Toxins

AbstractSnakebite envenoming is a neglected tropical disease that causes substantial mortality and morbidity globally. The puff adder (Bitis arietans) and saw-scaled viper (Echis romani) have cytotoxic venoms that cause permanent injury via tissue-destructive dermonecrosis around the bite site. Identification of cytotoxic toxins within these venoms will allow development of targeted treatments, such as small molecule inhibitors or monoclonal antibodies to prevent snakebite morbidity. Venoms from both species were fractionated using gel filtration chromatography, and a combination of cell-based cytotoxicity approaches, SDS-PAGE gel electrophoresis, and enzymatic assays were applied to identify venom cytotoxins in the resulting fractions. Our results indicated that snake venom metalloproteinase (SVMP) toxins are predominately responsible for causing cytotoxic effects across both venoms, but that the PII subclass of SVMPs are likely the main driver of cytotoxicity following envenoming by B. arietans, whilst the structurally distinct PIII subclass of SVMPs are responsible for conveying this effect in E. romani venom. Identification of distinct SVMPs as the primary cytotoxicity-causing toxins in these two African viper venoms will facilitate the future design and development of novel therapeutics targeting these medically important venoms, which in turn could help to mitigate the severe life and limb threatening consequences of tropical snakebite. Key ContributionSVMP toxins were identified as the primary cytotoxicity-causing toxins in the venoms of the puff adder (Bitis arietans) and saw-scaled viper (Echis romani); PII and PIII SVMPs, respectively. This cytotoxicity can be prevented using the metalloproteinase-inhibiting chelator EDTA, suggesting targeted drugs/antibodies may be a viable option for future treatment.

Venom expressed by the nearly 50,000 species of spiders on Earth largely remains an untapped reservoir of a diverse array of biomolecules with potential for pharmacological and agricultural applications. A large fraction of the noxious components of spider venoms are a functionally diverse family of structurally related polypeptides with an inhibitor cystine knot (ICK) motif. The cysteine-rich nature of these toxins makes structural elucidation difficult, and most studies have focused on venom components from the small handful of medically relevant spider species such as the highly aggressive Brazilian wandering spider Phoneutria nigriventer. To alleviate difficulties associated with the study of ICK toxins in spiders, we devised a comprehensive approach to explore the evolutionary patterns that have shaped ICK functional diversification using venom gland transcriptomes and proteomes from phylogenetically distinct lineages of wandering spiders and their close relatives. We identified 626 unique ICK toxins belonging to seven topological elaborations. Phylogenetic tests of episodic diversification revealed distinct regions between cysteine residues that demonstrated differential evidence of positive or negative selection, which may have structural implications towards the specificity and efficacy of these toxins. Increased taxon sampling and whole genome sequencing will provide invaluable insights to further understand the evolutionary processes that have given rise to this diverse class of toxins.

Background and PurposePeptide toxins isolated from animal venom are potent and selective modulators of ion channels, and promising therapeutic leads. Due to intricate disulphide bridge patterns, they are often challenging to produce in standard laboratory settings, which limits engineering approaches to manipulate their structure-function properties. Given the low cost, wide accessibility, and versatility of recombinant expression systems for protein production, we set out to establish a straightforward high-yield strategy across a broad panel of peptide toxins from snakes, spiders and scorpions. Experimental Approach13 toxin DNA sequences were genetically fused to the C-terminus of either bivalent or monovalent human IgG1 antibody fragment crystallisable (Fc) domain sequences and expressed recombinantly from mammalian Expi293F cells. Affinity-purified proteins were evaluated by SDS-PAGE and size-exclusion chromatography (SEC). Function was assessed by Ca2+ flux assays on CN21 cells, or whole-cell electrophysiology on human embryonic kidney (HEK293T) cells, Chinese hamster ovary (CHO) cells, or dorsal root ganglion (DRG) neurons. Immunocytochemistry using HEK293T cells and mouse DRG neurons assessed Fc-toxin fusion binding. Key ResultsMonovalent Fc-toxin fusions consistently yielded 1-6 mg of pure, non-proteolytically cleaved protein from 20-70 ml cultures for several toxin types, including three-finger toxins from snakes, inhibitory cystine knot (ICK) toxins from spiders, and -toxins from scorpions, substantially surpassing the performance of unfused toxins or bivalent Fc-toxin fusions which gave low or no yield. Snake toxins targeting nicotinic acetylcholine receptors retained high single digit nanomolar inhibitory potency. Spider and scorpion toxins targeting the voltage-gated Na+ channel Nav1.7 retained pharmacological function and selectivity across a panel of five Nav subtypes, albeit with reduced potencies that did not exceed [~]70 nM. Conclusions and ImplicationsWe present a strategy for straightforward robust production of pure, monodisperse, and functional animal venom-derived toxins. This lowers the barrier to toxin production in a standard laboratory setting for follow-on engineering purposes.

