Cross-linked forms of the isolated N-terminal domain of the lethal factor are potent inhibitors of anthrax toxin

The proteins that comprise anthrax toxin self-assemble at the mammalian cell surface into a series of toxic complexes, each containing a heptameric form of protective antigen (PA) plus up to a total of three molecules of the enzymatic moieties of the toxin (lethal factor [LF] and edema factor [EF]). These complexes are trafficked to the endosome, where the PA heptamer forms a pore in the membrane under the influence of low pH, and bound LF and EF unfold and translocate through the pore to the cytosol. To explore the hypothesis that the PA pore can translocate multiple, cross-linked polypeptides simultaneously, we cross-linked LF(N), the N-terminal domain of LF, via an introduced cysteine at its N or C terminus and characterized the products. Both dimers and trimers of LF(N) retained the ability to bind to PA pores and block ion conductance, but they were unable to translocate across the membrane, even at high voltages or with a transmembrane pH gradient. The multimers were remarkably potent inhibitors of toxin action in mammalian cells (20- to 50-fold more potent than monomeric LF(N)) and in a zebrafish model system. These findings show that the PA pore cannot translocate multimeric, cross-linked polypeptides and demonstrate a new approach to generating potent inhibitors of anthrax toxin.     

Expression and immunogenicity of pertussis toxin S1 subunit-tetanus toxin fragment C fusions in Salmonella typhi vaccine strain CVD 908.

Salmonella typhi vaccine strain CVD 908 can deliver heterologous antigens to the host immune system following mucosal immunization. Stable expression of foreign proteins in Salmonella cells often requires antigen-specific engineering strategies. Fusion of antigens to stabilizing proteins has proven to be a successful strategy for rescuing otherwise unstable proteins. We designed plasmids to allow the fusion of antigens to the amino terminus or carboxyl terminus of fragment C of tetanus toxin, separated by a 4-amino-acid hinge region. Towards the ultimate goal of developing a live oral diphtheria-pertussis-tetanus vaccine, we used these plasmids to stably express the S1 subunit of pertussis toxin in CVD 908. Driven by the anaerobically inducible nirB promoter, the S1 subunit alone was expressed poorly in Salmonella cytoplasm. In contrast, hybrid proteins with S1 fused to either the amino or carboxyl terminus of fragment C were expressed at a high level in CVD 908 and were recognized in Western blot (immunoblot) analysis by monoclonal antibodies directed to S1 and to fragment C. Mice were immunized by the oral or intranasal routes with CVD 908 derivatives harboring these recombinant plasmids. All fusion proteins elicited serum antibody responses to fragment C following intranasal immunization, whereas oral inoculation did not. The configuration of antigens constituting the fusion was critical; S1 fused to the amino terminus of fragment C was less effective than S1 fused to the carboxyl terminus in generating anti-fragment C antibodies. CVD 908 expressing truncated S1 fused to the carboxyl terminus of fragment C elicited neutralizing serum pertussis antitoxin following intranasal immunization of mice.     

Anthrax toxin

Anthrax is primarily a disease of herbivores caused by gram-positive, aerobic, spore-forming Bacillus anthracis. Humans are accidental hosts through the food of animal origin and animal products. Anthrax is prevelant in most parts of the globe, and cases of anthrax have been reported from almost every country. Three forms of the disease have been recognized: cutaneous (through skin), gastrointestinal (through alimentary tract), and pulmonary (by inhalation of spores). The major virulence factors of Bacillus anthracis are a poly-D glutamic acid capsule and a three-component protein exotoxin. The genes coding for the toxin and the enzymes responsible for capsule production are carried on plasmid pXO1 and pXO2, respectively. The three proteins of the exotoxin are protective antigen (PA, 83 kDa), lethal factor (LF, 90 kDa), and edema factor (EF, 89 kDa). The toxins follow the A-B model with PA being the B moeity and LF/EF, the alternative A moeities. LF and EF are individually nontoxic, but in combination with PA form two toxins causing different pathogenic responses in animals and cultured cells. PA + LF forms the lethal toxin and PA + EF forms the edema toxin. During the process of intoxication, PA binds to the cell surface receptor and is cleaved at the sequence RKKR (167) by cell surface proteases such as furin generating a cell-bound, C-terminal 63 kDa protein (PA63). PA63 possesses a binding site to which LF or EF bind with high affinity. The complex is then internalized by receptor-mediated endocytosis. Acidification of the vesicle leads to instertion of PA63 into the endosomal membrane and translocation of LF/EF across the bilayer into the cytosol where they exert their toxic effects. EF has a calcium- and calmodulin-dependent adenylate cyclase activity. Recent reports indicate that LF is a protease that cleaves the amino terminus of mitogen-activated protein kinase kinases 1 and 2 (MAPKK1 and 2), and this cleavage inactivates MAPKK1 and thus inhibits the mitogen-activated protein kinase signal transduction pathway. We describe in detail the studies so far done on unraveling the molecular mechanisms of pathogenesis of Bacillus anthracis.     

