Anthrax Protective Antigen Research Articles

Anthrax Vaccination, Gulf War Illness, and Human Leukocyte Antigen (HLA).

We report on a highly significant, positive association between anthrax vaccination and occurrence of Gulf War Illness (GWI) in 111 Gulf War veterans (42 with GWI and 69 controls). GWI was diagnosed in 47.1% of vaccinated veterans but only in 17.2% of non-vaccinated veterans (Pearson χ2 = 7.08, p = 0.008; odds ratio = 3.947; relative risk = 2.617), with 1.6x higher GWI symptom severity in vaccinated veterans (p = 0.007, F-test in analysis of covariance). Next, we tested the hypothesis that the susceptibility to GWI following anthrax vaccination could be due to inability to make antibodies against the anthrax protective antigen (PA), the key protein contained in the vaccine. Since the first step in initiating antibody production would be the binding of PA peptide fragments (typically 15-amino acid long [15-mer]) to peptide-binding motifs of human leukocyte antigen (HLA) Class II molecules, we assessed the binding-motif affinities of such HLA specific molecules to all linear 15-mer peptide fragments of the anthrax PA. We identified a total of 58 HLA Class II alleles carried by the veterans in our sample and found that, of those, 18 (31%) were present in the vaccinated group that did not develop GWI but were absent from the vaccinated group who developed GWI. Remarkably, in silico analyses revealed very high binding affinities of peptide-binding motifs of those 18 HLA alleles with fragments of anthrax vaccine PA, leading to the successful production of anti-PA antibodies. Conversely, the absence of these protective HLA alleles points to a reduced ability to develop antibodies against PA, thus resulting in harmful PA persistence and development of GWI.

Efficacy of therapeutically administered gepotidacin in a rabbit model of inhalational anthrax.

Bacillus anthracis is a Gram-positive Centers for Disease Control and Prevention category "A" biothreat pathogen. Without early treatment, inhalation of anthrax spores with progression to inhalational anthrax disease is associated with high fatality rates. Gepotidacin is a novel first-in-class triazaacenaphthylene antibiotic that inhibits bacterial DNA replication by a distinct mechanism of action and is being evaluated for use against biothreat and conventional pathogens. Gepotidacin selectively inhibits bacterial DNA replication via a unique binding mode and has in vitro activity against a collection of B. anthracis isolates including antibacterial-resistant strains, with the MIC90 ranging from 0.5 to 1 µg/mL. In vivo activity of gepotidacin was also evaluated in the New Zealand White rabbit model of inhalational anthrax. The primary endpoint was survival, with survival duration and bacterial clearance as secondary endpoints. The trigger for treatment was the presence of anthrax protective antigen in serum. New Zealand White rabbits were dosed intravenously for 5 days with saline or gepotidacin at 114 mg/kg/d to simulate a dosing regimen of 1,000 mg intravenous (i.v.) three times a day (TID) in humans. Gepotidacin provided a survival benefit compared to saline control, with 91% survival (P-value: 0.0001). All control animals succumbed to anthrax and were found to be blood- and organ culture-positive for B. anthracis. The novel mode of action, in vitro microbiology, preclinical safety, and animal model efficacy data, which were generated in line with Food and Drug Administration Animal Rule, support gepotidacin as a potential treatment for anthrax in an emergency biothreat situation.

Visualization of immune pathways that enhance the neutralizing antibody response to vaccines after primary immunization.

