Anthrax Exotoxin Research Articles

Anthrax toxin channel: What we know based on over 30years of research.

Protective antigen channel is the central component of the deadly anthrax exotoxin responsible for binding and delivery of the toxin's enzymatic lethal and edema factor components into the cytosol. The channel, which is more than three times longer than the lipid bilayer membrane thickness and has a 6-Å limiting diameter, is believed to provide a sophisticated unfoldase and translocase machinery for the foreign protein transport into the host cell cytosol. The tripartite toxin can be reengineered, one component at a time or collectively, to adapt it for the targeted cancer therapeutic treatments. In this review, we focus on the biophysical studies of the protective antigen channel-forming activity, small ion transport properties, enzymatic factor translocation, and blockage comparing it with the related clostridial binary toxin channels. We address issues linked to the anthrax toxin channel structural dynamics and lipid dependence, which are yet to become generally recognized as parts of the toxin translocation machinery.

Nanoporous Hydrogels for the Observation of Anthrax Exotoxin Translocation Dynamics.

The ability to observe lethal anthrax exotoxins translocating through size-constricting nanopores in vitro, combined with detailed sequence and structural data, has aided in elucidated mechanisms of exotoxin cell entry and toxicity. However, due to limited observations of anthrax exotoxins translocating through protective antigen nanopores in vitro and the instability of protective antigen-functionalized suspended lipid bilayers, questions remain regarding the native mechanisms of cell entry. Nanoporous hydrogel membranes offer a robust tool for studying protein translocation with ensemble measurements that complement conventional single-molecule translocation measurements. Here, we utilize nanoporous hydrogel membranes to assess the translocation of full-length anthrax lethal and edema factors through nanopores similar in diameter to protective antigen translocons. We find that, relative to globular serum and other proteins that do not translocate natively through nanopores, anthrax exotoxins demonstrate significantly reduced barriers to pore entry. Computed free-energy barriers to the unfolding of proteins and the dissociation of macromolecular complexes are generally found to coincide with translocation. Finally, a nanopore-blocking strategy is developed that utilizes nonspecific synthetic peptide constructs and effectively prevents LF translocation within the nanoporous hydrogel.

Deletion of a putative NlpC/P60 endopeptidase BAS1812 affects germination, long-term survival and endospore formation in Bacillus anthracis.

Bacillus anthracis, an aetiologic agent of the zoonotic disease anthrax, encodes a putative NlpC/P60 endopeptidase BAS1812. It harbours a signal peptide, three bacterial SH3 domains and an NlpC/P60 family domain. Previous studies showed that BAS1812 is immunogenic in infected hosts and is a potential biomarker for anthrax treatment. To date, however, little information is known about its function and involvement in anthrax pathogenesis. Here we describe the phenotypic effect of BAS1812 deletion in B. anthracis Sterne strain. Transcriptional analysis showed that BAS1812 expression in a host-like environment was enhanced at the end of log phase, started to diminish after entry to stationary phase and increased again late in stationary phase. The constructed BAS1812 mutant showed impaired long-term survival in the stationary growth phase, less resilience to detergent, lesser endospore formation and delayed germination. The mutant also showed diminished ability to degrade peptidoglycan, but its ability to produce anthrax exotoxins was not affected. We hypothesize that BAS1812 is a cell wall hydrolase involved in biological activities related to maintaining cell wall integrity, sporulation and spore germination.

Affinity binding of antibodies to supermacroporous cryogel adsorbents with immobilized protein A for removal of anthrax toxin protective antigen

Polymeric cryogels are efficient carriers for the immobilization of biomolecules because of their unique macroporous structure, permeability, mechanical stability and different surface chemical functionalities. The aim of the study was to demonstrate the potential use of macroporous monolithic cryogels for biotoxin removal using anthrax toxin protective antigen (PA), the central cell-binding component of the anthrax exotoxins, and covalent immobilization of monoclonal antibodies. The affinity ligand (protein A) was chemically coupled to the reactive hydroxyl and epoxy-derivatized monolithic cryogels and the binding efficiencies of protein A, monoclonal antibodies to the cryogel column were determined. Our results show differences in the binding capacity of protein A as well as monoclonal antibodies to the cryogel adsorbents caused by ligand concentrations, physical properties and morphology of surface matrices. The cytotoxicity potential of the cryogels was determined by an in vitro viability assay using V79 lung fibroblast as a model cell and the results reveal that the cryogels are non-cytotoxic. Finally, the adsorptive capacities of PA from phosphate buffered saline (PBS) were evaluated towards a non-glycosylated, plant-derived human monoclonal antibody (PANG) and a glycosylated human monoclonal antibody (Valortim®), both of which were covalently attached via protein A immobilization. Optimal binding capacities of 108 and 117 mg/g of antibody to the adsorbent were observed for PANG attached poly(acrylamide-allyl glycidyl ether) [poly(AAm-AGE)] and Valortim® attached poly(AAm-AGE) cryogels, respectively, This indicated that glycosylation status of Valortim® antibody could significantly increase (8%) its binding capacity relative to the PANG antibody on poly(AAm-AGE)-protien-A column (p < 0.05). The amounts of PA which remained in the solution after passing PA spiked PBS through PANG or Valortim bound poly(AAm-AGE) cryogel were significantly (p < 0.05) decreased relative to the amount of PA remained in the solution after passing through unmodified as well as protein A modified poly(AAm-AGE) cryogel columns, indicates efficient PA removal from spiked PBS over 60 min of circulation. The high adsorption capacity towards anthrax toxin PA of the cryogel adsorbents indicated potential application of these materials for treatment of Bacillus anthracis infection.

