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

## TL;DR

Extracellular vesicles and their RNA cargo enable communication between human and bacterial cells, influencing gene expression and immune responses.

## Contribution

This study reveals bidirectional cross-kingdom communication via EVs and RNA cargo between human and bacterial cells.

## Key findings

- Bacterial EVs induce species-specific gene expression changes in human cells, including immune-related genes like CCL20 and CXCL8.
- Human-derived EVs and miR-192-5p affect bacterial growth and interactions, with lipid packaging modulating miRNA-bacteria associations.
- EV RNA cargo contributes to cross-kingdom signaling, highlighting the role of EVs in host-microbe interactions.

## Abstract

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.

L. casei, E. faecalis, and P. mirabilis produce BEVs, which accumulate within Caco-2 cells upon incubation. L. casei-derived BEVs positively affect the viability of Caco-2 cells. Incubation of Caco-2 cells with BEVs leads to changes in the expression of immune response-related genes such as CCL20, CXCL8, or CXCL10.BEVs carry RNAs, and the RNA cargo varies between BEVs from the different bacterial species. Comparison of Caco-2 gene deregulation following incubation with BEVs or transfection with BEV-RNA highlights component-specific effects.E. faecalis shows modestly altered growth in the presence of Caco-2 EVs. MiRNA-192-5p is frequently detected in EVs from Caco-2 cells. P. mirabilis interacts with synthetic miR-192-5p, and the interaction of L. casei and E. faecalis with this miRNA can be altered by lipid-based packaging of the miRNA.

L. casei, E. faecalis, and P. mirabilis produce BEVs, which accumulate within Caco-2 cells upon incubation. L. casei-derived BEVs positively affect the viability of Caco-2 cells. Incubation of Caco-2 cells with BEVs leads to changes in the expression of immune response-related genes such as CCL20, CXCL8, or CXCL10.

BEVs carry RNAs, and the RNA cargo varies between BEVs from the different bacterial species. Comparison of Caco-2 gene deregulation following incubation with BEVs or transfection with BEV-RNA highlights component-specific effects.

E. faecalis shows modestly altered growth in the presence of Caco-2 EVs. MiRNA-192-5p is frequently detected in EVs from Caco-2 cells. P. mirabilis interacts with synthetic miR-192-5p, and the interaction of L. casei and E. faecalis with this miRNA can be altered by lipid-based packaging of the miRNA.