细菌YfiBNR三元系统的结构与功能研究

Over the late state of colonization, many baterial can form biofilm to evade host immunity system and and antibiotics ,that cause severe drug resistance.It is well documented that biofilm is highly coupled to the level cyclic diguanylate(c-di-GMP), an universal second messenger in bacteria. Recently research indicated yfiBNR operon encodes a tripartite signaling module that modulates intracellular c-di-GMP levels in response to signals received in the periplasm. YfiN is an inner membrane-localized protein with a cytosolic domain containg a HAMP domain and a C-terminal GGDEF domain. YfiN is repressed by the periplasmatic protein YfiR. YfiB is an OmpA/Pal-like outer-membrane lipoprotein that can active YfiN by sequestering YfiR. a SCVs phenotype appears when YfiN is activated or YfiR repression is alleviated..It has been reported that the sequestration of YfiR and activation of YfiN may be induced by a YfiB-mediated cell wall stress sensing mechanism and/or by redox driven misfold of YfiR, thus the YfiN diguanylate cyclase activity for c-di-GMP production is provoked. Previous mutagenesis studies reveal a number of key activation residues in YfiB, mainly located at the first helix at the predicted OmpA like domain. The YfiN interacting residues of YfiR is also revealed by compensatory mutagenesis studies, which are located at a C-terminal region of YfiR. However, being lack of the structural information, it is still unclear that which oligomeric state of YfiB is functional in cell, if YfiB sequesters YfiR through direct protein-protein contact or if any additional component mediates this interaction, how does YfiR interact with the periplasmic PAS domain of YfiN, and how the signal is transducted..The aim of our proposal is to determine the three dimentional structures of the YfiBNR system by crystallographic analysis, in conjugation with functional studies, reveal the structural mechanism of the YfiBNR system and provide structural basis for the development of antibiotic strategy.

许多病菌在长期感染的后期，可以形成生物被膜来抵抗免疫系统和抗生素，导致严重的耐药性。细菌被膜的产生与第二信使 （c-di-GMP）水平上调密切相关。近年在绿脓杆菌等革兰氏阴性菌中鉴定了c-di-GMP的一个上游三元调节系统-YfiBNR。 YfiN是内膜蛋白，它与周质腔中YfiR相互作用并被YfiR抑制。YfiB是一个外膜蛋白，通过与YfiR作用而释放被抑制的YfiN，从而激活YfiN，产生c-di-GMP。至今除了YfiN 的胞内结构域，该信号系统其他蛋白的结构均未得到解析。 由于缺乏结构信息，该系统的调控机制方面有许多问题尚不清楚 ，特别是蛋白被激活后发生怎样的构象变化，蛋白之间的如何作用以及信号具体如何传递机制等。我们拟利用结构生物学的手段，对YfiBNR的各个蛋白及复合物开展结构生物学研究。通过结构分析，并且结合功能试验，阐明作用机制，为设计新型抗菌药物提供思路。

许多病原菌会通过形成生物被膜来逃避宿主的免疫系统和抗菌药物，从而造成耐药性。研究表明，生物被膜的形成与第二信使环二鸟苷酸c-di-GMP密切相关。本课题在执行期间，针对来自铜绿假单胞菌以及霍乱弧菌的生物被膜密切相关的信号输入调控系统（YfiBNR 系统 以及 CdgH）开展结构和功能研究。该项研究一共解析了7个相关蛋白及蛋白复合物的结构，并开展相应的功能实验，在国际刊物发表研究论文4篇。本课题所取得的研究结果，对c-di-GMP 相关的信号输入输出调节机理研究提供了一系列新的观点，还对以细菌被膜为标靶的抗菌研究提供了新的结构依据和思路。