Clostridium difficile Toxin Gene Regulation

描述（由申请人提供）：艰难梭菌是抗生素相关性结肠炎的主要病原体，也是假膜性结肠炎的唯一已知原因，假膜性结肠炎是一种潜在的致命疾病。由弓形虫a和弓形虫b基因编码的两个大的毒素蛋白位于一个19 kb的致病性胰岛内，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， TxeR是一种在相同致病位点编码的新型RNA聚合酶sigma因子。然而，表达水平受到细胞生长状态和环境条件的强烈调节。例如，对于在肉汤培养基中生长的细胞，毒素基因转录仅限于固定期细胞，并被快速代谢的碳源（如葡萄糖）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。本提案旨在确定控制毒素合成的分子机制，以响应环境信号。候选调节蛋白，基于比较基因组学，将被专门测试其参与这种调节。这些蛋白包括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可以介导所讨论的各种调节作用。此外，将基于亲和层析对相关调节蛋白进行一般的、无偏的搜索。第四种蛋白，TcdC，将测试其作为TxeR拮抗剂的潜在活性。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，以及每个小组在使该实验系统适合详细的分子遗传学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.