunity.     

Anthrax lethal toxin enhances cytokine-induced VCAM-1 expression on human endothelial cells

Vascular endothelial dysfunction is thought to play a prominent role in systemic anthrax pathogenesis. We examined the effect of anthrax lethal toxin (LTx), a key virulence factor of Bacillus anthracis, on the expression of vascular cell adhesion molecule-1 (VCAM-1) on normal and cytokine-stimulated human lung microvascular endothelial cells. Confluent endothelial monolayers were treated with lethal factor (LF), protective antigen (PA), or both (LTx) in the presence or absence of tumor necrosis factor-alpha (TNFalpha). LTx enhanced cytokine-induced VCAM-1 expression and monocyte adhesion. LTx alone had no effect on VCAM-1 expression. LF, PA or the combination of a catalytically inactive mutant LF and PA failed to enhance cytokine-induced VCAM-1 expression. Treatment with inhibitors of mitogen-activated protein kinase kinases (MEKs) and mitogen-activated protein kinases did not reproduce the VCAM-1 enhancement effect of LTx, a known MEK metalloprotease, suggesting LTx-mediated MEK cleavage may not be a contributing factor.     

Anthrax edema toxin cooperates with lethal toxin to impair cytokine secretion during infection of dendritic cells

Bacillus anthracis secretes two critical virulence factors, lethal toxin (LT) and edema toxin (ET). In this study, we show that murine bone marrow-derived dendritic cells (DC) infected with B. anthracis strains secreting ET exhibit a very different cytokine secretion pattern than DC infected with B. anthracis strains secreting LT, both toxins, or a nontoxinogenic strain. ET produced during infection selectively inhibits the production of IL-12p70 and TNF-alpha, whereas LT targets IL-10 and TNF-alpha production. To confirm the direct role of the toxins, we show that purified ET and LT similarly disrupt cytokine secretion by DC infected with a nontoxinogenic strain. These effects can be reversed by specific inhibitors of each toxin. Furthermore, ET inhibits in vivo IL-12p70 and IFN-gamma secretion induced by LPS. These results suggest that ET produced during infection impairs DC functions and cooperates with LT to suppress the innate immune response. This may represent a new strategy developed by B. anthracis to escape the host immune response.     

Maturation modulates caspase-1-independent responses of dendritic cells to Anthrax lethal toxin

Anthrax lethal toxin (LT) contributes to the immune evasion strategy of Bacillus anthracis by impairing the function of cells of the immune system, such as macrophages and dendritic cells (DCs). Macrophages from certain inbred mice strains undergo rapid death upon LT treatment mediated by caspase-1 activation dependent on Nalp1b, an inflammasome component. Rapid LT-induced death is however, not observed in macrophages from human and many mouse strains. Here, we focused on the responses of various murine DCs to LT. Using a variety of knockout mice, we found that depending on the mouse strain, death of bone marrow-derived DCs and macrophages was mediated either by a fast Nalp1b and caspase-1-dependent, or by a slow caspase-1-independent pathway that was triggered by the impairment of MEK1/2 pathways. Caspase-1-independent death was observed in cells of different genetic backgrounds and interestingly occurred only in immature DCs. Maturation, triggered by different types of stimuli, led to full protection of DCs. These studies illustrate that the cellular damage inflicted by LT depends not only on the innate responses but also on the maturation stage of the cell, which modulates the more general caspase-1-independent responses.     

