| With the widespread appearance of antibiotic-resistant bacteria, there is an increasing demand for novel strategies to control infectious diseases. Bacteria can adapt by mutation to the selective pressure imposed by antibiotics,which has resulted in a significantly increased incidence of antibiotic resistance. Hence, the development of new approaches to the treatment of bacterial infections constitutes a focal point of research. The discovery of bacterial communication systems (quorum sensing, QS), which orchestrate important temporal events in the infection process, has afforded a novel opportunity to ameliorate bacterial infection without interfering with growth. Recent research showed that the bacteria will attenuate bacterial pathogenecity on the quorum sensing system mutation condition. Therefore, the interference with QS of bacteria has no selective pressure to bacteria,and is of great significance for the research and development of new antimicrobial drugs. In the present study, our aim is to find some high-performance quorum sensing inhibitors (QSI). Two quorum sensing inhibitors screen system PAO-MW1pW-1 and PAO-MW1pW-4 were developed and some culture extracts of marine microbe and a compound library were tested.Two translational fusion vectors, pW-1 and pW-4, which harbor the lasB promoter fused to the gfp gene and the lasB promoter fused to the levansucrase-encoding gene sacB, respectly, were constructed. Two screening systems, PAO-MW1pW-1 and PAO-MW1pW-4, were created by electrophoration of pW-1 and pW-4 into PAO-MW1 (lasI~- , rhlI~- ). PAO-MW1pW-1 can achieve screening by fluorescence microplate reader at 0.5μM 3-O-C12-HSL concentration. PAO-MW1pW-4 can be used directly visualization plate screening, which lead to cell death in the presence of 0.2μM 3-O-C12-HSL, 0.2μM C4-HSL and 6% sucrose. But the presence of a QSI compound can rescue the cells. In order to improve the sensitivity of PAO-MW1pW-4, a viable ways of high-sensitivity TTC staining method was set up, which can better distinguish between live and dead bacteria. The strains of marine microbe from the mud sample of Jiaozhou Bay were isolated and their culture extracts were prepared. Quorum sensing inhibitory activity was monitored by above two screening systems and Chromobacterium violaceum CV026. The culture extracts of three fungus were found to inhibit P. aeruginosa quorum sensing and one extracts of actinomycetes were found to have significant quorum sensing inhibitory activity in C. violaceum. The actinomycetes was assigned to the genus Streptomyces based on its 16S rDNA sequence. Further investigation revealed that the extract could inhibit the quorum sensing-controlled violacein and proteases production of C. violaceum in a concentration-dependent manner.PAO-MW1pW-1 and PAO-MW1pW-4 screening systems were used to examine a compound library. The results reveal that farnesol can effectively inhibit Pseudomonas aeruginosa las quorum sensing system. The effect of farnesol in PAO1 mutation of pqsA reveals that its inhibition of las quorum sensing system has no relation to PQS system. Further investigations demonstrate that its mechanism is competitive binding specific site with 3-O-C12-HSL signaling molecule to inhibit las system and its related genes, such as toxA and swarming behavior. Analysis of the activity of farnesol analogues shows that the three-dimensional conformation of double bonds of hydrophobic long chain and methyl side-chain is necessary.In conclusion, we constructed PAO-MW1pW-1 and PAO-MW1pW-4 screening systems. Quorum sensing inhibitory activity was monitored by the two screening systems and Chromobacterium violaceum CV026. Three fungus extracts were found to inhibit P. aeruginosa quorum sensing and one actinomycetes extract was shown to block C. violaceum quorum sensing in the culture extracts of marine microbe. The compound farnesol was revealed to inhibit Pseudomonas aeruginosa las quorum sensing system in a compound library. And the mechanism of farnesol was also investigated. Our work has practical significance in finding new approaches to the treatment of bacterial infections. |
