Functional analysis of alpha5beta1 integrin and lipid rafts in invasion of epithelial cells by Porphyromonas gingivalis using fluorescent beads coated with bacterial membrane vesicles.
Porphyromonas gingivalis, a periodontal pathogen, was previously suggested to exploit alpha5beta1 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-beta-cyclodextrin (MbetaCD) 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 MbetaCD treatment, downstream events leading to actin polymerization were abolished; however, alpha5beta1 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 MbetaCD. These results suggest that alpha5beta1 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.
Tsuda
Kayoko
K
Department of Oral Frontier Biology, Osaka University Graduate School of Dentistry, Suita-Osaka, Japan.
Furuta
Nobumichi
N
Inaba
Hiroaki
H
Kawai
Shinji
S
Hanada
Kentaro
K
Yoshimori
Tamotsu
T
Amano
Atsuo
A
D000199:Actins / Q000187:drug effects / Q000378:metabolism; D001422:Bacterial Adhesion / Q000502:physiology*; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism; D004847:Epithelial Cells / Q000502:physiology*; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D039081:Integrin alpha5beta1 / Q000378:metabolism*; D021962:Membrane Microdomains / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008863:Microspheres; D016966:Porphyromonas gingivalis / Q000166:cytology / Q000502:physiology*; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D013107:Sphingolipids / Q000378:metabolism; D013499:Surface Properties; D047392:beta-Cyclodextrins / Q000494:pharmacology; D020764:cdc42 GTP-Binding Protein / Q000187:drug effects / Q000502:physiology; D020830:rac1 GTP-Binding Protein / Q000187:drug effects / Q000502:physiology
D000199:Actins; D005456:Fluorescent Dyes; D039081:Integrin alpha5beta1; D013107:Sphingolipids; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D002784:Cholesterol; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein