Search

Abstract The bacterium Clostridium botulinum, well-known for producing botulinum neurotoxins, which cause the severe paralytic illness known as botulism, produces C2 toxin, a binary AB-toxin with ADP-ribosyltranferase activity. C2 toxin possesses two separate protein components, an enzymatically active A-component C2I and the binding and translocation B-component C2II. After proteolytic activation of C2II to C2IIa, the heptameric structure binds C2I and is taken up via receptor-mediated endocytosis into the target cells. Due to acidification of endosomes, the C2IIa/C2I complex undergoes conformational changes and consequently C2IIa forms a pore into the endosomal membrane and C2I can translocate into the cytoplasm, where it ADP-ribosylates G-actin, a key component of the cytoskeleton. This modification disrupts the actin cytoskeleton, resulting in the collapse of cytoskeleton and ultimately cell death. Here, we show that the serine-protease inhibitor α1-antitrypsin (α1AT) which we identified previously from a hemofiltrate library screen for PT from Bordetella pertussis is a multitoxin inhibitor. α1AT inhibits intoxication of cells with C2 toxin via inhibition of binding to cells and inhibition of enzyme activity of C2I. Moreover, diphtheria toxin and an anthrax fusion toxin are inhibited by α1AT. Since α1AT is commercially available as a drug for treatment of the α1AT deficiency, it could be repurposed for treatment of toxin-mediated diseases.

Many bacteria act pathogenic by the release of AB-type protein toxins that efficiently enter human or animal cells and act as enzymes in their cytosol. This leads to disturbed cell functions and the clinical symptoms characteristic for the individual toxin. Therefore, molecules that directly target and neutralize these toxins provide promising novel therapeutic options. Here, we found that the FDA-approved drug disulfiram (DSF), used for decades to treat alcohol abuse, protects cells from intoxication with diphtheria toxin (DT) from Corynebacterium diphtheria, the causative agent of diphtheria, lethal toxin (LT) from Bacillus anthracis, which contributes to anthrax, and C2 enterotoxin from Clostridium botulinum when applied in concentrations lower than those found in plasma of patients receiving standard DSF treatment for alcoholism (up to 20 µM). Moreover, this inhibitory effect is increased by copper, a known enhancer of DSF activity. LT and C2 are binary toxins, consisting of two non-linked proteins, an enzyme (A) and a separate binding/transport (B) subunit. To act cytotoxic, their proteolytically activated B subunits PA63 and C2IIa, respectively, form barrelshaped heptamers that bind to their cellular receptors and form complexes with their respective A subunits LF and C2I. The toxin complexes are internalized via receptor-mediated endocytosis and in acidified endosomes, PA63 and C2IIa form pores in endosomal membranes, which facilitate translocation of LF and C2I into the cytosol, where they act cytotoxic. In DT, A and B subunits are located within one protein, but DT also forms pores in endosomes that facilitate translocation of the A subunit. If cell binding, membrane translocation, or substrate modification is inhibited, cells are protected from intoxication. Our results implicate that DSF neither affects cellular binding nor the catalytic activity of the investigated toxins to a relevant extend, but interferes with the toxin pore-mediated translocation of the A subunits of DT, LT and C2 toxin, as demonstrated by membranetranslocation assays and toxin pore conductivity experiments in the presence or absence of DSF. Since toxin translocation across intracellular membranes represents a central step during cellular uptake of many bacterial toxins, DSF might neutralize a broad spectrum of medically relevant toxins. [ABSTRACT FROM AUTHOR]

Many bacteria act pathogenic by the release of AB-type protein toxins that efficiently enter human or animal cells and act as enzymes in their cytosol. This leads to disturbed cell functions and the clinical symptoms characteristic for the individual toxin. Therefore, molecules that directly target and neutralize these toxins provide promising novel therapeutic options. Here, we found that the FDA-approved drug disulfiram (DSF), used for decades to treat alcohol abuse, protects cells from intoxication with diphtheria toxin (DT) from Corynebacterium diphtheria, the causative agent of diphtheria, lethal toxin (LT) from Bacillus anthracis, which contributes to anthrax, and C2 enterotoxin from Clostridium botulinum when applied in concentrations lower than those found in plasma of patients receiving standard DSF treatment for alcoholism (up to 20 µM). Moreover, this inhibitory effect is increased by copper, a known enhancer of DSF activity. LT and C2 are binary toxins, consisting of two non-linked proteins, an enzyme (A) and a separate binding/transport (B) subunit. To act cytotoxic, their proteolytically activated B subunits PA63 and C2IIa, respectively, form barrel-shaped heptamers that bind to their cellular receptors and form complexes with their respective A subunits LF and C2I. The toxin complexes are internalized via receptor-mediated endocytosis and in acidified endosomes, PA63 and C2IIa form pores in endosomal membranes, which facilitate translocation of LF and C2I into the cytosol, where they act cytotoxic. In DT, A and B subunits are located within one protein, but DT also forms pores in endosomes that facilitate translocation of the A subunit. If cell binding, membrane translocation, or substrate modification is inhibited, cells are protected from intoxication. Our results implicate that DSF neither affects cellular binding nor the catalytic activity of the investigated toxins to a relevant extend, but interferes with the toxin pore-mediated translocation of the A subunits of DT, LT and C2 toxin, as demonstrated by membrane-translocation assays and toxin pore conductivity experiments in the presence or absence of DSF. Since toxin translocation across intracellular membranes represents a central step during cellular uptake of many bacterial toxins, DSF might neutralize a broad spectrum of medically relevant toxins.

