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Abstract: We report the comparative proteomic and antivenomic characterization of the venoms of subspecies cascavella and collilineatus of the Brazilian tropical rattlesnake Crotalus durissus. The venom proteomes of C. d. collilineatus and C. d. cascavella comprise proteins in the range of 4–115kDa belonging to 9 and 8 toxin families, respectively. Collilineatus and cascavella venoms contain 20–25 main toxins belonging to the following protein families: disintegrin, PLA2, serine proteinase, cysteine-rich secretory protein (CRISP), vascular endothelial growth factor-like (VEGF), l-amino acid oxidase, C-type lectin-like, and snake venom metalloproteinase (SVMP). As judged by reverse-phase HPLC and mass spectrometry, cascavella and collilineatus share about 90% of their venom proteome. However, the relative occurrence of the toxin families departs among the two C. durissus subspecies venoms. The most notable difference is the presence of the myotoxin crotamine in some C. d. collilineatus specimens (averaging 20.8% of the total proteins of pooled venom), which is absent in the venom of C. d. cascavella. On the other hand, the neurotoxic PLA2 crotoxin represents the most abundant protein in both C. durissus venoms, comprising 67.4% of the toxin proteome in C. d. collilineatus and 72.5% in C. d. cascavella. Myotoxic PLA2s are also present in the two venoms albeit in different relative concentrations (18.1% in C. d. cascavella vs. 4.6% in C. d. collilineatus). The venom composition accounts for the clinical manifestations caused by C. durissus envenomations: systemic neurotoxicity and myalgic symptoms and coagulation disturbances, frequently accompanied by myoglobinuria and acute renal failure. The overall compositions of C. d. subspecies cascavella and collilineatus venoms closely resemble that of C. d. terrificus, supporting the view that these taxa can be considered geographical variations of the same species. Pooled venom from adult C.d. cascavella and neonate C.d. terrificus lack crotamine, whereas this skeletal muscle cell membrane depolarizing inducing myotoxin accounts for ∼20% of the total toxins of venom pooled from C.d. collilineatus and C.d. terrificus from Southern Brazil. The possible relevance of the observed venom variability among the tropical rattlesnake subspecies was assessed by antivenomics using anti-crotalic antivenoms produced at Instituto Butantan and Instituto Vital Brazil. The results revealed that both antivenoms exhibit impaired immunoreactivity towards crotamine and display restricted (∼60%) recognition of PLA2 molecules (crotoxin and D49-myotoxins) from C. d. cascavella and C. d. terrificus venoms. This poor reactivity of the antivenoms may be due to a combination of factors: on the one hand, an inappropriate choice of the mixture of venoms for immunization and, on the other hand, the documented low immunogenicity of PLA2 molecules. C. durissus causes most of the lethal snakebite accidents in Brazil. The implication of the geographic variation of venom composition for the treatment of bites by different C. durissus subspecies populations is discussed. [Copyright &y& Elsevier]

Abstract: Crotalus durissus rattlesnakes are responsible for the most lethal cases of snakebites in Brazil. Crotalus durissus collilineatus subspecies is related to a great number of accidents in Southeast and Central West regions, but few studies on its venom composition have been carried out to date. In an attempt to describe the transcriptional profile of the C. durissus collilineatus venom gland, we generated a cDNA library and the sequences obtained could be identified by similarity searches on existing databases. Out of 673 expressed sequence tags (ESTs) 489 produced readable sequences comprising 201 singletons and 47 clusters of two or more ESTs. One hundred and fifty reads (60.5%) produced significant hits to known sequences. The results showed a predominance of toxin-coding ESTs instead of transcripts coding for proteins involved in all cellular functions. The most frequent toxin was crotoxin, comprising 88% of toxin-coding sequences. Crotoxin B, a basic phospholipase A2 (PLA2) subunit of crotoxin, was represented in more variable forms comparing to the non-enzymatic subunit (crotoxin A), and most sequences coding this molecule were identified as CB1 isoform from Crotalus durissus terrificus venom. Four percent of toxin-related sequences in this study were identified as growth factors, comprising five sequences for vascular endothelial growth factor (VEGF) and one for nerve growth factor (NGF) that showed 100% of identity with C. durissus terrificus NGF. We also identified two clusters for metalloprotease from PII class comprising 3% of the toxins, and two for serine proteases, including gyroxin (2.5%). The remaining 2.5% of toxin-coding ESTs represent singletons identified as homologue sequences to cardiotoxin, convulxin, angiotensin-converting enzyme inhibitor and C-type natriuretic peptide, Ohanin, crotamin and PLA2 inhibitor. These results allowed the identification of the most common classes of toxins in C. durissus collilineatus snake venom, also showing some unknown classes for this subspecies and even for C. durissus species, such as cardiotoxins and VEGF. [Copyright &y& Elsevier]