Snakebite envenoming is a neglected tropical disease that causes over 100,000 deaths annually. Envenomings result in variable pathologies, but systemic neurotoxicity is among the most serious and is currently only treated with difficult to access and variably efficacious commercial antivenoms. Venom-induced neurotoxicity is often caused by -neurotoxins antagonising the muscle-type nicotinic acetylcholine receptor (nAChR), a ligand-gated ion channel. Discovery of therapeutics targeting -neurotoxins is hampered by relying on binding assays that do not reveal restoration of receptor activity or more costly and/or lower throughput electrophysiology-based approaches. Here, we report the validation of a screening assay for nAChR activation using immortalised TE671 cells expressing the {gamma}-subunit containing muscle-type nAChR and a fluorescent dye that reports changes in cell membrane potential. Assay validation using traditional nAChR agonists and antagonists, which either activate or block ion fluxes, was consistent with previous studies. We then characterised antagonism of the nAChR by a variety of elapid snake venoms that cause muscle paralysis in snakebite victims, before defining the toxin-inhibiting activities of commercial antivenoms, and new types of snakebite therapeutic candidates, namely monoclonal antibodies, decoy receptors, and small molecules. Our findings show robust evidence of assay uniformity across 96-well plates and highlight the amenability of this approach for the future discovery of new snakebite therapeutics via screening campaigns. The described assay therefore represents a useful first-step approach for identifying -neurotoxins and their inhibitors in the context of snakebite envenoming, and it should provide wider value for studying modulators of nAChR activity from other sources.

All the members of the phylum Cnidaria are characterized by the production of venom in specialized structures, the nematocysts. Venom of jellyfish (Medusozoa) and sea anemones (Anthozoa) has been investigated since the 1970s, revealing a remarkable molecular diversity. Specifically, sea anemones harbour a rich repertoire of neurotoxic peptides, some of which have been developed in drug leads. However, venoms of the vast majority of Anthozoa species remain uncharacterized, particularly in the class Octocorallia. To fill this gap, we applied a proteo-transcriptomic approach to investigate the venom composition in Eunicella singularis, a gorgonian species common in Mediterranean hard-bottom benthic communities. Our results highlighted the peculiarities of the venom of E. singularis with respect to sea anemones, which is reflected in the presence of several toxins with novel folds, worthy of functional characterization. A comparative genomic survey across the octocoral radiation allowed us to generalize these findings and provided insights into the evolutionary history, molecular diversification patterns and putative adaptive roles of venom toxins. A comparison of whole-body and nematocyst proteomes revealed the presence of different cytolytic toxins inside and outside the nematocysts. Two instances of differential maturation patterns of toxin precursors were also identified, highlighting the intricate regulatory pathways underlying toxin expression.

