Membrane cholesterol plays an important role in enteropathogen adhesion and the activation of innate immunity via flagellin-TLR5 signaling.

Lipid rafts are cholesterol- and sphingolipid-rich ordered microdomains distributed in the plasma membrane that participates in mammalian signal transduction pathways. To determine the role of lipid rafts in mediating interactions between enteropathogens and intestinal epithelial cells, membrane cholesterol was depleted from Caco-2 and IPEC-J2 cells using methyl-ß-cyclodextrin. Cholesterol depletion significantly reduced Escherichia coli and Salmonella enteritidis adhesion and invasion into intestinal epithelial cells. Complementation with exogenous cholesterol restored bacterial adhesion to basal levels. We also evaluated the role of lipid rafts in the activation of Toll-like receptor 5 signaling by bacterial flagellin. Depleting membrane cholesterol reduced the ability of purified recombinant E. coli flagellin to activate TLR5 signaling in intestinal cells. These data suggest that both membrane cholesterol and lipid rafts play important roles in enteropathogen adhesion and contribute to the activation of innate immunity via flagellin-TLR5 signaling.

Both flagella and F4 fimbriae from F4ac+ enterotoxigenic Escherichia coli contribute to attachment to IPEC-J2 cells in vitro.

The role of flagella in the pathogenesis of F4ac+ Enterotoxigenic Escherichia coli (ETEC) mediated neonatal and post-weaning diarrhea (PWD) is not currently understood. We targeted the reference C83902 ETEC strain (O8:H19:F4ac+ LT+ STa+ STb+), to construct isogenic mutants in the fliC (encoding the major flagellin protein), motA (encoding the flagella motor), and faeG (encoding the major subunit of F4 fimbriae) genes. Both the ΔfliC and ΔfaeG mutants had a reduced ability to adhere to porcine intestinal epithelial IPEC-J2 cells. F4 fimbriae expression was significantly down-regulated after deleting fliC, which revealed that co-regulation exists between flagella and F4 fimbriae. However, there was no difference in adhesion between the ΔmotA mutant and its parent strain. These data demonstrate that both flagella and F4 fimbriae are required for efficient F4ac+ ETEC adhesion in vitro.

Contribution of flagellin subunit FliC to piglet epithelial cells invasion by F18ab E. coli.

Flagellar structures contribute to the virulence of multiple gastrointestinal pathogens either as the effectors of motility, as adhesins, or as a secretion apparatus for virulence factors. Escherichia coli F18ab variant strains are associated with edema disease (ED) in pig industries worldwide. These strains use flagella to increase the efficiency of epithelial cell invasion. In this study, we aimed to elucidate the mechanism by which flagella contribute to F18ab E. coli invasion. To explore the role of flagella in the invasion process, we performed invasion assays with either flagellated and motile, flagellated but non-motile, or non-flagellated non-motile bacteria. We observed that flagellated but non-motile bacteria invade piglet epithelial cells even more efficiently than the parent wild-type (WT) strain in vitro. By contrast, the non-flagellated bacteria have significantly reduced invasion as compared with the parent strain. These results demonstrate that flagella function mainly as adhesins to enhance the ability of F18ab E. coli to target piglet epithelial cells.