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 role as active participants in cross-kingdom communication remains incompletely understood. Here, we reveal that human colon cells and both Gram-positive and Gram-negative gut bacteria engage in species-specific, EV-mediated molecular dialogue, driven in part by RNA cargo. We show that bacterial EVs (BEVs) induce distinct transcriptomic responses in human cells, and that BEV-RNA independently causes similar effects. Conversely, we demonstrate that human EVs and highly abundant miR-192-5p are differentially internalized by bacteria, affecting their physiology. Our findings support a conceptual model in which EVs function as directional messengers that shape host-microbiome 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.