弧菌FadR调控蛋白所介导的脂肪酸信号感应的新机制

FadR is a type of global regulator with a diverse set of members exclusively distributed in Ɣ-proteobacteria. Unlike E. coli FadR, the paradigm member, Vibrio FadR orthologues exhibit the strikingly-superior ability to sense the available fatty acids in ecological niches. However, the molecular mechanism underlying this unusual scenario is not well understood. Here we report this case. Three types of different Vibrio FadR proteins were expressed and purified. Both electrophoretic mobility shift assay (EMSA) and surface plasmon resonance (SPR) validated binding of Vibrio FadR orthologues DNA targets. Isothermal titration calorimetry (ITC)-based analyses showed that binding of Vibrio FadR to the palmitoyl-CoA ligand is different from that seen with E. coli counterpart. Crystal structure of V. alginolyticus FadR revealed an extra helix-rich domain formed by the C-terminal 40-redisue insert. Unexpectedly, subsequent complex structural analyses of V. alginolyticus FadR bound to palmitoyl-CoA revealed that distinct from the E. coli FadR with only one ligand site, Vibrio FadR has one more ligand-binding site constituted mainly by this 40-redisue insert. Furthermore, the ligand binding to V. alginolyticus FadR leads to more seriously conformational alteration in N-terminal DNA-binding domain than that of E. coli FadR, which fits well to its super-sensing to long chain fatty acids. Apparently, Vibrio FadR having two ligand-binding sites represents a new mechanism for fatty acid sensing. Given the role of FadR in virulence of certain species of Vibrionaceae, it is rational that development of small molecule drugs/inhibitors targeting the FadR-mediated fatty acid sensing might be a promising approach/therapeutics against Vibrio infections.

弧菌FadR表现出与众不同的脂肪酸感应能力，但其分子机制不详。本项目的实施将试图回答这一科学问题。通过制备不同弧菌来源的FadR蛋白，运用凝胶阻滞和表面等离子共振技术等研究手段，揭示弧菌FadR特异性的DNA结合能力，阐明其对弧菌脂代谢的调节机制。等温滴定量热法将指出弧菌FadR与棕榈酸辅酶A配体分子的互作模式。弧菌FadR蛋白及其与配体结合物晶体结构的解析则将给出直观证据，绘制出其配体分子结合位点。初步晶体学分析结果显示：弧菌FadR多出的配体分子结合位点与其40个氨基酸的插入相关，符合我们的预期设想。该发现将代表病原细菌脂肪酸感应的一种崭新机制。鉴于弧菌fadR基因在其致病性中的作用，该感应机制将有潜力作为一个新抗菌靶点用于小分子药物（抑制剂）设计。

最近，霍乱弧菌FadR蛋白与油酸辅酶A的复合物结构研究提示存在额外的配体分子结合位点。然而我们对霍乱弧菌的近亲--溶藻弧菌的脂代谢及其调控网络了解甚少。本项目的开展不仅揭示了溶藻弧菌的FadR蛋白的晶体结构，而且勾勒了其与棕榈酰辅酶A配体分子的复合物结构。上述2类弧菌FadR/不同配体的复合物结构比对分析有力地说明了其双配体结合模式，这不同于经典的大肠杆菌FadR单一配体结合方式。事实上，等热滴定实验亦证实了弧菌FadR蛋白与配体结合比为2：1。SPR与EMSA地实验结果表明弧菌FadR可与fabA相互作用，且该相互作用可激活fabA的表达。基因敲除与小鼠定植实验阐明了弧菌FadR蛋白是一个致病相关的因子，而且第2个配体结合位点对于其完成成功感染至关重要。因此本课题提供了为脂代谢参与细菌感染过程提供了有力的证据。鉴于弧菌FadR调控蛋白不同寻常地携带了2个配体分子的结合位点。本课题进而制备了不同弧菌的FadR蛋白，并用体外和体内的手段比较研究了其蛋白特征。弧菌的一个辅助性fad基因VC2105携带了一个疑似的FadR结合位点,很可能编码一个硫酯酶。基于EMSA与SPR的实验结果，证实了VC2105启动子确实能够与FadR互作。体内与体外的实验证据进一步证实了FadR是VC2105基因的抑制子。基因敲除实验显示：VC2105不同于另一个脂肪酸代谢基因fadD基因，并不影响细菌的生长及其相关毒力因子基因的表达。由此可见，VC2105基因仅仅是一个辅助性的弧菌脂代谢相关的成员。此外，本课题亦研究了脂代谢的产物类脂A,发现其酰基乙醇胺修饰与粘菌素的耐药密切相关。同时发现了一大类不同的质粒可携带并传播类脂A的修饰酶基因mcr-1.