Luminescent Reporters and Their Applications for the Characterization of Signals and Signal-Mimics that Alter LasR-Mediated Quorum Sensing
In many pathogenic bacteria, quorum sensing (QS) controls expression of genes that are involved in virulence, production and resistance to antibiotics, formation and maintenance of microbial multicellular consortia on biotic and abiotic surfaces of medical and industrial importance. N-acyl homoserine lactones (AHL) are the best characterized quorum sensing signals in Gram-negative bacteria. Interference with AHL-mediated QS, therefore, is considered an attractive strategy for controlling virulence in pathogens. The search for AHL signals and their mimics has been facilitated by the development of sensitive bioassays, in which QS reporters luminesce in response to AHL signals. These bioassays have already led to the identification of dozens of compounds with QS modulating activities. The characterization of the mode of action of QS signals and their mimics requires follow-up biochemical studies. Here, we describe a set of luminescent reporters, which could be used in high, medium or low throughput format, for the discovery and validation of agonists or antagonists of the Las QS system of Pseudomonas aeruginosa. These nearly isogenic reporters contain truncations or point mutations in the AHL binding domain of the AHL receptor LasR, as well as mutations in the promoter for the gene encoding LasI AHL synthase. We also developed reporters for documenting the regulation of lasI and lasR promoters. The use of these reporters significantly streamlines identification and characterization of the Las QS signal agonists and antagonists prior to biochemical experiments. To test the usefulness of these reporters, we carried out bioassays with patulin, a known inhibitor of Las QS.
Key wordsCell-to-cell signaling rsaL Quorum sensing inhibition Quorum sensing signal-mimic LuxR
The development of the reporters and experiments presented in this manuscript were supported by USDA NRI grant # 2007-35319-18158 and funding from Protect Our Reefs Foundation.
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