ABSTRACT The Gram-positive bacterium Bacillus anthracis is the causative agent of anthrax and a bioterrorism threat worldwide. As a crucial second messenger in many bacterial species, cyclic di-AMP (c-di-AMP) modulates various key processes for bacterial homeostasis and pathogenesis. Overaccumulation of c-di-AMP alters cellular growth and reduces anthrax toxin expression as well as virulence in Bacillus anthracis by unresolved underlying mechanisms. In this report, we discovered that c-di-AMP binds to a series of receptors involved in potassium uptake in B. anthracis. By analyzing Kdp and Ktr mutants for osmotic stress, gene expression, and anthrax toxin expression, we also showed that c-di-AMP inhibits Kdp operon expression through binding to the KdpD and ydaO riboswitch; up-regulating intracellular potassium promotes anthrax toxin expression in c-di-AMP accumulated B. anthracis. Decreased anthrax toxin expression at high c-di-AMP occurs through the inhibition of potassium uptake. Understanding the molecular basis of how potassium uptake affects anthrax toxin has the potential to provide new insight into the control of B. anthracis.IMPORTANCEThe bacterial second messenger cyclic di-AMP (c-di-AMP) is a conserved global regulator of potassium homeostasis. How c-di-AMP regulates bacterial virulence is unknown. With this study, we provide a link between potassium uptake and anthrax toxin expression in Bacillus anthracis. c-di-AMP accumulation might inhibit anthrax toxin expression by suppressing potassium uptake.

An immunosensor for the assay of toxic biological warfare agents is a biosensor suitable for detecting hazardous substances such as aflatoxin, botulinum toxin, ricin, Shiga toxin, and others. The application of immunosensors is used in outdoor assays, point-of-care tests, as a spare method for more expensive devices, and even in the laboratory as a standard analytical method. Some immunosensors, such as automated flow-through analyzers or lateral flow tests, have been successfully commercialized as tools for toxins assay, but the research is ongoing. New devices are being developed, and the use of advanced materials and assay techniques make immunosensors highly competitive analytical devices in the field of toxic biological warfare agents assay. This review summarizes facts about current applications and new trends of immunosensors regarding recent papers in this area. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Treatments for advanced and recurrent ovarian cancer remain a challenge due to a lack of potent, selective, and effective therapeutics. Here, we developed the basis for a transformative anticancer strategy based on anthrax toxin that has been engineered to be selectively activated by the catalytic power of zymogen-activating proteases on the surface of malignant tumor cells to induce cell death. Exposure to the engineered toxin is cytotoxic to ovarian tumor cell lines and ovarian tumor spheroids derived from patient ascites. Preclinical studies demonstrate that toxin treatment induces tumor regression in several in vivo ovarian cancer models, including patient-derived xenografts, without adverse side effects, supportive of progression toward clinical evaluation. These data lay the groundwork for developing therapeutics for treating women with late-stage and recurrent ovarian cancers, utilizing a mechanism distinct from current anticancer therapies. [ABSTRACT FROM AUTHOR]

ABSTRACT: Objectives: Bacillus toyonensis is widespread in nature. Multidrug-resistant B. toyonensis strain 4HC1 was isolated from polyethylene submerged in the water column near a beach in Øygarden, Norway. We analysed the whole genome sequence of strain 4HC1 in order to understand the genetic basis of the observed phenotypic antibiotic resistance. Methods: Whole-genome sequencing of B. toyonensis strain 4HC1 was performed on Illumina MiSeq platform using 2 × 300 bp chemistry. The genome sequence was assembled using SPAdes v.3.13.0 and was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). Results: The draft genome of strain 4HC1 is 6 156 259 bp (133 contigs) in size with a GC content of 34.95%. The genome comprises 6089 protein-coding genes, 86 tRNAs and 24 rRNAs. Strain 4HC1 is resistant to cefotaxime, trimethoprim and ampicillin and carries various antibiotic resistance genes (ARGs), including several β-lactamases, aminoglycoside 6-adenylyltransferase, a TetM family tetracycline resistance gene, two different tetracycline efflux pumps, and a bleomycin resistance gene. Several virulence genes including genes involved in immune evasion, iron acquisition and toxins were also detected in strain 4HC1. Conclusion: The draft genome sequence of B. toyonensis strain 4HC1 released here shows the presence of various ARGs and virulence genes in a multidrug-resistant strain isolated from marine plastic.

