Bacterial polyphosphates in sepsis

败血症中的细菌多磷酸盐

• 批准号: 10573217
• 负责人: Markus Bosmann
• 金额: $ 53.71万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-22 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10573217
• 关键词: Address; Affinity Chromatography; Agonist; Alpha Granule; Bacteremia; Bacteria; Bacterial Infections; Binding Proteins; Biological; Blood Platelets; Blood coagulation; Bradykinin; Cells; Cellular Indexing of Transcriptomes and Epitopes by Sequencing; Chemicals; Coagulation Process; Colon; Complement Activation; Data; Environment; Escherichia coli; Event; FDA approved; Future; Glycolysis; Gnotobiotic; Growth; Heterogeneity; Host Defense; Human; Immune; Immune response; Immunity; Incubated; Infection; Inflammation; Integration Host Factors; Intervention; Intestinal Mucosa; Invaded; Knowledge; Label; Length; Light; Link; Macrophage; Mammalian Cell; Measures; Mediating; Metabolic; Metabolism; Modeling; Molecular; Molecular Chaperones; Mononuclear; Morbidity - disease rate; Mucous Membrane; Mus; Natural Immunity; Nutrient; Organism; Orthophosphate; Outcome; Pathogenesis; Patients; Peritoneal; Peritoneal Sepsis; Phagocytes; Pharmaceutical Preparations; Phenotype; Polymers; Polyphosphate kinase; Polyphosphates; Prevention; Production; Proteins; Proteomics; Reaction; Recombinants; Reporting; Research; Research Project Grants; Role; Saccharomyces cerevisiae; Sampling; Sepsis; Severities; Shapes; Signal Pathway; Signal Transduction; Source; Sterility; Stress; Surrogate Endpoint; TLR4 gene; Testing; Therapeutic; Titrations; Work; arginase; bacterial metabolism; cecal ligation puncture; chemokine; cytokine; fighting; host-microbe interactions; improved; inflammatory milieu; inorganic phosphate; insight; mast cell; monocyte; mortality; mutant; neutrophil; novel; pathogen; polymicrobial sepsis; proteogenomics; recruit; response; response to injury; transcriptome

Project Summary: Sepsis remains a leading cause of morbidity and mortality with almost 50 million cases per year worldwide. In the absence of FDA-approved drugs, there is a high demand for better insights into the host- microbe interactions that define the molecular pathogenesis of sepsis. Polyphosphates are linear polymers of inorganic phosphate (Pi) residues that are present in all living organisms. The metabolism of bacteria accumulates long-chains of polyphosphates (Pi: n≥1,000) in contrast to the short-chain polyphosphates (Pi: n<100) typically found in mammalian cells. The biologic effects are dependent on chain length. Emerging data suggest that short-chain polyphosphates modulate blood coagulation and inflammation, while the role of long- chain, bacteria-derived, polyphosphates in sepsis is an understudied research field. Our preliminary work suggests that neutralization of polyphosphates or bacterial polyphosphate deficiency improves survival of peritoneal sepsis induced by cecum ligation and puncture (CLP) in mice. In sterile macrophage cultures, long- chain polyphosphates modulate LPS/TLR4-induced macrophage polarization, iNOS expression and immuno- metabolism. Here, we propose to test the central hypothesis that bacterial polyphosphates are lethal metabolites in sepsis because of their detrimental interference with the innate host response to infection. To shed light into the biological activities of polyphosphates, we propose to address 3 specific aims: (1) To study the effects of polyphosphate neutralization, we will use a recombinant exopolyphosphatase (PPX) protein and characterize its activities on the host response to polymicrobial CLP sepsis. A single-cell proteogenomics approach (CITE-Seq) will aim to capture the heterogeneity/polarization of invading professional phagocytes as a function of polyphosphates. The polyphosphates will be measured in sepsis samples of mice and humans. (2) To characterize the direct interference of polyphosphates with the functions of cultured macrophages, we will combine bacterial TLR agonists with synthetic polyphosphates of different chain length. It will be studied if polyphosphates curb STAT/IRF signaling pathways for modulating iNOS, L-arginase, cytokines/chemokines, and metabolic reprogramming (OXPHOS, glycolysis). In addition, affinity purification combined with label-free proteomics will aim for the identification of novel polyphosphate targeted proteins in macrophages; to better understand the mechanisms how polyphosphates interfere with phagocyte responses in sepsis. (3) In gnotobiotic mice, monocolonized with a polyphosphate-deficient E. coli mutant (Δppk), we will investigate how bacteria- derived polyphosphates shape innate immunity before and after monomicrobial CLP sepsis. Peritoneal and intestinal mucosal macrophages will be characterized and compared for their functions, transcriptome plasticity and immuno-metabolic phenotypes. This research project will provide novel insights into the unexplored activities of bacterial polyphosphates within the networks of host-pathogen interactions of sepsis and may ultimately advance strategies for therapeutic reversal of maladaptive inflammatory milieus.

