RESUMEN

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-1ß, 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.

Asunto(s)

Citocinas , Necrosis , Piel , Humanos , Piel/patología , Piel/efectos de los fármacos , Animales , Citocinas/metabolismo , Citocinas/genética , Mordeduras de Serpientes/patología , Venenos Elapídicos/toxicidad , Venenos de Víboras/toxicidad , Inflamación/patología , Inflamación/inducido químicamente , Viperidae , Quimiocinas/metabolismo , Quimiocinas/genética

RESUMEN

Snakebite envenoming is a neglected tropical disease that causes substantial mortality and morbidity globally. The venom of African spitting cobras often causes permanent injury via tissue-destructive dermonecrosis at the bite site, which is ineffectively treated by current antivenoms. To address this therapeutic gap, we identified the etiological venom toxins in Naja nigricollis venom responsible for causing local dermonecrosis. While cytotoxic three-finger toxins were primarily responsible for causing spitting cobra cytotoxicity in cultured keratinocytes, their potentiation by phospholipases A2 toxins was essential to cause dermonecrosis in vivo. This evidence of probable toxin synergism suggests that a single toxin-family inhibiting drug could prevent local envenoming. We show that local injection with the repurposed phospholipase A2-inhibiting drug varespladib significantly prevents local tissue damage caused by several spitting cobra venoms in murine models of envenoming. Our findings therefore provide a therapeutic strategy that may effectively prevent life-changing morbidity caused by snakebite in rural Africa.

Asunto(s)

Acetatos , Venenos Elapídicos , Indoles , Cetoácidos , Necrosis , Mordeduras de Serpientes , Animales , Mordeduras de Serpientes/tratamiento farmacológico , Ratones , Humanos , Acrilamidas/farmacología , Fosfolipasas A2/metabolismo , Naja , Elapidae , Queratinocitos/efectos de los fármacos , Piel/efectos de los fármacos , Piel/patología , Reposicionamiento de Medicamentos

RESUMEN

New treatments that circumvent the pitfalls of traditional antivenom therapies are critical to address the problem of snakebite globally. Numerous snake venom toxin inhibitors have shown promising cross-species neutralization of medically significant venom toxins in vivo and in vitro. The development of high-throughput approaches for the screening of such inhibitors could accelerate their identification, testing, and implementation and thus holds exciting potential for improving the treatments and outcomes of snakebite envenomation worldwide. Energetics-based proteomic approaches, including thermal proteome profiling and proteome integral solubility alteration (PISA) assays, represent "deep proteomics" methods for high throughput, proteome-wide identification of drug targets and ligands. In the following study, we apply thermal proteome profiling and PISA methods to characterize the interactions between venom toxin proteoforms in Crotalus atrox (Western Diamondback Rattlesnake) and the snake venom metalloprotease (SVMP) inhibitor marimastat. We investigate its venom proteome-wide effects and characterize its interactions with specific SVMP proteoforms, as well as its potential targeting of non-SVMP venom toxin families. We also compare the performance of PISA thermal window and soluble supernatant with insoluble precipitate using two inhibitor concentrations, providing the first demonstration of the utility of a sensitive high-throughput PISA-based approach to assess the direct targets of small molecule inhibitors for snake venom.

Asunto(s)

Venenos de Crotálidos , Crotalus , Proteoma , Proteómica , Animales , Crotalus/metabolismo , Proteoma/metabolismo , Proteómica/métodos , Metaloproteasas/antagonistas & inhibidores , Metaloproteasas/metabolismo , Ácidos Hidroxámicos/farmacología , Venenos de Serpiente/metabolismo

RESUMEN

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.