Abstract Background To increase the size of the druggable proteome, it would be highly desirable to devise efficient methods to translocate designed binding proteins to the cytosol, as they could specifically target flat and hydrophobic protein-protein interfaces. If this could be done in a manner dependent on a cell surface receptor, two layers of specificity would be obtained: one for the cell type and the other for the cytosolic target. Bacterial protein toxins have naturally evolved such systems. Anthrax toxin consists of a pore-forming translocation unit (protective antigen (PA)) and a separate protein payload. When engineering PA to ablate binding to its own receptor and instead binding to a receptor of choice, by fusing a designed ankyrin repeat protein (DARPin), uptake in new cell types can be achieved. Results Prepore-to-pore conversion of redirected PA already occurs at the cell surface, limiting the amount of PA that can be administered and thus limiting the amount of delivered payload. We hypothesized that the reason is a lack of a stabilizing interaction with wild-type PA receptor. We have now reengineered PA to incorporate the binding domain of the anthrax receptor CMG2, followed by a DARPin, binding to the receptor of choice. This construct is indeed stabilized, undergoes prepore-to-pore conversion only in late endosomes, can be administered to much higher concentrations without showing toxicity, and consequently delivers much higher amounts of payload to the cytosol. Conclusion We believe that this reengineered system is an important step forward to addressing efficient cell-specific delivery of proteins to the cytosol.

An immunosensor for the assay of toxic biological warfare agents is a biosensor suitable for detecting hazardous substances such as aflatoxin, botulinum toxin, ricin, Shiga toxin, and others. The application of immunosensors is used in outdoor assays, point-of-care tests, as a spare method for more expensive devices, and even in the laboratory as a standard analytical method. Some immunosensors, such as automated flow-through analyzers or lateral flow tests, have been successfully commercialized as tools for toxins assay, but the research is ongoing. New devices are being developed, and the use of advanced materials and assay techniques make immunosensors highly competitive analytical devices in the field of toxic biological warfare agents assay. This review summarizes facts about current applications and new trends of immunosensors regarding recent papers in this area.

The Gram-positive bacterium Bacillus anthracis is the causative agent of anthrax and a bioterrorism threat worldwide. As a crucial second messenger in many bacterial species, cyclic di-AMP (c-di-AMP) modulates various key processes for bacterial homeostasis and pathogenesis. Overaccumulation of c-di-AMP alters cellular growth and reduces anthrax toxin expression as well as virulence in Bacillus anthracis by unresolved underlying mechanisms. In this report, we discovered that c-di-AMP binds to a series of receptors involved in potassium uptake in B. anthracis . By analyzing Kdp and Ktr mutants for osmotic stress, gene expression, and anthrax toxin expression, we also showed that c-di-AMP inhibits Kdp operon expression through binding to the KdpD and ydaO riboswitch; up-regulating intracellular potassium promotes anthrax toxin expression in c-di-AMP accumulated B. anthracis . Decreased anthrax toxin expression at high c-di-AMP occurs through the inhibition of potassium uptake. Understanding the molecular basis of how potassium uptake affects anthrax toxin has the potential to provide new insight into the control of B. anthracis. IMPORTANCEThe bacterial second messenger cyclic di-AMP (c-di-AMP) is a conserved global regulator of potassium homeostasis. How c-di-AMP regulates bacterial virulence is unknown. With this study, we provide a link between potassium uptake and anthrax toxin expression in Bacillus anthracis . c-di-AMP accumulation might inhibit anthrax toxin expression by suppressing potassium uptake., Competing Interests: The authors declare no conflict of interest.

Anthrax infection is associated with severe illness and high mortality. Protective antigen (PA) is the central component of the anthrax toxin, which is one of two major virulence factors of Bacillus anthracis, the causative agent of anthrax disease. Upon endocytosis, PA opens a pore in the membranes of endosomes, through which the cytotoxic enzymes of the toxin are extruded. The PA pore is formed by a cooperative conformational change in which the membrane‐penetrating loops of PA associate, forming a hydrophobic rim that pierces the membrane. Due to its crucial role in anthrax progression, PA is an important target for monoclonal antibody‐based therapy. cAb29 is a highly effective neutralizing antibody against PA. Here, the cryo‐EM structure of PA in complex with the Fab portion of cAb29 was determined. It was found that cAb29 neutralizes the toxin by clamping the membrane‐penetrating loop of PA to the static surface‐exposed loop of the D3 domain of the same subunit, thereby preventing pore formation. These results provide the structural basis for the antibody‐based neutralization of PA and bring into focus the membrane‐penetrating loop of PA as a target for the development of better anti‐anthrax vaccines. [ABSTRACT FROM AUTHOR]

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.

Translocation is essential to the anthrax toxin mechanism. Protective antigen (PA), the binding component of this AB toxin, forms an oligomeric pore that translocates lethal factor (LF) or edema factor, the active components of the toxin, into the cell. Structural details of the translocation process have remained elusive despite their biological importance. To overcome the technical challenges of studying translocation intermediates, we developed a method to immobilize, transition, and stabilize anthrax toxin to mimic important physiological steps in the intoxication process. Here, we report a cryoEM snapshot of PApore translocating the N-terminal domain of LF (LFN). The resulting 3.3 Å structure of the complex shows density of partially unfolded LFN near the canonical PApore binding site. Interestingly, we also observe density consistent with an α helix emerging from the 100 Å β barrel channel suggesting LF secondary structural elements begin to refold in the pore channel. We conclude the anthrax toxin β barrel aids in efficient folding of its enzymatic payload prior to channel exit. Our hypothesized refolding mechanism has broader implications for pore length of other protein translocating toxins. [ABSTRACT FROM AUTHOR]

