In Vivo Properties of Pseudomonas Aeruginosa: Outer Membrane Components and their Antigenicity

Abstract

This study involved an investigation of bacterial properties in vivo with particular reference to the outer membrane (OM) antigens of such bacteria and their recognition by host immunoglobulins during infection. Pseudomonas aeruginosa was recovered directly, without subculture, from the infected wounds of burn patients and the bacterial OM antigens analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The results indicated that the in vivo bacteria expressed high molecular weight OM proteins not present in cells of the same isolate cultivated in complex media, unless depleted of iron. Immunoblotting techniques further demonstrated recognition of the iron-regulated membrane proteins (IRMPs) by antibodies present in the patient serum and locally in wound tissue fluid, suggesting their potential as vaccine candidates. Differences between lipopolysaccharide (LPS) antigens from in vivo and in vitro grown bacteria were additionally observed following analysis by SDS-PAGE. Methods were devised to purify the IRMPs in a form relatively free from LPS. These included detergent extraction to solubilize the proteins and gel filtration procedures to separate the IRMPs from other OM proteins and subsequently to remove LPS. An investigation was made of the rabbit humoral immune response to P. aeruginosa growing as an adherent microcolony on the surface of inert materials (used in medical prostheses) and implanted into the peritoneum. The resulting localized infection was chronic in nature and involved large numbers of bacteria . Electron microscopy studies revealed that the adherent cells were embedded in an extensive glycocalyx partly composed of exopolysaccharide. Crossed immunoelectrophoresis demonstrated that in this biofilm mode of growth the bacteria avoided evoking a strong immune response, perhaps due to masking of key antigens by the glycocalyx. This was in contrast to bacteria in an acute disseminated peritonitis model, which elicited a high titre of antibodies, notably to the LPS antigen. Moreover, antibodies to the IRMPs were produced at an early stage of the disseminated infection. The results may help to explain the persistence of infections associated with medical prostheses such as peritoneal dialysis catheters.