Lethal Toxin Cytotoxicity Research Articles

Role of a Small Molecule in the Modulation of Cell Death Signal Transduction Pathways.

Inflammasomes activate caspase-1 in response to molecular signals from pathogens and other dangerous stimuli as a part of the innate immune response. A previous study discovered a small-molecule, 4-fluoro- N'-[1-(2-pyridinyl)ethylidene]benzohydrazide, which we named DN1, that reduces the cytotoxicity of anthrax lethal toxin (LT). We determined that DN1 protected cells irrespectively of LT concentration and reduced the pathogenicity of an additional bacterial exotoxin and several viruses. Using the LT cytotoxicity pathway, we show that DN1 does not prevent LT internalization and catalytic activity or caspase-1 activation. Moreover, DN1 does not affect the proteolytic activity of host cathepsin B, which facilitates the cytoplasmic entry of toxins. PubChem Bioactivities lists two G protein-coupled receptors (GPCR), type-1 angiotensin II receptor and apelin receptor, as targets of DN1. The inhibition of phosphatidylinositol 3-kinase, phospholipase C, and protein kinase B, which are downstream of GPCR signaling, synergized with DN1 in protecting cells from LT. We hypothesize that DN1-mediated antagonism of GPCRs modulates signal transduction pathways to induce a cellular state that reduces LT-induced pyroptosis downstream of caspase-1 activation. DN1 also reduced the susceptibility of Drosophila melanogaster to toxin-associated bacterial infections. Future experiments will aim to further characterize how DN1 modulates signal transduction pathways to inhibit pyroptotic cell death in LT-sensitive macrophages. DN1 represents a novel chemical probe to investigate host cellular mechanisms that mediate cell death in response to pathogenic agents.

Serological Correlate of Protection in Guinea Pigs for a Recombinant Protective Antigen Anthrax Vaccine Produced from Bacillus brevis

ObjectiveRecombinant protective antigen (rPA) is the active pharmaceutical ingredient of a second generation anthrax vaccine undergoing clinical trials both in Korea and the USA. By using the rPA produced from Bacillus brevis pNU212 expression system, correlations of serological immune response to anthrax protection efficacy were analyzed in a guinea pig model. MethodsSerological responses of rPA anthrax vaccine were investigated in guinea pigs that were given single or two injections (interval of 4 weeks) of various amounts of rPA combined with aluminum hydroxide adjuvant. Guinea pigs were subsequently challenged by the intramuscular injection with 30 half-lethal doses (30LD50) of virulent Bacillus anthracis spores. Serum antibody titers were determined by anti-PA IgG ELISA and the ability of antibodies to neutralize the cytotoxicity of lethal toxin on J774A.1 cell was measured through the toxin neutralizing antibody (TNA) assay. ResultsTo examine correlations between survival rate and antibody titers, correlation between neutralizing antibody titers and the extent of protection was determined. Toxin neutralization titers of at least 1176 were sufficient to confer protection against a dose of 30LD50 of virulent anthrax spores of the H9401 strain. Such consistency in the correlation was not observed from those antibody titers determined by ELISA. ConclusionNeutralizing-antibody titers can be used as a surrogate marker.

The Poly-γ- d -Glutamic Acid Capsule of Bacillus anthracis Enhances Lethal Toxin Activity

The poly-γ-D-glutamic acid (PGA) capsule is one of the major virulence factors of Bacillus anthracis, which causes a highly lethal infectious disease. The PGA capsule disguises B. anthracis from immune surveillance and allows its unimpeded growth in the host. The PGA capsule recently was reported to be associated with lethal toxin (LT) in the blood of experimentally infected animals (M. H. Cho, et al., Infect. Immun. 78:387-392, 2010). The effect of PGA, either alone or in combination with LT, on macrophages, which play an important role in the progression of anthrax disease, has not been thoroughly investigated. In this study, we investigated the effect of PGA on LT cytotoxicity using the mouse macrophage cell line J774A.1. PGA produced a concentration-dependent enhancement of the cytotoxicity of LT on J774A.1 cells through an enhancement in the binding and accumulation of protective antigen to its receptors. The increase of LT activity was confirmed using Western blot analysis, which showed that the combination of PGA and LT produced a greater degree of degradation of mitogen-activated protein kinase kinases and an increased level of the activation of the proform of caspase-1 to its processed form compared to the effects of LT alone. In addition, mice that received a tail vein injection of both PGA and LT had a significantly increased rate of death compared to that of mice injected with LT alone. PGA had no effect when added to cultures or administered to mice in the absence of LT. These results emphasize the importance of PGA in the pathogenesis of anthrax infection.