We demonstrate experimentally that anthrax toxin complexes rupture artificial lipid bilayer membranes when isolated from the blood of infected animals. When the solution pH is temporally acidified to mimic that process in endosomes, recombinant anthrax toxin forms an irreversibly bound complex, which also destabilizes membranes. The results suggest an alternative mechanism for the translocation of anthrax toxin into the cytoplasm. [ABSTRACT FROM AUTHOR]

Background: To increase the size of the druggable proteome, it would be highly desirable to devise efficient methods to translocate designed binding proteins to the cytosol, as they could specifically target flat and hydrophobic protein-protein interfaces. If this could be done in a manner dependent on a cell surface receptor, two layers of specificity would be obtained: one for the cell type and the other for the cytosolic target. Bacterial protein toxins have naturally evolved such systems. Anthrax toxin consists of a pore-forming translocation unit (protective antigen (PA)) and a separate protein payload. When engineering PA to ablate binding to its own receptor and instead binding to a receptor of choice, by fusing a designed ankyrin repeat protein (DARPin), uptake in new cell types can be achieved. Results: Prepore-to-pore conversion of redirected PA already occurs at the cell surface, limiting the amount of PA that can be administered and thus limiting the amount of delivered payload. We hypothesized that the reason is a lack of a stabilizing interaction with wild-type PA receptor. We have now reengineered PA to incorporate the binding domain of the anthrax receptor CMG2, followed by a DARPin, binding to the receptor of choice. This construct is indeed stabilized, undergoes prepore-to-pore conversion only in late endosomes, can be administered to much higher concentrations without showing toxicity, and consequently delivers much higher amounts of payload to the cytosol. Conclusion: We believe that this reengineered system is an important step forward to addressing efficient cell-specific delivery of proteins to the cytosol. [ABSTRACT FROM AUTHOR]

AIM: Bacillus anthracis is a causative agent of zoonotic anthrax disease. In the last years, significant progress in therapy and diagnosis of anthrax was made. Concurrently, knowledge about anthrax progression, molecular pathology and release of anthrax toxin during the disease has improved. This review covers the recent progress in this fi eld. METHODS: In this review, specifications of B. anthracis, anthrax disease, medical and biomedical countermeasures and diagnostic tools were surveyed. The actual literature was summarized and relevance of the microorganism as a biological warfare agent and the ways how to reduce its impact including therapeutic protocols were written and discussed. RESULTS: Currently, the microorganism is considered one of the top biological warfare agents due to lethality, long term stability of spores, easy dissemination and production. The recent research is focused on countermeasures suitable for reduction of consequences by a misuse of the microorganism in form of biological weapon (Tab. 3, Fig. 1, Ref. 101). Text in PDF www.elis.sk. [ABSTRACT FROM AUTHOR]

Background Human-use probiotics have recently been associated with clinical infections and antibiotic resistance transfer, raising public concern over their safety. However, despite their extensive application in aquaculture and animal husbandry, the safety of animal-use probiotics remains poorly described. Methods We evaluated the safety of 92 animal-use probiotics from China. The pattern of spread of pathogens from probiotics and the consequent public health implications were also examined by conducting in-field genomic surveillance at 2 farms. Results A total of 123 probiotic Bacillus species isolates were obtained from 92 brands of probiotics, of which 45 isolates were resistant to antibiotics. Notably, 33.7% of probiotic products were contaminated with life-threatening pathogens such as Klebsiella pneumoniae. Genomic surveillance at a chicken farm identified an anthrax toxin–positive Bacillus cereus strain in a probiotic product used as a feed supplement, which was transferred into the groundwater and to a nearby fish farm. Following up retrospective analysis of the surveillance data during 2015–2018 in 3 provinces retrieved 2 B. cereus strains from human with intestinal anthrax symptoms and confirmed the transmission of B. cereus from farm to human. Surveillance of anthrax toxin revealed that cya was detected in 8 of 31 farms. Conclusions This study provides the first national safety survey of animal-use probiotics in China and confirms the spillover effects of probiotics from the farms to human. These results suggest that the large-scale application of pathogen-containing probiotics leads to the transfer of pathogens, with worrisome implications for public health. Good Manufacturing Practice should be implemented during the production of all probiotics. Animal-use probiotic products are frequently contaminated with viable pathogenic bacteria. This study revealed that virulent probiotic organisms and contaminating pathogens were colonized with farm animals and shed into the environment, which facilitated the transfer of pathogens to humans. [ABSTRACT FROM AUTHOR]

