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）： 金黄色葡萄球菌由结合法定人数的新型系统调节 通过信号转导感测。 自动诱导分子是肽 那可能具有循环结构。 PI开发了一个假设的模型 关于自动诱导的工作方式。 预肽是在细胞内合成的， 然后排出。 在排泄过程中，将肽裂解和环旋。 什么时候 达到足够高的浓度，肽与受体结合 AGRC，可能形成共价键。 这改变了 AGRC，启动一个激活Arga并引线的信号转导级联 诱导该系统控制的毒力基因。 这 金黄色葡萄球菌的自动诱导肽可以抑制基因表达 在其他类型的金黄色葡萄球菌中，这一发现可能解释了某些菌株如何 金黄色葡萄球菌可以防止其他人定居粘膜部位。 pi 建议以系统的方式测试此模型。 1。PI将确定处理和分泌的机制 通过遵循完整细胞中的中间体来肽。 尝试 纯化参与该反应的蛋白质，以便在体外 可以开发系统。 2。PI将开发一种生成最终形式的方法 自动诱导剂，他认为这可能是用八肽的二肽 锡骨人债券。 将使用NMR和X射线晶体学的组合 证明自动引诱器的结构并探测 诱导者受体复合物。 3。PI将使用标记的自动引诱器来确定 该受体蛋白上的该配体Agrc。 PI希望这种分析 配体之间可能发生的共价债券将促进 和受体。 该地区针对更密集的地区的位置 调查将通过域交换和缺失分析获得。 4。PI注意到ArgBCD序列似乎是嵌合，带有 一些高度保守和一些高度可变的区域。 他将确定 序列多样化是如何通过与异源创建菌株来产生的 基因并使用AGR-TETK融合来选择获得功能突变体或 从同源基因组开始，然后使用SPA-TETK融合来选择损失 功能。 仅检查该地区内的突变。 5。PI注意到S的某些生物型之间可能存在关系。 金黄色和自动诱导仪的分组。 他将确定是否 生物型，例如摄起蛋白模式，脉冲场特征和酶 电泳。 来自已知地点和疾病的大量菌株 状态将被筛选。 这首先是Arg可能 规范相关因素，其次是它可以提供新的见解 进入许多金黄色葡萄球菌的表观组织向量的因素 菌株。 6。PI将使用三种不同的动物模型来检验假设 通过自动引诱剂从另一个 拉紧。 成对的不同菌株将在这些模型中竞争， 测试不同的金黄色葡萄球菌属性，例如能够定居皮肤和 能够感染深层组织的能力。 在这些模型中也将测试突变体 确定自动诱导系统的重要性，并尝试 制作以检测动物中AGR基因的表达。