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 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 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 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.     

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.     

A receptor-based switch that regulates anthrax toxin pore formation

Cellular receptors can act as molecular switches, regulating the sensitivity of microbial proteins to conformational changes that promote cellular entry. The activities of these receptor-based switches are only partially understood. In this paper, we sought to understand the mechanism that underlies the activity of the ANTXR2 anthrax toxin receptor-based switch that binds to domains 2 and 4 of the protective antigen (PA) toxin subunit. Receptor-binding restricts structural changes within the heptameric PA prepore that are required for pore conversion to an acidic endosomal compartment. The transfer cross-saturation (TCS) NMR approach was used to monitor changes in the heptameric PA-receptor contacts at different steps during prepore-to-pore conversion. These studies demonstrated that receptor contact with PA domain 2 is weakened prior to pore conversion, defining a novel intermediate in this pathway. Importantly, ANTXR2 remained bound to PA domain 4 following pore conversion, suggesting that the bound receptor might influence the structure and/or function of the newly formed pore. These studies provide new insights into the function of a receptor-based molecular switch that controls anthrax toxin entry into cells.     

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.     

Binding of filamentous actin to anthrax toxin receptor 1 decreases its association with protective antigen

ANTXR1 is a type I membrane protein that binds the protective antigen (PA) component of anthrax toxin. The cytosolic domain of ANTXR1 has a novel actin-binding region that influences the interaction of the ectodomain with PA. Here, we have investigated features of the cytosolic domain of ANTXR1 that reduce the association of the receptor with PA. We mutated a stretch of conserved acidic amino acids adjacent to the actin-binding region and found that the mutation increased the affinity for monomeric actin in vitro. ANTXR1 bearing this mutation exhibited increased association with the cytoskeleton and bound less PA compared to the wild-type receptor, confirming the inverse correlation between the two interactions. To determine whether binding of actin is sufficient to regulate the ectodomain, we replaced the actin-binding region of ANTXR1 with that from the yeast protein abp140 and with the WH2 domain of WAVE2. Although both of these domains bound monomeric actin in vitro, only the sequence from abp140 reduced binding of PA to a hybrid receptor. The actin binding regions of ANTXR1 and abp140, but not the WH2 domain, colocalized with actin stress fibers, which suggested that filamentous actin regulates ANTXR1. Consistent with this notion, disruption of actin filaments using latrunculin A increased the amount of PA bound to cells. This work provides evidence that cytoskeletal dynamics regulate ANTXR1 function.     

A viral nanoparticle with dual function as an anthrax antitoxin and vaccine

The recent use of Bacillus anthracis as a bioweapon has stimulated the search for novel antitoxins and vaccines that act rapidly and with minimal adverse effects. B. anthracis produces an AB-type toxin composed of the receptor-binding moiety protective antigen (PA) and the enzymatic moieties edema factor and lethal factor. PA is a key target for both antitoxin and vaccine development. We used the icosahedral insect virus Flock House virus as a platform to display 180 copies of the high affinity, PA-binding von Willebrand A domain of the ANTXR2 cellular receptor. The chimeric virus-like particles (VLPs) correctly displayed the receptor von Willebrand A domain on their surface and inhibited lethal toxin action in in vitro and in vivo models of anthrax intoxication. Moreover, VLPs complexed with PA elicited a potent toxin-neutralizing antibody response that protected rats from anthrax lethal toxin challenge after a single immunization without adjuvant. This recombinant VLP platform represents a novel and highly effective, dually-acting reagent for treatment and protection against anthrax.     

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.     

Cholesterol depletion induces ANTXR2-dependent activation of MMP-2 via ERK1/2 phosphorylation in neuroglioma U251 cell

Cholesterol is a critical component of lipid rafts implicated in regulating multiple signal transduction. The anthrax toxin receptor 2 (ANTXR2) is a type I membrane protein acting as the second receptor for the anthrax toxin. In this study, we first investigated the association between cholesterol and ANTXR2. We provided the evidence that cholesterol depletion by methyl-beta-cyclodextrin (MβCD) promoted ANTXR2 expression in U251 neuroglioma cell, which was reversed by cholesterol supplement. MβCD-induced ANTXR2 up-regulation contributed to ERK1/2 phosphorylation, which was responsible for MT1-MMP and MMP-2 activation. Our data suggested that cellular cholesterol regulated ANTXR2-dependent activation of MMP-2 via ERK1/2 phosphorylation in neuroglioma U251 cell.     