Protective antigen-mediated antibody response against a heterologous protein produced in vivo by Bacillus anthracis

Bacillus anthracis secretes a lethal toxin composed of two proteins, the lethal factor (LF) and the protective antigen (PA), which interact within the host or in vitro at the surfaces of eukaryotic cells. Immunization with attenuated B. anthracis strains induces an antibody response against PA and LF. The LF-specific response is potentiated by the binding of LF to PA. In this study, we investigated the capacity of PA to increase the antibody response against a foreign antigen. We constructed a chimeric gene encoding the PA-binding part of LF (LF254) fused to the C fragment of tetanus toxin (ToxC). The construct was introduced by allelic exchange into the locus encoding LF. Two recombinant B. anthracis strains secreting the hybrid protein LF254-ToxC were generated, one in a PA-producing background and the other in a PA-deficient background. Mice were immunized with spores of the strains, and the humoral response and protection against tetanus toxin were assessed. The B. anthracis strain producing both PA and LF254-ToxC induced significantly higher antibody titers and provided better protection against a lethal challenge with tetanus toxin than did its PA-deficient counterpart. Thus, PA is able to potentiate protective immunity against a heterologous antigen, demonstrating the potential of B. anthracis recombinant strains for use as live vaccine vehicles.     

Proteolytic activation of bacterial toxins by eukaryotic cells is performed by furin and by additional cellular proteases.

Before intoxication can occur, anthrax toxin protective antigen (PA), Pseudomonas exotoxin A (PE), and diphtheria toxin (DT) must be activated by proteolytic cleavage at specific amino acid sequences. Previously, it was shown that PA and DT can be activated by furin. In Chinese hamster ovary (CHO) cells, wild-type (RKKR) and cleavage site mutants of PA, each administered with a modified form of anthrax toxin lethal factor (the N terminus of lethal factor fused to PE domain III), had the following potencies: RKKR (wild type) (concentration causing 50% cell death [EC50] = 12 ng/ml) > or = RAAR (EC50 = 18 ng/ml) > FTKR (EC50 = 24 ng/ml) > STRR (EC50 = 49 ng/ml). In vitro cleavage of PA and cleavage site mutants of PA by furin demonstrated that native PA (RKKR) and PA with the cleavage sequence RAAR are substrates for furin. To characterize eukaryotic proteases that play a role in activating bacterial toxins, furin-deficient CHO cells were selected after chemical mutagenesis. Furin-deficient cells were resistant to PE, whose cleavage site, RQPR, constitutes a furin recognition site and to all PA cleavage site mutants, but were sensitive to DT (EC50 = 2.9 ng/ml) and PA (EC50 = 23 ng/ml), whose respective cleavage sites, RKKR and RVRR, contain additional basic residues. Furin-deficient cells that were transfected with the furin gene regained sensitivity to PE and PA cleavage site mutants. These studies provide evidence that furin can activate the three toxins and that one or more additional proteases contribute to the activation of DT and PA.     

Anthrax toxin characterization

The anthrax toxin comprises three proteins. When they work together, they can kill humans, especially after spores of the bacteria have been inhaled. One anthrax protein, called protective antigen (PA), chaperones the two other toxins into human or animal cells and shields them from the body's immune system. The second, lethal factor (LF), destroys the white blood cells that hosts send in defence. The third toxin molecule, edema factor (EF), hijacks the signaling system in the body. This disrupts the energy balance of cells and leads to them accumulating fluid and complete destroy of cells.     

