Danger in the Canopy. Comparative Proteomics and Bioactivities of the Venoms of the South American Palm Pit Viper Bothrops bilineatus Subspecies bilineatus and smaragdinus and Antivenomics of B. b. bilineatus (Rondônia) Venom against the Brazilian Pentabothropic Antivenom.

We report a structural and functional proteomics characterization of venoms of the two subspecies (Bothrops bilineatusbilineatus and B. b. smaragdinus) of the South American palm pit viper from the Brazilian state of Rondônia and B. b. smaragdinus from Perú. These poorly known arboreal and mostly nocturnal generalist predators are widely distributed in lowland rainforests throughout the entire Amazon region, where they represent an important cause of snakebites. The three B. bilineatus spp. venom samples exhibit overall conserved proteomic profiles comprising components belonging to 11 venom protein classes, with PIII (34-40% of the total venom proteins) and PI (8-18%) SVMPs and their endogenous tripeptide inhibitors (SVMPi, 8-10%); bradykinin-potentiating-like peptides (BBPs, 10.7-15%); snake venom serine proteinases (SVSP, 5.5-14%); C-type lectin-like proteins (CTL, 3-10%); phospholipases A2 (PLA2, 2.8-7.6%); cysteine-rich secretory proteins (CRISP, 0.9-2.8%); l-amino acid oxidases (LAO, 0.9-5%) representing the major components of their common venom proteomes. Comparative analysis of the venom proteomes of the two geographic variants of B. b. smaragdinus with that of B. b. bilineatus revealed that the two Brazilian taxa share identical molecules between themselves but not with Peruvian B. b. smaragdinus, suggesting hybridization between the geographically close, possibly sympatric, Porto Velho (RO, BR) B. b. smaragdinus and B. b. bilineatus parental populations. However, limited sampling does not allow determining the frequency of this event. The toxin arsenal of the South American palm pit vipers may account for the in vitro recorded collagenolytic, caseinolytic, PLA2, l-amino acid oxidase, thrombin-like and factor X-activating activities, and the clinical features of South American palm pit viper envenomings, i.e., local and progressively ascending pain, shock and loss of consciousness, spontaneous bleeding, and profound coagulopathy. The remarkable cross-reactivity of the Brazilian pentabothropic SAB antivenom toward the heterologous B. b. bilineatus venom suggests that the paraspecific antigenic determinants should have been already present in the venom of the last common ancestor of the Bothrops ″jararaca″ and ″taeniatus″ clades, about 8.5 Mya in the mid-late Miocene epoch of the Cenozoic era. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifiers PXD020043, PXD020026, and PXD020013.

Venomics and antivenomics of the poorly studied Brazil's lancehead, Bothrops brazili (Hoge, 1954), from the Brazilian State of Pará.

BACKGROUND: The Brazil's lancehead, Bothrops brazili, is a poorly studied pit viper distributed in lowlands of the equatorial rainforests of southern Colombia, northeastern Peru, eastern Ecuador, southern and southeastern Venezuela, Guyana, Suriname, French Guiana, Brazil, and northern Bolivia. Few studies have been reported on toxins isolated from venom of Ecuadorian and Brazilian B. brazili. The aim of the present study was to elucidate the qualitative and quantitative protein composition of B. brazili venom from Pará (Brazil), and to carry out a comparative antivenomics assessment of the immunoreactivity of the Brazilian antibothropic pentavalent antivenom [soro antibotrópico (SAB) in Portuguese] against the venoms of B. brazili and reference species, B. jararaca. METHODS: We have applied a quantitative snake venomics approach, including reverse-phase and two-dimensional electrophoretic decomplexation of the venom toxin arsenal, LC-ESI-MS mass profiling and peptide-centric MS/MS proteomic analysis, to unveil the overall protein composition of B. brazili venom from Pará (Brazil). Using third-generation antivenomics, the specific and paraspecific immunoreactivity of the Brazilian SAB against homologous (B. jararaca) and heterologous (B. brazili) venoms was investigated. RESULTS: The venom proteome of the Brazil's lancehead (Pará) is predominantly composed of two major and three minor acidic (19%) and two major and five minor basic (14%) phospholipase A2 molecules; 7-11 snake venom metalloproteinases of classes PI (21%) and PIII (6%); 10-12 serine proteinases (14%), and 1-2 L-amino acid oxidases (6%). Other toxins, including two cysteine-rich secretory proteins, one C-type lectin-like molecule, one nerve growth factor, one 5'-nucleotidase, one phosphodiesterase, one phospholipase B, and one glutaminyl cyclase molecule, represent together less than 2.7% of the venom proteome. Third generation antivenomics profile of the Brazilian pentabothropic antivenom showed paraspecific immunoreactivity against all the toxin classes of B. brazili venom, with maximal binding capacity of 132.2 mg venom/g antivenom. This figure indicates that 19% of antivenom's F(ab')2 antibodies bind B. brazili venom toxins. CONCLUSION: The proteomics outcome contribute to a deeper insight into the spectrum of toxins present in the venom of the Brazil's lancehead, and rationalize the pathophysiology underlying this snake bite envenomings. The comparative qualitative and quantitative immunorecognition profile of the Brazilian pentabothropic antivenom toward the venom toxins of B. brazili and B. jararaca (the reference venom for assessing the bothropic antivenom's potency in Brazil), provides clues about the proper use of the Brazilian antibothropic polyvalent antivenom in the treatment of bites by the Brazil's lancehead.

