Proteomic analysis of cell surface proteins from Clostridium difficile
Proteomic analysis of cell surface proteins from Clostridium difficile
复制标题
艰难梭菌细胞表面蛋白的蛋白质组学分析
作者：
A. Wright;R. Wait;S. Begum;B. Crossett;J. Nagy;K. Brown;N. Fairweather
艰难梭菌是一种导致大肠疾病的细菌，特别是在用抗生素治疗后。这种细菌产生两种毒素（A和B），这两种毒素是导致这种疾病的病理学原因。此外，先前已鉴定出许多与粘附至肠道相关的细菌毒力因子，包括细胞壁蛋白Cwp66、高分子量表面层蛋白（HMW‐SLP）和鞭毛。由于基因组序列预测了许多其他细胞壁相关蛋白，我们研究了细胞壁提取物中蛋白质的多样性，目的是确定进一步的毒力因子。我们已经使用了许多方法来去除与艰难梭菌细胞壁相关的蛋白质。 使用低pH甘氨酸处理和细胞壁的溶菌酶消化获得的两种所得提取物已通过二维电泳和质谱进行了详细分析。已鉴定出119个点，包括49种不同的蛋白质。两个表面层蛋白（SLP）是最丰富的蛋白质，我们还发现了鞭毛的成分。有趣的是，我们还确定了许多HMW‐SLP的旁系同源物的表达，这些可能代表了进一步研究毒力因子的靶点。
Clostridium difficile is a bacterium that causes disease of the large intestine, particularly after treatment with antibiotics. The bacterium produces two toxins (A and B) that are responsible for the pathology of the disease. In addition, a number of bacterial virulence factors associated with adhesion to the gut have previously been identified, including the cell wall protein Cwp66, the high‐molecular weight surface layer protein (HMW‐SLP) and the flagella. As the genome sequence predicts many other cell wall associated proteins, we have investigated the diversity of proteins in cell wall extracts, with the aim of identifying further virulence factors. We have used a number of methods to remove the proteins associated with the cell wall of C. difficile. Two of the resulting extracts, obtained using low pH glycine treatment and lysozyme digestion of the cell wall, have been analysed in detail by two‐dimensional electrophoresis and mass spectrometry. One hundred and nineteen spots, comprising 49 different proteins, have been identified. The two surface layer proteins (SLPs) are the most abundant proteins, and we have also found components of the flagellum. Interestingly, we have also determined that a number of paralogs of the HMW‐SLP are expressed, and these could represent targets for further investigation as virulence factors.