Comparison of Four Methods of RNA Extraction and cDNA Synthesis from The Venom of Peruvian Snakes of the Genus Bothrops of Clinical Importance.

RNA purification and cDNA synthesis represents the starting point for molecular analyses of snake venom proteins-enzymes. Usually, the sacrifice of snakes is necessary for venom gland extraction to identify protein-coding transcripts; however, the venom can be used as a source of transcripts. Although there are methods for obtaining RNA from venom, no comparative analysis has been conducted in the Bothrops genus. In the present study, we compared four commercial methods for RNA purification and cDNA synthesis from venom (liquid, lyophilized, or long-term storage) of four clinically relevant species of Peruvian Bothrops. Our results show that the TRIzol method presents the highest yield of RNA purified from venom (59 ± 11 ng/100 µL or 10 mg). The SuperScript First-Strand Synthesis System kit produced high amounts of cDNA (3.2 ± 1.2 ng cDNA/ng RNA), and the highest value was from combination with the Dynabeads mRNA DIRECT kit (4.8 ± 2.0 ng cDNA/ng RNA). The utility of cDNA was demonstrated with the amplification of six relevant toxins: thrombin-like enzymes, P-I and P-III metalloproteinases, acid and basic phospholipases A2, and disintegrins. To our knowledge, this is the first comparative study of RNA purification and cDNA synthesis methodologies from Bothrops genus venom.

Total-evidence phylogeny and evolutionary morphology of New World pitvipers (Serpentes: Viperidae: Crotalinae).

Crotalines (pitvipers) in the Americas are distributed from southern Canada to southern Argentina, and are represented by 13 genera and 163 species that constitute a monophyletic group. Their phylogenetic relationships have been assessed mostly based on DNA sequences, while morphological data have scarcely been used for phylogenetic inquiry. We present a total-evidence phylogeny of New World pitvipers, the most taxon/character comprehensive phylogeny to date. Our analysis includes all genera, morphological data from external morphology, cranial osteology and hemipenial morphology, and DNA sequences from mitochondrial and nuclear genes. We performed analyses with parsimony as an optimality criterion, using different schemes for character weighting. We evaluated the contribution of the different sources of characters to the phylogeny through analyses of reduced datasets and calculation of weighted homoplasy and retention indexes. We performed a morphological character analysis to identify synapomorphies for the main clades. In terms of biogeography, our results support a single colonization event of the Americas by pitvipers, and a cladogenetic event into a Neotropical clade and a North American/Neotropical clade. The results also shed light on the previously unstable position of some taxa, although they could not sufficiently resolve the position of Bothrops lojanus, which may lead to the paraphyly of either Bothrops or Bothrocophias. The morphological character analyses demonstrated that an important phylogenetic signal is contained in characters related to head scalation, the jaws and the dorsum of the skull, and allowed us to detect morphological convergences in external morphology associated with arboreality.

Action of BjussuMP-II, a snake venom metalloproteinase isolated from Bothrops jararacussu venom, on human neutrophils.

Snake venom metalloproteinases (SVMPs) are enzymatic proteins present in large amounts in snake venoms presenting proteolytic, hemorrhagic, and coagulant activities. BjussuMP-II, a class P-I SVMP, isolated from the Bothrops jararacussu snake venom does not have relevant hemorrhagic activity but presents fibrinolytic, fibrinogenolytic, antiplatelet, gelatinolytic, and collagenolytic action. This study aimed to verify the action of BjussuMP-II on human neutrophil functionality focusing on the lipid bodies formation and hydrogen peroxide production, the release of dsDNA through colorimetric and microscopic assays, and cytokines by immunoenzymatic assays. Results showed that BjussuMP-II at concentrations of 1.5 up to 50 µg/mL for 24 h is not toxic to human neutrophils using an MTT assay. Under non-cytotoxic concentrations, BjussuMP-II can induce an increase in the formation of lipid bodies, production of hydrogen peroxide and cytokines [tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and interleukin-8 (IL-8)] liberation and, the release of dsDNA to form NETs. Taken together, the data obtained show for the first time that BjussuMP-II has a pro-inflammatory action and activates human neutrophils that can contribute to local damage observed in snakebite victims.

