Iron-Regulated Surface Antigens of Pseudomonas Aeroginosa

Abstract

The iron uptake systems of pathogenic bacteria provide potential targets for immunological intervention. Essential components of the iron-uptake system of Pseudomonas aeruginosa and other Gram-negative bacteria are the high molecular weight iron-regulated membrane proteins (IRMPs) which are expressed in the outer membrane (OM), and which function as receptors for siderophore-iron complexes. Antibodies which bind to IRMPs in the intact bacterial cell will possibly block iron uptake and therefore exert a bacteriostatic effect upon the invading organism. In order to study the potential of anti-IRMP antibodies as immunotherapeutic agents, the production of specific antibodies was required. Selection of antibodies specific for the protein components of Gram-negative bacteria has, however, been severely restricted due to the response to the immunodominant molecule lipopolysaccharide (LPS). To circumvent the response to LPS, a purification strategy was developed to minimise the LPS contamination in the preparation for immunisation. The strategy involved selection of a rough mutant of P. aeruginosa, selective solubilisation of IRMPs and gel-filtration or ion-exchange chromatographic separation. Additionally, a purification technique involving excision of these antigens from SDS-PAGE gels was applied. Polyclonal and monoclonal antibodies reacting with outer membranes and whole bacterial cells were generated. Monoclonal antibodies which reacted specifically with the IRMPs and protein D were selected, and these antibodies were used in the study of the complexity and expression of IRMPs. Investigation of the surface exposure of these antigens determined using immunofluorescence and immunogold labelling techniques indicated that the IRMPs and protein D were surface exposed immunogens, which were more accessible to antibody in a rough strain of P. aeruginosa. Polyclonal and monoclonal antibodies to these surface exposed antigens were found to be cross-reactive with OM antigens in other bacterial species and with antigenic determinants expressed by representative strains of the seventeen serotypes of P. aeruginosa. A selection of the monoclonal antibodies generated were effective at agglutinating whole bacterial cells. IRMPs were confirmed in immunoblotting assays to be important immunogens, as they were expressed in vivo and recognised early by the host in both pleural cavity and Otitis externa infections. The protective activities of polyclonal and monoclonal antibodies to IRMPs were evaluated in an experimental intraperitoneal challenge model. Polyclonal antibodies were found to provide significant protection against challenge with a homologous strain of P. aeruginosa.