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 drives protective antigen oligomerization and stabilizes the heptameric and octameric oligomer by a similar mechanism

Background

Anthrax toxin is comprised of protective antigen (PA), lethal factor (LF), and edema factor (EF). These proteins are individually nontoxic; however, when PA assembles with LF and EF, it produces lethal toxin and edema toxin, respectively. Assembly occurs either on cell surfaces or in plasma. In each milieu, PA assembles into a mixture of heptameric and octameric complexes that bind LF and EF. While octameric PA is the predominant form identified in plasma under physiological conditions (pH 7.4, 37°C), heptameric PA is more prevalent on cell surfaces. The difference between these two environments is that the anthrax toxin receptor (ANTXR) binds to PA on cell surfaces. It is known that the extracellular ANTXR domain serves to stabilize toxin complexes containing the PA heptamer by preventing premature PA channel formation—a process that inactivates the toxin. The role of ANTXR in PA oligomerization and in the stabilization of toxin complexes containing octameric PA are not understood.

Methodology

Using a fluorescence assembly assay, we show that the extracellular ANTXR domain drives PA oligomerization. Moreover, a dimeric ANTXR construct increases the extent of and accelerates the rate of PA assembly relative to a monomeric ANTXR construct. Mass spectrometry analysis shows that heptameric and octameric PA oligomers bind a full stoichiometric complement of ANTXR domains. Electron microscopy and circular dichroism studies reveal that the two different PA oligomers are equally stabilized by ANTXR interactions.

Conclusions

We propose that PA oligomerization is driven by dimeric ANTXR complexes on cell surfaces. Through their interaction with the ANTXR, toxin complexes containing heptameric and octameric PA oligomers are similarly stabilized. Considering both the relative instability of the PA heptamer and extracellular assembly pathway identified in plasma, we propose a means to regulate the development of toxin gradients around sites of infection during anthrax pathogenesis.     

Anthrax oedema toxin induces anthrax toxin receptor expression in monocyte-derived cells

Bacillus anthracis, the causative agent of anthrax, secretes two bipartite toxins that help the bacterium evade the immune system and contribute directly to pathogenesis. Both toxin catalytic moieties, lethal factor (LF) and oedema factor (OF), are internalized into the host-cell cytosol by a third factor, protective antigen (PA), which binds to cellular anthrax toxin receptors (ANTXRs). Oedema factor is an adenylate cyclase that impairs host defences by raising cellular cAMP levels. Here we demonstrate that oedema toxin (PA + OF) induces an increase in ANTXR expression levels in macrophages and dendritic cells resulting in an increased rate of toxin internalization. Furthermore, we show that increases in ANTXR mRNA levels depends on the ability of OF to increase cAMP levels, is mediated through protein kinase A-directed signalling and is monocyte-lineage-specific. To our knowledge, this is the first report of a bacterial toxin inducing host target cells to increase toxin receptor expression.     

LRP5 and LRP6 are not required for protective antigen-mediated internalization or lethality of anthrax lethal toxin

Anthrax toxin (AnTx) plays a key role in the pathogenesis of anthrax. AnTx is composed of three proteins: protective antigen (PA), edema factor, and lethal factor (LF). PA is not toxic but serves to bind cells and translocate the toxic edema factor or LF moieties to the cytosol. Recently, the low-density lipoprotein receptor-related protein LRP6 has been reported to mediate internalization and lethality of AnTx. Based on its similarity to LRP6, we hypothesized that LRP5 may also play a role in cellular uptake of AnTx. We assayed PA-dependent uptake of anthrax LF or a cytotoxic LF fusion protein (FP59) in cells and mice harboring targeted deletions of Lrp5 or Lrp6. Unexpectedly, we observed that uptake was unaltered in the presence or absence of either Lrp5 or Lrp6 expression. Moreover, we observed efficient PA-mediated uptake into anthrax toxin receptor (ANTXR)-deficient Chinese hamster ovary cells (PR230) that had been stably engineered to express either human ANTXR1 or human ANTXR2 in the presence or absence of siRNA specific for LRP5 or LRP6. Our results demonstrate that neither LRP5 nor LRP6 is necessary for PA-mediated internalization or lethality of anthrax lethal toxin.     

