Development of a high-throughput in vitro screening method for the assessment of cell-damaging activities of snake venoms

Snakebite envenoming is a globally important public health issue that has devastating consequences on human health and well-being, with annual mortality rates between 81,000 and 138,000. Snake venoms may cause different pathological effects by altering normal physiological processes such as nervous transfer and blood coagulation. In addition, snake venoms can cause severe (local) tissue damage that may result in life-long morbidities, with current estimates pointing towards an additional 450,000 individuals that suffer from permanent disabilities such as amputations, contractions and blindness. Despite such high morbidity rates, research to date has been mainly focusing on neurotoxic and haemotoxic effects of snake venoms and considerably less on venom-induced tissue damage. The molecular mechanisms underlaying this pathology include membrane disruption and extracellular matrix degradation. This research describes methods used to study the (molecular) mechanisms underlaying venom-induced cell- and tissue damage. A selection of cellular bioassays and fluorescent microscopy were used to study cell-damaging activities of snake venoms in multi-well plates, using both crude and fractionated venoms. A panel of 10 representative medically relevant snake species was used, which cover a large part of the geographical regions most heavily affected by snakebite. The study comprises both morphological data as well as quantitative data on cell metabolism and viability, which were measured over time. Based on this data, a distinction could be made in the ways by which viper and elapid venoms exert their effects on cells. We further made an effort to characterise the bioactive compounds causing these effects, using a combination of liquid chromatography methods followed by bioassaying and protein identification using proteomics. The outcomes of this study might prove valuable for better understanding venom-induced cell- and tissue-damaging pathologies and could be used in the process of developing and improving snakebite treatments

Application of an Extracellular Matrix-Mimicking Fluorescent Polymer for the Detection of Proteolytic Venom Toxins

The cytotoxicity caused by snake venoms is a serious medical problem that greatly contributes to the morbidity observed in snakebite patients. The cytotoxic components found in snake venoms belong to a variety of toxin classes and may cause cytotoxic effects by targeting a range of molecular structures, including cellular membranes, the extracellular matrix (ECM) and the cytoskeleton. Here, we present a high-throughput assay (384-well plate) that monitors ECM degradation by snake venom toxins via the application of fluorescent versions of model ECM substrates, specifically gelatin and collagen type I. Both crude venoms and fractionated toxins of a selection of medically relevant viperid and elapid species, separated via size-exclusion chromatography, were studied using the self-quenching, fluorescently labelled ECM–polymer substrates. The viperid venoms showed significantly higher proteolytic degradation when compared to elapid venoms, although the venoms with higher snake venom metalloproteinase content did not necessarily exhibit stronger substrate degradation than those with a lower one. Gelatin was generally more readily cleaved than collagen type I. In the viperid venoms, which were subjected to fractionation by SEC, two (B. jararaca and C. rhodostoma, respectively) or three (E. ocellatus) active proteases were identified. Therefore, the assay allows the study of proteolytic activity towards the ECM in vitro for crude and fractionated venoms

Mud in the blood: Novel potent anticoagulant coagulotoxicity in the venoms of the Australian elapid snake genus Denisonia (mud adders) and relative antivenom efficacy

Due to their potent coagulotoxicity, Australian elapid venoms are unique relative to non-Australian members of the Elapidae snake family. The majority of Australian elapids possess potent procoagulant venom, while only a few species have been identified as possessing anticoagulant venoms. The majority of research to-date has concentrated on large species with range distributions overlapping major city centres, such as brown snakes (Pseudonaja spp.) and taipans (Oxyuranus spp.). We investigated the venom from the poorly studied genus Denisonia and documented anticoagulant activities that were differentially potent on amphibian, avian, and human plasmas. Both species were potently anticoagulant upon amphibian plasma, consistent with these snakes preying upon frogs as their primary food source. While D. devisi was only relatively weakly active on avian and human plasma, D. maculata was potently anticoagulant to amphibian, avian, and human plasma. The mechanism of anticoagulant action was determined to be the inhibition of prothrombin activation by Factor Xa by blocking the formation of the prothrombinase complex. Fractionation of D. maculata venom followed by MS sequencing revealed that the toxins responsible were Group I phospholipase A. As no antivenom is produced for this species or its near relatives, we examined the ability of Seqirus Australian snake polyvalent antivenom to neutralise the anticoagulant effects, with this antivenom shown to be effective. These results contribute to the body of knowledge regarding adaptive evolution of venom, revealing a unique taxon-specific anticoagulant effect for D. devisi venom. These results also reveal the potential effects and mechanisms behind envenomation by the potently acting D. maculata venom on human plasma, while the discovery of the efficacy of an available antivenom provides information crucial to the design of snakebite management strategies