Role of the N-Terminal Amino Acid of Bacillus anthracis Lethal Factor in Lethal Toxin Cytotoxicity and Its Effect on the Lethal Toxin Neutralization Assay

The cytotoxic activity of lethal factor (LF), a critical reagent used in the cell-based lethal toxin neutralization assay to assess anthrax vaccines, was shown to depend on the identity of its N-terminal amino acid, which plays a role in the targeting of LF to the proteasome for degradation. These results demonstrate that care must be taken to ensure that LF preparations used in standardized cell-based assays are not altered at their N-terminal ends.

Chemical Genetic Screening Identifies Critical Pathways in Anthrax Lethal Toxin-Induced Pathogenesis

Anthrax lethal toxin (LT)-induced cell death via mitogen-activated protein kinase kinase (MAPKK) cleavage remains questionable. Here, a chemical genetics approach was used to investigate what pathways mediate LT-induced cell death. Several small molecules were found to protect macrophages from anthrax LT cytotoxicity and MAPKK from cleavage by lethal factor (LF), without inhibiting LF enzymatic activity or cellular proteasome activity. Interestingly, the compounds activated MAPK-signaling molecules, induced proinflammatory cytokine production, and inhibited LT-induced macrophage apoptosis in a concentration-dependent manner. We propose that induction of antiapoptotic responses by MAPK-dependent or -independent pathways and activation of host innate responses may protect macrophages from anthrax LT-induced cell death. Altering host responses through a chemical genetics approach can help identify critical cellular pathways involved in the pathogenesis of anthrax and can be exploited to further explore host-pathogen interactions.

Selection of human antibodies against cell surface-associated oligomeric anthrax protective antigen

The protective antigen (PA83) of Bacillus anthracis is the dominant antigen in natural and vaccine-induced immunity to anthrax infection. Three human single-chain variable fragments (scFvs) against cell bound PA were isolated from an antibody phage display library. Specifically, the antibodies were evaluated for their ability to bind to cell bound heptameric PA and ultimately protect against the cytotoxicity of lethal toxin. In total, all three scFvs possessed neutralizing activity against the cytotoxic effects of lethal toxin in a macrophage lysis assay. The Kd values of the Fabs were determined, interestingly their protective effects did not parallel their affinities; hence, a simple binding argument alone to PA63 cannot be used as the distinguishing feature for the prediction of their neutralization abilities. Immunofluorescent microscopy experiments were conducted and provided strong evidence for Fab binding to oligomeric PA on the cell surface and thus a plausible mechanism for the toxin neutralization activity that was observed. The results of this study presented herein suggest that our antibodies compete with LF–PA cell surface interactions, and thus may provide potential application of human antibodies as passive immunization prophylactics in cases of B. anthracis exposure and infection.

Search for Cyclodextrin-Based Inhibitors of Anthrax Toxins: Synthesis, Structural Features, and Relative Activities

Recently, using structure-inspired drug design, we demonstrated that aminoalkyl derivatives of beta-cyclodextrin inhibited anthrax lethal toxin action by blocking the transmembrane pore formed by the protective antigen (PA) subunit of the toxin. In the present study, we evaluate a series of new beta-cyclodextrin derivatives with the goal of identifying potent inhibitors of anthrax toxins. Newly synthesized hepta-6-thioaminoalkyl and hepta-6-thioguanidinoalkyl derivatives of beta-cyclodextrin with alkyl spacers of various lengths were tested for the ability to inhibit cytotoxicity of lethal toxin in cells as well as to block ion conductance through PA channels reconstituted in planar bilayer lipid membranes. Most of the tested derivatives were protective against anthrax lethal toxin action at low or submicromolar concentrations. They also blocked ion conductance through PA channels at concentrations as low as 0.1 nM. The activities of the derivatives in both cell protection and channel blocking were found to depend on the length and chemical nature of the substituent groups. One of the compounds was also shown to block the edema toxin activity. It is hoped that these results will help to identify a new class of drugs for anthrax treatment, i.e., drugs that block the pathway for toxin translocation into the cytosol, the PA channel.

