Molecular dissection of internalization of Porphyromonas gingivalis by cells using fluorescent beads coated with bacterial membrane vesicle.
Porphyromonas gingivalis is one of the causative agents of adult periodontitis, and has been reported to be internalized by nonphagocytic epithelial cells. However, the mechanism for the internalization remains unclear. In the present study, we addressed this issue using fluorescent beads coated with bacterial membrane vesicles (MVs) that retain surface components of P. gingivalis. We established an assay system in which we could easily quantify the bead internalization to cells. MVs-coated beads were internalized by HeLa cells in kinetics similar to that of living bacteria. The internalization depended on dynamin but not clathrin. The beads were internalized through the actin-mediated pathway that is controlled by phosphatidylinositol (PI) 3-kinase. The dynamics of microtubule assembly and disassembly was also required. Further, the treatment of cells with cholesterol-binding reagents significantly inhibited bead internalization, and the internalized beads were apparently colocalized with ganglioside GM1 and caveolin-1, which suggest the involvement of the lipid raft in the process. These results suggest that P. gingivalis accomplishes its internalization utilizing membrane lipid raft and cytoskeletal functions of the target cells.
Tsuda
Kayoko
K
Graduate School of Medical Science, Kanazawa University, Kanazawa, Ishikawa 920-1192, Japan.
Amano
Atsuo
A
Umebayashi
Kyohei
K
Inaba
Hiroaki
H
Nakagawa
Ichiro
I
Nakanishi
Yoshinobu
Y
Yoshimori
Tamotsu
T
D008841:Actin Cytoskeleton / Q000502:physiology; D000199:Actins / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing; D000730:Androstadienes / Q000494:pharmacology; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000502:physiology*; D051242:Caveolin 1 / Q000032:analysis; D002462:Cell Membrane / Q000502:physiology*; D002867:Chromones / Q000494:pharmacology; D002966:Clathrin / Q000502:physiology; D015638:Cytochalasin D / Q000494:pharmacology; D034285:Dynamin II / Q000502:physiology*; D004705:Endocytosis; D005372:Filipin / Q000494:pharmacology; D005456:Fluorescent Dyes; D005677:G(M1) Ganglioside / Q000032:analysis; D006367:HeLa Cells / Q000382:microbiology* / Q000502:physiology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008563:Membrane Lipids / Q000502:physiology; D021962:Membrane Microdomains / Q000187:drug effects / Q000502:physiology*; D008870:Microtubules / Q000187:drug effects / Q000502:physiology*; D009025:Morpholines / Q000494:pharmacology; D015739:Nocodazole / Q000494:pharmacology; D009761:Nystatin / Q000494:pharmacology; D017239:Paclitaxel / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins / Q000235:genetics / Q000502:physiology*; D016966:Porphyromonas gingivalis / Q000737:chemistry* / Q000648:ultrastructure; D011993:Recombinant Fusion Proteins / Q000502:physiology; D017384:Sequence Deletion; D013844:Thiazoles / Q000494:pharmacology; D053778:Thiazolidines; D014404:Tubulin / Q000378:metabolism; D000077191:Wortmannin
D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; D000730:Androstadienes; D019086:Bridged Bicyclo Compounds, Heterocyclic; D002135:Calcium-Binding Proteins; D051242:Caveolin 1; D002867:Chromones; D002966:Clathrin; D015638:Cytochalasin D; D034285:Dynamin II; D005372:Filipin; D005456:Fluorescent Dyes; D005677:G(M1) Ganglioside; D047908:Intracellular Signaling Peptides and Proteins; D008563:Membrane Lipids; D009025:Morpholines; D015739:Nocodazole; D009761:Nystatin; D017239:Paclitaxel; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D013844:Thiazoles; D053778:Thiazolidines; D014404:Tubulin; D000077191:Wortmannin; C486206:EPS15 protein, human; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C037067:latrunculin A