PPE25蛋白在分枝杆菌感染中性粒细胞中的作用

目的 研究PPE25蛋白在耻垢分枝杆菌（M.smegmatis，MS）感染中性粒细胞（polymorphonuclear neutrophil，PMN）时所起的作用。方法 利用异源表达结核分枝杆菌（M.tuberculosis, Mtb）PPE25蛋白的重组MS（MS-ppe25组）感染人PMN，以空载体MS（MS-vec组）为对照菌，观察2种MS的菌落形成、单菌落大小、生长曲线，以了解PPE25蛋白对MS生长的影响；PMN感染MS后，以菌落形成单位(colony forming unit，CFU)计数了解细菌活力的变化；检测乳酸脱氢酶（lactate dehydrogenase，LDH）释放百分比，以观察重组MS对PMN死亡的影响，流式细胞仪检测活性氧（reactive oxygen species, ROS），硝酸还原酶法检测一氧化氮（nitric oxide，NO）水平，ELISA检测细胞因子白细胞介素（IL）、肿瘤坏死因子-α（tumor necrosis factor-α,TNF-α）的变化，分析PPE25蛋白在MS感染PMN中的作用。结果 PPE25蛋白对MS生长、菌落形成及单菌落大小无影响； MS感染PMN 2、6、12 h，MS-ppe25组CFU、LDH释放百分比均高于MS-vec组，两组LDH释放百分比差异有统计学意义（P<0.05）；MS感染PMN细胞后2 h， MS-vec组ROS、NO水平较MS-ppe25组高（P<0.01）； MS-ppe25组PMN细胞表达的TNF-α各时点均高于对照组（P<0.01），IL-1β在MS感染6 h后均高于对照组（P<0.01）。结论 PPE25蛋白能增加MS在PMN内的生存、诱导细胞坏死、抑制ROS和NO的表达，改变细胞因子的分泌，有利于病原体的播散及逃避宿主免疫。
Objective To determine the role of PPE25 in the infection of M. smegmatis (MS) in polymorphonuclear neutrophils (PMNs). Methods In MS-ppe25 group, PPE25 was expressed in non-pathogenic fast-growing M. tuberculosis (Mtb) that infected PMNs. The empty vector MS (MS-vec group) was served as control. Their colony formation was observed, including the size and growth curves of single colonies. The colony forming unit (CFU) indicated bacterial vitality. The percentage of lactate dehydrogenase (LDH) release measured PMN death. The role of PPE25 protein in MS infections was analyzed by reactive oxygen species (ROS) detected by flow cytometry, nitric oxide (NO) level detected by nitrate reductase, cytokine interleukin (IL) and tumor necrosis factor-α (TNF-α) detected by ELISA. Results PPE25 protein had no effect on MS growth, colony formation and the size of single colonies. MS-infected PMN had higher percentages of CFU and LDH release 2, 6, and 12 h after infections compared with the MS-vec group (P<0.05). MS-infected PMN also had lower levels of ROS and NO levels 2 h after infections (P<0.01), consistently higher levels of TNF-α (P<0.01), and higher levels of IL-1β infusion 6 h after MS infections (P<0.01). Conclusion PPE25 protein increases the survival of MS in PMN, induces cell necrosis, inhibits the expressions of ROS and NO, and changes the secretion of cytokines, which helps spread of the pathogen by evading host immunity.