The Cloning and Characterization of a Three-Finger Toxin Homolog (NXH8) from the Coralsnake Micrurus corallinus That Interacts with Skeletal Muscle Nicotinic Acetylcholine Receptors.

Coralsnakes (Micrurus spp.) are the only elapids found throughout the Americas. They are recognized for their highly neurotoxic venom, which is comprised of a wide variety of toxins, including the stable, low-mass toxins known as three-finger toxins (3FTx). Due to difficulties in venom extraction and availability, research on coralsnake venoms is still very limited when compared to that of other Elapidae snakes like cobras, kraits, and mambas. In this study, two previously described 3FTx from the venom of M. corallinus, NXH1 (3SOC1_MICCO), and NXH8 (3NO48_MICCO) were characterized. Using in silico, in vitro, and ex vivo experiments, the biological activities of these toxins were predicted and evaluated. The results showed that only NXH8 was capable of binding to skeletal muscle cells and modulating the activity of nAChRs in nerve-diaphragm preparations. These effects were antagonized by anti-rNXH8 or antielapidic sera. Sequence analysis revealed that the NXH1 toxin possesses eight cysteine residues and four disulfide bonds, while the NXH8 toxin has a primary structure similar to that of non-conventional 3FTx, with an additional disulfide bond on the first loop. These findings add more information related to the structural diversity present within the 3FTx class, while expanding our understanding of the mechanisms of the toxicity of this coralsnake venom and opening new perspectives for developing more effective therapeutic interventions.

The cloning and characterization of a three-finger toxin homolog (NXH8) from the Coralsnake Micrurus corallinus that interacts with skeletal muscle nicotinic acetylcholine receptors

Coralsnakes (Micrurus spp.) are the only elapids found throughout the Americas. They are recognized for their highly neurotoxic venom, which is comprised of a wide variety of toxins, including the stable, low-mass toxins known as three-finger toxins (3FTx). Due to difficulties in venom extraction and availability, research on coralsnake venoms is still very limited when compared to that of other Elapidae snakes like cobras, kraits, and mambas. In this study, two previously described 3FTx from the venom of M. corallinus, NXH1 (3SOC1_MICCO), and NXH8 (3NO48_MICCO) were characterized. Using in silico, in vitro, and ex vivo experiments, the biological activities of these toxins were predicted and evaluated. The results showed that only NXH8 was capable of binding to skeletal muscle cells and modulating the activity of nAChRs in nerve–diaphragm preparations. These effects were antagonized by anti-rNXH8 or antielapidic sera. Sequence analysis revealed that the NXH1 toxin possesses eight cysteine residues and four disulfide bonds, while the NXH8 toxin has a primary structure similar to that of non-conventional 3FTx, with an additional disulfide bond on the first loop. These findings add more information related to the structural diversity present within the 3FTx class, while expanding our understanding of the mechanisms of the toxicity of this coralsnake venom and opening new perspectives for developing more effective therapeutic interventions.

Consecutive envenomation of two men bitten by the same coral snake (Micrurus corallinus).

Objective: To report two patients who developed systemic neurotoxicity after consecutive bites by the same coral snake.Case report: Two previously healthy men (32-year-old and 34-year-old) found a coral snake in a woodpile while collecting wood for a barbecue. During the barbecue, both men became drunk and "played" with the snake, believing that they were handling a false coral snake, and were bitten within a few minutes of each other. Both patients were admitted to a referral tertiary care hospital (175 km from where the bites occurred) 16 hours and 19 hours postbite; both showed similar features of envenomation: palpebral ptosis, muscle weakness, dysphagia, and generalized myalgia. No fang marks or local pain were detected in either case. The patients were successfully treated with Brazilian coral snake antivenom (Fab´2) and discharged one-day postadmission, with improvement of myasthenia, but still showing palpebral ptosis. The offending snake was identified as a 42-cm-long Micrurus corallinus. During follow-up, both patients reported a transitory loss of taste that lasted approximately 3-4 weeks postbite.Conclusion: Consecutive bites by the same coral snake may cause systemic neurotoxicity (acute myasthenia) in more than one person, as well as transitory loss of taste, an underreported complication of snakebites.

Being a bright snake: testing aposematism and mimicry in a neotropical forest

Based on color patterns and behavioral similarities, venomous coral snake Micrurus corallinus (Elapidae) may act as a model for two polymorphic species, Erythrolamprus aesculapii (Dipsadidae) and Micrurus decoratus (Elapidae). Plasticine replicas were used to investigate the aposematism of these coloration patterns and whether these species may be part of mimetic complexes in two Atlantic Forest localities in Southeast Brazil. Coral replicas were more avoided when set upon a white background, evincing that the pattern may act aposematically in contrast with light substrates. Birds attacked all four patterns equally during the mimicry experiments. Birds of prey, known to be effective in predating snakes, are quite abundant in the study areas, which may have led to this lack of avoidance. Accordingly, they predated more adult‐sized replicas, which could be more dangerous. Interestingly, opossum avoided the Micrurus corallinus and Erythrolamprus aesculapii replicas that resembled the model. This suggests that opportunistic predators, as the opossum may be important selective agents in mimicry complexes.

