Enterococcus faecium WEFA23-Derived Surface Layer Protein OTC Prevents Listeria monocytogenes Invasion by Strengthening Intestinal Barrier Function and Modulating Immune Responses.

Listeria monocytogenes (L. monocytogenes) is a major foodborne pathogen which can invade intestinal epithelial cells and cause severe systemic infection. Probiotics, as well as their surface layer proteins, hold broad promise for enhancing intestinal barrier function and defending against pathogenic invasion. In the present study, the antagonistic effects of surface layer protein ornithine carbamoyltransferase (OTC) from Enterococcus faecium (E. faecium) WEFA23 against L. monocytogenes were systematically evaluated in vitro in human intestinal epithelial Caco-2 cells, including assessments of anti-adhesion and anti-invasion capacity, inflammatory cytokine responses, intestinal barrier integrity, and transcriptomic changes, by comparing the effects of wild-type E. faecium WEFA23 and a previously constructed E. faecium WEFA23 otc gene knockout strain (E. faecium WEFA23 otc-/-). The results demonstrated that E. faecium WEFA23 achieved significant stronger anti-adhesion and anti-invasion capacity of L. monocytogenes (p < 0.05) in the presence of OTC, potentially through increasing tight junction protein expression, regulating inflammatory cytokines, and modulating the virulence factors of the pathogen. To elucidate the potential mechanism of the inhibitory effect of OTC protein, RNA-seq was performed. The results revealed that the significantly regulated core differentially expressed genes (DEGs), including ADCY2, OARI3, CCL5, and CXCL9, were found to be involved in γ-aminobutyric acid (GABA)-ergic synapse, calcium, and toll-like receptor signaling pathways. These findings demonstrated that OTC is involved in blocking Listeria invasion and revealed the function of the OTC from E. faecium WEFA23 in antimicrobial and intestinal mucosal defense, providing a conceptual foundation for the development of new probiotic intervention strategies in anti-infection.