Disruption of Pseudomonas aeruginosa quorum sensing using high-affinity antibodies derived from immunised sheep

Abstract number: P564

Palliyil S.S., Broadbent I.D., Charlton K.A., Porter A.J.

Objectives:Pseudomonas aeruginosa is an opportunistic human pathogen which, like many other Gram-negative pathogens, employs quorum sensing – regulated virulence factors to establish an infection in its host. The bacterium is notorious for antibiotic resistance and causes life-threatening infections in immunocompromised and cystic fibrosis patients, and also accounts for 11% of nosocomial infections worldwide. Quorum sensing in Ps. aeruginosa populations is controlled by low molecular weight (hapten) signalling molecules known as homoserine lactones (HSLs). Previous work in our lab has demonstrated that sheep are capable of producing high affinity, high specificity anti-hapten antibodies, so we sought to develop anti-HSL antibodies derived from immunised sheep and explore their potential as novel antibacterial agents.

Methods: Derivatives of Ps. aeruginosa HSL molecules were synthesised, conjugated to suitable carrier proteins, and used to elicit an immune response in sheep. Post immunisation, sheep polyclonal sera were analysed using immunoassays to determine the level of specific immune response. Bioassays were also employed to demonstrate the abilities of the sera to block Ps. aeruginosa quorum sensing.

Results: The ability of immunised sheep polyclonal serum to bind specifically to quorum sensing molecules has been demonstrated by a series of in vitro immunoassays and bioassays. Immunisation with a mixture of three HSL subgroups resulted in high antibody titre polyclonal serum which showed nanomolar affinities to the free form of HSLs when applied in a competition format.

Conclusions: Antibodies are an attractive method for controlling bacterial virulence and biofilm formation in Gram-negative bacteria, and at the same time these 'antipathogenic' drugs are less likely to develop resistance in bacteria compared to conventional antibiotics.

The results from preliminary experiments are highly encouraging and demonstrate the possibility of isolating nanomolar affinity anti-HSL antibodies from immunised sheep. Phage display antibody libraries are currently being constructed from sheep peripheral blood lymphocytes, and will be screened in due course for anti-HSL monoclonal antibodies.