Anthrax lethal toxin disrupts intestinal barrier function and causes systemic infections with enteric bacteria

A variety of intestinal pathogens have virulence factors that target mitogen activated protein kinase (MAPK) signaling pathways, including Bacillus anthracis. Anthrax lethal toxin (LT) has specific proteolytic activity against the upstream regulators of MAPKs, the MAPK kinases (MKKs). Using a murine model of intoxication, we show that LT causes the dose-dependent disruption of intestinal epithelial integrity, characterized by mucosal erosion, ulceration, and bleeding. This pathology correlates with an LT-dependent blockade of intestinal crypt cell proliferation, accompanied by marked apoptosis in the villus tips. C57BL/6J mice treated with intravenous LT nearly uniformly develop systemic infections with commensal enteric organisms within 72 hours of administration. LT-dependent intestinal pathology depends upon its proteolytic activity and is partially attenuated by co-administration of broad spectrum antibiotics, indicating that it is both a cause and an effect of infection. These findings indicate that targeting of MAPK signaling pathways by anthrax LT compromises the structural integrity of the mucosal layer, serving to undermine the effectiveness of the intestinal barrier. Combined with the well-described immunosuppressive effects of LT, this disruption of the intestinal barrier provides a potential mechanism for host invasion via the enteric route, a common portal of entry during the natural infection cycle of Bacillus anthracis.     

Anthrax lethal toxin disrupts the endothelial permeability barrier through blocking p38 signaling

Exposure to anthrax causes life-threatening disease through the action of the toxin produced by the Bacillus anthracis bacteria. Lethal factor (LF), an anthrax toxin component which causes severe vascular leak and edema, is a protease which specifically degrades MAP kinase kinases (MKK). We have recently shown that p38 MAP kinase activation leading to HSP27 phosphorylation augments the endothelial permeability barrier. We now show that treatment of rat pulmonary microvascular endothelial cells with anthrax lethal toxin (LeTx), which is composed of LF and the protective antigen, increases endothelial barrier permeability and gap formation between endothelial cells through disrupting p38 signaling. LeTx treatment increases MKK3b degradation and in turn decreases p38 activity at baseline as well as after activation of p38 signaling. Consequently, LeTx treatment decreases activation of the p38 substrate kinase, MK2, and the phosphorylation of the latter's substrate, HSP27. LeTx treatment disrupts other signaling pathways leading to suppression of Erk-mediated signaling, but these effects do not correlate with LeTx-induced barrier compromise. Overexpressing phosphomimicking (pm)HSP27, which protects the endothelial permeability barrier against LeTx, blocks LeTx inactivation of p38 and MK2, but it does not block MKK3b degradation or Erk inactivation. Our results suggest that LeTx might cause vascular leak through inactivating p38-MK2-HSP27 signaling and that activating HSP27 phosphorylation specifically restores p38 signaling and blocks anthrax LeTx toxicity. The fact that barrier integrity could be restored by pmHSP27 overexpression without affecting degradation of MKK3b, or inactivation of Erk, suggests a specific and central role for p38-MK2-HSP27 in endothelial barrier permeability regulation.     

Anthrax toxin receptor 2 determinants that dictate the pH threshold of toxin pore formation

The anthrax toxin receptors, ANTXR1 and ANTXR2, act as molecular clamps to prevent the protective antigen (PA) toxin subunit from forming pores until exposure to low pH. PA forms pores at pH approximately 6.0 or below when it is bound to ANTXR1, but only at pH approximately 5.0 or below when it is bound to ANTXR2. Here, structure-based mutagenesis was used to identify non-conserved ANTXR2 residues responsible for this striking 1.0 pH unit difference in pH threshold. Residues conserved between ANTXR2 and ANTXR1 that influence the ANTXR2-associated pH threshold of pore formation were also identified. All of these residues contact either PA domain 2 or the neighboring edge of PA domain 4. These results provide genetic evidence for receptor release of these regions of PA as being necessary for the protein rearrangements that accompany anthrax toxin pore formation.     

