Screening And Research On Inhibitors Of Pseudomonas Quinolone Signal System

Clinical treatment of bacterial infections is a serious challenge. Some pathogens can adapt the selective pressure imposed from antibiotics by mutation. Such infections are possible through the production of virulence factors, many of which are regulated by cell-to-cell communication termed as quorum sensing (QS). Inhibition on quorum sensing can effectively reduce the infection, while no resistance is caused. Quorum sensing inhibitors are expected to inhibit the quorum sensing in the pathogens. Pseudomonas aeruginosa causes acute and chronic infections, especially in the immunocompromised individuals such as cystic fibrosis patients. Pseudomonas quinolone signal system plays an important role on the regulatory networks and the signals can take part in the communications between bacterial species, while a few researches focused on it. The purpose of this study is constructing an effective screening system for the detection of Pseudomonas quinolone signal system inhibitors from Traditional Chinese medicine extracts. These quorum sensing inhibitors (QSI) may be potential agents for the treatment of bacterial infections.In this study, there is a selector named PQSI1 for inhibitors on Pseudomonas quinolone signal system. The accepted strain is Pseudomonas aeruginosa PAOI. The expression of sacB gene is regulated by the promoter of PpqsA, which is 344bp long and activated by PQS-PqsR complex. The strains are unable to grow in the presence of PQS and sucrose unless functional QSIs are present. The presence of QSI compounds is detected by measuring the cell density; the higher density represents the more efficiency of inhibitor activity. This PQSI1 screening system can be used for high-throughput screening quorum sensing inhibitors. Meanwhile, PQSI2 is also constructed. PQSI2 is P. aeruginosa PAOI harboring pPQSI2, containing 439bp PpqsA and sacB. PQSI2 is used for separation and purification of quorum sensing inhibitors by bioautographic Thin-layer chromatography assay.Ten Traditional Chinese medicine extracts are found to exhibit quorum sensing inhibitory activity by PQSI1 from more than fifty Traditional Chinese medicine extracts. Quorum sensing inhibitor is isolated from Notopterygium incisum by PQSI2. By means of Mass Spectrometry and Spectroscopic method (1H NMR, 13C NMR), the structure of the bioactive compound is elucidated as falcarindiol. When growth is not affected, falcarindiol can reduce virulence factors such as elastase, pyocyanin and rhamnolipid. Pyocyanin production is reduced by falcarindiol up to 70% at 10 μM. The formation of biofilm is reduced by falcarindiol up to 60% at 20 μM and swarming motility is also attenuated by falcarindiol. Real-time PCR showed that falcarindiol can effectively inhibit the gene expression of las, rhl and pqs quorum sensing systems in P. aeruginosa.In this study, screening models for quorum sensing inhibitors named PQSI1 and PQSI2 are constructed. Falcarindiol isolated from N. incisum is shown quorum sensing inhibitory activity on quorum sensing in P.aeruginosa for the first time. Elastase activity, rhamnolipids and pyocyanin are attenuated by falcarindiol at 10μM. Biofilm formation and swarming motility are also attenuated by falcarindiol. This study contributes to the treatments on P.aeruginosa.