Efficient neutralization of anthrax toxin by chimpanzee monoclonal antibodies against protective antigen.

Four single-chain variable fragments (scFvs) against protective antigen (PA) and 2 scFvs against lethal factor (LF) of anthrax were isolated from a phage display library generated from immunized chimpanzees. Only 2 scFvs recognizing PA (W1 and W2) neutralized the cytotoxicity of lethal toxin in a macrophage lysis assay. Full-length immunoglobulin G (IgG) of W1 and W2 efficiently protected rats from anthrax toxin challenge. The epitope recognized by W1 and W2 was conformational and was formed by C-terminal amino acids 614–735 of PA. W1 and W2 each bound to PA with an equilibrium dissociation constant of 4×10-11 mol/L to 5 × 10−11 mol/L, which is an affinity that is 20–100-fold higher than that for the interaction of the receptor and PA. W1 and W2 inhibited the binding of PA to the receptor, suggesting that this was the mechanism of protection. These data suggest that W1 and W2 chimpanzee monoclonal antibodies may serve as PA entry inhibitors for use in the emergency prophylaxis against and treatment of anthrax

BRAF status and mitogen-activated protein/extracellular signal-regulated kinase kinase 1/2 activity indicate sensitivity of melanoma cells to anthrax lethal toxin

Anthrax lethal toxin, composed of protective antigen and lethal factor, was tested for cytotoxicity to human melanoma cell lines and normal human cells. Eleven of 18 melanoma cell lines were sensitive to anthrax lethal toxin (IC(50) < 400 pmol/L) and 10 of these 11 sensitive cell lines carried the V599E BRAF mutation. Most normal cell types (10 of 15) were not sensitive to anthrax lethal toxin and only 5 of 15 normal human cell types were sensitive to anthrax lethal toxin (IC(50) < 400 pmol/L). These cells included monocytes and a subset of endothelial cells. In both melanoma cell lines and normal cells, anthrax toxin receptor expression levels did not correlate with anthrax lethal toxin cytotoxicity. Furthermore, an anthrax toxin receptor-deficient cell line (PR230) did not show any enhanced sensitivity to anthrax lethal toxin when transfected with anthrax toxin receptor. Anthrax lethal toxin toxicity correlated with elevated phosphorylation levels of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) 1/2 in both melanoma cell lines and normal cells. Anthrax lethal toxin-sensitive melanoma cell lines and normal cells had higher phospho-MEK1/2 levels than anthrax lethal toxin-resistant melanoma cell lines and normal tissue types. U0126, a specific MEK1/2 inhibitor, was not toxic to anthrax lethal toxin-resistant melanoma cell lines but was toxic to 8 of 11 anthrax lethal toxin-sensitive cell lines. These results show that anthrax lethal toxin toxicity correlates with elevated levels of active MEK1/2 pathway but not with anthrax toxin receptor expression levels in both normal and malignant tissues. Anthrax lethal toxin may be a useful therapeutic for melanoma patients, especially those carrying the V599E BRAF mutation with constitutive activation of the mitogen-activated protein kinase pathway.

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.

Validation of the anthrax lethal toxin neutralization assay

A validation of the performance characteristics of a toxin neutralization assay is presented. This in vitro assay measures the functional ability of antisera, containing antibodies to anthrax lethal toxin, to specifically protect J774A.1 cells against Bacillus anthracis lethal toxin cytotoxicity. This colormetric assay is based upon the reduction of MTT by living cells. Human and rabbit antisera produced against anthrax vaccine absorbed (AVA) were used to validate the assay. Results showed a high level of repeatability and reproducibility, particularly for a bio-assay. Inter-assay variability in absorbance values was the most prominent negative finding however, an acceptable level was demonstrated with a ratio [neutralization ratio (NR)] of the test serum 50% effective dose (ED 50) to the reference standard ED 50. Accuracy was maintained, even in samples with minimal neutralizing capacity, and linearity was noted when sample dilutions resulted in accurate prediction of the Y maxand Y min. Specificity tests demonstrated that normal sera did not have an observable effect on the ability of the reference standard to neutralize toxin. The assay remained stable against time, temperature, and freeze/thaw effects on the reference standards, but not on the toxin. The assay also remained stable against media and solution storage effects. Cell passage number and cell plating density were two critical parameters identified during the robustness studies that may be responsible for inter-assay variability in absorbance values. The work was performed in accordance with the FDA's Bioanalytical Method Validation Guidance for Industry and the FDA's Good Laboratory Practice for Nonclinical Laboratory Studies (21 CFR Part 58).

