Kenneth A. Bradley

Identification of the cellular receptor for anthrax toxin

The tripartite toxin secreted by Bacillus anthracis, the causative agent of anthrax, helps the bacterium evade the immune system and can kill the host during a systemic infection. Two components of the toxin enzymatically modify substrates within the cytosol of mammalian cells: oedema factor (OF) is an adenylate cyclase that impairs host defences through a variety of mechanisms including inhibiting phagocytosis; lethal factor (LF) is a zinc-dependent protease that cleaves mitogen-activated protein kinase kinase and causes lysis of macrophages. Protective antigen (PA), the third component, binds to a cellular receptor and mediates delivery of the enzymatic components to the cytosol. Here we describe the cloning of the human PA receptor using a genetic complementation approach. The receptor, termed ATR (anthrax toxin receptor), is a type I membrane protein with an extracellular von Willebrand factor A domain that binds directly to PA. In addition, a soluble version of this domain can protect cells from the action of the toxin.

Human capillary morphogenesis protein 2 functions as an anthrax toxin receptor

Bacillus anthracis secretes two bipartite toxins thought to be involved in anthrax pathogenesis and resulting death of the host. The current model for intoxication is that protective antigen (PA) toxin subunits bind a single group of cell-surface anthrax toxin receptors (ATRs), encoded by the tumor endothelial marker 8 (TEM8) gene. The ATR/TEM8-PA interaction is mediated by the receptors extracellular domain related to von Willebrand factor type A or integrin inserted domains (VWA/I domains). A metal ion-dependent adhesion site (MIDAS) located within this domain of the ATR/TEM8 protein chelates a divalent cation critical for PA binding. In this report, we identify a second PA receptor encoded by capillary morphogenesis gene 2 (CMG2), which has 60% amino acid identity to ATR/TEM8 within the VWA/I domain, as well as a conserved MIDAS motif. A recombinant CMG2 protein bound PA and mediated toxin internalization when expressed on receptor-deficient cells. Binding between the CMG2 VWA/I domain and PA was shown to be direct and metal-dependent, although the cation specificity of this interaction is different than that observed with ATR/TEM8. Northern blot analysis revealed that CMG2 is widely expressed in human tissues, indicating that this receptor is likely to be relevant for disease pathogenesis. Finally, a soluble version of the CMG2 VWA/I domain inhibited intoxication of cells expressing endogenous toxin receptors when it was added to PA at a 3:1 ratio. These studies distinguish CMG2 as a second anthrax toxin receptor and identify a potent antitoxin that may prove useful for the treatment of anthrax.

Use of a Rapid Cytotoxicity Screening Approach to Engineer a Safer Zinc Oxide Nanoparticle through Iron Doping

The establishment of verifiably safe nanotechnology requires the development of assessment tools to identify hazardous nanomaterial properties that could be modified to improve nanomaterial safety. While there is a lot of debate of what constitutes appropriate safety screening methods, one approach is to use the assessment of cellular injury pathways to collect knowledge about hazardous material properties that could lead to harm to humans and the environment. We demonstrate the use of a multiparameter cytotoxicity assay that evaluates toxic oxidative stress to compare the effects of titanium dioxide (TiO(2)), cerium oxide (CeO(2)), and zinc oxide (ZnO) nanoparticles in bronchial epithelial and macrophage cell lines. The nanoparticles were chosen on the basis of their volume of production and likelihood of spread to the environment. Among the materials, dissolution of ZnO nanoparticles and Zn(2+) release were capable of ROS generation and activation of an integrated cytotoxic pathway that includes intracellular calcium flux, mitochondrial depolarization, and plasma membrane leakage. These responses were chosen on the basis of the compatibility of the fluorescent dyes that contemporaneously assess their response characteristics by a semiautomated epifluorescence procedure. Purposeful reduction of ZnO cytotoxicity was achieved by iron doping, which changed the material matrix to slow Zn(2+) release. In summary, we demonstrate the utility of a rapid throughput, integrated biological oxidative stress response pathway to perform hazard ranking of a small batch of metal oxide nanoparticles, in addition to showing how this assay can be used to improve nanosafety by decreasing ZnO dissolution through Fe doping.

