Anthrax Protective Antigen Research Articles

Humoral responses to independent vaccinations are correlated in healthy boosted adults

BackgroundRoughly half of U.S. adults do not receive recommended booster vaccinations, but protective antibody levels are rarely measured in adults. Demographic factors, vaccination history, and responses to other vaccinations could help identify at-risk individuals. We sought to characterize rates of seroconversion and determine associations of humoral responses to multiple vaccinations in healthy adults. MethodsHumoral responses toward measles, mumps, tetanus toxoid, pertussis, hepatitis B surface antigen, and anthrax protective antigen were measured by ELISA in post-immunization samples from 1465 healthy U.S. military members. We examined the effects of demographic and clinical factors on immunization responses, as well as assessed correlations between vaccination responses. ResultsSubsets of boosted adults did not have seroprotective levels of antibodies toward measles (10.4%), mumps (9.4%), pertussis (4.7%), hepatitis B (8.6%) or protective antigen (14.4%) detected. Half-lives of antibody responses were generally long (>30 years). Measles and mumps antibody levels were correlated (r=0.31, p<0.001), but not associated with select demographic features or vaccination history. Measles and mumps antibody levels also correlated with tetanus antibody response (r=0.11, p<0.001). ConclusionsVaccination responses are predominantly robust and vaccine specific. However, a small but significant portion of the vaccinated adult population may not have quantitative seroprotective antibody to common vaccine-preventable infections.

Potentiation of anthrax vaccines using protective antigen-expressing viral replicon vectors

DNA vaccines require improvement for human use because they are generally weak stimulators of the immune system in humans. The efficacy of DNA vaccines can be improved using a viral replicon as vector to administer antigen of pathogen. In this study, we comprehensively evaluated the conventional non-viral DNA, viral replicon DNA or viral replicon particles (VRP) vaccines encoding different forms of anthrax protective antigen (PA) for specific immunity and protective potency against anthrax. Our current results clearly suggested that these viral replicon DNA or VRP vaccines derived from Semliki Forest virus (SFV) induced stronger PA-specific immune responses than the conventional non-viral DNA vaccines when encoding the same antigen forms, which resulted in potent protection against challenge with the Bacillus anthracis strain A16R. Additionally, the naked PA-expressing SFV replicon DNA or VRP vaccines without the need for high doses or demanding particular delivery regimens elicited robust immune responses and afforded completely protective potencies, which indicated the potential of the SFV replicon as vector of anthrax vaccines for use in clinical application. Therefore, our results suggest that these PA-expressing SFV replicon DNA or VRP vaccines may be suitable as candidate vaccines against anthrax.

Detection of anthrax protective antigen (PA) using europium labeled anti-PA monoclonal antibody and time-resolved fluorescence

Inhalation anthrax is a rare but acute infectious disease following adsorption of Bacillus anthracis spores through the lungs. The disease has a high fatality rate if untreated, but early and correct diagnosis has a significant impact on case patient recovery. The early symptoms of inhalation anthrax are, however, non-specific and current anthrax diagnostics are primarily dependent upon culture and confirmatory real-time PCR. Consequently, there may be a significant delay in diagnosis and targeted treatment. Rapid, culture-independent diagnostic tests are therefore needed, particularly in the context of a large scale emergency response.The aim of this study was to evaluate the ability of monoclonal antibodies to detect anthrax toxin proteins that are secreted early in the course of B. anthracis infection using a time-resolved fluorescence (TRF) immunoassay. We selected monoclonal antibodies that could detect protective antigen (PA), as PA83 and also PA63 and LF in the lethal toxin complex. The assay reliable detection limit (RDL) was 6.63×10−6μM (0.551ng/ml) for PA83 and 2.51×10−5μM (1.58ng/ml) for PA63. Despite variable precision and accuracy of the assay, PA was detected in 9 out of 10 sera samples from anthrax confirmed case patients with cutaneous (n=7), inhalation (n=2), and gastrointestinal (n=1) disease. Anthrax Immune Globulin (AIG), which has been used in treatment of clinical anthrax, interfered with detection of PA. This study demonstrates a culture-independent method of diagnosing anthrax through the use of monoclonal antibodies to detect PA and LF in the lethal toxin complex.

