Fungal extracellular vesicles reshape Lactobacillus rhamnosus vesicles to enhance lysosomal homeostasis and resolve intestinal inflammation.

Extracellular vesicles (EVs) are emerging mediators of intercellular and interkingdom communication. While bacterial EVs directly modulate host health, whether exogenous vesicles can reprogram gut microbial vesiculation and bioactivity remains unclear. Using a defined in vitro conditioning system, ScEV exposure altered EV production by Lactobacillus rhamnosus, generating ScEV-conditioned L. rhamnosus vesicles (ScEV-LrEVs). Comparative proteomics and lipidomics revealed extensive remodeling of vesicle cargo, including ~60-fold enrichment of hydroxylated ceramide Cer(t18:0/24:0(2OH)) in ScEV-LrEVs. Functionally, ScEV-LrEVs showed enhanced anti-inflammatory activity in LPS-stimulated macrophages, accompanied by improved lysosomal function and coordinated changes in mTOR-TFEB-related lysosome-autophagy markers. In a DSS-induced colitis model, ScEV-LrEVs alleviated mucosal injury, reduced immune-cell infiltration, and reinforced epithelial barrier integrity, accompanied by restoration of lysosomal function and coordinated changes in mTOR-TFEB-related markers. Ceramide-enriched EVs partially recapitulated the anti-inflammatory and lysosome-restorative activity of ScEV-LrEVs. Together, these findings uncover a cross-kingdom vesicle communication axis whereby fungal EV exposure remodels the molecular and functional properties of gut microbial vesicles, accompanied by enhanced lysosomal homeostasis and protection against intestinal inflammation.