Does the administration of pilocarpine prior to venom milking influence the composition of Micrurus corallinus venom?

Considering that the scarcity of venom represents a huge challenge for biochemical and functional studies of Micrurus species (coral snakes), in this report we describe for the first time the influence of pilocarpine administration prior to venom milking on the yield and protein composition of Micrurus corallinus venom. The administration of pilocarpine resulted in an increase of about 127% in the volume of venom milked, with similar protein content. Venoms showed similar protein bands distribution and intensity by SDS-PAGE and equivalents RP-HPLC profiles. Our proteomic analysis showed that venoms milked in the presence and absence of pilocarpine presented comparable protein profiles, in terms of protein composition and relative abundance. The toxins identified were assigned to 13 protein families and represent the most complete M. corallinus venom proteome described so far, in terms of number of protein families identified. Our data indicate that the administration of pilocarpine prior to venom milking increases the venom yield and does not change significantly the venom composition of M. corallinus. The employment of pilocarpine represents a useful approach to increase the yield of venom not only for Micrurus species, but also for other genera of snakes with limitations regarding the amount of venom available. SIGNIFICANCE: In this report, we evaluated the influence of pilocarpine administration prior to venom milking in the overall composition of M. corallinus venom. We showed that the use of pilocarpine 10min before M. corallinus venom milking increases venom yield by ~127%. Not only the volume of venom obtained is higher, but also the protein concentration of both venoms is similar, opposing the idea that a more diluted venom is obtained as a result of pilocarpine administration, observed in non-front-fanged snakes. Shotgun proteomics analysis revealed that venom milked with and without the use of this drug showed similar overall protein composition and relative abundances. In addition, our proteomic approach allowed the identification of 13 toxin families in M. corallinus venom, representing the most complete M. corallinus venom proteome described so far. Moreover, two of these toxin families were identified for the first time in the venom of this species. Thus, considering the scarcity of Micrurus venom for biochemical and functional studies, we highlighted the usefulness of pilocarpine administration prior to venom milking to increase the venom yield of these snakes.

Avaliação da expressão gênica diferencial e da histopatologia de diferentes órgãos de camundongos em resposta ao envenenamento por Micrurus corallinus / Analysis of differential gene expression and histopathology in mice tissues in response to the envenoming caused by Micrurus corallinus

Snakes of the Elapidae family possess highly toxic venoms due to the presence of lethal neurotoxins. These venoms are rich, particularly, in two neurotoxic families of proteins: α-neurotoxins and phospholipases A2 (PLA2s). In the American continent, the Elapidae family is represented by the coral snakes group, which includes three genera (Micrurus, Leptomicrurus and Micruroides). Micrurus genus is the most specious among them and M. frontalis and M.corallinus are the most important species in Brazil in terms of public health. Their venoms are employed for the antivenoms production used in micruric envenoming. In the present work, we used RNA-Seq to gain deeper knowledge on the effects of M. corallinus venom, local and systemically, in a mice model injected with sublethal doses of the venom. Tissue samples from the brain, kidney, liver, spleen, heart, diaphragm and both gastrocnemius muscles were collected for total RNA isolation, at 8 and 24 hours after venom injection. Then, cDNA libraries were prepared and the sequencing was run in an Illumina HiSeq1500 instrument. The data obtained from the sequencing were processed by suitable bioinformatic programs. Those genes that changed their expression for a two-fold change cutoff (FDR ≤ 0.05) were submitted to enrichment analyses so as to investigate the affected pathways and networks. On the other hand, histopathological analyses were carried in order to help in the results interpretation. In terms of differential gene expression, the results showed a strong response in the right gastrocnemius, local of venom inoculation. The enrichment analyses identified a myriad of genes involved in pathways and networks related to cell adhesion, chemotaxis and inflammation processes, mainly at 8 hours. These findings were consistent with the histopathological observations, which showed an important leukocyte infiltrate and edema. At 24 hours, in addition to the identification of several pathways and networks involved in inflammatory processes, others related to cell cycle were also noticed, indicating the activation of satellite cells, responsible for the restoration of muscle tissue homeostasis. The systemic effects were more discrete and the liver was the organ exhibiting the greatest alterations among the other tissues and organs studied. Enrichment analyses of liver samples pointed out, at both times, the IL-6 signaling pathway as the most relevant one. In this pathway, normally activated in the presence of inflammatory processes, several up-regulated transcripts coding for acute phase proteins were identified. In addition, toxicity enrichment analyses revealed that a set of genes involved in the xenobiotics metabolism was down-regulated. The other samples studied did not show relevant results in the context of the experimental conditions. High-throughput technologies have been poorly exploited in the “toxicovenomic” field so far. Here, through the combination of a modern technology, RNA-seq, a powerful tool for the study of transcriptomes, bioinformatics programs and a traditional approach, such as histology, it was possible to obtain a comprehensive view of the molecular mechanisms underlying the pathological effects in response to venom of M. corallinus. Keywords: Envenoming, Micrurus corallinus,