Cytotoxic activity of Bacillus anthracis protective antigen observed in a macrophage cell line overexpressing ANTXR1

Anthrax toxin protective antigen (PA) binds cell surface receptors (e.g. ANTXR1,2), forms heptameric pores, and translocates lethal factor (LF) or oedema factor (OF) into the cytoplasm of mammalian cells. In the current study, we sought to determine how receptor levels influence these events, by examining PA heptamer stability and related processes in macrophages that overexpress ANTXR1 (RAW 264.7ANTXR1). In these experiments, PA-oligomers demonstrated an extended half-life in RAW 264.7ANTXR1 macrophages, with SDS-resistant heptamers detected up to 10 h following treatment, while levels of PA-oligomers declined within 3 h in control cells. RAW 264.7ANTXR1 macrophages were also more sensitive to lethal toxin, a combination of PA and LF. Surprisingly, we found that PA alone was cytotoxic to RAW 264.7ANTXR1 cells. Further analysis found that PA cytotoxicity required direct interaction with ANTXR1, oligomerization, channel formation, endosomal acidification, and was independent of the ANTXR1 cytoplasmic tail. PA intoxication of RAW 264.7ANTXR1 macrophages resulted in caspase-3 activation, with corresponding DNA fragmentation and proteolytic cleavage of poly-ADP-ribose polymerase, as well as activation of Bid, suggesting cell death occurred via apoptosis. Overall, results from the current study suggest that receptor levels dictate the extent of PA oligomer stability, and shifts in this normal process can lead to cell death via apoptosis in the absence of toxin catalytic subunits.     

Cytotoxicity of Anthrax Lethal Toxin to Human Acute Myeloid Leukemia Cells Is Nonapoptotic and Dependent on Extracellular Signal-Regulated Kinase 1/2 Activity

In this study, we attempt to target the mitogen-activated protein kinase (MAPK) pathway in acute myeloid leukemia (AML) cells using a recombinant anthrax lethal toxin (LeTx). LeTx consists of protective antigen (PrAg) and lethal factor (LF). PrAg binds cells, is cleaved by furin, oligomerizes, binds three to four molecules of LF, and undergoes endocytosis, releasing LF into the cytosol. LF cleaves MAPK kinases, inhibiting the MAPK pathway. We tested potency of LeTx on a panel of 11 human AML cell lines. Seven cell lines showed cytotoxic responses to LeTx. Cytotoxicity of LeTx was mimicked by the specific mitogen-activated protein/extracellular signal-regulated kinase kinase 1/2 (MEK1/2) inhibitor U0126, indicating that LeTx-induced cell death is mediated through the MEK1/2-extracellular signal-regulated kinase (ERK1/2) branch of the MAPK pathway. The four LeTx-resistant cell lines were sensitive to the phosphatidylinositol 3-kinase inhibitor LY294002. Co-treatment of AML cells with both LeTx and LY294002 did not lead to increased sensitivity, showing a lack of additive/synergistic effects when both pathways are inhibited. Flow cytometry analysis of MAPK pathway activation revealed the presence of phospho-ERK1/2 only in LeTx-sensitive cells. Staining for Annexin V/propidium iodide and active caspases showed an increase in double-positive cells and the absence of caspase activation following treatment, indicating that LeTx-induced cell death is caspase-independent and nonapoptotic. We have shown that a majority of AML cell lines are sensitive to the LF-mediated inhibition of the MAPK pathway. Furthermore, we have demonstrated that LeTx-induced cytotoxicity in AML cells is nonapoptotic and dependent on phospho-ERK1/2 levels.     

The Structure of Tumor Endothelial Marker 8 (TEM8) Extracellular Domain and Implications for Its Receptor Function for Recognizing Anthrax Toxin

Anthrax toxin, which is released from the Gram-positive bacterium Bacillus anthracis, is composed of three proteins: protective antigen (PA), lethal factor (LF), and edema factor (EF). PA binds a receptor on the surface of the target cell and further assembles into a homo-heptameric pore through which EF and LF translocate into the cytosol. Two distinct cellular receptors for anthrax toxin, TEM8/ANTXR1 and CMG2/ANTXR2, have been identified, and it is known that their extracellular domains bind PA with low and high affinities, respectively. Here, we report the crystal structure of the TEM8 extracellular vWA domain at 1.7 Å resolution. The overall structure has a typical integrin fold and is similar to that of the previously published CMG2 structure. In addition, using structure-based mutagenesis, we demonstrate that the putative interface region of TEM8 with PA (consisting of residues 56, 57, and 154–160) is responsible for the PA-binding affinity differences between the two receptors. In particular, Leu56 was shown to be a key factor for the lower affinity of TEM8 towards PA compared with CMG2. Because of its high affinity for PA and low expression in normal tissues, an isolated extracellular vWA domain of the L56A TEM8 variant may serve as a potent antitoxin and a potential therapeutic treatment for anthrax infection. Moreover, as TEM8 is often over-expressed in tumor cells, our TEM8 crystal structure may provide new insights into how to design PA mutants that preferentially target tumor cells.     

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.     