19F Nuclear Magnetic Resonance and Crystallographic Studies of 5-Fluorotryptophan-Labeled Anthrax Protective Antigen and Effects of the Receptor on Stability

The anthrax protective antigen (PA) is an 83 kDa protein that is one of three protein components of the anthrax toxin, an AB toxin secreted by Bacillus anthracis. PA is capable of undergoing several structural changes, including oligomerization to either a heptameric or octameric structure called the prepore, and at acidic pH a major conformational change to form a membrane-spanning pore. To follow these structural changes at a residue-specific level, we have conducted initial studies in which we have biosynthetically incorporated 5-fluorotryptophan (5-FTrp) into PA, and we have studied the influence of 5-FTrp labeling on the structural stability of PA and on binding to the host receptor capillary morphogenesis protein 2 (CMG2) using 19F nuclear magnetic resonance (NMR). There are seven tryptophans in PA, but of the four domains in PA, only two contain tryptophans: domain 1 (Trp65, -90, -136, -206, and -226) and domain 2 (Trp346 and -477). Trp346 is of particular interest because of its proximity to the CMG2 binding interface, and because it forms part of the membrane-spanning pore. We show that the 19F resonance of Trp346 is sensitive to changes in pH, consistent with crystallographic studies, and that receptor binding significantly stabilizes Trp346 to both pH and temperature. In addition, we provide evidence that suggests that resonances from tryptophans distant from the binding interface are also stabilized by the receptor. Our studies highlight the positive impact of receptor binding on protein stability and the use of 19F NMR in gaining insight into structural changes in a high-molecular weight protein.

In silico prediction of interaction between flavonoids of propolis of honey bee and anthrax protective antigen

In silico prediction of interaction between flavonoids of propolis of honey bee and anthrax protective antigen

Surface plasmon resonance measurements of plasma antibody avidity during primary and secondary responses to anthrax protective antigen

Establishment of humoral immunity against pathogens is dependent on events that occur in the germinal center and the subsequent induction of high-affinity neutralizing antibodies. Quantitative assays that allow monitoring of affinity maturation and duration of antibody responses can provide useful information regarding the efficacy of vaccines and adjuvants. Using an anthrax protective antigen (rPA) and alum model antigen/adjuvant system, we describe a methodology for monitoring antigen-specific serum antibody concentration and avidity by surface plasmon resonance during primary and secondary immune responses. Our analyses showed that following a priming dose in mice, rPA-specific antibody concentration and avidity increases over time and reaches a maximal response in about six weeks, but gradually declines in the absence of antigenic boost. Germinal center reactions were observed early with maximal development achieved during the primary response, which coincided with peak antibody avidity responses to primary immunization. Boosting with antigen resulted in a rapid increase in rPA-specific antibody concentration and five-fold increase in avidity, which was not dependent on sustained GC development. The described methodology couples surface plasmon resonance-based plasma avidity measurements with germinal center analysis and provides a novel way to monitor humoral responses that can play a role in facilitating vaccine and adjuvant development.

Substrate Cleavage Profiling Suggests a Distinct Function of Bacteroides fragilis Metalloproteinases (Fragilysin and Metalloproteinase II) at the Microbiome-Inflammation-Cancer Interface

Enterotoxigenic anaerobic Bacteroides fragilis is a significant source of inflammatory diarrheal disease and a risk factor for colorectal cancer. Two distinct metalloproteinase types (the homologous 1, 2, and 3 isoforms of fragilysin (FRA1, FRA2, and FRA3, respectively) and metalloproteinase II (MPII)) are encoded by the B. fragilis pathogenicity island. FRA was demonstrated to be important to pathogenesis, whereas MPII, also a potential virulence protein, remained completely uncharacterized. Here, we, for the first time, extensively characterized MPII in comparison with FRA3, a representative of the FRA isoforms. We employed a series of multiplexed peptide cleavage assays to determine substrate specificity and proteolytic characteristics of MPII and FRA. These results enabled implementation of an efficient assay of MPII activity using a fluorescence-quenched peptide and contributed to structural evidence for the distinct substrate cleavage preferences of MPII and FRA. Our data imply that MPII specificity mimics the dibasic Arg↓Arg cleavage motif of furin-like proprotein convertases, whereas the cleavage motif of FRA (Pro-X-X-Leu-(Arg/Ala/Leu)↓) resembles that of human matrix metalloproteinases. To the best of our knowledge, MPII is the first zinc metalloproteinase with the dibasic cleavage preferences, suggesting a high level of versatility of metalloproteinase proteolysis. Based on these data, we now suggest that the combined (rather than individual) activity of MPII and FRA is required for the overall B. fragilis virulence in vivo.