We examined the relationship between the association of a vaccine antigen with immune cells in secondary lymphoid organs shortly after immunization and the resulting neutralizing antibody response induced by that antigen using three antigenic forms of anthrax protective antigen (PA) that induce qualitatively different antibody responses. The three PA forms used were wild-type PA, which binds to anthrax toxin receptors and elicits a robust antibody response that includes both neutralizing and non-neutralizing antibodies; a receptor-binding-deficient (RBD) mutant form of PA, which does not bind cellular receptors and elicits only barely detectable antibody responses; and an engineered chimeric form of PA, which binds cholera toxin receptors and elicits a robust total antibody response but a poor neutralizing antibody response. We found that both wild-type PA and the PA chimera associated with immune cells in secondary lymphoid organs after immunization, but the RBD mutant PA exhibited minimal association, revealing a relationship between antigen binding to toxin receptors on immune cells after immunization and subsequent antibody responses. A portion of wild-type PA that bound to immune cells was cell surface-associated and maintained its native conformation. Much lower amounts of conformationally intact PA chimera were associated with immune cells after immunization, correlating with the lower neutralizing antibody response elicited by the PA chimera. Thus, binding of an antigen to receptors on immune cells in secondary lymphoid organs after immunization and maintenance of conformational integrity of the cell-associated antigen help dictate the magnitude of the resulting neutralizing antibody response, but not necessarily the total antibody response.IMPORTANCEMany vaccines protect by the induction of antibodies that neutralize the action of the pathogen. Here, we followed the fate of three antigenic forms of a vaccine antigen in secondary lymphoid organs after immunization to investigate events leading to a robust neutralizing antibody response. We found that the magnitude of the neutralizing antibody response, but not the total antibody response, correlates with the levels of conformationally intact antigen associated with immune cells in secondary lymphoid organs after primary immunization. We believe that these results provide important insights into the genesis of neutralizing antibody responses induced by vaccine antigens and may have implications for vaccine design.

Structural characterization of PCP-Ⅰ from Poria as vaccine adjuvant and its hydrolytic oligosaccharide

Poria is an important medical herb in clinic. The authors isolated a polysaccharide(PCP-Ⅰ) from Poria in previous studies, which is composed of galactose, mannose, fucose and glucose. PCP-Ⅰ exhibited significant adjuvant effects on H1N1 influenza vaccine, hepatitis B surface antigen and anthrax protective antigen, and its adjuvant activity was stronger than aluminium adjuvant. However, little is known about the chemical structure of PCP-Ⅰ at present. In this study, weak acid hydrolysis was used to obtain the backbone oligosaccharide of PCP-Ⅰ. Then periodate oxidation, Smith degradation, methylation analysis, Fourier transform infrared spectroscopy(FT-IR), nuclear magnetic resonance(NMR) and gas chromatography-mass spectrometry(GC-MS) were performed to investigate the chemical structural features of PCP-Ⅰ and its hydrolytic oligosaccharide(PCP-Ⅰ-hy-1). These results suggested that the backbone of PCP-Ⅰ was composed of galactose with α anomeric carbon and β anomeric carbon. The linking residues of galactan are(1→),(l→6) and(1→2,6).

Research on Detection of Ultra-Low Concentration Anthrax Protective Antigen Using Graphene Field-Effect Transistor Biosensor.

Protective antigen (PA) is an important biomarker for the early diagnosis of anthrax, and the accurate detection of protective antigen under extremely low concentration conditions has always been a hot topic in the biomedical field. To complete the diagnosis of anthrax in a timely manner, it is necessary to detect PA at extremely low concentrations, as the amount of PA produced in the early stage of anthrax invasion is relatively small. Graphene field-effect transistor (Gr-FET) biosensors are a new type of material for preparing biosensors, with the advantages of a short detection time and ultra-low detection limit. The effect of different concentrations of diluents on the affinity of PA monoclonal antibodies was determined via an ELISA experiment. Combined with the Debye equation, 0.01 × PBS solution was finally selected as the diluent for the experiment. Then, a PA monoclonal antibody was selected as the bio-recognition element to construct a Gr-FET device based on CVD-grown graphene, which was used to detect the concentration of PA while recording the response time, linear range, detection limit, and other parameters. The experimental results showed that the biosensor could quickly detect PA, with a linear range of 10 fg/mL to 100 pg/mL and a detection limit of 10 fg/mL. In addition, the biosensor showed excellent specificity and repeatability. By constructing a Gr-FET device based on CVD-grown graphene and selecting a PA monoclonal antibody as the bio-recognition element, a highly sensitive, specific, and repeatable Gr-FET biosensor was successfully prepared for detecting extremely low concentrations of anthrax protective antigen (PA). This biosensor is expected to have a wide range of applications in clinical medicine and biological safety monitoring.

