Heat shock and prolonged heat stress attenuate neurotoxin and sporulation gene expression in group I Clostridium botulinum strain ATCC 3502

Foodborne pathogenic bacteria are exposed to a number of environmental stresses during food processing, storage, and preparation, and in the human body. In order to improve the safety of food, the understanding of molecular stress response mechanisms foodborne pathogens employ is essential. Many response mechanisms that are activated during heat shock may cross-protect bacteria against other environmental stresses. To better understand the molecular mechanisms Clostridium botulinum, the causative agent of botulism, utilizes during acute heat stress and during adaptation to stressfully high temperature, the C. botulinum Group I strain ATCC 3502 was grown in continuous culture at 39°C and exposed to heat shock at 45°C, followed by prolonged heat stress at 45°C to allow adaptation of the culture to the high temperature. Growth in continuous culture was performed to exclude secondary growth phase effects or other environmental impacts on bacterial gene transcription. Changes in global gene expression profiles were studied using DNA microarray hybridization. During acute heat stress, Class I and III heat shock genes as well as members of the SOS regulon were activated. The neurotoxin gene botA and genes encoding the neurotoxin-associated proteins were suppressed throughout the study. Prolonged heat stress led to suppression of the sporulation machinery whereas genes related to chemotaxis and motility were activated. Induced expression of a large proportion of prophage genes was detected, suggesting an important role of acquired genes in the stress resistance of C. botulinum. Finally, changes in the expression of a large number of genes related to carbohydrate and amino acid metabolism indicated remodeling of the cellular metabolism.食源性致病菌在食品加工、贮藏、制备以及宿主体内会遭遇多种环境胁迫。为提升食品安全性，阐明食源性致病菌所采用的分子胁迫应答机制至关重要。许多在热激过程中被激活的应答机制，可使细菌获得对其他环境胁迫的交叉保护能力。为更深入地解析肉毒梭菌（Clostridium botulinum，肉毒中毒的病原菌）在急性热胁迫以及适应极端高温过程中所利用的分子机制，本研究以I型肉毒梭菌菌株ATCC 3502为实验材料，先在39℃下开展连续培养，随后将其置于45℃进行热激处理，继而在45℃下进行持续热胁迫以使培养物适应高温环境。采用连续培养方式，旨在排除细菌次级生长阶段效应及其他环境因素对细菌基因转录的干扰。本研究通过DNA微阵列杂交（DNA microarray hybridization）技术，分析了全基因组基因表达谱的变化情况。在急性热胁迫阶段，I类、III类热休克基因以及SOS调控子（SOS regulon）的成员均被激活。神经毒素基因botA与编码神经毒素相关蛋白的基因在本研究全程均处于表达抑制状态。持续热胁迫会抑制芽孢形成相关系统的表达，而趋化性与运动相关基因的表达则被激活。研究检测到大量前噬菌体（prophage）基因的表达上调，这表明外源获得基因在肉毒梭菌的胁迫抗性中发挥着重要作用。最后，大量碳水化合物与氨基酸代谢相关基因的表达变化，提示细胞代谢发生了重塑。