Evolution and Biological Roles of Three-Finger Toxins in Snake Venoms

Snake venoms are complex mixtures of many enzymatic and non-enzymatic proteins, as well as small peptides. Several major venom protein superfamilies, including three-finger toxins, phospholipases A2, serine proteinases, metalloproteinases, proteinase inhibitors and lectins, are found in almost all snake venoms, from front-fanged viperids (vipers and pit vipers) and elapids (cobras, mambas, sea snakes, etc.) to rear-fanged colubrids. However, these proteins vary in abundance and functionality between species. Variation in snake venom composition is attributed to both differences in the expression levels of toxin encoding genes and occurrence of amino acid sequence polymorphisms. Documenting intraspecific venom variation has both clinical (antiserum development) and biological (predator and prey coevolution) implications. Venom is primarily a trophic adaptation and as such, the evolution and abundance of venom proteins relates directly to prey capture success and organism natural history. Without this biologically relevant perspective, proteomic and transcriptomic approaches could produce simply a list of proteins, peptides, and transcripts. It is therefore important to consider the presence and evolution of venom proteins in terms of their biological significance to the organism. Three-finger toxins (3FTx) comprise a particularly common venom protein superfamily that contributes significantly to differences in envenomation symptomology, toxicity, and overall venom composition. Three-finger toxins are non-enzymatic proteins that maintain a common molecular scaffold, and bind to different receptors/acceptors and exhibit a wide variety of biological effects. These toxins are the main lethal neurotoxins in some snake venoms and are currently the only known venom proteins associated with prey-specific toxicity. This dissertation has four major objectives: (i) to examine 3FTxs in front-fanged Elapidae and rear-fanged snake venoms for prey-specific toxicity, (ii) to examine differences in 3FTx expression within rear-fanged snake venom glands, (iii) to determine if mRNA transcripts obtained from crude venoms can be utilized for molecular evolutionary studies and venom proteomic studies, and (iv) to determine if a transcriptomic and proteomic integrated approach can more thoroughly characterize differences in rear-fanged snake venom composition. Three-finger toxins were isolated from the venom of the front-fanged Naja kaouthia (Family Elapidae; Monocled Cobra) and rear-fanged Spilotes (Pseustes) sulphureus (Family Colubridae; Amazon Puffing Snake) using chromatographic techniques, and toxicity assays were performed to evaluate prey specificity. Despite various 3FTxs being present in abundance within N. kaouthia venom, only one 3FTx (alpha-cobratoxin) demonstrated lethal toxicity (\u3c5 \u3eµg/g) toward both NSA mice (Mus musculus) and House Geckos (Hemidactylus frenatus). For P. sulphureus, the most abundant 3FTx (sulmotoxin A), a heterodimeric complex, displayed prey-specific toxicity towards House Geckos, and the second most abundant 3FTx (sulmotoxin B) displayed prey-specific toxicity towards mice. This demonstrates how a relatively simple venom with toxins dominated by one venom protein superfamily (3FTXs) can still allow for the targeting of a diversity of prey. Venom gland toxin transcriptomes and crude venom transcriptomes were obtained via individual transcripts with 3’RACE (Rapid Amplification of cDNA Ends) and next- generation sequencing to evaluate the abundance, diversity, and molecular evolution of 3FTxs. Venom protein gene expression within rear-fanged snake venom glands revealed trends towards either viper-like expression, dominated by snake venom metalloproteinases, or elapid-like expression, dominated by 3FTxs. For non-conventional 3FTxs transcripts within these glands and within crude venom, approximately 32% of 3FTx amino acid sites were under positive selection, and approximately 20% of sites were functionally critical and conserved. RNA isolated from crude venom demonstrated to be a successful approach to obtain venom protein transcripts for molecular evolutionary analyses, resulting in a novel approach without the need to sacrifice snakes for tissue. The use of a combined venom gland transcriptome with proteomic approaches aided in characterizing venom composition from previously unstudied rear-fanged snake venoms. This dissertation represents an important step in the incorporation of multiple high-throughput characterization methods and the addition of multiple assays to explore the biological roles of toxins, in particular 3FTxs, within these venoms

