CLEAVAGE OF SHIGELLA SURFACE PROTEIN VIRG OCCURS AT A SPECIFIC SITE, BUT THE SECRETION IS NOT ESSENTIAL FOR INTRACELLULAR SPREADING

The large plasmid-encoded outer membrane protein VirG (IcsA) of Shigel la flexneri is essential for bacterial spreading by eliciting polar de position of filamentous actin (F-actin) in the cytoplasm of epithelial cells'. Recent studies have indicated that VirG is located at one pol e an the surface of the bacterium and secreted into the culture supern atant and that in host cells it is localized along the length of the F -actin tail. The roles of these VirG phenotypes in bacterial spreading still remain to be elucidated. In this study, we examined the surface -exposed portion of the VirG protein by limited trypsin digestion of S . flexneri YSH6000 and determined the sites for VirG processing during secretion into the culture supernatant. Our results indicated that th e 85-kDa amino-terminal portion of VirG is located on the external sid e of the outer membrane, while the 37-kDa carboxy-terminal portion is embedded in it, The VirG cleavage required for release of the 85-kDa p rotein into the culture supernatant occurred at the Arg-Arg bond at po sitions 758 to 759. VirG-specific cleavage was observed iri Shigella s pecies and enteroinvasive Escherichia coli, which requires an as yet u nidentified protease activity governed by the virB gene on the large p lasmid. To investigate whether the VirG-specific cleavage occurring in extracellular and intracellular bacteria is essential for VirG functi on in bacterial :spreading, the Arg-Arg cleavage site was modified to an Arg-Asp or Asp-Asp bond. The virG mutants thus constructed were cap able of unipolar deposition of VirG on the bacterial surface but were unable to cleave VirG under in vitro or in vivo conditions. However, t hese mutants were still capable of eliciting aggregation of F-actin at one pole, spreading into adjacent cells, and giving rise to a positiv e Sereny test. Therefore, the ability to cleave and secrete VirG in Sh igella species is not a prerequisite for intracellular spreading.