Department of Chemical and Biochemical Engineering and Institute for Environmental Technology and Industry, Pusan National University, Busan 609-735, Korea.
Biotechnol Bioeng. 2010 May 1;106(1):119-26. doi: 10.1002/bit.22672.
QscR is a quorum-sensing (QS) signal receptor that controls expression of virulence genes in the prevalent opportunistic pathogen, Pseudomonas aeruginosa. Unlike the previously reported LuxR-type QS receptor proteins, that is, LasR and TraR, QscR can be obtained as an apo-protein that can reversibly form an active complex in vitro with its cognate signal molecule, 3-oxododecanoyl-homoserine lactone (3OC12-HSL), and subsequently bind to target promoter DNA sequences. To search for potential QS inhibitors, an in vitro gel retardation assay was developed using the purified QscR. Both the in vitro assay and the in vivo cell-based assay using QscR-overproducing recombinant strains were applied in the screening process. Furanones were chosen for testing the activity as QS inhibitors because they have been reported to strongly inhibit expression of QS-related genes in Agrobacterium tumefaciens. Among more than a hundred furanones tested, three compounds showed strong and dose-dependent inhibitory effects on QscR in both assays. One compound in particular, designated as F2, could completely inhibit the 3OC12-HSL-dependent QscR activity in vitro at a concentration of 50-fold molar excess over 3OC12-HSL. However, with the furanones F3 and F4, which are structurally similar to F2 but with a nitro group instead of the amine moiety, significantly decreased activities were observed. These results suggest that (i) the in vitro assay is a sensitive and reliable tool for screening QS inhibitors, and (ii) furanones are potentially important QS inhibitors for many LuxR-type receptor proteins.
QscR 是一种群体感应 (QS) 信号受体，可控制流行的机会性病原体铜绿假单胞菌中毒力基因的表达。与先前报道的 LuxR 型 QS 受体蛋白（即 LasR 和 TraR）不同，QscR 可以作为无辅基蛋白获得，该蛋白可在体外与同源信号分子 3-氧代十二酰基高丝氨酸内酯 (3OC12-HSL) 可逆地形成活性复合物，随后与靶启动子 DNA 序列结合。为了寻找潜在的 QS 抑制剂，使用纯化的 QscR 开发了一种体外凝胶阻滞测定法。体外测定法和使用过量表达重组菌株的 QscR 的体内细胞测定法都应用于筛选过程。呋喃酮因其已被报道强烈抑制根癌农杆菌中与 QS 相关的基因表达而被选为测试 QS 抑制剂活性的物质。在测试的一百多种呋喃酮中，有三种化合物在两种测定法中均对 QscR 表现出强烈的、浓度依赖性抑制作用。一种化合物，特别指定为 F2，在 50 倍摩尔过量的 3OC12-HSL 存在下，可完全抑制体外 3OC12-HSL 依赖性 QscR 活性。然而，与呋喃酮 F3 和 F4 相比，它们的结构与 F2 相似，但具有硝基取代胺基部分，观察到活性显著降低。这些结果表明：(i) 体外测定法是筛选 QS 抑制剂的一种敏感且可靠的工具；(ii) 呋喃酮可能是许多 LuxR 型受体蛋白的重要 QS 抑制剂。
