Affiliation:
1. Institute of Aquaculture Torre de la Sal
2. Institute of Parasitology
3. University of South Bohemia in České Budějovice
4. Instituto de Salud Carlos III
Abstract
Abstract
Background: Helminth extracellular vesicles (EVs) are known to have a three-way communication role among parasitic helminths, their host and host-associated microbiota. They are considered biological containers carrying virulence factors, being therefore appealing as therapeutic and prophylactic target candidates. This study aims to describe and characterise EVs secreted by the monogenean Sparicotyle chrysophrii (Microcotylidae), a blood-feeding gill parasite of the gilthead seabream (Sparus aurata) inflicting 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 EV location and ultrastructure was studied using transmission electron microscopy in high-pressure frozen adult S. chrysophrii specimens. Moreover, EVs were isolated and purified from adult S. chrysophrii (N=200) using a newly developed ultracentrifugation-size-exclusion chromatography protocol for Monogenea, 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 microvesicles were observed in the parasite’s haptoral region syncytial layer lining the clamps. Moreover, isolated and purified nanoparticles presented a mean size of 251.8 ± 2.2 nm (± SD), and yielded 1.71×108 ± 4.55×107 particles·mL-1 (mean ± SD).
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. From the identified proteins, 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 in helminths.
Conclusions: We have unambiguously demonstrated the secretion of EVs by S. chrysophrii inferring their biogenesis machinery at a genomic and transcriptomic level, ultrastructural location in the parasite, and analysing their protein composition. The identification of several candidate therapeutic targets among EV’s protein repertoire, offers opportunities for target-based drug discovery and vaccine development approaches, for the first time in Monogenea and a fish-ectoparasite model.
Publisher
Research Square Platform LLC
Reference110 articles.
1. Diseases of cultured marine fishes caused by Platyhelminthes (Monogenea, Digenea, Cestoda);Ogawa K;Parasitology,2015
2. Mapping the knowledge of the main diseases affecting sea bass and sea bream in Mediterranean;Muniesa A;Transbound Emerg Dis,2020
3. Monogenean Sparicotyle chrysophrii: The major pathogen of the Mediterranean gilthead seabream aquaculture;Mladineo I;Rev Aquac,2023
4. A stratified compartmental model for the transmission of Sparicotyle chrysophrii (Platyhelminthes: Monogenea) in gilthead seabream (Sparus aurata) fish farms;Stella E;R Soc Open Sci,2023
5. Sitjà-Bobadilla A, de Felipe MC, Alvarez-Pellitero P. In vivo and in vitro treatments against Sparicotyle chrysophrii (Monogenea: Microcotylidae) parasitizing the gills of gilthead sea bream (Sparus aurata L.). Aquaculture. 2006;261:856–64.