Differences in PLA2 Constitution Distinguish the Venom of Two Endemic Brazilian Mountain Lanceheads, Bothrops cotiara and Bothrops fonsecai.

Interspecific differences in snake venom compositions can result from distinct regulatory mechanisms acting in each species. However, comparative analyses focusing on identifying regulatory elements and patterns that led to distinct venom composition are still scarce. Among venomous snakes, Bothrops cotiara and Bothrops fonsecai represent ideal models to complement our understanding of the regulatory mechanisms of venom production. These recently diverged species share a similar specialized diet, habitat, and natural history, but each presents a distinct venom phenotype. Here, we integrated data from the venom gland transcriptome and miRNome and the venom proteome of B. fonsecai and B. cotiara to better understand the regulatory mechanisms that may be acting to produce differing venom compositions. We detected not only the presence of similar toxin isoforms in both species but also distinct expression profiles of phospholipases A2 (PLA2) and some snake venom metalloproteinases (SVMPs) and snake venom serine proteinases (SVSPs) isoforms. We found evidence of modular expression regulation of several toxin isoforms implicated in venom divergence and observed correlated expression of several transcription factors. We did not find strong evidence for miRNAs shaping interspecific divergence of the venom phenotypes, but we identified a subset of toxin isoforms whose final expression may be fine-tuned by specific miRNAs. Sequence analysis on orthologous toxins showed a high rate of substitutions between PLA2s, which indicates that these toxins may be under strong positive selection or represent paralogous toxins in these species. Our results support other recent studies in suggesting that gene regulation is a principal mode of venom evolution across recent timescales, especially among species with conserved ecotypes.

Tracking the recruitment and evolution of snake toxins using the evolutionary context provided by the Bothrops jararaca genome.

Venom is a key adaptive innovation in snakes, and how nonvenom genes were co-opted to become part of the toxin arsenal is a significant evolutionary question. While this process has been investigated through the phylogenetic reconstruction of toxin sequences, evidence provided by the genomic context of toxin genes remains less explored. To investigate the process of toxin recruitment, we sequenced the genome of Bothrops jararaca, a clinically relevant pitviper. In addition to producing a road map with canonical structures of genes encoding 12 toxin families, we inferred most of the ancestral genes for their loci. We found evidence that 1) snake venom metalloproteinases (SVMPs) and phospholipases A2 (PLA2) have expanded in genomic proximity to their nonvenomous ancestors; 2) serine proteinases arose by co-opting a local gene that also gave rise to lizard gilatoxins and then expanded; 3) the bradykinin-potentiating peptides originated from a C-type natriuretic peptide gene backbone; and 4) VEGF-F was co-opted from a PGF-like gene and not from VEGF-A. We evaluated two scenarios for the original recruitment of nontoxin genes for snake venom: 1) in locus ancestral gene duplication and 2) in locus ancestral gene direct co-option. The first explains the origins of two important toxins (SVMP and PLA2), while the second explains the emergence of a greater number of venom components. Overall, our results support the idea of a locally assembled venom arsenal in which the most clinically relevant toxin families expanded through posterior gene duplications, regardless of whether they originated by duplication or gene co-option.

The absence of thrombin-like activity in Bothrops erythromelas venom is due to the deletion of the snake venom thrombin-like enzyme gene.

Snake venom thrombin-like enzymes (SVTLEs) are serine proteinases that clot fibrinogen. SVTLEs are distributed mainly in venoms from snakes of the Viperidae family, comprising venomous pit viper snakes. Bothrops snakes are distributed throughout Central and South American and are responsible for most venomous snakebites. Most Bothrops snakes display thrombin-like activity in their venoms, but it has been shown that some species do not present it. In this work, to understand SVTLE polymorphism in Bothrops snake venoms, we studied individual samples from two species of medical importance in Brazil: Bothrops jararaca, distributed in Southeastern Brazil, which displays coagulant activity on plasma and fibrinogen, and Bothrops erythromelas, found in Northeastern Brazil, which lacks direct fibrinogen coagulant activity but shows plasma coagulant activity. We tested the coagulant activity of venoms and the presence of SVTLE genes by a PCR approach. The SVTLE gene structure in B. jararaca is similar to the Bothrops atrox snake, comprising five exons. We could not amplify SVTLE sequences from B. erythromelas DNA, except for a partial pseudogene. These genes underwent a positive selection in some sites, leading to an amino acid sequence diversification, mostly in exon 2. The phylogenetic tree constructed using SVTLE coding sequences confirms that they are related to the chymotrypsin/kallikrein family. Interestingly, we found a B. jararaca specimen whose venom lacked thrombin-like activity, and its gene sequence was a pseudogene with SVTLE structure, presenting nonsense and frameshift mutations. Our results indicate an association of the lack of thrombin-like activity in B. jararaca and B. erythromelas venoms with mutations and deletions of snake venom thrombin-like enzyme genes.

