Visualizing intestinal colonization of GFP-displayed Lactobacillus plantarum PO23 and its effects on intestinal morphology and immunity in Paralichthys olivaceus.

Visualization of probiotic intestinal colonization is essential for clarifying their adhesion characteristics and mucosal immunomodulatory mechanisms. GFP surface display enables real-time visualization of probiotic attachment, distribution, and migration dynamics in living hosts. In our previous study, a highly adhesive potential probiotic strain, Lactobacillus plantarum PO23 (LP-PO23), was isolated from the intestine of flounder (Paralichthys olivaceus). To precisely track intestinal adhesion and elucidate its probiotic mechanisms, we constructed a visualized LP-PO23 strain by surface-displaying GFP using the LPXTG anchoring motif (designated LP-GFP), and assessed its probiotic functions in flounder. SDS-PAGE and Western blotting confirmed successful GFP expression, while immunofluorescence (IF) and immunoelectron microscopy (IEM) verified its surface display. The LP-GFP strain exhibited stable GFP expression and surface display. LP-GFP was orally administered to flounder at 1 × 109 CFU/g feed for one week, and the colonization dynamics of LP-GFP in the foregut, midgut and hindgut were assessed post-feeding using plate counting, IF and PCR. Results showed that LP-GFP effectively colonized the flounder intestine, with the highest abundance in the midgut, followed by the foregut and then the hindgut. Histological analysis further revealed that LP-GFP improved intestinal villus morphology and mucosal integrity. Moreover, LP-GFP significantly upregulated the pro-inflammatory cytokines IL-1β and TNFα at early time points, followed by increased expression of the anti-inflammatory cytokine IL-10 and the tight junction protein occludin, indicating a biphasic immune response and enhanced intestinal barrier function. These findings provide a visualizable and traceable probiotic tool for in vivo research on intestinal colonization and immune regulation in flounder.