Identification of the full scope of the CodY regulon in Clostridioides difficile

艰难梭菌中 CodY 调节子的完整范围鉴定

• 批准号: 10318205
• 负责人: BORIS R BELITSKY
• 金额: $ 8.25万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-12-15 至 2023-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10318205
• 关键词: Address; Affect; Affinity Chromatography; Anaerobic Bacteria; Animals; Bacillus subtilis; Binding; Binding Sites; Cells; ChIP-seq; Clostridium difficile; Code; Complement; Coupled; DNA; DNA Microarray Chip; Data; Detection; Disease; Environment; Epidemic; Foundations; Gene Expression; Genes; Genetic; Genetic Transcription; Genomics; Germination; Gram-Positive Bacteria; Growth; Human; In Vitro; Individual; Infection; Intestines; Knowledge; Laboratories; Location; Masks; Massive Parallel Sequencing; Mediating; Metabolic; Metabolic Pathway; Metabolism; Microarray Analysis; Mutation; Nucleotides; Organism; Pathogenicity; Positioning Attribute; Production; Proteins; Pseudomembranous Colitis; Recurrence; Regulation; Regulator Genes; Regulon; Reproduction spores; Resolution; Ribotypes; Role; Sigma Factor; Site; Time; Toxin; Untranslated RNA; Virulence; Virulent; Work; alpha Toxin; antibiotic-associated diarrhea; cell type; experimental study; gene complementation; gene repression; genome-wide; in vivo; mouse model; mutant; novel; novel strategies; pathogen; premature; prevent; tool; transcription termination; transcriptome sequencing; whole genome

ABSTRACT Clostridioides (formerly Clostridium) difficile is a Gram-positive, sporulating, anaerobic bacterium that can cause severe disease, including antibiotic-associated diarrhea and pseudomembranous colitis, in humans. Pathogenic C. difficile produces at least two potent toxins, TcdA and TcdB, which cause major intestinal damage to the host. Formation of spores by C. difficile is critical for survival of the organism in the environment, initiation of infection (normally caused by spores), and recurrence of the disease due to re-germination of spores formed in the gut of infected humans or animals and their ability to cause a new round of infection. A global transcriptional regulator CodY appears to be the most important regulator of toxin-encoding genes, and codY mutants of C. difficile produce more toxins and are more virulent than wild-type strains in a mouse model of infection. CodY also controls expression of genes involved in sporulation, and codY mutants sporulate more efficiently. Moreover, CodY affects expression of multiple metabolic pathways that are likely to be important for growth and virulence. Although CodY is known to directly repress the tcdR gene, encoding a toxin-specific sigma factor, the mechanism of CodY’s effect on toxin gene expression has not been fully established. The direct targets of CodY that control C. difficile sporulation gene expression and spore formation remain completely unknown. To understand in full the mechanisms of CodY’s effect on C. difficile virulence, sporulation, and metabolism, it is essential to determine the full scope of the direct CodY targets via identification at single- nucleotide detail of the entire complement of CodY-binding sites. Therefore, we propose to determine the entire set of genes that are able to interact with CodY directly either in vivo or in vitro. Using a novel approach, in vitro DNA affinity purification coupled with massively parallel sequencing (IDAP-Seq), we are now able to visualize CodY-binding sites in vitro on a genome-wide basis at near single-nucleotide resolution and rank the sites according to their relative strengths. In addition, we will use ChIP-Seq experiments, also at near single- nucleotide resolution, to define the genome-wide set of sites that interact with CodY in vivo. Comparing the data generated by these approaches with the results of in vivo expression analysis (RNA-Seq) will allow us to (a) identify direct and indirect targets of CodY regulation; (b) determine whether the breadth of the CodY regulon is significantly greater than presently known; (c) uncover potential targets of CodY responsible for its effect on sporulation; (d) find the genes that are potentially subject to dual control by CodY and other regulators; and (e) establish whether premature transcription termination within coding sequences is involved in CodY-mediated regulation of toxin-encoding and other C. difficile genes. The precise information on the locations and relative strengths of CodY-binding sites for critical genes will provide the foundation for future research on the detailed mechanisms by which CodY regulates individual genes.

抽象的 艰难梭菌 (Clostridium difficile) 是一种革兰氏阳性、产孢子、厌氧细菌， 可导致人类严重疾病，包括抗生素相关性腹泻和伪膜性结肠炎。 致病性艰难梭菌产生至少两种强效毒素，TcdA 和 TcdB，可导致肠道严重损伤。 对主机造成损害。艰难梭菌孢子的形成对于生物体在环境中的生存至关重要， 感染的开始（通常由孢子引起），以及由于孢子的重新萌发而导致疾病复发 受感染的人类或动物肠道中形成的孢子及其引起新一轮感染的能力。一个 全局转录调节因子 CodY 似乎是毒素编码基因最重要的调节因子，并且 在小鼠模型中，艰难梭菌的 codY 突变体比野生型菌株产生更多毒素且毒性更强 的感染。 CodY 还控制与孢子形成有关的基因的表达，并且 codY 突变体产生更多的孢子 高效。此外，CodY 影响多种代谢途径的表达，这些途径可能对 生长和毒力。尽管 CodY 已知可直接抑制 tcdR 基因，编码毒素特异性 sigma因子，CodY影响毒素基因表达的机制尚未完全确定。这 CodY 控制艰难梭菌孢子形成基因表达和孢子形成的直接靶点仍然存在 完全未知。 全面了解 CodY 对艰难梭菌毒力、孢子形成和生长的影响机制 代谢，有必要通过单点鉴定来确定直接 CodY 靶标的全部范围。 CodY 结合位点的整个互补序列的核苷酸细节。因此，我们建议确定 能够在体内或体外直接与 CodY 相互作用的整套基因。使用新颖的方法， 体外 DNA 亲和纯化结合大规模并行测序 (IDAP-Seq)，我们现在能够 以接近单核苷酸的分辨率在全基因组范围内体外可视化 CodY 结合位点，并对 根据网站的相对优势。此外，我们将使用 ChIP-Seq 实验，也是在近单 核苷酸分辨率，以确定体内与 CodY 相互作用的全基因组位点集。比较 这些方法生成的数据以及体内表达分析（RNA-Seq）的结果将使我们能够 (a) 确定 CodY 监管的直接和间接目标； (b) 确定CodY的宽度是否 调节子明显大于目前已知的； (c) 发现 CodY 的潜在目标 对孢子形成的影响； (d) 找到可能受到 CodY 和其他基因双重控制的基因 监管机构； (e) 确定是否涉及编码序列内的过早转录终止 CodY 介导的毒素编码基因和其他艰难梭菌基因的调节。有关的准确信息 关键基因 CodY 结合位点的位置和相对强度将为未来奠定基础 研究CodY调节个体基因的详细机制。