Bladder cancer is the sixth most common cancer in the United States, and it exhibits an alarming 70% recurrence rate. Thus, the development of more efficient antibladder cancer approaches is a high priority. Accordingly, this work provides the basis for a transformative anticancer strategy that takes advantage of the unique characteristics of the bladder. Unlike mucin‐shielded normal bladder cells, cancer cells are exposed to the bladder lumen and overexpress EGFR. Therefore, we used an EGF‐conjugated anthrax toxin that after targeting EGFR was internalized and triggered apoptosis in exposed bladder cancer cells. This unique agent presented advantages over other EGF‐based technologies and other toxin‐derivatives. In contrast to known agents, this EGF‐toxin conjugate promoted its own uptake via receptor microclustering even in the presence of Her2 and induced cell death with a LC50 < 1 nM. Furthermore, our data showed that exposures as short as ≈3 min were enough to commit human (T24), mouse (MB49) and canine (primary) bladder cancer cells to apoptosis. Exposure of tumor‐free mice and dogs with the agent resulted in no toxicity. In addition, the EGF‐toxin was able to eliminate cells from human patient tumor samples. Importantly, the administration of EGF‐toxin to dogs with spontaneous bladder cancer, who had failed or were not eligible for other therapies, resulted in ~30% average tumor reduction after one treatment cycle. Because of its in vitro and in vivo high efficiency, fast action (reducing treatment time from hours to minutes) and safety, we propose that this EGF–anthrax toxin conjugate provides the basis for new, transformative approaches against bladder cancer. What's new? Bladder cancer is hard to treat and after surgery has a notoriously high recurrence rate (70%). Here the authors propose a new therapeutic strategy combining epidermal growth factor (EGF) with anthrax toxin. Since bladder cancer cells are exposed to urine and carry high levels of EGF receptor, the toxin after intravesical application was specifically taken up by cancer cells and induced rapid apoptosis, regardless of whether cancer cells expressed another EGF receptor, Her2, or not. This underscores the broad potential of the approach for treatment of bladder tumors in the future. [ABSTRACT FROM AUTHOR]

Anthrax toxin has evolved to translocate its toxic cargo proteins to the cytosol of cells carrying its cognate receptor. Cargo molecules need to unfold to penetrate the narrow pore formed by its membrane-spanning subunit, protective antigen (PA). Various alternative cargo molecules have previously been tested, with some showing only limited translocation efficiency, and it may be assumed that these were too stable to be unfolded before passing through the anthrax pore. In this study, we systematically and quantitatively analyzed the correlation between the translocation of various designed ankyrin repeat proteins (DARPins) and their different sizes and thermodynamic stabilities. To measure cytosolic uptake, we used biotinylation of the cargo by cytosolic BirA, and we measured cargo equilibrium stability via denaturant-induced unfolding, monitored by circular dichroism (CD). Most of the tested DARPin cargoes, including target-binding ones, were translocated to the cytosol. Those DARPins, which remained trapped in the endosome, were confirmed by CD to show a high equilibrium stability. We could pinpoint a stability threshold up to which cargo DARPins still get translocated to the cytosol. These experiments have outlined the requirements for translocatable binding proteins, relevant stability measurements to assess translocatable candidates, and guidelines to further engineer this property if needed.

Background: Protective antigen of anthrax toxin, after touching the cell receptors, plays an important role in the pathogenesis of toxin. The purpose of this study was to investigate the interaction of anthrax toxin protective antigen and four great combination propolis included caffeic acid, benzyl caffeate, cinnamic acid and kaempferol using the softwares and bioinformatics web servers. Methods: Three-dimensional structure of protective antigen (receptor) obtains from Protein Data Bank (PDB). Four of the main components from propolis were selected as ligand and their 3D-structures were obtained from ChemSpider and ZINC compound database. The interaction of each ligand and receptor was assessed by SwissDock server (http://www.swissdock.ch/) and BSP-SLIM server (http://zhanglab.ccmb.med.umich.edu/BSP-SLIM). Docking results appears with Fullfitness numbers (in kcal/mol). Identification of amino acids involved in ligand and receptor interaction, was performed using the Chimera software; UCSF Chimera program (http://www.cgl.ucsf.edu/). Results: The results of interaction between propolis components and protective antigen by BSP-SLIM server showed that the most interaction was related with benzyl caffeate, caffeic acid, kaempferol and cinnamic acid, respectively. Results for the desired ligand Interaction with protective antigen genes using SwissDock server showed that the caffeic acid had ΔG equals -9.10 kcal/mol and FullFitness equal to -993.16 kcal/mol respectively. The analysis of interaction between ligands with amino-acids of protective antigen indicated that the interaction of Caffeic acid whit Glutamic acid 117 had energy -15.5429 kcal/mol. Conclusion: Finding strong and safe inhibitors for anthrax toxin is very useful method for inhibiting its toxicity to cell. In this study the binding ability of four flavonoids to protective antigen was studied. Glutamic acid 117 is very effective in protective antigen binding and cell receptor and subsequent in virulent of anthrax toxin. Effective interaction of caffeic acid in propolis and glutamic acid 117 can be as useful in preventing the toxic effect on cell. According to our results, all four flavonoids tested in this study have binding activity to protective antigen and are good choices for fighting against anthrax.

