Planchon Stella, Chambon Christophe, Desvaux Mickaël, Chafsey Ingrid, Leroy Sabine, Talon Régine, Hébraud Michel
INRA Centre de Clermont-Ferrand/Theix, UR454 Microbiologie, Equipe Qualité et Sécurité des Aliments, and Plate-Forme Protéomique, 63122 Saint-Genès Champanelle, France.
J Proteome Res. 2007 Sep;6(9):3566-80. doi: 10.1021/pr070139+. Epub 2007 Jul 17.
Staphylococcus xylosus is a saprophytic bacterium commonly found on skin of mammals but also used for its organoleptic properties in manufacturing of fermented meat products. This bacterium is able to form biofilms and to colonize biotic or abiotic surfaces, processes which are mediated, to a certain extent, by cell-envelope proteins. Thus, the present investigation aimed at evaluating and adapting different existing methods for cell-envelope subproteome analyses of the strain S. xylosus C2a. The protocol selected consisted initially of a lysostaphin treatment producing protoplasts and giving a fraction I enriched in cell wall proteins. A second fraction enriched in membrane proteins was then efficiently recovered by a procedure involving delipidation with a mixture of tributyl phosphate, methanol, and acetone and solubilization with a buffer containing ASB14. Proteins were separated using two-dimensional gel electrophoresis (2-DE) and identified using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). A total of 168 protein spots was identified corresponding to 90 distinct proteins. To categorize and analyze these proteomic data, a rational bioinformatic approach was carried out on proteins identified within cell envelope of S. xylosus C2a. Thirty-four proteins were predicted as membrane-associated with 91% present, as expected, within fraction II enriched in membrane proteins: 24 proteins were predicted as membranal, 3 as lipoproteins, and 7 as components of membrane protein complex. Eighteen out of 25 (72%) proteins predicted as secreted were indeed identified in fraction I enriched in cell wall proteins: 6 proteins were predicted as secreted via Sec translocon, and the remaining 19 proteins were predicted as secreted via unknown secretion system. Eighty-one percent (25/31) of proteins predicted as cytoplasmic were found in fraction II: 8 were clearly predicted as interacting temporarily with membrane components. By coupling conventional 2-DE and bioinformatic analysis, the approach developed allows fractionating, resolving, and analyzing a significant and important set of cell envelope proteins from a coagulase-negative staphylococcus, that is, S. xylosus C2a.
木糖葡萄球菌是一种腐生细菌，常见于哺乳动物的皮肤上，但因其感官特性也被用于发酵肉制品的生产。这种细菌能够形成生物膜并在生物或非生物表面定殖，这些过程在一定程度上是由细胞膜蛋白介导的。因此，本研究旨在评估和调整现有的不同方法，用于木糖葡萄球菌C2a菌株细胞膜亚蛋白质组的分析。所选方案最初包括用溶葡萄球菌素处理以产生原生质体，并得到富含细胞壁蛋白的组分I。然后，通过用磷酸三丁酯、甲醇和丙酮的混合物进行脱脂以及用含有ASB14的缓冲液溶解的程序，有效地回收了富含膜蛋白的第二个组分。使用二维凝胶电泳（2-DE）分离蛋白质，并使用基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）进行鉴定。总共鉴定出168个蛋白点，对应90种不同的蛋白质。为了对这些蛋白质组数据进行分类和分析，对在木糖葡萄球菌C2a细胞膜内鉴定出的蛋白质采用了合理的生物信息学方法。预测有34种蛋白质与膜相关，其中91%如预期的那样存在于富含膜蛋白的组分II中：24种蛋白质被预测为膜蛋白，3种为脂蛋白，7种为膜蛋白复合物的组分。预测为分泌型的25种蛋白质中有18种（72%）确实在富含细胞壁蛋白的组分I中被鉴定出来：6种蛋白质被预测为通过Sec转运体分泌，其余19种蛋白质被预测为通过未知分泌系统分泌。预测为细胞质型的蛋白质中有81%（25/31）存在于组分II中：8种被明确预测为与膜成分暂时相互作用。通过结合传统的2-DE和生物信息学分析，所开发的方法能够对来自凝固酶阴性葡萄球菌即木糖葡萄球菌C2a的一组重要的细胞膜蛋白进行分级分离、解析和分析。
