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

摘要 艰难梭菌（Clostridioides）（以前称为梭状芽孢杆菌（Clostridium difficile））是革兰氏阳性、形成孢子的厌氧细菌， 可导致严重的疾病，包括人类腹泻和伪膜性结肠炎。 病原C.艰难梭菌产生至少两种有效的毒素，TcdA和TcdB，其引起主要的肠道疾病。 对宿主的伤害。C.艰难梭菌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），以及由于细菌的重新萌发而导致的疾病复发。 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 全局转录调节子CodY似乎是毒素编码基因的最重要的调节子， codY突变体。在小鼠模型中，艰难梭菌比野生型菌株产生更多的毒素并且毒性更强 感染CodY还控制与孢子形成有关的基因的表达，并且codY突变体产生更多孢子。 有效地此外，CodY影响多个代谢途径的表达，这些代谢途径可能对 生长和毒性。虽然已知CodY直接抑制tcdR基因，编码毒素特异性的 虽然CodY对毒素基因表达的影响机制尚未完全确定，但它对毒素基因表达的影响机制尚未完全确定。的 控制C.艰难梭菌孢子形成基因表达和孢子形成仍然存在 完全未知 为全面了解CodY对C.艰难梭菌的毒力、孢子形成和 代谢，它是必要的，以确定直接CodY目标的全部范围，通过鉴定在单一的， 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。使用一种新颖的方法， 体外DNA亲和纯化与大规模平行测序（IDAP-Seq）相结合，我们现在能够 在体外以接近单核苷酸的分辨率在全基因组的基础上可视化CodY结合位点， 根据他们的相对优势。此外，我们将使用ChIP-Seq实验，也是在近单- 核苷酸分辨率，以确定在体内与CodY相互作用的全基因组位点集。比较 通过这些方法产生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监管的广度 调节子明显大于目前已知的;（c）发现CodY的潜在靶点，负责其 （d）发现可能受到CodY和其他生物因子双重控制的基因， 调节子;以及（e）确定是否涉及编码序列内的过早转录终止 在CodY介导的毒素编码和其他C. difficile基因关于这方面的确切信息 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奠定基础。 研究CodY调节单个基因的详细机制。

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.