| Enteropathogens, including pathogenic E. coli, Salmonella, Campylobacter jejuni enteritis, Listeria monocytogenes and Vibrio cholerae are the main causes of human/animal serious diarrhea, emesis, dehydration and hemorrhagic colitis. The first and most important step for a enteropathogen infecting the host is to adhere, colonize and invade the intestal epithelium effectively. Lipid rafts, which refers to ordered microdomains distributed in theplasma membrane, enriches of cholesterol, glycosphingolipids, glycosylphosphatidylinositol (GPI)-anchored proteins, sphingomyelin and phospholipids with unsaturated acyl chains. lipid rafts participate in large amounts of biological processes, including signal transduction, apoptosis, cholesterol transport, membrane traffic andprotein sorting. It has been reported that lipid rafts contributes viruse to invading host cells, and bacteria colonizing to cells in some references.Methyl-β-cyclodextrin (MβCD), which has high affinity to cholesterol, and could effectively deplete cholesterol from cell membrane to destroy the lipid rafts but not membrane. In this study, firstly, we analyzed the cytotoxicity of methyl-β-cyclodextrin (MβCD) to two different originated intestinal epithelial cells. Cell proliferation reagent WST-1was used to determine the viability of human colonic adenocarcinoma cell line Caco-2and porcine neonatal jejunal epithelial cell line IPEC-J2treating with different concentrations and time of MβCD. The results showed that<2mM MβCD has no significant cytotoxicity to either Caco-2or IPEC-J2cells. And in this dose of2mM,1h was the optimal time for treating cells. Then, we removed the cholesterol from membrane of two originated intestinal epithelial cell lines by using2mM MβCD, and exogenous cholesterol was added in for1h in the complement group. Cell cultured with normal media was regard as the control group. ETEC H10407, K88ac+ETEC C83902, F18ab+F107/86and S.enteritidis50336were used for adhering and invasing the Caco-2cells and IPEC-J2cells, to determine the role of cholesterol played in the enteropathogen adhering, colonizing and invading to intestinal epithelial cells. The results showed that, after removing the membrane cholesterol, the adhesion of ETEC H10407and S.enteritidis50336to Caco-2respectively decreased23%and33%as compared with control, as well as the invasion of H10407and50336to Caco-2decreased30%and25%, respectively. The adhesion of K88ac+ETEC C83902, F18ab+F107/86and S.enteritidis50336to IPEC-J2respectively decreased29%,23%and27%as compared with control, and the invasion of F18ab+F107/86and50336to IPEC-J2decreased21%and31%, respectively. The adhesion and invasion of all those enteropathogens to cholesterol-removing Caco-2and IPEC-J2cells restored to normal level after adding exogenous cholesterol for complement. Our study had revealed the function of membrane chelosterol in the enteropathogen adhering, colonizing and invading to intestinal epithelial cells, consummated the function of lipid rafts, and provided theoretical basis for further investigation of pathogenic mechanism of enteropathogens. |
