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

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.

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。

布鲁氏菌病是由布鲁氏菌引起的一种人畜共患疾病，不仅每年造成的经济损失巨大，而且还对人体健康造成严重威胁。本项目采用多学科交叉的方法对布鲁氏菌DNA进行研究，从中发现能够活化TLR9的REPs，并探讨其经TLR9信号通路调节机体免疫反应的分子机制。首先，选择已公布的牛种、羊种布鲁氏菌基因组，采用生物信息学方法共搜索到羊种布鲁氏菌2200个REPs、牛种布鲁氏菌1857个REPs。分别从牛种、羊种布鲁氏菌REPs中选择出回文结构较好的15条和12条序列进行合成，筛选获得5条牛种布鲁氏菌REPs和8条羊种布鲁氏菌REPs诱导巨噬细胞分泌IFN-α。利用TLR9-siRNA沉默TLR9可显著降低牛种活性REP序列M5和羊种活性REP序列M6诱导的巨噬细胞IFN-α的分泌，提示布鲁氏菌REPs在巨噬细胞中经TLR9信号通路介导IFN-α的分泌，进而调节机体的免疫反应。接下来采用分子模拟技术观察REP序列与TLR9的相互作用，发现超过20个氨基酸残基参与了布鲁氏菌REP序列与TLR9的结合，包括Lys95、 Lys181、Lys207、 Arg256、Lys292 和 Lys338等。进一步发现布鲁氏菌REP序列显著上调TLR9 mRNA和蛋白质表达水平，并活化IκB/NF-κB、MAPK信号通路。最后，发现布鲁氏菌REP序列作用DC细胞后，其表面分子CD40、CD86及MHCⅡ 表达增高, DC细胞的内吞能力下降，细胞培养上清液中IL-6、TNF-α浓度升高，提示布鲁氏菌REP序列调节DC细胞成熟。综上所述，布鲁氏菌DNA中存在具有免疫调节活性的REPs序列，活性REPs序列通过影响TLR9信号通路及DC细胞的成熟来调节机体免疫-炎症反应。研究成果不仅有助于以REPs为靶点控制布鲁氏菌感染引起的免疫失衡，为布鲁氏菌病的防治提供新思路，还有助于发现新的TLR9调节剂先导结构，具有一定的应用前景。