MITGARD: an automated pipeline for mitochondrial genome assembly in eukaryotic species using RNA-seq data.

MOTIVATION: Over the past decade, the field of next-generation sequencing (NGS) has seen dramatic advances in methods and a decrease in costs. Consequently, a large expansion of data has been generated by NGS, most of which have originated from RNA-sequencing (RNA-seq) experiments. Because mitochondrial genes are expressed in most eukaryotic cells, mitochondrial mRNA sequences are usually co-sequenced within the target transcriptome, generating data that are commonly underused or discarded. Here, we present MITGARD, an automated pipeline that reliably recovers the mitochondrial genome from RNA-seq data from various sources. The pipeline identifies mitochondrial sequence reads based on a phylogenetically related reference, assembles them into contigs, and extracts a complete mtDNA for the target species. RESULTS: We demonstrate that MITGARD can reconstruct the mitochondrial genomes of several species throughout the tree of life. We noticed that MITGARD can recover the mitogenomes in different sequencing schemes and even in a scenario of low-sequencing depth. Moreover, we showed that the use of references from congeneric species diverging up to 30 million years ago (MYA) from the target species is sufficient to recover the entire mitogenome, whereas the use of species diverging between 30 and 60 MYA allows the recovery of most mitochondrial genes. Additionally, we provide a case study with original data in which we estimate a phylogenetic tree of snakes from the genus Bothrops, further demonstrating that MITGARD is suitable for use on biodiversity projects. MITGARD is then a valuable tool to obtain high-quality information for studies focusing on the phylogenetic and evolutionary aspects of eukaryotes and provides data for easily identifying a sample using barcoding, and to check for cross-contamination using third-party tools.

Bothrops bilineatus: An Arboreal Pitviper in the Amazon and Atlantic Forest.

The two-striped forest-pitviper (Bothrops bilineatus) is an arboreal snake that is currently represented by two subspecies (B. b. bilineatus and B. b. smaragdinus) that comprise a species complex, and its distribution is in the Amazon and the Atlantic Forest. The rarity of encounters with this snake is reflected in the low occurrence of cases of snakebites throughout its geographic distribution and the resulting low number of published clinical reports. However, in some areas, B. bilineatus proves to be more frequent and causes envenomations in a greater proportion. Herein, we review the main aspects of the species complex B. bilineatus, including its biology, ecology, taxonomy, morphology, genetic and molecular studies, geographic distribution, conservation status, venom, pathophysiology and clinical aspects, and epidemiology. In addition, the different antivenoms available for the treatment of envenomations caused by B. bilineatus are presented along with suggestions for future studies that are needed for a better understanding of the snakebites caused by this snake.

Size Matters: An Evaluation of the Molecular Basis of Ontogenetic Modifications in the Composition of Bothrops jararacussu Snake Venom.

Ontogenetic changes in venom composition have been described in Bothrops snakes, but only a few studies have attempted to identify the targeted paralogues or the molecular mechanisms involved in modifications of gene expression during ontogeny. In this study, we decoded B. jararacussu venom gland transcripts from six specimens of varying sizes and analyzed the variability in the composition of independent venom proteomes from 19 individuals. We identified 125 distinct putative toxin transcripts, and of these, 73 were detected in venom proteomes and only 10 were involved in the ontogenetic changes. Ontogenetic variability was linearly related to snake size and did not correspond to the maturation of the reproductive stage. Changes in the transcriptome were highly predictive of changes in the venom proteome. The basic myotoxic phospholipases A2 (PLA2s) were the most abundant components in larger snakes, while in venoms from smaller snakes, PIII-class SVMPs were the major components. The snake venom metalloproteinases (SVMPs) identified corresponded to novel sequences and conferred higher pro-coagulant and hemorrhagic functions to the venom of small snakes. The mechanisms modulating venom variability are predominantly related to transcriptional events and may consist of an advantage of higher hematotoxicity and more efficient predatory function in the venom from small snakes.

