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Anthrax lethal toxin (LT) is a protease that activates the NLRP1b inflammasome sensor in certain rodent strains. Unlike better-studied sensors, relatively little is known about the priming requirements for NLRP1b. In this study, we investigate the rapid and striking priming-independent LT-induced release of IL-1β in mice within hours of toxin challenge. We find IL-1β release to be a NLRP1b- and caspase-1-dependent, NLRP3 and caspase-11-independent event that requires both neutrophils and peptidyl arginine deiminiase-4 (PAD4) activity. The simultaneous LT-induced IL-18 response is neutrophil-independent. Bone marrow reconstitution experiments in mice show toxin-induced IL-1β originates from hematopoietic cells. LT treatment of neutrophils in vitro did not induce IL-1β, neutrophil extracellular traps (NETs), or pyroptosis. Although platelets interact closely with neutrophils and are also a potential source of IL-1β, they were unable to bind or endocytose LT and did not secrete IL-1β in response to the toxin. LT-treated mice had higher levels of cell-free DNA and HMGB1 in circulation than PBS-treated controls, and treatment of mice with recombinant DNase reduced the neutrophil- and NLRP1-dependent IL-1β release. DNA sensor AIM2 deficiency, however, did not impact IL-1β release. These data, in combination with the findings on PAD4, suggest a possible role for in vivo NETs or cell-free DNA in cytokine induction in response to LT challenge. Our findings suggest a complex interaction of events and/or mediators in LT-treated mice with the neutrophil as a central player in induction of a profound and rapid inflammatory response to toxin.

Anthrax disease is caused by a toxin consisting of protective antigen (PA), lethal factor, and edema factor. Antibodies against PA have been shown to be protective against the disease. Variable domains of camelid heavy chain-only antibodies (VHHs) with affinity for PA were obtained from immunized alpacas and screened for anthrax neutralizing activity in macrophage toxicity assays. Two classes of neutralizing VHHs were identified recognizing distinct, non-overlapping epitopes. One class recognizes domain 4 of PA at a well characterized neutralizing site through which PA binds to its cellular receptor. A second neutralizing VHH (JKH-C7) recognizes a novel epitope. This antibody inhibits conversion of the PA oligomer from "pre-pore" to its SDS and heat-resistant "pore" conformation while not preventing cleavage of full-length 83-kDa PA (PA83) by cell surface proteases to its oligomer-competent 63-kDa form (PA63). The antibody prevents endocytosis of the cell surface-generated PA63 subunit but not preformed PA63 oligomers formed in solution. JKH-C7 and the receptor-blocking VHH class (JIK-B8) were expressed as a heterodimeric VHH-based neutralizing agent (VNA2-PA). This VNA displayed improved neutralizing potency in cell assays and protected mice from anthrax toxin challenge with much better efficacy than the separate component VHHs. The VNA protected virtually all mice when separately administered at a 1:1 ratio to toxin and protected mice against Bacillus anthracis spore infection. Thus, our studies show the potential of VNAs as anthrax therapeutics. Due to their simple and stable nature, VNAs should be amenable to genetic delivery or administration via respiratory routes.

Anthrax toxin protective antigen (PA) delivers its effector proteins into the host cell cytosol through formation of an oligomeric pore, which can assume heptameric or octameric states. By screening a highly directed library of PA mutants, we identified variants that complement each other to exclusively form octamers. These PA variants were individually nontoxic and demonstrated toxicity only when combined with their complementary partner. We then engineered requirements for activation by matrix metalloproteases and urokinase plasminogen activator into two of these variants. The resulting therapeutic toxin specifically targeted cells expressing both tumor associated proteases and completely stopped tumor growth in mice when used at a dose far below that which caused toxicity. This scheme for obtaining intercomplementing subunits can be employed with other oligomeric proteins and potentially has wide application.