Asunto(s)

Biología Computacional , Venenos Elapídicos , Animales , Venenos Elapídicos/química , Venenos Elapídicos/análisis , Elapidae , Cromatografía Líquida con Espectrometría de Masas

RESUMEN

BACKGROUND: Venom systems are ideal models to study genetic regulatory mechanisms that underpin evolutionary novelty. Snake venom glands are thought to share a common origin, but there are major distinctions between venom toxins from the medically significant snake families Elapidae and Viperidae, and toxin gene regulatory investigations in elapid snakes have been limited. Here, we used high-throughput RNA-sequencing to profile gene expression and microRNAs between active (milked) and resting (unmilked) venom glands in an elapid (Eastern Brown Snake, Pseudonaja textilis), in addition to comparative genomics, to identify cis- and trans-acting regulation of venom production in an elapid in comparison to viperids (Crotalus viridis and C. tigris). RESULTS: Although there is conservation in high-level mechanistic pathways regulating venom production (unfolded protein response, Notch signaling and cholesterol homeostasis), there are differences in the regulation of histone methylation enzymes, transcription factors, and microRNAs in venom glands from these two snake families. Histone methyltransferases and transcription factor (TF) specificity protein 1 (Sp1) were highly upregulated in the milked elapid venom gland in comparison to the viperids, whereas nuclear factor I (NFI) TFs were upregulated after viperid venom milking. Sp1 and NFI cis-regulatory elements were common to toxin gene promoter regions, but many unique elements were also present between elapid and viperid toxins. The presence of Sp1 binding sites across multiple elapid toxin gene promoter regions that have been experimentally determined to regulate expression, in addition to upregulation of Sp1 after venom milking, suggests this transcription factor is involved in elapid toxin expression. microRNA profiles were distinctive between milked and unmilked venom glands for both snake families, and microRNAs were predicted to target a diversity of toxin transcripts in the elapid P. textilis venom gland, but only snake venom metalloproteinase transcripts in the viperid C. viridis venom gland. These results suggest differences in toxin gene posttranscriptional regulation between the elapid P. textilis and viperid C. viridis. CONCLUSIONS: Our comparative transcriptomic and genomic analyses between toxin genes and isoforms in elapid and viperid snakes suggests independent toxin regulation between these two snake families, demonstrating multiple different regulatory mechanisms underpin a venomous phenotype.

Asunto(s)

Crotalus , MicroARNs , Toxinas Biológicas , Serpientes Venenosas , Viperidae , Humanos , Animales , Elapidae/genética , Venenos de Serpiente/química , Venenos de Serpiente/genética , Venenos de Serpiente/metabolismo , Venenos Elapídicos/química , Venenos Elapídicos/genética , Venenos Elapídicos/metabolismo , Viperidae/genética , Viperidae/metabolismo , Transcriptoma , Factores de Transcripción/metabolismo , MicroARNs/genética , MicroARNs/metabolismo

RESUMEN

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.

Asunto(s)

Aedes , Animales , Bovinos , Humanos , Aedes/metabolismo , Glicoproteínas/metabolismo , Proteínas de Transporte Vesicular , Mosquitos Vectores , Colesterol/metabolismo , Esteroles/química , Relación Estructura-Actividad

RESUMEN

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.

Asunto(s)

Aedes , Fiebre Chikungunya , Virus Chikungunya , Dengue , Animales , Transcriptoma , Aedes/genética , Virus Chikungunya/genética , Fiebre Chikungunya/genética , Mosquitos Vectores/genética , Dengue/genética

RESUMEN

Snake venoms are complex mixtures of toxins that differ on interspecific (between species) and intraspecific (within species) levels. Whether venom variation within a group of closely related species is explained by the presence, absence and/or relative abundances of venom toxins remains largely unknown. Taipans (Oxyuranus spp.) and brown snakes (Pseudonaja spp.) represent medically relevant species of snakes across the Australasian region and provide an excellent model clade for studying interspecific and intraspecific venom variation. Using liquid chromatography with ultraviolet and mass spectrometry detection, we analyzed a total of 31 venoms covering all species of this monophyletic clade, including widespread localities. Our results reveal major interspecific and intraspecific venom variation in Oxyuranus and Pseudonaja species, partially corresponding with their geographical regions and phylogenetic relationships. This extensive venom variability is generated by a combination of the absence/presence and differential abundance of venom toxins. Our study highlights that venom systems can be highly dynamical on the interspecific and intraspecific levels and underscores that the rapid toxin evolvability potentially causes major impacts on neglected tropical snakebites.

