Extracellular vesicles and their RNA cargo facilitate bidirectional cross-kingdom communication between human and bacterial cells [smallRNA-Seq]

Extracellular vesicles (EVs), released by both eukaryotic and prokaryotic cells, have emerged as key mediators of cell-to-cell communication. Recent advances highlight their crucial role in cross-kingdom communication, bridging the microbial world with human biology. Here, we investigated the molecular mechanisms underlying EV-mediated bidirectional communication within the gastrointestinal ecosystem. Using a model that includes human colon cells and both Gram-positive and Gram-negative gut bacteria, we reveal an intricate exchange of information between these kingdoms. Our analysis uncovered highly specific responses of host cells to bacterial EVs (BEVs) and BEV-RNA cargo, including uptake rates by human cells, impact on human cell viability, and alterations in their transcriptomic landscape. In parallel, we discovered that host-derived EVs and miR-192-5p are internalized by gut bacteria, leading to changes in their growth pattern. These findings highlight the precision with which EVs and their RNA cargo mediate interkingdom communication. Our results underscore the importance of tailored, context-specific analyses for understanding the scope of EV-mediated interactions in complex biological systems. Overall design: Sequencing of smallRNAs isolated from EVs purified from Caco-2 cell culture supernatant (conditioned medium) and fresh cell culture medium (unconditioned medium). EVs were isolated via ultracentrifugation and purified using size-exclusion chromatography. RNA was isolated from EVs and used for smallRNA-seq.