title: Salmonella typhimurium SifA effector protein requires its membrane-anchoring C-terminal hexapeptide for its biological function.
abstract: SifA is a Salmonella typhimurium effector protein that is translocated across the membrane of the Salmonella-containing vacuole by the Salmonella pathogenicity island 2-encoded type III secretion system. SifA is necessary for the formation of Salmonella-induced filaments and for the maintenance of the vacuolar membrane enclosing the pathogen. We have investigated the role of the C-terminal hexapeptide of SifA as a potential site for membrane anchoring. An S. typhimurium strain carrying a deletion of the sequence encoding this hexapeptide (sifA Delta 6) was found to be attenuated for systemic virulence in mice. In mouse macrophages, sifA Delta 6 mutant bacteria displayed a reduced association with vacuolar markers, similar to that of sifA null mutant bacteria, and exhibited a dramatic replication defect. Expression of SifA in epithelial cells results in the mobilization of lysosomal glycoproteins in large vesicular structures and Sif-like tubules. This process requires the presence of the C-terminal hexapeptide domain of SifA. Ectopic expression of truncated or mutated versions of SifA affecting the C-terminal hexapeptide revealed a strong correlation between the membrane binding capability and the biological activity of the protein. Finally, the eleven C-terminal residues of SifA are shown to be sufficient to target the Aequorea green fluorescent protein to membranes. Altogether, our results indicate that membrane anchoring of SifA requires its C-terminal hexapeptide domain, which is important for the biological function of this bacterial effector.
authors.affiliation: Centre d'Immunologie de Marseille-Luminy, Case 906, 13288 Marseille Cedex 9, France.
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Boucrot
authors.forename: Carmen R
authors.initials: CR
authors.lastname: Beuzón
authors.forename: David W
authors.initials: DW
authors.lastname: Holden
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Gorvel
authors.forename: Stéphane
authors.initials: S
authors.lastname: Méresse
mesh_terms: D000818:Animals; D001426:Bacterial Proteins; D001665:Binding Sites; D002460:Cell Line; D002470:Cell Survival; D017353:Gene Deletion; D006023:Glycoproteins / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012486:Salmonella typhimurium / Q000378:metabolism*; D014162:Transfection
chemical_list: D001426:Bacterial Proteins; D006023:Glycoproteins; D008164:Luminescent Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; C421862:SifA protein, Salmonella; D049452:Green Fluorescent Proteins