The designation, synthesis, and affinity determination of affinity peptide for anthrax protective antigen.

Detection of anthrax protective antigen is an effective way to diagnose anthracnose, and it plays an important part in the treatment of anthracnose. Affinity peptides, as a miniature biological recognition element, can quickly and effectively detect anthrax protective antigens. Based on computer-aided design technology (CAD), we have herein developed an affinity peptide design strategy for the detection of anthrax protective antigens. Firstly, six high-value mutation sites were determined based on the molecular docking between the template peptide and the receptor, and then the multi-site mutation of amino acids was carried out in order to establish a virtual peptide library. The library was selected by using molecular dynamics simulation and the best designed affinity peptide (code: P24) was found. The theoretical affinity with P24 peptide has increased by 19.8% compared with template peptide. Finally, the affinity with P24 peptide was measured by SPR technology to reach the nanomole level, which verified the effectiveness of the design strategy. The newly designed affinity peptide is expected to be used in the diagnosis of anthracnose.

Chloroquine-analogues block anthrax protective antigen channels in steady-state and kinetic studies

The tripartite anthrax toxin from Bacillus anthracis represents the prototype of A-B type of toxins, where the effector A (an enzymatic subunit) is transported with the help of a binding component B into a target cell. Anthrax toxin consists of three different molecules, two effectors, lethal factor (LF) and edema factor (EF) and the binding component also known as protective antigen (PA). PA forms heptamers or octamers following binding to host cell’s receptors and mediates the translocation of the effectors into the cytosol via the endosomal pathway. The cation-selective PA63-channel is able to reconstitute in lipid membranes and can be blocked by chloroquine and other heterocyclic compounds. This suggests that the PA63-channel contains a binding site for quinolines. In this study, we investigated the structure-function relationship of different quinolines for the block of the PA63-channel. The affinity of the different chloroquine analogues to the PA63-channel as provided by the equilibrium dissociation constant was measured using titrations. Some quinolines had a much higher affinity to the PA63-channel than chloroquine itself. We also performed ligand-induced current noise measurements using fast Fourier transformation to get insight in the kinetics of the binding of some quinolines to the PA63-channel. The on-rate constants of ligand binding were around 108 M−1·s−1 at 150 mM KCl and were only little dependent on the individual quinoline. The off-rates varied between 4 s−1 and 160 s−1 and depended much more on the structure of the molecules than the on-rate constants. The possible use of the 4-aminoquinolines as a therapy is discussed.

New formulation of a recombinant anthrax vaccine stabilised with structurally modified plant viruses.

Anthrax is a disease caused by Bacillus anthracis. The most promising approach to the development of anthrax vaccine is use of the anthrax protective antigen (PA). At the same time, recombinant PA is a very unstable protein. Previously, the authors have designed a stable modified recombinant anthrax protective antigen with inactivated proteolytic sites and substituted deamidation sites (rPA83m). As a second approach to recombinant PA stabilisation, plant virus spherical particles (SPs) were used as a stabiliser. The combination of these two approaches was shown to be the most effective. Here, the authors report the results of a detailed study of the stability, immunogenicity and protectiveness of rPA83m + SPs compositions. These compositions were shown to be stable, provided high anti-rPA83m antibody titres in guinea pigs and were able to protect them from a fully virulent 81/1 Bacillus anthracis strain. Given these facts, the formulation of rPA83m + SPs compositions is considered to be a prospective anthrax vaccine candidate.

