Bacterial polyphosphates in sepsis

脓毒症仍然是发病率和死亡率的主要原因，每年有近5000万例病例。 年全球。在缺乏FDA批准的药物的情况下，人们迫切需要更好地了解宿主- 微生物之间的相互作用定义了脓毒症的分子发病机制。多磷酸盐是线性聚合物， 无机磷酸盐（Pi）残留物存在于所有生物体中。细菌的新陈代谢 与短链多磷酸盐（Pi：n≥ 1，000）相比， n<100），通常存在于哺乳动物细胞中。生物学效应取决于链长。新出现的数据 表明短链多磷酸盐调节血液凝固和炎症，而长链多磷酸盐的作用 脓毒症中的细菌来源的多磷酸盐链是一个未充分研究的研究领域。我们的前期工作 表明，多磷酸盐或细菌多磷酸盐缺乏症的中和提高了生存， 盲肠结扎穿孔法（CLP）腹腔感染。在无菌巨噬细胞培养中，长- 链多磷酸盐调节LPS/TLR 4诱导的巨噬细胞极化、iNOS表达和免疫反应。 新陈代谢.在这里，我们建议测试的中心假设，细菌多磷酸盐是致命的代谢产物 在败血症中，因为它们有害地干扰宿主对感染的先天性反应。照亮 多磷酸盐的生物活性，我们提出了解决3个具体目标：（1）研究的影响， 为了中和多磷酸盐，我们将使用重组外聚磷酸酶（PPX）蛋白并表征其 活性对宿主对多微生物CLP脓毒症的反应。单细胞蛋白质组学方法（CITE-Seq） 目的是捕获入侵的专职吞噬细胞的异质性/极化， 多磷酸盐将在小鼠和人类的脓毒症样本中测量多磷酸盐。(2)到 为了表征多磷酸盐对培养的巨噬细胞功能的直接干扰，我们将 联合收割机将细菌TLR激动剂与不同链长的合成多磷酸盐组合。将研究，如果 多磷酸盐抑制STAT/IRF信号传导途径，用于调节iNOS、L-腺苷酸酶、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的多磷酸盐靶向蛋白质; 了解脓毒症中多磷酸盐如何干扰吞噬细胞反应的机制。(3)定菌 小鼠，单菌落与多磷酸盐缺陷型E.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微生物CLP脓毒症之前和之后形成先天免疫。腹膜和 肠粘膜巨噬细胞将表征并比较它们的功能、转录组可塑性 和免疫代谢表型。这项研究项目将为未探索的活动提供新的见解 细菌多磷酸盐在败血症的宿主-病原体相互作用网络中的作用， 治疗性逆转适应不良炎症环境的先进策略。

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.