Helminthic host defense peptides (HDP) are pleiotropic, multifunctional effector molecules of helminth immunity, efficient against Gram-negative and Gram-positive bacteria. Among them, anisaxin-2S (A-2S), membranolytic cecropin-like HDPs produced by the zoonotic nematodes of the genus Anisakis, shows remarkable efficacy even against multidrug-resistant Gram-negative bacteria, yet its immunomodulatory, antiproliferative and antiviral properties have not been elucidated. Therefore, we tested A-2S immunomodulation in the common carp (Cyprinus carpio) blood cells exposed to two pathogens, the zoonotic bacterium Aeromonas hydrophila and the fish parasite Sphaerospora molnari, and in carp in vivo challenged with the parasite. Furthermore, the A-2S antiproliferative activity was tested in vitro in human bladder and lung cancer cell line, while the antiviral protection was tested in common carp brain cell culture exposed to carp rhabdovirus, alloherpesvirus and paramyxovirus, and in a human immortalized myelogenous leukemia cell line infected with tick-borne encephalitis virus. A-2S exerts an immunostimulatory effect on fish blood cells through upregulation of cytokine expression, with the proinflammatory or anti-inflammatory repertoire conditioned by the presence or absence of co-stimulatory antigen. Surprisingly, in the majority of assays conducted, red blood cells demonstrate equal or even stronger regulation of innate immunity genes compared to white blood cells, along with a more extensive repertoire of differentially expressed markers. In contrast, A-2S has only a limited anticancer activity in human bladder cancer and lung adenocarcinoma cells and limited antiviral activity against the three fish viruses and a human tick-borne encephalitis virus. This study provides the first evidence of red blood cell and platelet immunomodulation by an antimicrobial peptide and highlights the induction of a cytokine repertoire. However, future research should address the study's limitations, including the need for longer in vitro assays (e.g., 3-4 days), testing different white blood cell lineages, to better understand antigen-processing interactions, and evaluating the anticipated adaptive immune response. Powerful antimicrobial activity of A-2S, coupled with immunostimulatory properties, warrant further pursuing of preclinical trials with this anisaxin.

• Klíčová slova
• anisaxin, antimicrobial peptide, immunomodulation, red blood cells, white blood cells,
• MeSH
• antivirové látky * farmakologie   MeSH
• cekropiny * farmakologie   MeSH
• imunologické faktory * farmakologie   MeSH
• imunomodulace MeSH
• kapři * imunologie   parazitologie   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nemoci ryb * imunologie   MeSH
• proliferace buněk účinky léků   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antivirové látky * MeSH
• cekropiny * MeSH
• imunologické faktory * MeSH

BACKGROUND: Helminth extracellular vesicles (EVs) are known to have a three-way communication function among parasitic helminths, their host and the host-associated microbiota. They are considered biological containers that may carry virulence factors, being therefore appealing as therapeutic and prophylactic target candidates. This study aims to describe and characterise EVs secreted by Sparicotyle chrysophrii (Polyopisthocotyla: Microcotylidae), a blood-feeding gill parasite of gilthead seabream (Sparus aurata), causing significant economic losses in Mediterranean aquaculture. METHODS: To identify proteins involved in extracellular vesicle biogenesis, genomic datasets from S. chrysophrii were mined in silico using known protein sequences from Clonorchis spp., Echinococcus spp., Fasciola spp., Fasciolopsis spp., Opisthorchis spp., Paragonimus spp. and Schistosoma spp. The location and ultrastructure of EVs were visualised by transmission electron microscopy after fixing adult S. chrysophrii specimens by high-pressure freezing and freeze substitution. EVs were isolated and purified from adult S. chrysophrii (n = 200) using a newly developed ultracentrifugation-size-exclusion chromatography protocol for Polyopisthocotyla, and EVs were characterised via nanoparticle tracking analysis and tandem mass spectrometry. RESULTS: Fifty-nine proteins involved in EV biogenesis were identified in S. chrysophrii, and EVs compatible with ectosomes were observed in the syncytial layer of the haptoral region lining the clamps. The isolated and purified nanoparticles had a mean size of 251.8 nm and yielded 1.71 × 108 particles · mL-1. The protein composition analysis identified proteins related to peptide hydrolases, GTPases, EF-hand domain proteins, aerobic energy metabolism, anticoagulant/lipid-binding, haem detoxification, iron transport, EV biogenesis-related, vesicle-trafficking and other cytoskeletal-related proteins. Several identified proteins, such as leucyl and alanyl aminopeptidases, calpain, ferritin, dynein light chain, 14-3-3, heat shock protein 70, annexin, tubulin, glutathione S-transferase, superoxide dismutase, enolase and fructose-bisphosphate aldolase, have already been proposed as target candidates for therapeutic or prophylactic purposes. CONCLUSIONS: We have unambiguously demonstrated for the first time to our knowledge the secretion of EVs by an ectoparasitic flatworm, inferring their biogenesis machinery at a genomic and transcriptomic level, and by identifying their location and protein composition. The identification of multiple therapeutic targets among EVs' protein repertoire provides opportunities for target-based drug discovery and vaccine development for the first time in Polyopisthocotyla (sensu Monogenea), and in a fish-ectoparasite model.

• Klíčová slova
• Drug target candidates, Ectosomes, Electron microscopy, Exosomes, Monogenea, Peptidases, Polyopisthocotyla, Prophylactic target candidates,
• MeSH
• extracelulární vezikuly * MeSH
• mořan zlatý * parazitologie   MeSH
• ploštěnci * MeSH
• proteomika MeSH
• Trematoda * MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH