Extracellular vesicles and their RNA cargo facilitate bidirectional cross-kingdom communication between human and bacterial cells.

While extracellular vesicles (EVs) are established mediators of intra-species signaling, their contribution to cross-kingdom communication remains incompletely understood. Here, we investigate the EV-mediated interactions between human colon epithelial cells and both Gram-positive and Gram-negative gut bacteria. We show that bacterial EVs (BEVs) derived from Lacticaseibacillus casei, Enterococcus faecalis, and Proteus mirabilis induce distinct transcriptomic changes in Caco-2 cells depending on the bacterial species, with up to ~6,000 differentially expressed genes, including CCL20, CXCL8, or CXCL10. Transfection of BEV-derived RNA independently induces a subset of similar effects, indicating that the EV-mediated communication is partially driven by the RNA cargo. Conversely, we demonstrate that bacteria interact with Caco-2-derived EVs and miR-192-5p, which is highly abundant (~36.4-fold higher) in EVs isolated from conditioned medium compared with EVs from unconditioned medium, with modest effects on bacterial growth. Furthermore, we show that lipid-based packaging of miR-192-5p modulates its association with the bacteria. Our findings support a conceptual model in which EVs and their RNA cargo contribute to species-dependent host-microbe interactions. This study introduces a framework for understanding EVs as cross-kingdom regulators and underscores the importance of tailored, context-specific analyses for understanding the scope of EV-mediated interactions in microbiome-host homeostasis and disease.