Quantitative Proteomic Analysis of the Heat Stress Response in <i>Clostridium difficile</i> 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（Clostridium difficile）是一种严重的医院获得性病原菌，其全球流行率呈逐年上升趋势。如今后基因组技术可用于深入解析这一重要人类病原菌的进化与多样性，但学界对艰难梭菌的适应能力仍知之甚少。本研究采用iTRAQ标记结合二维液相色谱-串联质谱（2D-LC–MS/MS）驱动的蛋白质组学技术，探究艰难梭菌630株对轻度但具有临床相关性的热应激的响应。研究得到了经统计学验证的447种蛋白质的功能注释列表，借此重构了包括糖酵解、γ-氨基丁酸代谢以及肽聚糖生物合成在内的核心代谢通路。热应激下共有约49种蛋白质的表达量发生显著变化：包括GroEL、GroES、DnaK、Clp蛋白酶以及HtpG在内的经典热休克蛋白均出现上调，同时还有多种应激诱导型红氧还蛋白以及与蛋白质修饰相关的蛋白质（如脯氨酰异构酶和脯氨酸消旋酶）的表达也发生了改变。鞭毛丝蛋白FliC与SecA1前蛋白转位酶的表达量均出现下调，这可能是为了节约能量。氢化酶与多种氧化还原酶的表达上调，表明热应激下这些酶类参与的电子传递通量发生了改变。本研究是首次针对艰难梭菌630株热应激响应的比较蛋白质组学分析，可弥补现有蛋白质组学数据集以及单张微阵列应激响应比较分析的不足。本研究为该病原菌构建了基准蛋白质组，通过解析模拟宿主发热的温度升高过程中其独特的功能与适应过程，进一步加深了对该病原菌生理与代谢机制的理解。