Volume 271, Number 36, Issue of September 6, 1996 pp. 21878-21885
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Functional Analysis of Shigella VirG Domains Essential for Interaction with Vinculin and Actin-based Motility

(Received for publication, April 30, 1996, and in revised form, June 25, 1996)

Toshihiko Suzuki , Shinsuke Saga and Chihiro Sasakawa

From the Department of Bacteriology, Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108, Japan and the  Department of Morphology, Institute for Developmental Research, Aichi Human Service Center, 7-1-3-8, Kamiya-cho, Kasugai, Aichi 480-03, Japan

The VirG (IcsA) protein of Shigella is required for recruitment of host actin filament (F-actin) by intracellularly motile bacteria. An N-terminal 80-kDa VirG portion (-domain) is exposed on the bacterial surface, while the following C-terminal 37-kDa portion (-core) is embedded in the outer membrane. Here, we report that the surface exposed -domain of VirG possesses two distinct functional domains; one is the N-terminal two-thirds portion of the -domain which is required for eliciting F-actin assembly on the bacteria in infected cells, and the other one is the rest of the C-terminal portion of the VirG -domain, which is essential for the asymmetric distribution of VirG on the bacterial surface. Furthermore, we found that vinculin, an actin-binding cytoskeletal protein, accumulates on the surface of bacteria expressing VirG in infected cells, and that the distribution of vinculin coincided with the distribution of VirG and assembled F-actin. The vinculin accumulation depended on the expression of the -domain VirG portion required for F-actin assembly, but the recruitment of vinculin on Shigella appeared prior to the appearance of F-actin in the infected cells. Analysis of proteins interacting with VirG using Xenopus laevis eggs extracts revealed that vinculin was a protein that bound to the -domain portion. This was further confirmed using purified chicken gizzard vinculin, in that the 95-kDa vinculin head part, but not the 30-kDa tail part, directly bound to the -domain portion. These results suggest a possible role for vinculin in recruitment of F-actin to the VirG moiety exposed on Shigella in infected mammalian cells.

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