De novo venom gland transcriptomics of Calliophis bivirgata flaviceps: uncovering the complexity of toxins from the Malayan blue coral snake

The Malayan blue coral snake, Calliophis bivirgata flaviceps, is a medically important venomous snake in Southeast Asia. However, the complexity and diversity of its venom genes remain little explored. Methods: To address this, we applied high-throughput next-generation sequencing to profile the venom gland cDNA libraries of C. bivirgata flaviceps. The transcriptome was de novo assembled, followed by gene annotation, multiple sequence alignment and analyses of the transcripts. Results: A total of 74 non-redundant toxin-encoding genes from 16 protein families were identified, with 31 full-length toxin transcripts. Three-finger toxins (3FTx), primarily delta-neurotoxins and cardiotoxin-like/cytotoxin-like proteins, were the most diverse and abundantly expressed. The major 3FTx (Cb_FTX01 and Cb_FTX02) are highly similar to calliotoxin, a delta-neurotoxin previously reported in the venom of C. bivirgata. This study also revealed a conserved tyrosine residue at position 4 of the cardiotoxin-like/cytotoxin-like protein genes in the species. These variants, proposed as Y-type CTX-like proteins, are similar to the H-type CTX from cobras. The substitution is conservative though, preserving a less toxic form of elapid CTX-like protein, as indicated by the lack of venom cytotoxicity in previous laboratory and clinical findings. The ecological role of these toxins, however, remains unclear. The study also uncovered unique transcripts that belong to phospholipase A2 of Groups IA and IB, and snake venom metalloproteinases of PIII subclass, which show sequence variations from those of Asiatic elapids. Conclusion: The venom gland transcriptome of C. bivirgata flaviceps from Malaysia was de novo assembled and annotated. The diversity and expression profile of toxin genes provide insights into the biological and medical importance of the species.(AU)

De novo venom gland transcriptomics of Calliophis bivirgata flaviceps: uncovering the complexity of toxins from the Malayan blue coral snake

The Malayan blue coral snake, Calliophis bivirgata flaviceps, is a medically important venomous snake in Southeast Asia. However, the complexity and diversity of its venom genes remain little explored. Methods: To address this, we applied high-throughput next-generation sequencing to profile the venom gland cDNA libraries of C. bivirgata flaviceps. The transcriptome was de novo assembled, followed by gene annotation, multiple sequence alignment and analyses of the transcripts. Results: A total of 74 non-redundant toxin-encoding genes from 16 protein families were identified, with 31 full-length toxin transcripts. Three-finger toxins (3FTx), primarily delta-neurotoxins and cardiotoxin-like/cytotoxin-like proteins, were the most diverse and abundantly expressed. The major 3FTx (Cb_FTX01 and Cb_FTX02) are highly similar to calliotoxin, a delta-neurotoxin previously reported in the venom of C. bivirgata. This study also revealed a conserved tyrosine residue at position 4 of the cardiotoxin-like/cytotoxin-like protein genes in the species. These variants, proposed as Y-type CTX-like proteins, are similar to the H-type CTX from cobras. The substitution is conservative though, preserving a less toxic form of elapid CTX-like protein, as indicated by the lack of venom cytotoxicity in previous laboratory and clinical findings. The ecological role of these toxins, however, remains unclear. The study also uncovered unique transcripts that belong to phospholipase A2 of Groups IA and IB, and snake venom metalloproteinases of PIII subclass, which show sequence variations from those of Asiatic elapids. Conclusion: The venom gland transcriptome of C. bivirgata flaviceps from Malaysia was de novo assembled and annotated. The diversity and expression profile of toxin genes provide insights into the biological and medical importance of the species.(AU)

Proteome of fraction from Tityus serrulatus venom reveals new enzymes and toxins

Tityus serrulatus venom (Ts venom) is a complex mixture of several compounds with biotechnological and therapeutical potentials, which highlights the importance of the identification and characterization of these components. Although a considerable number of studies have been dedicated to the characterization of this complex cocktail, there is still a limitation of knowledge concerning its venom composition. Most of Ts venom studies aim to isolate and characterize their neurotoxins, which are small, basic proteins and are eluted with high buffer concentrations on cation exchange chromatography. The first and largest fraction from carboxymethyl cellulose-52 (CMC-52) chromatography of Ts venom, named fraction I (Fr I), is a mixture of proteins of high and low molecular masses, which do not interact with the cation exchange resin, being therefore a probable source of components still unknown of this venom. Thus, the present study aimed to perform the proteome study of Fraction I from Ts venom, by high resolution mass spectrometry, and its biochemical characterization, by the determination of several enzymatic activities. Methods: Fraction I was obtained by a cation exchange chromatography using 50 mg of crude venom. This fraction was subjected to a biochemical characterization, including determination of L-amino acid oxidase, phospholipase, hyaluronidase, proteases activities and inhibition of angiotensin converting enzyme (ACE) activity. Fraction I was submitted to reduction, alkylation and digestion processes, and the tryptic digested peptides obtained were analyzed in a Q-Exactive Orbitrap mass spectrometer. Data analysis was performed by PEAKS 8.5 software against NCBI database. Results: Fraction I exhibits proteolytic activity and it was able to inhibit ACE activity. Its proteome analysis identified 8 different classes of venom components, among them: neurotoxins (48%), metalloproteinases (21%), hypotensive peptides (11%), cysteine-rich venom protein (9%), antimicrobial peptides (AMP), phospholipases and other enzymes (chymotrypsin and lysozymes) (3%) and phosphodiesterases (2%). Conclusions: The combination of a proteomic and biochemical characterization strategies leads us to identify new components in the T. serrulatus scorpion venom. The proteome of venom´s fraction can provide valuable direction in the obtainment of components in their native forms in order to perform a preliminary characterization and, consequently, to promote advances in biological discoveries in toxinology.(AU)