Spider venoms are a unique source of bioactive peptides, many of which display remarkable biological stability and neuroactivity. Phoneutria nigriventer, often referred to as the Brazilian wandering spider, banana spider or "armed" spider, is endemic to South America and amongst the most dangerous venomous spiders in the world. There are 4,000 envenomation accidents with P. nigriventer each year in Brazil, which can lead to symptoms including priapism, hypertension, blurred vision, sweating, and vomiting. In addition to its clinical relevance, P. nigriventer venom contains peptides that provide therapeutic effects in a range of disease models. In this study, we explored the neuroactivity and molecular diversity P. nigriventer venom using fractionation-guided high-throughput cellular assays coupled to proteomics and multi-pharmacology activity to broaden the knowledge about this venom and its therapeutic potential and provide a proof-of-concept for an investigative pipeline to study spider-venom derived neuroactive peptides. We coupled proteomics with ion channel assays using a neuroblastoma cell line to identify venom compounds that modulate the activity of voltage-gated sodium and calcium channels, as well as the nicotinic acetylcholine receptor. Our data revealed that P. nigriventer venom is highly complex compared to other neurotoxin-rich venoms and contains potent modulators of voltage-gated ion channels which were classified into four families of neuroactive peptides based on their activity and structures. In addition to the reported P. nigriventer neuroactive peptides, we identified at least 27 novel cysteine-rich venom peptides for which their activity and molecular target remains to be determined. Our findings provide a platform for studying the bioactivity of known and novel neuroactive components in the venom of P. nigriventer and other spiders and suggests that our discovery pipeline can be used to identify ion channel-targeting venom peptides with potential as pharmacological tools and to drug leads.

Increase of infections caused by microorganisms resistant to conventional antibiotics is a health problem in Brazil and worldwide. The search for new molecules capable of inhibiting the growth of pathogens is a challenge for researchers, who find in venoms a rich source of biomolecules, including antimicrobial peptides (AMPs). The Brazilian scorpion, Tityus serrulatus, is one of the species that cause serious accidents; its venom is rich in neurotoxins that have been well characterized, highlighting their activities on channels (especially sodium and potassium). In this study we identifified and characterized one AMPs in T. serrulatus venom. After milking, the venom was fractioned by high performance liquid chromatography and the fractions were tested by liquid growth inhibition assay, the minimum inhibitory concentration (MIC) against Escherichia coli, Micrococcus luteus, Candida albicans and Aspergillus niger. The fraction identified with antimicrobial activity was analyzed by electrophoresis and mass spectrometry and this AMP (with molecular mass 6.882 kDa) has a similar amino acid sequence to TsTX-{kappa} beta, a neurotoxin that acts on ion channels. The TsTX-{kappa} beta in this study was identified by de novo sequencing. This peptide showed activity against all microorganisms tested. At high concentrations, this peptide, showed hemolytic activity against human erythrocytes. This is a new function described for this peptide, the identification of antimicrobial activity in a neurotoxin already known. Key ContributionMultifunction: antimicrobial and hemolytic activity associated to TsTX-{kappa} beta, a neurotoxin that acts on potassium channels.

African trypanosomes, such as Trypanosoma brucei, are flagellated protozoa which proliferate in mammals and cause a variety of diseases in people and animals. In a mammalian host, the external face of the African trypanosome plasma membrane is covered by a densely packed coat formed of variant surface glycoprotein (VSG), which counteracts the host adaptive immune response by antigenic variation. The VSG is attached to the external face of the plasma membrane by covalent attachment of the C-terminus to a glycosylphosphatidylinositol. As the trypanosome grows, newly synthesised VSG is added to the plasma membrane by vesicle fusion to the flagellar pocket, the sole location of exo- and endocytosis. Snake venoms contain dozens of components including proteases and phospholipases. Here, we investigated the effect of Naja nigricollis on T. brucei with the aim of describing the response of the trypanosome to hydrolytic attack on the VSG. We found no evidence for VGS hydrolysis however N. nigricollis venom caused: (i) an enlargement of the flagellar pocket, (ii) the Rab11 positive endosomal compartments to adopt an abnormal dispersed localisation, and (iii) a cell cycle arrest prior to cytokinesis. A single protein family, the phospholipases A2s present in N. nigricollis venom, was necessary and sufficient for the effects. This study provides new molecular insight into T. brucei biology and possibly describes mechanisms that could be exploited for T. brucei targeting.

