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 Pag C (S. typhimurium). Structural models for these proteins predict eight membrane-spanning domains alternating with hydrophilic inner and oute r loops. When expressed in Escherichia coli, Rck and Ail, but not PagC , confer high-level resistance to the bactericidal activity of complem ent as well as the ability to adhere to and invade mammalian cell line s. To identify functional domains of Rck, we made and screened random mutations in Rck for decreased bioactivity. We found that a single ami no acid substitution (glycine to aspartic acid) in the putative third outer loop greatly reduced Rck-mediated serum resistance and eukaryoti c cell invasion. We then constructed two chimeric proteins between Rck and PagC. Substitution of the C-terminal half of Rck with the corresp onding PagC fragment containing both the third and the fourth outer lo ops abolishes the Rck-mediated serum resistance and invasion phenotype s. Substitution of Rck with a smaller C-terminal portion of PagC conta ining the fourth outer loop did not affect the invasive phenotype or s erum resistance. These data reveal that the third putative outer membr ane loop region is important for the virulence-associated properties o f the Rck protein and suggest a similarity between the mechanism of se rum resistance and epithelial cell invasion involving the same domain of Rck.