Characterization of the Mechanism of Glycine induced Germination of Clostridium difficile Spores

甘氨酸诱导艰难梭菌孢子萌发机制的表征

• 批准号: 9189972
• 负责人: Travis Joseph Kochan
• 金额: $ 3.45万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9189972
• 关键词: Address; Affect; Aftercare; Amino Acids; Anaerobic Bacteria; Antibiotics; Bacillus (bacterium); Bacteria; Bacterial Spores; Binding; Calcium; Cessation of life; Clinical; Clostridium difficile; Community Hospitals; Data; Desiccation; Development; Diarrhea; Disease; Drug Design; Environment; Enzyme Precursors; Ethanol; Figs - dietary; Foundations; Genes; Germination; Glycine; Goals; Heating; Hydration status; Hydrolysis; Infection; Knowledge; Lead; Location; LytA enzyme; Mediating; Membrane; Methods; Modeling; Outcome; Oxygen; Pathogenesis; Pathway interactions; Patients; Peptide Hydrolases; Process; Property; Proteins; Recurrence; Rehydrations; Reproduction spores; Research; Resistance; Role; Signal Transduction; Subtilisins; Taurine Cholate; Testing; Toxin; Water; Work; antimicrobial drug; bile salts; dipicolinic acid; genome sequencing; gut microbiota; improved; improved outcome; insight; mouse model; new therapeutic target; novel; novel strategies; protein function; receptor; small molecule; therapeutic development; transmission process

Project Summary Clostridium difficile (Cd), a Gram-positive anaerobe, is a leading cause of both hospital- and community- acquired antibiotic associated diarrhea. Cd infection (CDI) typically occurs after treatment with broad-spectrum antibiotics disrupts the normal gut microbiota, allowing for Cd colonization. While CDI is a toxin-mediated disease, its capacity for transmission and, therefore, disease is dependent on spore formation and germination. Cd spores become metabolically active (germinate) when receptors packaged within the spore recognize host-specific molecules (e.g., bile salts, glycine) known as germinants. Currently, the mechanism of Cd germination is not completely understood. Many of the proteins required for germination in other spore forming bacteria, including known germinant receptors, are not encoded in the genomes of sequenced Cd strains. The receptor for the bile salt taurocholate (Tc), CspC, has been identified. However, the receptor(s) for the required co-germinant, glycine, remains unknown. There is a fundamental gap in our understanding of the mechanism controlling the requirement of glycine for efficient germination of Cd spores. Addressing this gap has the potential for the development of therapeutics that can improve the outcome of CDI and could provide new targets for disruption of persistent infections, reducing the burden of recurrent CDI. The central hypothesis is that glycine is sensed in the environment through specific interactions with receptors within the spore. These interactions trigger core rehydration and subsequent release of calcium-dipicolinic acid (Ca-DPA). In turn, Ca-DPA transduces the germination signal from the spore core inducing cortex hydrolysis by mature SleC. Guided by substantial preliminary data, this hypothesis will be tested in the following two specific aims: 1) Define the role of proteins essential for Tc-Glycine induced germination. The applicant has identified a protein that is highly expressed during sporulation that is essential for glycine-induced germination, termed GsgA. The goal of this aim is to define the role of GsgA in Cd germination and pathogenesis in a murine model. 2) Characterization of the mechanism of Tc-CaDPA induced germination. The applicant has discovered a novel method to induce efficient germination in Cd. By using Tc and CaDPA the applicant is able to circumvent the need for glycine. The goal of this aim is to elucidate the mechanism for Tc-CaDPA induced germination. The proposed research is significant because it will provide insight into the unknown mechanisms of Cd germination. Ultimately, this knowledge will provide an important foundation for the use of novel therapeutic targets that may improve outcomes for patients with recurrent CDI.

项目摘要 艰难梭菌（CD）是一种革兰氏阳性厌氧菌，是医院和医院的主要原因 社区获得的抗生素相关腹泻。 CD感染（CDI）通常发生在 用广谱抗生素治疗会破坏正常的肠道菌群，从而允许CD 殖民化。尽管CDI是一种毒素介导的疾病，但其传播的能力，因此 疾病取决于孢子的形成和发芽。 CD孢子变为代谢活性 （发芽）当包装在孢子中的受体识别宿主特异性分子时（例如，胆汁 盐，甘氨酸）称为发芽剂。目前，CD发芽的机制不是完全 理解。在其他孢子形成细菌中发芽所需的许多蛋白质，包括 已知的发芽受体未在测序CD菌株的基因组中编码。受体 对于胆汁盐牛排（TC），已鉴定出CSPC。但是， 所需的共晶甘氨酸仍然未知。我们的理解存在根本差距 控制甘氨酸对CD孢子有效发芽的要求的机制。 解决这一差距具有开发治疗剂的潜力，可以改善 CDI的结果，可以为破坏持续感染的新目标提供新的目标，从而减少 经常性CDI的负担。中心假设是通过 与孢子中受体的特定相互作用。这些相互作用触发核心补液，并 随后释放二吡啶酸钙（CA-DPA）。反过来，CA-DPA转导发芽 来自孢子核的信号通过成熟的Slec诱导皮质水解。在实质性的指导下 初步数据，该假设将在以下两个具体目的中进行检验：1）定义 TC-甘氨酸诱导发芽必不可少的蛋白质。申请人已经确定了一种蛋白质 在散发过程中高度表达，这对于甘氨酸诱导的发芽至关重要，称为GSGA。 该目标的目的是确定GSGA在鼠中CD发芽和发病机理中的作用 模型。 2）TC-CADPA诱导发芽机理的表征。申请人有 发现了一种新的方法来诱导CD中有效发芽。通过使用TC和CADPA 申请人能够规避甘氨酸的需求。这个目标的目的是阐明 TC-CADPA诱导发芽的机制。拟议的研究很重要，因为它将 提供有关CD发芽未知机制的见解。最终，这些知识将 为使用新型治疗靶标提供了重要的基础，以改善结果 对于复发性CDI的患者。