Probiotic Extracellular Vesicles: Bridging Lacticaseibacillus casei and the Intestinal Epithelium

The mechanisms underlying the health benefits of probiotics remain incompletely understood. Among the proposed mediators, bacterial extracellular vesicles (EVs) have been proposed as mediators of interkingdom communication between probiotic bacteria and their hosts. In this study, we show that Lacticaseibacillus casei BL23 releases higher EV levels during late stationary phase, with a predominant population of ∼113 nm. Scanning electron microscopy (SEM) confirmed the ultrastructural morphology of the EV preparations. Proteomic analysis revealed enrichment of proteins associated with probiotic function, including P40 and P75, as well as adhesion-related moonlighting proteins such as glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and enolase implicated in interactions with intestinal mucus and epithelial cells. L. casei BL23-derived EVs showed dose-dependent interactions with mucin and with differentiated Caco-2 epithelial monolayers in vitro. Using Transwell systems, we further demonstrated that intact EVs cross these polarized monolayers within 240 min. Super-resolution microscopy further revealed time-dependent uptake and intracellular trafficking of fluorescently-labeled EVs. Together, these findings, obtained in vitro, are consistent with a role for probiotic-derived EVs as postbiotics capable of mediating microbiota-host interactions at the intestinal barrier, providing the experimental basis for future validation in more complex physiological models.