In vitro and in vivo activities of recombinant anthrax protective antigen co-expressed with thioredoxin in Escherichia coli

Because of the central role it plays in the formation of lethal toxin and edema toxin, protective antigen (PA) is the principal target for the development of vaccines against anthrax. In the present study, we explored and compared the in vitro and in vivo activities of recombinant anthrax protective antigen (rPA) and receptor binding domain of protective antigen (PA4). As a result, the fully soluble rPA and PA4 proteins were successfully expressed in Escherichia coli by co-expression with thioredoxin (Trx), and the rPA was active in forming cytotoxic lethal toxins, indicating that the rPA protein retains a functionally biological activity. Furthermore, immunization with rPA protein induced stronger PA-specific immune responses in mice than PA4 protein. The protection elicited by immunization with PA4 suggests the presence of common neutralizing epitopes between rPA and PA4, but the immunization with rPA protein induced stronger neutralizing antibodies and protective levels against challenge with the B. anthracis strain A16R than the PA4 protein. The sera neutralizing antibodies titers correlated well with anti-PA group ELISA antibodies titers and the in vivo protective potency. Based on the results of cell cytotoxicity assays and the observed immune responses and protective potency, we concluded that the soluble rPA protein retains the in vitro and in vivo functionally biological activity and can be developed into a highly effective human subunit vaccine candidate against anthrax.

A Chemically Synthesized Capture Agent Enables the Selective, Sensitive, and Robust Electrochemical Detection of Anthrax Protective Antigen

We report on a robust and sensitive approach for detecting protective antigen (PA) exotoxin from Bacillus anthracis in complex media. A peptide-based capture agent against PA was developed by improving a bacteria display-developed peptide into a highly selective biligand through in situ click screening against a large, chemically synthesized peptide library. This biligand was coupled with an electrochemical enzyme-linked immunosorbent assay utilizing nanostructured gold electrodes. The resultant assay yielded a limit of detection of PA of 170 pg/mL (2.1 pM) in buffer, with minimal sensitivity reduction in 1% serum. The powdered capture agent could be stably stored for several days at 65 °C, and the full electrochemical biosensor showed no loss of performance after extended storage at 40 °C. The engineered stability and specificity of this assay should be extendable to other cases in which biomolecular detection in demanding environments is required.

The Use of Serological Tests (I.F.A.T. and iELISA) for Evaluation of Specific Antibodies Level in Vaccinated and Nonvaccinated Animals Against Anthrax

Various diseases, especially infectious ones, have caused untold harm to humanity in centuries. Among these, special attention should be paid to infectious diseases, especially anthrax, which reduces a lot the number of heads and livestock products. One of the most important tools to combat this infection are vaccinations. Today there are different kinds of vaccines against anthrax. The aim of this study was the use of IFA tests and iELISA to determine the efficacy of vaccines against anthrax. For the implementation of the IFA test bacterial emulsions prepared with bacterial strains of B. anthracis Stern 34F2 and Pasteur were used as antigens. As for determining the level of antibodies in the serum by iELISA was used Anthrax Protective antigen IgG ELISA kit. Sera of sheep and goat vaccinated with capsular and noncapsular vaccines were analyzed with both methods 21 days, 9 months and 12 months after vaccination. The results obtained show that sheep respond better to the vaccine than goat. The index of IgG anti-PA antibodies between vaccines, determined by ELISA, varies from 3.3 to 1.6 and 3.85 to 1.94, respectively, in the sera of animals vaccinated with noncapsular and capsular vaccines 21 days and 12 months after vaccination. For evaluation of vaccine immunogenicity, techniques of IgG anti-PA ELISA and indirect immunofluorescence (IFAT) can be used as alternative methods of control by the State Laboratory of Control of Veterinary Bioproducts. DOI: 10.5901/ajis.2013.v2n9p642

