A thermo-resistant and RNase-sensitive cargo from extracellular vesicles modifies the behaviour of enterobacteria.

Extracellular vesicles (EVs) recently emerged as important players in the pathophysiology of parasitic infections. While the protist parasite  can produce EVs, their role in giardiasis remains obscure. can disrupt gut microbiota biofilms and transform commensal bacteria into invasive pathobionts at sites devoid of colonizing trophozoites via unknown mechanisms. We hypothesized that EVs could modify gut bacterial behaviour via a novel mode of trans-kingdom communication. Our findings indicate that EVs exert bacteriostatic effects on HB101 and TW1, increasing their swimming motility. EVs also decreased the biofilm-forming ability of HB101 but not by TW1, supporting the hypothesis that these effects are, at least in part, bacteria-selective. HB101 and TW1 exhibited increased adhesion/invasion onto small intestine epithelial cells when exposed to EVs. EVs labelled with PKH67 revealed colocalization with HB101 and TW1 bacterial cells. Small RNA sequencing revealed a high abundance of ribosomal RNA (rRNA)- and transfer RNA (tRNA)-derived small RNAs, short-interfering RNAs (siRNAs) and micro-RNAs (miRNAs) within EVs. Proteomic analysis of EVs uncovered the presence of RNA chaperones and heat shock proteins that can facilitate the thermal stability of EVs and its sRNA cargo, as well as protein-modifying enzymes. In vitro, RNase heat-treatment assays showed that total RNAs in EVs, but not proteins, are responsible for modulating bacterial swimming motility and biofilm formation. small RNAs of EVs, but not proteins, were responsible for the increased bacterial adhesion to intestinal epithelial cells induced upon exposure to EVs. Together, the findings indicate that EVs contain a heat-stable, RNase-sensitive cargo that can trigger the development of pathobiont characteristics in Enterobacteria, depicting a novel trans-kingdom cross-talk in the gut.