Assessing Target Specificity of the Small Molecule Inhibitor MARIMASTAT to Snake Venom Toxins: A Novel Application of Thermal Proteome Profiling.

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.

Snake venom gene expression is coordinated by novel regulatory architecture and the integration of multiple co-opted vertebrate pathways.

Understanding how regulatory mechanisms evolve is critical for understanding the processes that give rise to novel phenotypes. Snake venom systems represent a valuable and tractable model for testing hypotheses related to the evolution of novel regulatory networks, yet the regulatory mechanisms underlying venom production remain poorly understood. Here, we use functional genomics approaches to investigate venom regulatory architecture in the prairie rattlesnake and identify cis-regulatory sequences (enhancers and promoters), trans-regulatory transcription factors, and integrated signaling cascades involved in the regulation of snake venom genes. We find evidence that two conserved vertebrate pathways, the extracellular signal-regulated kinase and unfolded protein response pathways, were co-opted to regulate snake venom. In one large venom gene family (snake venom serine proteases), this co-option was likely facilitated by the activity of transposable elements. Patterns of snake venom gene enhancer conservation, in some cases spanning 50 million yr of lineage divergence, highlight early origins and subsequent lineage-specific adaptations that have accompanied the evolution of venom regulatory architecture. We also identify features of chromatin structure involved in venom regulation, including topologically associated domains and CTCF loops that underscore the potential importance of novel chromatin structure to coevolve when duplicated genes evolve new regulatory control. Our findings provide a model for understanding how novel regulatory systems may evolve through a combination of genomic processes, including tandem duplication of genes and regulatory sequences, cis-regulatory sequence seeding by transposable elements, and diverse transcriptional regulatory proteins controlled by a co-opted regulatory cascade.

Snakes on a plain: biotic and abiotic factors determine venom compositional variation in a wide-ranging generalist rattlesnake.

BACKGROUND: Snake venoms are trophic adaptations that represent an ideal model to examine the evolutionary factors that shape polymorphic traits under strong natural selection. Venom compositional variation is substantial within and among venomous snake species. However, the forces shaping this phenotypic complexity, as well as the potential integrated roles of biotic and abiotic factors, have received little attention. Here, we investigate geographic variation in venom composition in a wide-ranging rattlesnake (Crotalus viridis viridis) and contextualize this variation by investigating dietary, phylogenetic, and environmental variables that covary with venom. RESULTS: Using shotgun proteomics, venom biochemical profiling, and lethality assays, we identify 2 distinct divergent phenotypes that characterize major axes of venom variation in this species: a myotoxin-rich phenotype and a snake venom metalloprotease (SVMP)-rich phenotype. We find that dietary availability and temperature-related abiotic factors are correlated with geographic trends in venom composition. CONCLUSIONS: Our findings highlight the potential for snake venoms to vary extensively within species, for this variation to be driven by biotic and abiotic factors, and for the importance of integrating biotic and abiotic variation for understanding complex trait evolution. Links between venom variation and variation in biotic and abiotic factors indicate that venom variation likely results from substantial geographic variation in selection regimes that determine the efficacy of venom phenotypes across populations and snake species. Our results highlight the cascading influence of abiotic factors on biotic factors that ultimately shape venom phenotype, providing evidence for a central role of local selection as a key driver of venom variation.

The origins and evolution of chromosomes, dosage compensation, and mechanisms underlying venom regulation in snakes.

Here we use a chromosome-level genome assembly of a prairie rattlesnake (Crotalus viridis), together with Hi-C, RNA-seq, and whole-genome resequencing data, to study key features of genome biology and evolution in reptiles. We identify the rattlesnake Z Chromosome, including the recombining pseudoautosomal region, and find evidence for partial dosage compensation driven by an evolutionary accumulation of a female-biased up-regulation mechanism. Comparative analyses with other amniotes provide new insight into the origins, structure, and function of reptile microchromosomes, which we demonstrate have markedly different structure and function compared to macrochromosomes. Snake microchromosomes are also enriched for venom genes, which we show have evolved through multiple tandem duplication events in multiple gene families. By overlaying chromatin structure information and gene expression data, we find evidence for venom gene-specific chromatin contact domains and identify how chromatin structure guides precise expression of multiple venom gene families. Further, we find evidence for venom gland-specific transcription factor activity and characterize a complement of mechanisms underlying venom production and regulation. Our findings reveal novel and fundamental features of reptile genome biology, provide insight into the regulation of snake venom, and broadly highlight the biological insight enabled by chromosome-level genome assemblies.

