Sun Yu, Xu Bin, Zhuge Xiangkai, Tang Fang, Wang Xuhang, Gong Qianwen, Chen Rui, Xue Feng, Dai Jianjun
MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China.
Key Laboratory of Animal Bacteriology, Ministry of Agriculture, Nanjing Agricultural University, Nanjing, China.
Front Cell Infect Microbiol. 2021 Jan 25;10:592906. doi: 10.3389/fcimb.2020.592906. eCollection 2020.
Extraintestinal pathogenic (ExPEC) causes bloodstream infections in humans and animals. Complement escape is a prerequisite for bacteria to survive in the bloodstream. Factor H (FH) is an important regulatory protein of the complement system. In this study, ExPEC was found to bind FH from serum. However, the mechanisms of ExPEC binding to FH and then resistance to complement-mediated attacks remain unclear. Here, a method that combined desthiobiotin pull-down and liquid chromatography-tandem mass spectrometry was used to identify the FH-binding membrane proteins of ExPEC. Seven identified proteins, which all were carbohydrate metabolic enzymes (CMEs), including acetate kinase, fructose-bisphosphate aldolase, fumarate reductase flavoprotein subunit, L-lactate dehydrogenase, dihydrolipoamide dehydrogenase, phosphoenolpyruvate synthase, and pyruvate dehydrogenase, were verified to recruit FH from serum using GST pull-down and ELISA plate binding assay. The ELISA plate binding assay determined that these seven proteins bind to FH in a dose-dependent manner. Magnetic beads coupled with any one of seven proteins significantly reduced the FH recruitment of ExPEC ( < 0.05) Subsequently, immunofluorescence, colony blotting, and Western blotting targeting outer membrane proteins determined that these seven CMEs were located on the outer membrane of ExPEC. Furthermore, the FH recruitment levels and C3b deposition levels on bacteria were significantly increased and decreased in an FH-concentration-dependent manner, respectively ( < 0.05). The FH recruitment significantly enhanced the ability of ExPEC to resist the opsonophagocytosis of human macrophage THP-1 in an FH-concentration-dependent manner ( < 0.05), which revealed a new mechanism for ExPEC to escape complement-mediated killing. The identification of novel outer membrane-displayed CMEs which played a role in the FH recruitment contributes to the elucidation of the molecular mechanism of ExPEC pathogenicity.
肠外致病性大肠杆菌（ExPEC）可引起人和动物的血流感染。补体逃逸是细菌在血液中存活的先决条件。补体因子H（FH）是补体系统的一种重要调节蛋白。在本研究中，发现ExPEC可与血清中的FH结合。然而，ExPEC与FH结合并进而抵抗补体介导攻击的机制仍不清楚。在此，采用脱硫生物素下拉法与液相色谱-串联质谱联用的方法来鉴定ExPEC的FH结合膜蛋白。通过谷胱甘肽S-转移酶（GST）下拉法和酶联免疫吸附测定（ELISA）板结合试验验证，鉴定出的7种蛋白质均为碳水化合物代谢酶（CMEs），包括乙酸激酶、果糖二磷酸醛缩酶、延胡索酸还原酶黄素蛋白亚基、L-乳酸脱氢酶、二氢硫辛酰胺脱氢酶、磷酸烯醇丙酮酸合酶和丙酮酸脱氢酶，它们可从血清中募集FH。ELISA板结合试验确定这7种蛋白质以剂量依赖的方式与FH结合。与7种蛋白质中的任何一种偶联的磁珠均显著降低了ExPEC对FH的募集（P<0.05）。随后，针对外膜蛋白的免疫荧光、菌落印迹和蛋白质印迹分析确定这7种CMEs位于ExPEC的外膜上。此外，细菌上的FH募集水平和C3b沉积水平分别以FH浓度依赖的方式显著增加和降低（P<0.05）。FH募集以FH浓度依赖的方式显著增强了ExPEC抵抗人巨噬细胞THP-1调理吞噬作用的能力（P<0.05），这揭示了ExPEC逃避补体介导杀伤的一种新机制。对在FH募集中起作用的新型外膜展示CMEs的鉴定有助于阐明ExPEC致病性的分子机制。
