A fragment of the Salmonella typhimurium virulence plasmid containing the rck locus, when cloned in the recombinant cosmid pADE016, was shown previously to confer high-level complement resistance on both rough and smooth Escherichia coli, SalmoneUa minnesota, and S. typhimurium and was associated with the production of an outer membrane protein. The outer membrane proteins (OMPs) of gram-negative bacteria provide specialized functions enabling the organisms to interact with the environment, promoting survival. For pathogenic bacteria, a number of specific proteins required for virulence are located in the outer membrane. Gram-negative organisms causing invasive disease must resist the bactericidal action of complement present in normal human serum (22,46). The outer membrane is the initial site of interaction between complement and the bacterial cell, and both the structure of the lipopolysaccharide (LPS) and the presence of specific OMPs have been shown to be critical determinants of bacterial resistance to complement (22). In Salmonella spp., the number and length of the LPS side chains (O antigen) play a major role in complement resistance, since long-chain LPS leads to complement activation at a distance from the cell surface and precludes insertion of the membrane attack complex in the outer membrane (23, 24).Several genes encoded by Salmonella virulence plasmids have been implicated in serum resistance. These plasmids, 50 to 100 kb in size, are present in several invasive Salmonella serotypes, and all appear to contain a common 8.4-kb region required for progressive infection of the reticuloendothelial system, leading to septicemia and death in experimental animals (14). Genetic loci specifying serum resistance map outside this core virulence region and include a traT allele (42), a determinant affecting LPS structure (26), and the rsk region, which enhances the ability of smooth S. typhimurium to grow in serum when the virulence plasmid is integrated in the chromosome (50, 51).A distinct complement resistance locus was cloned from the S. typhimurium virulence plasmid by J. Hackett and colleagues (15). These workers showed that pADE016, containing a 2.4-kb ClaI-PstI fragment in a multicopy cosmid vector, confers high-level complement resistance to a serumsensitive Escherichia coli K-12 strain and to the rough, plasmid-cured S. typhimurium derivative of the wild-type parent strain (15). In addition, they found that expression of the serum resistance phenotype by pADE016 was associated with the production of an OMP. We have recently investigated the mechanisms by which this locus (designated rck, for resistance to complement killing) mediates serum resistance in gram-negative bacteria (17). These studies suggest that the rck gene product prevents complement-mediated bacteriolysis by inhibiting effective insertion of the terminal C5b-9 membrane attack complex into the gram-negative outer membrane, a mechanism of complement resistance similar to that noted in serum-resistant strains of Neisseria gonorrhoeae (2...

We find that pADEO16, a recombinant cosmid carrying the rck gene of the Salmonella typhimurium virulence plasmid, when cloned into either rough or smooth Escherichia coli and Salmonella strains, confers high level resistance to the bactericidal activity of pooled normal human serum. The rek gene encodes a 17-kD outer membrane protein that is homologous to a family of virulence-associated outer membrane proteins, including pagC and Ail. Complement depletion, C3 and C5 binding, and membrane-bound C3 cleavage products are similar in strains with and without rck. Although a large difference in C9 binding was not seen, trypsin cleaved 55.7% of bound '"I-C9 counts from rough S. typhimunium with pADEO16, whereas only 26.4% were released from S. typhimurium with K2011, containing a mutation in rck. The majority ofC9 extracted from rck strain membranes sediments at a lower molecular weight than in strains without rek, suggesting less C9 polymerization. Furthermore, SDS-PAGE analysis of gradient peak fractions indicated that the slower sedimenting C9-containing complexes in rck strains did not contain polymerized C9 typical of the tubular membrane attack complex. These results indicate that complement resistance mediated by Rck is associated with a failure to form fully polymerized tubular membrane attack complexes. (J. Clin. Invest. 1992.90:953-964.)

BALB/c mice were infected orally with Salmonella dublin strains Lane and LD842, an isogenic derivative of the former that is avirulent because it was cured of its 80-kilobase-pair virulence plasmid pSDL2. Both strains colonized the intestine and invaded Peyer's patches with equivalent efficiency. However, the parent strain multiplied in mesenteric nodes and in the spleen; the plasmid-cured strain reached these organs, but the infection was low grade and remained relatively static until the mice developed active immunity and cured themselves. The histological response to plasmid-free strain LD842 was mononuclear, whereas the virulent parent strain produced abscesses. Sublethal irradiation of the mice before infection with strain LD842 prevented the mononuclear infiltrate in the liver and made the animals susceptible. Thus the virulence plasmid of S. dublin allows multiplication within the reticuloendothelial system and does not have any effect on the organism's ability to colonize the intestine or invade Peyer's patches.

Virulence-associated phenotypes of an outer membrane protein gene family of members of the family Enterobacteriaceae were compared by means of pBR322 constructs transformed into Escherichia coli HB101. rck (Salmonella typhimurium) and ail (Yersinia enterocolitica) promote serum resistance and eukaryotic cell invasion, properties not shared by other members of the gene family, pagC, ompX, and lom.