Short abstractConsidering that there are still many species little-studied, this work aimed to analyze the venom of the spider Avicularia juruensis searching for antimicrobial peptides. Using reverse-phase high-performance liquid chromatography, microbial growth inhibition assay, transcriptomics, and proteomics approaches we identified three antimicrobial peptides: Avilin, Juruin_2, and Juruenine. All of them showed similarities with neurotoxins that act on ion channels and, probably, they have the ICK motif. The study of animal venoms is of great importance to carry out the characterization of unknown components and that may have a biotechnological application, in special venoms from spiders that are from less studied families. Spiders are the most successful group of venomous animals, comprising more than 50,350 species distributed in all terrestrial habitats. One strategy that facility their broad distribution is the production of elaborate venoms, which are composed of inorganic salts, organic molecules with low molecular mass, free amino acids, small polypeptides, linear peptides, nucleotides, disulfide-rich peptides, enzymes, and high molecular mass proteins. Considering that there are still many species little-studied, this work aimed to analyze the venom of the mygalomorph spider Avicularia juruensis searching for new antimicrobial peptides. Using reverse-phase high-performance liquid chromatography, microbial growth inhibition assay, transcriptomics, and proteomics approaches we identified three antimicrobial peptides that were named Avilin, Juruin_2, and Juruenine. All of them showed similarities with neurotoxins that act on ion channels and, probably, they have the ICK motif in their structure. The ICK fold seems to be conserved in several venomous animal lineages and presents elevated functional diversity, as well as gives stability to the toxins. The study of animal venoms is of great importance to carry out the characterization of unknown components and that may have a biotechnological application (like the antimicrobial peptides), in special venoms from spiders that are from less studied families.

BackgroundThe venom of Bothrops lanceolatus, a viperid species endemic to the Lesser Antillean Island of Martinique, induces a unique clinical manifestation, i.e., thrombosis. Previous clinical observations indicate that thromboses are more common in patients bitten by juvenile specimens. There is a need to develop an experimental model of this effect in order to study the mechanisms involved. Methodology/principal findingsThe venoms of juvenile and adult specimens of B. lanceolatus were compared by (a) describing their proteome, (b) assessing their ability to induced thrombosis in a mouse model, and (c) evaluating their in vitro procoagulant activity and in vivo hemostasis alterations. Venom proteomes of juvenile and adult specimens were highly similar. When injected by the intraperitoneal (i.p.) route, the venom of juvenile specimens induced the formation of abundant thrombi in the pulmonary vasculature, whereas this effect was less frequent in the case of adult venom. Thrombosis was not abrogated by the metalloproteinase inhibitor Batimastat. Both venoms showed a weak in vitro procoagulant effect on citrated mouse plasma and bovine fibrinogen. When administered intravenously (i.v.) venoms did not affect classical clotting tests (prothrombin time and activated partial thromboplastin time) but caused a partial drop in fibrinogen concentration. The venom of juvenile specimens induced partial alterations in some rotational thromboelastometry parameters after i.v. injection. No alterations in coagulation tests were observed when venoms were administered i.p., but juvenile and adult venoms induced a marked thrombocytopenia. Conclusions/significanceAn experimental model of the thrombotic effect induced by B. lanceolatus venom was developed. This effect is more pronounced in the case of venom of juvenile specimens, despite the observation that juvenile and adult venom proteomes are similar. Adult and juvenile venoms do not induce a consumption coagulopathy characteristic of other Bothrops sp venoms. Both venoms induce a conspicuous thrombocytopenia. This experimental model reproduces the main clinical findings described in these envenomings and should be useful to understand the mechanisms of this thrombotic effect. Author summaryEnvenomings by the viperid species Bothrops lanceolatus, endemic of the Caribbean Island of Martinique, are characterized by a unique thrombotic effect responsible for infarcts in various organs. Until now, no experimental in vivo models of this effect have been described. In this study, we developed a mouse model of thrombosis by using the intraperitoneal route of venom injection. The venom of juvenile specimens of B. lanceolatus induced the formation of abundant thrombi in the lungs, whereas the effect was much less pronounced with the venom of adult specimens. This difference in the ability of juvenile and adult venoms occurs despite both venoms having highly similar proteomic profiles. Both adult and juvenile venoms showed a weak in vitro procoagulant effect on plasma and fibrinogen, underscoring a thrombin-like (pseudo-procoagulant) activity. In vivo, the venoms did not affect the classical clotting tests (prothrombin time and activated partial thromboplastin time) but induced a partial drop in fibrinogen concentration and limited alterations in rotational thromboelastometry parameters when injected by the i.v. route. In contrast, few alterations of these parameters were observed after i.p. injection of venoms, in conditions in which thrombosis occurred, hence evidencing the lack of a consumption coagulopathy. After i.p. injection both venoms induced a pronounced thrombocytopenia. This experimental model reproduces some of the main clinical manifestations of envenoming by this species. This model can be used to identify the toxins responsible for the thrombotic effect, to study the mechanism(s) of thrombosis and to assess the preclinical efficacy of antivenoms.