Four core structures capable of providing sub-nanomolar inhibitors of anthrax lethal factor (LF) were evaluated by comparing the potential for toxicity, physicochemical properties, in vitro ADME profiles, and relative efficacy in a rat lethal toxin (LT) model of LF intoxication. Poor efficacy in the rat LT model exhibited by the phenoxyacetic acid series (3) correlated with low rat microsome and plasma stability. Specific molecular interactions contributing to the high affinity of inhibitors with a secondary amine in the C2-side chain were revealed by x-ray crystallography.

The anthrax edema toxin (ET) of Bacillus anthracis is composed of the receptor-binding component protective antigen (PA) and of the adenylyl cyclase catalytic moiety, edema factor (EF). Uptake of ET into cells raises intracellular concentrations of the secondary messenger cyclic AMP, thereby impairing or activating host cell functions. We report here on a new consequence of ET action in vivo . We show that in mouse models of toxemia and infection, serum PA concentrations were significantly higher in the presence of enzymatically active EF. These higher concentrations were not caused by ET-induced inhibition of PA endocytosis; on the contrary, ET induced increased PA binding and uptake of the PA oligomer in vitro and in vivo through upregulation of the PA receptors TEM8 and CMG2 in both myeloid and nonmyeloid cells. ET effects on protein clearance from circulation appeared to be global and were not limited to PA. ET also impaired the clearance of ovalbumin, green fluorescent protein, and EF itself, as well as the small molecule biotin when these molecules were coinjected with the toxin. Effects on injected protein levels were not a result of general increase in protein concentrations due to fluid loss. Functional markers for liver and kidney were altered in response to ET. Concomitantly, ET caused phosphorylation and activation of the aquaporin-2 water channel present in the principal cells of the collecting ducts of the kidneys that are responsible for fluid homeostasis. Our data suggest that in vivo , ET alters circulatory protein and small molecule pharmacokinetics by an as-yet-undefined mechanism, thereby potentially allowing a prolonged circulation of anthrax virulence factors such as EF during infection.

Bacillus anthracis is the causative agent of anthrax, and the tripartite anthrax toxin is an essential element of its pathogenesis. Edema factor (EF), a potent adenylyl cyclase, is one of the toxin components. In this work, anti-EF monoclonal antibodies (MAb) were produced following immunization of mice, and four of the antibodies were fully characterized. MAb 3F2 has an affinity of 388 pM, was most effective for EF detection, and appears to be the first antibody reported to neutralize EF by binding to the catalytic C B domain. MAb 7F10 shows potent neutralization of edema toxin activity in vitro and in vivo ; it targets the N-terminal protective antigen binding domain. The four MAb react with three different domains of edema factor, and all were able to detect purified edema factor in Western blot analysis. None of the four MAb cross-reacted with the lethal factor toxin component. Three of the four MAb protected mice in both a systemic edema toxin challenge model and a subcutaneous spore-induced foreleg edema model. A combination of three of the MAb also significantly delayed the time to death in a third subcutaneous spore challenge model. This appears to be the first direct evidence that monoclonal antibody-mediated neutralization of EF alone is sufficient to delay anthrax disease progression.

Three chimpanzee Fabs reactive with lethal factor (LF) of anthrax toxin were isolated and converted into complete monoclonal antibodies (MAbs) with human γ1 heavy-chain constant regions. In a macrophage toxicity assay, two of the MAbs, LF10E and LF11H, neutralized lethal toxin (LT), a complex of LF and anthrax protective antigen (PA). LF10E has the highest reported affinity for a neutralizing MAb against LF (dissociation constant of 0.69 nM). This antibody also efficiently neutralized LT in vitro, with a 50% effective concentration (EC 50 ) of 0.1 nM, and provided 100% protection of rats against toxin challenge with a 0.5 submolar ratio relative to LT. LF11H, on the other hand, had a slightly lower binding affinity to LF (dissociation constant of 7.4 nM) and poor neutralization of LT in vitro (EC 50 of 400 nM) and offered complete protection in vivo only at an equimolar or higher ratio to toxin. Despite this, LF11H, but not LF10E, provided robust synergistic protection when combined with MAb W1, which neutralizes PA. Epitope mapping and binding assays indicated that both LF10E and LF11H recognize domain I of LF (amino acids 1 to 254). Although domain I is responsible for binding to PA, neither MAb prevented LF from binding to activated PA. Although two unique MAbs could protect against anthrax when used alone, even more efficient and broader protection should be gained by combining them with anti-PA MAbs.

