Functional Analysis of α5β1 Integrin and Lipid Rafts in Invasion of Epithelial Cells by Porphyromonas gingivalis using Fluorescent Beads Coated with Bacterial Membrane Vesicles
Functional Analysis of α5β1 Integrin and Lipid Rafts in Invasion of Epithelial Cells by Porphyromonas gingivalis using Fluorescent Beads Coated with Bacterial Membrane Vesicles
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DOI：
10.1247/csf.08012
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发表时间：
2008-01-01
影响因子：
1.5
通讯作者：
Amano, Atsuo
中科院分区：
文献类型：
--
作者：
Tsuda, Kayoko;Furuta, Nobumichi;Amano, Atsuo
以前认为牙龈卟啉单胞菌是一种牙周病原体，利用α 5 β 1整合素和脂筏侵入宿主细胞。然而，目前尚不清楚这些宿主成分在细菌入侵过程中的功能作用是否彼此不同。在本研究中，我们利用涂有细菌膜囊泡（MV珠）的荧光珠分析了牙龈假单胞菌侵袭的机制。包括甲基- β -环糊精（M β - CD）在内的胆固醇消耗试剂可显著抑制MV珠进入上皮细胞，但对珠粘附细胞的效果较差。在缺乏鞘脂（脂筏的组成部分）的CHO细胞中，珠粒的进入也被取消，而粘附受到的影响可以忽略不计。M β CD处理后，导致肌动蛋白聚合的下游事件被消除；然而，α 5 β 1整合素被招募到附着在细胞表面的小珠上。显性阴性Rho GTPase Rac1消除了细胞对小珠的吞噬，而显性阴性Cdc42则没有。在细胞与小珠相互作用后，Rac1被发现转移到脂筏部分，这被M β CD抑制。这些结果表明，独立于脂筏的α 5 β 1整合素促进牙龈卟噬菌与上皮细胞的粘附，而随后的摄取过程需要脂筏成分来组织肌动蛋白，即Rho GTPase Rac1。
Porphyromonas gingivalis, a periodontal pathogen, was previously suggested to exploit alpha 5 beta 1 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 (M beta CD) 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 M beta CD treatment, downstream events leading to actin polymerization were abolished; however, alpha 5 beta 1 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 M beta CD. These results suggest that alpha 5 beta 1 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.