Staphylococcus aureus is a bacterial pathogen responsible for a diverse spectrum of human and animal diseases. Most S. aureus isolates are microencapsulated, i.e., they produce ureonic acid-containing extracellular polysaccharides that can be detected biochemically or serologically in bacterial cell extracts. Although there are 11 capsular serotypes, types 5 and 8 predominate, compromising about 22% and 53%, respectively, of all isolates. These organisms are poorly phagocytosed and killed by human leukocytes in vitro in the absence of specific capsular antibodies. Long term objectives of this study are to determine if antibodies to the S. aureus capsule play a role in immunity to staphylococcal infections and to expand our fundamental knowledge concerning the biology and immunochemistry of the staphylococcal capsule. Specifically, capsular antibodies will be measured in acute and convalescent sera from patients with an invasive S. aureus infection (bacteremia). Capsule-binding antibody levels will be measured using an enzyme-linked immunosorbant assay (ELISA) on plates coated with purified S. aureus capsular polysaccharide. Opsonic antibody titers will be determined using an in vitro opsonophagocytic bacterial killing assay with the patient's S. aureus isolate as the target organism. Antibody levels in patients will be compared with antibody levels in normal, healthy individuals and in patients with bacteremias due to other organisms. To evaluate type 5 and 8 capsular polysaccharides as protective immunogens against staphylococcal infection, a rat model of catheter-associated endocarditis will be used. Rats will be immunized with purified capsular polysaccharide and their capsular antibody response evaluated by ELISA. Control and immunized animals will be challenged with microencapsulated S. aureus and protection against endocarditis assessed. Protection will be defined as a significant rise in the bacterial ID(50) in immunized animals compared with controls and a significant drop in the numbers of viable bacteria recovered from the bloods, vegetations, and catheters of immunized animals. If rats are protected by active immunization, passive immunization studies will be performed using either polyclonal or monoclonal antibodies specific for the capsule. Because there have been many conflicting reports on the stability of S. aureus capsule expression in vitro and in vivo, a study of type 5 and 8 capsule production will be performed on bacteria serially passaged on laboratory media, as well as on bacteria recovered from acute and chronic infections in mice. Capsule expression will be evaluated qualitatively by immunoblot and quantitatively by rocket immunoelectrophoresis. Finally, mutants have been identified that produce a serologically altered type 5 microcapsule. Capsular polysaccharide will be purified from one of the mutants and its biochemical structure deduced. The relative virulence of the mutant strains compared with the wild type will be assessed by in vitro opsonophagocytic assays and the in vivo model of endocarditis in rats.