This study describes the isolation and characterization of a neutralizing monoclonal antibody (mAb) against anthrax edema factor, EF13D. EF13D neutralized edema toxin (ET)-mediated cyclic AMP (cAMP) responses in cells and protected mice from both ET-induced footpad edema and systemic ET-mediated lethality. The antibody epitope was mapped to domain IV of EF. The mAb was able to compete with calmodulin (CaM) for EF binding and displaced CaM from EF-CaM complexes. EF-mAb binding affinity (0.05–0.12 nM) was 50- to 130-fold higher than that reported for EF-CaM. This anti-EF neutralizing mAb could potentially be used alone or with an anti-PA mAb in the emergency prophylaxis and treatment of anthrax infection.

Inflammasomes are large cytoplasmic multiprotein complexes that activate caspase-1 in response to diverse intracellular danger signals. Inflammasome components termed nucleotide-binding oligomerization domain–like receptor (NLR) proteins act as sensors for pathogen-associated molecular patterns, stress, or danger stimuli. We discovered that arsenicals, including arsenic trioxide and sodium arsenite, inhibited activation of the NLRP1, NLRP3, and NAIP5/NLRC4 inflammasomes by their respective activating signals, anthrax lethal toxin, nigericin, and flagellin. These compounds prevented the autoproteolytic activation of caspase-1 and the processing and secretion of IL-1β from macrophages. Inhibition was independent of protein synthesis induction, proteasome-mediated protein breakdown, or kinase signaling pathways. Arsenic trioxide and sodium arsenite did not directly modify or inhibit the activity of preactivated recombinant caspase-1. Rather, they induced a cellular state inhibitory to both the autoproteolytic and substrate cleavage activities of caspase-1, which was reversed by the reactive oxygen species scavenger N-acetylcysteine but not by reducing agents or NO pathway inhibitors. Arsenicals provided protection against NLRP1-dependent anthrax lethal toxin–mediated cell death and prevented NLRP3-dependent neutrophil recruitment in a monosodium urate crystal inflammatory murine peritonitis model. These findings suggest a novel role in inhibition of the innate immune response for arsenical compounds that have been used as therapeutics for a few hundred years.

Bacillus anthracis , the causative agent of anthrax, manifests its pathogenesis through the action of two secreted toxins. The bipartite lethal and edema toxins, a combination of lethal factor or edema factor with the protein protective antigen, are important virulence factors for this bacterium. We previously developed small-molecule inhibitors of lethal factor proteolytic activity (LFIs) and demonstrated their in vivo efficacy in a rat lethal toxin challenge model. In this work, we show that these LFIs protect against lethality caused by anthrax infection in mice when combined with subprotective doses of either antibiotics or neutralizing monoclonal antibodies that target edema factor. Significantly, these inhibitors provided protection against lethal infection when administered as a monotherapy. As little as two doses (10 mg/kg) administered at 2 h and 8 h after spore infection was sufficient to provide a significant survival benefit in infected mice. Administration of LFIs early in the infection was found to inhibit dissemination of vegetative bacteria to the organs in the first 32 h following infection. In addition, neutralizing antibodies against edema factor also inhibited bacterial dissemination with similar efficacy. Together, our findings confirm the important roles that both anthrax toxins play in establishing anthrax infection and demonstrate the potential for small-molecule therapeutics targeting these proteins.