Extracellular phospholipase production of oral Candida albicans isolates from smokers, diabetics, asthmatics, denture wearers and healthy individuals following brief exposure to polyene, echinocandin and azole antimycotics

Objective Candida albicans is the primary causative agent of oral candidosis, and one of its key virulent attributes is considered to be its ability to produce extracellular phospholipases that facilitate cellular invasion. Oral candidosis can be treated with polyenes, and azoles, and the more recently introduced echinocandins. However, once administered, the intraoral concentration of these drugs tend to be sub-therapeutic and rather transient due to factors such as the diluent effect of saliva and cleansing effect of the oral musculature. Hence, intra-orally, the pathogenic yeasts may undergo a brief exposure to antifungal drugs. We, therefore, evaluated the phospholipase production of oral C. albicans isolates following brief exposure to sub-therapeutic concentrations of the foregoing antifungals. Materials and methods Fifty C. albicans oral isolates obtained from smokers, diabetics, asthmatics using steroid inhalers, partial denture wearers and healthy individuals were exposed to sub-therapeutic concentrations of nystatin, amphotericin B, caspofungin, ketoconazole and fluconazole for one hour. Thereafter the drugs were removed and the phospholipase production was determined by a plate assay using an egg yolk-agar medium. Results The phospholipase production of these isolates was significantly suppressed with a percentage reduction of 10.65, 12.14, 11.45 and 6.40% following exposure to nystatin, amphotericin B, caspofungin and ketoconazole, respectively. This suppression was not significant following exposure to fluconazole. Conclusions Despite the sub-therapeutic, intra oral, bioavailability of polyenes, echinocandins and ketoconazole, they are likely to produce a persistent antifungal effect by suppressing phospholipase production, which is a key virulent attribute of this common pathogenic yeast.(AU)

In vitro activity of phospholipase A 2 and of peptides from Crotalus durissus terrificus venom against amastigote and promastigote forms of Leishmania (L) infantum chagasi

Background American visceral leishmaniasis is caused by the intracellular parasiteLeishmania (L.) infantum chagasi, and transmitted by the sand fly Lutzomyia longipalpis. Since treatment is based on classical chemotherapeutics with significant side effects, the search for new drugs remains the greatest global challenge. Thus, this in vitro study aimed to evaluate the leishmanicidal effect ofCrotalus durissus terrificus venom fractions on promastigote and amastigote forms of Leishmania (L.) infantum chagasi. Methods Phospholipase A 2 (PLA 2 ) and a pool of peptide fraction (<3 kDa) were purified from Crotalusvenom. Furthermore, promastigotes and peritoneal macrophages of mice infected by amastigotes were exposed to serial dilutions of the PLA 2 and peptides at intervals varying between 1.5625 μg/mL and 200 μg/mL. Both showed activity against promastigotes that varied according to the tested concentration and the time of incubation (24, 48 and 72 h). Results MTT assay for promastigotes showed IC 50 of 52.07 μg/mL for PLA2 and 16.98 μg/mL for the peptide fraction of the venom. The cytotoxicity assessment in peritoneal macrophages showed IC50 of 98 μg/mL and 16.98 μg/mL for PLA 2 and peptide by MTT assay, respectively. In peritoneal macrophages infected by Leishmania (L.) infantum chagasi amastigotes, the PLA 2 stimulated growth of parasites, and at higher doses reduced growth by 23 %. The peptide fraction prevented 43 % of the intracellular parasite growth at a dose of 16.98 μg/mL, demonstrating the toxicity of this dose to macrophages. Both fractions stimulated H 2 O 2 production by macrophages but only PLA 2 was able to stimulate NO production. Conclusion We have demonstrated the in vitro leishmanicidal activity of the PLA2 and peptide fraction ofCrotalus venom. The results encourage further studies to describe the metabolic pathways involved in cell death, as well as the prospecting of molecules with antiparasitic activity present in the peptide fraction of Crotalus durissus terrificus venom.(AU)

