Surface-layer proteins of Enterococcus faecium WEFA23 inhibit Listeria monocytogenes-induced inflammation via TLR2-mediated NF-κB and MAPK signalling in RAW 264.7 cells.

Listeria monocytogenes infection, which has a higher mortality rate than other foodborne pathogens, is a major concern in global food safety. Among various interventions, lactic acid bacteria and their metabolites have gained significant attention. Our previous study showed that the surface layer proteins (SLPs) of Enterococcus faecium WEFA23, isolated from the gastrointestinal tract of newborn infants, exhibited strong inhibitory activity against L. monocytogenes CMCC57007; however, the underlying mechanism remained unclear. In this study, the immunomodulatory effects of E. faecium WEFA23 SLPs against L. monocytogenes infection were investigated in RAW 264.7 macrophage cells, focusing on phagocytic and bactericidal activity, as well as cytokine production. Furthermore, LC-MS/MS analysis indicated that ornithine carbamoyltransferase (OTC) could be a functional component of the SLPs. Consequently, otc gene knockout and heterologous expression strains were constructed. The results showed that deletion of the otc gene eliminated the inhibitory activity, while recombinant OTC maintained a satisfactory inhibitory effect. Mechanistically, both SLPs and purified OTC suppressed the activation of the NF-κB/MAPK signalling pathways, which were likely mediated through TLR2. Overall, our findings provide a scientific basis for the application of SLPs and E. faecium in food systems to prevent pathogenic infections.