Toxins

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.

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.

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.

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.

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.

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.

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.

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.

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.

Diphtheria toxin (DT) is the archetype of bacterial exotoxins implicated in human diseases and has played a central role in defining the field of toxinology since its discovery in 1888. Despite being one of the most extensively characterized bacterial toxins, the origins and molecular evolution of DT host specialization remain unknown. Here, we determined high-resolution structures of two recently discovered distant homologs of DT. These DT-like proteins from non-human associated Streptomyces albireticuli (17% identity to DT) and Seinonella peptonophila (20% identity to DT) display remarkable structural similarity to DT enabling a comparative investigation into DTs unique toxicity toward mammalian cells. We find that the individual domains of DT-like toxins retain two critical features of DTs activity: full catalytic function and ability to translocate across mammalian cell membranes. However, we show that receptor-binding, pH-dependent pore-formation and proteolytic release of the cytotoxic enzyme into the cytosol are not optimized for human cell physiology and thus unable to efficiently deliver the cytotoxic cargo into human hosts. Our work provides structural insights into DTs evolutionary history, and implies key transitions required for the emergence of human-specificity of a major bacterial exotoxin with an important history in human disease.

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.

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.

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.

In this work we describe the isolation of a new isoform L-amino acid oxidase (LAAO) referred to as Balt-LAAO-II from Bothrops alternatus snake venom, which was highly purified using a combination of molecular exclusion (Sephadex G-75) and RP-HPLC chromatographics steps. When analyzed by SDS-PAGE, the purified Balt-LAAO-II presented a molecular weight of [~]66 kDa. The N-terminal amino acid sequence and internal peptide sequences showed close structural homology to other snake venom L-amino acid oxidases. This enzyme induces in vitro cytotoxicity on cultured human leukemic HL60 cells. Cells were grown in RPMI medium and were incubated with isoform Balt-LAAO-II (1, 10 and 100 g/mL) for up to 72 h. All three concentrations of venom markedly decreased the cell viability from 6 h onwards based on the staining with propidium iodide, the reduction of 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and the uptake of neutral red. Flow cytometry showed that all isoform Balt-LAAO-II and whole venom concentrations induced apoptosis after 2-6 h of incubation. Morphological analysis of cells incubated with isoform Balt-LAAO-II and whole venom showed cell rounding and lysis that increased with the venom concentration and duration of incubation. These results show that isoform Balt-LAAO-II from venom Bothrops alternatus is cytotoxic to cultured HL60 cells and suggest that this damage may involve apoptotic and oxidative stress pathways.

Interactions between predators and prey are often characterized by strong selection pressures that shape extreme physiological adaptations. Venom resistance in large-bodied South American opossums (Clade Didelphini) is a striking example, as these marsupials prey on venomous snakes and exhibit remarkable resistance to their venom. While resistance is well documented in Didelphini, relatively little is known about venom resistance in the smaller, more diverse members of Didelphidae, which inhabit the same regions and encounter the same predators. Here, we investigate venom resistance in the small-bodied opossum, Monodelphis domestica, through multi-level physiological assays, examining responses to purified venom components and whole venom from sympatric and allopatric vipers. Our results show M. domestica resists venom-induced disruptions to blood coagulation, retains platelet function in the presence of platelet-disrupting venoms, and inhibits snake venom metalloproteinases. Unexpectedly, we find that M. domestica von Willebrand Factor (VWF) requires increased shear force to elongate, a previously unknown aspect of opossum blood physiology that may contribute to venom resistance and may have relevance to human coagulopathies. These findings expand the extent of venom resistance beyond large-bodied Didelphini, suggesting it is a widespread trait in South American marsupials and providing new insights into venom-mammal coevolution. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/634112v2_ufig1.gif" ALT="Figure 1"> View larger version (67K): org.highwire.dtl.DTLVardef@1efb74forg.highwire.dtl.DTLVardef@ebcb82org.highwire.dtl.DTLVardef@7ddef2org.highwire.dtl.DTLVardef@1b81569_HPS_FORMAT_FIGEXP M_FIG C_FIG