Receptor-Directed Chimeric Toxins Created by Sortase-Mediated Protein Fusion

Chimeric protein toxins that act selectively on cells expressing a designated receptor may serve as investigational probes and/or antitumor agents. Here, we report use of the enzyme sortase A (SrtA) to create four chimeric toxins designed to selectively kill cells bearing the tumor marker HER2. We first expressed and purified: (i) a receptor recognition-deficient form of diphtheria toxin that lacks its receptor-binding domain and (ii) a mutated, receptor-binding-deficient form of anthrax-protective antigen. Both proteins carried at the C terminus the sortase recognition sequence LPETGG and a H₆ affinity tag. Each toxin protein was mixed with SrtA plus either of two HER2-recognition proteins--a single-chain antibody fragment or an Affibody--both carrying an N-terminal G₅ tag. With wild-type SrtA, the fusion reaction between the toxin and receptor-recognition proteins approached completion only after several hours, whereas with an evolved form of the enzyme, SrtA*, the reaction was virtually complete within 5 minutes. The four fusion toxins were purified and shown to kill HER2-positive cells in culture with high specificity. Sortase-mediated ligation of binary combinations of diverse natively folded proteins offers a facile way to produce large sets of chimeric proteins for research and medicine.

Monitoring the Kinetics of the pH-Driven Transition of the Anthrax Toxin Prepore to the Pore by Biolayer Interferometry and Surface Plasmon Resonance

Domain 2 of the anthrax protective antigen (PA) prepore heptamer unfolds and refolds during endosome acidification to generate an extended 100 Å β barrel pore that inserts into the endosomal membrane. The PA pore facilitates the pH-dependent unfolding and translocation of bound toxin enzymic components, lethal factor (LF) and/or edema factor, from the endosome to the cytoplasm. We constructed immobilized complexes of the prepore with the PA-binding domain of LF (LFN) to monitor the real-time prepore to pore kinetic transition using surface plasmon resonance and biolayer interferometry (BLI). The kinetics of this transition increased as the solution pH was decreased from 7.5 to 5.0, mirroring acidification of the endosome. Once it had undergone the transition, the LFN-PA pore complex was removed from the BLI biosensor tip and deposited onto electron microscopy grids, where PA pore formation was confirmed by negative stain electron microscopy. When the soluble receptor domain (ANTRX2/CMG2) binds the immobilized PA prepore, the transition to the pore state was observed only after the pH was lowered to early (pH 5.5) or late (pH 5.0) endosomal pH conditions. Once the pore formed, the soluble receptor readily dissociated from the PA pore. Separate binding experiments with immobilized PA pores and the soluble receptor indicate that the receptor has a weakened propensity to bind to the transitioned pore. This immobilized anthrax toxin platform can be used to identify or validate potential antimicrobial lead compounds capable of regulating and/or inhibiting anthrax toxin complex formation or pore transitions.

Engineering Biomaterial-Associated Complement Activation to Improve Vaccine Efficacy

The complement system plays an important role in innate and adaptive immunity, which suggests that complement activation could be exploited as a potential strategy for vaccine adjuvants. Here we explored the potential of chitosan-based microparticles (CS-NH2 MPs) as a vaccine adjuvant with an active surface for complement activation due to the abundance of amino groups. In vaccination studies, using recombinant anthrax protective antigen as a model antigen, compared with the control microparticles (amino-cross-linked MPs), we found that microparticles (MPs) with abundant amino groups significantly enhanced higher antigen-specific IgG titers in vivo and enhanced the production of IL-4 and IFN-γ with ex vivo restimulation. Furthermore, proliferative responses of splenocytes to ex vivo antigen restimulation were enhanced following immunization with MPs with amino groups. Overall, these results indicated that CS-NH2 MPs with a high surface density of amino groups were favorable for complement activation and immune responses. Our data provide further design principles for studies on complement-activating MPs as a vaccine platform.

