百日咳鲍特菌铁离子相关分泌蛋白研究

A study of iron-related proteins secreted by Bordetella pertussis

目的探讨儿童呼吸道致病菌之一百日咳鲍特菌不同铁离子培养条件下分泌蛋白质组的差异以及与其致病的关系。方法百日咳鲍特菌分别在加铁、加铁螯合剂和加铁加镁的包-姜氏培养基中培养,于对数生长期(培养48h)收集细菌,分别用三氯乙酸-丙酮沉淀法制备分泌蛋白,制备的分泌蛋白做MALDI-TOF MS质谱分析,并进行分泌蛋白的细菌亚细胞定位和分子信号肽预测。结果加铁培养从47h开始细菌生长进入平台期,加铁离子螯合剂组细菌随铁离子螯合剂的增加而趋于不生长状态,加铁加镁组细菌在培养至47h时缓慢生长;百日咳鲍特菌在3种培养条件下的蛋白亚细胞定位分别为:加铁组胞质蛋白326个,内膜蛋白61个,分泌蛋白3个,外膜蛋白13个,周质蛋白33个,未知蛋白201个;加铁螯合剂组胞质蛋白466个,外膜蛋白21个,周质蛋白63个,内膜蛋白66个,分泌蛋白3个,未知蛋白160个;加铁加镁组胞质蛋白601个,内膜蛋白97个,分泌蛋白5个,外膜蛋白26个,周质蛋白7个,未知蛋白185个。结论通过亚细胞定位和分子信号肽预测,锁定百日咳鲍特菌6个分泌蛋白,其中3个分泌蛋白核糖核苷二磷酸激酶(NDPK)、假定输出蛋白和血清三型菌毛亚单位是本研究首次发现的百日咳鲍特菌分泌蛋白。

Objective To explore the potential relationship between iron-related proteins secreted by Bordetella pertussis under different culturing conditions and the mechanisms of its virulence in children. Methods.pertussis was cultured on Bordet-Gengou Agar Medium containing iron,on similar medium containing an iron-chelating agent,and on similar medium containing iron and magnesium..pertussis was grown to the logarithmic growth phase and then harvested at47 hours.Trichloroacetic acid acetone precipitation was used to collect secreted proteins,and then these collected proteins were identified using MALDI-TOF MS.Subcellular localization and the secretory signal peptides of the identified proteins secreted by.pertussis were predicted and analyzed. Results When cultured with iron,.pertussis growth began to enter the plateau phase after 47 h.When cultured with an ion-chelating agent,.pertussis tended not to grow as the concentration of the ion-chelating agent increased.When cultured with iron and magnesium,.pertussis grew slowly for 47 h.When.pertussis was cultured under the 3conditions,its iron-related proteins were located in different subcellular locations.When.pertussis was cultured with iron,326 cytoplasm proteins,61 inner membrane proteins,3secreted proteins,13 outer membrane proteins,and 33 periplasmic proteins were identified.When.pertussis was cultured with an iron-chelating agent,466 cytosolic proteins,21 outer membrane proteins,63 periplasmic proteins,66 inner membrane proteins,3secreted proteins,and 160 unknown proteins were identified.When.pertussis was cultured with iron and magnesium,601 cytoplasmic proteins,97 membrane proteins,5secreted proteins,26 outer membrane proteins,7periplasmic proteins,and 185 unknown proteins were identified. Conclusion Prediction of the subcellular localization and secretory signal peptides of secreted proteins focused on 6key secreted proteins,including 3nucleoside diphosphate kinases（NDPKs）,apresumed export protein,and a subunit of type III fimbriae.This study is the first to identify these proteins secreted by.pertussis.