幽门螺杆菌26695及其不同克拉霉素耐药水平衍生株的比较蛋白质组学分析

Comparative Proteomics Analysis of Helicobacter pylori 26695 and its Derived Isolates that Resistant to Different Concentrations of Clarithromycin

背景:目前克拉霉素耐药机制的研究主要集中于对幽门螺杆菌(H.pylori)23S rRNA基因的分析,其他基因与克拉霉素耐药的关系在国内外尚未见报道。目的:应用蛋白质组学技术分析不同克拉霉素浓度下耐药H.pylori菌株的发生机制,以期发现新的克拉霉素耐药相关基因。方法:以H.pylori 26695为出发菌株,在含克拉霉素平皿上选择耐药突变体,提取全菌蛋白后通过双向凝胶电泳(2-DE)分离蛋白,银染后应用Image Master 2D Elite软件比较不同浓度克拉霉素耐药株和野生株的蛋白表达差异,应用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)进行鉴定,并在NCBI的H.pylori蛋白数据库中搜索理论上酶解肽段能与之相匹配的蛋白。结果:共10个蛋白点在耐药株与野生株以及不同浓度耐药株间有明显差异,其中3个蛋白点为DNA指导的RNA多聚酶α亚单位,其表达量和等电点在耐药株与野生株间存在明显差异;1个蛋白点为黄素氧化还原蛋白,其在耐药株中的表达量上调;2个蛋白点为30S核糖体蛋白S1,其在耐药株中的表达量下调;3个蛋白点为过氧化氢酶,其在耐药株中的表达量上调;1个蛋白点为细胞分裂抑制剂,其在低浓度克拉霉素耐药株中的表达量较野生株上调,而在高浓度克拉霉素耐药株中的表达量较野生株下调。结论:不同浓度克拉霉素耐药株的耐药机制有不同的相关靶点,为研究H.pylori 23S rRNA以外克拉霉素的耐药机制提供了线索。

Background： It has been reported that clarithromycin resistance is associated with Helicobacter pylori （H. pylori） 23S rRNA. Other genes associated with clarithromycin resistance have not been reported either at home or abroad. Aims： Proteomics was used to analyze the mechanisms on different concentrations of clarithromycin resistance H. pylori strains, so as to detect the novel gene associated with clarithromycin resistance. Methods： Clarithromycin resistance strain was selected from the H. pylori 26695 strain by serial passage technique in vitro. Protein samples were prepared and separated by two-dimensional electrophoresis （2-DE）. After silver staining, expression of proteins between the different concentrations of clarithromycin resistance strains and wide-type H. pylori 26695 was analyzed by Image Master 2D Elite, and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry （MALDI-TOF-MS）. The Helicobacter proteins of the NCBI database were searched for protein identification. Results： Ten protein spots were obviously different between H. pylori 26695 and the clarithromycin resistance strains as well as between the different concentrations of clarithromycin resistance strains. Three spots were DNA-directed RNA polymerase alpha subunit, its expression and isoelectric point were different between the clarithromycin resistance strains and H. pylori 26695. One was flavodoxin, up-regulated in clarithromycin resistance strains. Two were 30S ribosomal protein SI, down-regulated in clarithromycin resistance strains. Three were catalase, up-regulated in clarithromycin resistance strains. One was cell division inhibitor, up-regulated in lowclarithromycin resistance strain and down-regulated in high-clarithromycin resistance strain. Conclusions： The mechanism of H. pylori resistant to clarithromycin are different in the different concentrations of clarithromycin resistance strains, which provide new clues for studying the resistance mechanism except H. pylori 23S rRNA.