CCT chaperonin complex is required for efficient delivery of anthrax toxin into the cytosol of host cells

Bacterial toxins have evolved successful strategies for coopting host proteins to access the cytosol of host cells. Anthrax lethal factor (LF) enters the cytosol through pores in the endosomal membrane formed by anthrax protective antigen. Although in vitro models using planar lipid bilayers have shown that translocation can occur in the absence of cellular factors, recent studies using intact endosomes indicate that host factors are required for translocation in the cellular environment. In this study, we describe a high-throughput shRNA screen to identify host factors required for anthrax lethal toxin-induced cell death. The cytosolic chaperonin complex chaperonin containing t-complex protein 1 (CCT) was identified, and subsequent studies showed that CCT is required for efficient delivery of LF and related fusion proteins into the cytosol. We further show that knockdown of CCT inhibits the acid-induced delivery of LF and the fusion protein LFN-Bla (N terminal domain of LF fused to β-lactamase) across the plasma membrane of intact cells. Together, these results suggest that CCT is required for efficient delivery of enzymatically active toxin to the cytosol and are consistent with a direct role for CCT in translocation of LF through the protective antigen pore.

Structure-based non-canonical amino acid design to covalently crosslink an antibody–antigen complex

Engineering antibodies to utilize non-canonical amino acids (NCAA) should greatly expand the utility of an already important biological reagent. In particular, introducing crosslinking reagents into antibody complementarity determining regions (CDRs) should provide a means to covalently crosslink residues at the antibody-antigen interface. Unfortunately, finding the optimum position for crosslinking two proteins is often a matter of iterative guessing, even when the interface is known in atomic detail. Computer-aided antibody design can potentially greatly restrict the number of variants that must be explored in order to identify successful crosslinking sites. We have therefore used Rosetta to guide the introduction of an oxidizable crosslinking NCAA, l-3,4-dihydroxyphenylalanine (l-DOPA), into the CDRs of the anti-protective antigen scFv antibody M18, and have measured crosslinking to its cognate antigen, domain 4 of the anthrax protective antigen. Computed crosslinking distance, solvent accessibility, and interface energetics were three factors considered that could impact the efficiency of l-DOPA-mediated crosslinking. In the end, 10 variants were synthesized, and crosslinking efficiencies were generally 10% or higher, with the best variant crosslinking to 52% of the available antigen. The results suggest that computational analysis can be used in a pipeline for engineering crosslinking antibodies. The rules learned from l-DOPA crosslinking of antibodies may also be generalizable to the formation of other crosslinked interfaces and complexes.

Abstract 5076: Identification and anti-angiogenic effects of small molecule inhibitors of TEM8.

Abstract Tumor endothelial marker 8 (TEM8) is a gene upregulated on colonic tumor endothelium compared to quiescent normal endothelium. Inhibition of TEM8 using antibodies potently inhibits the growth of a variety of tumor types in mice. This inhibition of tumor growth is accompanied by a reduction in microvessel density within the tumors. Thus, TEM8 appears to represent a tumor specific endothelial marker that has a functional role in tumor angiogenesis. TEM8 is also a receptor for anthrax toxin. In the current study, we used chemical libraries to identify small molecule inhibitors of TEM8 that may have efficacy as inhibitors of tumor angiogenesis and anthrax toxin. We conducted a fluorescence resonance energy transfer (FRET) based screen of over 270,000 small molecules in order to identify molecules that inhibit the interaction of TEM8 with its well-known ligand anthrax protective antigen (PA). We describe here the identification and anti-angiogenic effects of small molecules that bind to TEM8. From the initial screen 280 molecules inhibited TEM8/PA interaction by greater than 50%. A secondary screen involved the elucidation of IC50 values for these 280 compounds as inhibitors of the TEM8/PA interaction. Twelve compounds had measurable IC50s in the micromolar range. The anti-angiogenic effects of these compounds were studied in vitro. Of the 12 compounds tested in a human microvascular endothelial cell (HMVEC) proliferation assay, 3 compounds significantly inhibited endothelial cell proliferation after 24 hours (P &amp;lt;0.05), and 5 compounds significantly inhibited proliferation at 72 hours (P &amp;lt;0.05). None of the compounds exhibited measurable cytotoxicity. One of the five compounds tested for effects on HMVEC migration, significantly inhibited migration on a fibronectin coated-surface by 78% (P&amp;lt;0.01). We have identified a number of small molecule inhibitors of TEM8, some of which display anti-angiogenic effects in vitro. Should these inhibitors also display potent anti-angiogenic effects in vivo, they may serve as lead compounds for the future development of TEM8 therapeutic small molecules. Future studies will focus on determining the effects of these molecules on tumor growth and angiogenesis in mouse models. Citation Format: Lorna Cryan, Marinya Roznik, Aaron McBride, Robert D'Amato, Kenneth Christensen, Michael S. Rogers. Identification and anti-angiogenic effects of small molecule inhibitors of TEM8. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5076. doi:10.1158/1538-7445.AM2013-5076

