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：脓毒症仍然是发病和死亡的主要原因，每年有近 5000 万例脓毒症 年全球。在缺乏 FDA 批准的药物的情况下，迫切需要更好地了解宿主 - 定义脓毒症分子发病机制的微生物相互作用。聚磷酸盐是线性聚合物 所有生物体中都存在无机磷酸盐 (Pi) 残留物。细菌的新陈代谢 与短链聚磷酸盐 (Pi: n≥1,000) 相比，积累长链聚磷酸盐 (Pi: n≥1,000) n<100）通常存在于哺乳动物细胞中。生物效应取决于链长度。新兴数据 表明短链聚磷酸盐调节血液凝固和炎症，而长链聚磷酸盐的作用 脓毒症中的细菌源性多磷酸盐是一个尚未得到充分研究的研究领域。我们的前期工作 表明多磷酸盐的中和或细菌多磷酸盐缺乏可提高细菌的存活率 小鼠盲肠结扎穿刺（CLP）诱发腹膜败血症。在无菌巨噬细胞培养物中，长 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，我们建议检验细菌多磷酸盐是致命代谢物的中心假设 在脓毒症中，因为它们对宿主对感染的先天反应产生有害干扰。照亮 为了研究多磷酸盐的生物活性，我们建议解决 3 个具体目标：（1）研究 多磷酸中和，我们将使用重组外多磷酸酶（PPX）蛋白并表征其 活性对宿主对多种微生物 CLP 脓毒症反应的影响。单细胞蛋白质组学方法 (CITE-Seq) 将旨在捕获入侵专业吞噬细胞的异质性/极化作为函数 聚磷酸盐。将在小鼠和人类的败血症样本中测量多磷酸盐。 (2) 至 表征多磷酸盐对培养巨噬细胞功能的直接干扰，我们将 将细菌 TLR 激动剂与不同链长的合成聚磷酸盐结合起来。将会研究如果 多磷酸盐抑制 STAT/IRF 信号通路，调节 iNOS、L-精氨酸酶、细胞因子/趋化因子、 和代谢重编程（OXPHOS、糖酵解）。此外，亲和纯化结合无标记 蛋白质组学的目标是鉴定巨噬细胞中新型多磷酸盐靶向蛋白质；为了更好 了解多磷酸盐如何干扰脓毒症中吞噬细胞反应的机制。 (3) 在知生中 小鼠，单克隆有缺乏多磷酸盐的大肠杆菌突变体（Δppk），我们将研究细菌如何- 衍生的多磷酸盐在单一微生物 CLP 脓毒症前后塑造先天免疫。腹膜和 将表征并比较肠粘膜巨噬细胞的功能、转录组可塑性 和免疫代谢表型。该研究项目将为未探索的活动提供新颖的见解 脓毒症宿主-病原体相互作用网络中细菌多磷酸盐的变化，最终可能 治疗逆转适应不良的炎症环境的先进策略。