Crotalus oreganus concolor: Envenomation Case with Venom Analysis and a Diagnostic Conundrum of Myoneurologic Symptoms.

A case of midget-faded rattlesnake (Crotalus oreganus concolor) envenomation of an adult male professional herpetologist occurred in a rural setting and resulted in an array of venom induced myoneurologic symptoms. The patient experienced blurry vision, total body paresthesia, dyspnea, chest tightness, and waves of spastic muscle movements of the hands and feet that resembled tetany. It was not apparent whether these symptoms were potentially venom induced or were related to stress-induced physiologic responses. Local envenomation effects were minimal, and coagulation parameters remained within normal limits. Antivenom was not administered per patient concerns related to a history of acute allergic reactions to antivenom. Venom was collected from the Crotalus oreganus concolor responsible for the bite, and analysis revealed the presence of high levels of myotoxins (SR calcium pump antagonists) and concolor toxin, a presynaptic neurotoxin that can have myotoxic effects and cause respiratory paralysis; several serine proteinases associated with coagulopathies were also present in the venom profile.

A plethora of rodents: Rattlesnake predators generate unanticipated patterns of venom resistance in a grassland ecosystem.

Predation has the potential to impart strong selective pressures on organisms within their environments, resulting in adaptive changes in prey that minimize risk of predation. Pressures from venomous snakes present an exceptional challenge to prey, as venom represents a unique chemical arsenal evolutionarily tailored to incapacitate prey. In response, venom resistance has been detected in various snake prey species, and to varying degrees. This study analyzes venom resistance in an eastern Colorado grassland habitat, where the Prairie Rattlesnake (Crotalus viridis) and Desert Massasauga Rattlesnake (Sistrurus tergeminus edwardsii) co-occur with a suite of grassland rodents. We test for venom resistance across rodent and snake pairings using two geographically distant field sites to determine the role of 1) predation pressure and trophic ecology, and 2) sympatric and allopatric patterns of venom resistance. Resistance was measured using serum-based metalloproteinase inhibition assays to determine potential inhibition of proteolytic activity, augmented by median lethal dose (LD50) assays on rodent species to assess toxicity of crude venoms. Resistance is present in several rodent species, with strong resistance present in populations of Eastern Woodrat (Neotoma floridana), Ord's Kangaroo Rat (Dipodomys ordii), and Northern Grasshopper Mouse (Onychomys leucogaster). Resistance is less developed in other species, including the House Mouse (Mus musculus) and Plains Pocket Mouse (Perognathus flavescens). An unexpected differential is present, where Lincoln County Kangaroo Rats are highly resistant to venom of co-occurring Prairie Rattlesnakes yet are sensitive to an allopatric population of Prairie Rattlesnakes in Weld County. Lincoln Co. Northern Grasshopper Mice also demonstrate extremely elevated resistance to Weld Co. Prairie Rattlesnake venoms, and they may possess resistance mechanisms for myotoxin a, an abundant component of Weld Co. C. v viridis venoms. This study illustrates the complexity of venom resistance in biological communities that can exist when incorporating multiple species interactions. Future studies aimed at characterizing resistance mechanisms at the molecular level will provide a more detailed physiological context for understanding mechanisms by which resistance to venoms occurs.

Diverse Gene Regulatory Mechanisms Alter Rattlesnake Venom Gene Expression at Fine Evolutionary Scales.