Snakebite Therapeutics Based on Endogenous Inhibitors from Vipers

Venomous snakebite is a major human health issue in many countries and has been categorized as a neglected tropical disease by the World Health Organization. Venomous snakes have evolved to produce venom, which is a complex mixture of toxic proteins and peptides, both enzymatic and nonenzymatic in nature. In this current era of high-throughput technologies, venomics projects, which include genome, transcriptome, and proteome analyses of various venomous species, have been conducted to characterize divergent venom phenotypes and the evolution of venom-related genes. Additionally, venomics can also inform about mechanisms of toxin production, storage, and delivery. Venomics can guide antivenom and therapeutic strategies against envenomations and identify new toxin-derived drugs/tools. One potentially promising drug development direction is the use of endogenous inhibitors present in snake venom glands and serum that could be useful for snakebite therapeutics. These inhibitors suppress the activity of venom proteases, enzymatic proteins responsible for the irreversible damage from snakebite. This book chapter will focus on insights from venomous snake adaptations, such as the evolution of venom proteases to generate diverse activities and snake natural resistance to inhibit activity, and how this information can inform and have applications in the treatment of venomous snakebite

総会抄録

<p><b>Aligned Middle American Rattlesnake (<i>Crotalus simus tzabcan</i>) C-type lectins (A) and serine proteases (B).</b> A) Four unique venom-based C-type lectin transcripts (asterisks) were identified for <i>C</i>. <i>s</i>. <i>tzabcan</i> and aligned to other crotaline species. Identical nucleotide sequences are shaded and corresponding GenBank accession numbers are as follows: Crotalus_adamanteus (AEJ31974.1), Deinagkistrodon_acutus (AAM22790.1), Crotalus_d_terrificus (Q719L8.1), and Crotalus_o_helleri (AEU60004.1). B) Venom-based serine proteases cDNA sequences (asterisks) were also obtained from <i>C</i>. <i>s</i>. <i>tzabcan</i> and were aligned with toxins from several other species; identical nucleotide sequences are shaded, and the catalytic triad composed of Ser195, Asp102, and His57 associated with thrombin-like activity in snake venom serine proteases are identified (arrowheads). Isoform 3 from <i>C</i>. <i>s</i>. <i>tzabcan</i> is a partial sequence. GenBank accession numbers are as follows: Agkistrodon_p_leucostoma (HQ270466.1), Bothrops_asper (DQ247724.1), Crotalus_d_terrificus7 (EU360954.1), Crotalus_d_terrificus4 (EU360952.1), Crotalus_d_terrificus3 (EU360951.1), Crotalus_d_durissus (DQ164401.1), Sistrurus_c_edwardsi (DQ464239.1), Trimeresurus_mucrosquamatus (X83225.1), Crotalus_adamanteus (HQ414118.1), Calloselasma_rhodostoma (L07308.1), Deinagkistrodon_acutus (AY861382.1), Trimeresurus_stejnegeri (AF545575.1), and Crotalus_atrox (AF227153.1).</p

Niemann–Pick Type C2 Proteins in Aedes aegypti: Molecular Modelling and Prediction of Their Structure–Function Relationships

Aedes aegypti is a major vector that transmits arboviruses through the saliva injected into the host. Salivary proteins help in uninterrupted blood intake and enhance the transmission of pathogens. We studied Niemann–Pick Type C2 (NPC2) proteins, a superfamily of saliva proteins that play an important role in arbovirus infections. In vertebrates, a single conserved gene encodes for the NPC2 protein that functions in cholesterol trafficking. Arthropods, in contrast, have several genes that encode divergent NPC2 proteins. We compared the sequences of 20 A. aegypti NPC2 proteins to the cholesterol-binding residues of human and bovine, and fatty-acid-binding residues of ant NPC2 protein. We identified four mosquito NPC2 proteins as potential sterol-binding proteins. Two of these proteins (AAEL006854 and/or AAEL020314) may play a key role in ecdysteroid biosynthesis and moulting. We also identified one mosquito NPC2 protein as a potential fatty-acid-binding protein. Through molecular modelling, we predicted the structures of the potential sterol- and fatty-acid-binding proteins and compared them to the reference proteins

