Biosynthesis of Non-Native Autoinducing Peptides

项目摘要 群体感应（Quorum sensing，QS）是同一物种的细菌在高浓度下协调行为的过程。 人口密度在许多病原菌中，QS系统用于调节毒力。这项建议 重点关注在几种革兰氏阳性病原体中发现的辅助基因调节器（agr）型QS系统， 包括金黄色葡萄球菌、表皮葡萄球菌、单核细胞增生李斯特菌和梭菌 很难Agr型QS系统含有四种蛋白质AgrA-D，它们一起产生并响应于一种蛋白质。 自诱导肽（AIP）信号。我将研究两种蛋白质，AgrB和AgrD，它们负责信号传导， 生物合成在该途径中，肽前体AgrD被AgrB和细胞外蛋白酶加工 产生AIP信号在目标1中，我将突变S的AIP区域。金黄色葡萄球菌AgrD，检测AIP是否 信号还在产生。通过一轮又一轮的突变，我将确定肽的位置和方向 容忍程度变化。嵌入在这种方法中，以了解AIP的基本机制 处理是两个额外的目标，其中一个已经实现。首先，我测试了 天然系统产生非天然AIP类似物，其充当S.金黄色葡萄球菌QS， 证明了两种高效的S.金黄色葡萄球菌QS可以用我的 系统其次，通过揭示AIP信号的哪些残基可以突变而不损失加工， 可以测试产生的非天然AIP类似物抑制S.金黄色葡萄球菌，可能发现 新的和更有效的抑制剂。对于目标2，我将设计一种非致病性细菌， 目的1中生物合成的QS抑制剂。然后，我将测试益生菌菌株的能力，以防止S。金黄色 秀丽隐杆线虫模型中的致病性。Aim 3将继续研究AgRD的加工， 将焦点转移到研究最后一步，其中细胞外蛋白酶切割AgrD肽， 产生最终AIP信号。这个过程的一个奥秘是，AIP信号，即使是在一个单一的物种，往往 不同的是它们的蛋白水解位点。为了发现是什么驱动了这种变异性，我将对AgrD进行靶向突变， 并比较天然AgrD序列与突变序列的蛋白水解位点。运用这些知识，我 然后将生物合成设计的AIP类似物，所述设计的AIP类似物结合来自两种或更多种天然AIP信号的联合收割机特征。 这三个目标将大大增加我们对AIP生物合成的理解，并提供一种新的 途径有价值的化学工具抑制agr型QS系统在主要的人类病原体。

PROJECT SUMMARY Quorum sensing (QS) is the process by which bacteria of the same species coordinate behavior at a high population density. In many pathogenic bacteria, QS systems are used to regulate virulence. This proposal focuses on the accessory gene regulator (agr)-type QS systems found in a several Gram-positive pathogens, including Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes, and Clostridioides difficile. Agr-type QS systems contain four proteins, AgrA-D, that together produce and respond to an autoinducing peptide (AIP) signal. I will study the two proteins, AgrB and AgrD, that are responsible for signal biosynthesis. In this pathway, the peptide precursor AgrD is processed by AgrB and an extracellular protease to produce the AIP signal. In Aim 1, I will mutate the AIP region of S. aureus AgrD, testing to see if the AIP signal is still produced. Through iterative rounds of mutation, I will determine where in the peptide and to what extent variation is tolerated. Imbedded within this approach to understand the basic mechanisms of AIP processing is two additional goals, one of which has already been realized. First, I have tested the ability of the native system to produce non-native AIP analogs that act as potent inhibitors of S. aureus QS and have demonstrated that two highly potent pan-group inhibitors of S. aureus QS can be biosynthesized using my system. Second, by uncovering which residues of the AIP signal can be mutated without a loss of processing, I can test the non-native AIP analogs produced for their ability to inhibit QS in S. aureus, potentially discovering new and more potent inhibitors. For Aim 2, I will engineer a non-pathogenic bacterium to constitutively express the QS inhibitors biosynthesized in Aim 1. Then I will test the probiotic strain’s ability to prevent S. aureus pathogenicity in a Caenorhabditis elegans model. Aim 3 will continue to study the processing of AgrD but will switch the focus to investigating the final step, wherein an extracellular protease cleaves the AgrD peptide to yield final AIP signal. One mystery of this process is that AIP signals, even those within a single species, often differ in their proteolysis site. To discover what drives this variability, I will make targeted mutations to AgrD and compare proteolysis sites for the native AgrD sequence to mutant sequences. Applying this knowledge, I will then biosynthesize designer AIP analogs that combine features from two or more native AIP signals. Together these three aims will significantly increase our understanding of AIP biosynthesis and provide a novel pathway to valuable chemical tools for inhibiting agr-type QS systems in major human pathogens.