Erythrocyte haemotoxicity profiling of snake venom toxins after nanofractionation

Snakebite is classified as a priority Neglected Tropical Disease by the World Health Organization. Understanding the pathology of individual snake venom toxins is of great importance when developing more effective snakebite therapies. Snake venoms may induce a range of pathologies, including hemolytic activity. Although snake venom-induced erythrocyte lysis is not the primary cause of mortality, hemolytic activity can greatly debilitate victims and contributes to systemic hemotoxicity. Current assays designed for studying hemolytic activity are not suitable for rapid screening of large numbers of toxic compounds. Consequently, in this study, a high-throughput hemolytic assay was developed that allows profiling of erythrocyte lysis, and was validated using venom from a number of medically important snake species (Calloselasma rhodostoma, Daboia russelii, Naja mossambica, Naja nigricollis and Naja pallida). The assay was developed in a format enabling direct integration into nanofractionation analytics, which involves liquid chromatographic separation of venom followed by high-resolution fractionation and subsequent bioassaying (and optional proteomics analysis), and parallel mass spectrometric detection. Analysis of the five snake venoms via this nanofractionation approach involving hemolytic assaying provided venom-cytotoxicity profiles and enabled identification of the toxins responsible for hemolytic activity. Our results show that the elapid snake venoms (Naja spp.) contained both direct and indirect lytic toxins, while the viperid venoms (C. rhodostoma and D. russelii) only showed indirect lytic activities, which required the addition of phospholipids to exert cytotoxicity on erythrocytes. The hemolytic venom toxins identified were mainly phospholipases A2 and cytotoxic three finger toxins. Finally, the applicability of this new analytical method was demonstrated using a conventional snakebite antivenom treatment and a small-molecule drug candidate to assess neutralization of venom cytotoxins

A non-lethal method for studying scorpion venom gland transcriptomes, with a review of potentially suitable taxa to which it can be applied

Scorpion venoms are mixtures of proteins, peptides and small molecular compounds with high specificity for ion channels and are therefore considered to be promising candidates in the venoms-to-drugs pipeline. Transcriptomes are important tools for studying the composition and expression of scorpion venom. Unfortunately, studying the venom gland transcriptome traditionally requires sacrificing the animal and therefore is always a single snapshot in time. This paper describes a new way of generating a scorpion venom gland transcriptome without sacrificing the animal, thereby allowing the study of the transcriptome at various time points within a single individual. By comparing these venom-derived transcriptomes to the traditional whole-telson transcriptomes we show that the relative expression levels of the major toxin classes are similar. We further performed a multi-day extraction using our proposed method to show the possibility of doing a multiple time point transcriptome analysis. This allows for the study of patterns of toxin gene activation over time a single individual, and allows assessment of the effects of diet, season and other factors that are known or likely to influence intraindividual venom composition. We discuss the gland characteristics that may allow this method to be successful in scorpions and provide a review of other venomous taxa to which this method may potentially be successfully applied

Terrestrial venomous animals, the envenomings they cause, and treatment perspectives in the Middle East and North Africa

