PEPTIDE AUTOINDUCERS OF STAPHYLOCOCCAL PATHOGENICITY

描述（改编自申请人摘要）： 金黄色葡萄球菌受一种新的系统调节， 通过信号转导进行传感。 自诱导分子是肽 其可以具有环状结构。 私家侦探建立了一个假设模型 自动归纳的原理 前肽在细胞内合成， 然后排泄。 在排泄过程中，肽被切割和环化。 当 当达到足够高的浓度时， AgrC，可能形成共价键。 这改变了 AgrC启动信号转导级联，激活ArgA并导致 诱导由该系统控制的毒力基因。 的 一种类型的S.金黄色葡萄球菌可抑制基因表达 在其他类型的S.金黄色葡萄球菌，这一发现可能解释了一些菌株 S.金黄色葡萄球菌可以防止其他细菌在粘膜部位定植。 的PI 他建议以系统的方式测试这个模型。 1. PI将确定处理和分泌的机制， 肽的后续中间体在完整的细胞。 将尝试 纯化参与该反应的蛋白质， 系统可以开发。 2. PI将开发一种生产最终形式的方法， 自诱导物，他认为这可能是一个八肽环化与一个 硫酯键 将使用NMR和X射线晶体学的组合 为了证明自诱导物的结构， 诱导物-受体复合物 3. PI将使用标记的自诱导剂来确定 受体蛋白AgrC上的配体 PI希望这种分析 将通过配体之间可能发生的共价键来促进 和受体。 该地区的定位目标更密集 将通过域交换和缺失分析获得调查结果。 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。 他将决定 序列多样化是如何通过创造具有异源性的菌株而产生的 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，并使用spa-tetK融合来选择丢失 的功能。 仅检查区域内的突变。 5. 主要研究者注意到了沙门氏菌某些生物型之间的可能关系。 金黄色葡萄球菌和自身诱导物的分组。 他将决定是否 生物型，如外蛋白模式，脉冲场签名，和酶 电泳 来自已知地点和疾病的大量菌株 将对各州进行筛选。 这一点的重要性首先在于，arg可以 调节相关因素，第二，它可以提供新的见解 许多S.金黄色 菌株 6. PI将使用三种不同的动物模型来检验假设 S.金黄色葡萄球菌agr基因通过自诱导剂从另一个 株 不同菌株对将在这些模型中竞争， 测试不同的S.金黄色葡萄球菌的属性，如定殖皮肤的能力， 感染深层组织的能力。 突变体也将在这些模型中进行测试 确定自动感应系统的重要性，并尝试将 用于检测动物体内agr基因的表达。

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.