Asunto(s)

Mordeduras de Serpientes , Toxinas Biológicas , Animales , Venenos Elapídicos/genética , Filogenia , Elapidae/genética , Venenos de Serpiente , Serpientes , Antivenenos

RESUMEN

Successful completion of the dengue virus (DENV) life cycle in its mosquito vectors is important for efficient human-mosquito-human cycle of transmission, but the virus-mosquito interactions that underpin this critical event are poorly defined. To understand the virus-host interactions that determine viral infection by Aedes aegypti, the principal DENV vector, the authors compared transcriptomic changes in the head/thorax of the mosquito after intrathoracic infection with the wild-type DENV2 16681 strain and its attenuated derivative, PDK53. Using high-throughput RNA-sequencing, the authors identified 1,629 differentially expressed genes (DEGs) during 16681 infection, compared with only 22 DEGs identified during PDK53 infection, indicating that 16681 infection triggers a more robust host transcriptomic response compared with PDK53 infection. The authors further found that 16681 infection, but not PDK53 infection, altered metabolism in these heads/thoraces. Altogether, our findings reveal differential regulation of metabolic processes during wild-type and attenuated DENV infection, and suggest the need for future work to study the role of metabolic processes in determining DENV infection and replication in its mosquito vectors.

Asunto(s)

Aedes , Virus del Dengue , Dengue , Aedes/genética , Animales , Virus del Dengue/genética , Humanos , Mosquitos Vectores/genética , Transcriptoma

RESUMEN

Arboviruses such as dengue (DENV), Zika (ZIKV) and chikungunya (CHIKV) viruses infect close to half a billion people per year, and are primarily transmitted through Aedes aegypti bites. Infection-induced changes in mosquito salivary glands (SG) influence transmission by inducing antiviral immunity, which restricts virus replication in the vector, and by altering saliva composition, which influences skin infection. Here, we profiled SG proteome responses to DENV serotype 2 (DENV2), ZIKV and CHIKV infections by using high-resolution isobaric-tagged quantitative proteomics. We identified 218 proteins with putative functions in immunity, blood-feeding or related to the cellular machinery. We observed that 58, 27 and 29 proteins were regulated by DENV2, ZIKV and CHIKV infections, respectively. While the regulation patterns were mostly virus-specific, we separately depleted four uncharacterized proteins that were upregulated by all three viral infections to determine their effects on these viral infections. Our study suggests that gamma-interferon responsive lysosomal thiol-like (GILT-like) has an anti-ZIKV effect, adenosine deaminase (ADA) has an anti-CHIKV effect, salivary gland surface protein 1 (SGS1) has a pro-ZIKV effect and salivary gland broad-spectrum antiviral protein (SGBAP) has an antiviral effect against all three viruses. The comprehensive description of SG responses to three global pathogenic viruses and the identification of new restriction factors improves our understanding of the molecular mechanisms influencing transmission.

Asunto(s)

Aedes/fisiología , Aedes/virología , Virus Chikungunya/inmunología , Virus del Dengue/inmunología , Interacciones Huésped-Patógeno/inmunología , Glándulas Salivales/fisiología , Glándulas Salivales/virología , Virus Zika/inmunología , Aedes/clasificación , Animales , Cromatografía Liquida , Biología Computacional/métodos , Resistencia a la Enfermedad , Femenino , Filogenia , Proteómica/métodos , Espectrometría de Masas en Tándem

RESUMEN

INTRODUCTION: Snake venoms contain many protein and peptide isoforms with high levels of sequence variation, even within a single species. AREAS COVERED: 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. EXPERT OPINION: 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.

Asunto(s)

Colubridae , Transcriptoma , Animales , Colubridae/genética , Humanos , Proteoma , Proteómica , Venenos de Serpiente

RESUMEN

Quaking (QKI) controls RNA metabolism in many biological processes including innate immunity, where its roles remain incompletely understood. To illuminate these roles, we performed genome scale transcriptome profiling in QKI knockout cells with or without poly(I:C) transfection, a double-stranded RNA analog that mimics viral infection. Analysis of RNA-sequencing data shows that QKI knockout upregulates genes induced by interferons, suggesting that QKI is an immune suppressor. Furthermore, differential splicing analysis shows that QKI primarily controls cassette exons, and among these events, we noted that QKI silences splicing of the extra domain A (EDA) exon in fibronectin (FN1) transcripts. QKI knockout results in elevated production and secretion of FN1-EDA protein, which is a known activator of interferons. Consistent with an upregulation of the interferon response in QKI knockout cells, our results show reduced production of dengue virus-2 and Japanese encephalitis virus in these cells. In conclusion, we demonstrate that QKI downregulates the interferon system and attenuates the antiviral state.

