Biosynthesis of Non-Native Autoinducing Peptides

非天然自诱导肽的生物合成

• 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
• 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

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 人口密度。在许多致病细菌中，QS 系统用于调节毒力。这个提议 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 艰难梭菌。 Agr 型 QS 系统包含四种蛋白质 AgrA-D，它们共同产生并响应 自诱导肽（AIP）信号。我将研究负责信号的两种蛋白质 AgrB 和 AgrD 生物合成。在此途径中，肽前体 AgrD 由 AgrB 和细胞外蛋白酶加工 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 信号仍然产生。通过迭代轮次的突变，我将确定肽中的位置以及突变的内容 变化程度是可以容忍的。嵌入此方法中以了解 AIP 的基本机制 处理是另外两个目标，其中之一已经实现。首先，我测试了我的能力 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 证明可以使用我的方法生物合成两种高效的金黄色葡萄球菌 QS 泛群抑制剂 系统。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 新的、更有效的抑制剂。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 秀丽隐杆线虫模型中的致病性。目标 3 将继续研究 AgrD 的加工，但将 将焦点转移到研究最后一步，其中细胞外蛋白酶将 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，即使是同一物种内的信号，也经常会发出信号。 它们的蛋白水解位点不同。为了找出导致这种变异性的原因，我将对 AgrD 进行有针对性的突变 并比较天然 AgrD 序列与突变序列的蛋白水解位点。应用这些知识，我 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。