Abstract 3890: Antiangiogenic small molecule antagonists of the anthrax toxin receptor CMG2.

Abstract We have previously shown that the anthrax protective antigen mutant PASSSR can inhibit endothelial cell migration, VEGF and bFGF driven angiogenesis in the corneal micropocket assay, and tumor growth. However anthrax toxin has three reported receptors (CMG2, TEM8, and β1 integrin) that might mediate antiangiogenic effects. Knockdown of β1 integrin in endothelial cells had no effect on PASSSR inhibition of migration and circulating levels of PASSSR in mice are too low for significant integrin occupancy. Using soluble extracellular domain constructs fused to the antibody Fc region to improve pharmacokinetics, we observed that CMG2-Fc inhibits angiogenesis, while TEM8-Fc does not. Similarly, treatment of mice undergoing the corneal micropocket assay with the SR8F7C7 anti-CMG2 antibody resulted in dose-dependent inhibition of bFGF-induced corneal neovascularization, while the L2 anti-TEM8 antibody had no effect at a dose that has potent antitumor activity. Thus, CMG2 blockade, but not TEM8 blockade, can inhibit corneal angiogenesis. To assess the role of CMG2 in angiogenesis in vivo without pharmacologic intervention, we performed the corneal micropocket assay on knockout mice, and observed that knockout mice exhibit a dramatic decrease in angiogenesis vs. wild-type controls. Also, PASSSR does not further inhibit corneal angiogenesis in CMG2−/− mice. This is consistant with the ex vivo observation that CMG2 knockdown reduces the ability of HMVEC to migrate to serum and knockdown cells are no longer inhibited by PASSSR. To allow identification of CMG2 antagonists, we developed a high-throughput FRET-based screening assay. Validation of the assay suggested that tannic acid may inhibit this protein. However, further analysis demonstrated that this activity is attributable not to tannic acid, but instead to a hydrolysis product (PGG) that contaminated our tannic acid sample. We found that PGG inhibits CMG2 with an IC50 of 300 nM, a value consistent with serum levels achieved by antiangiogenic and antitumor dosing. PGG also inhibited endothelial cell migration and angiogenesis in the corneal micropocket assay, demonstrating that this compound can inhibit not only CMG2-PA binding, but also angiogenesis. Further screening of small molecule libraries identified several additional compounds that inhibit endothelial cell migration and angiogenesis in the corneal micropocket assay. In addition, we identified one compound that stimulates both activities. This important result reveals that CMG2 modulators can both stimulate and inhibit angiogenesis, ruling out indirect effects (such as compound toxicity) as a general explanation for the observed effects on angiogenesis. These results demonstrate small molecule modulation of CMG2 and identify potential pharmacoactive leads. Together, these data strongly suggest that small molecule CMG2 antagonists can be used as antiangiogenic therapies. Citation Format: Lorna M. Cryan, P. Christine Ackroyd, Shugeng Cao, Jon Clardy, Kenneth A. Christensen, Michael S. Rogers. Antiangiogenic small molecule antagonists of the anthrax toxin receptor CMG2. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3890. doi:10.1158/1538-7445.AM2013-3890

