Bacterial extracellular vesicles and direct microbial contact drive species-specific corneal epithelial reprogramming.

BACKGROUND: Extracellular vesicles (EVs) are important mediators of host-microorganism communication at epithelial surfaces. However, their role in shaping corneal epithelial responses to ocular-associated bacteria remains poorly understood. Here, we investigated the species-specific effects of Pseudomonas aeruginosa and Staphylococcus epidermidis on human corneal epithelial cells, focusing on bacterial extracellular vesicles (BEVs), direct microbial contact, and epithelial exosomal remodeling. METHODS: Human corneal epithelial cells were exposed to BEVs or co-cultured with P. aeruginosa or S. epidermidis. Transcriptomic, proteomic, and extracellular vesicle RNA analyses were performed together with functional assays evaluating epithelial metabolic activity, motility, and apoptosis. RESULTS: BEVs from P. aeruginosa induced a strong pro-inflammatory epithelial response associated with IL-17, TNF, and NF-κB signaling, extracellular matrix remodeling, and increased cell motility. Direct bacterial contact further amplified these effects, triggering extensive transcriptional reprogramming and remodeling of epithelial exosomal cargo, including enrichment of proteasome-associated proteins and apoptosis-related microRNAs. Functional apoptosis assays confirmed that exosomes released following P. aeruginosa exposure acquired a modest but significant pro-apoptotic activity. In contrast, S. epidermidis induced a substantially milder transcriptional response characterized by limited differential gene expression, preferential detection of regulatory non-coding RNAs, and distinct exosomal remodeling associated with cell cycle and signaling pathways. Notably, responses induced by BEVs and direct bacterial contact showed limited overlap, indicating complementary mechanisms of epithelial regulation. CONCLUSIONS: Together, these findings demonstrate that BEVs and direct microbial contact act as complementary and species-specific drivers of corneal epithelial reprogramming. Our results further support a model in which BEVs function as early signaling mediators, whereas epithelial-derived exosomes contribute to the propagation and modulation of these responses across the corneal epithelium.