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Functional and phylogenetic importance of antimicrobial peptides and enzymes in spider venoms

English title Functional and phylogenetic importance of antimicrobial peptides and enzymes in spider venoms
Applicant Nentwig Wolfgang
Number 162564
Funding scheme Project funding (Div. I-III)
Research institution Abteilung Synökologie Institut für Ökologie und Evolution Universität Bern
Institution of higher education University of Berne - BE
Main discipline Zoology
Start/End 01.10.2015 - 31.08.2019
Approved amount 474'000.00
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Keywords (10)

spider phylogeny; RTA clade; proteome analysis; venom composition; envenomation process; antimicrobial peptides; bioassays; mass spectrometry; enzymes; transcriptome analysis

Lay Summary (German)

Lead
Spinnengift besteht aus vielen einzelnen Substanzen, die im Laufe der Evolution dieser Tiere immer wieder ersetzt wurden. In diesem Projekt untersuchen wir, welche Substanzen nachgewiesen werden können und welche Bedeutung diese für die Wirkung des Giftes haben.
Lay summary

Spinnengift besteht aus vielen Komponenten, die zu 3 Gruppen gehören: niedermolekulare Substanzen, Peptide mit Cystein-Doppelbindungen und Enzyme. Diese Substanzen kommen vermutlich in allen Spinnengiften vor. Des Weiteren enthalten einige Gifte Acylpolyamine, antimikrobielle Peptide und grosse Neurotoxine. Innerhalb der Evolution von Spinnengiften wurden diese Substanzen ständig modifiziert und einige Gruppen ersetzt. Es ist kaum bekannt, welche Auswirkungen dies für den Gifteinsatz durch die Spinne und ihre Phylogenie hat.

 

In diesem Projekt wollen wir antimikrobielle Peptide und Enzyme analysieren. Kationische a-helikale antimikrobielle Peptide zerstören Membrane und verstärken Neurotoxine. Sie sind nur von wenigen Familien im RTA-Clade bekannt, wolfsspinnenverwandte Familien. Wir gehen davon aus, dass die Erfindung von antimikrobiellen Peptiden eine Besonderheit dieser Familien ist und wollen dies analysieren. Enzyme sind bisher nur aus dem Gift weniger Spinnen bekannt und sie bauen die extrazelluläre Matrix oder Zellmembranen ab. Hierdurch können sie die Wirkung der eigentlichen Neurotoxine verstärken. Wir vermuten, dass Enzymen eine wichtige Rolle bei der Giftverstärkung haben.

 

Unser Forschungsansatz basiert auf Transkriptomanalysen von Giftdrüsen von etwa 50 Arten aus 30 Familien. Hierzu setzen wir verschiedene Analysemethoden ein (u.a. mRNA Strukturanalysen von antimikrobiellen Peptiden und Enzymen, in Kombination mit LC-MS/MS), führen zytolytische und Enzym-Substrat Assays durch und verarbeiten die Daten mit Bioinformatik-Ansätzen. Unser Ziel ist, über Kenntnisse zur Verteilung und funktionellen Bedeutung von antimikrobiellen Peptiden und Enzymen im Spinnengift vieler Arten und Familien innerhalb des RTA-Clades mehr über die Evolution von Spinnengift zu erfahren.

