Functional Analysis of α5β1 Integrin and Lipid Rafts in Invasion of Epithelial Cells by Porphyromonas gingivalis using Fluorescent Beads Coated with Bacterial Membrane Vesicles

摘要

References(30) Cited-By(23) Porphyromonas gingivalis, a periodontal pathogen, was previously suggested to exploit α5β1 integrin and lipid rafts to invade host cells. However, it is unknown if the functional roles of these host components are distinct from one another during bacterial invasion. In the present study, we analyzed the mechanisms underlying P. gingivalis invasion, using fluorescent beads coated with bacterial membrane vesicles (MV beads). Cholesterol depletion reagents including methyl-β-cyclodextrin (MβCD) drastically inhibited the entry of MV beads into epithelial cells, while they were less effective on bead adhesion to the cells. Bead entry was also abolished in CHO cells deficient in sphingolipids, components of lipid rafts, whereas adhesion was negligibly influenced. Following MβCD treatment, downstream events leading to actin polymerization were abolished; however, α5β1 integrin was recruited to beads attached to the cell surface. Dominant-negative Rho GTPase Rac1 abolished cellular engulfment of the beads, whereas dominant-negative Cdc42 did not. Following cellular interaction with the beads, Rac1 was found to be translocated to the lipid rafts fraction, which was inhibited by MβCD. These results suggest that α5β1 integrin, independent of lipid rafts, promotes P. gingivalis adhesion to epithelial cells, while the subsequent uptake process requires lipid raft components for actin organization, with Rho GTPase Rac1.