Proteomic characterization of extracellular vesicles released by third stage larvae of the zoonotic parasite Anisakis pegreffii (Nematoda: Anisakidae)

Introduction Anisakis pegreffii is a sibling species within the A. simplex (s.l.) complex requiring marine homeothermic (mainly cetaceans) and heterothermic (crustaceans, fish, and cephalopods) organisms to complete its life cycle. It is also a zoonotic species, able to accidentally infect humans (anisakiasis). To investigate the molecular signals involved in this host-parasite interaction and pathogenesis, the proteomic composition of the extracellular vesicles (EVs) released by the third-stage larvae (L3) of A. pegreffii, was characterized. Methods Genetically identified L3 of A. pegreffii were maintained for 24 h at 37°C and EVs were isolated by serial centrifugation and ultracentrifugation of culture media. Proteomic analysis was performed by Shotgun Analysis. Results and discussion EVs showed spherical shaped structure (size 65-295 nm). Proteomic results were blasted against the A. pegreffii specific transcriptomic database, and 153 unique proteins were identified. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis predicted several proteins belonging to distinct metabolic pathways. The similarity search employing selected parasitic nematodes database revealed that proteins associated with A. pegreffii EVs might be involved in parasite survival and adaptation, as well as in pathogenic processes. Further, a possible link between the A. pegreffii EVs proteins versus those of human and cetaceans’ hosts, were predicted by using HPIDB database. The results, herein described, expand knowledge concerning the proteins possibly implied in the host-parasite interactions between this parasite and its natural and accidental hosts.

[1]  Chelsea N. Davis,et al.  Special considerations for studies of extracellular vesicles from parasitic helminths: A community‐led roadmap to increase rigour and reproducibility , 2023, Journal of extracellular vesicles.

[2]  M. Salvemini,et al.  Rat and fish peripheral blood leukocytes respond distinctively to Anisakis pegreffii (Nematoda, Anisakidae) crude extract , 2022, Frontiers in Cellular and Infection Microbiology.

[3]  H. Sugiyama,et al.  Anisakiasis Annual Incidence and Causative Species, Japan, 2018–2019 , 2022, Emerging infectious diseases.

[4]  A. Buck,et al.  Organoids as tools to investigate gastrointestinal nematode development and host interactions , 2022, Frontiers in Cellular and Infection Microbiology.

[5]  Alfredo López,et al.  Distribution and genetic diversity of Anisakis spp. in cetaceans from the Northeast Atlantic Ocean and the Mediterranean Sea , 2022, Scientific Reports.

[6]  A. Cortés,et al.  Parasitic helminths and the host microbiome - a missing 'extracellular vesicle-sized' link? , 2022, Trends in parasitology.

[7]  M. Santoro,et al.  De novo transcriptome assembly and annotation of the third stage larvae of the zoonotic parasite Anisakis pegreffii , 2022, BMC Research Notes.

[8]  B. Arcà,et al.  A miRNAs catalogue from third-stage larvae and extracellular vesicles of Anisakis pegreffii provides new clues for host-parasite interplay , 2022, Scientific Reports.

[9]  M. Podolska,et al.  Drug efficacy on zoonotic nematodes of the Anisakidae family: new metabolic data , 2022, Parasitology.

[10]  S. Cavallero,et al.  What Do In Vitro and In Vivo Models Tell Us about Anisakiasis? New Tools Still to Be Explored , 2022, Pathogens.

[11]  Maciej Kochanowski,et al.  Proteomic Profiling and In Silico Characterization of the Secretome of Anisakis simplex Sensu Stricto L3 Larvae , 2022, Pathogens.

[12]  Á. Kittel,et al.  Formation of a protein corona on the surface of extracellular vesicles in blood plasma , 2021, Journal of extracellular vesicles.

[13]  R. Maizels,et al.  Helminth extracellular vesicles: Interactions with the host immune system , 2021, Molecular immunology.

[14]  V. Carbone,et al.  Molecular and evolutionary basis for survival, its failure, and virulence factors of the zoonotic nematode Anisakis pegreffii. , 2021, Genomics.

[15]  P. Bork,et al.  eggNOG-mapper v2: Functional Annotation, Orthology Assignments, and Domain Prediction at the Metagenomic Scale , 2021, bioRxiv.

[16]  A. Marcilla,et al.  Overview of the interaction of helminth extracellular vesicles with the host and their potential functions and biological applications. , 2021, Molecular immunology.