Monoclonal antibodies directed against protective antigen of Bacillus anthracis enhance lethal toxin activity in vivo

Protective antigen (PA) from Bacillus anthracis binds to cellular receptors, combines with lethal factor (LF) forming lethal toxin (LeTx), and facilitates the translocation of LF into the cytosol. LeTx is cytotoxic for J774A.1 cells, a murine macrophage cell line, and causes death of Fisher 344 rats when injected intravenously. PA is also the major protective component in anthrax vaccines. Antibody-dependent enhancement has been reported for several viral diseases, a bacterial infection, and for B. anthracis LeTx in vitro cytotoxicity. Further screening of our 73 PA monoclonal antibodies (mAbs) identified a total of 17 PA mAbs that enhanced in vitro cytotoxicity at suboptimal concentrations of LeTx. A competitive binding enzyme-linked immunosorbent assay showed that these 17 PA mAbs identified eight different antigenic regions on PA. Eight of the 17 PA mAbs that enhanced LeTx in vitro cytoxicity were examined for their activity in vivo. Of the eight mAbs that were injected intravenously with a sublethal concentration of LeTx into male Fisher 344 rats, four mAbs enhanced the lethality of LeTx and resulted in the death of animals, whereas control animals did not succumb to intoxication. This is the first demonstration that PA mAbs can enhance LeTx intoxication in vivo.     

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.     

Delivery of nucleic acid into mammalian cells by anthrax toxin

Gene delivery vehicles based on receptor-mediated endocytosis offer an attractive long-term solution as they might overcome the limitations of toxicity and cargo capacity inherent to many viral gene delivery systems. The protective antigen component of anthrax toxin bind to specific receptors and deliver lethal factor or edema factor into the cytosol of mammalian cells. The N-terminal 254 amino acids of LF (LF(1-254)) binds to PA and, when fused to heterologous proteins, delivers such proteins into the cytosol. However, so far no attempt has been made to use the anthrax toxin system for the intracellular delivery of DNA. In the present study, LF(1-254) of anthrax toxin was fused to the DNA-binding domain of GAL4 protein. The fusion protein (LF(254)-GAL4DBD) showed both PA binding as well as DNA-binding activity in solution. The complex of fusion protein with plasmid DNA containing a reporter gene (luciferase or green fluorescent protein) along with PA delivered plasmid DNA into the cytosol of COS-1 cells. These results suggest that anthrax toxin components can be used as a non-viral system for the efficient delivery of DNA into the cytosol of mammalian cells.     

Anthrax lethal factor cleaves the N-terminus of MAPKKS and induces tyrosine/threonine phosphorylation of MAPKS in cultured macrophages

The lethal toxin (LeTx) of Bacillus anthracis is the major virulence factor responsible for the death of infected animals and for cytolysis of cultured macrophages. Its catalytic component, LF, contains the characteristic zinc-binding motif of metalloproteases, it binds zinc and indirect evidence suggests that this hydrolytic activity is essential for LeTx cytotoxicity ( Limpel et al . 1994 ; Kochi et al . 1994 ). To identify substrates of LF, we have used the yeast two-hybrid system, employing an LF inactive mutant as bait. This approach has led to the identification of the MAP kinase kinases (MAPKKs) Mek1 and Mek2 as proteins capable of specific interaction with LF. LF cleaves Mek1 and Mek2 within their N-terminus in vitro and in vivo , hydrolysing a Pro8-Ile9 and a Pro10-Arg11 peptide bond in Mek1 and Mek2, respectively ( Vitale et al . 1998 ), similarly to that found with a different approach by Duesbery et al . (1998) . The removal of the amino terminus of MAPKKs eliminates the 'docking site' involved in the specific interaction with MAPKs and interferes with the phospho-activation of the MAPKs ERK1 and ERK2, which become phosphorylated in cultured macrophages following toxin challenge. We are currently investigating the relevance of MAPKKs cleavage for LeTx cytotoxicity and the consequences for the activity of the MAP pathway.     

Anthrax toxin components stimulate chemotaxis of human polymorphonuclear neutrophils

Effects of the three-component toxin of Bacillus anthracis on chemotaxis of human polymorphonuclear leukocytes (PMN) were investigated in an effort to determine the basis of the reported antiphagocytic effect of the toxin. The three toxin components, edema factor (EF), protective antigen (PA), and lethal factor (LF), were tested alone and in various combinations for their effect on PMN chemotaxis under agarose to formyl peptides and zymosan-activated serum. No component was active alone; combinations of EF + PA, LF + PA, and EF + LF + PA markedly stimulated chemotaxis (directed migration), but had little or no effect on unstimulated random migration. The toxin components were not themselves chemoattractants. EF in combination with PA had previously been identified as an adenylate cyclase in Chinese hamster ovary (CHO) cells. We found that EF + PA produced detectable cyclic adenosine 3'-5'monophosphate (cAMP) in PMN, but the level of cAMP was less than 1% of that produced in CHO cells by EF + PA, and in PMN by other bacterial adenylate cyclases. LF + PA (which stimulated chemotaxis to an equivalent extent) had no effect on cAMP levels. Thus, the enhancement of chemotaxis by anthrax toxin (at least by LF + PA) does not seem to be related to adenylate cyclase activity.     

