Introduction Pseudomonas aeruginosa is a major opportunistic pathogen in cystic fibrosis, wound and nosocomial infections due to its high levels of intrinsic and acquired resistance to antibiotics, presenting a serious problem to public health. The Pqs quorum sensing (QS) system from this organism has been shown to play a key role in the control of virulence. Using a high-throughput screening method we identified, within a 7,000 library of natural and synthetic compounds, four that seem to inhibit the PqsR QS regulator from P. aeruginosa which controls the expression of the pqsABCDE genes required for the biosynthesis of the 2-alkyl-4-quinolones (AQs) and the switch-on of virulence. The aim of this study was to further characterise the impact of these compounds on the expression of genes and traits controlled by the Pqs system and assess their cytotoxicity.

1 Centre for Biomolecular Sciences, School of Life Sciences, University of Nottingham, Nottingham, United Kingdom 2 Greenpharma S. A. S., Orléans, France

Conclusions We have characterised four compounds which can inhibit psqA expression with IC50s in the nM range. These compounds also inhibit pyocyanin production and lecA expression. Their low cytotoxicity detected suggests they may be suitable for further pre-clinical studies.

Discovery of novel small-molecules for the inhibition of Pseudomonas aeruginosa quinolone signalling

Ivan Lafayette1, Jean-Frédéric Dubern1, Quoc Tuan Do2, James Lazenby1, Nigel Halliday1, Stephan Heeb1, Paul Williams1, and Miguel Cámara1

lecA/lecB expression The impact of the 4 compounds on the expression of the lecA and lecB lectin genes, involved in biofilm formation, were tested using the P. aeruginosa bioreporters PA01-N CTX::PlecA-lux (figure 3) and the CTX::PlecB-lux (figure 4) bioreporters using 10 and 100 µM of each compound. Whilst all compounds showed clear inhibition of lecA expression this was not the case for lecB with the exception of GPS104.

pqsA expression The P. aeruginosa PA01-L bioreporter CTX::PpqsA-lux, was used to monitor pqsA expression (figure 1), and CTX::PKm-lux, as a metabolic control (figure 2). They were were incubated with 10 and 100 µM of each compound and the bioluminescence output determined. All compounds showed strong inhibition of pqsA expression at both concentrations.

Figure 3

Table 2

Cytotoxicity Jurkat T lymphocyte cells at 8x105 cells/ml were exposed to 8 different concentrations of the 4 compounds for 20 hours (37°C, 5% CO2). Cytotoxicity was measured using a LSRII flow cytometer and used nucleic acid binding dye 7-Aminoactinomycin D as a measure of cell death. The results suggest that these compounds have low toxicity (table 3).

Pyocyanin production Three P. aeruginosa strains differing in virulence potential were used: PA01-N, PA14 and SED2-LES (epidemic strain). Results show that even at IC50 concentrations there is a variable reduction in pyocyanin production with the exception of GPS102 showing an increase (table 2).

Table 1

Table 3

Figure 4

Figure 1 Figure 2

IC50 determination The IC50s on the inhibition of pqsA expression were determined using the P. aeruginosa CTX::PpqsA-lux bioreporter in two different strains PA01-N and PA14, the latter containing two pathogenic islands (PAPI-1 and PAPI-2) absent from PA01-L. IC50 scores were higher for PA14 than PA01-L (table 1).