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

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 使用广谱抗生素治疗会扰乱正常的肠道微生物区系，从而导致CD 殖民主义。虽然CDI是一种毒素介导的疾病，但它的传播能力，因此， 病害依赖于孢子的形成和萌发。CD孢子在代谢上变得活跃起来 (萌发)当包裹在孢子内的受体识别寄主特定的分子(例如胆汁)时 盐、甘氨酸)称为发芽剂。目前，Cd萌发的机制还不完全清楚。 明白了。其他芽胞形成细菌萌发所需的许多蛋白质，包括 已知的萌发受体，在测序的CD菌株的基因组中没有编码。感受器 对于胆盐，牛磺胆酸盐(TC)已被鉴定为CSPC。然而，该受体(S) 所需的共同发芽剂甘氨酸仍不清楚。我们在认识上有一个根本性的差距 控制甘氨酸对CD孢子有效萌发的需要的机制。 解决这一差距具有开发治疗学的潜力，可以改善 CDI的结果，并可为中断持续感染提供新的目标，减少 复发性CDI的负担。中心假设是甘氨酸在环境中通过 与孢子内的受体发生特定的相互作用。这些相互作用会触发岩心再水化和 随后释放钙-二吡啶甲酸(Ca-DPA)。反过来，Ca-DPA转导种子萌发 来自孢子核的信号诱导成熟的SLEC的皮层水解物。以实质为指导 初步数据显示，这一假设将在以下两个具体目标中得到检验：1)确定 TC-甘氨酸诱导萌发所必需的蛋白质。申请人已经鉴定出一种蛋白质，它是 在产孢期高表达，是甘氨酸诱导萌发所必需的，称为GsgA。 其目的是确定GsgA在CD萌发和小鼠发病机制中的作用 模特。2)TC-CaDPA诱导种子萌发机理的研究。申请人有 发现了一种诱导Cd高效萌发的新方法。通过使用Tc和CaDPA， 申请者能够避开甘氨酸的需求。这一目标的目的是阐明 TC-CaDPA诱导种子萌发的机理这项拟议的研究意义重大，因为它将 洞察Cd萌发的未知机制。最终，这一知识将 为使用可能改善结果的新的治疗靶点提供重要的基础 适用于复发的CDI患者。

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.