Biosynthesis of Non-Native Autoinducing Peptides

项目总结 群体感应(QS)是同一物种的细菌在较高水平上协调行为的过程 人口密度。在许多致病细菌中，QS系统被用来调节毒力。这项建议 重点介绍了在几种革兰氏阳性病原菌中发现的辅助基因调节(AGR)型QS系统， 包括金黄色葡萄球菌、表皮葡萄球菌、单核细胞增多性李斯特菌和梭状芽孢杆菌 艰难抉择。AGR型QS系统包含四种蛋白质，AGRA-D，它们共同产生并响应一种 自身诱导肽(AIP)信号。我将研究负责信号传递的两种蛋白质，agRB和agRG D 生物合成。在这个途径中，多肽前体agrD被agrB和胞外蛋白水解酶加工。 以产生AIP信号。在目标1中，我将突变金黄色葡萄球菌agrD的AIP区域，测试看看AIP是否 信号仍在产生。通过反复几轮的突变，我将确定多肽的位置和目标 可以容忍范围的变化。嵌入此方法以了解AIP的基本机制 处理是另外两个目标，其中一个已经实现。首先，我已经测试了 天然系统产生非天然AIP类似物，作为金黄色葡萄球菌QS的有效抑制剂，并具有 证明了金黄色葡萄球菌QS的两种高效泛基抑制剂可以用我的 系统。第二，通过揭示AIP信号的哪些残基可以在不损失处理的情况下突变，即 可以测试生产的非天然AIP类似物抑制金黄色葡萄球菌中QS的能力，潜在地发现 新的更有效的抑制剂。对于目标2，我将设计一种非致病细菌来结构性表达 在目标1中生物合成的QS抑制剂。然后我将测试益生菌菌株预防金黄色葡萄球菌的能力 秀丽隐杆线虫模型的致病性。Aim 3将继续研究AgD的加工，但将 将焦点转移到最后一步的研究上，在这一步中，细胞外蛋白水解酶将农业D肽裂解为 发出最终AIP信号。这一过程的一个谜团是，AIP信号，即使是单个物种的信号，通常 它们的蛋白分解部位不同。为了发现这种变异性的驱动因素，我将对agrD进行定向突变 并将天然的agrD序列的蛋白水解点与突变序列进行比较。应用这些知识，我 然后将生物合成设计者AIP类似物，将来自两个或更多原生AIP信号的特征结合在一起。 这三个目标加在一起将大大增加我们对AIP生物合成的理解，并提供一种新的 在人类主要病原体中获得抑制agr型qs系统的有价值的化学工具的途径。

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.