Insertion of anthrax protective antigen into liposomal membranes: effects of a receptor

Protective antigen (PA), the receptor-binding component of anthrax toxin, heptamerizes and inserts into the endosomal membrane at acidic pH, forming a pore that mediates translocation of the enzymic components of the toxin to the cytosol. When the heptameric pre-insertion form of PA (the prepore) is acidified in solution, it rapidly loses the ability to insert into membranes. To maximize insertion into model membranes, we examined two ways to bind the protein to large unilamellar vesicles (LUV). One involved attaching a His tag to the von Willebrand factor A domain of one of the PA receptors, ANTXR2, and using this protein as a bridge to bind PA to LUV containing a nickel-chelating lipid. The other involved using a His tag fused to the C terminus of PA to bind the protein directly to LUV containing the same lipid. Both ways enhanced pore formation at pH 5.0 strongly and about equally, as measured by the release of K+. Controls showed that pore formation in this system faithfully reproduced that in vivo. We also showed that binding unmodified ANTXR2 von Willebrand factor A to the prepore in solution enhanced its pore forming activity by slowing its inactivation at acidic pH. These findings indicate that an important role of PA receptors is to promote partitioning of PA into the bilayer by maintaining the prepore close to the target membrane and presumably in the optimal orientation as it undergoes the acidic pH-dependent conformational transition to the pore.     

The Disulfide Bond Cys255-Cys279 in the Immunoglobulin-Like Domain of Anthrax Toxin Receptor 2 Is Required for Membrane Insertion of Anthrax Protective Antigen Pore

Anthrax toxin receptors act as molecular clamps or switches that control anthrax toxin entry, pH-dependent pore formation, and translocation of enzymatic moieties across the endosomal membranes. We previously reported that reduction of the disulfide bonds in the immunoglobulin-like (Ig) domain of the anthrax toxin receptor 2 (ANTXR2) inhibited the function of the protective antigen (PA) pore. In the present study, the disulfide linkage in the Ig domain was identified as Cys255-Cys279 and Cys230-Cys315. Specific disulfide bond deletion mutants were achieved by replacing Cys residues with Ala residues. Deletion of the disulfide bond C255-C279, but not C230-C315, inhibited the PA pore-induced release of the fluorescence dyes from the liposomes, suggesting that C255-C279 is essential for PA pore function. Furthermore, we found that deletion of C255-C279 did not affect PA prepore-to-pore conversion, but inhibited PA pore membrane insertion by trapping the PA membrane-inserting loops in proteinaceous hydrophobic pockets. Fluorescence spectra of Trp59, a residue adjacent to the PA-binding motif in von Willebrand factor A (VWA) domain of ANTXR2, showed that deletion of C255-C279 resulted in a significant conformational change on the receptor ectodomain. The disulfide deletion-induced conformational change on the VWA domain was further confirmed by single-particle 3D reconstruction of the negatively stained PA-receptor heptameric complexes. Together, the biochemical and structural data obtained in this study provides a mechanistic insight into the role of the receptor disulfide bond C255-C279 in anthrax toxin action. Manipulation of the redox states of the receptor, specifically targeting to C255-C279, may become a novel strategy to treat anthrax.     

Interactions between anthrax toxin receptors and protective antigen

Since the anthrax mail attacks of 2001, much has been learned about the interactions between anthrax toxin and its receptors. Two distinct cellular receptors for anthrax toxin have been identified and are designated capillary morphogenesis protein 2 (CMG2) and anthrax toxin receptor/tumor endothelial marker 8 (ATR/TEM8). The molecular details of the toxin-receptor interactions have been revealed through crystallographic, biochemical and genetic studies. In addition, a novel pathway by which anthrax toxin enters cells is starting to be uncovered.     

Anthrax toxin receptor 2 mediates Bacillus anthracis killing of macrophages following spore challenge

Initiation of inhalation anthrax is believed to involve phagocytosis of Bacillus anthracis spores by alveolar macrophages, followed by spore germination within the phagolysosome. In order to establish a systemic infection, it is predicted that bacilli then escape from the macrophage and replicate extracellularly. Mechanisms utilized by B. anthracis to escape from the macrophage are not well characterized, but a role for anthrax toxin has been proposed. Here we report the isolation of an anthrax toxin-resistant cell line (R3D) following chemical mutagenesis of toxin-sensitive RAW 264.7 murine macrophage cells. Both R3D and RAW 264.7 cells phagocytize spores of a B. anthracis Sterne strain. However, RAW 264.7 cells are killed following spore challenge, whereas R3D cells survive. Resistance to toxin and spore challenge correlates with loss of expression of anthrax toxin receptor 2 (ANTXR2/CMG-2). When R3D cells are complemented with cDNA encoding either murine ANTXR2 or human anthrax toxin receptor 1 (ANTXR1/TEM-8), toxin and spore challenge susceptibility are restored, indicating that over-expression of either ANTXR can confer susceptibility to anthrax spore challenge. Taken together, these results indicate that anthrax toxin expression by the germinated spore enables B. anthracis killing of the macrophage from within.     