The Middle East and Northern Africa, collectively known as the MENA region, are inhabited by a plethora of venomous animals that cause up to 420,000 bites and stings each year. To understand the resultant health burden and the key variables affecting it, this review describes the epidemiology of snake, scorpion, and spider envenomings primarily based on heterogenous hospital data in the MENA region and the pathologies associated with their venoms. In addition, we discuss the venom composition and the key medically relevant toxins of these venomous animals, and, finally, the antivenoms that are currently in use to counteract them. Unlike Asia and sub-Saharan Africa, scorpion stings are significantly more common (approximately 350,000 cases/year) than snakebites (approximately 70,000 cases/year) and present the most significant contributor to the overall health burden of envenomings, with spider bites being negligible. However, this review also indicates that there is a substantial lack of high-quality envenoming data available for the MENA region, rendering many of these estimates speculative. Our understanding of the venoms and the toxins they contain is also incomplete, but already presents clear trends. For instance, the majority of snake venoms contain snake venom metalloproteinases, while sodium channel-binding toxins and potassium channel-binding toxins are the scorpion toxins that cause most health-related challenges. There also currently exist a plethora of antivenoms, yet only few are clinically validated, and their high cost and limited availability present a substantial health challenge. Yet, some of the insights presented in this review might help direct future research and policy efforts toward the appropriate prioritization of efforts and aid the development of future therapeutic solutions, such as next-generation antivenoms

Terrestrial venomous animals, the envenomings they cause, and treatment perspectives in the Middle East and North Africa

The Middle East and Northern Africa, collectively known as the MENA region, are inhabited by a plethora of venomous animals that cause up to 420,000 bites and stings each year. To understand the resultant health burden and the key variables affecting it, this review describes the epidemiology of snake, scorpion, and spider envenomings primarily based on heterogenous hospital data in the MENA region and the pathologies associated with their venoms. In addition, we discuss the venom composition and the key medically relevant toxins of these venomous animals, and, finally, the antivenoms that are currently in use to counteract them. Unlike Asia and sub-Saharan Africa, scorpion stings are significantly more common (approximately 350,000 cases/year) than snakebites (approximately 70,000 cases/year) and present the most significant contributor to the overall health burden of envenomings, with spider bites being negligible. However, this review also indicates that there is a substantial lack of high-quality envenoming data available for the MENA region, rendering many of these estimates speculative. Our understanding of the venoms and the toxins they contain is also incomplete, but already presents clear trends. For instance, the majority of snake venoms contain snake venom metalloproteinases, while sodium channel–binding toxins and potassium channel–binding toxins are the scorpion toxins that cause most health-related challenges. There also currently exist a plethora of antivenoms, yet only few are clinically validated, and their high cost and limited availability present a substantial health challenge. Yet, some of the insights presented in this review might help direct future research and policy efforts toward the appropriate prioritization of efforts and aid the development of future therapeutic solutions, such as next-generation antivenoms

Comparison of morphological data and quantitative data for three venoms that have varying effects on the cells.

(A) Morphological data represented by immunofluorescent microscopy images showing morphology of RPTEC/TERT1cells after 24 hours exposure. Images were captured using confocal microscopy with 10X magnification. H342 staining is shown in blue, and PI in orange. 0.1% Triton T-100 was used as a positive control. Yellow stars represent wells in which activity was observed. Scale bar represents 200 μm. (B) Quantitative data of four cellular bioassays shown as bar graphs, with the activity of the venom represented relative to negative control (0 μg/mL). Live cell count (orange); cell surface area (grey); resazurin reduction activity (blue); ATP level (black). Increasing venom concentrations on the X-axes (in μg/mL) and percentage relative to negative control on the Y-axes. Measurements are presented as the mean of three individual experiments (N = 3), error bars depict SD; ‘*’ represents a statistically significant difference when compared to negative control, two-tailed test, p < 0.05 (Bonferroni-corrected).</p

Graphical overview of the bioassay workflow.

After injection of the venom, chromatographic separation by HPLC is performed, followed by high-resolution fractionation on 96- or 384-well plates for subsequent cellular bioassaying and protein identification using proteomics as described by Slagboom et al. [27]. Image created using www.biorender.com.</p

Toxins identified by nano-LC-MS/MS following tryptic digestion of fractionated toxins from the venom of <i>N</i>. <i>mossambica</i>.

Abbreviations: prot_acc, protein accession number; prot_score, protein score; prot_cover, protein coverage; prot_desc, protein description; prot_seq, protein sequence; pep_seq, peptide sequence. (XLSX)</p