[17]  M. Santoro,et al.  Insights into the role of deep-sea squids of the genus Histioteuthis (Histioteuthidae) in the life cycle of ascaridoid parasites in the Central Mediterranean Sea waters , 2021, Scientific Reports.

[18]  G. Bosi,et al.  Immunohistopathological response against anisakid nematode larvae and a coccidian in Micromesistius poutassou from NE Atlantic waters , 2021, Journal of Helminthology.

[19]  P. Denuncio,et al.  Genetic identification of Anisakis spp. (Nematoda: Anisakidae) from cetaceans of the Southwestern Atlantic Ocean: ecological and zoogeographical implications , 2021, Parasitology Research.

[20]  A. Levsen,et al.  Anisakis simplex (s.s.) larvae (Nematoda: Anisakidae) hidden in the mantle of European flying squid Todarodes sagittatus (Cephalopoda: Ommastrephidae) in NE Atlantic Ocean: Food safety implications. , 2020, International journal of food microbiology.

[21]  Z. Gardian,et al.  Extracellular vesicles secreted by model tapeworm Hymenolepis diminuta: biogenesis, ultrastructure and protein composition. , 2020, International journal for parasitology.

[22]  Alex Warwick Vesztrocy,et al.  OMA orthology in 2021: website overhaul, conserved isoforms, ancestral gene order and more , 2020, Nucleic Acids Res..

[23]  Ren Zhang,et al.  DEG 15, an update of the Database of Essential Genes that includes built-in analysis tools , 2020, Nucleic Acids Res..

[24]  M. Carrera,et al.  Proteomic Insights into the Biology of the Most Important Foodborne Parasites in Europe , 2020, Foods.

[25]  Tiziano Flati,et al.  ELIXIR-IT HPC@CINECA: high performance computing resources for the bioinformatics community , 2020, BMC Bioinformatics.

[26]  A. Marcilla,et al.  The protein and microRNA cargo of extracellular vesicles from parasitic helminths - current status and research priorities. , 2020, International journal for parasitology.

[27]  A. Stensballe,et al.  Fluorescent Labeling of Helminth Extracellular Vesicles Using an In Vivo Whole Organism Approach , 2020, Biomedicines.

[28]  L. Putignani,et al.  Mass Spectrometry Based-Proteomic Analysis of Anisakis spp.: A Preliminary Study towards a New Diagnostic Tool , 2020, Genes.

[29]  G. Nascetti,et al.  A novel nuclear marker and development of an ARMS-PCR assay targeting the metallopeptidase 10 (nas 10) locus to identify the species of the Anisakis simplex (s. l.) complex (Nematoda, Anisakidae) , 2020, Parasite.

[30]  Lars Malmström,et al.  Extracellular Vesicle-Contained microRNA of C. elegans as a Tool to Decipher the Molecular Basis of Nematode Parasitism , 2020, Frontiers in Cellular and Infection Microbiology.

[31]  G. Nascetti,et al.  Differences in Gene Expression Profiles of Seven Target Proteins in Third-Stage Larvae of Anisakis simplex (Sensu Stricto) by Sites of Infection in Blue Whiting (Micromesistius poutassou) , 2020, Genes.

[32]  J. Leavenworth,et al.  Angiostrongylus cantonensis Galectin-1 interacts with Annexin A2 to impair the viability of macrophages via activating JNK pathway , 2020, Parasites & Vectors.

[33]  M. Podolska,et al.  Effect of freezing on the metabolic status of L3 larvae of Anisakis simplex s. s. , 2020, Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases.

[34]  G. Nascetti,et al.  Gene expression profiles of antigenic proteins of third stage larvae of the zoonotic nematode Anisakis pegreffii in response to temperature conditions , 2019, Parasite.

[35]  I. Bočina,et al.  Interplay between proinflammatory cytokines, miRNA, and tissue lesions in Anisakis-infected Sprague-Dawley rats , 2019, PLoS neglected tropical diseases.

[36]  Ross S Hall,et al.  Exploration of extracellular vesicles from Ascaris suum provides evidence of parasite–host cross talk , 2019, Journal of extracellular vesicles.

[37]  Sebastian Maurer-Stroh,et al.  AllerCatPro—prediction of protein allergenicity potential from the protein sequence , 2019, Bioinform..

[38]  T. Geary,et al.  Helminth extracellular vesicles in host-parasite interactions. , 2018, Current opinion in microbiology.

[39]  Jing Xu,et al.  Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines , 2018, Journal of Extracellular Vesicles.