Snakebite envenoming affects more than 250,000 people annually in sub-Saharan Africa. Envenoming by Dispholidus typus (boomslang) results in venom induced consumption coagulopathy, whereby highly abundant prothrombin-activating snake venom metalloproteinases (SVMPs) consume clotting factors and deplete fibrinogen. The only available treatment for D. typus envenoming is the monovalent SAIMR Boomslang antivenom. Treatment options are urgently required because this antivenom is often difficult to source and, at $6,000/vial, typically unaffordable for most snakebite patients. We therefore investigated the in vitro and in vivo preclinical efficacy of four SVMP inhibitors to neutralise the effects of D. typus venom; the matrix metalloproteinase inhibitors marimastat and prinomastat, and the metal chelators dimercaprol and DMPS. The venom of D. typus exhibited an SVMP-driven procoagulant phenotype in vitro. Marimastat and prinomastat demonstrated equipotent inhibition of the SVMP-mediated procoagulant activity of the venom in vitro, whereas dimercaprol and DMPS showed considerably lower potency. However, when tested in preclinical murine models of envenomation, DMPS and marimastat demonstrated partial protection against venom lethality, demonstrated by prolonged survival times of experimental animals, whereas dimercaprol and prinomastat failed to confer any protection at the doses tested. The results presented here demonstrate that DMPS and marimastat show potential as novel small molecule-based therapeutics for D. typus snakebite envenomation. These two drugs have been previously shown to be effective against Echis ocellatus venom induced consumption coagulopathy (VICC) in preclinical models, and thus we conclude that marimastat and DMPS may be valuable early intervention therapeutics to broadly treat VICC following snakebite envenoming in sub-Saharan Africa.

Snakebite claims 138,000 lives a year with an additional 400,000 patients left permanently disabled or disfigured1. Morbidity following envenoming includes the development of chronic wounds around the bite site. The understanding of the underlying pathophysiology of chronic snakebite wounds has been severely limited by the historical reliance on a preclinical model that only captures acute local envenoming pathology. Through the application of three medically important snake venoms (Echis ocellatus, Bothrops atrox and Naja nigricollis) to a recently developed preclinical model of chronic wounds, we have been able to characterise key features of venom wounds. We have been able to show that venom wounds share consistencies with non-venom induced preclinical wounds, and also display unique characteristics such as extracellular matrix degradation and eosinophilic infiltrate. This model will not only serve to increase our understanding the underlying pathophysiology of venom wounds, but will also provide a platform for exploring therapeutic interventions to reduce or resolve snakebite wounds.

Snake venoms contain diverse mixtures of toxins that evolved to incapacitate prey, but in humans they cause extensive pathology following snakebite envenomation. In viper venom, some of the most potent toxins are the haemorrhagic and coagulopathic snake venom metalloproteinases (SVMPs). Because venoms contain a SVMP cocktail, and due to their cytotoxicity, SVMP characterizations have been hampered by the lack of purified enzymes. By incorporating their prodomain, which blocks the active SVMP site, we overcame their cytotoxicity and enabled recombinant production of zymogens from all three structurally variable SVMP classes (PI, PII and PIII) using our baculovirus/insect cell expression system. Zymogens were auto-activated by incubation with Zn2+ ions, resulting in prodomain cleavage, PII disintegrin cleavage and PIII prodomain proteolysis. Auto-activated SVMPs were characterized using protein substrate degradation, platelet aggregation and blood coagulation assays, benchmarked to native venom-purified SVMP. Our recombinant zymogen production protocol is generically applicable for the expression of SVMPs, unlocking biomedical use in haematology, and discovery of novel snakebite therapeutics.