Venomics and antivenomics of the poorly studied Brazil's lancehead, Bothrops brazili (Hoge, 1954), from the Brazilian State of Pará

Background: The Brazils lancehead, Bothrops brazili, is a poorly studied pit viper distributed in lowlands of the equatorial rainforests of southern Colombia, northeastern Peru, eastern Ecuador, southern and southeastern Venezuela, Guyana, Suriname, French Guiana, Brazil, and northern Bolivia. Few studies have been reported on toxins isolated from venom of Ecuadorian and Brazilian B. brazili. The aim of the present study was to elucidate the qualitative and quantitative protein composition of B. brazili venom from Pará (Brazil), and to carry out a comparative antivenomics assessment of the immunoreactivity of the Brazilian antibothropic pentavalent antivenom [soro antibotrópico (SAB) in Portuguese] against the venoms of B. brazili and reference species, B. jararaca. Methods: We have applied a quantitative snake venomics approach, including reverse-phase and two-dimensional electrophoretic decomplexation of the venom toxin arsenal, LC-ESI-MS mass profiling and peptide-centric MS/MS proteomic analysis, to unveil the overall protein composition of B. brazili venom from Pará (Brazil). Using third-generation antivenomics, the specific and paraspecific immunoreactivity of the Brazilian SAB against homologous (B. jararaca) and heterologous (B. brazili) venoms was investigated. Results: The venom proteome of the Brazils lancehead (Pará) is predominantly composed of two major and three minor acidic (19%) and two major and five minor basic (14%) phospholipase A2 molecules; 7-11 snake venom metalloproteinases of classes PI (21%) and PIII (6%); 10-12 serine proteinases (14%), and 1-2 L-amino acid oxidases (6%). Other toxins, including two cysteine-rich secretory proteins, one C-type lectin-like molecule, one nerve growth factor, one 5'-nucleotidase, one phosphodiesterase, one phospholipase B, and one glutaminyl cyclase molecule, represent together less than 2.7% of the venom proteome...(AU)

Venomics and antivenomics of the poorly studied Brazil's lancehead, Bothrops brazili (Hoge, 1954), from the Brazilian State of Pará