Receptor-enhanced immunogenicity of anthrax protective antigen is primarily mediated by capillary morphogenesis Protein-2

Anthrax protective antigen (PA), the receptor-binding component of anthrax toxin, elicits toxin-neutralizing antibodies which provide protection against anthrax disease. PA binds to two mammalian receptors, capillary morphogenesis protein-2 (CMG2) and tumor endothelial marker-8 (TEM8). We previously observed that binding of PA to its receptors plays a role in eliciting a strong toxin-neutralizing antibody response. In this study, we examined the roles that individual receptors play in mediating the toxin-neutralizing antibody response. Mice immunized with PA that binds preferentially to CMG2 elicited a toxin-neutralizing antibody response similar to that elicited by wild-type PA, whereas the antibody response elicited by PA that binds preferentially to TEM8 was significantly lower. Also, the toxin-neutralizing antibody response elicited by wild-type PA in CMG2-null mice was found to be significantly lower than that induced in CMG2-sufficient mice, further supporting a predominant role for the CMG2 receptor in mediating a protective antibody response to PA.

Structure of the Anthrax Protective Antigen D425A Dominant Negative Mutant Reveals a Stalled Intermediate State of Pore Maturation

The tripartite protein complex produced by anthrax bacteria (Bacillus anthracis) is a member of the AB family of β-barrel pore-forming toxins. The protective antigen (PA) component forms an oligomeric prepore that assembles on the host cell surface and serves as a scaffold for binding of lethal and edema factors. Following endocytosis, the acidic environment of the late endosome triggers a pH-induced conformational rearrangement to promote maturation of the PA prepore to a functional, membrane spanning pore that facilitates delivery of lethal and edema factors to the cytosol of the infected host. Here, we show that the dominant-negative D425A mutant of PA stalls anthrax pore maturation in an intermediate state at acidic pH. Our 2.7 Å cryo-EM structure of the intermediate state reveals structural rearrangements that involve constriction of the oligomeric pore combined with an intramolecular dissociation of the pore-forming module. In addition to defining the early stages of anthrax pore maturation, the structure identifies asymmetric conformational changes in the oligomeric pore that are influenced by the precise configuration of adjacent protomers.

Capillary morphogenesis gene 2 (CMG2) mediates growth factor-induced angiogenesis by regulating endothelial cell chemotaxis.

Anthrax protective antigen (PA) is a potent inhibitor of pathological angiogenesis with an unknown mechanism. In anthrax intoxication, PA interacts with capillary morphogenesis gene 2 (CMG2) and tumor endothelial marker 8 (TEM8). Here, we show that CMG2 mediates the antiangiogenic effects of PA and is required for growth-factor-induced chemotaxis. Using specific inhibitors of CMG2 and TEM8 interaction with natural ligand, as well as mice with the CMG2 or TEM8 transmembrane and intracellular domains disrupted, we demonstrate that inhibiting CMG2, but not TEM8 reduces growth-factor-induced angiogenesis in the cornea. Furthermore, the antiangiogenic effect of PA was abolished when the CMG2, but not the TEM8, gene was disrupted. Binding experiments demonstrated a broad ligand specificity for CMG2 among extracellular matrix (ECM) proteins. Ex vivo experiments demonstrated that CMG2 (but not TEM8) is required for PA activity in human dermal microvascular endothelial cell (HMVEC-d) network formation assays. Remarkably, blocking CMG2-ligand binding with PA or CRISPR knockout abolishes endothelial cell chemotaxis but not chemokinesis in microfluidic migration assays. These effects are phenocopied by Rho inhibition. Because CMG2 mediates the chemotactic response of endothelial cells to peptide growth factors in an ECM-dependent fashion, CMG2 is well-placed to integrate growth factor and ECM signals. Thus, CMG2 targeting is a novel way to inhibit angiogenesis.

