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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.

Anthrax is a severe, although rather rare, infectious disease that is caused by the Gram-positive, spore-forming bacterium Bacillus anthracis. The infectious form is the spore and the major virulence factors of the bacterium are its poly-γ-D-glutamic acid capsule and the tripartite anthrax toxin. The discovery of the anthrax toxin receptors in the early 2000s has allowed in-depth studies on the mechanisms of anthrax toxin cellular entry and translocation from the endocytic compartment to the cytoplasm. The toxin generally hijacks the endocytic pathway of CMG2 and TEM8, the two anthrax toxin receptors, in order to reach the endosomes. From there, the pore-forming subunit of the toxin inserts into endosomal membranes and enables translocation of the two catalytic subunits. Insertion of the pore-forming unit preferentially occurs in intraluminal vesicles rather than the limiting membrane of the endosome, leading to the translocation of the enzymatic subunits in the lumen of these vesicles. This has important consequences that will be discussed. Ultimately, the toxins reach the cytosol where they act on their respective targets. Target modification has severe consequences on cell behavior, in particular on cells of the immune system, allowing the spread of the bacterium, in severe cases leading to host death. Here we will review the literature on anthrax disease with a focus on the structure of the toxin, how it enters cells and its immunological effects.

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 1 , 2 ; lethal factor (LF) is a zinc-dependent protease that cleaves mitogen-activated protein kinase kinase and causes lysis of macrophages 3 , 4 , 5 . 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.

•A novel safe and efficacious anthrax live vaccine based on the Sterne strain was generated.•This vaccine exhibits disruption of htrA and cya genes and mutation of the lef gene.•The vaccine retains its ability to elicit protective immune response in guinea pigs and rabbits.•The vaccine exhibits an extended therapeutic window therefore may be compatible with human use. We recently reported the development of a novel, next-generation, live attenuated anthrax spore vaccine based on disruption of the htrA (High Temperature Requirement A) gene in the Bacillus anthracis Sterne veterinary vaccine strain. This vaccine exhibited a highly significant decrease in virulence in murine, guinea pig and rabbit animal models yet preserved the protective value of the parental Sterne strain. Here, we report the evaluation of additional mutations in the lef and cya genes, encoding for the toxin components lethal factor (LF) and edema factor (EF), to further attenuate the SterneΔhtrA strain and improve its compatibility for human use. Accordingly, we constructed seven B. anthracis Sterne-derived strains exhibiting different combinations of mutations in the htrA, cya and lef genes. The various strains were indistinguishable in growth in vitro and in their ability to synthesise the protective antigen (PA, necessary for the elicitation of protection). In the sensitive murine model, we observed a gradual increase (ΔhtrA<ΔhtrAΔcya<ΔhtrAΔlef<ΔhtrAΔlefΔcya) in attenuation – up to 108-fold relative to the parental Sterne vaccine strain. Most importantly, all various SterneΔhtrA derivative strains did not differ in their ability to elicit protective immunity in guinea pigs. Immunisation of guinea pigs with a single dose (109 spores) or double doses (>107spores) of the most attenuated triple mutant strain SterneΔhtrAlefMUTΔcya induced a robust immune response, providing complete protection against a subsequent respiratory lethal challenge. Partial protection was observed in animals vaccinated with a double dose of as few as 105spores. Furthermore, protective immune status was maintained in all vaccinated guinea pigs and rabbits for at least 40 and 30weeks, respectively.

•AVP booster doses elicit significant increases in anti-PA & anti-LF IgG concentrations.•TNA NF50 response to AVP boosters increases as time interval between doses increases.•JCVI extended interval between AVP booster doses from 1 to 10 years on basis of results.•Anti-PA & anti-LF IgG contribute independently to anthrax lethal toxin neutralisation. We undertook a Phase 4 clinical trial to assess the effect of time interval between booster doses on serological responses to AVP. The primary objective was to evaluate responses to a single booster dose in two groups of healthy adults who had previously received a complete 4-dose primary course. Group A had received doses on schedule while Group B had not had one for ≥2 years. Secondary objectives were to evaluate the safety and tolerability of AVP booster doses, and to gain information on correlates of protection to aid future anthrax vaccine development. Blood samples were taken on Day 1 before dosing, and on Days 8, 15, 29 and 120, to measure Toxin Neutralisation Assay (TNA) NF50 values and concentrations of IgG antibodies against Protective Antigen (PA), Lethal Factor (LF) and Edema Factor (EF) by ELISA. For each serological parameter, fold changes from baseline following the trial AVP dose were greater in Group B than Group A at every time-point studied. Peak responses correlated positively with time since last AVP dose (highest values being observed after intervals of ≥10 years), and negatively with number of previous doses (highest values occurring in individuals who had received a primary course only). In 2017, having reviewed these results, the Joint Committee on Vaccination and Immunisation (JCVI) updated UK anthrax vaccination guidelines, extending the interval between routine AVP boosters from one to 10 years. Booster doses of AVP induce significant IgG responses against the three anthrax toxin components, particularly PA and LF. Similarly high responses were observed in TNA, a recognised surrogate for anthrax vaccine efficacy. Analysis of the 596 TNA results showed that anti-PA and anti-LF IgG make substantial independent contributions to neutralisation of anthrax lethal toxin. AVP may therefore have advantages over anthrax vaccines that depend on generating immunity to PA alone.