Comparative compositional and functional analyses of Bothrops moojeni specimens reveal several individual variations.

Snake venoms are complex protein mixtures with different biological activities that can act in both their preys and human victims. Many of these proteins play a role in prey capture and in the digestive process of these animals. It is known that some snakes are resistant to the toxicity of their own venom by mechanisms not yet fully elucidated. However, it was observed in the Laboratory of Herpetology of Instituto Butantan that some Bothrops moojeni individuals injured by the same snake species showed mortalities caused by envenoming effects. This study analyzed the biochemical composition of 13 venom and plasma samples from Bothrops moojeni specimens to assess differences in their protein composition. Application of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed distinct venom protein profiles, but very homogeneous plasma profiles. Western Blotting (WB) was performed with plasma samples, which were submitted to incubation with the respective venom. Some individuals showed an immunorecognized band zone around 25 kDa, indicating interaction between the same individual plasma and venom proteins. Crossed-WB assay using non-self-plasma and venom showed that this variability is due to venom protein composition instead of plasma composition. These venoms presented higher caseinolytic, collagenolytic and coagulant activities than the venoms without these regions recognized by WB. Mass spectrometry analyses performed on two individuals revealed that these individuals present, in addition to higher protein concentrations, other exclusive proteins in their composition. When these same two samples were tested in vivo, the results also showed higher lethality in these venoms, but lower hemorrhagic activity than in the venoms without these regions recognized by WB. In conclusion, some Bothrops moojeni specimens differ in venom composition, which may have implications in envenomation. Moreover, the high individual venom variability found in this species demonstrates the importance to work with individual analyses in studies involving intraspecific venom variability and venom evolution.

Deep Profiling of the Cleavage Specificity and Human Substrates of Snake Venom Metalloprotease HF3 by Proteomic Identification of Cleavage Site Specificity (PICS) Using Proteome Derived Peptide Libraries and Terminal Amine Isotopic Labeling of Substrates (TAILS) N-Terminomics.

Snakebite is a major medical concern in many parts of the world with metalloproteases playing important roles in the pathological effects of Viperidae venoms, including local tissue damage, hemorrhage, and coagulopathy. Hemorrhagic Factor 3 (HF3), a metalloprotease from Bothrops jararaca venom, induces local hemorrhage and targets extracellular matrix (ECM) components, including collagens and proteoglycans, and plasma proteins. However, the full substrate repertoire of this metalloprotease is unknown. We report positional proteomic studies identifying >2000 N-termini, including neo-N-termini of HF3 cleavage sites in mouse embryonic fibroblast secretome proteins. Terminal amine isotopic labeling of substrates (TAILS) analysis identified a preference for Leu at the P1' position among candidate HF3 substrates including proteins of the ECM and focal adhesions and the cysteine protease inhibitor cystatin-C. Interestingly, 190 unique peptides matched to annotated cleavage sites in the TopFIND N-termini database, suggesting that these cleavages occurred at a site prone to cleavage or might have been generated by other proteases activated upon incubation with HF3, including caspases-3 and -7, cathepsins D and E, granzyme B, and MMPs 2 and 9. Using Proteomic identification of cleavage site specificity (PICS), a tryptic library derived from THP-1 monocytic cells was used as HF3 substrates for identifying protease cleavage sites and sequence preferences in peptides. A total of 799 unique cleavage sites were detected and, in accordance with TAILS analysis using native secreted protein substrates of MEF cells, revealed a clear preference for Leu at P1'. Taken together, these results greatly expand the known substrate degradome of HF3 and reveal potential new targets, which may serve as a basis to better elucidate the complex pathophysiology of snake envenomation.

Functional variability of Bothrops atrox venoms from three distinct areas across the Brazilian Amazon and consequences for human envenomings.