Generation of Protein-Phosphorodiamidate Morpholino Oligomer Conjugates for Efficient Cellular Delivery via Anthrax Protective Antigen.

Phosphorodiamidate morpholino oligomers (PMOs) offer great promise as therapeutic agents for translation blocking or splice modulation due to their high stability and affinity for target sequences. However, in spite of their neutral charge as compared to natural oligonucleotides or phosphorothioate analogs, they still show little permeability for cellular membranes, highlighting the need for effective cytosolic delivery strategies. In addition, the implementation of strategies for efficient cellular targeting is highly desirable to minimize side effects and maximize the drug dose at its site of action. Anthrax toxin is a three-protein toxin of which the pore-forming protein anthrax protective antigen (PA) can be redirected to a receptor of choice and lethal factor (LF), one of the two substrate proteins, can be coupled to various cargoes for efficient cytosolic cargo delivery. In this protocol, we describe the steps to produce the proteins and protein conjugates required for cytosolic delivery of PMOs through the cation-selective pore generated by anthrax protective antigen. The method relies on the introduction of a unique cysteine at the C-terminal end of a truncated LF (aa 1-254), high-yield expression of the (truncated) toxin proteins in E. coli, functionalization of a PMO with a maleimide group and coupling of the maleimide-functionalized PMO to the unique cysteine on LF by maleimide-thiol conjugation chemistry. Through co-administration of PA with LF-PMO conjugates, an efficient cytosolic delivery of PMOs can be obtained.

Label-Free and Rapid Detection of Anthrax Protective Antigen by Surface-Enhanced Raman Scattering on Au Nanorods

Aiming at the requirement of ultra-sensitive on-site rapid detection of anthrax protective antigens (PA), we proposed a label-free detection scheme based on surface-enhanced Raman Scattering (SERS) on Au nanorods (AuNRs) substrate that we fabricated. The Raman spectrum of anthrax protective antigen was reported for the first time. The limit of detection (LOD) of PA on the AuNRs was characterized to be 100 pg/mL. To simulate the actual detection of PA in human serum albumin (HSA), the label-free detection of HSA based on the AuNRs SERS substrate was studied in this paper. The characteristic peaks of the obtained HSA spectrum are consistent with the previously reported literature. The SERS label-free detection of PA in HSA solution has also been achieved. The limit of detection of the PA in HSA solution can also reach 100 pg/mL, which is much lower than the PA concentration of 100 μg/mL in human serum after anthrax infection. A good linear relationship between the peak intensity of the mixture spectrum and the logarithm of the concentration of PA was obtained with the correlation coefficient to be 0.9769. Using Principal Component Analysis (PCA) method, the SERS data of PA, HSA and the mixture can be classified into three completely separated areas without overlap. The entire testing process will cost only about eight minutes, which is much faster than the mature technology, such as Enzyme-linked Immunosorbent Assay (ELISA).

Efficacy assessment of a triple anthrax chimeric antigen as a vaccine candidate in guinea pigs: challenge test with Bacillus anthracis 17 JB strain spores

Context Bacillus anthracis secretes a tripartite toxin comprising protective antigen (PA), edema factor (EF), and lethal factor (LF). The human anthrax vaccine is mainly composed of the anthrax protective antigen (PA). Considerable efforts are being directed towards improving the efficacy of vaccines because the use of commercial anthrax vaccines (human/veterinary) is associated with several limitations. Objective In this study, a triple chimeric antigen referred to as ELP (gene accession no: MT590758) comprising highly immunogenic domains of PA, LF, and EF was designed, constructed, and assessed for the immunization capacity against anthrax in a guinea pig model. Materials and methods Immunization was carried out considering antigen titration and immunization protocol. The immunoprotective efficacy of the ELP was evaluated in guinea pigs and compared with the potency of veterinary anthrax vaccine using a challenge test with B. anthracis 17JB strain spores. Results The results demonstrated that the ELP antigen induced strong humoral responses. The T-cell response of the ELP was found to be similar to PA, and showed that the ELP could protect 100%, 100%, 100%, 80% and 60% of the animals from 50, 70, 90, 100 and 120 times the minimum lethal dose (MLD, equal 5 × 105 spore/ml), respectively, which killed control animals within 48 h. Discussion and conclusions It is concluded that the ELP antigen has the necessary requirement for proper immunization against anthrax and it can be used to develop an effective recombinant vaccine candidate against anthrax.

