Quantitative Proteomic Analysis of the Heat Stress Response in Clostridium difficile Strain 630

Clostridium difficile is a serious nosocomial pathogen whose prevalence worldwide is increasing. Postgenomic technologies can now be deployed to develop understanding of the evolution and diversity of this important human pathogen, yet little is known about the adaptive ability of C. difficile. We used iTRAQ labeling and 2D-LC–MS/MS driven proteomics to investigate the response of C. difficile 630 to a mild, but clinically relevant, heat stress. A statistically validated list of 447 proteins to which functional roles were assigned was generated, allowing reconstruction of central metabolic pathways including glycolysis, γ-aminobutyrate metabolism, and peptidoglycan biosynthesis. Some 49 proteins were significantly modulated under heat stress: classical heat shock proteins including GroEL, GroES, DnaK, Clp proteases, and HtpG were up-regulated in addition to several stress inducible rubrerythrins and proteins associated with protein modification, such as prolyl isomerases and proline racemase. The flagellar filament protein, FliC, was down-regulated, possibly as an energy conservation measure, as was the SecA1 preprotein translocase. The up-regulation of hydrogenases and various oxidoreductases suggests that electron flux across these pools of enzymes changes under heat stress. This work represents the first comparative proteomic analysis of the heat stress response in C. difficile strain 630, complementing the existing proteomics data sets and the single microarray comparative analysis of stress response. Thus we have a benchmark proteome for this pathogen, leading to a deeper understanding of its physiology and metabolism informed by the unique functional and adaptive processes used during a temperature upshift mimicking host pyrexia.

艰难梭菌是一种严重的医院获得性病原体，其全球流行率呈上升趋势。通过后基因组技术，如今我们可以深入探究这一重要的人体病原体的进化与多样性，然而关于艰难梭菌的适应性知之甚少。本研究采用iTRAQ标记和二维液相色谱-串联质谱（2D-LC–MS/MS）驱动的蛋白质组学方法，研究了艰难梭菌630株对一种温和但具有临床相关性的热应激的反应。我们生成了一份经统计学验证的447个蛋白质列表，并为其分配了功能角色，从而重建了包括糖酵解、γ-氨基丁酸代谢和肽聚糖生物合成在内的中心代谢途径。在大约49个蛋白质在热应激下发生了显著调控：除了经典的热休克蛋白（如GroEL、GroES、DnaK、Clp蛋白酶和HtpG）被上调外，还有多个应激诱导的红色素蛋白和与蛋白质修饰相关的蛋白（如脯氨酸异构酶和脯氨酸消旋酶）也被上调。鞭毛丝蛋白FliC的下调可能是一种能量节约措施，同样SecA1前蛋白转运酶也呈现下调趋势。氢化酶和各种氧化还原酶的上调表明，这些酶库中的电子流在热应激下发生改变。这项工作代表了艰难梭菌630株热应激反应的首次比较蛋白质组学分析，补充了现有的蛋白质组数据集以及单一微阵列应激反应比较分析。因此，我们为该病原体建立了一个基准蛋白质组，这有助于更深入地理解其生理学和代谢学，这些理解源于在模拟宿主发热的温度上升过程中所使用的独特功能和适应性过程。