Use of a High Throughput Screening Approach Coupled With In Vivo Zebrafish Embryo Screening to Develop Hazard Ranking for Engineered Nanomaterials

Because of concerns about the safety of a growing number of engineered nanomaterials (ENM), it is necessary to develop high-throughput screening and in silico data transformation tools that can speed up in vitro hazard ranking. Here, we report the use of a multiparametric, automated screening assay that incorporates sublethal and lethal cellular injury responses to perform high-throughput analysis of a batch of commercial metal/metal oxide nanoparticles (NP) with the inclusion of a quantum dot (QD1). The responses chosen for tracking cellular injury through automated epifluorescence microscopy included ROS production, intracellular calcium flux, mitochondrial depolarization, and plasma membrane permeability. The z-score transformed high volume data set was used to construct heat maps for in vitro hazard ranking as well as showing the similarity patterns of NPs and response parameters through the use of self-organizing maps (SOM). Among the materials analyzed, QD1 and nano-ZnO showed the most prominent lethality, while Pt, Ag, SiO2, Al2O3, and Au triggered sublethal effects but without cytotoxicity. In order to compare the in vitro with the in vivo response outcomes in zebrafish embryos, NPs were used to assess their impact on mortality rate, hatching rate, cardiac rate, and morphological defects. While QDs, ZnO, and Ag induced morphological abnormalities or interfered in embryo hatching, Pt and Ag exerted inhibitory effects on cardiac rate. Ag toxicity in zebrafish differed from the in vitro results, which is congruent with this materials designation as extremely dangerous in the environment. Interestingly, while toxicity in the initially selected QD formulation was due to a solvent (toluene), supplementary testing of additional QDs selections yielded in vitro hazard profiling that reflect the release of chalcogenides. In conclusion, the use of a high-throughput screening, in silico data handling and zebrafish testing may constitute a paradigm for rapid and integrated ENM toxicological screening.

Cutting Edge: Resistance to Bacillus anthracis Infection Mediated by a Lethal Toxin Sensitive Allele of Nalp1b/Nlrp1b

Pathogenesis of Bacillus anthracis is associated with the production of lethal toxin (LT), which activates the murine Nalp1b/Nlrp1b inflammasome and induces caspase-1–dependent pyroptotic death in macrophages and dendritic cells. In this study, we investigated the effect of allelic variation of Nlrp1b on the outcome of LT challenge and infection by B. anthracis spores. Nlrp1b allelic variation did not alter the kinetics or pathology of end-stage disease induced by purified LT, suggesting that, in contrast to previous reports, macrophage lysis does not contribute directly to LT-mediated pathology. However, animals expressing a LT-sensitive allele of Nlrp1b showed an early inflammatory response to LT and increased resistance to infection by B. anthracis. Data presented here support a model whereby LT-mediated activation of Nlrp1b and subsequent lysis of macrophages is not a mechanism used by B. anthracis to promote virulence, but rather a protective host-mediated innate immune response.

Retrocyclins Kill Bacilli and Germinating Spores of Bacillus anthracis and Inactivate Anthrax Lethal Toxin

