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，是医院和医院感染的主要原因。 社区获得性抗生素相关性腹泻。Cd感染（CDI）通常发生在 用广谱抗生素治疗破坏了正常的肠道微生物群，使镉 殖民化虽然CDI是一种毒素介导的疾病，但其传播能力，因此， 疾病依赖于孢子的形成和萌发。Cd孢子变得代谢活跃 （萌发）当包装在孢子内的受体识别宿主特异性分子（例如，胆汁 盐、甘氨酸）作为发芽剂。目前，镉的萌发机理尚不完全清楚， 明白其他孢子形成细菌中萌发所需的许多蛋白质，包括 已知的萌发受体，在测序的Cd菌株的基因组中不编码。受体 对于胆盐牛磺胆酸盐（Tc），CspC已经被鉴定。然而，用于所述细胞的受体（一个或多个）是不稳定的。 所需的助萌发剂甘氨酸仍然未知。在我们的理解中存在着根本性的差距 探讨了镉孢子高效萌发所需甘氨酸的控制机制。 解决这一差距有可能开发出可以改善患者症状的治疗方法。 CDI的结果，并可以提供新的目标，破坏持续感染，减少 经常性CDI的负担。中心假设是甘氨酸在环境中通过 与孢子内受体的特异性相互作用。这些相互作用触发核心再水化， 随后释放钙-吡啶二羧酸（Ca-DPA）。反过来，Ca-DPA转导萌发 来自孢子核的信号诱导成熟SleC水解皮层。在实质性指导下 初步数据，这一假设将在以下两个具体目标进行测试：1）定义的作用， Tc-甘氨酸诱导萌发所必需的蛋白质。申请人已经鉴定了一种蛋白质， 在孢子形成过程中高度表达，对甘氨酸诱导的萌发至关重要，称为GsgA。 本研究的目的是确定GsgA在小鼠镉萌发和发病机制中的作用， 模型2)Tc-CaDPA诱导种子萌发机制的研究申请人已经 发现了一种新的方法来诱导有效的发芽镉。通过使用Tc和CaDPA， 申请人能够避免对甘氨酸的需要。这一目标的目的是阐明 Tc-CaDPA诱导种子萌发的机制。这项研究意义重大，因为它将 提供深入了解镉发芽的未知机制。最终，这些知识将 为使用可能改善结果的新型治疗靶点提供重要基础 对于复发性CDI的患者。