Alagely Ali, Rajamani Sathish, Teplitski Max
Soil and Water Sciences Department, Genetics Institute, Institute of Food and Agricultural Sciences (IFAS), University of Florida, Gainesville, FL, USA.
Methods Mol Biol. 2011;692:113-30. doi: 10.1007/978-1-60761-971-0_9.
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.
在许多致病细菌中，群体感应（QS）控制着与毒力、抗生素产生及抗性、在具有医学和工业重要性的生物和非生物表面上形成和维持微生物多细胞聚集体相关的基因表达。N-酰基高丝氨酸内酯（AHL）是革兰氏阴性细菌中表征最为充分的群体感应信号。因此，干扰AHL介导的群体感应被认为是控制病原体毒力的一种有吸引力的策略。灵敏生物测定法的发展促进了对AHL信号及其模拟物的搜寻，在这些生物测定法中，群体感应报告基因会响应AHL信号而发光。这些生物测定法已经导致鉴定出了数十种具有群体感应调节活性的化合物。对群体感应信号及其模拟物作用模式的表征需要后续的生化研究。在此，我们描述了一组发光报告基因，它们可用于高通量、中通量或低通量形式，用于发现和验证铜绿假单胞菌Las群体感应系统的激动剂或拮抗剂。这些近乎同基因的报告基因在AHL受体LasR的AHL结合结构域中包含截短或点突变，以及编码LasI AHL合酶基因的启动子中的突变。我们还开发了用于记录lasI和lasR启动子调控的报告基因。在进行生化实验之前，使用这些报告基因可显著简化Las群体感应信号激动剂和拮抗剂的鉴定和表征。为了测试这些报告基因的实用性，我们用棒曲霉素（一种已知的Las群体感应抑制剂）进行了生物测定。