Anthrax edema toxin requires influx of calcium for inducing cyclic AMP toxicity in target cells

The anthrax edema toxin comprises two proteins: protective antigen and edema factor. Anthrax protective antigen binds to the receptors on the surface of target cells and facilitates the entry of edema factor into these target cells. Edema factor (EF) is an adenylate cyclase that catalyzes the synthesis of cyclic AMP (cAMP) in the cytosol of the host cells. In this study, we examined the requirement of extracellular calcium for anthrax edema toxin-induced toxicity in host cells. The cAMP response generated by edema toxin was analyzed in a variety of cells, including CHO, macrophage-like RAW264.7, human neutrophils, and human lymphocytes. Our investigations reveal that after EF reaches the cell cytosol, a rapid influx of calcium is triggered in the host cell that has a pivotal role in determining the cAMP response of the affected cells. Although the cAMP response generated by edema toxin in different cell types varied in intensity and in the time of initiation, the influx of calcium invariably preceded cAMP accumulation. Agents that blocked the uptake of calcium also inhibited edema toxin-induced accumulation of cAMP in the host cells. This is the first report that demonstrates that edema toxin induces accumulation of cAMP in lymphocytes. By accumulating cAMP, a potent inhibitor of immune cell function, edema toxin may actually be poisoning the immune system and thus facilitating the survival of the bacteria in the host.     

A role for PACE4 in the proteolytic activation of anthrax toxin protective antigen.

Several bacterial protein toxins require activation by eukaryotic proteases. Previous studies have shown that anthrax toxin protective antigen (PA), Pseudomonas exotoxin A (PE), and diphtheria toxin (DT) are cleaved by furin C-terminal to the sequences RKKR, RQPR, and RVRR, respectively. Because furin-deficient cells retain some sensitivity to PA and DT, it is evident that other cellular proteases can activate these toxins. Whereas furin has been shown to require arginine residues at positions -1 and -4 for substrate recognition, another protease with an activity which could substitute for furin in toxin activation, the furin-related protease PACE4, requires basic residues in the -1, -2, and -4 positions of the substrate sequence. To examine the relative roles of furin and PACE4 in toxin activation, we used furin-deficient CHO cells (FD11 cells) transfected with either the furin (FD11/furin cells) or PACE4 (FD11/PACE4 cells) gene. Mutant PA proteins containing the cleavage sequence RAAR or KR were cytotoxic toward cells expressing only PACE4. In vitro cleavage data demonstrated that PACE4 can recognize RAAR and, to a much lesser extent, KR and RR. When extracts from PACE4-transfected cells were used as a source of proteases, PACE4 had minimal activity, indicating that it had been partially inactivated or did not remain associated with the cell membranes. Cleavage of iodinated PA containing the sequence RKKR or RAAR was detected on the surface of all cell types tested, but cleavage of a dibasic sequence was detected only intracellularly and only in cells that expressed furin or PACE4. The data provide evidence that PACE4 is present at the exterior of cells, that it plays a role in the proteolytic activation of anthrax toxin PA, and that PACE4 can activate substrates at the sequence RAAR or KR.     

Oligomerization of anthrax toxin protective antigen and binding of lethal factor during endocytic uptake into mammalian cells

The protective antigen (PA) protein of anthrax toxin binds to a cellular receptor and is cleaved by cell surface furin to produce a 63-kDa fragment (PA63). The receptor-bound PA63 oligomerizes to a heptamer and acts to translocate the catalytic moieties of the toxin, lethal factor (LF) and edema factor (EF), from endosomes to the cytosol. In this report, we used nondenaturing gel electrophoresis to show that each PA63 subunit in the heptamer can bind one LF molecule. Studies using PA immobilized on a plastic surface showed that monomeric PA63 is also able to bind LF. The internalization of PA and LF by cells was studied with radiolabeled and biotinylated proteins. Uptake was relatively slow, with a half-time of 30 min. The number of moles of LF internalized was nearly equal to the number of moles of PA subunit internalized. The essential role of PA oligomerization in LF translocation was shown with PA protein cleaved at residues 313-314. The oligomers formed by these proteins during uptake into cells were not as stable when subjected to heat and detergent as were those formed by native PA. The results show that the structure of the toxin proteins and the kinetics of proteolytic activation, LF binding, and internalization are balanced in a way that allows each PA63 subunit to internalize an LF molecule. This set of proteins has evolved to achieve highly efficient internalization and membrane translocation of the catalytic components, LF and EF.     

Major histocompatibility complex class I presentation of exogenous soluble tumor antigen fused to the B-fragment of Shiga toxin

Targeting exogenous antigen into the MHC class I-restricted presentation pathway is a prerequisite for the induction of cytotoxic T lymphocytes (CTL) which have been shown to represent an important component of the protective and therapeutic immune response to viral infections and tumors. In this study, we produced recombinant proteins composed of the receptor-binding non-toxic B-fragment of bacterial Shiga toxin derived from Shigella dysenteriae associated with an epitope from a model tumor antigen, Mage 1. We show that Shiga B-Mage 1 fusion proteins carrying an active or inactive endoplasmic reticulum retrieval signal (the C-terminal peptides KDEL or KDELGL, respectively) could be presented by peripheral blood mononuclear cells in an MHC class I-restricted manner to Mage 1-specific CTL. After pulsing B lymphoblastoid cells or dendritic cells with Shiga B-Mage 1 fusion protein, activation of the MHC class I-restricted Mage 1-specific CTL was also demonstrated. In further analysis, we showed that treatment with brefeldin A or paraformaldehyde fixation of Epstein-Barr virus-transformed B cells prevented the presentation of the Mage 1 T cell epitope, which excluded extracellular processing of the antigen. Immunofluorescence analysis also revealed that the Shiga B-Mage 1 fusion protein was largely excluded from Lamp-2-positive lysosomal structures. Therefore, the ability of Shiga toxin B-fragment to target dendritic cells and B cells and to direct antigen into the exogenous class I-restricted pathway makes it an attractive non-living and non-toxic vaccine vector.     

Role of toxin functional domains in anthrax pathogenesis

We investigated the role of the functional domains of anthrax toxins during infection. Three proteins produced by Bacillus anthracis, the protective antigen (PA), the lethal factor (LF), and the edema factor (EF), combine in pairs to produce the lethal (PA+LF) and edema (PA+EF) toxins. A genetic strategy was developed to introduce by allelic exchange specific point mutations or in-frame deletions into B. anthracis toxin genes, thereby impairing either LF metalloprotease or EF adenylate cyclase activity or PA functional domains. In vivo effects of toxin mutations were analyzed in an experimental infection of mice. A tight correlation was observed between the properties of anthrax toxins delivered in vivo and their in vitro activities. The synergic effects of the lethal and edema toxins resulted purely from their enzymatic activities, suggesting that in vivo these toxins may act together. The PA-dependent antibody response to LF induced by immunization with live B. anthracis was used to follow the in vivo interaction of LF and PA. We found that the binding of LF to PA in vivo was necessary and sufficient for a strong antibody response against LF, whereas neither LF activity nor binding of lethal toxin complex to the cell surface was required. Mutant PA proteins were cleaved in mice sera. Thus, our data provide evidence that, during anthrax infection, PA may interact with LF before binding to the cell receptor. Immunoprotection studies indicated that the strain producing detoxified LF and EF, isogenic to the current live vaccine Sterne strain, is a safe candidate for use as a vaccine against anthrax.     