Cellular and Physiological Effects of Anthrax Exotoxin and Its Relevance to Disease

Bacillus anthracis, the causative agent of anthrax, secretes a tri-partite exotoxin that exerts pleiotropic effects on the host. The purification of the exotoxin components, protective antigen, lethal factor, and edema factor allowed the rapid characterization of their physiologic effects on the host. As molecular biology matured, interest focused on the molecular mechanisms and cellular alterations induced by intoxication. Only recently have researchers begun to connect molecular and cellular knowledge back to the broader physiological effects of the exotoxin. This review focuses on the progress that has been made bridging molecular knowledge back to the exotoxin’s physiological effects on the host.

Protective properties of the receptor domain of anthrax exotoxin protective antigen depend from the orientation of its presentation on nanoparticles

Polysterene particles bearing on its surface recombinant protein receptor domain of protective antigen anthrax exotoxin, exposed in different orientations have been constructed. Particles with exposed COOH region of receptor domain induced the highest protective immunity in mice anthrax model (95%). We revealed that immunization with these particles causes a specific induction of Th1-response, characterized by increased levels ofcytokine TNF-alpha and IL-2.

Inter-α Inhibitor Proteins

Human inter-α inhibitor proteins are endogenous human plasma proteins that function as serine protease inhibitors. Inter-α inhibitor proteins can block the systemic release of proteases in sepsis and block furin-mediated assembly of protective antigen, an essential stop in the intracellular delivery of the anthrax exotoxins, lethal toxin and edema toxin. Inter-α inhibitor proteins administered on hour or up to 24 h after spore challenge with Bacillus anthracis Sterne strain protected mice from lethality if administered with antimicrobial therapy (P < 0.001). These human plasma proteins possess combined actions against anthrax as general inhibitors of excess serine proteases in sepsis and specific inhibitors of anthrax toxin assembly. Inter-α inhibitor proteins could represent a novel adjuvant therapy for the treatment of established anthrax infection.

Plasmid-based vaccination with candidate anthrax vaccine antigens induces durable type 1 and type 2 T-helper immune responses

The current anthrax vaccine imparts protective immunity by generating a humoral immune response against a single antigen, the PA exotoxin subunit. While this response neutralizes the two anthrax exotoxins and protects the recipient from toxin-related mortality, the recipient is not protected from spore germination, infection, and/or bacteremia. Moreover, protective immunity against PA must be generated via a lengthy injection schedule and maintained by a yearly booster. In an effort to improve upon the current vaccine formulation, we screened six of seven known virulence factors encoded by Bacillus anthracis epigenetic elements pXO1 and pXO2 as well as the major surface proteins EA1 and SAP. Screening was carried out in conjunction with a plasmid-based technology known for its ability to generate type 1 and type 2 T-helper responses. Long-term high level antibody titers were generated against the products of eag (EA1), sap (SAP), and the capA capsule synthesis subunit in vivo. Further analysis of PA- and EA1-vaccinated mice demonstrated antigen-specific type 1 helper responses including IFN-gamma secretion and lysis of EA1- or PA-loaded macrophages; further, an EA1 T-cell epitope was identified. The results demonstrate that anthrax antigens other than PA might be suitable for the generation of durable immune responses against anthrax.

Catalytically inactive anthrax toxin(s) are potential prophylactic agents

The anthrax exotoxin, which is a key mediator of anthrax related pathogenesis, is composed of two separate toxins formed by pairwise combinations of three proteins that are encoded on the pXO1 plasmid of Bacillus anthracis. Lethal toxin is composed of protective antigen (PA) combined with lethal factor (LF) while edema toxin is composed of PA and edema factor (EF). The present study found that the catalytic mutants of LF (LF E687A) and EF (EF H351A) competitively inhibited lethal toxin and edema toxin-mediated activity in vitro and lethality in vivo and were non-toxic to sensitive cell lines when combined with PA. While PA combined with EF H351A was non-lethal in mice, PA combined with LF E687A was of reduced virulence. Full protection of mice against a lethal toxin challenge required injection of mice with PA combined with both LF E687A and EF H351A. The potential use of these full-length, biologically inactive mutant proteins combined with PA as prophylactics or therapeutics is discussed.

