Corneal epithelial signals remodel bacterial physiology and extracellular vesicle cargo in Pseudomonas aeruginosa and Staphylococcus epidermidis

Host–microbe communication at the ocular surface is bidirectional, yet the reciprocal effects of corneal epithelial signals on bacterial physiology remain poorly understood. Here, we investigated how corneal epithelial-derived exosomes and bacteria–epithelium co-culture influence Pseudomonas aeruginosa , a major opportunistic ocular pathogen, and Staphylococcus epidermidis , a common member of the ocular surface microbiota. Bacterial responses were assessed by transcriptomic and functional analyses, while bacterial extracellular vesicles released during epithelial interaction were characterized by proteomics and RNA sequencing. Corneal epithelial-derived exosomes reduced bacterial growth and increased epithelial adhesion in both species, while enhancing biofilm formation in P. aeruginosa . Bacteria–epithelium co-culture induced substantially broader responses than exposure to epithelial-derived exosomes alone. In P. aeruginosa , epithelial interaction reorganized quorum-sensing, motility, iron-acquisition, phenazine and biofilm-associated pathways, resulting in reduced motility and pyocyanin production, increased pyoverdine production and enhanced biofilm formation. In S. epidermidis , epithelial interaction was associated with reduced expression of several virulence factors associated with cytotoxicity and inflammatory responses, upregulation of genes associated with surface interaction and protein export, and decreased biofilm biomass. In both species, epithelial interaction also induced extensive remodeling of bacterial extracellular vesicle protein and RNA cargo, which did not simply mirror the corresponding intracellular bacterial profiles. These findings demonstrate that corneal epithelial signals actively drive species-specific bacterial adaptation and vesicular remodeling. Together with previous evidence of bacteria-to-epithelium signaling, they support a dynamic model of bidirectional interkingdom communication at the ocular surface.