炭疽毒素及其细胞受体的研究进展

炭疽毒素由 3种蛋白组成 :保护性抗原 (protectiveantigen ,PA)、致死因子 (lethalfactor,LF)和水肿因子 (edemafactor ,EF) .综述炭疽毒素研究的最新进展 .主要介绍炭疽毒素的关键致病因子———LF的结构与功能 ,炭疽毒素膜转运成分PA的结构及其受体 (anthraxtoxinreceptor ,ATR)和其cDNA克隆的结构 ,并讨论了在炭疽的治疗、预防和毒素在肿瘤治疗中的可能应用 .     

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 lethal factor activates K(+) channels to induce IL-1β secretion in macrophages

Anthrax lethal toxin (LeTx) is a virulence factor of Bacilillus anthracis that is a bivalent toxin, containing lethal factor (LF) and protective Ag proteins, which causes cytotoxicity and altered macrophage function. LeTx exposure results in early K(+) efflux from macrophages associated with caspase-1 activation and increased IL-1β release. The mechanism of this toxin-induced K(+) efflux is unknown. The goals of the current study were to determine whether LeTx-induced K(+) efflux from macrophages is mediated by toxin effects on specific K(+) channels and whether altered K(+)-channel activity is involved in LeTx-induced IL-1β release. Exposure of macrophages to LeTx induced a significant increase in the activities of two types of K(+) channels that have been identified in mouse macrophages: Ba(2+)-sensitive inwardly rectifying K(+) (Kir) channels and 4-aminopyridine-sensitive outwardly rectifying voltage-gated K(+) (Kv) channels. LeTx enhancement of both Kir and Kv required the proteolytic activity of LF, because exposure of macrophages to a mutant LF-protein (LF(E687C)) combined with protective Ag protein had no effect on the currents. Furthermore, blocking Kir and Kv channels significantly decreased LeTx-induced release of IL-1β. In addition, retroviral transduction of macrophages with wild-type Kir enhanced LeTx-induced release of IL-1β, whereas transduction of dominant-negative Kir blocked LeTx-induced release of IL-1β. Activation of caspase-1 was not required for LeTx-induced activation of either of the K(+) channels. These data indicate that a major mechanism through which LeTx stimulates macrophages to release IL-1β involves an LF-protease effect that enhances Kir and Kv channel function during toxin stimulation.     

Proteolytic activation of receptor-bound anthrax protective antigen on macrophages promotes its internalization

Immunofluorescence and other methods have been used to probe the self-assembly and internalization of the binary toxin, anthrax lethal toxin (LeTx), in primary murine macrophages. Proteolytic activation of protective antigen (PA; 83 kDa, the B moiety of the toxin) by furin was the rate-limiting step in internalization of LeTx and promoted clearance of PA from the cell surface. A furin-resistant form of PA remained at the cell surface for at least 90 min. Oligomerization of receptor-bound PA63, the 63 kDa active fragment of PA, was manifested by its conversion to a pronase-resistant state, characteristic of the heptameric prepore form in solution. That oligomerization of PA63 triggers toxin internalization is supported by the observation that PA20, the complementary 20 kDa fragment of PA, inhibited clearance of nicked PA. The PA63 prepore, with or without lethal factor (LF), cleared slowly from the cell surface. These studies show that proteolytic cleavage of PA, in addition to permitting oligomerization and LF binding, also promotes internalization of the protein. The relatively long period of activation and internalization of PA at the cell surface may reflect adaptation of this binary toxin that maximizes self-assembly.     

Production and proteolytic assay of lethal factor from Bacillus anthracis

Bacillus anthracis is the causative agent of anthrax. The major virulence factors are a poly-D-glutamic acid capsule and three-protein component exotoxin, protective antigen (PA, 83 kDa), lethal factor (LF, 90 kDa), and edema factor (EF, 89 kDa), respectively. These three proteins individually have no known toxic activities, but in combination with PA form two toxins (lethal toxin or edema toxin), causing different pathogenic responses in animals and cultured cells. In this study, we constructed and produced rLF as a form of GST fusion protein in Escherichia coli. rLF was rapidly purified through a single affinity purification step to near homogeneity. Furthermore, we developed an in vitro immobilized proteolytic assay of LF under the condition containing full-length native substrate, MEK1, rather than short synthetic peptide. The availability of full-length substrate and of an immobilized LF assay could facilitate not only the in-depth investigation of structure-function relationship of the enzyme toward its substrate but also wide spectrum screening of inhibitor collections based on the 96-well plate system.     

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.