Outer membrane (OM) protein F (porin) was purified by extraction from polyacrylamide gels of cell envelope proteins of the Pseudomonas aeruginosa PA01 strain. Mice were immunized intramuscularly with 10 micrograms of protein F preparation on days 1 and 14 and then subjected to burn and challenge on day 28. Protein F immunization afforded significant protection above that provided by PA01 lipopolysaccharide (LPS) immunization against subsequent challenge with six of six heterologous LPS immunotype strains of P. aeruginosa. By an ELISA, the murine immune response revealed an IgG titer of 5,120 to protein F by day 30. Immunoblot analysis of antisera from protein F-immunized mice revealed bands with both protein F and protein H of cell envelopes of all immunotypes tested. Active immunization with OM protein H did not, however, afford significant protection to mice in this burned mouse model. These data show the efficacy of OM protein F as a protective vaccine in a murine model representative of human infection.

Results from analytical ultracentrifugal analysis, Sephadex gel filtration, isopycnic density-gradient centrifugation, and polyacrylamide disc-gel electrophoresis revealed that ethylenediaminetetraacetic acid liberated a protein–lipopolysaccharide complex from cell walls of Pseudomonas aeruginosa with an estimated molecular weight of not less than 160 000 and probably about one million. Electron microscopy of this complex revealed spherules and rodlets. The diameter of the former was approximately 70 ± 10 Å while the dimensions of the latter were 70 ± 10 Å × 200 ± 50 Å. The rodlets appeared to be composed of three or more spherules arranged in a chain-like fashion. Electron microscopy of protein-free lipopolysaccharide revealed predominantly hollow spheres from 300 Å to 1500 Å in diameter, morphologically resembling membrane sacculi. It is proposed that the protein–lipopolysaccharide complex, but not the protein-free lipopolysaccharide, is representative of the in situ form of native endotoxin.

No abstract

The outer membrane protein F (porin) from the PAO1 strain of Pseudomonas aeruginosa was purified by two different methods. One procedure involved separation by column chromatography of proteins extracted from isolated outer membranes, whereas the other involved extraction from gels after slab polyacrylamide gel electrophoresis of proteins extracted from cell envelopes. Both procedures yielded protein F preparations which successfully immunized mice from subsequent challenge with the PAO1 strain. The protein F preparations contained small quantities of lipopolysaccharide (LPS). This level of LPS contamination protected immunized mice from challenge with the homologous LPS serotype strain. However, immunization of mice with protein F preparations from the PAO1 strain also afforded protection against challenge with two different LPS serotype strains. This protective ability was lost when the protein F preparation was treated with papain before use as a vaccine. These observations support the conclusion that protein F has protective ability, which is not due to LPS contamination, when given as a vaccine. After immunization with the protein F preparation, mice showed an increase in antibody titer to the purified protein F preparation by enzyme-linked immunosorbent assay. Mice were protected passively by administration of rabbit antisera raised to the protein F preparation. These results indicate that the protein F preparation elicits a specific humoral antibody response in immunized animals. Our results suggest that purified protein F has potential as an effective vaccine for P. aeruginosa.