HAART therapy does not reduce the proteinase and phospholipase secretion by oral Candida albicans isolated from HIV-positive patients

Production of exoenzymes, specifically the proteinase and phospholipase, is considered one of the most important of pathogenicity mechanisms of C. albicans, which is crucial for tissue invasion. This study aimed at evaluating the production of these exoenzymes in 50 oral C. albicans isolates from HIV-positive (HIV+) patients treated with highly active anti-retroviral therapy (HAART), and from 50 control individuals. For testing the production of phospholipase and proteinase, the culture media containing egg yolk and bovine albumin were used, respectively. The results were obtained by measuring the diameter of the colony and divided by the diameter of colony plus the precipitation zone, defined as Pz. Data were statistically analyzed by Students t test (5). Statistically significant difference (p = 0.001) was observed between the mean values of Pz for proteinase in isolates from HIV+ patients (Pz = 0.358±0.295) and from control group (Pz = 0.660±0.370). The same results were observed for phospholipase production (Pz = 0.399±0.227 for HIV+ group; Pz =0.635±0.292 control group). Both enzymes were highly produced by C. albicans isolated from HIV+ patients when compared with those secreted by C. albicans obtained from control group, suggesting that HAART did not reduce the secretion of these enzymes by this pathogenic fungus infecting HIV+ patients.(AU)

Functional and structural characterization of phospholipases A2 isolated from Bothrops asper snake venom in Panamá

Envenoming by Bothrops snakes is the most serious type of envenoming from the medical and economic point of view in Central America. Bothrops asper is responsible for 90% of the snakebites registered in Panamá every year. Despite its medical and economic relevance, only the venom of Costa Rican and Guatemalan populations of this species has been studied to some detail, and there is very little information on intraspecies variability in venom composition and toxicity. In this study the crude venom of B. asper from Panamá was characterized and its pharmacological and biochemistry activities were investigated with standard laboratory assays. Furthermore, we described the isolation, functional and structural characterization of four basic phospholipases A2, namely MTX-I, MTX-II, MTX-III, MTX-IV, and a new acid phospholipase A2 called Basp-I-PLA2. The proteins were isolated from the crude venom by a combination of two chromatographic steps, using ion-exchange chromatography on CM-Sepharose (0.05 M NH4HCO3 pH 8.1 buffer), and hydrophobic chromatography on Phenyl-Sepharose (0.05 M Tris-HCl pH 7.4), followed by concentration gradient from 4 to 0 M NaCl at 25°C in the same buffer. Analyses of phospholipids hydrolyzed by these enzymes have shown that all phospholipases belong to type A2. The acidic isoform demonstrated more catalytic activity than basic PLA2s. This enzyme was more active on substrates such as phosphotidylcholine and phosphatidylglycerol. The isoelectric focusing evidenced pIs between 8.1 to 8.3 for MTXs and 4.6 for the isoform Basp-I-PLA2. The molecular weight was estimated by mass spectrometry to be: MTX-1 = 14,156.5; MTX-2 = 14,249.5 and MTX-3 = 14,253.0 and Basp-I-PLA2 = 14,246.0.8 Da.(AU)

Clinical and biochemical manifestation produced by scorpion (Hemiscorpius lepturus) venom in experimental animals

Several studies have been published about the clinical and biochemical manifestations produced by the venom of scorpions of the Buthidae family, but very few reports have indicated the manifestations induced by the venom of the Scorpionidae family. Hemiscorpius lepturus is an important scorpion species present in the south and southwestern part of Iran, causing morbidity and mortality in children and adults. For the present study, H. lepturus venom was extracted by electric shock and subcutaneously injected (6.3mg/kg) into a group of six rabbits. Blood collection was carried out before and three hours after venom injection for determination of osmotic fragility and levels of blood sugar, alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), creatine phosphokinase (CPK) and alkaline phosphatase (ALP). In vitro studies were also carried out to verify the osmotic fragility of red blood cells (RBCs) exposed to venom concentrations ranging from 0-90µg/2ml blood. Results showed the extreme effect of this venom on the lysis of RBCs both in vitro and in vivo. Venom injection caused significant (p>0.001) increase in ALT, AST, LDH and blood sugar levels. There was also an increase in CPK, and ALP levels after venom injection; however, it was not statistically significant. All animals died four hours after having received the venom. The current study revealed that the neurological effect of H. lepturus venom is similar to that of scorpions of the Buthidae family. However, they differ in RBCs lysis, which was highly significant when induced by H. lepturus venom, probably due to the presence of a type of phospholipase in this venom. Further studies are needed to provide a clearer view of the mechanism of action of H. lepturus venom.(AU)