As serpentes da família Elapidae possuem venenos altamente neurotóxicos. Estes venenos são ricos, particularmente, em duas famílias de proteínas neurotóxicas: α- neurotoxinas e fosfolipases A2 (PLA2), as quais agem pré e pós-sinapticamente na junção neuromuscular, respectivamente. No continente Americano, a família Elapidae está representada pelo grupo das cobras corais, que inclui três gêneros (Micrurus, Leptomicrurus e Micruroides), sendo o gênero Micrurus o que exibe o maior número de espécies. No Brasil, M. frontalis e M. corallinus são as espécies de maior importância médica, e, em consequência, as usadas para a produção dos soros hiperimunes utilizados nos acidentes micrúricos. O presente trabalho teve como objetivo aprofundar os conhecimentos dos efeitos, locais e sistêmicos, do veneno de M. corallinus quando inoculado em doses subletais em camundongos heterogêneos, por meio de uma análise de expressão gênica diferencial com o uso de RNA-Seq. Para tal, a partir do RNA extraído do cérebro, rim, fígado, baço, coração, diafragma e ambos os músculos gastrocnêmios, em 8 e 24 horas de tratamento, foram preparadas bibliotecas de cDNA e sequenciadas no equipamento Illumina HiSeq1500. Os dados obtidos do sequenciamento foram processados por uma bateria de programas bioinformáticos e os resultados daqueles genes que apresentaram mudanças na expressão gênica foram submetidos a análises de enriquecimento no intuito de investigar as vias e redes moleculares afetadas. Em outra frente, foram realizadas análises histopatológicas para auxiliar na interpretação dos resultados. Em termos de expressão gênica diferencial, os resultados indicaram uma forte resposta no gastrocnêmio direito, lugar da inoculação do veneno. No tratamento de 8 horas, as análises de enriquecimento identificaram uma miríade de genes envolvidos em vias e redes relacionadas a processos de adesão celular, quimiotaxia e inflamação. Estes achados foram condizentes com as observações realizadas por meio das análises histopatológicas, as quais evidenciaram um importante infiltrado leucocitário e edema. Em 24 horas, além de terem sido identificadas várias vias e redes envolvidas em processos inflamatórios, também foram notadas outras relacionadas ao ciclo celular, indicando a ativação das células satélites, responsáveis pelo restabelecimento da homeostase do tecido muscular. Os efeitos sistêmicos foram muito mais discretos, sendo o fígado o órgão que exibiu as maiores alterações dentre os outros tecidos e órgãos avaliados. As análises de enriquecimento das amostras do fígado indicaram, em ambos os tempos, a via de sinalização da IL-6 como a de maior relevância. Nesta via, ativada em presença de processos inflamatórios, foram identificados vários transcritos de proteínas de fase aguda regulados positivamente. Além disso, as análises de toxicidade revelaram que um conjunto de genes envolvidos no metabolismo de xenobióticos encontrou-se regulado negativamente. As outras amostras estudadas não mostraram resultados relevantes no contexto das condições experimentais. As tecnologias High-throughput têm sido pouco exploradas na área da “toxicovenômica”. Neste sentido, adotamos a combinação de uma moderna tecnologia, RNA-seq, poderosa ferramenta para o estudo de transcriptomas, sofisticados programas de bioinformática e uma abordagem tradicional, como a histologia, para obter uma visão abrangente dos mecanismos moleculares subjacentes aos efeitos patológicos desencadeados pelo veneno de M. corallinus.

Does the administration of pilocarpine prior to venom milking influence the composition of Micrurus corallinus venom?

Considering that the scarcity of venom represents a huge challenge for biochemical and functional studies of Micrurus species (coral snakes), in this report we describe for the first time the influence of pilocarpine administration prior to venom milking on the yield and protein composition of Micrurus corallinus venom. The administration of pilocarpine resulted in an increase of about 127% in the volume of venom milked, with similar protein content. Venoms showed similar protein bands distribution and intensity by SDS-PAGE and equivalents RP-HPLC profiles. Our proteomic analysis showed that venoms milked in the presence and absence of pilocarpine presented comparable protein profiles, in terms of protein composition and relative abundance. The toxins identified were assigned to 13 protein families and represent the most complete M. corallinus venom proteome described so far, in terms of number of protein families identified. Our data indicate that the administration of pilocarpine prior to venom milking increases the venom yield and does not change significantly the venom composition of M. corallinus. The employment of pilocarpine represents a useful approach to increase the yield of venom not only for Micrurus species, but also for other genera of snakes with limitations regarding the amount of venom available.