In recent years, anthrax toxin has been reengineered to act as a highly specific antiangiogenic cancer therapeutic, shown to kill tumors in animal models. This has been achieved by modifying protective antigen (PA) so that its activation and toxicity require the presence of two proteases, matrix metalloproteinase (MMP) and urokinase plasminogen activator (uPA), which are upregulated in tumor microenvironments. These therapeutics consist of intercomplementing PA variants, which are individually nontoxic, but form functional toxins upon complementary oligomerization. Here, we have created a dual‐protease requiring PA targeting system which utilizes bismaleimide cross‐linked PA (CLPA) rather than the intercomplementing PA variants. Three different CLPA agents were tested and, as expected, found to exclusively form octamers. Two of the CLPA agents have in vitro toxicities equal to those of previous intercomplementing agents, while the third CLPA agent had compromised in vitro cleavage and was significantly less cytotoxic. We hypothesize this difference was due to steric hindrance caused by cross‐linking two PA monomers in close proximity to the PA cleavage site. Overall, this work advances the development and use of the PA and LF tumor‐targeting system as a practical cancer therapeutic, as it provides a way to reduce the drug components of the anthrax toxin drug delivery system from three to two, which may lower the cost and simplify testing in clinical trials. HIGHLIGHT: Previously, anthrax toxin has been reengineered to act as a highly specific antiangiogenic cancer therapeutic. Here, we present a version, which utilizes bismaleimide cross‐linked protective antigen (PA) rather than intercomplementing PA variants. This advances the development of anthrax toxin as a practical cancer therapeutic as it reduces the components of the drug delivery system to two, which may lower the cost and simplify testing in clinical trials. [ABSTRACT FROM AUTHOR]

The antibiotic bacitracin (Bac) inhibits cell wall synthesis of gram-positive bacteria. Here, we discovered a totally different activity of Bac: the neutralization of bacterial exotoxins. Bac prevented intoxication of mammalian cells with the binary enterotoxins Clostridiumbotulinum C2, C. perfringens, C. difficile transferase (CDT), and Bacillus anthracis lethal toxin. The transport (B) subunits of these toxins deliver their respective enzyme (A) subunits into cells. Following endocytosis, the B subunits form pores in membranes of endosomes, which mediate translocation of the A subunits into the cytosol. Bac inhibited formation of such B pores in lipid bilayers in vitro and in living cells, thereby preventing translocation of the A subunit into the cytosol. Bac preserved the epithelial integrity of toxin-treated CaCo-2 monolayers, a model for the human gut epithelium. In conclusion, Bac should be discussed as a therapeutic option against infections with medically relevant toxin-producing bacteria, including C. difficile and B. anthracis, because it inhibits bacterial growth and neutralizes the secreted toxins. [ABSTRACT FROM AUTHOR]

Abstract Anthrax toxin receptor 1 (ANTXR1) is a transmembrane protein with an extracellular domain which is deeply associated with the process of bone formation and plays an important role in angiogenesis. However, there have been no reports investigating the effects of ANTXR1 on bone metabolism mediated by the two types of bone cells, osteoclasts, and osteoblasts. The aim of this study is to reveal the role of ANTXR1 in the differentiation and function of osteoclasts and osteoblasts. We found that ANTXR1 positively regulated the receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclast differentiation and bone resorption with no effects on osteoblast differentiation by performing gain- and loss-of-function studies. During ANTXR1-mediated regulation of osteoclastogenesis, phosphorylation of early signal transducers such as c-Jun N-terminal kinase (JNK), Akt, inhibitor of kappa B (IκB), and phospholipase C gamma 2 (PLCγ2) was affected, which in turn altered the mRNA and protein levels of c-Fos and nuclear factor of activated T cells cytoplasmic 1 (NFATc1). In addition, genetic manipulation of ANTXR1 in bone marrow macrophages (BMMs) modulated the capillary-like tube formation in HUVECs via secretion of two angiogenic factors, matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor-A (VEGF-A). These results elucidated the importance of ANTXR1 in osteoclast differentiation and functional activity, as well as, osteoclast-mediated angiogenesis of endothelial cells. Taken together, we propose that ANTXR1 might be a promising candidate for gene therapy for bone metabolic diseases and further, might potentially serve as an important biomarker in the field of bone metastasis associated with vascularization. Graphical abstract Image 1 Highlights • We first investigated the effects and the mechanisms of Antxr1 in osteoclast differentiation and bone resorbing activity. • ANTXR1 positively regulated the RANKL-induced osteoclast differentiation and bone resorption. • ANTXR1 knockdown inhibited c-Fos and NFATc1 expression by de-phosphorylation of JNK, Akt, IκB, and PLCg2. • Genetic manipulation of ANTXR1 modulated the capillary-like tube formation by HUVECs via secretion of MMP-9 and VEGF-A. • ANTXR1 might be a promising candidate for gene therapy for bone metabolic diseases. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Anthrax is a fatal infectious disease which can affect animals and humans alike. Anthrax outbreaks occur periodically in animals, and they are of particular concern in herbivores, due to substantial economic consequences associated with animal death. The purpose of this study is to develop optimal control interventions that focus on vaccinating susceptible animals and/or removing infected carcasses. Our mathematical goal is to minimize the infectious animal population while reducing the cost of interventions. Optimal control interventions are derived theoretically, and numerical results with conclusions are presented. [ABSTRACT FROM AUTHOR]

Over the last few decades, proteins and peptides have become increasingly more common as FDA-approved drugs, despite their inefficient delivery due to their inability to cross the plasma membrane. In this context, bacterial two-component systems, termed AB toxins, use various protein-based membrane translocation mechanisms to deliver toxins into cells, and these mechanisms could provide new insights into the development of bio-based drug delivery systems. These toxins have great potential as therapies both because of their intrinsic properties as well as the modular characteristics of both subunits, which make them highly amenable to conjugation with various drug classes. This review focuses on the therapeutical approaches involving the internalization mechanisms of three representative AB toxins: botulinum toxin type A, anthrax toxin, and cholera toxin. We showcase several specific examples of the use of these toxins to develop new therapeutic strategies for numerous diseases and explain what makes these toxins promising tools in the development of drugs and drug delivery systems.