Asunto(s)

Virus del Dengue/crecimiento & desarrollo , Virus de la Encefalitis Japonesa (Especie)/crecimiento & desarrollo , Fibronectinas/genética , Interferón Tipo I/inmunología , Empalme del ARN/genética , Proteínas de Unión al ARN/metabolismo , Células A549 , Línea Celular Tumoral , Virus del Dengue/inmunología , Virus de la Encefalitis Japonesa (Especie)/inmunología , Perfilación de la Expresión Génica , Humanos , Inmunidad Innata/genética , Inmunidad Innata/inmunología , Interferón Tipo I/genética , Poli I-C/inmunología , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN/genética , Transcriptoma/genética , Regulación hacia Arriba/genética

RESUMEN

Arbovirus infection of Aedes aegypti salivary glands (SGs) determines transmission. However, there is a dearth of knowledge on SG immunity. Here, we characterized SG immune response to dengue, Zika and chikungunya viruses using high-throughput transcriptomics. We also describe a transcriptomic response associated to apoptosis, blood-feeding and lipid metabolism. The three viruses differentially regulate components of Toll, Immune deficiency (IMD) and c-Jun N- terminal Kinase (JNK) pathways. However, silencing of the Toll and IMD pathway components showed variable effects on SG infection by each virus. In contrast, regulation of the JNK pathway produced consistent responses in both SGs and midgut. Infection by the three viruses increased with depletion of the activator Kayak and decreased with depletion of the negative regulator Puckered. Virus-induced JNK pathway regulates the complement factor, Thioester containing protein-20 (TEP20), and the apoptosis activator, Dronc, in SGs. Individual and co-silencing of these genes demonstrate their antiviral effects and that both may function together. Co-silencing either TEP20 or Dronc with Puckered annihilates JNK pathway antiviral effect. Upon infection in SGs, TEP20 induces antimicrobial peptides (AMPs), while Dronc is required for apoptosis independently of TEP20. In conclusion, we revealed the broad antiviral function of JNK pathway in SGs and showed that it is mediated by a TEP20 complement and Dronc-induced apoptosis response. These results expand our understanding of the immune arsenal that blocks arbovirus transmission.

Asunto(s)

Aedes/inmunología , Apoptosis , Fiebre Chikungunya/inmunología , Proteínas del Sistema Complemento/inmunología , Dengue/inmunología , Sistema de Señalización de MAP Quinasas , Glándulas Salivales/inmunología , Infección por el Virus Zika/inmunología , Aedes/virología , Animales , Fiebre Chikungunya/metabolismo , Fiebre Chikungunya/prevención & control , Fiebre Chikungunya/virología , Virus Chikungunya/inmunología , Proteínas del Sistema Complemento/metabolismo , Dengue/metabolismo , Dengue/prevención & control , Dengue/virología , Virus del Dengue/inmunología , Femenino , Interacciones Huésped-Patógeno , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Insectos Vectores/inmunología , Insectos Vectores/virología , Glándulas Salivales/virología , Transcriptoma , Replicación Viral , Virus Zika/inmunología , Infección por el Virus Zika/metabolismo , Infección por el Virus Zika/prevención & control , Infección por el Virus Zika/virología

RESUMEN

The genera Ophiophagus and Naja comprise part of a clade of snakes referred to as cobras, dangerously venomous front-fanged snakes in the family Elapidae responsible for significant human mortality and morbidity throughout Asia and Africa. We evaluated venom enzyme variation for eleven cobra species and three N. kaouthia populations using SDS-PAGE venom fingerprinting and numerous enzyme assays. Acetylcholinesterase and PLA2 activities were the most variable between species, and PLA2 activity was significantly different between Malaysian and Thailand N. kaouthia populations. Venom metalloproteinase activity was low and significantly different among most species, but levels were identical for N. kaouthia populations; minor variation in venom L-amino acid oxidase and phosphodiesterase activities were seen between cobra species. Naja siamensis venom lacked the α-fibrinogenolytic activity common to other cobra venoms. In addition, venom from N. siamensis had no detectable metalloproteinase activity and exhibited an SDS-PAGE profile with reduced abundance of higher mass proteins. Venom profiles from spitting cobras (N. siamensis, N. pallida, and N. mossambica) exhibited similar reductions in higher mass proteins, suggesting the evolution of venoms of reduced complexity and decreased enzymatic activity among spitting cobras. Generally, the venom proteomes of cobras show highly abundant three-finger toxin diversity, followed by large quantities of PLA2s. However, PLA2 bands and activity were very reduced for N. haje, N. annulifera and N. nivea. Venom compositionalenzy analysis provides insight into the evolution, diversification and distribution of different venom phenotypes that complements venomic data, and this information is critical for the development of effective antivenoms and snakebite treatment.

