Analysis of Oesophagostomum dentatum excretory-secretory molecules and extracellular vesicles uncovers a repertoire of microRNAs shared with A. Suum and T. Suis and ES containing Paz and Piwi domain proteins implicated in RNAi signaling

Soil-transmitted helminths (STH) are prevalent in pigs, with the nodular worm, Oesophagostomum dentatum, causing a chronic infection that affects animal welfare and productivity. Unlike the two other dominant soil-transmitted helminths of pigs, Ascaris suum and Trichuris suis, O. dentatum promotes immune responses that deviate from the normal parasite-induced type 2 helper (Th2) response causing long-term infection with minimally acquired immunity. O. dentatum induces a proinflammatory Type 1 helper(Th1)-like response followed by a delayed non-protective Th2 response. The mechanism underlying this disparate response is unclear. Intestinal parasites interact with the host via excreted-secreted molecules and here we investigate the protein and microRNA content of the infectious L3 and adult stage of O. dentatum focusing particularly on identifying the content of extracellular vesicles (EVs). Using Cryo-EM, nanoparticle tracking analysis and silver staining, we confirmed the presence of EVs in adult worm ES. Proteomic analysis revealed prototypical ES proteins, including SCP-like proteins, transthyretin-like proteins, and peptidases, proteases and hydrolases. Interestingly, a DEAD-Box RNA helicase was found unique to EVs, while a Paz domain protein and a Piwi domain protein was found in the adult worm ES that shows sequence similarity to these domains in the exWAGO protein found in H. bakeri EVs. Pathway enrichment analyses revealed proteins involved in nutrient metabolism, and the proteasome. To understand which microRNAs O. dentatum parasites secrete, we annotated the microRNA complement from small RNA-seq libraries from the ES, EVs and EV-depleted SN using microRNA prediction tools (MirMachine and MirMiner). We identified 44 precursor microRNAs and 88 mature microRNAs in the O. dentatum ES microRNA complement. Quantification revealed 22, 44, and 48 mature microRNAs in L3 EVs, adult EVs and adult SN, respectively, with substantial overlap between L3 and adult EVs (n = 22, 37 % of shared set) and between adult EVs and SN (n = 58, 75 % of shared set). Differential analysis identified 11 microRNAs significantly differentially abundant between sample types. To explore potential conservation of EV microRNAs across nematode species, we here, for the first time, compare the microRNA family profiles of O. dentatum adult EVs with those of A. suum and T. suis. We found eight shared microRNA families that are known to be highly abundant in the worms themselves: LET-7, MIR-10, MIR-34, MIR-9, MIR-305, MIR-54, MIR-216, and MIR-750. Their diverging microRNA profiles might underlie their species-specific differences in parasitism. Taken together, this study expands the current knowledge of excretory-secretory molecules in non-model parasitic nematodes and confirms the presence of proteins involved in RNAi processing in the ES of the clade V nematode, O. dentatum. AUTHOR SUMMARY: Parasitic nematodes pose a significant challenge to animal health and productivity, particularly in livestock. Among these, Oesophagostomum dentatum establishes chronic infections in pigs with minimally acquired immunity, with an underlying mechanism that remains poorly understood. In this study, we uncovered the excretory-secretory (ES) molecules and extracellular vesicles (EVs) released by O. dentatum, which are thought to play a key role in host-parasite interactions at the gut interphase. Using advanced proteomic and RNA sequencing approaches, we characterized the proteins and microRNAs associated with these compartments. Our findings confirmed the presence of EVs in adult worm ES and identified an EV protein profile significantly enriched in nutrient metabolic and proteasomal pathways, as well as an RNA helicase in the EVs and Paz and Piwi domain proteins in the ES that might be involved in argonaute RNA-mediated silencing. We also annotated the first microRNA complement of O. dentatum ES, identifying 88 mature microRNAs, with a substantial overlap between L3 and adult worm EVs. Comparative analysis with other parasitic nematodes revealed eight conserved microRNA families also generally known as highly abundant microRNAs in nematodes. By characterizing the secreted proteins and microRNAs of this non-model parasitic nematode, our study lays the groundwork for future research into host immune modulation and survival strategies of parasitic nematodes.