Clostridium difficile Toxin Gene Regulation

描述(申请人提供)：艰难梭菌是抗生素相关性结肠炎的主要病原体，也是伪膜性结肠炎的唯一已知原因，伪膜性结肠炎是一种潜在的致命疾病。由毒素A和毒素B基因编码的两个大的毒素蛋白位于一个19kb的致病胰岛内，是主要的毒力因子。申请人实验室之前的工作表明，TOX基因的转录完全依赖于TxeR，这是一种新的RNA聚合酶西格玛因子，编码在相同的致病基因位点内。然而，表达水平受到细胞生长状态和环境条件的强烈调控。例如，对于在肉汤培养基中生长的细胞，TOX基因的转录仅限于稳定期细胞，并受到葡萄糖等快速代谢碳源的抑制。影响毒素合成的其他因素是生物素和某些氨基酸的可用性以及培养温度。本提案试图确定控制毒素合成以响应环境信号的分子机制。基于比较基因组学的候选调控蛋白将接受专门的测试，以确定它们是否参与了这种调控。这些蛋白质包括Cody、CCPA和VirR，它们在枯草芽孢杆菌或产气荚膜梭菌中的同源物已被证明介导了所讨论的调节效应的类型。此外，将根据亲和层析对相关调节蛋白进行一般性、无偏见的搜索。第四种蛋白质TcdC将作为TxeR的拮抗剂进行潜在活性测试。该项目利用了三个合作研究小组在艰难梭菌生物化学、遗传学和生理学方面的专业知识，以及每个小组在使这一实验系统能够进行详细的分子遗传分析方面取得的最新进展。

DESCRIPTION (provided by applicant): Clostridium difficile is the principal causative agent of antibiotic-associated colitis and the only known cause of pseudomembranous colitis, a potentially lethal disease. Two large toxin proteins, encoded by the toxA and toxB genes, which lie within a 19 kb pathogenicity islet, are the primary virulence factors. Previous work from the applicants' laboratories has shown that transcription of the tox genes depends entirely on TxeR, a novel RNA polymerase sigma factor encoded within the same pathogenicity locus. The level of expression is strongly modulated, however, by the growth state of the cells and by environmental conditions. For instance, for cells growing in broth medium, tox gene transcription is restricted to stationary phase cells and is repressed by rapidly metabolizable carbon sources, such as glucose. Other factors that influence toxin synthesis are the availability of biotin and certain amino acids and the temperature of cultivation. The present proposal seeks to determine the molecular mechanisms that control toxin synthesis in response to environmental signals. Candidate regulatory proteins, based on comparative genomics, will be specifically tested for their participation in such regulation. These proteins include CodY, CcpA and VirR, whose homologs in Bacillus subtilis or Clostridium perfringens have been shown to mediate the types of regulatory effects in question. In addition, general, unbiased searches for the relevant regulatory proteins will be carried out based on affinity chromatography. A fourth protein, TcdC, will be tested for its potential activity as an antagonist of TxeR. The project makes use of the expertise in C. difficile biochemistry, genetics and physiology of the three collaborating research groups and recent advances made by each of the groups in making this experimental system amenable to detailed molecular genetic analysis.