噬菌体调节S. aureus诱导TLR2信号转导通路的分子机制

Staphylococcus aureus (S. aureus) is recognized as one of the most major pathogens causing bovine mastitis. Bacteriophage is a new potential antimicrobial that deal with resistant pathogen. The previous study found that phage vB_SauM_JS25 could significantly inhibit intracellular S. aureus colonization, and can reduced S. aureus-induced the expression of TNF-α, IL-1β, and IL-6 mediated by Toll-like receptor 2 (TLR2) pathway. In this study, bovine mammary epithelial cells (MAC-T) will be stimulated with TLR2 ligand-LTA instead of bacterial infection, and then infected with phage, quantitative analysis will be utilized to detect the expression of several cytokines, the level of proteins in MAPK (p38, JNK and ERK1/2) and NF-κB (p65, IκBα) pathway, which helps to understand the phage regulates innate immune signaling pathways induced by S. aureus. The Ig-like domain in phage genome will be analyzed by PSI-BLAST, then the phage capsid proteins containing Ig-like domain will be expressed by eukaryotic expression system. After transfected with recombinant plasmid, we will measure the levels and phosphorylation of MAPK and NF-κB and its impact on the colonization of S. aureus, that could clarify which protein of phage regulates S. aureus induced innate immune. Furtherly we will verify the function of screened phage proteins in the mouse mammary. These results will indicate the molecular mechanism of phage regulates TLR2 signal transduction pathways induced by S. aureus, enrich antibacterial mechanism of the phage and provide new ideas for the prevention of bovine mastitis.

金黄色葡萄球菌（S. aureus）是目前影响我国奶牛乳房炎的重要致病菌。噬菌体作为一种新型的抗耐药致病菌的潜在抗菌剂正受到广泛关注。前期研究发现噬菌体vB_SauM_JS25能显著抑制胞内S. aureus定殖，且能通过Toll样受体2（TLR2）途径下调S. aureus诱导的IL-6等细胞因子的表达。本研究拟采用牛乳腺上皮细胞系 MAC-T，以TLR2配体LTA代替细菌感染，通过对IL-6等细胞因子及TLR2信号通路中MAPK和NF-κB相关磷酸化蛋白的表达定量检测，明确噬菌体调节S. aureus诱导的先天性免疫信号通路。在此基础上，利用生物信息学分析含Ig样结构域的噬菌体蛋白，通过体内外实验测定其对TLR2信号通路中MAPK和NF-κB及其对S. aureus定殖的影响，进一步探索噬菌体调控S. aureus诱导TLR2信号通路的功能蛋白，丰富噬菌体抗感染机制理论基础。

金黄色葡萄球菌（S.aureus）是目前影响我国奶牛乳房炎的重要致病菌之一。噬菌体因能有效裂解细菌被作为一种新型的对付耐药致病菌的潜在“抗菌剂”和“抗生素替代品”。因此，噬菌体在哺乳动物细胞内的抗感染研究，无论是对基础研究还是对潜在的应用都具重要价值。本研究通过荧光标记追踪S.aureus噬菌体vB_SauM_JS25（简称JS25），证实其能内化进入MAC-T奶牛乳腺上皮细胞系，抑制胞内S.aureus增殖；且发现噬菌体JS25能显著抑制S.aureus诱导的炎性细胞因子和趋化因子的表达。为了排除噬菌体的裂菌作用，采用大肠杆菌脂多糖（LPS）代替宿主菌S.aureus建立炎症模型，结果显示噬菌体JS25同样能显著抑制LPS诱导的炎性细胞因子的表达，同时抑制LPS诱导的核转录因子NF-κB p65的磷酸化，明确NF-κB信号通路参与了噬菌体的抗炎作用。利用生物信息学软件分析噬菌体JS25蛋白序列，筛选出含Ig样结构域的噬菌体功能蛋白2个，通过原核表达得到噬菌体重组蛋白，检测结果表明含Ig样功能域噬菌体蛋白与细胞骨架蛋白存在共定位，显示出对MAC-T细胞的黏附性，且可抑制MAC-T细胞中LPS诱导的TLR4表达，这意味着噬菌体重组蛋白可能具有免疫调节潜能。本研究挖掘出对MAC-T细胞具有黏附性的噬菌体蛋白，提供最直接的S.aureus噬菌体与上皮细胞互作的证据，丰富了噬菌体抗感染机制理论基础。