Variability in the composition of snake venoms occurs in different taxa and is usually correlated to snake fitness. Here, we compared B. atrox venoms from three different geographic regions across the Brazilian Amazon and found remarkable functional differences particularly between venoms from two populations separated by the Amazon River, in specimens born, raised and maintained under the same conditions at Instituto Butantan serpentary. Venom from Presidente Figueiredo snakes induced stronger dermonecrosis, but was less procoagulant and lethal to mice; these activities were correlated to the presence of a PI-class SVMP and absence of a SVSP in the venom, respectively. Venom from São Bento snakes was more hemorrhagic, killed mice more efficiently, but induced lower signs of dermonecrosis, which was correlated to the higher proportion of SVMPs and the absence of a PI-class SVMP isoform. Belterra snakes, a reference of wild snakes, presented venoms with intermediate phenotypes. Commercial Bothrops antivenom was effective in neutralizing all biological activities evaluated in this study, including dermonecrosis and pro-coagulant, which are relevant for human snakebite accidents by B. atrox. Functional differences correlated to snake fitness may also imply in different symptomatology for B. atrox snakebite patients and deserve special attention from clinical toxicologists.

cDNA cloning, heterologous expression, protein folding and immunogenic properties of a phospholipase A2 from Bothrops ammodytoides venom.

A mRNA transcript that codes for a phospholipase (PLA2) was isolated from a single venom gland of the Bothrops ammodytoides viper. The PLA2 transcript was cloned onto a pCR®2.1-TOPO vector and subsequently expressed heterologously in the E. coli strain M15, using the pQE30 vector. The recombinant phospholipase was named rBamPLA2_1, and is composed of an N-terminal fusion protein of 16 residues, along with 122 residues from the mature protein that includes 14 cysteines that form 7 disulfide bonds. Following bacterial expression, rBamPLA2_1 was obtained from inclusion bodies and extracted using a chaotropic agent. rBamPLA2_1 had an experimental molecular mass of 15,692.5 Da that concurred with its theoretical molecular mass. rBamPLA2_1 was refolded in in vitro conditions and after refolding, three main protein fractions with similar molecular masses, were identified. Although, the three fractions were considered to represent different oxidized cystine isoforms, their secondary structures were comparable. All three recombinant isoforms were active on egg-yolk phospholipid and recognized similar cell membrane phospholipids to be native PLA2s, isolated from B. ammodytoides venom. A mixture of the three rBamPLA2_1 cystine isoforms was used to immunize a horse in order to produce serum antibodies (anti-rBamPLA2_1), which partially inhibited the indirect hemolytic activity of B. ammodytoides venom. Although, anti-rBamPLA2_1 antibodies were not able to recognize crotoxin, a PLA2 from the venom of a related but different viper genus, Crotalus durissus terrificus, they recognized PLA2s in other venoms from regional species of Bothrops.

Genotoxic effects of BnSP-6, a Lys-49 phospholipase A2 (PLA2) homologue from Bothrops pauloensis snake venom, on MDA-MB-231 breast cancer cells.

Herein we evaluated the genotoxic effects of BnSP-6, a Lys-49 phospholipase A2 (PLA2) from Bothrops pauloensis, on breast cancer cells. BnSP-6 was able to induce a higher cytotoxic and genotoxic activity in MDA-MB-231 cells, when compared to MCF10A (a non-tumorigenic breast cell line), suggesting that this protein presented a possible preference for cancer cells. BnSP-6 inhibited MDA-MB-231 proliferation at 24, 48 and 72 h. In addition, BnSP-6 induced significant increase in the percentage of TUNEL-positive cells, a marker of DNA damage. To obtain novel insight into the direct DNA damage interference in MDA-MB-231 survival and proliferation, we evaluated cell cycle progression. BnSP-6 produced a significant decrease in 2N (G1) and an increase in the G2/M phase and this capacity is likely related to the modulation of expression of progression cell cycle-associated genes (CCND1, CCNE1, CDC25A, CHEK2, E2F1, CDH-1 and NF-kB). Taken together, these results indicate that BnSP-6 induces DNA damage in breast cancer cells and is an attractive model for developing innovative therapeutic agents against breast cancer.

Anti-platelet aggregation activity of two novel acidic Asp49-phospholipases A2 from Bothrops brazili snake venom.

