Identification of a domain in Rck, a product of the Salmonella typhimurium virulence plasmid, required for both serum resistance and cell invasion.
Rck is encoded on the Salmonella typhimurium virulence plasmid and is a member of a family of related 17- to 19-kDa outer membrane proteins of Enterobacteriaceae, including Ail (Yersinia enterocolitica) and PagC (S. typhimurium). Structural models for these proteins predict eight membrane-spanning domains alternating with hydrophilic inner and outer loops. When expressed in Escherichia coli, Rck and Ail, but not PagC, confer high-level resistance to the bactericidal activity of complement as well as the ability to adhere to and invade mammalian cell lines. To identify functional domains of Rck, we made and screened random mutations in Rck for decreased bioactivity. We found that a single amino acid substitution (glycine to aspartic acid) in the putative third outer loop greatly reduced Rck-mediated serum resistance and eukaryotic cell invasion. We then constructed two chimeric proteins between Rck and PagC. Substitution of the C-terminal half of Rck with the corresponding PagC fragment containing both the third and the fourth outer loops abolishes the Rck-mediated serum resistance and invasion phenotypes. Substitution of Rck with a smaller C-terminal portion of PagC containing the fourth outer loop did not affect the invasive phenotype or serum resistance. These data reveal that the third putative outer membrane loop region is important for the virulence-associated properties of the Rck protein and suggest a similarity between the mechanism of serum resistance and epithelial cell invasion involving the same domain of Rck.
Cirillo
D M
DM
Department of Medicine, University of California, La Jolla 92032, USA.
Heffernan
E J
EJ
Wu
L
L
Harwood
J
J
Fierer
J
J
Guiney
D G
DG
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000502:physiology; D001483:Base Sequence; D001770:Blood Bactericidal Activity; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D017354:Point Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012486:Salmonella typhimurium / Q000276:immunology* / Q000472:pathogenicity; D013329:Structure-Activity Relationship; D014774:Virulence
D001425:Bacterial Outer Membrane Proteins; D011993:Recombinant Fusion Proteins