Anthrax toxin complexes: heptameric protective antigen can bind lethal factor and edema factor simultaneously

The 83 kDa protective antigen (PA(83)) component of anthrax toxin, after proteolytic activation, self-associates to form ring-shaped heptamers ([PA(63)](7)) that bind and aid delivery of the Edema Factor (EF) and Lethal Factor (LF) components to the cytosol. Here we show using fluorescence (F?rster) resonance energy transfer that a molecule of [PA(63)](7) can bind EF and LF simultaneously. We labeled EF and LF with an appropriate donor/acceptor pair and found quenching of the donor and an increase in sensitized emission of the acceptor when, and only when, a mixture of the labeled proteins was combined with [PA(63)](7). Addition of unlabeled PA(63)-binding domain of LF to the mixture competitively displaced labeled EF and LF, causing a loss of energy transfer. In view of the known maximum occupancy of 3 ligand molecules per [PA(63)](7), these findings indicate that PA, EF, and LF can form mixtures of liganded toxin complexes containing both EF and LF.     

Anthrax toxin protective antigen: low-pH-induced hydrophobicity and channel formation in liposomes

To probe the role of the protective antigen (PA) component of anthrax toxin in toxin entry into animals cells, we examined the membrane channel-forming properties and hydrophobicity of intact and trypsin-cleaved forms of the protein at various pH values. At neutral pH neither form caused release of entrapped K+ from unilamellar lipid vesicles. At pH values below 6.0, however, K+ was rapidly released upon addition of either the nicked PA (PAN) or the 63 kDa tryptic fragment of PA (PA63), which has been implicated in the toxin entry process. Under the same conditions intact PA exhibited only weak channel-forming activity, and PA20, the complementary tryptic fragment, showed no such activity. Both PA and PA63 exhibited enhanced hydrophobicity at acidic pH values, but the enhancement was greater and the pH threshold higher with PA63. Our findings indicate that proteolytic removal of PA20 from intact PA enables the residual protein, PA63, to adopt a conformation at mildly acidic pH values that permits it to insert readily and form channels in membranes. Thus acidic conditions within endocytic vesicles may trigger membrane insertion of PA63, which in turn promotes translocation of ligated effector moieties, edema factor or lethal factor, across the vesicle membrane into the cytosol.     

Anthrax lethal toxin has direct and potent inhibitory effects on B cell proliferation and immunoglobulin production

Protective host immune responses to anthrax infection in humans and animal models are characterized by the development of neutralizing Abs against the receptor-binding anthrax protective Ag (PA), which, together with the lethal factor (LF) protease, composes anthrax lethal toxin (LT). We now report that B cells, in turn, are targets for LT. Anthrax PA directly binds primary B cells, resulting in the LF-dependent cleavage of the MAPK kinases (MAPKKs) and disrupted signaling to downstream MAPK targets. Although not directly lethal to B cells, anthrax LT treatment causes severe B cell dysfunction, greatly reducing proliferative responses to IL-4-, anti-IgM-, and/or anti-CD40 stimulation. Moreover, B cells treated with anthrax LT in vitro or isolated from mice treated with anthrax LT in vivo have a markedly diminished capacity to proliferate and produce IgM in response to TLR-2 and TLR-4 ligands. The suppressive effects of anthrax LT on B cell function occur at picomolar concentrations in vitro and at sublethal doses in vivo. These results indicate that anthrax LT directly inhibits the function of B cells in vitro and in vivo, revealing a potential mechanism through which the pathogen could bypass protective immune responses.     

Site directed mutagenesis of Histidine residues in Anthrax toxin lethal factor binding domain reduces toxicity

Anthrax lethal toxin is a mixture of protective antigen (PA, 735 AA) and lethal factor (LF, 776 AA). Earlier studies have shown that 254 residues of lethal factor are sufficient for PA binding to cause internalization (Arora N and Leppla SH, J Biol Chem 268: 3334-3342, 1993). The present study was undertaken to determine residues which are important for binding of LF to PA. LF modification with diethyl pyrocarbonate (DEPC, modifies histidine residue primarily) results in the loss of binding and toxicity in mammalian cells. There are nine histidine residues in the binding domain. To locate the important residue(s), site-directed mutagenesis of these histidines were performed by recombinant methods. Replacement of His42 with Gly42 destablizes the protein and hence it could not be purified. His35 when mutagenized to Gly35 (mLF-DTA) diminishes the toxicity by 20 fold. Time dependent studies show that binding of mLF-DTA was reduced at shorter incubations and longer incubations taper off this difference. Gel shift assay suggested 8-10% less binding of mLF-DTA as compared to LF-DTA. In conclusion His35 is important for binding and His42 is critical and confers proper conformation for LF binding to PA.     