项目摘要：败血症仍然是发病率和死亡率的主要原因，每个病例近5000万例 全球。在没有FDA批准的药物的情况下，对宿主的更好见解的需求很高 - 定义败血症分子发病机理的微生物相互作用。多磷酸盐是线性聚合物 无机磷酸盐（PI）保留了所有生物体中存在的。细菌的代谢 与短链多磷酸盐（PI：：PI：：PI：：PI：：PI： n <100）通常在哺乳动物细胞中发现。生物学效应取决于链长。新兴数据 表明短链元磷酸盐调节血液凝结和感染，而长长的作用 败血症中的链，细菌衍生的多磷酸盐是一个理解的研究领域。我们的初步工作 表明多磷酸或多磷酸细菌缺乏的中和改善了 小鼠塞卡姆结扎和穿刺（CLP）诱导的腹膜败血症。在无菌巨噬细胞培养物中，长期 链多磷酸盐调节LPS/TLR4诱导的巨噬细胞极化，iNOS表达和免疫 - 代谢。在这里，我们建议测试中心假设，即细菌多磷酸是致命的代谢物 在败血症中，由于它们对先天宿主对感染的反应有害。将照明到 多磷酸盐的生物学活性，我们建议解决3个具体目的：（1）研究 多磷酸神经化，我们将使用重组外聚磷酸酶（PPX）蛋白质并表征其 宿主对多数型CLP败血症的反应。单细胞蛋白质组学方法（cite-seq） 旨在捕获入侵专业吞噬细胞的异质性/偏光性 多磷酸盐。多磷酸盐将在小鼠和人类的败血症样品中进行测量。 （2）至 表征多磷酸盐与培养巨噬细胞功能的直接干扰，我们将 将细菌TLR激动剂与具有不同链长的合成多磷酸盐结合在一起。如果是 多磷酸盐曲线stat/irf信号传导途径，用于调节iNOS，l-精氨酸酶，细胞因子/趋化因子，趋化因子， 和代谢重编程（Oxphos，糖酵解）。另外，亲和力纯化与无标签的结合 蛋白质组学将旨在鉴定巨噬细胞中新型的多磷酸盐靶向蛋白。更好 理解多磷酸盐如何干扰败血症的吞噬细胞反应的机制。 （3）在gnotobiotic中 小鼠，用多磷酸盐的大肠杆菌突变体（Δppk）单卵子，我们将研究细菌 - 衍生的多磷酸盐在单晶CLP败血症之前和之后塑造先天免疫。腹膜和 肠粘膜巨噬细胞将被表征并比较其功能，转录组可塑性 和免疫代谢表型。该研究项目将为意外活动提供新颖的见解 败血症宿主 - 病原体相互作用网络中的细菌多磷酸 适应不良炎症环境的热逆转策略。