During active multiple sclerosis (MS), red blood cells (RBCs) harvested from patients reportedly display increased osmotic fragility and increased cellular volume (macrocytosis). The cause of these abnormalities remains unknown. We have previously proposed that Clostridium perfringens epsilon toxin (ETX) may be a blood-borne trigger for newly forming MS lesions based on its tropism for blood-brain barrier vasculature and CNS myelin. Recently, Gao et al. have reported that ETX binds to and damages human RBCs, leading to hemolysis. Moreover, the authors suggest that purinergic nucleotide (P2) receptor activation amplifies the hemolytic process. Here, we confirm that ETX indeed causes human-specific RBC lysis. However, our data suggest that the hemolytic process is mediated by metal-catalyzed oxidation of the swell-induced, nucleotide-sensitive ICln chloride channel. We use spectrophotometry, flow cytometry and Western blotting to show that ETX targets human RBCs and T lymphocytes via their shared expression of Myelin and Lymphocyte protein (MAL); a protein shown to be both necessary and sufficient for ETX binding and toxicity. ETX likely triggers T cells to release redox-active heavy metals, Cu+ and Fe3+, via the lysosomal exocytosis pathway, while RBCs likely release these heavy metals via ETX pore formation within the RBC membrane. Extracellular Cu+ and Fe3+ may then amplify hemolysis by oxidizing a previously identified heavy metal-binding site within the ICln channel pore, thus deregulating its normal conductance. Elucidating the precise mechanism of ETX-mediated hemolysis may shed light on the underlying etiology of MS, as it would explain why MS RBC abnormalities occur during active disease.\n\nIMPORTANCEDuring active MS, numerous reports suggest that circulating RBCs are larger than normal and fragment more easily. The exact trigger(s) for these RBC abnormalities and for newly forming MS lesions remains unidentified. We have proposed that ETX, secreted by the gut bacterium Clostridium perfringens, may be an environmental trigger for newly forming MS lesions. Indeed, ETX has been shown to breakdown the BBB, enter the brain and damage the myelin sheath. Because ETX is typically spread through the circulatory system, we wished to determine how the toxin affects human blood. Provocatively, there has been a recent report that ETX produces cellular abnormalities in human RBCs, reminiscent of what has been described during active MS. In our study, we sought to elucidate the precise mechanism for how ETX causes RBC damage. In addition to triggering BBB breakdown and CNS demyelination, ETX might also explain why RBCs appear abnormal during MS attacks.

BackgroundChronic myeloid leukemia and acute lymphoblastic leukemia are significant malignancies requiring novel therapeutic agents. This study aimed to identify cytotoxic components of the venom of the spider Enoploctenus cyclothorax against K562 and NALM-6 cell lines. MethodsVenom was extracted, lyophilizated and incubated with cultured K562 and NALM-6 cells at concentrations ranging from 0.1 to 100 {micro}g/mL for 24 and 48 hours. MTT assay for cell viability was assessed to measure cytotoxicity. Venom fractionation started with centrifugation on AMICON(R) filters in a cutting edge of 10 kDa which separates low-(LW) and high-molecular weight (HW) fractions. The active fraction was fractionated using reversed-phase HPLC and the most active compound was purified and analyzed via mass spectrometry to determine its molecular weight. ResultsCytotoxic activity was observed in both cell lines at concentrations of 10, 30, and 100 {micro}g/mL E. cyclothorax. The centrifugation revealed that antineoplastic activity was concentrated in HW fraction. Subsequent chromatography isolated fraction F7 (specifically F7c) as the most potent. Mass spectrometry characterized F7c as a mild hydrophobic polypeptide with a molecular mass of approximately 6270 Da. ConclusionA polypeptide of approximately 6270 Da isolated from E. cyclothorax venom demonstrates significant in vitro cytotoxic properties against leukemic lineages. Further sequencing and synthesis are required to advance the pharmacological bioprospecting of this potential antineoplastic agent.