Protection against anthrax lethal toxin challenge by genetic immunization with a plasmid encoding the lethal factor protein

The ability of genetic vaccination to protect against a lethal challenge of anthrax toxin was evaluated. BALB/c mice were immunized via gene gun inoculation with eucaryotic expression vector plasmids encoding either a fragment of the protective antigen (PA) or a fragment of lethal factor (LF). Plasmid pCLF4 contains the N-terminal region (amino acids [aa] 10 to 254) of Bacillus anthracis LF cloned into the pCI expression plasmid. Plasmid pCPA contains a biologically active portion (aa 175 to 764) of B. anthracis PA cloned into the pCI expression vector. One-micrometer-diameter gold particles were coated with plasmid pCLF4 or pCPA or a 1:1 mixture of both and injected into mice via gene gun (1 microg of plasmid DNA/injection) three times at 2-week intervals. Sera were collected and analyzed for antibody titer as well as antibody isotype. Significantly, titers of antibody to both PA and LF from mice immunized with the combination of pCPA and pCLF4 were four to five times greater than titers from mice immunized with either gene alone. Two weeks following the third and final plasmid DNA boost, all mice were challenged with 5 50% lethal doses of lethal toxin (PA plus LF) injected intravenously into the tail vein. All mice immunized with pCLF4, pCPA, or the combination of both survived the challenge, whereas all unimmunized mice did not survive. These results demonstrate that DNA-based immunization alone can provide protection against a lethal toxin challenge and that DNA immunization against the LF antigen alone provides complete protection.     

Neutralizing activity of vaccine-induced antibodies to two Bacillus anthracis toxin components, lethal factor and edema factor

Anthrax vaccine adsorbed (AVA; BioThrax), the current FDA-licensed human anthrax vaccine, contains various amounts of the three anthrax toxin components, protective antigen (PA), lethal factor (LF), and edema factor (EF). While antibody to PA is sufficient to mediate protection against anthrax in animal models, it is not known if antibodies to LF or EF contribute to protection in humans. Toxin-neutralizing activity was evaluated in sera from AVA-vaccinated volunteers, all of whom had antibody responses to LF and EF, as well as PA. The contribution of antibodies to LF and EF was assessed using mouse macrophage J774A.1 cells by examining neutralization of LF-induced lysis using alamarBlue reduction and neutralization of EF-induced cyclic AMP increases by enzyme-linked immunosorbent assay. Antibody responses to LF and EF were low compared to those to PA, and the amount of LF or EF in the assay could exceed the amount of antibodies to LF or EF. Higher titers were seen for most individuals when the LF or EF concentration was limiting compared to when LF or EF was in excess, initially suggesting that antibody to LF or EF augmented protection. However, depletion of LF and EF antibodies in sera did not result in a significant decrease in toxin neutralization. Overall, this study suggests that AVA-induced LF and EF antibodies do not significantly contribute to anthrax toxin neutralization in humans and that antibodies to PA are sufficient to neutralize toxin activity.     

Cytotoxic T-Lymphocyte Epitopes Fused to Anthrax Toxin Induce Protective Antiviral Immunity

We have investigated the use of the protective antigen (PA) and lethal factor (LF) components of anthrax toxin as a system for in vivo delivery of cytotoxic T-lymphocyte (CTL) epitopes. During intoxication, PA directs the translocation of LF into the cytoplasm of mammalian cells. Here we demonstrate that antiviral immunity can be induced in BALB/c mice immunized with PA plus a fusion protein containing the N-terminal 255 amino acids of LF (LFn) and an epitope from the nucleoprotein (NP) of lymphocytic choriomeningitis virus. We also demonstrate that BALB/c mice immunized with a single LFn fusion protein containing NP and listeriolysin O protein epitopes in tandem mount a CTL response against both pathogens. Furthermore, we show that NP-specific CTL are primed in both BALB/c and C57BL/6 mice when the mice are immunized with a single fusion containing two epitopes, one presented by Ld and one presented by Db. The data presented here demonstrate the versatility of the anthrax toxin delivery system and indicate that this system may be used as a general approach to vaccinate outbred populations against a variety of pathogens.     