Asunto(s)

Acetilcolinesterasa/metabolismo , Antivenenos/metabolismo , Venenos Elapídicos/enzimología , Elapidae/metabolismo , Fosfolipasas A2/metabolismo , Proteoma/metabolismo , África , Animales , Asia , Venenos Elapídicos/toxicidad , Elapidae/clasificación , Especificidad de la Especie

RESUMEN

The CAP protein superfamily (Cysteine-rich secretory proteins (CRISPs), Antigen 5 (Ag5), and Pathogenesis-related 1 (PR-1) proteins) is widely distributed, but for toxinologists, snake venom CRISPs are the most familiar members. Although CRISPs are found in the majority of venoms, very few of these proteins have been functionally characterized, but those that have been exhibit diverse activities. Snake venom CRISPs (svCRISPs) inhibit ion channels and the growth of new blood vessels (angiogenesis). They also increase vascular permeability and promote inflammatory responses (leukocyte and neutrophil infiltration). Interestingly, CRISPs in lamprey buccal gland secretions also manifest some of these activities, suggesting an evolutionarily conserved function. As we strive to better understand the functions that CRISPs serve in venoms, it is worth considering the broad range of CRISP physiological activities throughout the animal kingdom. In this review, we summarize those activities, known crystal structures and sequence alignments, and we discuss predicted functional sites. CRISPs may not be lethal or major components of venoms, but given their almost ubiquitous occurrence in venoms and the accelerated evolution of svCRISP genes, these venom proteins are likely to have functions worth investigating.

Asunto(s)

Proteínas de Reptiles , Venenos de Serpiente , Animales , Cisteína , Evolución Molecular , Humanos , Unión Proteica , Proteínas de Reptiles/química , Proteínas de Reptiles/genética , Proteínas de Reptiles/toxicidad , Venenos de Serpiente/química , Venenos de Serpiente/genética , Venenos de Serpiente/toxicidad

RESUMEN

Studying animal toxin evolution requires sequences of these proteins and peptides, and transcript sequences allow for the construction of cladograms and evaluation of selection pressures from nonsynonymous and synonymous nucleotide mutation ratios. In addition, these translated sequences can be useful as custom databases for peptide identifications within venoms and for better proteomic quantification. Obtaining these transcripts is achieved by sequencing cDNA originating from venom gland tissue or venom. This chapter provides the methodology for (1) targeted sequencing of transcripts from a single venom protein family (RNA isolation and 3'RACE [rapid amplification of cDNA ends]), (2) generation of a venom gland transcriptome with next-generation sequencing (NGS) technology (de novo transcriptome assembly, toxin transcript identification, quantification, and positive selection analysis), and (3) combined high-throughput proteomics to identify secreted venom components. Transcriptomics has become fundamental for studying toxin evolution, but it creates many challenges for scientists who are unfamiliar with working with RNA, managing large NGS datasets and executing the required programs, particularly considering that there is an overabundance of available software in this field and not all perform optimally for venom gland transcriptome assembly. This chapter provides one pipeline for the integration of both low- and high-throughput transcriptomics with proteomics to characterize venoms.