[40]  T. Whiteside,et al.  Harmonization of exosome isolation from culture supernatants for optimized proteomics analysis , 2018, PloS one.

[41]  Dongyou Liu Anisakis , 2018, Handbook of Foodborne Diseases.

[42]  I. Bočina,et al.  Molecular and Cellular Response to Experimental Anisakis pegreffii (Nematoda, Anisakidae) Third-Stage Larval Infection in Rats , 2018, Front. Immunol..

[43]  M. Santoro,et al.  Helminth parasites of the dwarf sperm whale Kogia sima (Cetacea: Kogiidae) from the Mediterranean Sea, with implications on host ecology. , 2018, Diseases of aquatic organisms.

[44]  M. Blaxter,et al.  Functional insights into the infective larval stage of Anisakis simplex s.s., Anisakis pegreffii and their hybrids based on gene expression patterns , 2018, BMC Genomics.

[45]  Jia Xu,et al.  Molecular characterization of Trichinella spiralis galectin and its participation in larval invasion of host’s intestinal epithelial cells , 2018, Veterinary Research.

[46]  A. Loukas,et al.  Immunobiology of parasitic worm extracellular vesicles , 2018, Immunology and cell biology.

[47]  M. Field,et al.  Hookworm Secreted Extracellular Vesicles Interact With Host Cells and Prevent Inducible Colitis in Mice , 2018, Front. Immunol..

[48]  A. Santoni,et al.  Drug-Induced Senescent Multiple Myeloma Cells Elicit NK Cell Proliferation by Direct or Exosome-Mediated IL15 Trans-Presentation , 2018, Cancer Immunology Research.

[49]  M. Zamanian,et al.  Profiling extracellular vesicle release by the filarial nematode Brugia malayi reveals sex-specific differences in cargo and a sensitivity to ivermectin , 2018, PLoS neglected tropical diseases.

[50]  M. Field,et al.  Characterization of Trichuris muris secreted proteins and extracellular vesicles provides new insights into host–parasite communication , 2018, Journal of extracellular vesicles.

[51]  M. Salvemini,et al.  Tissue-specific transcriptomes of Anisakis simplex (sensu stricto) and Anisakis pegreffii reveal potential molecular mechanisms involved in pathogenicity , 2018, Parasites & Vectors.

[52]  G. Caracciolo,et al.  Tumor-Derived Microvesicles Modulate Antigen Cross-Processing via Reactive Oxygen Species-Mediated Alkalinization of Phagosomal Compartment in Dendritic Cells , 2017, Front. Immunol..

[53]  G. Pierce,et al.  Population genetic structure of the parasite Anisakis simplex (s. s.) collected in Clupea harengus L. from North East Atlantic fishing grounds , 2017, Fisheries Research.

[54]  G. Nascetti,et al.  IgE sensitization to Anisakis pegreffii in Italy: Comparison of two methods for the diagnosis of allergic anisakiasis , 2017, Parasite immunology.

[55]  K. Buchmann,et al.  Excretory/secretory products of anisakid nematodes: biological and pathological roles , 2017, Acta Veterinaria Scandinavica.

[56]  Qinping Zhao,et al.  Immunization with recombinant schistosome adenylate kinase 1 partially protects mice against Schistosoma japonicum infection , 2017, Parasitology Research.

[57]  G. Nascetti,et al.  Anisakis pegreffii (Nematoda: Anisakidae) in European anchovy Engraulis encrasicolus from the Mediterranean Sea: Fishing ground as a predictor of parasite distribution , 2017, Fisheries Research.

[58]  Jüergen Cox,et al.  The MaxQuant computational platform for mass spectrometry-based shotgun proteomics , 2016, Nature Protocols.

[59]  Bindu Nanduri,et al.  HPIDB 2.0: a curated database for host–pathogen interactions , 2016, Database J. Biol. Databases Curation.

[60]  C. Cantacessi,et al.  The Anisakis Transcriptome Provides a Resource for Fundamental and Applied Studies on Allergy-Causing Parasites , 2016, PLoS neglected tropical diseases.

[61]  L. Baum,et al.  Galectins and Immune Responses-Just How Do They Do Those Things They Do? , 2016, Annual review of immunology.

[62]  R. Maizels,et al.  Exosomes and Other Extracellular Vesicles: The New Communicators in Parasite Infections , 2015, Trends in parasitology.