Phospholipases A2 (PLA2s) are important enzymes present in snake venoms and are related to a wide spectrum of pharmacological effects, however the toxic potential and therapeutic effects of acidic isoforms have not been fully explored and understood. Due to this, the present study describes the isolation and biochemical characterization of two new acidic Asp49-PLA2s from Bothrops brazili snake venom, named Braziliase-I and Braziliase-II. The venom was fractionated in three chromatographic steps: ion exchange, hydrophobic interaction and reversed phase. The isoelectric point (pI) of the isolated PLA2s was determined by two-dimensional electrophoresis, and 5.2 and 5.3 pIs for Braziliase-I and II were observed, respectively. The molecular mass was determined with values ​​of 13,894 and 13,869Da for Braziliase-I and II, respectively. Amino acid sequence by Edman degradation and mass spectrometry completed 87% and 74% of the sequences, respectively for Braziliase-I and II. Molecular modeling of isolated PLA2s using acid PLA2BthA-I-PLA2 from B. jararacussu template showed high quality. Both acidic PLA2s showed no significant myotoxic activity, however they induced significant oedematogenic activity. Braziliase-I and II (100µg/mL) showed 31.5% and 33.2% of cytotoxicity on Trypanosoma cruzi and 26.2% and 19.2% on Leishmania infantum, respectively. Braziliase-I and II (10µg) inhibited 96.98% and 87.98% of platelet aggregation induced by ADP and 66.94% and 49% induced by collagen, respectively. The acidic PLA2s biochemical and structural characterization can lead to a better understanding of its pharmacological effects and functional roles in snakebites pathophysiology, as well as its possible biotechnological applications as research probes and drug leads.

Phylogeography of the Central American lancehead Bothrops asper (SERPENTES: VIPERIDAE).

The uplift and final connection of the Central American land bridge is considered the major event that allowed biotic exchange between vertebrate lineages of northern and southern origin in the New World. However, given the complex tectonics that shaped Middle America, there is still substantial controversy over details of this geographical reconnection, and its role in determining biogeographic patterns in the region. Here, we examine the phylogeography of Bothrops asper, a widely distributed pitviper in Middle America and northwestern South America, in an attempt to evaluate how the final Isthmian uplift and other biogeographical boundaries in the region influenced genealogical lineage divergence in this species. We examined sequence data from two mitochondrial genes (MT-CYB and MT-ND4) from 111 specimens of B. asper, representing 70 localities throughout the species' distribution. We reconstructed phylogeographic patterns using maximum likelihood and Bayesian methods and estimated divergence time using the Bayesian relaxed clock method. Within the nominal species, an early split led to two divergent lineages of B. asper: one includes five phylogroups distributed in Caribbean Middle America and southwestern Ecuador, and the other comprises five other groups scattered in the Pacific slope of Isthmian Central America and northwestern South America. Our results provide evidence of a complex transition that involves at least two dispersal events into Middle America during the final closure of the Isthmus.

New findings from the first transcriptome of the Bothrops moojeni snake venom gland.

Snakebites are a serious health problem in tropical countries. In Brazil, the genus Bothrops (Viperidae family) causes most of the ophidic accidents, characterized by proteolysis and haemorrhage. Snake venoms are rich sources of toxins with great therapeutic and biotechnological potential and omics approaches is a valuable tool for identification of new bioactive components in the venom. In this study, we described the first transcriptome of the venom gland of Bothrops moojeni snake, using the next-generation sequencing with the Illumina platform. We identified: (i) 20 venom components classes, among which metalloproteases were the most expressed ones, followed by serine proteases and phospholipases; and (ii) the 33 full-length amino acid sequences of toxins that have never been reported before in B. moojeni venom, such as one cysteine-rich secretory protein (Moojin), two hyaluronidases (BmooHyal-1 and BmooHyal-2), and one three-finger toxin (Bmoo-3FTx). Altogether, the transcripts identified herein represent a starting point for the analysis of structure-function relationships of toxins, which shall help develop novel biological tools and therapeutic drugs.

Molecular cloning and structural modelling of gamma-phospholipase A2 inhibitors from Bothrops atrox and Micrurus lemniscatus snakes.