Understanding and predicting the relationships between genotype and phenotype is often challenging, largely due to the complex nature of eukaryotic gene regulation. A step towards this goal is to map how phenotypic diversity evolves through genomic changes that modify gene regulatory interactions. Using the Prairie Rattlesnake (Crotalus viridis) and related species, we integrate mRNA-seq, proteomic, ATAC-seq and whole-genome resequencing data to understand how specific evolutionary modifications to gene regulatory network components produce differences in venom gene expression. Through comparisons within and between species, we find a remarkably high degree of gene expression and regulatory network variation across even a shallow level of evolutionary divergence. We use these data to test hypotheses about the roles of specific trans-factors and cis-regulatory elements, how these roles may vary across venom genes and gene families, and how variation in regulatory systems drive diversity in venom phenotypes. Our results illustrate that differences in chromatin and genotype at regulatory elements play major roles in modulating expression. However, we also find that enhancer deletions, differences in transcription factor expression, and variation in activity of the insulator protein CTCF also likely impact venom phenotypes. Our findings provide insight into the diversity and gene-specificity of gene regulatory features and highlight the value of comparative studies to link gene regulatory network variation to phenotypic variation.

The best of both worlds? Rattlesnake hybrid zones generate complex combinations of divergent venom phenotypes that retain high toxicity.

Studying the consequences of hybridization between closely related species with divergent traits can reveal patterns of evolution that shape and maintain extreme trophic adaptations. Snake venoms are an excellent model system for examining the evolutionary and ecological patterns that underlie highly selected polymorphic traits. Here we investigate hybrid venom phenotypes that result from natural introgression between two rattlesnake species that express highly divergent venom phenotypes: Crotalus o. concolor and C. v. viridis. Though not yet documented, interbreeding between these species may lead to novel venom phenotypes with unique activities that break the typical trends of venom composition in rattlesnakes. The characteristics of these unusual phenotypes could unveil the roles of introgression in maintaining patterns of venom composition and variation, including the near ubiquitous dichotomy between neurotoxic or degradative venoms observed across rattlesnakes. We use RADseq data to infer patterns of gene flow and hybrid ancestry between these diverged lineages and link these genetic data with analyses of venom composition, biological activity, and whole animal model toxicity tests to understand the impacts of introgression on venom composition. We find that introgressed populations express admixed venom phenotypes that do not sacrifice biological activity (lethal toxicity) or overall abundance of dominant toxins compared to parental venoms. These hybridized venoms therefore do not represent a trade-off in functionality between the typical phenotypic extremes but instead represent a unique combination of characters whose expression appears limited to the hybrid zone.

Feasibility of detecting snake envenomation biomarkers from dried blood spots.

Biofluid proteomics is a sensitive and high throughput technique that provides vast amounts of molecular data for biomarker discovery. More recently, dried blood spots (DBS) have gained traction as a stable, noninvasive, and relatively cheap source of proteomic data for biomarker identification in disease and injury. Snake envenomation is responsible for significant morbidity and mortality worldwide; however, much remains unknown about the systemic molecular response to envenomation and acquiring biological samples for analysis is a major hurdle. In this study, we utilized DBS acquired from a case of lethal rattlesnake envenomation to determine the feasibility of discovering biomarkers associated with human envenomation. We identified proteins that were either unique or upregulated in envenomated blood compared to non-envenomated blood and evaluated if physiological response pathways and protein markers that correspond to the observed syndromes triggered by envenomation could be detected. We demonstrate that DBS provide useful proteomic information on the systemic processes that resulted from envenomation in this case and find evidence for a massive and systemic inflammatory cascade, combined with coagulation dysregulation, complement system activation, hypoxia response activation, and apoptosis. We also detected potential markers indicative of lethal anaphylaxis, cardiac arrest, and brain death. Ultimately, DBS proteomics has the potential to provide stable and sensitive molecular data on envenomation syndromes and response pathways, which is particularly relevant in low-resource areas which may lack the materials for biofluid processing and storage.

Origins, genomic structure and copy number variation of snake venom myotoxins.