Protection of mammalian cells from diphtheria toxin by exogenous nucleotides.

Exogenous nucleotides were found to protect mammalian cells from the lethal effects of diphtheria toxin. Protective potency of a given nucleotide was base specific and phosphate chain length dependent. Full expression of protective potency required an intact nucleotide, but the effect did not appear to be mediated by nucleotide-induced phosphorylation. Nucleotides antagonized the binding of diphtheria toxin to its cell surface receptor in a manner that correlated with the degree of protection. It was concluded that cellular protection from diphtheria toxin by nucleotides results from inhibition of toxin-receptor binding and that nucleotides therefore may serve as valuable research tools for future studies.     

Cathepsin B-mediated Autophagy Flux Facilitates the Anthrax Toxin Receptor 2-mediated Delivery of Anthrax Lethal Factor into the Cytoplasm

Anthrax lethal toxin (LeTx) is a virulence factor secreted by Bacillus anthracis and has direct cytotoxic effects on most cells once released into the cytoplasm. The cytoplasmic delivery of the proteolytically active component of LeTx, lethal factor (LF), is carried out by the transporter component, protective antigen, which interacts with either of two known surface receptors known as anthrax toxin receptor (ANTXR) 1 and 2. We found that the cytoplasmic delivery of LF by ANTXR2 was mediated by cathepsin B (CTSB) and required lysosomal fusion with LeTx-containing endosomes. Also, binding of protective antigen to ANXTR1 or -2 triggered autophagy, which facilitated the cytoplasmic delivery of ANTXR2-associated LF. We found that whereas cells treated with the membrane-permeable CTSB inhibitor CA074-Me- or CTSB-deficient cells had no defect in fusion of LC3-containing autophagic vacuoles with lysosomes, autophagic flux was significantly delayed. These results suggested that the ANTXR2-mediated cytoplasmic delivery of LF was enhanced by CTSB-dependent autophagic flux.     

Membrane traffic and the cellular uptake of cholera toxin.

In nature, cholera toxin (CT) and the structurally related E. coli heat labile toxin type I (LTI) must breech the epithelial barrier of the intestine to cause the massive diarrhea seen in cholera. This requires endocytosis of toxin-receptor complexes into the apical endosome, retrograde transport into Golgi cisternae or endoplasmic reticulum (ER), and finally transport of toxin across the cell to its site of action on the basolateral membrane. Targeting into this pathway depends on toxin binding ganglioside GM1 and association with caveolae-like membrane domains. Thus to cause disease, both CT and LTI co-opt the molecular machinery used by the host cell to sort, move, and organize their cellular membranes and substituent components.     

Cytotoxicity of Bacillus thuringiensis Cry1Ab toxin depends on specific binding of the toxin to the cadherin receptor BT-R1 expressed in insect cells

The specific role of cadherin receptors in cytotoxicity involving Cry toxins of Bacillus thuringiensis and their interactions with cell membrane has not been defined. To elucidate the involvement of toxin-membrane and toxin-receptor interactions in cytotoxicity, we established a cell-based system utilizing High Five insect cells stably expressing BT-R1, the cadherin receptor for Cry1Ab toxin. Cry1Ab toxin is incorporated into cell membrane in both oligomeric and monomeric form. Monomeric toxin binds specifically to BT-R1 whereas incorporation of oligomeric toxin is nonspecific and lipid dependent. Toxin oligomers in the cell membrane do not produce lytic pores and do not kill insect cells. Rather, cell death correlates with binding of the Cry1Ab toxin monomer to BT-R1, which apparently activates a Mg2+-dependent cellular signaling pathway.     

Binding of anthrax toxin to its receptor is similar to alpha integrin-ligand interactions

The secreted protein toxin produced by Bacillus anthracis contributes to virulence of this pathogen and can cause many of the symptoms seen during an anthrax infection, including shock and sudden death. The cell-binding component of anthrax toxin, protective antigen, mediates entry of the toxin into cells by first binding directly to the extracellular integrin-like inserted (I) domain of the cellular anthrax toxin receptor, ATR. Here we report that this interaction requires an intact metal ion-dependent adhesion site (MIDAS) in the receptor as well as the presence of specific divalent cations. Also, we demonstrate that the toxin-receptor interaction is critically dependent on the Asp-683 carboxylate group of protective antigen, which projects from the receptor binding surface. We propose that this carboxylate group completes the coordination of the MIDAS metal of ATR, mimicking integrin-ligand interactions.