Three dimensional structure of the anthrax toxin translocon–lethal factor complex by cryo‐electron microscopy

We have visualized by cryo-electron microscopy (cryo-EM) the complex of the anthrax protective antigen (PA) translocon and the N-terminal domain of anthrax lethal factor (LF(N) inserted into a nanodisc model lipid bilayer. We have determined the structure of this complex at a nominal resolution of 16 Å by single-particle analysis and three-dimensional reconstruction. Consistent with our previous analysis of negatively stained unliganded PA, the translocon comprises a globular structure (cap) separated from the nanodisc bilayer by a narrow stalk that terminates in a transmembrane channel (incompletely distinguished in this reconstruction). The globular cap is larger than the unliganded PA pore, probably due to distortions introduced in the previous negatively stained structures. The cap exhibits larger, more distinct radial protrusions, previously identified with PA domain three, fitted by elements of the NMFF PA prepore crystal structure. The presence of LF(N), though not distinguished due to the seven-fold averaging used in the reconstruction, contributes to the distinct protrusions on the cap rim volume distal to the membrane. Furthermore, the lumen of the cap region is less resolved than the unliganded negatively stained PA, due to the low contrast obtained in our images of this specimen. Presence of the LF(N) extended helix and N terminal unstructured regions may also contribute to this additional internal density within the interior of the cap. Initial NMFF fitting of the cryoEM-defined PA pore cap region positions the Phe clamp region of the PA pore translocon directly above an internal vestibule, consistent with its role in toxin translocation.

Assembly of anthrax toxin pore: Lethal‐factor complexes into lipid nanodiscs

We have devised a procedure to incorporate the anthrax protective antigen (PA) pore complexed with the N-terminal domain of anthrax lethal factor (LFN ) into lipid nanodiscs and analyzed the resulting complexes by negative-stain electron microscopy. Insertion into nanodiscs was performed without relying on primary and secondary detergent screens. The preparations were relatively pure, and the percentage of PA pore inserted into nanodiscs on EM grids was high (∼43%). Three-dimensional analysis of negatively stained single particles revealed the LFN -PA nanodisc complex mirroring the previous unliganded PA pore nanodisc structure, but with additional protein density consistent with multiple bound LFN molecules on the PA cap region. The assembly procedure will facilitate collection of higher resolution cryo-EM LFN -PA nanodisc structures and use of advanced automated particle selection methods.

The Receptors that Mediate the Direct Lethality of Anthrax Toxin

Tumor endothelium marker-8 (TEM8) and capillary morphogenesis protein-2 (CMG2) are the two well-characterized anthrax toxin receptors, each containing a von Willebrand factor A (vWA) domain responsible for anthrax protective antigen (PA) binding. Recently, a cell-based analysis was used to implicate another vWA domain-containing protein, integrin β1 as a third anthrax toxin receptor. To explore whether proteins other than TEM8 and CMG2 function as anthrax toxin receptors in vivo, we challenged mice lacking TEM8 and/or CMG2. Specifically, we used as an effector protein the fusion protein FP59, a fusion between the PA-binding domain of anthrax lethal factor (LF) and the catalytic domain of Pseudomonas aeruginosa exotoxin A. FP59 is at least 50-fold more potent than LF in the presence of PA, with 2 μg PA + 2 μg FP59 being sufficient to kill a mouse. While TEM8−/− and wild type control mice succumbed to a 5 μg PA + 5 μg FP59 challenge, CMG2−/− mice were completely resistant to this dose, confirming that CMG2 is the major anthrax toxin receptor in vivo. To detect whether any toxic effects are mediated by TEM8 or other putative receptors such as integrin β1, CMG2−/−/TEM8−/− mice were challenged with as many as five doses of 50 μg PA + 50 μg FP59. Strikingly, the CMG2−/−/TEM8−/− mice were completely resistant to the 5-dose challenge. These results strongly suggest that TEM8 is the only minor anthrax toxin receptor mediating direct lethality in vivo and that other proteins implicated as receptors do not play this role.