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

摘要 艰难梭菌是一种革兰氏阳性、产孢子、厌氧细菌。 会导致严重的疾病，包括抗生素相关性腹泻和伪膜性结肠炎。 致病性艰难梭菌至少产生两种有效毒素TcdA和TcdB，这两种毒素会导致主要肠道 对主机的损坏。艰难梭菌形成的孢子对有机体在环境中的生存至关重要， 开始感染(通常由孢子引起)，以及因重新萌发而复发的疾病 在受感染的人或动物的肠道中形成的孢子及其引起新一轮感染的能力。一个 全球转录调控因子Cody似乎是毒素编码基因最重要的调控因子，并且 在小鼠模型中，艰难梭菌的Cody突变株比野生型菌株产生更多的毒素和更强的毒力 感染的可能性。Cody还控制与产孢子有关的基因的表达，而Cody突变体产孢量更多 效率很高。此外，Cody还影响多种代谢途径的表达，这些途径可能对 生长和毒力。虽然已知Cody直接抑制tcdR基因，编码一种毒素特异性的 Sigma因子，Cody对毒素基因表达的影响机制尚未完全确定。这个 控制艰难梭菌产孢子基因表达和孢子形成的Cody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、产孢量和产孢量的影响机制 在新陈代谢方面，必须通过在单细胞水平上的鉴定来确定直接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相互作用的一整套基因。使用一种新的方法， 体外DNA亲和纯化结合大规模并行测序(IDAP-Seq)，我们现在能够 在体外以接近单核苷酸分辨率的全基因组基础上可视化Cody结合位点，并对 网站根据它们的相对优势。此外，我们将使用芯片序列实验，也是在接近单- 核苷酸分辨率，以定义在体内与Cody相互作用的全基因组位置集。比较一下 通过这些方法产生的数据与体内表达分析(RNA-Seq)的结果将使我们能够 (A)确定Cody法规的直接和间接目标；(B)确定Cody法规的广度 Regon比目前已知的要大得多；(C)发现Cody负责其 对孢子形成的影响；(D)找出可能受到Cody和其他人双重控制的基因 调节器；以及(E)确定是否涉及编码序列内的提前转录终止 在Cody介导的毒素编码和其他艰难梭菌基因的调节中。关于这一点的准确信息 关键基因的Cody结合位点的位置和相对强度将为未来的研究提供基础 科迪调控单个基因的详细机制的研究。

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.