具有活化TLR9活性的布鲁氏菌DNA中REPs的确证及作用机制研究

布鲁氏菌病是由布鲁氏菌引起的一种人畜共患病,对人类健康造成严重威胁。TLR9是先天免疫中识别细菌和病毒非甲基化CpG DNA的主要受体，可介导机体对布鲁氏菌的清除，但机制不详。本项目拟依据革兰氏阴性菌DNA中基因外重复回文序列（REPs）具有活化TLR9活性的理论，结合国内外已公布的四种对人有致病性的布鲁氏菌的基因组信息，在课题组前期研究布鲁氏菌DNA中REPs分布及TLRs识别核酸配体分子机制的基础上，从布鲁氏菌DNA中筛选具有活化TLR9活性的REPs，探讨TLR9识别REPs的分子机制，并进一步观察活性REPs对TLR9信号通路及树突状细胞成熟和活化的影响，分析活性REPs的构效关系。研究结果不仅有助于揭示TLR9识别布鲁氏菌DNA的分子机制，为布鲁氏菌病的防治提供新思路和新靶点，而且有望发现新型具有活化TLR9活性的化合物，为感染性疾病、肿瘤等的药物研究提供先导结构。

Brucella is a genus of Gram-negative, facultative intracellular bacteria that can cause brucellosis, a zoonotic disease representing significant public health threats. Among Toll-like receptors (TLRs), TLR9 recognizes bacterial and viral unmethylated DNA containing repeated CG motifs，and plays an essential role in the clearance of intracellular Brucella. However, the underlying mechanism is still unclear. Recently, it has been demonstrated that the natural DNA sequences named repetitive extragenic palindromic (REP) sequences present in Gram-negative bacteria are able to produce innate immune system stimulation via TLR9. Additionally, complete genome sequences of Brucella melitensis, Brucella abortus, Brucella canis and Brucella suis are available. In our previous study, REPs were found in the genomes of four Brucella species as described above. We also investigated the recognition mechanism of nucleic acid ligands by TLRs. The aim of this work is to identify REP sequences as natural TLR9 ligands from Brucella DNA. Once we identify REP candidates with TLR9 agonistic activity, we will investigate the molecular recognition mechanism between TLR9 and REPs. Also, we will assess the effects of active REPs on the TLR9 signaling pathway and dendritic cells. Furthermore, structure-activity relationship of active REP candidates will be discussed. This work not only provides some new information which may be helpful for understanding the recognition mechanism of REPs by TLR9, but also provides excellent opportunities to identify novel targets for brucellosis treatment. Moreover, knowledge of REP sequences serving as TLR9 ligands is useful in the discovery of lead compounds for treatment of cancer and infectious diseases.