权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Study of actin-based cell-to-cell spreading of Shigella in infected epithelial cells

感染上皮细胞中志贺氏菌基于肌动蛋白的细胞间传播研究

基本信息

批准号：
12670250
负责人：
SUZUKI Toshihiko
金额：
$ 2.05万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670250/
关键词：
Shigella actin N-WASP profilin epitherial cells macrophages WASP 低分子量GTPase Arp2 3 complex

项目摘要

(1) Shigella, the causative agent of bacillary dysentery, are capable of directing its movement within host cells by exploiting actin dynamics. The VirG protein expressed at one pole of the bacterium can recruit neural Wiskott-Aldrich syndrome protein (N-WASP), a downstream effector of Cdc42. We investigated the role of Cdc42 in actin-based movement of Shigella. The several experiments using microinjection, in vitro motility assay in Xenopus egg extracts, and pyrene actin assay revealed that Cdc42 activity is involved in initiating actin nucleation mediated by VirG-N-WASP-actin-related protein (Arp) 2/3 complex formed on intracellular Shigella.(2) We showed that profilin I is essential to the rapid movement of Shigella in infected cells. Two mutants of profilin which failed to bind G-actin, or to bind with poly-L-proline, did not support fast moving of Shigella. The results indicate that profilin I associated with N-WASP is an essential host factor for sustaining efficient intra- and intercellular spreading of Shigella.(3) Since all of the WASP family proteins including N-WASP induce actin polymerization by recruiting Arp2/3 complex, we investigated their involvement in Shigella motility. We showed that VirG binds to N-WASP but not the other WASP family proteins. We observed that in hemtopoietic cells such as macrophages, polymorphonuclear leukocytes (PMNs) and platelets, WASP was predominantly expressed, while the expression of N-WASP was greatly suppressed. Indeed, unlike Listeria, Shigella was unable to move in macrophages at all, though the movement was restored as N-WASP was ectopically expressed. Thus, our findings demonstrate that N-WASP is a specific ligand of VirG, which determines the host cell type allowing actin-based spreading of Shigella.

(1)志贺氏菌是细菌性痢疾的病原菌，它能够利用肌动蛋白动力学来指导其在宿主细胞内的运动。在细菌的一极表达的VirG蛋白可以募集神经Wiskott-Aldrich综合征蛋白（N-WASP），Cdc 42的下游效应子。我们研究了Cdc 42在志贺菌肌动蛋白运动中的作用。通过显微注射、非洲爪蟾卵提取物体外运动试验和芘肌动蛋白试验等实验，发现Cdc 42参与了胞内志贺菌VirG-N-WASP-肌动蛋白相关蛋白（阿普）2/3复合物介导的肌动蛋白成核。(2)我们发现profilin I对志贺氏菌在感染细胞中的快速移动至关重要。两个不能与G-肌动蛋白结合或不能与聚-L-脯氨酸结合的profilin突变体不支持志贺菌的快速移动。结果表明，与N-WASP相关的profilin I是维持志贺氏菌有效的细胞内和细胞间传播的重要宿主因子。(3)由于所有的WASP家族蛋白，包括N-WASP诱导肌动蛋白聚合招募Arp 2/3复合物，我们研究了它们参与志贺氏菌运动。我们发现VirG与N-WASP结合，但不与其他WASP家族蛋白结合。我们观察到，在造血细胞如巨噬细胞、多形核白细胞（PMNs）和血小板中，WASP主要表达，而N-WASP的表达被极大地抑制。事实上，与李斯特菌不同，志贺氏菌根本无法在巨噬细胞中移动，尽管随着N-WASP异位表达，运动得以恢复。因此，我们的研究结果表明，N-WASP是一个特定的配体的VirG，这决定了宿主细胞类型，允许肌动蛋白为基础的传播志贺氏菌。