Attenuation of Pseudomonas aeruginosa virulence by quorum sensing inhibitors

Abstract

Traditional treatment of infectious diseases is based on compounds that kill or inhibit growth of bacteria. A major concern with this approach is the frequent development of resistance to antibiotics. The discovery of communication systems (quorum sensing systems) regulating bacterial virulence has afforded a novel opportunity to control infectious bacteria without interfering with growth. Compounds that can override communication signals have been found in the marine environment. Using Pseudomonas aeruginosa PAO1 as an example of an opportunistic human pathogen, we show that a synthetic derivate of natural furanone compounds can act as a potent antagonist of bacterial quorum sensing. We employed GeneChip microarray technology to identify furanone target genes and to map the quorum sensing regulon. The transcriptome analysis showed that the furanone drug specifically targeted quorum sensing systems and inhibited virulence factor expression. Application of the drug to P.aeruginosa biofilms increased bacterial susceptibility to tobramycin and SDS. In a mouse pulmonary infection model, the drug inhibited quorum sensing of the infecting bacteria and promoted their clearance by the mouse immune response.

Fig. 1. From algal metabolite to Pseudomonas drug. (A) Compound 2, a natural furanone compound isolated from (C) D.pulchra. (B) compound C-30, a synthetic furanone with enhanced QSI activity.

Fig. 2. Influence of furanone C-30 on growth and expression of virulence factors of P.aeruginosa PAO1. Cultures were grown in the absence (circles) or presence of 1 µM (squares) and 10 µM (triangles) furanone C-30. (A) Growth rate; (B) exoprotease activity; (C) pyoverdin activity; (D) chitinase activity. The data represent mean values of three independent experiments. Error bars represent the standard errors of the means.

Fig. 3. Effect of furanone C-30 on genome-wide gene expression profile of P.aeruginosa. Differential gene expression in planktonic cultures of P.aeruginosa PAO1 in response to 10 µM C-30 analyzed by microarrays. Positive values represent C-30-induced genes. Negative values indicate C-30-repressed genes. The dashed lines indicate 5-fold induction or repression. Genes significantly affected by C-30 are indicated. The color coding of the individual genes indicates which of the two QS systems are required for induction: blue, LasR-controlled genes (activated by addition of only OdDHL); gray, both LasR and RhlR are required for full expression (activated by OdDHL, and further induced by addition of BHL); yellow, RhlR-controlled genes (genes induced only by simultaneous addition of OdDHL and BHL). C-30-regulated genes recorded as non-QS-controlled are not colored. Data represent samples retrieved at an OD₆₀₀ of 2.0.

Fig. 4. Quorum-induced genes and C-30-repressed genes in P.aeruginosa. Quorum-induced genes identified in lasI rhlI mutant grown with or without added exogenous AHL signals. The transcription profile of the quorum-induced genes in P.aeruginosa PAO1 is shown together with the maximal repression caused by C-30. ¹The gene number and name are from the Pseudomonas Genome Project (www.pseudomonas.com). Genes previously reported as QS regulated are shown in bold type. ], genes organized in operon. The criteria for organization into putative operons were as follows: (i) genes are transcribed in the same orientation; (ii) intergenic regions are <200 bp; (iii) genes exhibit similar transcription profiles. ²Description from the Pseudomonas genome project. qsc gene names refer to the study by Whiteley et al. (1999); pqs gene names refer to the study by Gallagher et al. (2002).

Fig. 4. Quorum-induced genes and C-30-repressed genes in P.aeruginosa. Quorum-induced genes identified in lasI rhlI mutant grown with or without added exogenous AHL signals. The transcription profile of the quorum-induced genes in P.aeruginosa PAO1 is shown together with the maximal repression caused by C-30. ¹The gene number and name are from the Pseudomonas Genome Project (www.pseudomonas.com). Genes previously reported as QS regulated are shown in bold type. ], genes organized in operon. The criteria for organization into putative operons were as follows: (i) genes are transcribed in the same orientation; (ii) intergenic regions are <200 bp; (iii) genes exhibit similar transcription profiles. ²Description from the Pseudomonas genome project. qsc gene names refer to the study by Whiteley et al. (1999); pqs gene names refer to the study by Gallagher et al. (2002).

Fig. 5. Sensitivity of furanone C-30-treated P.aeruginosa biofilms to tobramycin. Scanning confocal laser microscopy (SCLM) photomicrographs of P.aeruginosa PAO1 biofilms grown in the absence (left panel) or presence (right panel) of 10 µM C-30. After 3 days, the biofilms were exposed to 100 µg/ml tobramycin for 24 h. Bacterial viability was assayed by staining using the LIVE/DEAD BacLight Bacterial Viability Kit: red areas are dead bacteria, and green areas are live bacteria. The biofilms were exposed to (A) no furanone and 100 µg/ml tobramycin, (B) 10 µM C-30 and 100 µg/ml tobramycin, (C) non-treated control and (D) 10 µM C-30 and no tobramycin.

Fig. 6. Inhibition of QS in vivo. Photomicrographs of mouse lung tissue infected with an E.coli-based dual-labeled AHL sensor. The strain expresses GFP in response to the presence of exogenous AHL signals and carries a dsred expression cassette to provide a red fluorescent tag on the sensor bacteria for simple identification in tissue samples. Mice carrying the sensor bacteria in the lungs were administered OHHL and furanone C-30 via intravenous injection. (A) No injection; (B) 200 µM OHHL; (C) 200 µM OHHL + 2 µg/g BW C-30 (corresponds to ∼10 µM); (D) 400 µM OHHL + 2 µg/g BW C-30; (E) 800 µM OHHL + 2 µg/g BW C-30; (F) 1200 µM OHHL + 2 µg/g BW C-30.

Fig. 7. Inhibition of P.aeruginosa QS in mouse lungs. Photomicrographs of mouse lung tissue infected with P.aeruginosa carrying the dual-labeled PA quorum sensor for detection of QS signaling and a red fluorescent tag for simple identification in tissue samples. Mice were administered C-30 via intravenous injection at time zero. Infected animals were killed in groups of three at the time points indicated and the lung tissue samples were examined by SCLM.

Fig. 8. Lung bacteriology. Healthy CBA/J mice (10 in each group) were infected with PAO1 as described in the text. Furanone C-30 (solid bars; concentration as indicated below the x-axis) or saline (open bars) were given three times a day (every 7 h) for 3 days. The mice were killed on day 7 after the bacterial challenge and the bacterial content of the infected lungs was determined as described in the text. Average values were plotted and the standard deviation is shown by error bars. The values were tested by means of an F-test (analysis of variance), and the p values for the 0.7, 0.4 and the 0.2 µM C-30 concentrations were 0.0007, 0.004 and 0.5, respectively.