Characterisation and evaluation of novel drug targets in Pseudomonas aeruginosa

Pseudomonas aeruginosa (PA) is a major human pathogen for clinical conditions including CF, COPD and chronic wounds/burns. It is intrinsically resistant to antibiotics and hence there is a clear clinical need to develop novel strategies and therapeutics for PA management in chronically ill patients where antibiotic therapy selects for multi-resistant organisms. We have performed a genome wide/multi-host screening for novel anti-virulence targets in PA where we have identified a number mutants showing normal growth but strongly attenuated in virulence both in vitro and in vivo. Some of the mutated genes have not been characterised before and the role in the biology of PA remains unknown. They do not have any homologues in the human genome which makes them ideal targets for the design of drugs with selective toxicity.

Our hypothesis is that by inhibiting the identified targets we can attenuate the virulence of multi-resistance PA isolates.
The project will focus on one of the targets and the above hypothesis will be addressed through a highly multidisciplinary approach including: the construction of in-frame deletions of the target gene in several clinical isolates and the testing of these mutants in a wide range of phenotypic assays including biofilm formation using confocal microscopy. For some of these targets there is a predicted function. To verify this function the target protein will be purified and relevant enzymatic assays performed. A search for inhibitors of the identifed enzymatic activity will be performed and the inhibitors identified tested for the attenuation of virulence in PA.

This project will also look at the regulation of the expression of the selected targets with a view to understand its expression patterns under different environmental conditions and hence its relevance in different host environments. Regulators of the expression of the target will be identified through the construction of a reporter system (normally lux-based) for the expression of the target gene and the screening of a transposon library, in the strain carrying the reporter system, for colonies showing differential expression of the gene under analysis. The mutated genes in the selected mutants will be identified and their role in the control of the target validated by constructing in-frame deletion mutants in the reporter strain.

The results from this project will set the grounds for consideration of the protein studied as a true therapeutic target in P. aeruginosa which can be taken into to pre-clinical and subsequently clinical phases.