Integration of transcriptomic and proteomic approaches for venom profiling

Snake venoms contain many protein and peptide isoforms with high levels of sequence variation, even within a single species. When characterizing venoms, peptide mass fingerprinting using databases built predominately from protein sequences originating from model organisms can be disadvantageous, especially when the intention is to document protein diversity. Therefore, the use of species-specific venom gland transcriptomes corrects for the absence of these unique peptide sequences in databases. The integration of transcriptomics and proteomics improves the accuracy of either approach alone for venom profiling. In this review, we highlight several examples, from both published and unpublished work in our lab, demonstrating how a combined venom gland transcriptome and proteome methodology allows for comprehensive characterization of venoms, including those from understudied rear-fanged snake species, and we provide recommendations for using these approaches. Article highlights: • Use of a species-specific venom gland transcriptome allows for more accurate proteomic quantification of venom components • Different databases bias proteomic results, and smaller databases increase detection sensitivity • Species-specific databases better detect unique peptide sequence

Experimental and computational aspects of bioactive proteins from animal venoms: an insight into pharmacological properties and drug discovery

Editoria

Investigating Snake-Venom-Induced Dermonecrosis and Inflammation Using an Ex Vivo Human Skin Model

Snakebite envenoming is a neglected tropical disease that causes >100,000 deaths and >400,000 cases of morbidity annually. Despite the use of mouse models, severe local envenoming, defined by morbidity-causing local tissue necrosis, remains poorly understood, and human-tissue responses are ill-defined. Here, for the first time, an ex vivo, non-perfused human skin model was used to investigate temporal histopathological and immunological changes following subcutaneous injections of venoms from medically important African vipers (Echis ocellatus and Bitis arietans) and cobras (Naja nigricollis and N. haje). Histological analysis of venom-injected ex vivo human skin biopsies revealed morphological changes in the epidermis (ballooning degeneration, erosion, and ulceration) comparable to clinical signs of local envenoming. Immunostaining of these biopsies confirmed cell apoptosis consistent with the onset of necrosis. RNA sequencing, multiplex bead arrays, and ELISAs demonstrated that venom-injected human skin biopsies exhibited higher rates of transcription and expression of chemokines (CXCL5, MIP1-ALPHA, RANTES, MCP-1, and MIG), cytokines (IL-1β, IL-1RA, G-CSF/CSF-3, and GM-CSF), and growth factors (VEGF-A, FGF, and HGF) in comparison to non-injected biopsies. To investigate the efficacy of antivenom, SAIMR Echis monovalent or SAIMR polyvalent antivenom was injected one hour following E. ocellatus or N. nigricollis venom treatment, respectively, and although antivenom did not prevent venom-induced dermal tissue damage, it did reduce all pro-inflammatory chemokines, cytokines, and growth factors to normal levels after 48 h. This ex vivo skin model could be useful for studies evaluating the progression of local envenoming and the efficacy of snakebite treatments

Studying Venom Toxin Variation Using Accurate Masses from Liquid Chromatography–Mass Spectrometry Coupled with Bioinformatic Tools

