Abstract
Extracellular vesicles (EVs) have emerged as a ubiquitous component of helminth excretory-secretory products that can deliver parasite molecules to host cells to elicit immunomodulatory effects. RNAs are one type of cargo molecule that can underpin EV functions, hence there is extensive interest in characterising the RNAs that are present in EVs from different helminth species. Here we outline methods for identifying all of the small RNAs (sRNA) in helminth EVs and address how different methodologies may influence the sRNAs detected. We show that different EV purification methods introduce relatively little variation in the sRNAs that are detected, and that different RNA library preparation methods yielded larger differences. We compared the EV sRNAs in the gastrointestinal nematode Heligmosomoides bakeri with those in EVs from the distantly related gastrointestinal nematode Trichuris muris, and found that many of the sRNAs in both organisms derive from repetitive elements or intergenic regions. However, only in H. bakeri do these RNAs contain a 5′ triphosphate, and Guanine (G) starting nucleotide, consistent with their biogenesis by RNA-dependent RNA polymerases (RdRPs). Distinct microRNA (miRNA) families are carried in EVs from each parasite, with H. bakeri EVs specific for miR-71, miR-49, miR-63, miR-259 and miR-240 gene families, and T. muris EVs specific for miR-1, miR-1822 and miR-252, and enriched for miR-59, miR-72 and miR-44 families, with the miR-9, miR-10, miR-80 and let-7 families abundant in both. We found a larger proportion of miRNA reads derive from the mouse host in T. muris EVs, compared with H. bakeri EVs. Our report underscores potential biases in the sRNAs sequenced based on library preparation methods, suggests specific nematode lineages have evolved distinct sRNA synthesis/export pathways, and highlights specific differences in EV miRNAs from H. bakeri and T. muris that may underpin functional adaptation to their host niches.
Highlights
In order to maintain long-term infections, helminths have evolved mechanisms to directly modify the host environment to favour their survival
Purification methods for helminth extracellular vesicles (EVs) vary between research groups and there is extensive interest in documenting whether and how these variables can impact the properties of EVs and the cargo molecules that are identified, as outlined for mammalian EV research (Théry et al, 2018)
Our results suggest that detection of the EV miRNA cargo is relatively consistent across different purification strategies for H. bakeri EVs, for example adding a sucrose flotation step after UC purification does not yield any difference in the miRNA detected compared
Summary
In order to maintain long-term infections, helminths have evolved mechanisms to directly modify the host environment to favour their survival. A growing body of literature demonstrates that diverse parasites have evolved EV cargos that promote parasite survival (Ofir-Birin and Regev-Rudzki, 2019). Many helminth species have been shown to release EVs that can have modulatory actions on host cells in vitro and in vivo (Coakley et al, 2015; Eichenberger et al, 2018a; Tritten and Geary, 2018).
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