Mutational analysis of the enzymatic domain of Clostridium difficile toxin B reveals novel inhibitors of the wild-type toxin

Toxin B (TcdB), a major Clostridium difficile virulence factor, glucosylates and inactivates the small GTP-binding proteins Rho, Rac, and Cdc42. In the present study we provide evidence that enzymatically inactive fragments of the TcdB enzymatic domain are effective intracellular inhibitors of native TcdB. Site-directed and deletion mutants of the TcdB enzymatic region (residues 1 to 556), lacking receptor binding and cell entry domains, were analyzed for attenuation of glucosyltransferase and glucosylhydrolase activity. Five of six derivatives from TcdB(1-556) were found to be devoid of enzymatic activity. In order to facilitate cell entry, mutants were genetically fused to lfn, which encodes the protective antigen binding region of anthrax toxin lethal factor and mediates the cell entry of heterologous proteins. In line with reduced enzymatic activity, the mutants also lacked cytotoxicity. Remarkably, pretreatment or cotreatment of cells with four of the mutants provided protection against the cytotoxic effects of native TcdB. Furthermore, a CHO cell line expressing enzymatically active TcdB(1-556) was also protected by the mutant-derived inhibitors, suggesting that inhibition occurred at an intracellular location. Protection also was afforded by the inhibitor to cells treated with Clostridium sordellii lethal toxin (TcsL), which uses the same cosubstrate as TcdB but shares Rac only as a common substrate target. Finally, the inhibitor did not provide protection against Clostridium novyi alpha-toxin (Tcnalpha), which shares similar substrates with TcdB yet uses a different cosubstrate. This is the first report to demonstrate that the potential exists to inhibit toxins at their intracellular site of action by using inactive mutants.     

Both CD4+ and CD8+ T cells respond to antigens fused to anthrax lethal toxin

The lethal toxin produced by Bacillus anthracis is a bipartite toxin in which the first protein, protective antigen (PA), transports the second protein, lethal factor, across the host cell membrane. We have previously shown that CD8(+) T-cell epitopes fused to a nontoxic derivative of lethal factor (LFn) are delivered into the host cell cytosol in a PA-dependent manner. Delivery of these antigens targets them to the intracellular major histocompatibility complex (MHC) class I processing and presentation pathway and leads to the stimulation of antigen-specific CD8(+) T cells in vivo. In this report, we describe the generation and characterization of LFn fusion proteins that include not only a CD8(+) T-cell epitope but also a CD4(+) T-cell epitope. We first show that these fusion proteins induce antigen-specific CD4(+) T-cell responses following incubation with dendritic cells in vitro or injection into mice. Stimulation of CD4(+) T cells by LFn fusion proteins does not require PA but is enhanced by PA in vitro. We also show that a single LFn fusion protein and PA can deliver antigen to both the MHC class II and the MHC class I pathways, resulting in the simultaneous induction of antigen-specific CD4(+) T cells and antigen-specific CD8(+) T cells in the same mouse. These results suggest that this toxin delivery system is capable of stimulating protective immune responses where effective immunization requires stimulation of both classes of T cells.     

Passive vaccination with a human monoclonal antibody: generation of antibodies and studies for efficacy in Bacillus anthracis infections

A major difficulty in creating human monoclonal antibodies is the lack of a suitable myeloma cell line to be used for fusion experiments. In order to create fully human monoclonal antibodies for passive immunization, the human mouse heteromyeloma cell line CB-F7 was evaluated. Using this cell line, we generated human monoclonal antibodies against Bacillus anthracis toxin components. Antibodies against protective antigen (PA) and against lethal factor (LF) were obtained using peripheral blood lymphocytes (PBLs) from persons vaccinated with the UK anthrax vaccine. PBL were fused with the cell line CB-F7. We obtained several clones producing PA specific Ig and one clone (hLF1-SAN) producing a monoclonal antibody (hLF1) directed against LF. The LF binding antibody was able to neutralize Anthrax toxin activity in an in vitro neutralization assay, and preliminary in vivo studies in mice also indicated a trend towards protection. We mapped the epitope of the antibody binding to LF by dot blot analysis and ELIFA using 80 synthetic LF peptides of 20 amino acid lengths with an overlapping range of 10 amino acids. Our results suggest the binding of the monoclonal antibody to the peptide regions 121-150 or 451-470 of LF. The Fab-fragment of the antibody hLF1 was cloned in Escherichia coli and could be useful as part of a fully human monoclonal antibody for the treatment of Anthrax infections. In general, our studies show the applicability of the CB-F7 line to create fully human monoclonal antibodies for vaccination.     