Here, we present empirical data documenting the siRNA-mediated protection of cells after Zika virus (ZIKV) infection. siRNAs were designed to target well-conserved sequences across the ZIKV genome. Several delivery technologies were utilized. After the electroporation of 100 nM siRNA into human hepatocyte-derived carcinoma (Huh7) cells, the Feron Zv-2 sequence (specific to the ZIKV NS3 gene) yielded a cell viability of 150.3% ± 7.4% (SEM: n = 4) (p = 0.0004) relative to the cells treated only with the virus (33.9% ± 12%, SEM: n = 4). Furthermore, 100 nM siRNA Feron Zv-4 (specific to ZIKV 3’UTR) resulted in 119.1% ± 11.2% cell viability (SEM: n = 4) relative to the control cells treated with ZIKV (p = 0.0021). The cells were electroporated with siRNA prior to ZIKV infection and viability was monitored four days after this. Additionally, two novel siRNA delivery systems were tested. The first utilized recombinant Bacillus anthracis PA83 (octomer-forming mutants), co-incubated with the N-terminal 255 amino acids of B. anthracis lethal factor (LFn) fused in-frame with the RNA binding domain for human protein kinase R (LFn-PKR) at a concentration of 50 µg/mL (each). Here, baby hamster kidney (BHK) cells, treated with 100 nM siRNA Feron Zv-1, yielded 79.0% ± 4.0% viability relative to the control (50.2% ± 1.7%, SEM: n = 3) three days after exposure to ZIKV (p = 0.0096). Finally, HeLa exosomes loaded with siRNA Feron-Zv2 were incubated with Huh7 cells prior to ZIKV infection. For the siRNA-exosome treated cells, a viability of 123% ± 46% (SEM: n = 18), relative to 8% ± 16% (SEM: n = 18) for the same concentration of control HeLa exosomes, was recorded (p = 0.0416). In each instance, 0.3 moI was used and cell viability monitored using the PierceTM Firefly Luciferase Glow Assay Kit by Thermo ScientificTM. Here, we show for the first time that siRNA can significantly reduce ZIKV-induced cell killing. Future work will require quantitating ZIKV mRNA in relation to siRNA treatment, as well as testing the siRNAs and delivery systems within more complex models.

Processing of certain viral proteins and bacterial toxins by host serine proteases is a frequent and critical step in virulence. The coronavirus spike glycoprotein contains three (S1, S2, and S2′) cleavage sites that are processed by human host proteases. The exact nature of these cleavage sites, and their respective processing proteases, can determine whether the virus can cross species and the level of pathogenicity. Recent comparisons of the genomes of the highly pathogenic SARS-CoV2 and MERS-CoV, with less pathogenic strains (e.g., Bat-RaTG13, the bat homologue of SARS-CoV2) identified possible mutations in the receptor binding domain and in the S1 and S2′ cleavage sites of their spike glycoprotein. However, there remains some confusion on the relative roles of the possible serine proteases involved for priming. Using anthrax toxin as a model system, we show that in vivo inhibition of priming by pan-active serine protease inhibitors can be effective at suppressing toxicity. Hence, our studies should encourage further efforts in developing either pan-serine protease inhibitors or inhibitor cocktails to target SARS-CoV2 and potentially ward off future pandemics that could develop because of additional mutations in the S-protein priming sequence in coronaviruses.

The nucleotide-free chaperonin GroEL is capable of capturing transient unfolded or partially unfolded states that flicker in and out of existence due to large-scale protein dynamic vibrational modes. In this work, three short vignettes are presented to highlight our continuing advances in the application of GroEL biosensor biolayer interferometry (BLI) technologies and includes expanded uses of GroEL as a molecular scaffold for electron microscopy determination. The first example presents an extension of the ability to detect dynamic pre-aggregate transients in therapeutic protein solutions where the assessment of the kinetic stability of any folded protein or, as shown herein, quantitative detection of mutant-type protein when mixed with wild-type native counterparts. Secondly, using a BLI denaturation pulse assay with GroEL, the comparison of kinetically controlled denaturation isotherms of various von Willebrand factor (vWF) triple A domain mutant-types is shown. These mutant-types are single point mutations that locally disorder the A1 platelet binding domain resulting in one gain of function and one loss of function phenotype. Clear, separate, and reproducible kinetic deviations in the mutant-type isotherms exist when compared with the wild-type curve. Finally, expanding on previous electron microscopy (EM) advances using GroEL as both a protein scaffold surface and a release platform, examples are presented where GroEL-protein complexes can be imaged using electron microscopy tilt series and the low-resolution structures of aggregation-prone proteins that have interacted with GroEL. The ability of GroEL to bind hydrophobic regions and transient partially folded states allows one to employ this unique molecular chaperone both as a versatile structural scaffold and as a sensor of a protein's folded states.

