Introduction We have previously identified patients colonised with Pseudomonas aeruginosa (PA), in whom strains isolated from their sputum cannot be killed by their serum, yet are fully sensitive to healthy control serum (HCS). Addition of inhibitory patient serum to HCS also impaired killing of PA.
Fractionation of patient serum identified that IgG was responsible for the inhibition. FACS demonstrated excessive binding of IgG2 from patient serum to autologous strains.
Visualisation of lipopolysaccharide (LPS) isolated from bacterial strains by silver staining, showed that strains from patients with inhibitory serum demonstrated detectable O antigen expression – a component of the LPS cell wall of PA.
We aimed to confirm this relationship between inhibitory IgG2 and LPS expression by PA.
Methods Anti-LPS antibodies were removed from inhibitory serum (S4) by binding LPS from PA isolated from patient sputum, to polymyxin-B agarose overnight. Inhibitory serum was then passed over the LPS bound column. Antibodies specific for LPS bound to the column, and the flow through fractions of serum were collected. Bound antibody was subsequently buffer exchanged into PBS.
LPS was isolated from a PA strain resistant to patient sera (B4) and attached to a 96 well plate. ELISA was performed by adding dilutions of patient or HCS to the plate followed by anti-human IgG2 conjugated to alkaline phosphotase. Results were derived 30 minutes after addition of developer.
Results LPS removal of IgG from the patient’s serum restored bacterial killing. Adding the eluted antibody to HCS impaired killing. This confirmed that inhibitory IgG2 is LPS specific. The patients’ serum that blocked bacterial killing had high titres of IgG2 to LPS compared to those from patients with bactericidal serum (fig. 1)
Conclusion We have established that PA strains deficient in LPS O antigen are generally sensitive to killing by patients’ serum, whilst the presence of O antigen leads to serum resistance mediated by IgG2.
Results indicate that PA LPS O antigen repeats are central to serum resistance of autologous strains but not normal HCS. Current data indicates that bacterial killing is impaired in subjects colonised with PA expressing O antigen due to overproduction of LPS specific IgG2.