Crotamine, myotoxin a and homologs are short peptides that often comprise major fractions of rattlesnake venoms and have been extensively studied for their bioactive properties. These toxins are thought to be important for rapidly immobilizing mammalian prey and are implicated in serious, and sometimes fatal, responses to envenomation in humans. While high quality reference genomes for multiple venomous snakes are available, the loci that encode myotoxins have not been successfully assembled in any existing genome assembly. Here, we integrate new and existing genomic and transcriptomic data from the Prairie Rattlesnake (Crotalus viridis viridis) to reconstruct, characterize, and infer the chromosomal locations of myotoxin-encoding loci. We integrate long-read transcriptomics (Pacific Bioscience's Iso-Seq) and short-read RNA-seq to infer gene sequence diversity and characterize patterns of myotoxin and paralogous ß-defensin expression across multiple tissues. We also identify two long non-coding RNA sequences which both encode functional myotoxins, demonstrating a newly discovered source of venom coding sequence diversity. We also integrate long-range mate-pair chromatin contact data and linked-read sequencing to infer the structure and chromosomal locations of the three myotoxin-like loci. Further, we conclude that the venom-associated myotoxin is located on chromosome 1 and is adjacent to non-venom paralogs. Consistent with this locus contributing to venom composition, we find evidence that the promoter of this gene is selectively open in venom gland tissue and contains transcription factor binding sites implicated in broad trans-regulatory pathways that regulate snake venoms. This study provides the best genomic reconstruction of myotoxin loci to date and raises questions about the physiological roles and interplay between myotoxin and related genes, as well as the genomic origins of snake venom variation.

The roles of balancing selection and recombination in the evolution of rattlesnake venom.

The origin of snake venom involved duplication and recruitment of non-venom genes into venom systems. Several studies have predicted that directional positive selection has governed this process. Venom composition varies substantially across snake species and venom phenotypes are locally adapted to prey, leading to coevolutionary interactions between predator and prey. Venom origins and contemporary snake venom evolution may therefore be driven by fundamentally different selection regimes, yet investigations of population-level patterns of selection have been limited. Here, we use whole-genome data from 68 rattlesnakes to test hypotheses about the factors that drive genomic diversity and differentiation in major venom gene regions. We show that selection has resulted in long-term maintenance of genetic diversity within and between species in multiple venom gene families. Our findings are inconsistent with a dominant role of directional positive selection and instead support a role of long-term balancing selection in shaping venom evolution. We also detect rapid decay of linkage disequilibrium due to high recombination rates in venom regions, suggesting that venom genes have reduced selective interference with nearby loci, including other venom paralogues. Our results provide an example of long-term balancing selection that drives trans-species polymorphism and help to explain how snake venom keeps pace with prey resistance.

Evidence that genomic incompatibilities and other multilocus processes impact hybrid fitness in a rattlesnake hybrid zone.

Hybrid zones provide valuable opportunities to understand the genomic mechanisms that promote speciation by providing insight into factors involved in intermediate stages of speciation. Here, we investigate introgression in a hybrid zone between two rattlesnake species (Crotalus viridis and Crotalus oreganus concolor) that have undergone historical allopatric divergence and recent range expansion and secondary contact. We use Bayesian genomic cline models to characterize genomic patterns of introgression between these lineages and identify loci potentially subject to selection in hybrids. We find evidence for a large number of genomic regions with biased ancestry that deviate from the genomic background in hybrids (i.e., excess ancestry loci), which tend to be associated with genomic regions with higher recombination rates. We also identify suites of excess ancestry loci that show highly correlated allele frequencies (including conspecific and heterospecific combinations) across physically unlinked genomic regions in hybrids. Our findings provide evidence for multiple multilocus evolutionary processes impacting hybrid fitness in this system.

Venomics of the Central American Lyre Snake Trimorphodon quadruplex (Colubridae: Smith, 1941) from Costa Rica.

