Differential protection by cell wall components of Lactobacillus amylovorus DSM 16698Tagainst alterations of membrane barrier and NF-kB activation induced by enterotoxigenic F4+ Escherichia coli on intestinal cells

Background: The role of Lactobacillus cell wall components in the protection against pathogen infection in the gut is still largely unexplored. We have previously shown that L. amylovorus DSM 16698T is able to reduce the enterotoxigenic F4+ Escherichia coli (ETEC) adhesion and prevent the pathogen-induced membrane barrier disruption through the regulation of IL-10 and IL-8 expression in intestinal cells. We have also demonstrated that L. amylovorus DSM 16698T protects host cells through the inhibition of NF-kB signaling. In the present study, we investigated the role of L. amylovorus DSM 16698T cell wall components in the protection against F4+ETEC infection using the intestinal Caco-2 cell line.Methods: Purified cell wall fragments (CWF) from L. amylovorus DSM 16698T were used either as such (uncoated, U-CWF) or coated with S-layer proteins (S-CWF). Differentiated Caco-2/TC7 cells on Transwell filters were infected with F4+ETEC, treated with S-CWF or U-CWF, co-treated with S-CWF or U-CWF and F4+ETEC for 2.5 h, or pre-treated with S-CWF or U-CWF for 1 h before F4+ETEC addition. Tight junction (TJ) and adherens junction (AJ) proteins were analyzed by immunofluorescence and Western blot. Membrane permeability was determined by phenol red passage. Phosphorylated p65-NF-kB was measured by Western blot.Results: We showed that both the pre-treatment with S-CWF and the co- treatment of S-CWF with the pathogen protected the cells from F4+ETEC induced TJ and AJ injury, increased membrane permeability and activation of NF-kB expression. Moreover, the U-CWF pre-treatment, but not the co-treatment with F4+ETEC, inhibited membrane damage and prevented NF-kB activation.Conclusions: The results indicate that the various components of L. amylovorus DSM 16698T cell wall may counteract the damage caused by F4+ETEC through different mechanisms. S-layer proteins are essential for maintaining membrane barrier function and for mounting an anti-inflammatory response against F4+ETEC infection. U-CWF are not able to defend the cells when they are infected with F4+ETEC but may activate protective mechanisms before pathogen infection