Detection of antibody to Bacillus anthracis recombinant PA in vaccinated animal serum

Anthrax is a worldwide zoonosis in animals and human. In Mongolia, the confirmed case of anthrax outbreak is reported every year over the past decade. The prevention and control measure of animal anthrax is vaccination using spore of attenuated Sterne strain, but horse does not get vaccinated in Mongolia. In this study, we constructed the recombinant plasmid for over expression of anthrax protective antigen (PA)/GST fusion protein in pGEX-6P-1 vector and purified the recombinant PA (r-PA) using glutathione Sepharose column under native and denaturing conditions. Since both forms of r-PA were recognized by specific antibody against PA, ELISA system to detect antibody titer in vaccinated bovine serum was constructed. Total of 890 vaccinated cattle serum were collected from 178 cattle at 0, 3, 5, 8 and 12 months’ post vaccination. As negative control, 200 cattle serum from Umnugovi aimag were selected which does not have anthrax foci. All serum was tested by rPA indirect ELISA and, antibody to PA were detected in vaccinated cattle serum but were not detected in negative serum. Therefore, rPA should be used in as monitoring of the anthrax vaccination.

Clindamycin Protects Nonhuman Primates Against Inhalational Anthrax But Does Not Enhance Reduction of Circulating Toxin Levels When Combined With Ciprofloxacin

Inhalational anthrax is rare and clinical experience limited. Expert guidelines recommend treatment with combination antibiotics including protein synthesis-inhibitors to decrease toxin production and increase survival, although evidence is lacking. Rhesus macaques exposed to an aerosol of Bacillus anthracis spores were treated with ciprofloxacin, clindamycin, or ciprofloxacin + clindamycin after becoming bacteremic. Circulating anthrax lethal factor and protective antigen were quantitated pretreatment and 1.5 and 12 hours after beginning antibiotics. In the clindamycin group, 8 of 11 (73%) survived demonstrating its efficacy for the first time in inhalational anthrax, compared to 9 of 9 (100%) with ciprofloxacin, and 8 of 11 (73%) with ciprofloxacin + clindamycin. These differences were not statistically significant. There were no significant differences between groups in lethal factor or protective antigen levels from pretreatment to 12 hours after starting antibiotics. Animals that died after clindamycin had a greater incidence of meningitis compared to those given ciprofloxacin or ciprofloxacin + clindamycin, but numbers of animals were very low and no definitive conclusion could be reached. Treatment of inhalational anthrax with clindamycin was as effective as ciprofloxacin in the nonhuman primate. Addition of clindamycin to ciprofloxacin did not enhance reduction of circulating toxin levels.

Anthrax Protective Antigen Retargeted with Single-Chain Variable Fragments Delivers Enzymes to Pancreatic Cancer Cells.

The nontoxic, anthrax protective antigen/lethal factor N-terminal domain (PA/LFN ) complex is an effective platform for translocating proteins into the cytosol of cells. Mutant PA (mPA) was recently fused to epidermal growth factor (EGF) to retarget delivery of LFN to cells bearing EGF receptors (EGFR), but the requirement for a known cognate ligand limits the applicability of this approach. Here, we render practical protective antigen retargeting to a variety of receptors with mPA single-chain variable fragment (scFv) fusion constructs. Our design enables the targeting of two pancreatic cancer-relevant receptors, EGFR and carcinoembryonic antigen. We demonstrate that fusion to scFvs does not disturb the basic functions of mPA. Moreover, mPA-scFv fusions enable cell-specific delivery of diphtheria toxin catalytic domain and Ras/Rap1-specific endopeptidase to pancreatic cancer cells. Importantly, mPA-scFv fusion-based treatments display potent cell-specific toxicity in vitro, opening fundamentally new routes toward engineered immunotoxins and providing a potential solution to the challenge of targeted protein delivery to the cytosol of cancer cells.

