Molecular dissection of internalization of Porphyromonas gingivalis by cells using fluorescent beads coated with bacterial membrane vesicle.
Molecular dissection of internalization of Porphyromonas gingivalis by cells using fluorescent beads coated with bacterial membrane vesicle.
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DOI：
10.1247/csf.30.81
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发表时间：
2005
影响因子：
1.5
通讯作者：
Kayoko Tsuda;A. Amano;K. Umebayashi;H. Inaba;I. Nakagawa;Y. Nakanishi;T. Yoshimori
中科院分区：
文献类型：
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作者：
Kayoko Tsuda;A. Amano;K. Umebayashi;H. Inaba;I. Nakagawa;Y. Nakanishi;T. Yoshimori
牙龈卟啉单胞菌是成人牙周炎的病原体之一，据报道可被非吞噬性上皮细胞内化。然而，内化的机制仍不清楚。在本研究中，我们使用涂有细菌膜囊泡（MV）的荧光珠来解决这个问题，这些膜囊泡保留牙龈卟啉单胞菌的表面成分。我们建立了一个测定系统，其中我们可以很容易地量化珠内化到细胞。MVs包被的珠粒被HeLa细胞内化的动力学类似于活细菌。内化依赖于发动蛋白而不是网格蛋白。珠粒通过由磷脂酰肌醇（PI）3-激酶控制的肌动蛋白介导的途径内化。还需要微管组装和拆卸的动力学。此外，用胆固醇结合试剂处理细胞显著抑制珠粒内化，并且内化的珠粒明显与神经节苷脂GM 1和小窝蛋白-1共定位，这表明脂质筏参与了该过程。这些结果表明，牙龈卟啉单胞菌利用靶细胞的膜脂筏和细胞骨架功能完成其内化。
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.