Phospholipases A2 inhibitors (PLIs) produced by venomous and non-venomous snakes play essential role in this resistance. These endogenous inhibitors may be classified by their fold in PLIα, PLIß and PLIγ. Phospholipases A2 (PLA2s) develop myonecrosis in snake envenomation, a consequence that is not efficiently neutralized by antivenom treatment. This work aimed to identify and characterize two PLIs from Amazonian snake species, Bothrops atrox and Micrurus lemniscatus. Liver tissues RNA of specimens from each species were isolated and amplified by RT-PCR using PCR primers based on known PLIγ gene sequences, followed by cloning and sequencing of amplified fragments. Sequence similarity studies showed elevated identity with inhibitor PLIγ gene sequences from other snake species. Molecular models of translated inhibitors' gene sequences resemble canonical three finger fold from PLIγ and support the hypothesis that the decapeptide (residues 107-116) may be responsible for PLA2 inhibition. Structural studies and action mechanism of these PLIs may provide necessary information to evaluate their potential as antivenom or as complement of the current ophidian accident treatment.

Lys49 myotoxin from the Brazilian lancehead pit viper elicits pain through regulated ATP release.

Pain-producing animal venoms contain evolutionarily honed toxins that can be exploited to study and manipulate somatosensory and nociceptive signaling pathways. From a functional screen, we have identified a secreted phospholipase A2 (sPLA2)-like protein, BomoTx, from the Brazilian lancehead pit viper (Bothrops moojeni). BomoTx is closely related to a group of Lys49 myotoxins that have been shown to promote ATP release from myotubes through an unknown mechanism. Here we show that BomoTx excites a cohort of sensory neurons via ATP release and consequent activation of P2X2 and/or P2X3 purinergic receptors. We provide pharmacological and electrophysiological evidence to support pannexin hemichannels as downstream mediators of toxin-evoked ATP release. At the behavioral level, BomoTx elicits nonneurogenic inflammatory pain, thermal hyperalgesia, and mechanical allodynia, of which the latter is completely dependent on purinergic signaling. Thus, we reveal a role of regulated endogenous nucleotide release in nociception and provide a detailed mechanism of a pain-inducing Lys49 myotoxin from Bothrops species, which are responsible for the majority of snake-related deaths and injuries in Latin America.

Functional proteomic analyses of Bothrops atrox venom reveals phenotypes associated with habitat variation in the Amazon.

Venom variability is commonly reported for venomous snakes including Bothrops atrox. Here, we compared the composition of venoms from B. atrox snakes collected at Amazonian conserved habitats (terra-firme upland forest and várzea) and human modified areas (pasture and degraded areas). Venom samples were submitted to shotgun proteomic analysis as a whole or compared after fractionation by reversed-phase chromatography. Whole venom proteomes revealed a similar composition among the venoms with predominance of SVMPs, CTLs, and SVSPs and intermediate amounts of PLA2s and LAAOs. However, when distribution of particular isoforms was analyzed by either method, the venom from várzea snakes showed a decrease in hemorrhagic SVMPs and an increase in SVSPs, and procoagulant SVMPs and PLA2s. These differences were validated by experimental approaches including both enzymatic and in vivo assays, and indicated restrictions in respect to antivenom efficacy to variable components. Thus, proteomic analysis at the isoform level combined to in silico prediction of functional properties may indicate venom biological activity. These results also suggest that the prevalence of functionally distinct isoforms contributes to the variability of the venoms and could reflect the adaptation of B. atrox to distinct prey communities in different Amazon habitats. BIOLOGICAL SIGNIFICANCE: In this report, we compared isoforms present in venoms from snakes collected at different Amazonian habitats. By means of a species venom gland transcriptome and the in silico functional prediction of each isoform, we were able to predict the principal venom activities in vitro and in animal models. We also showed remarkable differences in the venom pools from snakes collected at the floodplain (várzea habitat) compared to other habitats. Not only was this venom less hemorrhagic and more procoagulant, when compared to the venom pools from the other three habitats studied, but also this enhanced procoagulant activity was not efficiently neutralized by Bothrops antivenom. Thus, using a functional proteomic approach, we highlighted intraspecific differences in B. atrox venom that could impact both in the ecology of snakes but also in the treatment of snake bite patients in the region.