Anthrax lethal toxin blocks MAPK kinase-dependent IL-2 production in CD4+ T cells

Anthrax lethal toxin (LT) is a critical virulence factor that cleaves and inactivates MAPK kinases (MAPKKs) in host cells and has been proposed as a therapeutic target in the treatment of human anthrax infections. Despite the potential use of anti-toxin agents in humans, the standard activity assays for anthrax LT are currently based on cytotoxic actions of anthrax LT that are cell-, strain-, and species-specific, which have not been demonstrated to occur in human cells. We now report that T cell proliferation and IL-2 production inversely correlate with anthrax LT levels in human cell assays. The model CD4+ T cell tumor line, Jurkat, is a susceptible target for the specific protease action of anthrax LT. Anthrax LT cleaves and inactivates MAPKKs in Jurkat cells, whereas not affecting proximal or parallel TCR signal transduction pathways. Moreover, anthrax LT specifically inhibits PMA/ionomycin- and anti-CD3-induced IL-2 production in Jurkat cells. An inhibitor of the protease activity of anthrax LT completely restores IL-2 production by anthrax LT-treated Jurkat cells. Anthrax LT acts on primary CD4+ T cells as well, cleaving MAPKKs and leading to a 95% reduction in anti-CD3-induced proliferation and IL-2 production. These findings not only will be useful in the development of new human cell-based bioassays for the activity of anthrax LT, but they also suggest new mechanisms that facilitate immune evasion by Bacillus anthracis. Specifically, anthrax LT inhibits IL-2 production and proliferative responses in CD4+ T cells, thereby blocking functions that are pivotal in the regulation of immune responses.     

Anthrax edema toxin has cAMP-mediated stimulatory effects and high-dose lethal toxin has depressant effects in an isolated perfused rat heart model

While anthrax edema toxin produces pronounced tachycardia and lethal toxin depresses left ventricular (LV) ejection fraction in in vivo models, whether these changes reflect direct cardiac effects as opposed to indirect ones related to preload or afterload alterations is unclear. In the present study, the effects of edema toxin and lethal toxin were investigated in a constant pressure isolated perfused rat heart model. Compared with control hearts, edema toxin at doses comparable to or less than a dose that produced an 80% lethality rate (LD(80)) in vivo in rats (200, 100, and 50 ng/ml) produced rapid increases in heart rate (HR), coronary flow (CF), LV developed pressure (LVDP), dP/dt(max), and rate-pressure product (RPP) that were most pronounced and persisted with the lowest dose (P ≤ 0.003). Edema toxin (50 ng/ml) increased effluent and myocardial cAMP levels (P ≤ 0.002). Compared with dobutamine, edema toxin produced similar myocardial changes, but these occurred more slowly and persisted longer. Increases in HR, CF, and cAMP with edema toxin were inhibited by a monoclonal antibody blocking toxin uptake and by adefovir, which inhibits the toxin's intracellular adenyl cyclase activity (P ≤ 0.05). Lethal toxin at an LD(80) dose (50 ng/ml) had no significant effect on heart function but a much higher dose (500 ng/ml) reduced all parameters (P ≤ 0.05). In conclusion, edema toxin produced cAMP-mediated myocardial chronotropic, inotropic, and vasodilatory effects. Vasodilation systemically with edema toxin could contribute to shock during anthrax while masking potential inotropic effects. Although lethal toxin produced myocardial depression, this only occurred at high doses, and its relevance to in vivo findings is unclear.     