Clostridioides difficile is a leading cause of antibiotic-associated diarrhea and nosocomial infection in the United States. The symptoms of C. difficile infection (CDI) are associated with the production of two homologous protein toxins, TcdA and TcdB. The toxins are considered bona fide targets for clinical diagnosis as well as the development of novel prevention and therapeutic strategies. While there are extensive studies that document these efforts, there are several gaps in knowledge that could benefit from the creation of new research tools. First, we now appreciate that while TcdA sequences are conserved, TcdB sequences can vary across the span of circulating clinical isolates. An understanding of the TcdA and TcdB epitopes that drive broadly neutralizing antibody responses could advance the effort to identify safe and effective toxin-protein chimeras and fragments for vaccine development. Further, an understanding of TcdA and TcdB concentration changes in vivo can guide research into how host and microbiome-focused interventions affect the virulence potential of C. difficile. We have developed a panel of alpaca-derived nanobodies that bind specific structural and functional domains of TcdA and TcdB. We note that many of the potent neutralizers of TcdA bind epitopes within the delivery domain, a finding that could reflect roles of the delivery domain in receptor binding and/or the conserved role of pore-formation in the delivery of the toxin enzyme domains to the cytosol. In contrast, neutralizing epitopes for TcdB were found in multiple domains. The nanobodies were also used for the creation of sandwich ELISA assays that allow for quantitation of TcdA and/or TcdB in vitro and in the cecal and fecal contents of infected mice. We anticipate these reagents and assays will allow researchers to monitor the dynamics of TcdA and TcdB production over time, and the impact of various experimental interventions on toxin production in vivo. Author SummaryC. difficile (C. diff) is a leading cause of diarrhea and is recognized as an urgent threat by the Centers for Disease Control. Disease symptoms are caused by two large, similar, protein toxins, TcdA and TcdB. These toxins are drug targets and are also important for diagnosis. Despite their clear importance, the understanding of how to neutralize toxin activity is incomplete, and there are no freely available tools to quantify toxin concentration in research studies. To address these issues, we have developed nanobodies that bind and neutralize TcdA and TcdB and have also used these nanobodies to develop quantitative assays for TcdA and TcdB detection. Neutralization studies led us to discover that many of the potent neutralizers of TcdA bind epitopes within the delivery domain. This finding suggests either a role for the delivery domain in receptor binding or that the nanobodies block pore-formation and thereby inhibit delivery of the toxin enzyme domains to the cytosol. The availability of nanobody assays that can differentiate the quantities of TcdA from TcdB should permit a better understanding of toxin-specific effects and how toxin levels change over the course of infection.

Adenylate cyclase toxin (ACT) is one of the main virulence factors of Bordetella pertussis, with crucial role in colonization of human respiratory tract. ACT toxicity on target phagocytes results from translocation of its adenylate cyclase domain and production of high cAMP levels and from pore formation. Recently, we unveiled in ACT four cholesterol-recognition motifs involved in specific interaction with membrane cholesterol, which might stabilize membrane topology of critical helices for ACT activity. Here we explore an amphipathic peptide corresponding to ACT residues 454 to 487 containing one of such CRAC motifs. We show that P454-487 penetrates into DOPC vesicles as a long and tilted -helix, while in cholesterol presence experiments conformational changes that critically depend on the CRAC Phe-485 residue. Moreover, P454-487 is capable of blocking ACT toxicity on cells by outcompeting with the full-length toxin for membrane binding. We anticipate P454-487 may have potential clinical applicability in controlling Bordetella infection.

