The R6-5 plasmid-specified outer membrane protein, TraT protein, has previously been shown to mediate resistance to bacterial killing by serum. Colony hybridization with a 700 bp DNA fragment carrying most of the traT gene was used to examine the prevalence of traT in Gramnegative bacteria, particularly strains of Escherichia coli, isolated from clinical specimens. traT was found in isolates of E. coli, Salmonella, Shigella and Klebsiella, but not in Pseudomonas, Aeromonas or Plesiomonas, nor in the few isolates of Enterobacter, Proteus, Acinetobacter, Citrobacter, Serratia or Yersinia that were examined. It was detected in a significantly higher proportion of the E. coli strains isolated from the blood of patients with bacteraemia/septicaemia or from faeces of patients with enteric infections (5670%) than in that of strains isolated from normal faeces (20-40%). The incidence of traT in strains isolated from cases of urinary tract infections was variable. traT was found to be frequently associated with production of the K1 capsule and with the carriage of ColV plasmids, but not with the carriage of R plasmids, nor with serum resistance or the production of haemolysin.
Isogenic Escherichia coli strains, differing in their expression of K1 antigen and ColV plasmid, were studied for their ability to produce disease. Newborn rats were used to test the ability of these strains to colonize the intestine and to produce bacteremia and meningitis; adult rats were used to test their ability to produce urinary tract infection. Colonization of intestine and bladder by K1+ ColV+ E. coli was associated with rapid induction of bacteremia and higher mortalities compared with colonization with K1+ ColV- strains. These findings suggest that the ColV plasmid could play a role in the pathogenesis of human infections.
The presence of the outer membrane protein TraT, encoded by plasmid R6-5, reduces the sensitivity of Escherichia coli cells to phagocytosis by macrophages. This effect is independent of the bacterial capsule and is more evident in the presence of adsorbed normal human serum. The property of inhibiting phagocytosis is specifically abolished by anti-TraT protein antiserum and anti-TraT immunoglobulin G but not by Fab fragments. These results indicate that the TraT protein is a passive inhibitor of phagocytosis. Inhibition of phagocytosis is produced because the TraT protein antagonizes opsonization by complement, such that C3 deposition is reduced and altered in distribution.Phagocytosis and the bacteriostatic and bactericidal properties of the serum constitute the first line of defense against invasive bacterial infections. The antibacterial activity of blood is effective in eliminating most normally encountered transient bacteremias and is the result of a constellation of specific (e.g., antibodies) and nonspecific (e.g., complement, iron-binding proteins) factors (7,16).The ability of bacteria to avoid host defenses and become invasive rests largely in cell surface components which are important in the preliminary steps of the infective process and crucial in determining its outcome (35). The role of surface components such as capsules, peptidoglycan, proteins, pili, and 0-antigens in increasing the virulence of bacteria is well documented (34). Although most of these are encoded by genetic determinants located on the chromosome, recent reports testify to the importance of plasmidencoded gene product modifications to the cell surface, which alter bacterial virulence (13-15, 31, 33, 34).The Escherichia coli plasmid R6-5 carries a gene specifying resistance to the antibacterial activity of serum (31). This determinant, which has been identified as traT, is one of the genes of the tra operon, the genetic unit conferring conjugative ability upon the bacterial cell (1, 31). The traT gene directs the synthesis of a highly exposed outer membrane protein, which mediates resistance to serum. Since phagocytosis, the other main line of defense against invasive bacterial infections, involves complement, we explored the possibility that the TraT protein may confer upon bacteria the ability to resist phagocytosis. In this communication, we show that this is the case and that the protein interferes mainly with opsonization by the alternative pathway of complement by restricting and altering the pattern of C3 deposition. MATERIALS AND METHODSBacterial strains and plasmids. Bacterial strains and plasmids are listed in Table 1. Plasmid DNA, isolated by standard procedures, was introduced into bacterial strains by transformation or conjugation (45). Strains were stored at -20°C in 60% glycerol. Molecular cloning procedures. Restriction endonuclease * Corresponding author.cleavage of plasmid DNA, ligation reactions, transformations, screening, and analysis by agarose gel electrophoresis were performed as described (45,46). Media. ...
To study the relevance of the ColV plasmid and the capsular K1 antigen in the pathogenicity of Escherichia coli , isogenic strains that differ only in these characteristics were constructed. Studies with these variants demonstrated that the presence of the ColV plasmid increased the serum resistance of E. coli. This increase did not depend on the expression of the K1 antigen. This work also demonstrated that the presence of the K1 antigen protects E. coli from the bactericidal activity of serum. Studies using mouse peritoneal macrophages in the presence of normal serum indicated that the presence of K1 antigen protects E. coli from phagocytosis. Similar experiments with the K1 + strains performed in the presence of anti-K1 antibodies demonstrated that these antibodies opsonized these bacteria very efficiently in the absence of complement. The K1 − E. coli variants were efficiently phagocytized in the presence of normal human serum and absorbed human serum, indicating that they are able to be opsonized by complement deposited by activation of the alternative pathway of complement. Work using fluorescence microscopy confirmed that the K1 − strains are able to fix complement in the absence of antibody. It was also found that the presence of the ColV plasmid may interfere with phagocytosis of the E. coli K1 strains and deposition of complement on these cells. To test the relevance of the results of the in vitro experiments for disease, the pathogenicity of the strains was tested in mice. The results showed that the K1 antigen is the main determinant of pathogenicity of these strains and that the presence of ColV can modify the pathogenic potential of the E. coli K1 strains through a mechanism that does not depend on the production of colicin V.
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