Clostridium difficile Toxin Gene Regulation

艰难梭菌毒素基因调控

• 批准号: 6869008
• 负责人: ABRAHAM Lincoln SONENSHEIN
• 金额: $ 31.71万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-12-15 至 2009-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6869008
• 关键词: Clostridium difficile; RNase protection assay; bacterial genetics; bacterial proteins; bacterial toxins; biosynthesis; cues; environment; enzyme linked immunosorbent assay; gene expression; gene induction /repression; genetic regulation; genetic strain; western blottings

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.

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，也是伪膜性结肠炎（一种潜在致命疾病）的唯一已知原因。由 toxA 和 toxB 基因编码的两个大毒素蛋白位于 19 kb 致病性胰岛内，是主要毒力因子。申请人实验室先前的工作表明，tox基因的转录完全取决于TxeR，TxeR是在相同致病基因座内编码的新型RNA聚合酶σ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稳定期细胞，并被快速代谢的碳源（例如葡萄糖）抑制。影响毒素合成的其他因素包括生物素和某些氨基酸的可用性以及培养温度。本提案旨在确定响应环境信号控制毒素合成的分子机制。基于比较基因组学的候选调节蛋白将专门测试它们对此类调节的参与。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可以介导所讨论的调节效应类型。此外，将基于亲和层析对相关调节蛋白进行一般、公正的搜索。第四种蛋白质 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分析方面取得的最新进展。