θ-defensins are cyclic octadecapeptides encoded by the modified α-defensin genes of certain nonhuman primates. The recent demonstration that human α-defensins could prevent deleterious effects of anthrax lethal toxin in vitro and in vivo led us to examine the effects of θ-defensins on Bacillus anthracis (Sterne). We tested rhesus θ-defensins 1-3, retrocyclins 1-3, and several analogues of RC-1. Low concentrations of θ-defensins not only killed vegetative cells of B. anthracis (Sterne) and rendered their germinating spores nonviable, they also inactivated the enzymatic activity of anthrax lethal factor and protected murine RAW-264.7 cells from lethal toxin, a mixture of lethal factor and protective antigen. Structure-function studies indicated that the cyclic backbone, intramolecular tri-disulfide ladder, and arginine residues of θ-defensins contributed substantially to these protective effects. Surface plasmon resonance studies showed that retrocyclins bound the lethal factor rapidly and with high affinity. Retrocyclin-mediated inhibition of the enzymatic activity of lethal factor increased substantially if the enzyme and peptide were preincubated before substrate was added. The temporal discrepancy between the rapidity of binding and the slowly progressive extent of lethal factor inhibition suggest that post-binding events, perhaps in situ oligomerization, contribute to the antitoxic properties of retrocyclins. Overall, these findings suggest that θ-defensins provide molecular templates that could be used to create novel agents effective against B. anthracis and its toxins.

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

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

Anthrax toxin receptor proteins

Anthrax toxin is a key virulence factor for Bacillus anthracis, the causative agent of anthrax. Here we discuss what is known about the anthrax toxin receptor (ATR), the cellular receptor for anthrax toxin, and how this information is being used to develop treatments for anthrax as well as to understand aspects of cancer. ATR was identified recently as a type I transmembrane protein with unknown function that contains an extracellular integrin-like inserted (I) domain. The ATR I domain contains the toxin binding site, and a soluble form of this domain was shown to serve as an effective antitoxin to protect cultured cells from toxin action. ATR is encoded by the tumor endothelial marker 8 (TEM8) gene, which is selectively up-regulated during blood vessel formation and in tumor vasculature, raising the possibility that this protein normally functions in angiogenesis. Therefore, identification of the cellular receptor for anthrax toxin has made possible new avenues of research in the areas of anthrax pathogenesis, antitoxin development, and cancer biology.

Self-Organizing Map Analysis of Toxicity-Related Cell Signaling Pathways for Metal and Metal Oxide Nanoparticles

The response of a murine macrophage cell line exposed to a library of seven metal and metal oxide nanoparticles was evaluated via High Throughput Screening (HTS) assay employing luciferase-reporters for ten independent toxicity-related signaling pathways. Similarities of toxicity response among the nanoparticles were identified via Self-Organizing Map (SOM) analysis. This analysis, applied to the HTS data, quantified the significance of the signaling pathway responses (SPRs) of the cell population exposed to nanomaterials relative to a population of untreated cells, using the Strictly Standardized Mean Difference (SSMD). Given the high dimensionality of the data and relatively small data set, the validity of the SOM clusters was established via a consensus clustering technique. Analysis of the SPR signatures revealed two cluster groups corresponding to (i) sublethal pro-inflammatory responses to Al2O3, Au, Ag, SiO2 nanoparticles possibly related to ROS generation, and (ii) lethal genotoxic responses due to exposure to ZnO and Pt nanoparticles at a concentration range of 25-100 μg/mL at 12 h exposure. In addition to identifying and visualizing clusters and quantifying similarity measures, the SOM approach can aid in developing predictive quantitative-structure relations; however, this would require significantly larger data sets generated from combinatorial libraries of engineered nanoparticles.

Ion channel and toxin measurement using a high throughput lipid membrane platform

Measurements of ion channels are important for scientific, sensing and pharmaceutical applications. Reconstitution of ion channels into lipid vesicles and planar lipid bilayers for measurement at the single molecule level is a laborious and slow process incompatible with the high throughput methods and equipment used for sensing and drug discovery. A recently published method of lipid bilayer formation mechanically combines lipid monolayers self-assembled at the interfaces of aqueous and apolar phases. We have expanded on this method by vertically orienting these phases and using gravity as the driving force to combine the monolayers. As this method only requires fluid dispensation, it is trivially integrated with high throughput automated liquid-handling robotics. In a proof-of-concept demonstration, we created over 2200 lipid bilayers in 3h. We show single molecule measurements of technologically and physiologically relevant ion channels incorporated into lipid bilayers formed with this method.