PEPTIDE AUTOINDUCERS OF STAPHYLOCOCCAL PATHOGENICITY

葡萄球菌致病性肽自诱导剂

• 批准号: 6510804
• 负责人: Richard P. Novick
• 金额: $ 32.56万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-05-15 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6510804
• 关键词: Staphylococcus aureus; X ray crystallography; antigen presentation; bacteria infection mechanism; bacterial cytopathogenic effect; biological signal transduction; disease /disorder model; gene induction /repression; intermolecular interaction; nuclear magnetic resonance spectroscopy; peptides; protein binding; protein purification; pulsed field gel electrophoresis; receptor expression; tissue /cell culture; virulence

DESCRIPTION (Adapted from the applicant's abstract): The virulence genes of Staphylococcus aureus are regulated by a novel system that combines quorum sensing with signal transduction. The autoinducing molecules are peptides that may have a cyclic structure. The PI has developed a hypothetical model for how autoinduction works. A prepeptide is synthesized inside the cell, then excreted. During excretion, the peptide is cleaved and cyclized. When a high enough concentration is reached, the peptides bind to the receptor AgrC, probably forming a covalent bond. This changes the conformation of AgrC, starting a signal transduction cascade that activates ArgA and leads to the induction of virulence genes controlled by this system. The autoinducing peptides of one type of S. aureus can inhibit gene expression in other types of S. aureus, a finding that may explain how some strains of S. aureus can prevent others from colonizing a mucosal site. The PI proposes to test this model in a systematic fashion. 1. The PI will determine the mechanism of processing and secretion of the peptides by following intermediates in the intact cell. An attempt will be made to purify the proteins involved in this reaction so that an in vitro system can be developed. 2. The PI will develop a method for producing the final form of the autoinducer, which he believes may be an octapeptide cyclized with a thioester bond. A combination of NMR and X-ray crystallography will be used to prove the structure of the autoinducer and probe the structure of the inducer-receptor complex. 3. The PI will use labeled autoinducer to determine the binding site for this ligand on the receptor protein, AgrC. The PI hopes that this analysis will be facilitated by the covalent bond that may occur between the ligand and receptor. The location of the region to target for more intensive investigation will be obtained by domain swapping and deletion analysis. 4. The PI has noted that the argBCD sequences appear to be chimeric, with some highly conserved and some highly variable regions. He will determine how sequence diversification arises by creating strains with heterologous genes and use an agr-tetK fusion to select for gain of function mutants or start with homologous gene sets and use a spa-tetK fusion to select for loss of function. Only mutations within the region will be examined. 5. The PI has noted a possible relationship between some biotypes of S. aureus and the grouping of the autoinducers. He will determine whether biotypes such as exoprotein pattern, pulsed field signature, and enzyme electrophoresis. A large number of strains from known sites and disease states will be screened. The significance of this is first that arg may regulate the correlated factors and second that it may provide new insights into the factors behind the apparent tissue tropism of many S. aureus strains. 6. The PI will use three different animal models to test the hypothesis that cross inhibition of S. aureus agr genes by autoinducer from another strain. Pairs of different strains will be competed in these models, which test for different S. aureus attributes such as ability to colonize skin and ability to infect deep tissues. Mutants will also be tested in these models to determine the importance of the autoinduction system, and an attempt will be made to detect expression of agr genes in the animal.

描述(改编自申请人的摘要)：毒力基因 金黄色葡萄球菌受一种新的结合Quorum的系统调节 通过信号转导进行传感。自我诱导的分子是多肽 这可能具有循环结构。PI已经开发了一个假设模型 了解自动感应的工作原理。在细胞内合成一种前肽， 然后被排泄出来。在排泄过程中，多肽被切割和环化。什么时候 当达到足够高的浓度时，这些多肽就会与受体结合 AgC，可能形成共价键。这改变了构象 AGRC，启动信号转导级联激活ARGA和导联 该系统控制的毒力基因的诱导。这个 一种金黄色葡萄球菌自身诱导肽可抑制基因表达 在其他类型的金黄色葡萄球菌中，一项发现可能解释了一些菌株是如何 金黄色葡萄球菌可以防止其他细菌侵占黏膜部位。《少年派》 建议以系统的方式测试这一模型。 1.等电点将决定蛋白质的加工和分泌机制 通过跟随完整细胞中的中间产物来合成多肽。一次尝试将是 用来提纯参与这一反应的蛋白质，以便在体外 系统是可以开发的。 2.PI将制定一种方法来产生最终形式的 自动诱导剂，他认为它可能是一个环化了的八肽 硫酯键。将使用核磁共振和X射线结晶学相结合的方法 为了证明自动诱导器的结构，并探索自动诱导器的结构 诱导剂-受体复合体。 3.PI将使用标记的自动诱导剂来确定与 受体蛋白AGRC上的这一配体。公安部希望这一分析 将通过可能发生在配体之间的共价键来促进 和受体。以区域为目标的位置更加密集 调查将通过域名交换和删除分析获得。 4.PI注意到，argBCD序列似乎是嵌合的， 一些高度保守的区域和一些高度可变的区域。他会决定 如何通过创造具有异源的菌株来实现序列多样化 基因，并使用agr-tek融合来选择功能突变体或 从同源基因集开始，使用SpA-ETK融合来选择丢失 功能的一部分。只会检查该区域内的突变。 5.PI注意到一些生物型之间的可能关系。 金色和自动诱导器的分组。他将决定是否 生物类型，如外源蛋白模式、脉冲场特征和酶 电泳法。来自已知地点和疾病的大量菌株 各州将接受筛选。这一点的意义首先是Arg可能 调节相关因素；第二，它可能提供新的见解 许多金黄色葡萄球菌明显的组织嗜性背后的因素 菌株。 6.PI将使用三种不同的动物模型来检验这一假设 自体诱导剂对金黄色葡萄球菌agr基因的交叉抑制 紧张。不同的菌株对将在这些模型中竞争， 测试不同的金黄色葡萄球菌属性，如定植皮肤和 感染深层组织的能力。突变体也将在这些模型中进行测试。 以确定自动感应系统的重要性，并尝试 用来检测AGR基因在动物体内的表达。