Abstract
The ability of Shigella to spread within and between epithelial cells is a prerequisite for causing bacillary dysentery and requires the function encoded by the virG gene on the large plasmid. The outer membrane VirG (IcsA) protein is essential for bacterial spreading by eliciting polar deposition of filamentous actin (F-actin) in the cytoplasm of epithelial cells. Recent studies have indicated that an N-terminal 80-kDa VirG portion is exposed on the bacterial cell surface and released into the external medium, while the following 37-kDa C-terminal portion is embedded in the outer membrane, although little is known about the extracellular transport of the VirG protein. In this study, we attempted to elucidate the export pathway of VirG protein across the outer membrane and found that the C-terminal 37-kDa portion, termed VirG beta-core, serves as the self-transporter for the secretion of the preceding 80-kDa portion from the periplasmic side of the outer membrane to the external side. Indeed, foreign polypeptides such as MalE or PhoA covalently linked to the N terminus of VirG beta-core were transported to the external side of the outer membrane, and it was further shown that the folding structure of the passenger polypeptide at the periplasmic side of the outer membrane interferes with its translocation. Analysis of the secondary structure of VirG beta-core predicted that the critical structural property was a beta-barrel channel consisting of amphipathic anti-parallel transmembrane beta-strands, interspersed by hairpin turns and loops. These results thus strongly suggest that the secretion of VirG protein from Shigella is similar to the export system utilized by the IgA protease of Neisseria.
Highlights
Shigella are the causative agents of shigellosis, a disease which provokes a severe, bloody diarrhea in humans and primates
Processing of VirG Polypeptide—To confirm that cleavage occurred after the putative signal sequence of VirG protein was Extracellular Transport of VirG Protein in Shigella employed in crossing the cytoplasmic membrane [5], we utilized pD10, a cloned virG plasmid [3]
The immunoblots with VRG-120 showed that a large amount of VirG protein expressed in M94 carrying pD10 distributed in the outer membrane, with some in the cytoplasm, but was not present in the cytoplasmic membrane or in the periplasm (Fig. 2) [6, 19]
Summary
(Received for publication, August 14, 1995, and in revised form, October 6, 1995). From the Department of Bacteriology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108, Japan and the ‡Laboratorie de Microbiologie et de Genetique, Universite Louis-Pasteur, URA D-1481 CNRS, Institut de Botanique, 28, rue Goethe, 67083 Strasbourg, France. The outer membrane VirG (IcsA) protein of Shigella, comprising 1102 amino acids, has been shown to be responsible for the localized deposition of filamentous actin (F-actin) trailing from one pole of invading bacterial cells and extending in a filament through the host epithelial cytoplasm [4, 5]. This observed F-actin tail associated with the infecting bacteria apparently provides a motive force, since the accumulation of F-actin fibrils results in the formation of extracellular protrusions through which bacteria penetrate adjacent cells [4].
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