Abstract

BackgroundThe 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.MethodsPurified 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.ResultsWe 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.ConclusionsThe 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.

Highlights

  • The role of Lactobacillus cell wall components in the protection against pathogen infection in the gut is still largely unexplored

  • We have shown that L. amylovorus DSM 16698T prevented the F4+ETECinduced disruption of Tight junction (TJ) and cytoskeleton proteins in intestinal cells through IL-10-mediated signaling involving IL-8 down-regulation [13]

  • Different protection of membrane barrier integrity by L. amylovorus DSM 16698T S-cell wall fragments (CWF) and U-CWF To verify whether L. amylovorus S-layer protein (SLP) and/or U-CFW were crucial for protection against the barrier damage induced by F4+enteroxigenic Escherichia coli (ETEC), we analyzed the major TJ and adherens junction (AJ)

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Summary

Introduction

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 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. Lactobacillus amylovorus DSM 16698T, isolated from an unweaned piglet, is abundant in piglets [12] This strain has been shown to protect intestinal cells from membrane damage and inflammation induced by enteroxigenic Escherichia coli (ETEC) carrying F4 fimbriae [3, 13, 14]. F4+E. coli strains are major causative agents of enteric infections, diarrhoea and mortality in piglets [15]

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