Snake Venomics and Antivenomics of Bothrops diporus, a Medically Important Pitviper in Northeastern Argentina

Snake species within genus Bothrops are responsible for more than 80% of the snakebites occurring in South America. The species that cause most envenomings in Argentina, B. diporus, is widely distributed throughout the country, but principally found in the Northeast, the region with the highest rates of snakebites. The venom proteome of this medically relevant snake was unveiled using a venomic approach. It comprises toxins belonging to fourteen protein families,be ing dominated by PI- and PIII-SVMPs, PLA2 molecules, BPP-like peptides, L-amino acid oxidase and serine proteinases. This toxin profile largely explains the characteristic pathophysiological effects of bothropic snakebites observed in patients envenomed by B. diporus. Antivenomic analysis of the SAB antivenom (Instituto Vital Brazil) against the venom of B. diporus showed that this pentabothropic antivenom efficiently recognized all the venom proteins and exhibited poor affinity towards the small peptide (BPPs and tripeptide inhibitors of PIII-SVMPs) components of the venom.Fil: Gay, Claudia Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; ArgentinaFil: Sanz, Libia. Instituto de Biomedicina de Valencia; EspañaFil: Calvete, Juan J.. Instituto de Biomedicina de Valencia; EspañaFil: Pla, Davinia. Instituto de Biomedicina de Valencia; Españ

Venomics Study of <em>Protobothrops flavoviridis</em> Snake: How Venom Proteins Have Evolved and Diversified?

Venomics projects have been conducted to disclose the divergent profiles and evolution of various venomous animals. Here, we describe the venomics project including genome and transcriptome of habu snake, leading to drug discovery. Venomics project including the decoding of their whole genomes revealed partly a producing mechanism of various venom proteins including accelerated evolution and alternative splicing and how the toxic organisms have evolved from the nontoxic ones. In addition, the venomics analysis of transcriptomes and proteomes beyond species reveals the relationship between the geographical distribution and evolution of toxic organisms. The abundance of different gene products within a gene family caused by accelerated evolution and alternative splicing may contribute to expand the repertoire of effective weapons to prey capture accompanied with neofunctionalization

Mutual enlightenment: A toolbox of concepts and methods for integrating evolutionary and clinical toxinology via snake venomics and the contextual stance

Snakebite envenoming is a neglected tropical disease that may claim over 100,000 human lives annually worldwide. Snakebite occurs as the result of an interaction between a human and a snake that elicits either a defensive response from the snake or, more rarely, a feeding response as the result of mistaken identity. Snakebite envenoming is therefore a biological and, more specifically, an ecological problem. Snake venom itself is often described as a “cocktail”, as it is a heterogenous mixture of molecules including the toxins (which are typically proteinaceous) responsible for the pathophysiological consequences of envenoming. The primary function of venom in snake ecology is pre-subjugation, with defensive deployment of the secretion typically considered a secondary function. The particular composition of any given venom cocktail is shaped by evolutionary forces that include phylogenetic constraints associated with the snake’s lineage and adaptive responses to the snake’s ecological context, including the taxa it preys upon and by which it is predated upon. In the present article, we describe how conceptual frameworks from ecology and evolutionary biology can enter into a mutually enlightening relationship with clinical toxinology by enabling the consideration of snakebite envenoming from an “ecological stance”. We detail the insights that may emerge from such a perspective and highlight the ways in which the high-fidelity descriptive knowledge emerging from applications of -omics era technologies – “venomics” and “antivenomics” – can combine with evolutionary explanations to deliver a detailed understanding of this multifactorial health crisis.Ministerio de Ciencia e Innovacion/[BMC 2004-01432]//EspañaMinisterio de Ciencia e Innovacion/[BFU 2007-61563]//EspañaMinisterio de Ciencia e Innovacion/[BFU 2010-173730]//EspañaMinisterio de Ciencia e Innovacion/[BFU 2013-42833-P]//EspañaMinisterio de Ciencia e Innovacion/[BFU 2017-89103-P]//EspañaNorwegian Research Council/[No.287462.]/NFR/NoruegaNational Health and Medical Research Council/[Grant 13/093/002 AVRU]/AustraliaDBT/Wellcome Trust India Alliance/[IA/I/19/2/504647]//IndiaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

The emerging field of venom-microbiomics for exploring venom as a microenvironment, and the corresponding Initiative for Venom Associated Microbes and Parasites (iVAMP)

Venom is a known source of novel antimicrobial natural products. The substantial, increasing number of these discoveries have unintentionally culminated in the misconception that venom and venom-producing glands are largely sterile environments. Culture-dependent and -independent studies on the microbial communities in venom microenvironments reveal the presence of archaea, algae, bacteria, endoparasites, fungi, protozoa, and viruses. Venom-centric microbiome studies are relatively sparse to date and the adaptive advantages that venom-associated microbes might offer to their hosts, or that hosts might provide to venom-associated microbes, remain unknown. We highlight the potential for the discovery of venom-microbiomes within the adaptive landscape of venom systems. The considerable number of known, convergently evolved venomous animals juxtaposed with the comparatively few studies to identify microbial communities in venom provides new possibilities for both biodiversity and therapeutic discoveries. We present an evidence-based argument for integrating microbiology as part of venomics to which we refer to as venom-microbiomics. We also introduce iVAMP, the Initiative for Venom Associated Microbes and Parasites (https://ivamp-consortium.github.io/), as a growing consortium for interested parties to contribute and collaborate within this subdiscipline. Our consortium seeks to support diversity, inclusion and scientific collaboration among all researchers interested in this subdiscipline

One Size Fits All—Venomics of the Iberian Adder (Vipera seoanei, Lataste 1878) reveals low levels of venom variation across its distributional range

European vipers (genus Vipera) are medically important snakes displaying considerable venom variation, occurring at different levels in this group. The presence of intraspecific venom variation, however, remains understudied in several Vipera species. Vipera seoanei is a venomous snake endemic to the northern Iberian Peninsula and south-western France, presenting notable phenotypic variation and inhabiting several diverse habitats across its range. We analysed the venoms of 49 adult specimens of V. seoanei from 20 localities across the species’ Iberian distribution. We used a pool of all individual venoms to generate a V. seoanei venom reference proteome, produced SDS-PAGE profiles of all venom samples, and visualised patterns of variation using NMDS. By applying linear regression, we then assessed presence and nature of venom variation between localities, and investigated the effect of 14 predictors (biological, eco-geographic, genetic) on its occurrence. The venom comprised at least 12 different toxin families, of which five (i.e., PLA2, svSP, DI, snaclec, svMP) accounted for about 75% of the whole proteome. The comparative analyses of the SDS-PAGE venom profiles showed them to be remarkably similar across the sampled localities, suggesting low geographic variability. The regression analyses suggested significant effects of biological and habitat predictors on the little variation we detected across the analysed V. seoanei venoms. Other factors were also significantly associated with the presence/absence of individual bands in the SDS-PAGE profiles. The low levels of venom variability we detected within V. seoanei might be the result of a recent population expansion, or of processes other than directional positive selection

Modern venomics--Current insights, novel methods, and future perspectives in biological and applied animal venom research

Venoms have evolved >100 times in all major animal groups, and their components, known as toxins, have been fine-tuned over millions of years into highly effective biochemical weapons. There are many outstanding questions on the evolution of toxin arsenals, such as how venom genes originate, how venom contributes to the fitness of venomous species, and which modifications at the genomic, transcriptomic, and protein level drive their evolution. These questions have received particularly little attention outside of snakes, cone snails, spiders, and scorpions. Venom compounds have further become a source of inspiration for translational research using their diverse bioactivities for various applications. We highlight here recent advances and new strategies in modern venomics and discuss how recent technological innovations and multi-omic methods dramatically improve research on venomous animals. The study of genomes and their modifications through CRISPR and knockdown technologies will increase our understanding of how toxins evolve and which functions they have in the different ontogenetic stages during the development of venomous animals. Mass spectrometry imaging combined with spatial transcriptomics, in situ hybridization techniques, and modern computer tomography gives us further insights into the spatial distribution of toxins in the venom system and the function of the venom apparatus. All these evolutionary and biological insights contribute to more efficiently identify venom compounds, which can then be synthesized or produced in adapted expression systems to test their bioactivity. Finally, we critically discuss recent agrochemical, pharmaceutical, therapeutic, and diagnostic (so-called translational) aspects of venoms from which humans benefit.This work is funded by the European Cooperation in Science and Technology (COST, www.cost.eu) and based upon work from the COST Action CA19144 – European Venom Network (EUVEN, see https://euven-network.eu/). This review is an outcome of EUVEN Working Group 2 (“Best practices and innovative tools in venomics”) led by B.M.v.R. As coordinator of the group Animal Venomics until end 2021 at the Institute for Insectbiotechnology, JLU Giessen, B.M.v.R. acknowledges the Centre for Translational Biodiversity Genomics (LOEWE-TBG) in the programme “LOEWE – Landes-Offensive zur Entwicklung Wissenschaftlich-ökonomischer Exzellenz” of Hesse's Ministry of Higher Education, Research, and the Arts. B.M.v.R. and I.K. further acknowledge funding on venom research by the German Science Foundation to B.M.v.R. (DFG RE3454/6-1). A.C., A.V., and G.Z. were supported by the European Union's Horizon 2020 Research and Innovation program through Marie Sklodowska-Curie Individual Fellowships (grant agreements No. A.C.: 896849, A.V.: 841576, and G.Z.: 845674). M.P.I. is supported by the TALENTO Program by the Regional Madrid Government (2018-T1/BIO-11262). T.H.'s venom research is funded by the DFG projects 271522021 and 413120531. L.E. was supported by grant No. 7017-00288 from the Danish Council for Independent Research (Technology and Production Sciences). N.I. acknowledges funding on venom research by the Research Fund of Nevsehir Haci Bektas Veli University (project Nos. ABAP20F28, BAP18F26). M.I.K. and A.P. acknowledge support from GSRT National Research Infrastructure structural funding project INSPIRED (MIS 5002550). G.A. acknowledges support from the Slovenian Research Agency grants P1-0391, J4-8225, and J4-2547. G.G. acknowledges support from the Institute for Medical Research and Occupational Health, Zagreb, Croatia. E.A.B.U. is supported by a Norwegian Research Council FRIPRO-YRT Fellowship No. 287462

