Functional analysis of Shigella VirG domains essential for interaction with vinculin and actin-based motility.
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 (alpha-domain) is exposed on the bacterial surface, while the following C-terminal 37-kDa portion (beta-core) is embedded in the outer membrane. Here, we report that the surface exposed alpha-domain of VirG possesses two distinct functional domains; one is the N-terminal two-thirds portion of the alpha-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 alpha-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 alpha-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 alpha-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 alpha-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.
Suzuki
T
T
Department of Bacteriology, Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108, Japan.
Saga
S
S
Sasakawa
C
C
D000199:Actins / Q000378:metabolism*; D000818:Animals; D001426:Bacterial Proteins; D001483:Base Sequence; D002645:Chickens; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004405:Dysentery, Bacillary / Q000378:metabolism; D019085:Fluorescent Antibody Technique, Direct; D016368:Frameshift Mutation; D005982:Glutathione Transferase / Q000378:metabolism; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012763:Shigella flexneri / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000378:metabolism*; D016596:Vinculin / Q000378:metabolism*; D014982:Xenopus laevis
D000199:Actins; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C058483:virG protein, Shigella flexneri; D016596:Vinculin; D005982:Glutathione Transferase