The Brazil's lancehead, Bothrops brazili, is a poorly studied pit viper distributed in lowlands of the equatorial rainforests of southern Colombia, northeastern Peru, eastern Ecuador, southern and southeastern Venezuela, Guyana, Suriname, French Guiana, Brazil, and northern Bolivia. Few studies have been reported on toxins isolated from venom of Ecuadorian and Brazilian B. brazili. The aim of the present study was to elucidate the qualitative and quantitative protein composition of B. brazili venom from Pará (Brazil), and to carry out a comparative antivenomics assessment of the immunoreactivity of the Brazilian antibothropic pentavalent antivenom [soro antibotrópico (SAB) in Portuguese] against the venoms of B. brazili and reference species, B. jararaca. Methods: We have applied a quantitative snake venomics approach, including reverse-phase and two-dimensional electrophoretic decomplexation of the venom toxin arsenal, LC-ESI-MS mass profiling and peptide-centric MS/MS proteomic analysis, to unveil the overall protein composition of B. brazili venom from Pará (Brazil). Using third-generation antivenomics, the specific and paraspecific immunoreactivity of the Brazilian SAB against homologous (B. jararaca) and heterologous (B. brazili) venoms was investigated. Results: The venom proteome of the Brazil's lancehead (Pará) is predominantly composed of two major and three minor acidic (19%) and two major and five minor basic (14%) phospholipase A2 molecules; 7-11 snake venom metalloproteinases of classes PI (21%) and PIII (6%); 10-12 serine proteinases (14%), and 1-2 L-amino acid oxidases (6%). Other toxins, including two cysteine-rich secretory proteins, one C-type lectin-like molecule, one nerve growth factor, one 5'-nucleotidase, one phosphodiesterase, one phospholipase B, and one glutaminyl cyclase molecule, represent together less than 2.7% of the venom proteome. Third generation antivenomics profile of the Brazilian pentabothropic antivenom showed paraspecific immunoreactivity against all the toxin classes of B. brazili venom, with maximal binding capacity of 132.2 mg venom/g antivenom. This figure indicates that 19% of antivenom's F(ab')2 antibodies bind B. brazili venom toxins. Conclusion: The proteomics outcome contribute to a deeper insight into the spectrum of toxins present in the venom of the Brazil's lancehead, and rationalize the pathophysiology underlying this snake bite envenomings. The comparative qualitative and quantitative immunorecognition profile of the Brazilian pentabothropic antivenom toward the venom toxins of B. brazili and B. jararaca (the reference venom for assessing the bothropic antivenom's potency in Brazil), provides clues about the proper use of the Brazilian antibothropic polyvalent antivenom in the treatment of bites by the Brazil's lancehead.(AU)

Danger in the canopy. Comparative proteomics and bioactivities of the venoms of the South American palm pit viper bothrops bilineatus subspecies bilineatus and smaragdinus and antivenomics of B. b. bilineatus (Rondônia) venom against the Brazilian pentabothropic antivenom

We report a structural and functional proteomics characterization of venoms of the two subspecies (Bothrops bilineatusbilineatus and B. b. smaragdinus) of the South American palm pit viper from the Brazilian state of Rondônia and B. b. smaragdinus from Perú. These poorly known arboreal and mostly nocturnal generalist predators are widely distributed in lowland rainforests throughout the entire Amazon region, where they represent an important cause of snakebites. The three B. bilineatus spp. venom samples exhibit overall conserved proteomic profiles comprising components belonging to 11 venom protein classes, with PIII (34–40% of the total venom proteins) and PI (8–18%) SVMPs and their endogenous tripeptide inhibitors (SVMPi, 8–10%); bradykinin-potentiating-like peptides (BBPs, 10.7–15%); snake venom serine proteinases (SVSP, 5.5–14%); C-type lectin-like proteins (CTL, 3–10%); phospholipases A2 (PLA2, 2.8–7.6%); cysteine-rich secretory proteins (CRISP, 0.9–2.8%); l-amino acid oxidases (LAO, 0.9–5%) representing the major components of their common venom proteomes. Comparative analysis of the venom proteomes of the two geographic variants of B. b. smaragdinus with that of B. b. bilineatus revealed that the two Brazilian taxa share identical molecules between themselves but not with Peruvian B. b. smaragdinus, suggesting hybridization between the geographically close, possibly sympatric, Porto Velho (RO, BR) B. b. smaragdinus and B. b. bilineatus parental populations. However, limited sampling does not allow determining the frequency of this event. The toxin arsenal of the South American palm pit vipers may account for the in vitro recorded collagenolytic, caseinolytic, PLA2, l-amino acid oxidase, thrombin-like and factor X-activating activities, and the clinical features of South American palm pit viper envenomings, i.e., local and progressively ascending pain, shock and loss of consciousness, spontaneous bleeding, and profound coagulopathy. The remarkable cross-reactivity of the Brazilian pentabothropic SAB antivenom toward the heterologous B. b. bilineatus venom suggests that the paraspecific antigenic determinants should have been already present in the venom of the last common ancestor of the Bothrops ″jararaca″ and ″taeniatus″ clades, about 8.5 Mya in the mid-late Miocene epoch of the Cenozoic era. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifiers PXD020043, PXD020026, and PXD020013.

Comparative venomics of Brazilian coral snakes: Micrurus frontalis, Micrurus spixii spixii, and Micrurus surinamensis.

