High-throughput characterization of antimicrobial peptide-PhoPQ interactions

抗菌肽-PhoPQ 相互作用的高通量表征

• 批准号: 10378042
• 负责人: Jeffrey Jay Tabor
• 金额: $ 37.71万
• 依托单位: RICE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-25 至 2026-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10378042
• 关键词: Address; Amino Acids; Anti-Bacterial Agents; Bacteria; Binding; Biochemical; Biological; Biology; Cells; Cessation of life; Chemicals; Computer Analysis; DNA sequencing; Dangerousness; Enterobacteriaceae; Epithelial; Epithelial Cells; Escherichia coli; Family; Fluorescence-Activated Cell Sorting; Food Contamination; Future; Gene Expression; Genes; Genetic Engineering; Genetic Transcription; Goals; Human; Immune; Immune system; Infection; Ingestion; Innate Immune System; Invaded; Knowledge; Laboratories; Lead; Link; Location; Measures; Mediating; Membrane; Methods; Microbiology; Modeling; Mus; Mutation Analysis; Names; Organism; Pathogenicity; Pathway interactions; Peptide Library; Peptides; Physiological; Play; Process; Property; Reporter Genes; Resistance; Role; Salmonella typhimurium; Signal Transduction; Signal Transduction Pathway; Site; Specificity; Structure; Surface; Synthetic Peptide Libraries; System; Systemic infection; Technology; Therapeutic; Time; United States; Virulence; Work; antimicrobial; antimicrobial drug; antimicrobial peptide; base; biophysical analysis; biophysical properties; cathelicidin; contaminated water; cost; design; experimental study; gastrointestinal; in vivo; inhibitor; insight; intestinal epithelium; macrophage; mutant; new technology; next generation; novel; pathogen; pathogenic bacteria; pressure; prevent; promoter; protein-histidine kinase; reconstitution; response; sensor; sensor histidine kinase; synthetic peptide

Project Summary/Abstract The human immune system produces at least 140 different antimicrobial peptides (AMPs) to kill invading bacteria. However, pathogenic bacteria use specialized pathways called two-component systems (TCSs) to detect these AMPs and activate the expression of AMP-resistance and virulence genes. This response enables pathogens to survive immune attacks and mount deadly infections. Therefore, elucidating the mechanisms by which peptides interact with TCSs is critical to understanding how infections progress. This knowledge could also lead to the design of new antimicrobial drugs that interfere with TCS-mediated AMP sensing. Gram-negative Enterobacteriaceae, such as the common pathogen Salmonella Typhimurium, cause 200,000 infections and 10,000 deaths in the United States each year. The most important AMP-sensing TCS in Gram-negative Enterobacteriaceae is named PhoPQ. Here, the membrane bound histidine kinase PhoQ senses AMPs and responds by phosphorylating the cytoplasmic response regulator PhoP, which activates a gene expression response. Though its interactions with a small number of model AMPs have been characterized, little is known about the broader peptide binding and sensing capabilities of PhoQ. The major limitations have been the cost and time required to chemically synthesize peptides and characterize their effects on TCSs using traditional microbiological or biochemical methods. In preliminary work, we have developed a new technology named SLAY-TCS that combines bacterial peptide display, fluorescence-activated cell sorting, and next-generation DNA sequencing to measure how S. Typhimurium PhoQ responds to millions of peptides in a single experiment. Using SLAY-TCS, we have already revealed that PhoQ senses a far wider range of peptides than previously known. Here, we propose to use SLAY-TCS to characterize how S. Typhimurium PhoQ responds to nearly every AMP produced by the human immune system, and thousands of mutants thereof, in order to reveal the identities, sequence motifs, and biophysical properties of PhoQ-activating peptides (Aim 1). We will also combine this approach with PhoQ mutational analyses to reveal how PhoQ sensing specificity has evolved across diverse pathogens, which may have enabled them to adapt to different biogeographical locations in vivo (Aim 2). Finally, we will use SLAY-TCS to perform the first large-scale characterization of peptide inhibitors of PhoQ, and explore the efficacy of the strongest inhibitors we identify in preventing S. Typhimurium virulence in primary mouse macrophages (Aim 3). The work in Aim 3 will reveal mechanisms by which exogenously-delivered peptides can inhibit PhoQ, and could lead to the design of novel antimicrobial therapeutics based on modified peptides in the future. Taken together, this proposal will substantially enhance our understanding of how a dangerous family of bacteria causes infections in humans and accelerate the design of sorely-needed antimicrobial therapeutics. Finally, our approach could be extended to other peptide-sensing TCSs beyond PhoPQ in future studies. 1

项目摘要/摘要 人类免疫系统产生至少140种不同的抗菌肽(AMP)来杀死入侵 细菌。然而，病原菌使用称为双组分系统(TCS)的特殊途径来 检测这些AMP并激活AMP抗性和毒力基因的表达。此响应将启用 病原体在免疫攻击中存活下来，并引发致命的感染。因此，通过以下方式阐明其机制 哪些多肽与TCS相互作用，对于了解感染是如何发展的至关重要。这一知识可能会 这也导致了干扰TCS介导的AMP传感的新抗菌药物的设计。革兰氏阴性 肠杆菌科，如常见的病原体鼠伤寒沙门氏菌，导致20万人感染和 美国每年有1万人死亡。革兰氏阴性菌中最重要的AMP敏感TCS 肠杆菌科被命名为PhoPQ。在这里，膜结合的组氨酸激酶PhoQ感觉AMPS和 通过磷酸化细胞质反应调节因子Phop来进行反应，从而激活基因表达 回应。尽管它与少数模型AMP的相互作用已被描述，但鲜为人知 关于PhoQ更广泛的多肽结合和传感能力。主要的限制是成本 以及使用传统方法化学合成多肽并表征其对TCS的影响所需的时间 微生物或生化方法。在前期工作中，我们开发了一种名为 SLAY-TCS结合了细菌肽展示、荧光激活细胞分选和下一代 DNA测序以测量鼠伤寒沙门氏菌如何在一次实验中对数百万个多肽做出反应。 使用SLAY-TCS，我们已经揭示了PhoQ可以感知比以前更广泛的多肽 为人所知。在这里，我们建议使用SLAY-TCS来表征鼠伤寒沙门氏菌对几乎每一个 由人类免疫系统产生的AMP及其数千个突变体，为了揭示身份， 光Q激活肽的序列基序和生物物理性质(目标1)。我们还将结合这一点 用PhoQ突变分析的方法来揭示PhoQ传感特异性如何在不同的 病原体，这可能使它们能够适应体内不同的生物地理位置(目标2)。最后， 我们将使用SLAY-TCS对PhoQ的多肽抑制剂进行首次大规模表征，并探索 我们确定的最强抑制剂对鼠伤寒沙门氏菌原代毒力的抑制效果 巨噬细胞(目标3)。目标3中的工作将揭示外源递送多肽可以 抑制PhoQ，并可能导致基于修饰多肽的新型抗菌疗法的设计 未来。综上所述，这项建议将大大加强我们对一个危险的家庭如何 细菌会导致人类感染，并加速设计急需的抗菌疗法。 最后，在未来的研究中，我们的方法可以扩展到除PhoPQ之外的其他多肽敏感的TCS。 1