A miRNAs catalogue from third-stage larvae and extracellular vesicles of Anisakis pegreffii provides new clues for host-parasite interplay

Abstract

Anisakids are widespread marine parasites of medical, veterinary and economic relevance. They infect marine natural hosts but humans can accidentally acquire the fish-borne zoonosis anisakiasis by ingesting infected raw fishes or mollusks. Among the several species described, Anisakis pegreffii is one of the main etiological agent of the disease, in particular in the Mediterranean area. Despite the growing evidence of miRNAs involvement in host-parasite interplay, and the emerging role of exosomal microvesicles in shuttling them between different cell types (and sometime across species), no information on miRNAs from any Anisakis species is presently available. In this study we isolated extracellular vesicles (EVs) released by Anisakis pegreffii infective third-stage larvae (L3) and analyzed by RNA-seq small RNAs from both L3 and EVs. We showed by nanoparticle tracking analysis that L3 release in culture medium particles of size compatible with the one of extracellular vesicles. A catalogue of 156 miRNAs from A. pegreffii was compiled by sequence comparison to evolutionary close species and miRNA prediction software. Using differential expression analysis, we identified a small number of highly abundant miRNAs in larvae and extracellular vesicles fractions whose potential biological relevance may deserve future investigation. Finally, A. pegreffii miRNAs were compared to those described in other parasitic helminths and predicted targets among human genes were searched, suggesting their potential involvement during infection.

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

“…So far, the proteomic profiling of ESPs has been described for the species A. simplex (s.s.) ( Kochanowski et al., 2022 ), while no specific analysis is available for ESPs of A. pegreffii . Recently, EVs from the A. pegreffii L3 were shown to deliver miRNA ( Cavallero et al., 2022a ), while the proteomic information available for L3-EVs are referred to Anisakis sp. ( Boysen et al., 2020 ), although the object of that study was likely the species A. simplex (s.s.) being the larvae collected from Clupea harengus of the NE Atlantic Ocean, which has been found parasitised by that species ( Mattiucci et al., 2018b ).…”

“…So far, the proteomic composition of EVs have been reported in Anisakis spp. ( Boysen et al., 2020 ), while miRNAs have been studied and recorded in A. pegreffii ( Cavallero et al., 2022a ).…”

Re‐evaluation of certain aspects of the EFSA Scientific Opinion of April 2010 on risk assessment of parasites in fishery products, based on new scientific data. Part 1: ToRs1–3

“…Recent progress using omic methodologies have provided new information on the virulence and pathogenic repertoire of fish zoonotic parasites. It has been demonstrated that the L3 of A. pegreffii secrete these factors, either as free proteins in the secretome (Trumbić et al., 2021 ), or packaged in form of extracellular vesicles (EVs) (Boysen et al., 2020 ;Cavallero et al., 2022 ; Palomba, Rughetti, et al., 2023 ). These products have a role in the larval invasion mechanism and the immunomodulation of the host's response, in particular the evasion from the immune system.…”

“…These products have a role in the larval invasion mechanism and the immunomodulation of the host's response, in particular the evasion from the immune system. It has been suggested that the pathogenicity of A. pegreffii L3 is likely the result of the direct invasive capability of the larva, its released excretory secretory products (ESPs) (Trumbić et al., 2021 ), including those transported by EVs (Cavallero et al., 2022 ; Palomba, Rughetti, et al., 2023 ), as well as the intricate interplay of the host's immune response (Mehrdana et al., 2017 ). Among ESPs an array of peptidases and peptidase inhibitors are considered essential for larval moulting, feeding, tissue penetration and migration (Mladineo et al., 2023 ; Palomba, Rughetti, et al., 2023 ).…”

Anisakiasis and Anisakidae

“…The authors also propose further studies on extracellular vesicles, as recent studies have enabled their fluorescent labelling in Anisakis , facilitating work with them [ 33 ], which is very important due to their potential as a mechanism of communication and interaction between parasite and host. Their contents include DNA, proteins, and non-coding RNAs, such as microRNAs (already in evidence in A. pegreffii L3 [ 34 ]), which play relevant roles in all aspects of cellular life through post-transcriptional gene regulation, potentially promoting the degradation and/or translational repression of target messenger RNAs [ 35 ]. Thus, parasites could use their extracellular vesicles to potentially regulate host genes that, among other possible functions, could regulate host immune and inflammatory responses [ 32 ].…”