Modern venomics – Current insights, novel methods and future perspectives in biological and applied animal venom research

Venoms have evolved &gt;100 times in all major animal groups, and their components, known as toxins, have been fine-tuned over millions of years into highly effective biochemical weapons. There are many outstanding questions on the evolution of toxin arsenals, such as how venom genes originate, how venom contributes to the fitness of venomous species, and which modifications at the genomic, transcriptomic, and protein level drive their evolution. These questions have received particularly little attention outside of snakes, cone snails, spiders, and scorpions. Venom compounds have further become a source of inspiration for translational research using their diverse bioactivities for various applications. We highlight here recent advances and new strategies in modern venomics and discuss how recent technological innovations and multi-omic methods dramatically improve research on venomous animals. The study of genomes and their modifications through CRISPR and knockdown technologies will increase our understanding of how toxins evolve and which functions they have in the different ontogenetic stages during the development of venomous animals. Mass spectrometry imaging combined with spatial transcriptomics, in situ hybridization techniques, and modern computer tomography gives us further insights into the spatial distribution of toxins in the venom system and the function of the venom apparatus. All these evolutionary and biological insights contribute to more efficiently identify venom compounds, which can then be synthesized or produced in adapted expression systems to test their bioactivity. Finally, we critically discuss recent agrochemical, pharmaceutical, therapeutic, and diagnostic (so-called translational) aspects of venoms from which humans benefit

Chaperone-mediated native folding of a β-scorpion toxin in the periplasm of E.coli

Background: Animal neurotoxin peptides are valuable probes for investigating ion channel structure/function relationships and represent lead compounds for novel therapeutics and insecticides. However, misfolding and aggregation are common outcomes when toxins containing multiple disulfides are expressed in bacteria. Methods: The ß-scorpion peptide toxin Bj-xtrIT from Hottentotta judaica and four chaperone enzymes (DsbA, DsbC, SurA and FkpA) were co-secreted into the oxidizing environment of the E.coli periplasm. Expressed Bj-xtrIT was purified and analyzed by HPLC and FPLC chromatography. Its thermostability was assessed using synchrotron radiation circular dichroism spectroscopy and its crystal structure was determined. Results: Western blot analysis showed that robust expression was only achieved when cells co-expressed the chaperones. The purified samples were homogenous and monodisperse and the protein was thermostable. The crystal structure of the recombinant toxin confirmed that it adopts the native disulfide connectivity and fold. Conclusions: The chaperones enabled correct folding of the four-disulfide-bridged Bj-xtrIT toxin. There was no apparent sub-population of misfolded Bj-xtrIT, which attests to the effectiveness of this expression method. General Significance: We report the first example of a disulfide-linked scorpion toxin natively folded during bacterial expression. This method eliminates downstream processing steps such as oxidative refolding or cleavage of a fusion-carrier and therefore enables efficient production of insecticidal Bj-xtrIT. Periplasmic chaperone activity may produce native folding of other extensively disulfide-reticulated proteins including animal neurotoxins. This work is therefore relevant to venomics and studies of a wide range of channels and receptors