Rear-fanged colubrid snakes include hundreds of species globally that possess a Duvernoy's venom gland and often one-several enlarged rear maxillary teeth. We investigated the venom proteome of the Central American Lyre Snake (Trimorphodon quadruplex), a moderate-sized rear-fanged colubrid snake and the southernmost Trimorphodon, using a bottom-up proteomic approach coupled with enzyme and inhibitor assays, cytotoxicity assays and lethal toxicity assays. Several enzymes uncommonly observed in colubrid venoms were purified and characterized further. Trimorphodon quadruplex has a rather low complexity venome, typical of many rear-fanged snakes, but its venom contains L-amino acid oxidase, phospholipase A2, and a dimeric 3FTx, and 3FTxs dominate the proteome. Its PLA2 is catalytically quite active, but it lacks myotoxicity or acute toxicity; LAAO exhibits conserved structure and appears to be highly labile. Several P-III metalloproteinases are present and hydrolyze azocasein and the α-subunit of fibrinogen but lack hemorrhagic activity. Trimorphodon quadruplex produces venom and retains constriction, utilizing both chemically-mediated and mechanical feeding modes. SIGNIFICANCE: We demonstrate that T. quadruplex venom proteins are similar to those found in front-fanged snake species are present but show different biological activities. Our results underscore the importance of considering the biological roles of venoms from more than a mammal-centric perspective.

First reported case of thrombocytopenia from a Heterodon nasicus envenomation.

CONTEXT: The vast majority of the 2.5 million annual worldwide venomous snakebites are attributed to Viperidae or Elapidae envenomations. Of the nearly 2000 Colubridae species described, only a handful are known to cause medically significant envenomations. Considered medically insignificant, Heterodon nasicus (Western Hognose Snake) is a North American rear-fanged colubrid common in the legal pet trading industry. Previously reported cases of envenomations describe local pain, swelling, edema, and blistering. However, there are no reported cases of systemic or hematologic toxicity. CASE DETAILS: A 20-year-old female sustained a bite while feeding a captive H. nasicus causing local symptoms and thrombocytopenia. On day three after envenomation, the patient was seen in the emergency department for persistent pain, swelling, and blistering. At that time, she was found to have a platelet count of 90 × 109/L. Previous routine platelet counts ranged from 315 to 373 × 109/L during the prior two years. Local symptoms peaked on day seven post envenomation. Her local symptoms and thrombocytopenia improved on evaluation four months after envenomation. DISCUSSION: We report the first Heterodon nasicus envenomation causing both local toxicity and thrombocytopenia. Potential mechanisms based on H. nasicus venom composition are discussed in detail. Treatment is largely supportive. Bites by H. nascius should be evaluated by a toxicologist familiar with Colubridae species. This represents the first reported case of hematologic toxicity from envenomation by a North American colubrid snake.

Evidence for divergent patterns of local selection driving venom variation in Mojave Rattlesnakes (Crotalus scutulatus).

Snake venoms represent an enriched system for investigating the evolutionary processes that lead to complex and dynamic trophic adaptations. It has long been hypothesized that natural selection may drive geographic variation in venom composition, yet previous studies have lacked the population genetic context to examine these patterns. We leverage range-wide sampling of Mojave Rattlesnakes (Crotalus scutulatus) and use a combination of venom, morphological, phylogenetic, population genetic, and environmental data to characterize the striking dichotomy of neurotoxic (Type A) and hemorrhagic (Type B) venoms throughout the range of this species. We find that three of the four previously identified major lineages within C. scutulatus possess a combination of Type A, Type B, and a 'mixed' Type A + B venom phenotypes, and that fixation of the two main venom phenotypes occurs on a more fine geographic scale than previously appreciated. We also find that Type A + B individuals occur in regions of inferred introgression, and that this mixed phenotype is comparatively rare. Our results support strong directional local selection leading to fixation of alternative venom phenotypes on a fine geographic scale, and are inconsistent with balancing selection to maintain both phenotypes within a single population. Our comparisons to biotic and abiotic factors further indicate that venom phenotype correlates with fang morphology and climatic variables. We hypothesize that links to fang morphology may be indicative of co-evolution of venom and other trophic adaptations, and that climatic variables may be linked to prey distributions and/or physiology, which in turn impose selection pressures on snake venoms.