In Russia in the second half of the XIX century, research schools developing anthrax vaccines for agriculture were formed. According to their level, they are not inferior to similar Western schools. At the end of the 1930s, Soviet military scientists were the first in the world to create the anthrax vaccine on the basis of the spores of unencapsulated live strains of Bacillus anthracis for medical use. In the 1940–1960s, domestic scientists determined the principles of the development of anthrax vaccines, which enabled to avoid failures of their Western colleagues, when developing the vaccines capable of protecting the population from biological weapons with anthrax spores as the harmful agent. Russian military scientists in the 1990s managed to keep the vaccine strains of anthrax bacteria and to restore the technological capability for their manufacture, which helps to protect the population of the Russian Federation from natural anthrax outbreaks and from biological terror. At a time when there is a need in creating immunity in humans against infection caused by inhalation of anthrax spores, the most reasonable decision for the upcoming decades is to use the domestic combined vaccine that combines unencapsulated live strains of B.anthracis and anthrax toxin protective antigen.

The principal portal for anthrax infection in natural animal outbreaks is the digestive tract. Enteric exposure to anthrax, which is difficult to detect or prevent in a timely manner, could be exploited as an act of terror through contamination of human or animal food. Our group has developed a novel animal model of gastrointestinal (GI) anthrax for evaluation of disease pathogenesis and experimental therapeutics, utilizing vegetative Bacillus anthracis (Sterne strain) administered to A/J mice (a complement-deficient strain) by oral gavage. We hypothesized that a humanized recombinant monoclonal antibody (mAb) * that neutralizes the protective antigen (PA) component of B. anthracis lethal toxin (LT) and edema toxin (ET) could be an effective treatment. Although the efficacy of this anti-anthrax PA mAb has been shown in animal models of inhalational anthrax, its activity in GI infection had not yet been ascertained. We hereby demonstrate that passive immunotherapy with anti-anthrax PA mAb, administered at the same time as gastrointestinal exposure to B. anthracis, prevents lethal sepsis in nearly all cases (>90%), while a delay of up to forty-eight hours in treatment still greatly reduces mortality following exposure (65%). Moreover, passive immunotherapy protects against enteric invasion, associated mucosal injury and subsequent dissemination by gastrointestinal B. anthracis, indicating that it acts to prevent the initial stages of infection. * Expired raxibacumab being cycled off the Strategic National Stockpile; biological activity confirmed by in vitro assay.

Seneca Valley virus (SVV) is an oncolytic picornavirus with selective tropism for neuroendocrine cancers. SVV mediates cell entry by attachment to the receptor anthrax toxin receptor 1 (ANTXR1). Here we determine atomic structures of mature SVV particles alone and in complex with ANTXR1 in both neutral and acidic conditions, as well as empty "spent" particles in complex with ANTXR1 in acidic conditions by cryoelectron microscopy. SVV engages ANTXR1 mainly by the VP2 DF and VP1 CD loops, leading to structural changes in the VP1 GH loop and VP3 GH loop, which attenuate interprotomer interactions and destabilize the capsid assembly. Despite lying on the edge of the attachment site, VP2 D146 interacts with the metal ion in ANTXR1 and is required for cell entry. Though the individual substitution of most interacting residues abolishes receptor binding and virus propagation, a serineto- alanine mutation at VP2 S177 significantly increases SVV proliferation. Acidification of the SVV-ANTXR1 complex results in a major reconfiguration of the pentameric capsid assemblies, which rotate ~20° around the icosahedral fivefold axes to form a previously uncharacterized spent particle resembling a potential uncoating intermediate with remarkable perforations at both two- and threefold axes. These structures provide high-resolution snapshots of SVV entry, highlighting opportunities for anticancer therapeutic optimization. [ABSTRACT FROM AUTHOR]

Recently, the use of oncolytic viruses in cancer therapy has become a realistic therapeutic option. Seneca Valley Virus (SVV) is a newly discovered picornavirus, which has earned a significant reputation as a potent oncolytic agent. Anthrax toxin receptor 1 (ANTXR1), one of the cellular receptors for the protective antigen secreted by Bacillus anthracis, has been identified as the high-affinity cellular receptor for SVV. Here, we report the structure of the SVVANTXR1 complex determined by single-particle cryo-electron microscopy analysis at near-atomic resolution. This is an example of a shared receptor structure between a mammalian virus and a bacterial toxin. Our structure shows that ANTXR1 decorates the outer surface of the SVV capsid and interacts with the surface-exposed BC loop and loop II of VP1, "the puff" of VP2 and "the knob" of VP3. Comparison of the receptor-bound capsid structure with the native capsid structure reveals that receptor binding induces minor conformational changes in SVV capsid structure, suggesting the role of ANTXR1 as an attachment receptor. Furthermore, our results demonstrate that the capsid footprint on the receptor is not conserved in anthrax toxin receptor 2 (ANTXR2), thereby providing a molecular mechanism for explaining the exquisite selectivity of SVV for ANTXR1. [ABSTRACT FROM AUTHOR]