Abstract
The ability of epithelial barriers to perform as the first defense line against external damage derives from tight junctions, protein complexes that block microorganisms through the paracellular space. Indeed, disturbances of barrier permeability caused by bacterial metabolites and other inflammatory stimuli are the consequence of changes in protein expression in these complexes. Postbiotics, molecules derived from bacteria with beneficial effects on the host, improve barrier function through the activation of survival pathways in epithelial cells. Lacticaseibacillus rhamnosus GG secretes the muramidase p40, which protects intestinal barriers through an EGFR-dependent pathway. In this work, we cloned, expressed, and purified the recombinant p40 protein from L. rhamnosus GR-1 to evaluate its effect on cell viability, cell cytotoxicity, TEER, and protein levels of tight junctions, as well as EGFR activation via Western blot on HaCaT keratinocytes subjected to LPS. We found a novel mutation at residue 368 that does not change the structure of p40. Our protein also reduces the LPS-induced increase in cell cytotoxicity when it is added prior to this stimulus. Furthermore, although LPS did not cause changes in barrier function, p40 increased TEER and occludin expression in HaCaT, but unlike previous work with p40 from LGG, we found that recombinant p40 did not activate EGFR. This suggests that recombinant p40 enhances epithelial barrier function through distinct signaling pathways.