Inositol hexakisphosphate-dependent processing of Clostridium sordellii lethal toxin and Clostridium novyi alpha-toxin

Clostridium sordellii lethal toxin and Clostridium novyi α-toxin, which are virulence factors involved in the toxic shock and gas gangrene syndromes, are members of the family of clostridial glucosylating toxins. The toxins inactivate Rho/Ras proteins by glucosylation or attachment of GlcNAc (α-toxin). Here, we studied the activation of the autoproteolytic processing of the toxins by inositol hexakisphosphate (InsP(6)) and compared it with the processing of Clostridium difficile toxin B. In the presence of low concentrations of InsP(6) (<1 μM), toxin fragments consisting of the N-terminal glucosyltransferase (or GlcNAc-transferase) domains and the cysteine protease domains (CPDs) of C. sordellii lethal toxin, C. novyi α-toxin, and C. difficile toxin B were autocatalytically processed. The cleavage sites of lethal toxin (Leu-543) and α-toxin (Leu-548) and the catalytic cysteine residues (Cys-698 of lethal toxin and Cys-707 of α-toxin) were identified. Affinity of the CPDs for binding InsP(6) was determined by isothermal titration calorimetry. In contrast to full-length toxin B and α-toxin, autocatalytic cleavage and InsP(6) binding of full-length lethal toxin depended on low pH (pH 5) conditions. The data indicate that C. sordellii lethal toxin and C. novyi α-toxin are InsP(6)-dependently processed. However, full-length lethal toxin, but not its short toxin fragments consisting of the glucosyltransferase domain and the CPD, requires a pH-sensitive conformational change to allow binding of InsP(6) and subsequent processing of the toxin.     

Anthrax lethal toxin increases superoxide production in murine neutrophils via differential effects on MAPK signaling pathways

The combination of lethal factor and its receptor-binding partner, protective Ag, is termed lethal toxin (LT) and has critical pathogenic activity during infection with Bacillus anthracis. We herein report that anthrax LT binds and enters murine neutrophils, leading to the cleavage of mitogen-activated protein kinase kinase/MEK/MAPKK 1-4 and 6, but not mitogen-activated protein kinase kinase 5 and 7. Anthrax LT treatment of neutrophils disrupts signaling to downstream MAPK targets in response to TLR stimulation. Following anthrax LT treatment, ERK family and p38 phosphorylation are nearly completely blocked, but signaling to JNK family members persists in vitro and ex vivo. In contrast to previous reports involving human neutrophils, anthrax LT treatment of murine neutrophils increases their production of superoxide in response to PMA or TLR stimulation in vitro or ex vivo. Although this enhanced superoxide production correlates with effects due to the LT-induced blockade of ERK signaling, it requires JNK signaling that remains largely intact despite the activity of anthrax LT. These findings reveal a previously unrecognized mechanism through which anthrax LT supports a critical proinflammatory response of murine neutrophils.     

Internalization and processing of Bacillus anthracis lethal toxin by toxin-sensitive and -resistant cells

Anthrax lethal toxin consists of two separate proteins, protective antigen and lethal factor (LF). Certain macrophages and a mouse macrophage-like cell line, J774A.1, are lysed by low concentrations of lethal toxin. In contrast, another macrophage cell line, IC-21, and all other cell types tested were resistant to this toxin. To discover the basis for this difference, each step in the intoxication process was examined. No differences between sensitive and resistant cells were found in receptor binding or proteolytic activation of protective antigen, steps that are required prior to LF binding. To determine whether resistance results from a defect in translocation to the cytosol, we introduced LF into J774A.1 and IC-21 cells and a nonmacrophage cell line (L6 myoblast) by osmotic lysis of pinocytic vesicles. Only J774A.1 cells were lysed; no effect was observed in IC-21 and L6 cells. These results suggest that resistant cells either lack the intracellular target of LF or fail to process LF to an active form. The relatively low potency of LF introduced into J774A.1 cells by osmotic lysis suggests that protective antigen may also be required at a stage subsequent to endocytosis.