Skeletal muscle regeneration is often impaired after acute muscle damage induced by viperid snake venoms, such as that of Bothrops asper, a medically-relevant species in Latin America. It has been shown that traces of venom that remain in the damaged muscle affect myogenic cells in culture, raising the possibility of inhibition of these toxins during the regenerative process as a way to improve regeneration. Using a mouse model of myonecrosis and regeneration, we evaluated the effects of Varespladib (a phospholipase A2 inhibitor) or Marimastat (a metalloproteinase inhibitor) on muscle regeneration when administered intravenously 24 h after the onset of myonecrosis, i.e., after muscle damage has occurred. The regenerative process was evaluated 14 and 28 days after venom injection. Results show that Marimastat, or a combination of both inhibitors, improved the extent of skeletal muscle regeneration and reduced the extent of tissue fibrosis when compared to tissue from mice receiving venom and no inhibitors, as judged by qualitative and quantitative histological assessment. Results underscore the deleterious role of traces of venom components in the damaged muscle during muscle regeneration and suggest that the administration of metalloproteinase inhibitors, or a combination of metalloproteinase and phospholipase A2 inhibitors, even when muscle damage has developed, may be a therapeutic alternative for improving the extent of muscle regeneration.

Bacillus cereus is a Gram-positive bacterium widely distributed in food, soil, and plants. It is a spore-forming, facultative anaerobic bacterium known as a food-borne opportunistic human pathogen responsible for causing gastrointestinal and non-gastrointestinal infections, including wound-associated infections. In the present study, we report that among single-component, bi-component, and tripartite alpha pore-forming toxin (-PFT) producing bacteria, the tripartite NheABC toxin produced by B. cereus induced the maximum cell death in infected epithelial cells. Similar to its effects in 2D monolayers, NheABC exhibited potent cell toxicity in 3D spheroids and intestinal organoids, targeting their cell membrane and mitochondria. Moreover, using erythrocytes as a model system, we found that the cytolytic activity of NheABC is pH-dependent, and was markedly reduced at acidic pH (5.5). The pH-dependent biological activity of NheABC was further confirmed by a liposome leakage assay. Importantly, NheABC enhanced the colonization of B. cereus in a non-gastrointestinal murine wound infection model. Overall, our study highlights the role of pH in regulating NheABC-mediated cytotoxicity in mammalian cells, which may lead to the development of novel therapeutic strategies for managing B. cereus gastrointestinal and non-gastrointestinal infections.

Botulism is a life-threatening disease characterized by a descending flaccid paralysis caused by a protein neurotoxin (BoNT) released by different anaerobic bacterial species of the genus Clostridium. The paralysis results from blockade of neurotransmitter release from the terminals of peripheral cholinergic, skeletal and autonomic neurons exerted by BoNT through the cleavage of SNARE proteins, which are essential for neuroexocytosis. Here, we investigated the effect of different doses of BoNT serotypes A and B, the serotypes most commonly associated with human botulism, on enteric nervous system neurons which play an important role in gut health and physiology. We found that BoNT/A and BoNT/B enter cholinergic neurons where they cleave SNARE proteins even at doses that do not cause signs of flaccid neuroparalysis. However, these low BoNT doses favour the invasion and infection of the mouse body by Salmonella thyphimurium and Shigella flexneri. This may have significant animal health implications.

We determined echinocandin susceptibility and FKS1 genotypes of thirteen clinical isolates of Candida auris recovered from four patients at a tertiary care center in Salvador, Brazil. Three isolates were categorized as echinocandin-resistant and harbored a novel FKS1 mutation leading to an amino acid change W691L located downstream from hot-spot 1. When introduced to echinocandin-susceptible C. auris strains by CRISPR/Cas9, Fks1 W691L induced elevated MIC values to all echinocandins (ANF 16-32x; CAS >64x; MCF >64x).

The major virulence factors of Clostridioides difficile (C. difficile) are enterotoxin A (TcdA) and cytotoxin B (TcdB). The study of toxins is a crucial step in exploring the virulence of this pathogen. Currently, the toxin purification process is either laborious and time-consuming in C. difficile or performed in heterologous hosts. Therefore, we propose a streamlined method to obtain functional toxins in C. difficile. Two C. difficile strains were generated each harboring a sequence encoding a His-tag at the 3 end of C. difficile 630{Delta}erm tcdA or tcdB genes. Each toxin gene is expressed using the Ptet promoter inducible by anhydro-tetracycline. The purification yields were estimated to be 0.28 mg per liter and 0.1 mg per liter for rTcdA and rTcdB, respectively. In this study, we successfully developed a simple routine method that allows the production and purification of biologically rTcdA and rTcdB active toxins with similar activities compared to native toxins.