Anthrax Toxin

Anthrax is primarily a disease of herbivores caused by Gram-positive, aerobic, spore-forming Bacillus anthracis. Humans are accidental hosts through the food of animal origin and animal products. Anthrax is prevelant in most parts of the globe, and cases of anthrax have been reported from almost every country. Three forms of the disease have been recognized: cutaneous (through skin), gastrointestinal (through alimentary tract), and pulmonary (by inhalation of spores).

The major virulence factors of Bacillus anthracis are a poly-D glutamic acid capsule and a three-component protein exotoxin. The genes coding for the toxin and the enzymes responsible for capsule production are carried on plasmid pXO1 and pXO2, respectively. The three proteins of the exotoxin are protective antigen (PA, 83?kDa), lethal factor (LF, 90?kDa), and edema factor (EF, 89?kDa). The toxins follow the A-B model with PA being the B moeity and LF/EF, the alternative A moeities. LF and EF are individually nontoxic, but in combination with PA form two toxins causing different pathogenic responses in animals and cultured cells. PA + LF forms the lethal toxin and PA + EF forms the edema toxin. During the process of intoxication, PA binds to the cell surface receptor and is cleaved at the sequence RKKR (167) by cell surface proteases such as furin generating a cell-bound, C-terminal 63?kDa protein (PA63). PA63 possesses a binding site to which LF or EF bind with high affinity. The complex is then internalized by receptor-mediated endocytosis. Acidification of the vesicle leads to instertion of PA63 into the endosomal membrane and translocation of LF/EF across the bilayer into the cytosol where they exert their toxic effects. EF has a calcium- and calmodulin-dependent adenylate cyclase activity. Recent reports indicate that LF is a protease that cleaves the amino terminus of mitogen-activated protein kinase kinases 1 and 2 (MAPKK1 and 2), and this cleavage inactivates MAPKK1 and thus inhibits the mitogen-activated protein kinase signal transduction pathway. We describe in detail the studies so far done on unraveling the molecular mechanisms of pathogenesis of Bacillus anthracis.     

Toxicity of anthrax toxin is influenced by receptor expression

Anthrax toxin protective antigen (PA) binds to its cellular receptor, and seven subunits self-associate to form a heptameric ring that mediates the cytoplasmic entry of lethal factor or edema factor. The influence of receptor type on susceptibility to anthrax toxin components was examined using Chinese hamster ovary (CHO) cells expressing the human form of one of two PA receptors: TEM8 or CMG2. Unexpectedly, PA alone, previously believed to only mediate entry of lethal factor or edema factor, was found to be toxic to CHO-TEM8 cells; cells treated with PA alone displayed reduced cell growth and decreased metabolic activity. PA-treated cells swelled and became permeable to membrane-excluded dye, suggesting that PA formed cell surface pores on CHO-TEM8 cells. While CHO-CMG2 cells were not killed by wild-type PA, they were susceptible to the PA variant, F427A. Receptor expression also conferred differences in susceptibility to edema factor.     

Effects of anthrax toxin components on human neutrophils.

The virulence of Bacillus anthracis has been attributed to a tripartite toxin composed of three proteins designated protective antigen, lethal factor, and edema factor. The effects of the toxin components on phagocytosis and chemiluminescence of human polymorphonuclear neutrophils were studied in vitro. Initially, it was determined that the avirulent Sterne strain of B. anthracis (radiation killed) required opsonization with either serum complement or antibodies against the Sterne cell wall to be phagocytized. Phagocytosis of the opsonized Sterne cells was not affected by the individual anthrax toxin components. However, a combination of protective antigen and edema factor inhibited Sterne cell phagocytosis and blocked both particulate and phorbol myristate acetate-induced polymorphonuclear neutrophil chemiluminescence. These polymorphonuclear neutrophil effects were reversible upon removal of the toxin components. The protective antigen-edema factor combination also increased intracellular cyclic AMP levels. These studies suggest that two of the protein components of anthrax toxin, edema factor and protective antigen, increase host susceptibility to infection by suppressing polymorphonuclear neutrophil function and impairing host resistance.