Anthrax Vaccines

SUMMARYAnthrax, an uncommon disease in humans, is caused by a large bacterium, Bacillus anthracis. The risk of inhalation infection is the main indication for anthrax vaccination. Pre-exposure vaccination is provided by an acellular vaccine (anthrax vaccine adsorbed or AVA), which contains anthrax toxin elements and results in protective immunity after 3 to 6 doses. Anthrax vaccine precipitated (AVP) is administered at primovaccination in 3 doses with a booster dose after 6 months. To evoke and maintain protective immunity, it is necessary to administer a booster dose once at 12 months. In Russia, live spore vaccine (STI) has been used in a two-dose schedule. Current anthrax vaccines show considerable local and general reactogenicity (erythema, induration, soreness, fever). Serious adverse reactions occur in about 1% of vaccinations. New second-generation vaccines in current research programs include recombinant live vaccines and recombinant sub-unit vaccines.

Calcium-independent calmodulin binding and two-metal–ion catalytic mechanism of anthrax edema factor

Edema factor (EF), a key anthrax exotoxin, has an anthrax protective antigen-binding domain (PABD) and a calmodulin (CaM)-activated adenylyl cyclase domain. Here, we report the crystal structures of CaM-bound EF, revealing the architecture of EF PABD. CaM has N- and C-terminal domains and each domain can bind two calcium ions. Calcium binding induces the conformational change of CaM from closed to open. Structures of the EF-CaM complex show how EF locks the N-terminal domain of CaM into a closed conformation regardless of its calcium-loading state. This represents a mechanism of how CaM effector alters the calcium affinity of CaM and uncouples the conformational change of CaM from calcium loading. Furthermore, structures of EF-CaM complexed with nucleotides show that EF uses two-metal-ion catalysis, a prevalent mechanism in DNA and RNA polymerases. A histidine (H351) further facilitates the catalysis of EF by activating a water to deprotonate 3'OH of ATP. Mammalian adenylyl cyclases share no structural similarity with EF and they also use two-metal-ion catalysis, suggesting the catalytic mechanism-driven convergent evolution of two structurally diverse adenylyl cyclases.

A High-Affinity Monoclonal Antibody to Anthrax Protective Antigen Passively Protects Rabbits before and after Aerosolized Bacillus anthracis Spore Challenge

We have developed a therapeutic for the treatment of anthrax using an affinity-enhanced monoclonal antibody (ETI-204) to protective antigen (PA), which is the central cell-binding component of the anthrax exotoxins. ETI-204 administered preexposure by a single intravenous injection of a dose of between 2.5 and 10 mg per animal significantly protected rabbits from a lethal aerosolized anthrax spore challenge ( approximately 60 to 450 times the 50% lethal dose of Bacillus anthracis Ames). Against a similar challenge, ETI-204 administered intramuscularly at a 20-mg dose per animal completely protected rabbits from death (100% survival). In the postexposure setting, intravenous administration of ETI-204 provided protection 24 h (8 of 10) and 36 h (5 of 10) after spore challenge. Administration at 48 h postchallenge, when 3 of 10 animals had already succumbed to anthrax infection, resulted in the survival of 3 of 7 animals (43%) for the duration of the study (28 days). Importantly, surviving ETI-204-treated animals were free of bacteremia by day 10 and remained so until the end of the studies. Only 11 of 51 ETI-204-treated rabbits had positive lung cultures at the end of the studies. Also, rabbits that were protected from inhalational anthrax by administration of ETI-204 developed significant titers of PA-specific antibodies. Presently, the sole therapeutic regimen available to treat infection by inhalation of B. anthracis spores is a 60-day course of antibiotics that is effective only if administered prior to or shortly after exposure. Based upon results reported here, ETI-204 is an effective therapy for prevention and treatment of inhalational anthrax.

Chloroquine enhances survival in Bacillus anthracis intoxication.