A comparative venom proteomic analysis of the Brazilian southern coral snake, M. frontalis, the Amazon coral snake M. spixii spixii, and the aquatic coral snake M. surinamensis is reported. Venoms from M. frontalis and M. s. spixii were composed mainly (>90% of the total venom proteome) by 3FTxs and PLA2s in different proportions, and minor proteins from 2 to 5 protein families. Conversely, the aquatic coral snake expressed a streamlined (95%) 3FTx venom with low abundance (4.2%) of PLA2 molecules. A compositional-lethal activity for natural prey correlation analysis suggests that M. surinamensis venom may has evolved under strong pressure to quickly immobilize aquatic prey. On the other hand, venoms from M. frontalis and M. s. spixii, whose diet consist mainly of amphisbaenians and colubrid snakes, may have been shaped through balancing selection. Our work provides strong evidence for the occurrence in M. frontalis venom, but not in those from M. s. spixi and M. surinamensis, of a KUN-PLA2 complex homologue to heterodimeric venom toxins from some long-tailed monadal coral snakes that target acid-sensing receptors ASIC1a/2 evoking pain. The M. frontalis protein would represent the first example of a KUN-PLA2 heterodimer in a South American short-tailed triadal coral snake venom.

New insights into the phylogeographic distribution of the 3FTx/PLA2 venom dichotomy across genus Micrurus in South America.

Micrurus is a monophyletic genus of venomous coral snakes of the family Elapidae. The ~80 recognized species within this genus are endemic to the Americas, and are distributed from southeastern United States to northern Argentina. Although relatively few bites are recorded due to their reclusive nature, semi-fossorial habits, and their occurrence in sparsely populated areas, coral snakes possess powerful venoms that target the cholinergic system and, if early treatment is missed, can cause neuromuscular paralysis, respiratory failure, and death by asphyxiation within hours of envenoming. The to-date proteomically characterized 18 micrurine venoms exhibit a puzzling phenotypic dichotomy, characterized by the toxin arsenal being dominated either by pre-synaptically acting PLA2s or post-synaptic 3FTxs, and a general, but imperfect, distributional pattern of these venom phenotypes along the North-South axis of the American continent. The lack of perfect phylogenetic clustering suggests that phylogeny may not be the sole factor driving the evolution of the divergent venom phenotypes across Micrurus venoms. To shed new light on the origin and expression pattern of the 3FTx/PLA2 venom dichotomy, we have conducted a comparative proteomics analysis of venoms from the Brazilian ribbon coral snake, Micrurus lemniscatus carvalhoi, sourced from different localities in the Brazilian states of São Paulo; the Caatinga coral snake, M. ibiboboca, from central Bahia state (Brazil); two Micrurus specimens of uncertain taxonomy collected in the Brazilian states of Alagoas and Rio de Janeiro; and the Western ribbon coral snake, M. l. helleri, from Leticia, the southernmost town of the Colombian Department of Amazonas. Venoms from São Paulo and Rio de Janeiro showed 3FTx-predominant phenotypes, while in venoms from Leticia, Alagoas and Bahia PLA2s represented the major toxin family. Comparative venom proteomics suggests that both Micrurus venom phenotypes exhibit a high degree of toxin evolvability. Mapping the 3FTx/PLA2 dichotomy across the Americas points to a phylogeographic pattern for venom phenotypes consistent with, but more complex than, the North-South distribution hypothesis anticipated in previous investigations. BIOLOGICAL SIGNIFICANCE: New World coral snakes (Micrurus: Elapidae) produce potent venoms that target pre- and post-synaptically cholinergic nerve terminals resulting in neuromuscular paralysis, and in severe envenomings, may lead to death from asphyxiation by respiratory arrest. Presynaptic ß-neurotoxins of group IA PLA2 protein subfamily and postsynaptic α-neurotoxins with 3FTx fold are the major components (>80%) of coral snake venoms. Micrurine venoms exhibit a puzzling phenotypic venom dichotomy, characterized by the dominant expression of either α- or ß-neurotoxins. The distribution of these alternative compositional profiles has been fragmentarily studied both across Micrurus phylogeny and along the North-South axis of the genus radiation in the American continent, from southern United States to Northern Argentina. The unpredictability of the neurotoxin profile across the distribution range of the coral snakes represents a difficulty for applying the most appropriate treatment upon a coral snakebite. A deep knowledge of the phylogeographic distribution and the evolution of dichotomic Micrurus venoms would be useful for tracing the evolutionary path to their present day phenotypes, rationalizing the patchy cross-reactivity of current Micrurus antivenoms, and improving the efficacy of antivenoms to neutralize coral snake envenomings.