Abstract

Bacteria release flagellin that elicits innate responses via Toll-like receptor 5 (TLR5). Here, we investigated the fate of apically administrated full length flagellin from virulent and avirulent bacteria, along with truncated recombinant flagellin proteins in intestinal epithelial cells and cellular responses. Flagellin was internalized by intestinal epithelial cell (IEC) monolayers of IEC-18. Additionally, apically applied flagellin was internalized by polarized human Caco-2BBe and T-84 cells in a TLR5 dependent mechanism. More, flagellin exposure did not affect the integrity of intestinal monolayers. With immunofluorescent staining, internalized flagellin was detected in both early endosomes as well as lysosomes. We found that apical exposure of polarized Caco-2BBe and T-84 to flagellin from purified Salmonella, Escherichia coli O83:H1 (isolate from Crohn’s lesion) or avirulent E. coli K12 induced comparable levels of basolateral IL-8 secretion. A recombinant protein representing the conserved amino (N) and carboxyl (C) domains (D) of the flagellin protein (ND1/2ECHCD2/1) induced IL-8 secretion from IEC similar to levels elicited by full-length flagellins. However, a recombinant flagellin protein containing only the D3 hypervariable region elicited no IL-8 secretion in both cell lines compared to un-stimulated controls. Silencing or blocking TLR5 in Caco-2BBe cells resulted in a lack of flagellin internalization and decreased IL-8 secretion. Furthermore, apical exposure to flagellin stimulated transepithelial migration of neutrophils and dendritic cells. The novel findings in this study show that luminal-applied flagellin is internalized by normal IEC via TLR5 and co-localizes to endosomal and lysosomal compartments where it is likely degraded as flagellin was not detected on the basolateral side of IEC cultures.

Highlights

  • Various species of Gram-negative [1] and some Gram-positive [2] bacteria have thousands of motile hair-like structure called flagella extending from their outer membranes

  • Internalization of flagellin by polarized intestinal epithelial cell (IEC) In a pilot experiment, we examined whether flagellin was taken up by IEC

  • To study whether internalization of flagellin is through a transor para-cellular pathway, we first examined whether the AP exposure of human IEC to flagellin would affect the barrier integrity of the monolayer

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Summary

Introduction

Various species of Gram-negative [1] and some Gram-positive [2] bacteria have thousands of motile hair-like structure called flagella extending from their outer membranes. Flagella structures enable the bacteria to move through its aqueous environment, and attach to and invade host cells [I, 3–5]. Flagellin is the primary protein component that forms the flagellar structure. The flagellin protein folds to form a hairpin arrangement that has been divided into three domains [6,7,8,9]. The folding of the flagellin protein is such that domains 1 (D1) and 2 (D2) are discontinuous and are formed when residues in the amino terminus (N) and carboxyl terminus (C) are juxtaposed by the hairpin structure [6,7,8]. The linear arrangement of the domains is amino domain (ND), ND2, D3, carboxyl domain (CD) and CD1

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