Activation of Phospholipase C and Protein Kinase C is Required for Expression of Anthrax Lethal Toxin Cytotoxicity in J774A.1 Cells

Anthrax lethal toxin (LT) comprises two proteins: the protective antigen (PA) and the lethal factor (LF). The LT is cytotoxic to macrophage-like cell line J774A.1. Pre-treatment of these cells with neomycin, a phospholipase C inhibitor, protected them against anthrax LT cytotoxicity. Protection obtained with neomycin indicated that LT stimulates phospholipase C in these cells. It was found that levels of inositol 1,4,5-triphosphate (IP 3) dramatically increased in toxin-treated cells. The rise in IP 3 levels was proportional to the dose of LF that was allowed to bind to receptor-bound PA. By using protein kinase C (PKC) inhibitors, we found that the activation of PKC is required for mediating anthrax LT cytotoxicity. Activation of phospholipase C or PKC is not required for the binding of PA to the cell surface receptors or for the uptake or internalisation of the toxin. In this study, we demonstrate that the IP 3 signalling cascade is initiated by anthrax lethal toxin in J774A.1 cells. The second messengers generated during the cascade aid LF in mediating lethality only after its translocation into the cytosol.

Comparative efficacy of experimental anthrax vaccine candidates against inhalation anthrax in rhesus macaques

The authors examined the efficacy of Bacillus anthracis protective antigen (PA) combined with adjuvants as vaccines against an aerosol challenge of virulent anthrax spores in rhesus macaques. Adjuvants tested included i) aluminum hydroxide (Alhydrogel), ii) saponin QS-21 and iii) monophosphoryl lipid A (MPL) in squalene/ lecithin/Tween 80 emulsion (SLT). Animals were immunized once with either 50 μg of recombinant PA plus adjuvant, or with Anthrax Vaccine Adsorbed (AVA), the licensed human anthrax vaccine. The serological response to PA was measured by enzymelinked immunosorbent assay. Lymphocyte proliferation and serum neutralization of in vitro lethal toxin cytotoxicity were also assayed. In all vaccine groups, anti-PA IgM and IgG titers peaked at 2 weeks and 4–5 weeks postimmunization, respectively. Five weeks postimmunization, animals in all vaccine groups demonstrated PA-specific lymphocyte proliferation and sera that neutralized in vitro cytotoxicity. Six weeks after immunization, the animals were challenged by aerosol with approximately 93 LD 50 of virulent anthrax spores. Animals were bled daily for 1 week to monitor bacteremia, and deaths were recorded. Anti-PA ELISA titers in all groups of immunized animals were substantially increased 2 weeks after challenge. One dose of each vaccine provided significant protection ( > 90%) against inhalation anthrax in the rhesus macaques.

Protein synthesis is required for expression of anthrax lethal toxin cytotoxicity.

Anthrax lethal toxin, which is composed of two proteins, i.e., protective antigen and lethal factor, is cytolytic to mouse peritoneal macrophages and the macrophage-like cell line J774A.1. After exposure of cells to lethal toxin, inhibition of protein synthesis occurred only slightly before the onset of cytolysis. Thus, cell death did not appear to be due to inhibition of protein synthesis. However, prior treatment of J774A.1 cells with cycloheximide or puromycin, which inhibited protein synthesis, protected them completely against lethal toxin-induced cytolysis, which suggested that continuous protein synthesis is required for the expression of lethal toxin activity. Inhibition of protein synthesis had no appreciable effect on the binding of protective antigen to the cell surface receptor or on proteolytic cleavage of surface-bound protective antigen. Furthermore, inhibition of protein synthesis did not alter the uptake of toxin, which suggested that protein synthesis is required at a later stage of the intoxication process. The protection provided by inhibition of protein synthesis was effective, even up to 1 h after exposure to anthrax lethal toxin. The increased uptake of calcium observed in cells exposed to lethal toxin did not occur when they were protected by blocking protein synthesis. Identifying the protein(s) synthesized during the intoxication process may help to understand the mechanism of cell death produced by anthrax lethal toxin.