The intentional release of anthrax in the United States in 2001 resulted in 11 cases of inhalational disease, with an attendant mortality rate of 45%. Current therapeutic options for anthrax are limited; antimicrobials target only replicating organisms, thus allowing bacterial toxins to cause unchecked, devastating physiological derangements in the host. Novel approaches that target the cytotoxic effects of anthrax exotoxins are needed. Chloroquine (CQ), a commonly used antimalarial agent, endows anthrax-intoxicated murine peritoneal macrophages with a 50% and 35% marginal survival advantage at 2 and 4 h, respectively, over that of untreated control cells. The cell rescue is dose dependent and, at lower concentrations, results in delayed cell death. We subsequently studied the effect of CQ in BALB/c mice challenged with anthrax lethal toxin. CQ-treated mice demonstrated reduced tissue injury, as assessed by histopathological examination of the spleen and by peripheral blood differential cell count ratios. CQ significantly enhanced survival and may augment current treatment and prophylaxis options for this otherwise lethal infection.

Enhancement of anthrax lethal toxin cytotoxicity: a subset of monoclonal antibodies against protective antigen increases lethal toxin-mediated killing of murine macrophages.

We investigated the ability of using monoclonal antibodies (MAbs) against anthrax protective antigen (PA), an anthrax exotoxin component, to modulate exotoxin cytotoxic activity on target macrophage cell lines. Anthrax PA plays a critical role in the pathogenesis of Bacillus anthracis infection. PA is the cell-binding component of the two anthrax exotoxins: lethal toxin (LeTx) and edema toxin. Several MAbs that bind the PA component of LeTx are known to neutralize LeTx-mediated killing of target macrophages. Here we describe for the first time an overlooked population of anti-PA MAbs that, in contrast, function to increase the potency of LeTx against murine macrophage cell lines. The results support a possible mechanism of enhancement: binding of MAb to PA on the macrophage cell surface stabilizes the PA by interaction of MAb with macrophage Fcgamma receptors. This results in an increase in the amount of PA bound to the cell surface, which in turn leads to an enhancement in cell killing, most likely due to increased internalization of LF. Blocking of PA-receptor binding eliminates enhancement by MAb, demonstrating the importance of this step for the observed enhancement. The additional significance of these results is that, at least in mice, immunization with PA appears to elicit a poly-clonal response that has a significant prevalence of MAbs that enhance LeTx-mediated killing in macrophages.

Novel aspects of calmodulin target recognition and activation.

Several crystal and NMR structures of calmodulin (CaM) in complex with fragments derived from CaM-regulated proteins have been reported recently and reveal novel ways for CaM to interact with its targets. This review will discuss and compare features of the interaction between CaM and its target domains derived from the plasma membrane Ca2+-pump, the Ca2+-activated K+-channel, the Ca2+/CaM-dependent kinase kinase and the anthrax exotoxin. Unexpected aspects of CaM/target interaction observed in these complexes include: (a) binding of the Ca2+-pump domain to only the C-terminal part of CaM (b) dimer formation with fragments of the K+-channel (c) insertion of CaM between two domains of the anthrax exotoxin (d) binding of Ca2+ ions to only one EF-hand pair and (e) binding of CaM in an extended conformation to some of its targets. The mode of interaction between CaM and these targets differs from binding conformations previously observed between CaM and peptides derived from myosin light chain kinase (MLCK) and CaM-dependent kinase IIalpha (CaMKIIalpha). In the latter complexes, CaM engulfs the CaM-binding domain peptide with its two Ca2+-binding lobes and forms a compact, ellipsoid-like complex. In the early 1990s, a model for the activation of CaM-regulated proteins was developed based on this observation and postulated activation through the displacement of an autoinhibitory or regulatory domain from the target protein upon binding of CaM. The novel structures of CaM-target complexes discussed here demonstrate that this mechanism of activation may be less general than previously believed and seems to be not valid for the anthrax exotoxin, the CaM-regulated K+-channel and possibly also not for the Ca2+-pump.

Early Bacillus anthracis-macrophage interactions: intracellular survival survival and escape.

This study describes early intracellular events occurring during the establishment phase of Bacillus anthracis infections. Anthrax infections are initiated by dormant endospores gaining access to the mammalian host and becoming engulfed by regional macrophages (Mphi). During systemic anthrax, late stage events include vegetative growth in the blood to very high titres and the synthesis of the anthrax exotoxin complex, which causes disease symptoms and death. Experiments focus on the early events occurring during the first few hours of the B. anthracis infectious cycle, from endospore germination up to and including release of the vegetative cell from phagocytes. We found that newly vegetative bacilli escape from the phagocytic vesicles of cultured Mphi and replicate within the cytoplasm of these cells. Release from the Mphi occurs 4-6 h after endospore phagocytosis, timing that correlates with anthrax infection of test animals. Genetic analysis from this study indicates that the toxin plasmid pXO1 is required for release from the Mphi, whereas the capsule plasmid pXO2 is not. The transactivator atxA, located on pXO1, is also found to be essential for release, but the toxin genes themselves are not required. This suggests that Mphi release of anthrax bacilli is atxA regulated. The putative 'escape' genes may be located on the chromosome and/or on pXO1.