Predictive and fluorescent nanosensing experimental methods for evaluating anthrax protective antigen and lethal factor interactions for therapeutic applications

Recently, specific interaction of anthrax protective antigen domain 4 (PAD4) and lethal factor domain 1 (LFD1) have been considered for the design of novel diagnostic and therapeutic systems in medicine. In this study, theoretical and experimental approaches were used to monitor the interactions of PAD4 and LFD1. CLusPro server and Dimplot software were used to predict the interaction of these domains. Results, revealed interactive sites between PAD4 and LFD1 on loop regions of both C and N terminal of PAD4. In experimental methods, PAD4 and LFD1 were expressed in Escherichia coli and purified for usage in Magnetic Bead (MB) and Multi-Walled Carbon Nanotubes (MWCNTs) based bio-sensing platforms. In the magnetic-based system, the magnetic sedimentation of QD-PAD4 by MBs-LFD1 and the observation of the fluorescence spectrum related to QD-PAD4 in the precipitated materials confirmed the interaction of PAD4 with LFD1 protein. In the MWCNTs-based method, the QD-PAD4 fluorescence was quenched by absorption on MWCNTs. Upon the addition of LFD1, fluorescence emission was recovered, indicating interaction of LFD1 with QD-PAD4, which results the separation of QD-PAD4 from MWCNTs surfaces and fluorescence restoration. Finally, new approaches showed the interaction of PAD4 and LFD1, which can be used as an attractive model in medicine.

Dual-Readout Sandwich Immunoassay for Device-Free and Highly Sensitive Anthrax Biomarker Detection.

We report a dual-readout, AuNP-based sandwich immunoassay for the device-free colorimetric and sensitive scanometric detection of disease biomarkers. An AuNP-antibody conjugate serves as a signal transduction and amplification agent by promoting the reduction and deposition of either platinum or gold onto its surface, generating corresponding colorimetric or light scattering (scanometric) signals, respectively. We apply the Pt-based colorimetric readout of this assay to the discovery of a novel monoclonal antibody (mAb) sandwich pair for the detection of an anthrax protective antigen (PA83). The identified antibody pair detects PA83 down to 1 nM in phosphate-buffered saline and 5 nM in human serum, which are physiologically relevant concentrations. Reducing gold rather than platinum onto the mAb-AuNP sandwich enables scanometric detection of subpicomolar PA83 concentrations, over 3 orders of magnitude more sensitive than the colorimetric readout.

Cryo-EM structures reveal translocational unfolding in the clostridial binary iota toxin complex.

The iota toxin produced by Clostridium perfringens type E is a binary toxin comprising two independent polypeptides: Ia, an ADP-ribosyltransferase, and Ib, which is involved in cell binding and translocation of Ia across the cell membrane. Here we report cryo-EM structures of the translocation channel Ib-pore and its complex with Ia. The high-resolution Ib-pore structure demonstrates a similar structural framework to that of the catalytic ϕ-clamp of the anthrax protective antigen pore. However, the Ia-bound Ib-pore structure shows a unique binding mode of Ia: one Ia binds to the Ib-pore, and the Ia amino-terminal domain forms multiple weak interactions with two additional Ib-pore constriction sites. Furthermore, Ib-binding induces tilting and partial unfolding of the Ia N-terminal α-helix, permitting its extension to the ϕ-clamp gate. This new mechanism of N-terminal unfolding is crucial for protein translocation.