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 PLA 2 s 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 PLA 2 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-PLA 2 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-PLA 2 heterodimer in a South American short-tailed triadal coral snake venom., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The Micrurus genus is the American representative of Elapidae family. Micrurus spixii is endemic of South America and northern states of Brazil. Elapidic venoms contain neurotoxins that promote curare-mimetic neuromuscular blockage. In this study, biochemical and functional characterizations of M. spixii crude venom were performed and a new neurotoxic phospholipase A2 called MsPLA2-I was isolated. M. spixii crude venom caused severe swelling in the legs of tested mice and significant release of creatine kinase (CK) showing its myotoxic activity. Leishmanicidal activity against Leishmania amazonensis (IC50 1.24 μg/mL) was also observed, along with antiplasmodial activity against Plasmodium falciparum, which are unprecedented for Micrurus venoms. MsPLA2-I with a Mr 12,809.4 Da was isolated from the crude venom of M. spixii. The N-terminal sequencing of a fragment of 60 amino acids showed 80% similarity with another PLA2 from Micrurus altirostris. This toxin and the crude venom showed phospholipase activity. In a mouse phrenic nerve-diaphragm preparation, M. spixii venom and MsPLA2-I induced the blockage of both direct and indirect twitches. While the venom presented a pronounced myotoxic activity, MsPLA2-I expressed a summation of neurotoxic activity. The results of this study make M. spixii crude venom promising compounds in the exploration of molecules with microbicidal potential., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Prof. Dr. José R. Giglio (1934–2014) made a highly significant contribution to the field of Toxinology. During 48 years devoted to research and teaching Prof Giglio published more than 160 articles, with more than 4400 citations, in international journals, trained a vast amount of graduate and undergraduate students, and developed an international network of collaborators. Throughout these years, he worked with dedication and deep commitment to science, leaving an immortalized legacy. During his professional career he contributed mostly in the isolation, and biochemical and functional characterization of various protein toxins derived from animal venoms such as snakes, scorpions and spiders, in addition to his studies searching for alternative therapies for poisoning. Even after his departure, his presence and influence remains among his former students and in the outstanding legacy of his scientific contributions.

Despite preventing death after snakebites, there is little evidence that polyvalent antivenoms (PAVs) protect against myotoxicity and local damages. We evaluated antibothropic Brazilian PAVs from three manufacturers against the myotoxicity and hemorrhagic activity of Bothrops jararacussu and B. jararaca venoms, respectively, by using two protocols: preincubation of PAVs with venom, and i.v. pretreatment with PAVs, prior to the venom inoculation. In this investigation, we used doses of PAVs ranging from 0.4 to 4.0mL/mg of venom equivalent up to 10 times the amount recommended by the producers for the clinical practice in Brazil. In our preincubation protocol in vivo, PAVs antagonized myotoxicity of B. jararacussu venom by 40-95%, while our pretreatment protocol antagonized myotoxic activity by 0-60%. Preincubation of antivenoms with B. jararaca venom antagonized its hemorrhagic activity by 70-95%, while pretreatment antagonized hemorrhagic activity by 10-50%. Although all PAVs demonstrated partial antagonism against both venoms, the magnitude of these effects varied greatly among the manufactures. The results suggest that the current clinical doses of these PAVs may have negligible antimyotoxic effect.

