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

doi:10.1074/jbc.271.36.21878

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

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

^§ To whom correspondence should be addressed:
Dept. of Bacteriology, Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108, Japan.
Tel.: 81-3-5449-5252; Fax: 81-3-5449-5405 E-mail: sasakawa{at}ims.u-tokyo.ac.jp.

Abstract

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 (α-domain) is exposed on the bacterial surface, while the following C-terminal 37-kDa portion (β-core) is embedded in the outer membrane. Here, we report that the surface exposed α-domain of VirG possesses two distinct functional domains; one is the N-terminal two-thirds portion of the α-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 α-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 α-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 α-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 α-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.

Footnotes

↵* This work was supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists and a Grant-in-aid for Scientific Research from the Japanese Ministry of Education, Science, Sports and Culture. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

PCR

polymerase chain reaction

bp

base pair(s)

GST

glutathione S-transferase

FITC

fluorescein-isothiocyanate

PAGE

polyacrylamide gel electrophoresis.
↵²T. Suzuki, unpublished results.
- Received April 30, 1996.
- Revision received June 25, 1996.