Bacillus cereus group bacteria containing the anthrax toxin genes can cause fatal anthrax pneumonia in welders. Two welder’s anthrax cases identified in 2020 were investigated to determine the source of each patient’s exposure. Environmental sampling was performed at locations where each patient had recent exposure to soil and dust. Samples were tested for the anthrax toxin genes by real-time PCR, and culture was performed on positive samples to identify whether any environmental isolates matched the patient’s clinical isolate. A total of 185 environmental samples were collected in investigation A for patient A and 108 samples in investigation B for patient B. All samples from investigation B were real-time PCR-negative, but 14 (8%) samples from investigation A were positive, including 10 from patient A’s worksite and 4 from his work-related clothing and gear. An isolate genetically matching the one recovered from patient A was successfully cultured from a worksite soil sample. All welder’s anthrax cases should be investigated to determine the source of exposure, which may be linked to their worksite. Welding and metalworking employers should consider conducting a workplace hazard assessment and implementing controls to reduce the risk of occupationally associated illnesses including welder’s anthrax.

The protein acyl transferase ZDHHC5 was recently proposed to regulate trafficking in the endocytic pathway. Therefore, we explored the function of this enzyme in controlling the action of bacterial toxins. We found that ZDHHC5 activity is required for two very different toxins: the anthrax lethal toxin and the pore-forming toxin aerolysin. Both of these toxins have precursor forms, the protoxins, which can use the proprotein convertases Furin and PC7 for activation. We show that ZDHHC5 indeed affects the processing of the protoxins to their active forms. We found that Furin and PC7 can both be S-palmitoylated and are substrates of ZDHHC5. The impact of ZDHHC5 on Furin/PC7-mediated anthrax toxin cleavage is dual, having an indirect and a direct component. First, ZDHHC5 affects the homeostasis and trafficking of a subset of cellular proteins, including Furin and PC7, presumably by affecting the endocytic/recycling pathway. Second, while not inhibiting the protease activity per se, ZDHHC5-mediated Furin/PC7 palmitoylation is required for the cleavage of the anthrax toxin. Finally, we show that palmitoylation of Furin and PC7 promotes their association with plasma membrane microdomains. Both the receptor-bound toxin and the convertases are of very low abundance at the cell surface. Their encounter is unlikely on reasonable time scales. This work indicates that palmitoylation drives their encounter in specific domains, allowing processing and thereby intoxication of the cell.

Bacillus cereus is ubiquitous in the environment and a well-known causative agent of foodborne disease. Surprisingly, more and more emerging strains of atypical B. cereus have been identified and related to severe disease in humans and mammals such as chimpanzees, apes, and bovine. Recently, the atypical B. cereus isolates, which mainly derive from North America and Africa, have drawn great attention due to the potential risk of zoonosis. The cluster of B. cereus carries several anthrax-like virulent genes that are implicated in lethal disease. However, in non-mammals, the distribution of atypical B. cereus is still unknown. In this study, we conducted a retrospective screening of the 32 isolates of Bacillus spp. from diseased Chinese soft-shelled turtles from 2016 to 2020. To recognize the causative agent, we used various methods, such as sequencing analysis using PCR-amplification of the 16S rRNA gene, multiplex PCR for discriminating, and colony morphology by following previous studies. Furthermore, the digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values were calculated, respectively, below the 70 and 96% cutoff to define species boundaries. According to the summarized results, the pathogen is taxonomically classified as Bacillus tropicus str. JMT (previous atypical Bacillus cereus). Subsequently, analyses such as targeting the unique genes using PCR and visual observation of the bacteria under various staining techniques were implemented in our study. Our findings show that all (32/32, 100%) isolates in this retrospective screening share similar phenotypical properties and carry the protective antigen (PA), edema factor (EF), hyaluronic acid (HA), and exopolysaccharide (Bps) genes on their plasmids. In this study, the results indicate that the geographic distribution and host range of B. tropicus were previously underestimated.

The anthrax lethal toxin consists of protective antigen (PA) and lethal factor (LF). Understanding both the PA pore formation and LF translocation through the PA pore is crucial to mitigating and perhaps preventing anthrax disease. To better understand the interactions of the LF-PA engagement complex, the structure of the LFN-bound PA pore solubilized by a lipid nanodisc was examined using cryo-EM. CryoSPARC was used to rapidly sort particle populations of a heterogeneous sample preparation without imposing symmetry, resulting in a refined 17 Å PA pore structure with 3 LFN bound. At pH 7.5, the contributions from the three unstructured LFN lysine-rich tail regions do not occlude the Phe clamp opening. The open Phe clamp suggests that, in this translocation-compromised pH environment, the lysine-rich tails remain flexible and do not interact with the pore lumen region.

Anthrax represents a disease resulting from infection by toxin-secreting bacteria, Bacillus anthracis. This research aimed to identify new therapeutic targets to combat anthrax. We performed assays to assess cell viability, apoptosis, glycogen consumption, and compound uptake and release in hepatocytes and cardiomyocytes responding to anthrax toxins. Microarray analysis was carried out to identify the genes potentially involved in toxin-induced toxicity. Knockdown experiments were performed to validate the contributions of the identified genes. Our study showed that anthrax edema toxin (EdTx) and lethal toxin (LeTx) induced lethal damage in mouse liver and heart, respectively. Microarray assays showed that 218 genes were potentially involved in EdTx-mediated toxicity, and 18 genes were potentially associated with LeTx-mediated toxicity. Among these genes, the knockdown of Rgs1, Hcar2, Fosl2, Hcar2, Cxcl2, and Cxcl3 protected primary hepatocytes from EdTx-induced cytotoxicity. Plasminogen activator inhibitor 1 (PAI-1)-encoding Serpine1 constituted the most significantly upregulated gene in response to LeTx treatment in mouse liver. PAI-1 knockout mouse models had a higher tolerance to LeTx compared with wild-type counterparts, suggesting that PAI-1 is essential for LeTx-induced toxicity and might represent a therapeutic target in LeTx-induced tissue damage. These results provide potential therapeutic targets for combating anthrax-toxin-induced liver and heart damage.