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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 investigate the differences of proteome secreted by different Bordetella pertussis cultivars and their relationship with pathogenicity in children with respiratory pathogens. Methods Bordetella pertussis were cultured in iron-added, iron-chelated and iron-magnesium supplemented-ginger medium, respectively. Bacteria were harvested in logarithmic growth phase (cultured for 48h), respectively. Trichloroacetic acid-acetone precipitation The secreted protein was prepared and the prepared secreted protein was analyzed by MALDI-TOF MS. The secretory protein was analyzed by bacterial subcellular localization and molecular signal peptide. Results Bacteria grew into plateau with 47h incubation in iron-supplemented medium. Bacteria with ferric ion chelating agent tended to be non-growth with the increase of iron chelator. The bacteria with Fe-Mg-Mg group grew slowly at 47h. The subcellular localization of the bacteria under the three culture conditions were as follows: 326 fern exosomes, 61 endomembrane proteins, 3 secretory proteins, 13 outer membrane proteins, 33 periplasmic proteins, and unknown proteins There were 466 cytoplasmic proteins, 21 outer membrane proteins, 63 periplasmic proteins, 66 endometrial proteins, 3 secretory proteins and 160 unknown proteins in the group of iron chelator. 601, 97 intima proteins, 5 secretory proteins, 26 outer membrane proteins, 7 periplasmic proteins and 185 unknown proteins. Conclusion Through subcellular localization and molecular signal peptide prediction, six secretory proteins of Bordetella pertussis were blocked, of which three secreted protein ribonucleoside diphosphate kinase (NDPK), putative export protein and serogroup III pili subunit The first discovery of Bordetella pertussis secreted protein.