乳房链球菌感染后形成胞内感染，而ROS的产生对清除胞内微生物是必需的。在胞内感染形成的过程中病原能有效激发线粒体产生mROS，此效应受特异性TLRs调控。为探讨TLRs/mROS信号通路在宿主抗乳房链球菌感染过程中的作用，研究将乳房链球菌与其靶细胞（巨噬细胞及乳腺上皮细胞）共孵育，通过应用针对TLRs（TLR2/4）、TAK1及NADPH氧化酶的特异性拮抗剂或siRNA阻断/抑制相关信号通路，细胞水平评价TLRs及mROS在宿主抗乳房链球菌感染中的作用。检测内容包括TLRs信号通路上的关键接头蛋白，促炎症因子、ROS、mROS、胞内活菌数及细胞损伤等。最后用TLR2-/-,4-/-双基因敲除小鼠在体验证，达到全面阐明TLRs/mROS信号通路在宿主抗乳房链球菌感染过程中的作用的目的。研究的开展有助于理解宿主识别和清除乳房链球菌感染的天然免疫机制，对乳房链球菌的免疫调控及防治具有重要的意义。

Streptococcus uberis (S. uberis) is an intracellular pathogen of the mammary gland. Reactive oxygen species (ROS) are essential components of the innate immune response against intracellular pathogen. Mitochondria are stimulated to produce ROS during the infective process and are regulated by specific toll-like receptors (TLRs). In order to elucidate the role of TLRs/mROS signaling pathway in S. uberis infection, bacteria will be co-cultured with target cells (macrophages and mammary epithelial cells). Then, different inhibitors or specific small interfering RNA targeting TLRs (TLR2/4), TGF-β-activated kinase and NADPH oxidases are used to inhibit the relative signaling pathways, and evaluate the effects of TLRs, ROS and mROS on the bacteriocidal ability of the effector cells. The specific TLRs, key adaptor proteins, inflammatory factors, ROS, mROS, the number of intracellular bacteria and cell injury are quantified. Finally, an double gene knockout (TLR2-/-,4-/-) mouse model of S. uberis mastitis will be employed to demonstrate the in vivo antibacterial activity of TLRs/mROS signaling pathway. This study will contribute to a more thorough understanding of the mechanism(s) of immune recognition and clearance of S. uberis infection, and may offer new insights into the prevention and control of economically-important S. uberis mastitis.