Bacillus anthracis, the causative agent of anthrax disease, is lethal owing to the actions of two exotoxins: anthrax lethal toxin (LT) and oedema toxin (ET). The key tissue targets responsible for the lethal effects of these toxins are unknown. Here we generated cell-type-specific anthrax toxin receptor capillary morphogenesis protein-2 (CMG2)-null mice and cell-type-specific CMG2-expressing mice and challenged them with the toxins. Our results show that lethality induced by LT and ET occurs through damage to distinct cell types; whereas targeting cardiomyocytes and vascular smooth muscle cells is required for LT-induced mortality, ET-induced lethality occurs mainly through its action in hepatocytes. Notably, and in contradiction to what has been previously postulated, targeting of endothelial cells by either toxin does not seem to contribute significantly to lethality. Our findings demonstrate that B. anthracis has evolved to use LT and ET to induce host lethality by coordinately damaging two distinct vital systems. Cell-type-specific anthrax toxin receptor CMG2-null mice are generated and used to show that the Bacillus anthracis toxins lethal toxin (LT) and oedema toxin (ET) target distinct cell types; in contrast to previous suggestions, it is shown that endothelial cells are not key targets for either toxin and instead LT targets cardiomyocytes and vascular smooth muscle cells whereas ET targets hepatocytes. Bacillus anthracis produces two toxins — anthrax lethal toxin and oedema toxin — which are targeted to tissues by captor-associated protective antigen. The two toxins have essential but little understood roles in pathogenesis. Here, Shihui Liu and colleagues generate mice lacking cell-type-specific anthrax toxin receptor capillary morphogenesis protein-2 (CMG2) and use them to show that the two toxins target distinct cell types. Contrary to previous suggestions, endothelial cells are not key targets for either toxin. Rather, lethal toxin targets cardiomyocytes and vascular smooth muscle cells, and oedema toxin targets hepatocytes. Recognition that the anthrax toxins are specifically targeting the cardiovascular system and liver may suggest supportive therapies that would limit tissue damage and increase survival in human anthrax infections.

New anthrax lethal factor inhibitors (LFIs) were designed based upon previously identified potent inhibitors 1a and 2. Combining the new core structures with modifications to the C2-side chain yielded analogs with improved efficacy in the rat lethal toxin model. [BMCL ABSTRACT]

MyD88-deficient mice were previously shown to have increased susceptibility to Bacillus anthracis infection relative to wild-type animals. To determine the mechanism by which MyD88 protects against B. anthracis infection, knockout mice were challenged with nonencapsulated, toxigenic B. anthracis or with anthrax toxins. MyD88-deficient mice had increased susceptibility to B. anthracis and anthrax lethal toxin but not to edema toxin. Lethal toxin alone induced marked multifocal intestinal ulcers in the knockout animals, compromising the intestinal epithelial barrier. The resulting enteric bacterial leakage in the knockout animals led to peritonitis and septicemia. Focal ulcers and erosion were also found in MyD88-heterozygous control mice but with far lower incidence and severity. B. anthracis infection also induced a similar enteric bacterial septicemia in MyD88-deficient mice but not in heterozygous controls. We show that lethal toxin and B. anthracis challenge induce bacteremia as a result of intestinal damage in MyD88-deficient mice. These results suggest that loss of the intestinal epithelial barrier and enteric bacterial septicemia may contribute to sensitizing MyD88-deficient mice to B. anthracis and that MyD88 plays a protective role against lethal toxin-induced impairment of intestinal barrier.

