Pumirat Pornpan, Saetun Putita, Sinchaikul Supachok, Chen Shui-Tein, Korbsrisate Sunee, Thongboonkerd Visith
Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.
Biochim Biophys Acta. 2009 Jun;1794(6):898-904. doi: 10.1016/j.bbapap.2009.01.011. Epub 2009 Feb 11.
Burkholderia pseudomallei is a saprophyte found in soil and water. It is a difficult microorganism to kill and can survive in these environments for many years. Mechanisms for its adaptive response to environmental changes remain largely unknown. We performed a proteomics study to examine alterations in secreted proteins (secretome) under a salt stress (with 150 mM NaCl) compared to the normal cultured condition in LB broth. The culture supernatants were filtrated and precipitated with 50% ethanol. The isolated proteins were recovered, separated with 2-D PAGE, and visualized with SYPRO Ruby stain (n=5 gels for each group). Differentially expressed protein spots were identified by Q-TOF MS and/or MS/MS analyses. A total of 42 protein spots representing 37 unique proteins were identified as the altered proteins during the salt stress, including metabolic enzymes, transcription/translation regulators, potential virulence factors, chaperones, phage capsid proteins, drug resistance protein, solute transport regulator, and hypothetical proteins. The presence of secreted GroEL only after NaCl exposure was confirmed by Western blot analysis. The increased level (19-fold) of a beta-lactamase-like protein suggested that the NaCl-exposed bacterium might resist to beta-lactam antibiotics. Functional analysis revealed that the NaCl-exposed bacterium had significantly greater survival rate after a treatment with ceftazidime. Our study provided the first dataset of the secretome of B. pseudomallei and its alterations, which may lead to novel insights into adaptive response of B. pseudomallei during the salt stress.
类鼻疽伯克霍尔德菌是一种存在于土壤和水中的腐生菌。它是一种难以杀灭的微生物，能在这些环境中存活多年。其对环境变化的适应性反应机制在很大程度上仍不为人知。我们进行了一项蛋白质组学研究，以检测在盐胁迫（150 mM NaCl）下与在LB肉汤中正常培养条件相比，分泌蛋白（分泌组）的变化。将培养上清液过滤并用50%乙醇沉淀。回收分离的蛋白质，通过二维聚丙烯酰胺凝胶电泳进行分离，并用SYPRO Ruby染色可视化（每组n = 5块凝胶）。通过四极杆飞行时间质谱和/或串联质谱分析鉴定差异表达的蛋白点。共有42个蛋白点代表37种独特的蛋白质被鉴定为盐胁迫期间的变化蛋白，包括代谢酶、转录/翻译调节因子、潜在毒力因子、伴侣蛋白、噬菌体衣壳蛋白、耐药蛋白、溶质转运调节因子和假定蛋白。通过蛋白质免疫印迹分析证实仅在NaCl暴露后分泌型伴侣蛋白GroEL的存在。一种类β-内酰胺酶蛋白水平的升高（19倍）表明暴露于NaCl的细菌可能对β-内酰胺类抗生素具有抗性。功能分析显示，用头孢他啶处理后，暴露于NaCl的细菌存活率显著更高。我们的研究提供了类鼻疽伯克霍尔德菌分泌组及其变化的首个数据集，这可能为深入了解类鼻疽伯克霍尔德菌在盐胁迫期间的适应性反应带来新的见解。
