The ability of Pseudomonas aeruginosa to grow as a biofilm on both biotic and abiotic materials underlies its ability to establish chronic infections nd to persist despite antibiotic treatment and a host immune response. Therefore a better understanding of how biofilms form, grow and disperse addresses not only fundamental aspects of P. aeruginosa pathogenesis, but also identifies potential bacterial targets for anti-biofilm therapies. This application proposes the novel use of intrabodies in phenotypic screens to identify molecules that will inhibit biofilm formation, promote biofilm dispersal or disrupt biofil integrity. Our library of intrabodies-small, unmodified single-chain camelid antibodies raised against a highly diverse pool of P. aeruginosa antigens- can be inducibly expressed inside bacteria and targeted to the cytoplasm or periplasm. The ability to induce intrabody expression allows phenotypic screens for both biofilm dispersal as well as formation to be carried out, an advantage over genetic approaches. The active intrabody provides a tool for direct identification of the disrupted target-a significant advantage over small molecule screening approaches-as it can be used to bind and purify its target, and even identify the part of the target relevant o its function. Therefore, we hypothesize that the application of intrabody phenotypic screening to P. aeruginosa biofilm biology will identify novel targets involved in biofilm formation and maintenance inaccessible to genetic screens, and also generate intrabody reagents uniquely suited to studying the mechanism of target action and its potential for development as a therapeutic target.
Bacterial biofilms are involved in most device- and catheter-associated infections, as well as chronic infections of the lungs, skin and wounds and the heart valves. Bacteria in a biofilm are very resistant to antibiotics and the action of the immune system, so biofilm infections are very challenging to treat. This application describes the use of a new type of antibody ('intrabody') to identify molecules that might prevent or disrupt biofilms.