 
Direct link to Lay Summary Last update: 28.09.2015

Responsible applicant and co-applicants

Employees

Publications

Publication
The Dual Prey-Inactivation Strategy of Spiders—In-Depth Venomic Analysis of Cupiennius salei
Kuhn-Nentwig Lucia, Langenegger Nicolas, Heller Manfred, Koua Dominique, Nentwig Wolfgang (2019), The Dual Prey-Inactivation Strategy of Spiders—In-Depth Venomic Analysis of Cupiennius salei, in Toxins, 11(3), 167-167.
Distribution and medical aspects of Loxosceles rufescens, one of the most invasive spiders of the world (Araneae: Sicariidae)
NentwigWolfgang, PantiniPaolo, VetterRichard S. (2017), Distribution and medical aspects of Loxosceles rufescens, one of the most invasive spiders of the world (Araneae: Sicariidae), in Toxicon, 132, 19-28.
Peptidomic and transcriptomic profiling of four distinct spider venoms
Oldrati Vera, Koua Dominique, Allard Pierre-Marie, Hulo Nicolas, Arrell Miriam, Nentwig Wolfgang, Lisacek Frédérique, Wolfender Jean-Luc, Kuhn-Nentwig Lucia, Stöcklin Reto (2017), Peptidomic and transcriptomic profiling of four distinct spider venoms, in PLOS ONE, 12(3), e0172966-e0172966.
Spider neurotoxins, short linear cationic peptides and venom proteins classification improved by an automated competition between exhaustive profile HMM classifiers.
Koua D, Kuhn-Nentwig L (2017), Spider neurotoxins, short linear cationic peptides and venom proteins classification improved by an automated competition between exhaustive profile HMM classifiers., in Toxins, 9(245), 1-17.
Isolation, N-glycosylations and Function of a Hyaluronidase-Like Enzyme from the Venom of the Spider Cupiennius salei
Biner Olivier, Trachsel Christian, Moser Aline, Kopp Lukas, Langenegger Nicolas, Kämpfer Urs, von Ballmoos Christoph, Nentwig Wolfgang, Schürch Stefan, Schaller Johann, Kuhn-Nentwig Lucia (2015), Isolation, N-glycosylations and Function of a Hyaluronidase-Like Enzyme from the Venom of the Spider Cupiennius salei, in PLOS ONE, 10(12), e0143963-e0143963.
Venom of Cupiennius salei (Ctenidae)
Kuhn-Nentwig Lucia, Schaller Johann, Schürch Stefan, Nentwig Wolfgang (2015), Venom of Cupiennius salei (Ctenidae), Springer Netherlands, Dordrecht.

Collaboration

Group / person Country
Types of collaboration
Prof. Dr. Glen King, Inst Mol Bioscience, University of Queensland Australia (Oceania)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
- Exchange of personnel
Dr. Alexander Vasilevski, Russian Academy of Sciences, Moscow Russia (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
- Exchange of personnel
Prof. Dr. Jan Tytgat, Laboratory for Toxicology and Food Chemistry, University of Leuven Belgium (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Dr. Christian Trachsel, Functional Genomics Center Zurich Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Dr. Seraina Klopfstein, Natural History Museum Bern / later Basel Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Exchange of personnel
Prof. Lev G. Magazanik, Sechenov Inst Evolutionary Physiology + Biochemistry, RAS Russia (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Prof. Dr. F. Separovic, School of Chemistry, University of Melbourne, Parkville Australia (Oceania)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Dr. Volker Herzig, Inst Mol Bioscience, University of Queensland Australia (Oceania)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
- Exchange of personnel
Prof. Dr. Christian Kropf, Natural History Museum Bern Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
- Exchange of personnel
Dr. Schürch, Department of Chemistry & Biochemistry, University of Bern Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
- Exchange of personnel
Dr. Mónica Cunningham, Instituto de Investigaciones Bioquímicas, Fac. Ciencias Argentina (South America)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
2019 21st International Congress of Arachnology Talk given at a conference Kuhn-Nentwig: A dual prey-inactivation strategy 11.02.2019 Canterbury, New Zealand Langenegger Nicolas; Nentwig Wolfgang;
2019 21st International Congress of Arachnology Talk given at a conference Langenegger: Spider venom enzymes and the function of a specific protease from the venom of the spider Cupiennius salei 11.02.2019 Canterbury, New Zealand Langenegger Nicolas; Nentwig Wolfgang;
2019 GCB Symposium, Graduate School Poster Langenegger: The venom gland transcriptome and the venom proteome of the spider Cupiennius salei: A dual prey inactivation strategy to subdue the prey 31.01.2019 Bern, Switzerland Langenegger Nicolas;
2018 Vortragsreihe der Naturwissenschaftlichen Gesellschaft Winterthur Individual talk Nentwig: Ergebnisse moderner Forschung zu Spinnengift 26.10.2018 Winterthur, Switzerland Nentwig Wolfgang;
2018 Swiss Chemical Society fall meeting Talk given at a conference Langenegger: A spider venom protease responsible for maturing of neurotoxic peptide precursors 07.08.2018 Lausanne, Switzerland Langenegger Nicolas;
2017 19th World Congress of the International Society on Toxinology Talk given at a conference Langenegger: A venom neurotoxin precursor processing protease from the spider Cupiennius salei 25.10.2017 Haikou, China Nentwig Wolfgang; Langenegger Nicolas;
2017 European Congress of Arachnology Talk given at a conference Langenegger: The venom neurotoxin precursor processing protease from the spider Cupiennius salei 25.08.2017 Nottingham, Great Britain and Northern Ireland Nentwig Wolfgang; Urfer Karin; Langenegger Nicolas;
2016 Jahrestagung der Arachnologischen Gesellschaft Individual talk Nentwig: Spinnengift: Zusammensetzung und Wirkung 14.10.2016 Greifswald, Germany Nentwig Wolfgang;
2016 Arachnid workshop Talk given at a conference Kuhn-Nentwig: Venom of Cupiennius salei: Systematics, proteomics, transcriptomics, functionality 23.06.2016 Instituto Butantan, Sao Paulo, Brazil Nentwig Wolfgang;
2016 Sociedade Brasileira de Biochimica e Biologia Molecular Talk given at a conference Kuhn-Nentwig: Function of a hyaluronidase-like enzyme in the venom of spiders 19.06.2016 Natal, Brazil Nentwig Wolfgang;
Vortragsreihe der Naturforschenden Gesellschaft Basel Individual talk Nentwig: Evolutionäre Aspekte von Spinnengift 15.10.2015 Basel, Switzerland Nentwig Wolfgang;


