Biosynthesis of Non-Native Autoinducing Peptides

非天然自诱导肽的生物合成

基本信息

批准号：
10678113
负责人：
Danielle Lee Widner
金额：
$ 6.91万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-03-01 至 2026-02-28
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10678113
关键词：
Affect Amino Acids Anabolism Attenuated Bacteria Behavior Biology Caenorhabditis elegans Chemicals Clostridium difficile Creativeness Endopeptidases Engineering Environment Genetic Transcription Goals Image Infection Investigation Knowledge Laboratories Libraries Listeria monocytogenes Location Methods Modeling Molecular Mutate Mutation N-terminal Pathogenicity Pathway interactions Peptide Biosynthesis Peptide Hydrolases Peptide Signal Sequences Peptide Synthesis Peptides Population Density Probiotics Process Production Proteins Proteolysis Public Health Recording of previous events Regulator Genes Reporter Signal Transduction Site Specificity Staphylococcus aureus Staphylococcus epidermidis System Tail Testing Variant Virulence Work chemical synthesis clinically significant combat design extracellular fighting host colonization human pathogen inhibitor insight interest multi-drug resistant pathogen mutant nanomolar non-Native novel novel strategies pathogen pathogenic bacteria peptide I peptide analog prevent protein aminoacid sequence quorum sensing screening therapeutic target tool

项目摘要

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.

项目概要群体感应（QS）是同一物种的细菌在高水平下协调行为的过程在许多致病细菌中，QS系统被用来调节毒力。专注于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 Agr 型 QS 系统包含四种蛋白质 AgrA-D，它们一起产生并响应我将研究负责信号的两种蛋白质 AgrB 和 AgrD。在此途径中，肽前体 AgrD 由 AgrB 和细胞外蛋白酶加工。为了产生 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 AIP 是否有效。通过迭代的突变，信号仍然会产生，我将确定肽中的位置和内容。嵌入该方法中以了解 AIP 的基本机制。处理是另外两个目标，其中一个已经实现了，第一，我测试了能力。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，并具有金黄色葡萄球菌 QS 可以使用我的生物合成其次，通过揭示 AIP 信号的哪些残基可以在不损失处理的情况下发生突变，我可以测试所产生的非天然 AIP 类似物抑制金黄色葡萄球菌中 QS 的能力，可能会发现对于目标 2，我将设计一种非致病性细菌来组成型表达。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线虫模型中的致病性 Aim 3 将继续研究 AgrD 的加工过程。将重点转移到研究最后一步，同时细胞外蛋白酶将 AgrD 肽裂解为产生最终的 AIP 信号这一过程的一个谜团是 AIP 信号，即使是单个物种内的信号，通常也是如此。为了找出导致这种差异的原因，我将对 AgrD 进行靶向突变。并将天然 AgrD 序列的蛋白水解位点与突变序列进行比较，我应用这些知识。然后将生物合成设计师 AIP 类似物，这些类似物结合了两个或多个本地 AIP 信号的特征。这三个目标共同将显着增加我们对 AIP 生物合成的理解，并提供一种新颖的方法。抑制主要人类病原体中 agr 型 QS 系统的有价值的化学工具的途径。