This study provides a new methodology for the rapid analysis of numerous venom samples in an automated fashion. Here, we use LC-MS (Liquid Chromatography–Mass Spectrometry) for venom separation and toxin analysis at the accurate mass level combined with new in-house written bioinformatic scripts to obtain high-throughput results. This analytical methodology was validated using 31 venoms from all members of a monophyletic clade of Australian elapids: brown snakes (Pseudonaja spp.) and taipans (Oxyuranus spp.). In a previous study, we revealed extensive venom variation within this clade, but the data was manually processed and MS peaks were integrated into a time-consuming and labour-intensive approach. By comparing the manual approach to our new automated approach, we now present a faster and more efficient pipeline for analysing venom variation. Pooled venom separations with post-column toxin fractionations were performed for subsequent high-throughput venomics to obtain toxin IDs correlating to accurate masses for all fractionated toxins. This workflow adds another dimension to the field of venom analysis by providing opportunities to rapidly perform in-depth studies on venom variation. Our pipeline opens new possibilities for studying animal venoms as evolutionary model systems and investigating venom variation to aid in the development of better antivenoms

Midgut transcriptomic responses to dengue and chikungunya viruses in the vectors Aedes albopictus and Aedes malayensis

Dengue (DENV) and chikungunya (CHIKV) viruses are among the most preponderant arboviruses. Although primarily transmitted through the bite of Aedes aegypti mosquitoes, Aedes albopictus and Aedes malayensis are competent vectors and have an impact on arbovirus epidemiology. Here, to fill the gap in our understanding of the molecular interactions between secondary vectors and arboviruses, we used transcriptomics to profile the whole-genome responses of A. albopictus to CHIKV and of A. malayensis to CHIKV and DENV at 1 and 4 days post-infection (dpi) in midguts. In A. albopictus, 1793 and 339 genes were significantly regulated by CHIKV at 1 and 4 dpi, respectively. In A. malayensis, 943 and 222 genes upon CHIKV infection, and 74 and 69 genes upon DENV infection were significantly regulated at 1 and 4 dpi, respectively. We reported 81 genes that were consistently differentially regulated in all the CHIKV-infected conditions, identifying a CHIKV-induced signature. We identified expressed immune genes in both mosquito species, using a de novo assembled midgut transcriptome for A. malayensis, and described the immune architectures. We found the JNK pathway activated in all conditions, generalizing its antiviral function to Aedines. Our comprehensive study provides insight into arbovirus transmission by multiple Aedes vectors

Investigating Snake-Venom-Induced Dermonecrosis and Inflammation Using an Ex Vivo Human Skin Model

Snakebite envenoming is a neglected tropical disease that causes &gt;100,000 deaths and &gt;400,000 cases of morbidity annually. Despite the use of mouse models, severe local envenoming, defined by morbidity-causing local tissue necrosis, remains poorly understood, and human-tissue responses are ill-defined. Here, for the first time, an ex vivo, non-perfused human skin model was used to investigate temporal histopathological and immunological changes following subcutaneous injections of venoms from medically important African vipers (Echis ocellatus and Bitis arietans) and cobras (Naja nigricollis and N. haje). Histological analysis of venom-injected ex vivo human skin biopsies revealed morphological changes in the epidermis (ballooning degeneration, erosion, and ulceration) comparable to clinical signs of local envenoming. Immunostaining of these biopsies confirmed cell apoptosis consistent with the onset of necrosis. RNA sequencing, multiplex bead arrays, and ELISAs demonstrated that venom-injected human skin biopsies exhibited higher rates of transcription and expression of chemokines (CXCL5, MIP1-ALPHA, RANTES, MCP-1, and MIG), cytokines (IL-1β, IL-1RA, G-CSF/CSF-3, and GM-CSF), and growth factors (VEGF-A, FGF, and HGF) in comparison to non-injected biopsies. To investigate the efficacy of antivenom, SAIMR Echis monovalent or SAIMR polyvalent antivenom was injected one hour following E. ocellatus or N. nigricollis venom treatment, respectively, and although antivenom did not prevent venom-induced dermal tissue damage, it did reduce all pro-inflammatory chemokines, cytokines, and growth factors to normal levels after 48 h. This ex vivo skin model could be useful for studies evaluating the progression of local envenoming and the efficacy of snakebite treatments.</jats:p