Reconhecimento de frações do veneno de cobras corais brasileiras pelo soro antielapídico / Recognition of venom fractions from Brazilian coral snakes by antielapid serum

Objetivos: Investigar a capacidade do soro antielapídico produzido no Brasil na identificação de frações do veneno de seis espécies, incluindo as que constituem o pool de inoculação: Micrurus brasiliensis, M. corallinus, M. frontalis, M. lemniscatus, M. spixii e M. surinamensis. Métodos: As amostras utilizadas fazem parte do banco de venenos do Centro de Estudos e Pesquisas Biológicas, da Pontifícia Universidade Católica de Goiás. O soro antielapídico foi cedido pela Fundação Ezequiel Dias. As metodologias empregadas foram eletroforese e imunoblotting. Resultados: Foi demonstrada uma variabilidade toxinológica e uma capacidade também variável de reconhecimento desses componentes pelo soro antielapídico. A partir da técnica de western-blotting o soro antielapídico da Fundação Ezequiel Dias foi capaz de reconhecer a maioria, mas não todos os componentes presentes nos venenos analisados. Conclusões: Os resultados sugerem uma eficácia restrita do soro antielapídico já que o mesmo possui limitações quanto as espécies Amazônicas, o que reforça a necessidade de uma revisão dos estudos intra e interespecíficos dos venenos micrúricos.

Aims: To investigate whether the antielapid serum produced in Brazil could identify venom fractions from six species of Micrurus, including those in the inoculation pool: Micrurus brasiliensis, M. corallinus, M. frontalis, M. lemniscatus, M. spixii, and M. surinamensis. Methods: The samples belong to the venom bank of the Center for Biological Studies and Research from the Pontifícia Universidade Católica de Goiás, Brazil. The antielapid serum was granted by the Ezequiel Dias Foundation. Both electrophoresis and immunoblotting methods were used. Results: Variability in venom components and in the ability to recognize such components was demonstrated by the antielapid serum. Based on the western-blotting technique, the antielapid serum from Ezequiel Dias Foundation was able to recognize most, but not all the components present in the analyzed venoms. Conclusions: The results suggest restricted efficacy of the antielapid serum, due to its limitations against species from the Amazon region, reinforcing the need for a review of intraspecific and interspecific studies of Micrurus venoms.

Identification and characterization of B-cell epitopes of 3FTx and PLA(2) toxins from Micrurus corallinus snake venom.

The main goal of this work was to develop a strategy to identify B-cell epitopes on four different three finger toxins (3FTX) and one phospholipase A2 (PLA2) from Micrurus corallinus snake venom. 3FTx and PLA2 are highly abundant components in Elapidic venoms and are the major responsibles for the toxicity observed in envenomation by coral snakes. Overlapping peptides from the sequence of each toxin were prepared by SPOT method and three different anti-elapidic sera were used to map the epitopes. After immunogenicity analysis of the spot-reactive peptides by EPITOPIA, a computational method, nine sequences from the five toxins were chemically synthesized and antigenically and immunogenically characterized. All the peptides were used together as immunogens in rabbits, delivered with Freund's adjuvant for a first cycle of immunization and Montanide in the second. A good antibody response against individual synthetic peptides and M. corallinus venom was achieved. Anti-peptide IgGs were also cross-reactive against Micrurus frontalis and Micrurus lemniscatus crude venoms. In addition, anti-peptide IgGs inhibits the lethal and phospholipasic activities of M. corallinus crude venom. Our results provide a rational basis to the identification of neutralizing epitopes on coral snake toxins and show that their corresponding synthetic peptides could improve the generation of immuno-therapeutics. The use of synthetic peptide for immunization is a reasonable approach, since it enables poly-specificity, low risk of toxic effects and large scale production.

New reports on parasitism by Haplometroides buccicola (Digenea, Plagiorchiidae) in Brazilian snakes

The occurrence of Haplometroides buccicola (Digenea, Plagiorchiidae) in the esophagus of two Brazilian snakes is reported in the present study. The trematodes were collected from one Micrurus corallinus (Elapidae) and one Phalotris lativittatus (Colubridae); both snakes were found in Botucatu city, São Paulo State, Brazil. Morphological and morphometric analyses of the trematodes are presented. For the first time Micrurus corallinus has been recorded as a host for H. buccicola and this is the second time that P. lativittatus has been reported as a host for this trematode species.