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.

项目总结