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Patagonin - CRISP: Antimicrobial activity and source of antimicrobial molecules in Duvernoy's gland secretion - Philodryas patagoniensis snake

ABSTRACT: Snake venoms contain a variety of toxins with a range of biological activity. Among these are the cysteine-rich secreted proteins (CRISPs). The proteins of this family have masses of 20–30 kDa and display homologous amino acid sequences containing 16 cysteine residues, forming eight disulfide bonds. Some of these proteins have been explored, characterized, and described in terms of their activity; however, little is known of their range of activities. A search for new antimicrobial molecules is ongoing, as the number of microbial strains resistant to available antibiotics is increasing. We identified antimicrobial activity in the secretion of the Duvernoy’s gland of the rear-fanged Philodryas patagoniensis. Fractions of this venom were subjected to Reverse Phase – High Performance Liquid Chromatography and analyzed to determine their antimicrobial activity with a liquid broth inhibition assay. One of the fractions presented activity against a Gram-negative bacterium and a filamentous fungus. This fraction was analyzed with LC-MS/MS, and a protein of 24,848.8 Da was identified. Database searches allowed us to identify it as a CRISP due to the presence of some unique fragments in the molecule. We called it patagonin-CRISP, as the same protein in the venom of P. patagoniensis had previously been characterized as having a different biological activity. Patagonin-CRISP presented activity at very low concentrations and showed no cytotoxic activity. This is the first time that antimicrobial activity has been identified for P. patagoniensis venom or for a CRISP family protein.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Philodryas Patagoniensis

SUBMITTER: Juliana Badari

LAB HEAD: Pedro Ismael da Silva Junior

PROVIDER: PXD022842 | Pride | 2021-02-01

REPOSITORIES: Pride

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Project description:Spider venoms are a unique source of bioactive peptides displaying remarkable structural stability and potent neuroactivity. Phoneutria nigriventer, often referred to as Brazilian wandering spider, banana spider or “armed” spider, is endemic from South America and amongst the most dangerous venomous spiders in the world. Envenomation accidents with P. nigriventer often occur in Brazil with approximately 4,000 cases per year and which symptoms include priapism, hypertension, blurred vision, sweating, and vomiting, amongst others. Besides its clinical relevance, P. nigriventer venom comprises promising peptide drug leads providing therapeutic effects in a range of disease models. In this study, we further explored the neuroactivity and molecular diversity of the venom from P. nigriventer using fractionation-guided high-throughput cellular assays coupled to proteomics analysis and multi-pharmacology activity to broaden the knowledge and therapeutic potential of this venom, as well as a proof-of-concept for an investigative pipeline to study spider-venom derived neuroactive peptides. We applied ion channel assays in a neuroblastoma cell line to investigate modulation of voltage-gated sodium and calcium channels, and nicotinic acetylcholine α7 receptor. We then investigated the venom components using mass spectrometry to identify peptide masses and sequences associated to the observed neuromodulations. Our findings showed this venom is highly complex compared to other neurotoxin-rich venoms and comprises potent modulators of voltage-gated sodium and calcium channels which were classified into 4 families of neuroactive peptides based on their activities and structures. In addition to the reported P. nigriventer neuroactive peptides, we identified at least 27 novel cysteine-rich venom peptides in which neuroactivities are still to be elucidated in voltage-gated ion channels and other potential targets. Our findings provide a new basis for studying non-explored bioactivities of known and novel neuroactive components in P. nigriventer venom, and further supports the successful application of our discovery pipeline for identifying ion channel-targeting venom peptides with potential to become drug leads with diverse exploratory and therapeutic applications.

Project description:Assassin bugs (Hemiptera: Heteroptera: Reduviidae) are venomous insects that prey on invertebrates. Assassin bug venom has features in common with venoms from other animals, such as paralysing and lethal activity when injected, and a molecular composition that includes disulfide-rich peptide neurotoxins. Uniquely, this venom also has strong liquefying activity that has been hypothesised to facilitate feeding through the narrow channel of the proboscis—a structure inherited from sap- and phloem-feeding phytophagous hemipterans and adapted during the evolution of Heteroptera into a fang and feeding structure. However, further understanding of the function of assassin bug venom is impeded by the lack of proteomic studies detailing its molecular composition. In addition, the lack of knowledge regarding venoms of predaceous reduviids limits our understanding of how the venoms of the blood-feeding kissing bugs (Reduviidae: Triatominae) evolved to facilitate hematophagy. By using a combined transcriptomic/proteomic approach we show that the venom proteome of the harpactorine assassin bug Pristhesancus plagipennis includes a complex suite of >100 proteins comprising disulfide-rich peptides, CUB-domain proteins, cystatins, putative cytolytic toxins, triabin-like protein, odorant binding protein, serine proteases, catabolic enzymes, putative nutrient-binding proteins, plus eight families of proteins without homology to characterised proteins. Serine proteases, CUB domain proteins and other novel proteins in the 10–16 kDa mass range, as well as putative cytolytic toxins, were the most abundant venom components. Thus, in addition to putative neurotoxins, assassin bug venom includes a high proportion of enzymatic and cytolytic venom components well suited to tissue liquefaction. While some protein families such as lipocalin/triabins occur in the venoms of both predaceous and blood-feeding reduviids, the composition of venoms in these two groups differs markedly. These results provide insights into the venom evolution in the insect suborder Heteroptera.

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Patagonin - CRISP: Antimicrobial activity and source of antimicrobial molecules in Duvernoy's gland secretion - Philodryas patagoniensis snake

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