Communication with the public

Communication Title Media Place Year
Talks/events/exhibitions Spinnen - die tollsten Tiere überhaupt German-speaking Switzerland 2017

Awards

Title Year
1st Place Oral Student Presentation 21st International Congress of Arachnology, Canterbury, New Zealand, 10 - 15 Feb 2019 2019
Pierre Bonnet Award for the Promotion of Arachnology 21st International Congress of Arachnology, Canterbury, New Zealand, 10 - 15 Feb 2019 2019
1st Place Oral Student Presentation 19th World Congress of the International Society on Toxinology, Haikou, China, 24 - 31 Oct 2017 2017

Associated projects

Number Title Start Funding scheme
127500 Host defence peptides of the innate immune system and in the venom of the spider Cupiennius salei 01.12.2009 Project funding (Div. I-III)
113681 Antimicrobial peptides and the dual immune system of the spider Cupiennius salei 01.10.2006 Project funding (Div. I-III)

Abstract

SummarySpider venoms consist of many components which mainly can be attributed to three major functional groups present in (probably) all spider venoms: low molecular mass compounds, cysteine-knotted peptides (the classical neurotoxins), and enzymes. Further functional groups (low molecular mass acylpolyamines, antimicrobial peptides and large neurotoxic proteins) are restricted to one or a few spider families. Within the evolution of spiders and their venom, numerous modifications, new developments and replacements of venom compounds occurred but their overall importance for the envenomation process and possible implications within the phylogeny of spiders are not well investigated. With this proposal, we will focus on the importance of (1) antimicrobial peptides and of (2) venom enzymes. (1) Cationic a-helical antimicrobial peptides destroy membranes and enhance thereby the effect of neurotoxins. So far, they are only known from species in a few lycosid-related families and from Zodariidae, both belonging to the RTA clade. Since antimicrobial peptides could speed up the envenomation process considerably, we hypothesize that they are a new invention (autapomorphy) within the RTA clade which represents the evolutionary youngest spider families. (2) There are several enzymes known from the venom of single spider species, either attacking the extracellular matrix or cell membranes and matrix proteins. These enzymes may act as “spreading factor” but experimental evidence for spider venoms is lacking. We assume that enzymes in spider venoms contribute considerably to the envenomation process but different spider families or family groups may have found different solutions.Our approach includes transcriptomic and proteomic studies of 50 spider species from 30 families. Antimicrobial peptide identification from different venoms will be done by specific cytolytic assays. Transcriptome (mRNA structure of antimicrobial peptides and neurotoxins) analysis will be done in combination with LC-MS/MS. Enzyme identification and characterization of three key enzymes will be done by specific enzyme/substrate assays. Sequence data of enzymes from C. salei will be obtained by a combination of N-terminal Edman sequencing of purified enzymes and mass spectrometry based identification using Peaks mass spectrometry software in combination with the venom gland cDNA library. With these obtained sequence data, different BLASTpn/p/x searches will be performed against different spider venom gland libraries, resulting in cDNA data of the three enzymes. These data will be confirmed by LC-MS/MS and mass spectrometry based identification of pretreated (reduced/alkylated and enzymatically digested) venom samples. We use Drosophila bioassays to quantify spreading effects of enzymes in dependency on coinjected neurotoxin CSTx-1 and cytolytically acting cupiennin 1a.Our goal is to explore the abundance and diversity of antimicrobial peptides within spider families, specifically to establish them as a new development of the RTA clade. The presence/absence data of enzymes in spider venoms and knowledge of their substrate specificity will give as insight into the importance of enzymes for the envenomation process. Both will allow us to draw some general conclusions on the level of functional aspects of major venom compound groups within spiders, but also, by sequence comparison, to investigate evolutionary dynamics between families or family groups.
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