Lyngbyoic Acid, a "Tagged" Fatty Acid from a Marine Cyanobacterium, Disrupts Quorum Sensing in Pseudomonas aeruginosa. Pseudomonas aeruginosa

Quorum sensing (QS) is a mechanism of bacterial gene regulation in response to increases in population density. Production of small molecule QS signals, their accumulation within a diffusion-limited environment and their binding to the LuxR-type receptor trigger QS-controlled gene regulatory cascades. QS pathways mediated by acylhomoserine lactones (AHLs) in Gram-negative bacteria are the best studied. In Pseudomonas aeruginosa, for example, binding of AHLs to their cognate receptors (LasR, RhlR) controls production of virulence factors, pigments, antibiotics and other behaviors important for its interactions with eukaryotic hosts and other bacteria. We isolated a new small cyclopropane-containing fatty acid, lyngbyoic acid (1), as a major metabolite of the marine cyanobacterium, Lyngbya sp., collected off Fort Pierce, Florida. The structure of 1 was determined by NMR, MS and optical rotation. We screened 1 against four reporters based on AHL receptors from Vibrio fischeri (LuxR), Aeromonas hydrophila (AhyR), Agrobacterium tumefaciens (TraR) and P. aeruginosa (LasR) and found that 1 most strongly affected LasR. We show, by using a defined set of reporters, that compound 1 acts both through the AHL-binding site of LasR and independent of it. We also show that 1 reduces pyocyanin and LasB, both on the protein and transcript level, in wild-type P. aeruginosa, and that 1 directly inhibits LasB enzymatic activity. Conversely, dodecanoic acid (11) increased pyocanin and LasB, demonstrating that 1 is a “tagged” fatty acid potentially resistant to β-oxidation. Overall design: 1 mL Cultures of P. aeruginosa PAO1 were grown for 6h at 37 °C with shaking either in the presence of lyngbyoic acid (1 mM) or EtOH alone (10 μL) as a control. RNA samples were obtained from one treated and one control culture, and were reverse transcribed, labeled and fragmented according to Affymetrix's protocol. The two samples were hybridized in duplicate.

群体感应（Quorum sensing, QS）是细菌响应种群密度升高而启动的基因调控机制。细菌会合成小分子QS信号分子，这类信号在扩散受限环境中不断积累，并结合LuxR型受体（LuxR-type receptor），进而触发受QS调控的基因级联调控通路。在革兰氏阴性菌中，由酰基高丝氨酸内酯（acylhomoserine lactones, AHLs）介导的QS通路是当前研究最为深入的一类。例如，在铜绿假单胞菌（Pseudomonas aeruginosa）中，AHLs与其同源受体（LasR、RhlR）结合后，可调控毒力因子、色素、抗生素及其他与真核宿主和其他细菌互作相关的生理行为的产生。
我们从佛罗里达州皮尔斯堡海域采集的海洋蓝细菌鞘丝藻属（Lyngbya sp.）的主要代谢产物中，分离得到一种新型含环丙烷的脂肪酸——藻苔酸（lyngbyoic acid, 1）。该化合物的结构通过核磁共振波谱（Nuclear Magnetic Resonance, NMR）、质谱（Mass Spectrometry, MS）及旋光性分析得以确定。
我们以费氏弧菌（Vibrio fischeri, LuxR）、嗜水气单胞菌（Aeromonas hydrophila, AhyR）、根癌农杆菌（Agrobacterium tumefaciens, TraR）及铜绿假单胞菌（P. aeruginosa, LasR）来源的AHL受体为基础，构建了四种报告基因体系，对化合物1进行活性筛选，发现其对LasR的调控作用最为显著。
我们通过一组定制化的报告基因体系证实，化合物1既可通过LasR的AHL结合位点发挥调控作用，也可通过不依赖该位点的其他途径产生效果。我们还发现，化合物1可在蛋白和转录水平上，同时降低野生型铜绿假单胞菌的绿脓菌素（pyocyanin）与弹性蛋白酶LasB的表达，并且能够直接抑制LasB的酶活性。与之相反，十二烷酸（dodecanoic acid, 11）可上调绿脓菌素与LasB的表达，这表明化合物1是一种“标记型”脂肪酸，大概率具有抵抗β-氧化（β-oxidation）的特性。
实验整体设计如下：将1 mL铜绿假单胞菌PAO1培养液置于37℃振荡培养6h，实验组添加1 mM藻苔酸，对照组仅添加10 μL无水乙醇作为溶剂对照。分别收集实验组与对照组的RNA样品，按照Affymetrix公司的实验流程进行反转录、标记与片段化，两组样品均进行两次重复杂交实验。