[63]  M. Ramirez,et al.  Exosomes or microvesicles? Two kinds of extracellular vesicles with different routes to modify protozoan-host cell interaction , 2015, Parasitology Research.

[64]  B. Espinoza,et al.  The role of small heat shock proteins in parasites , 2015, Cell stress & chaperones (Print).

[65]  L. O’Driscoll,et al.  Biological properties of extracellular vesicles and their physiological functions , 2015, Journal of extracellular vesicles.

[66]  C. Théry,et al.  Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. , 2014, Annual review of cell and developmental biology.

[67]  G. Nascetti,et al.  Genetic and Morphological Approaches Distinguish the Three Sibling Species of the Anisakis simplex Species Complex, with a Species Designation as Anisakis berlandi n. sp. for A. simplex sp. C (Nematoda: Anisakidae) , 2014, The Journal of parasitology.

[68]  R. Maizels,et al.  The Secreted Triose Phosphate Isomerase of Brugia malayi Is Required to Sustain Microfilaria Production In Vivo , 2014, PLoS pathogens.

[69]  Paul W. Sternberg,et al.  Genome of the human hookworm Necator americanus , 2014, Nature Genetics.

[70]  H. D. del Portillo,et al.  Extracellular vesicles in parasitic diseases , 2014, Journal of extracellular vesicles.

[71]  Neil D. Rawlings,et al.  MEROPS: the database of proteolytic enzymes, their substrates and inhibitors , 2013, Nucleic Acids Res..

[72]  N. Maltsev,et al.  Microvesicles and intercellular communication in the context of parasitism , 2013, Front. Cell. Infect. Microbiol..

[73]  Aled Clayton,et al.  How pure are your vesicles? , 2013, Journal of extracellular vesicles.

[74]  A. Marcilla,et al.  Extracellular Vesicles from Parasitic Helminths Contain Specific Excretory/Secretory Proteins and Are Internalized in Intestinal Host Cells , 2012, PloS one.

[75]  Paul J. Harrison,et al.  Sizing and phenotyping of cellular vesicles using Nanoparticle Tracking Analysis , 2011, Nanomedicine : nanotechnology, biology, and medicine.

[76]  Soon-Cheol Ahn,et al.  Inhibition of dextran sulfate sodium (DSS)-induced intestinal inflammation via enhanced IL-10 and TGF-beta production by galectin-9 homologues isolated from intestinal parasites. , 2010, Molecular and biochemical parasitology.

[77]  Bindu Nanduri,et al.  HPIDB - a unified resource for host-pathogen interactions , 2010, BMC Bioinformatics.

[78]  M. Robles,et al.  University of Birmingham High throughput functional annotation and data mining with the Blast2GO suite , 2022 .

[79]  A. Valentini,et al.  GENETIC RELATIONSHIPS AMONG ANISAKIS SPECIES (NEMATODA: ANISAKIDAE) INFERRED FROM MITOCHONDRIAL COX2 SEQUENCES, AND COMPARISON WITH ALLOZYME DATA , 2006, The Journal of parasitology.

[80]  S. Sidhu,et al.  Phylogenetic analysis of the vertebrate galectin family. , 2004, Molecular biology and evolution.

[81]  R. Benítez,et al.  In vitro cultivation of Anisakis simplex: pepsin increases survival and moulting from fourth larval to adult stage , 2001, Parasitology.

[82]  J. Sakanari,et al.  Characterization of the serine protease and serine protease inhibitor from the tissue-penetrating nematode Anisakis simplex. , 1994, The Journal of biological chemistry.

[83]  J. Donelson,et al.  OvGalBP, a filarial antigen with homology to vertebrate galactoside-binding proteins. , 1994, Molecular and biochemical parasitology.

[84]  J. Knowles,et al.  To build an enzyme.... , 1991, Philosophical transactions of the Royal Society of London. Series B, Biological sciences.

[85]  G. Nascetti,et al.  Genetic structure of Anisakis physeteris, and its differentiation from the Anisakis simplex complex (Ascaridida: Anisakidae) , 1986, Parasitology.

[86]  B. Berland Nematodes from some Norwegian marine fishes , 1961 .

[87]  G. Nascetti,et al.  Molecular Epidemiology of Anisakis and Anisakiasis: An Ecological and Evolutionary Road Map. , 2018, Advances in parasitology.

[88]  G. Nascetti,et al.  Advances and trends in the molecular systematics of anisakid nematodes, with implications for their evolutionary ecology and host-parasite co-evolutionary processes. , 2008, Advances in parasitology.