The Cytotoxic Necrotizing Factor Y (CNFY) is produced by the gram-negative, enteric pathogen Yersinia pseudotuberculosis. The bacterial toxin belongs to a family of deamidases, which constitutively activate Rho GTPases, thereby balancing inflammatory processes. We identified heparan sulfate proteoglycans as essential host cell factors for intoxication with CNFY. Using flow cytometry, microscopy, knockout cell lines, pulsed electron–electron double resonance and bio-layer interferometry, we studied the role of glucosaminoglycans in the intoxication process of CNFY. To analyze toxin-glucosaminoglycan interaction we utilized a truncated CNFY (CNFY709-1014). Especially this C-terminal part of CNFY, which encompasses the catalytic activity, binds with high affinity to heparan sulfates. CNFY binding with the N-terminal domain to its protein receptor seems to induce a first conformational change supporting the interaction between the C-terminal domain and heparan sulfates, which seems sterically hindered in the full toxin. A second conformational change occurs by acidification of the endosome, probably allowing insertion of the hydrophobic regions of the toxin into the endosomal membrane. Our findings suggest that heparan sulfates play a major role for intoxication within the endosome, rather than being relevant for an interaction at the cell surface. Lastly, cleavage of heparin sulfate chains by heparanase is likely required for efficient uptake of the toxic enzyme into the cytosol of mammalian cells.Author Summary The RhoA deamidating Cytotoxic Necrotizing Factor Y (CNFY) from Yersinia pseudotuberculosis is a crucial virulence factor that is important for successful infection of mammalian cells by the pathogen. The mode of action by which CNFY is able to intoxicate cells can be divided into the following steps: Binding to the cell surface, internalization, translocation from the endosome to the cytosol and deamidation of RhoA. We show, that CNFY uses heparan sulfates to maximize the amount of molecules entering the cytosol. While not being necessary for toxin binding and uptake, the sugars hold a key role in the intoxication process. We show that CNFY undergoes a conformational change at a low endosomal pH, allowing the C-terminal domain to be released from the endosomal membrane by the action of heparanase. This study reveals new insights into the CNFY-host interaction and promotes understanding of the complex intoxication process of bacterial toxins.View Full Text

Cholesterol dependent cytolysins (CDCs) are a large family of pore forming toxins, produced by numerous gram-positive pathogens. CDCs depend on host membrane cholesterol for pore formation; some CDCs also require surface associated human CD59 (hCD59) for binding, conferring specificity for human cells. We purified a recombinant version of a putative CDC encoded in the genome of Streptococcus oralis subsp. tigurinus, tigurilysin (TGY), and used CRISPR/Cas9 to construct hCD59 knockout (KO) HeLa and JEG-3 cell lines. Cell viability assays with TGY on WT and hCD59 KO cells showed that TGY is a hCD59-dependent CDC. Two variants of TGY exist among S. oralis subsp. tigurinus genomes, only one of which is functional. We discovered that a single amino acid change between these two TGY variants determines its activity. Flow cytometry and oligomerization western blots revealed that the single amino acid difference between the two TGY isoforms disrupts host cell binding and oligomerization. Furthermore, experiments with hCD59 KO cells and cholesterol depleted cells demonstrated that TGY is fully dependent on both hCD59 and cholesterol for activity, unlike other known hCD59-dependent CDCs. Using full-length CDCs and toxin constructs differing only in the binding domain, we determined that having hCD59-dependence leads to increased lysis efficiency, conferring a potential advantage to organisms producing hCD59-dependent CDCs. IMPORTANCECholesterol dependent cytolysins (CDCs) are produced by a variety of disease-causing bacteria, and may play a significant role in pathogenesis. Understanding CDC mechanisms of action provides useful information for developing anti-virulence strategies against bacteria that utilize CDCs and other pore-forming toxins in pathogenesis. This study describes for the first time a novel human-specific CDC with an atypical pore forming mechanism compared to known CDCs. In addition, this study demonstrates that human-specificity potentially confers increased lytic efficiency to CDCs. These data provide a possible explanation for the selective advantage of developing hCD59-dependency in CDCs and the consequent host restriction.

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.