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

Anthrax lethal toxin (LT) is a bipartite protease-containing toxin and a key virulence determinant of Bacillus anthracis. In mice, LT causes the rapid lysis of macrophages isolated from certain inbred strains, but the correlation between murine macrophage sensitivity and mouse strain susceptibility to toxin challenge is poor. In rats, LT induces a rapid death in as little as 37 minutes through unknown mechanisms. We used a recombinant inbred (RI) rat panel of 19 strains generated from LT-sensitive and LT-resistant progenitors to map LT sensitivity in rats to a locus on chromosome 10 that includes the inflammasome NOD-like receptor (NLR) sensor, Nlrp1. This gene is the closest rat homolog of mouse Nlrp1b, which was previously shown to control murine macrophage sensitivity to LT. An absolute correlation between in vitro macrophage sensitivity to LT-induced lysis and animal susceptibility to the toxin was found for the 19 RI strains and 12 additional rat strains. Sequencing Nlrp1 from these strains identified five polymorphic alleles. Polymorphisms within the N-terminal 100 amino acids of the Nlrp1 protein were perfectly correlated with LT sensitivity. These data suggest that toxin-mediated lethality in rats as well as macrophage sensitivity in this animal model are controlled by a single locus on chromosome 10 that is likely to be the inflammasome NLR sensor, Nlrp1., Author Summary Inflammasomes are multiprotein cytoplasmic complexes that respond to a variety of danger signals by activating the host innate immune response. The sensor components of these complexes are NLR (NOD-like receptor) proteins. In this report, a recombinant inbred rat strain collection was used to genetically map anthrax lethal toxin (LT) susceptibility to a limited region of chromosome 10 containing one such sensor, Nlrp1. Similar to its mouse ortholog, Nlrp1b, which controls murine macrophage sensitivity to this toxin, the locus containing rat Nlrp1 was shown to control macrophage sensitivity to anthrax LT. However, unlike the situation in mice, where multiple genetic loci influence animal susceptibility to LT, the single chromosome 10 locus alone appears to control the rapid anthrax LT-induced death, which can occur in as little as 37 minutes. Sequencing of Nlrp1 from 12 rat strains identified polymorphisms which correlated perfectly with animal sensitivity to toxin. These polymorphisms were within the N-terminal 100-amino acid portion of Nlrp1, in an area of unknown function, which suggests that the N-terminus of rodent Nlrp1 could be an important functional domain.

Anthrax toxin, a major virulence factor of Bacillus anthracis , gains entry into target cells by binding to either of 2 von Willebrand factor A domain-containing proteins, tumor endothelium marker-8 (TEM8) and capillary morphogenesis protein-2 (CMG2). The wide tissue expression of TEM8 and CMG2 suggest that both receptors could play a role in anthrax pathogenesis. To explore the roles of TEM8 and CMG2 in normal physiology, as well as in anthrax pathogenesis, we generated TEM8- and CMG2-null mice and TEM8/CMG2 double-null mice by deleting TEM8 and CMG2 transmembrane domains. TEM8 and CMG2 were found to be dispensable for mouse development and life, but both are essential in female reproduction in mice. We found that the lethality of anthrax toxin for mice is mostly mediated by CMG2 and that TEM8 plays only a minor role. This is likely because anthrax toxin has approximately 11-fold higher affinity for CMG2 than for TEM8. Finally, the CMG2-null mice are also shown to be highly resistant to B. anthracis spore infection, attesting to the importance of both anthrax toxin and CMG2 in anthrax infections.

Anthrax lethal toxin (LT) induces vascular insufficiency in experimental animals through unknown mechanisms. In this study, we show that neuronal nitric oxide synthase (nNOS) deficiency in mice causes strikingly increased sensitivity to LT, while deficiencies in the two other NOS enzymes (iNOS and eNOS) have no effect on LT-mediated mortality. The increased sensitivity of nNOS−/− mice was independent of macrophage sensitivity to toxin, or cytokine responses, and could be replicated in nNOS-sufficient wild-type (WT) mice through pharmacological inhibition of the enzyme with 7-nitroindazole. Histopathological analyses showed that LT induced architectural changes in heart morphology of nNOS−/− mice, with rapid appearance of novel inter-fiber spaces but no associated apoptosis of cardiomyocytes. LT-treated WT mice had no histopathology observed at the light microscopy level. Electron microscopic analyses of LT-treated mice, however, revealed striking pathological changes in the hearts of both nNOS−/− and WT mice, varying only in severity and timing. Endothelial/capillary necrosis and degeneration, inter-myocyte edema, myofilament and mitochondrial degeneration, and altered sarcoplasmic reticulum cisternae were observed in both LT-treated WT and nNOS−/− mice. Furthermore, multiple biomarkers of cardiac injury (myoglobin, cardiac troponin-I, and heart fatty acid binding protein) were elevated in LT-treated mice very rapidly (by 6 h after LT injection) and reached concentrations rarely reported in mice. Cardiac protective nitrite therapy and allopurinol therapy did not have beneficial effects in LT-treated mice. Surprisingly, the potent nitric oxide scavenger, carboxy-PTIO, showed some protective effect against LT. Echocardiography on LT-treated mice indicated an average reduction in ejection fraction following LT treatment in both nNOS−/− and WT mice, indicative of decreased contractile function in the heart. We report the heart as an early target of LT in mice and discuss a protective role for nNOS against LT-mediated cardiac damage., Author Summary Injection of pure anthrax lethal toxin (LT) at the levels found in terminally-ill, anthrax-infected animals induces an atypical vascular collapse in several experimental animals without the associated hemorrhagic manifestations and disseminated intravascular coagulopathies seen in bacteria-induced shock states. Nevertheless, LT alone produces the pleural edema that is a classic manifestation of anthrax. All the histopathological changes previously observed in LT-treated mice can be explained as predictable sequelae of vascular insufficiency. In this report, electron microscopic analysis was used to identify rapid and striking pathological changes in endothelial cells, myocytes, mitochondria, and the sarcoplasmic reticulum cisternae in the hearts of LT-challenged mice. These morphological changes were paralleled by release of very high levels of multiple cardiac injury biomarkers. LT-induced pathological changes occurred earlier and with markedly higher severity in nNOS knockout mice compared to wild-type or iNOS or eNOS knockout mice, reflecting the well-established role of nNOS in maintenance of cardiac function, and suggesting that nitric oxide produced by nNOS counteracts the damage induced by LT.