Aqueous extract of Casearia sylvestris (Flacourtiaceae) has been shown to inhibit enzymatic and biological properties of some Bothrops and Crotalus venoms and their purified phospholipase A(2) (PLA(2)) toxins. In this work we evaluated the influence of C. sylvestris aqueous extract upon neuromuscular blocking and muscle damaging activities of some PLA(2)s (crotoxin from C. durissus terrificus, bothropstoxin-I from B. jararacussu, piratoxin-I from B. pirajai and myotoxin-II from B. moojeni) in mouse phrenic-diaphragm preparations. Crotoxin (0.5 microM) and all other PLA(2) toxins (1.0 microM) induced irreversible and time-dependent blockade of twitches. Except for crotoxin, all PLA(2) toxins induced significant muscle damage indices, assessed by microscopic analysis. Preincubation of bothropstoxin-I, piratoxin-I or myotoxin-II with C. sylvestris extract (1:5 (w/w), 30 min, 37 degrees C) significantly prevented the neuromuscular blockade of preparations exposed to the mixtures for 90 min; the extent of protection ranged from 93% to 97%. The vegetal extract also neutralized the muscle damage (protection of 80-95%). Higher concentration of the C. sylvestris extract (1:10, w/w) was necessary to neutralize by 90% the neuromuscular blockade induced by crotoxin. These findings expanded the spectrum of C. sylvestris antivenom activities, evidencing that it may be a good source of potentially useful PLA(2) inhibitors.

Many plants are used in traditional medicine as active agents against various effects induced by snakebite. Few attempts have been made however to identify the nature of plant natural products with anti-ophidian properties. Baccharis trimera (Less) DC (Asteraceae), known in Brazil as carqueja, has been popularly used to treat liver diseases, rheumatism, diabetes, as well as digestive, hepatic and renal disorders. The active component was identified as 7alpha-hydroxy-3,13-clerodadiene-16,15:18,19-diolide, C20H28O5, (clerodane diterpenoid, Bt-CD). We report now the anti-proteolytic and anti-hemorrhagic properties against snake venoms of a Bt-CD inhibitor from B. trimera. Bt-CD exhibited full inhibition of hemorrhage and proteolytic activity caused by Bothrops snake venoms. The inhibitor was able to neutralize the hemorrhagic, fibrinogenolytic and caseinolytic activities of class P-I and III metalloproteases isolated from B. neuwiedi and B. jararacussu venoms. No inhibition of the coagulant activity was observed. Bt-CD also partially inhibited the edema induced by other crude venoms, metalloproteases, basic and acidic phospholipases A2. To further elucidate the inhibitory specificity of Bt-CD against metalloproteases isolated from snake venoms, a deeper understanding of its structure and function is necessary. Furthermore, the potential use of these inhibitors to complement anti-venom as an alternative treatment of snakebite envenomations needs to be evaluated in future studies.

Two basic myotoxic PLA(2)s, namely BnpTX-I and II, were isolated from Bothrops neuwiedi pauloensis snake venom through three chromatographic steps: ion-exchange chromatography on CM-Sepharose, gel filtration on Sephadex G-50 and reverse phase HPLC on a C18 column. Both PLA(2)s showed a M(r) around 14,000 for the monomer and 28,000 for the dimer (as estimated by SDS-PAGE), pI approximately 7.8 and approximately 121 amino acid residues cross-linked by seven disulfide bonds. The N-terminal sequences revealed significant homology with Asp49 basic myotoxic PLA(2)s from other snake venoms. The catalytic and anticoagulant activities of BnpTX-I were higher than those of BnpTX-II. Both were able to induce cytotoxicity in vitro, as well as, myotoxicity, edema and lethality in mice. BnpTX-I also induced neurotoxic effect on mouse neuromuscular preparations and bactericidal activity on Eschericia coli and Staphylococcus aureus. After chemical modification of BnpTX-I with BPB or incubation with EDTA or Mn(2+) ions, the catalytic activity was completely abolished, while the toxic and pharmacological activities were partially reduced. Interaction with heparin inhibited the cytotoxic and bactericidal effects. Anti-BthTX-I, anti-BthTX-II and anti-115-129-C terminal antibodies strongly recognize both BnpTX-I and II. It is shown that the neurotoxic effect induced by B. neuwiedi pauloensis venom is due to the presence of myotoxic PLA(2)s. The data also corroborate the hypothesis of a partial dissociation between toxic and enzymatic domains. In addition, BnpTX-I displays a heparin binding C-terminal region, which is probably responsible for the cytotoxic and bactericidal effects.