Asunto(s)

Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Proteómica/métodos , Animales , Biología Computacional/métodos , Transcriptoma/genética , Ponzoñas/análisis

RESUMEN

Snake venoms are primarily composed of proteins and peptides, and these toxins have developed high selectivity to their biological targets. This makes venoms interesting for exploration into protein evolution and structure-function relationships. A single venom protein superfamily can exhibit a variety of pharmacological effects; these variations in activity originate from differences in functional sites, domains, posttranslational modifications, and the formations of toxin complexes. In this review, we discuss examples of how the major venom protein superfamilies have diversified, as well as how newer technologies in the omics fields, such as genomics, transcriptomics, and proteomics, can be used to characterize both known and unknown toxins.Because toxins are bioactive molecules with a rich diversity of activities, they can be useful as therapeutic and diagnostic agents, and successful examples of toxin applications in these areas are also reviewed. With the current rapid pace of technology, snake venom research and its applications will only continue to expand.

Asunto(s)

Evolución Molecular , Venenos de Serpiente/química , Venenos de Serpiente/farmacología , Animales , Genómica , Proteómica , Venenos de Serpiente/genética , Serpientes/genética , Transcriptoma

RESUMEN

Venom proteins evolve rapidly, and as a trophic adaptation are excellent models for predator-prey evolutionary studies. The key to a deeper understanding of venom evolution is an integrated approach, combining prey assays with analysis of venom gene expression and venom phenotype. Here, we use such an approach to study venom evolution in the Amazon puffing snake, Spilotes sulphureus, a generalist feeder. We identify two novel three-finger toxins: sulditoxin and sulmotoxin 1. These new toxins are not only two of the most abundant venom proteins, but are also functionally intriguing, displaying distinct prey-specific toxicities. Sulditoxin is highly toxic towards lizard prey, but is non-toxic towards mammalian prey, even at greater than 22-fold higher dosage. By contrast, sulmotoxin 1 exhibits the reverse trend. Furthermore, evolutionary analysis and structural modelling show highest sequence variability in the central loop of these proteins, probably driving taxon-specific toxicity. This is, to our knowledge, the first case in which a bimodal and contrasting pattern of toxicity has been shown for proteins in the venom of a single snake in relation to diet. Our study is an example of how toxin gene neofunctionalization can result in a venom system dominated by one protein superfamily and still exhibit flexibility in prey capture efficacy.

Asunto(s)

Colubridae/genética , Venenos de Serpiente/química , Venenos de Serpiente/toxicidad , Secuencia de Aminoácidos , Animales , Evolución Biológica , Colubridae/metabolismo , Expresión Génica , Lagartos , Ratones , Conformación Proteica , Venenos de Serpiente/genética

RESUMEN

High-throughput technologies were used to identify venom gland toxin expression and to characterize the venom proteomes of two rear-fanged snakes, Ahaetulla prasina (Asian Green Vine Snake) and Borikenophis portoricensis (Puerto Rican Racer). Sixty-nine complete toxin-coding transcripts from 12 venom protein superfamilies (A. prasina) and 50 complete coding transcripts from 11 venom protein superfamilies (B. portoricensis) were identified in the venom glands. However, only 18% (A. prasina) and 32% (B. portoricensis) of the translated protein isoforms were detected in the proteome of these venoms. Both venom gland transcriptomes and venom proteomes were dominated by P-III metalloproteinases. Three-finger toxins, cysteine-rich secretory proteins, and C-type lectins were present in moderate amounts, but other protein superfamilies showed very low abundances. Venoms contained metalloproteinase activity comparable to viperid snake venom levels, but other common venom enzymes were absent or present at negligible levels. Western blot analysis showed metalloproteinase and cysteine-rich secretory protein epitopes shared with the highly venomous Boomslang (Dispholidus typus). The abundance of metalloproteinases emphasizes the important trophic role of these toxins. Comprehensive, transcriptome-informed definition of proteomes and functional characterization of venom proteins in rear-fanged snake families help to elucidate toxin evolution and provide models for protein structure-function analyses.

Asunto(s)

Colubridae/metabolismo , Metaloproteasas/metabolismo , Proteómica/métodos , Venenos de Serpiente/análisis , Venenos de Serpiente/metabolismo , Transcriptoma/fisiología , Animales , Cromatografía Liquida , Colubridae/genética , Pruebas de Enzimas , Metaloproteasas/aislamiento & purificación , Proteoma/análisis , Proteoma/metabolismo , Venenos de Serpiente/química , Venenos de Serpiente/genética , Espectrometría de Masas en Tándem