Anthrax lethal factor (LF) is a Zn(+2)-dependent metalloprotease that cleaves several MAPK kinases and is responsible for the lethality of anthrax lethal toxin (LT). We observed that a recombinant LF (LF-HMA) which differs from wild type LF (LF-A) by the addition of two residues (His-Met) to the native Ala (A) terminus as a result of cloning manipulations has 3-fold lower potency toward cultured cells and experimental animals. We hypothesized that the "N-end rule", which relates the half-life of proteins in cells to the identity of their N-terminal residue, might be operative in the case of LF, so that the N-terminal residue of LF would determine the cytosolic stability and thereby the potency of LF. Mutational studies that replaced the native N-terminal residue of LF with known N-end rule stabilizing or destabilizing residues confirmed that the N-terminal residue plays a significant role in determining the potency of LT for cultured cells and experimental animals. The fact that a commercially-available LF preparation (LF-HMA) that is widely used in basic research studies and for evaluation of vaccines and therapeutics is 3-fold less potent than native LF (LF-A) should be considered when comparing published studies and in the design of future experiments.

Background Signals of danger and damage in the cytosol of cells are sensed by NOD-like receptors (NLRs), which are components of multiprotein complexes called inflammasomes. Inflammasomes activate caspase-1, resulting in IL-1-beta and IL-18 secretion and an inflammatory response. To date, the only known activator of rodent Nlrp1 is anthrax lethal toxin (LT), a protease secreted by the bacterial pathogen Bacillus anthracis. Although susceptibility of mouse macrophages to LT has been genetically linked to Nlrp1b, mice harbor two additional Nlrp1 paralogs in their genomes (Nlrp1a and Nlrp1c). However, little is known about their expression profile and sequence in different mouse strains. Furthermore, simultaneous expression of these paralogs may lead to competitional binding of Nlrp1b interaction partners needed for inflammasome activation, thus influencing macrophages susceptibility to LT. To more completely understand the role(s) of Nlrp1 paralogs in mice, we surveyed for their expression in a large set of LT-resistant and sensitive mouse macrophages. In addition, we provide sequence comparisons for Nlrp1a and report on previously unrecognized splice variants of Nlrp1b. Results Our results show that macrophages from some inbred mouse strains simultaneously express different splice variants of Nlrp1b. In contrast to the highly polymorphic Nlrp1b splice variants, sequencing of expressed Nlrp1a showed the protein to be highly conserved across all mouse strains. We found that Nlrp1a was expressed only in toxin-resistant macrophages, with the sole exception of expression in LT-sensitive CAST/EiJ macrophages. Conclusions Our data present a complex picture of Nlrp1 protein variations and provide a basis for elucidating their roles in murine macrophage function. Furthermore, the high conservation of Nlrp1a implies that it might be an important inflammasome sensor in mice.