Bacterial polyphosphates in sepsis

项目摘要：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 世界范围内的一年。在没有FDA批准的药物的情况下，对更好地了解宿主的需求很高- 定义败血症分子发病机制的微生物相互作用。聚磷酸盐是一种线性聚合物， 存在于所有生物体内的无机磷(PI)残留物。细菌的新陈代谢 聚磷酸盐(PI：N≥1,000)积累长链，而短链聚磷酸盐(PI：N)聚磷酸盐 N&lt；100)，通常存在于哺乳动物细胞中。生物效应依赖于链长。新兴数据 提示短链多聚磷酸调节血液凝固和炎症，而长链多磷酸的作用是 败血症中的链状、细菌来源的多聚磷酸盐是一个研究不足的领域。我们的前期工作 提示多聚磷酸盐的中和或细菌多聚磷酸盐缺乏症可提高 盲肠结扎穿孔(CLP)所致小鼠腹膜脓毒症在无菌巨噬细胞培养中，长期- 链状多聚磷酸对内毒素/TLR4诱导的巨噬细胞极化、iNOS表达和免疫功能的影响 新陈代谢。在这里，我们建议检验细菌聚磷酸盐是致命代谢物这一中心假设。 在脓毒症中，由于它们对先天宿主感染反应的有害干扰。照亮，照亮 关于聚磷酸盐的生物活性，我们建议解决三个具体目标：(1)研究 为了中和多聚磷，我们将使用重组胞外多聚磷酸酶(PPX)蛋白并鉴定其 多菌CLP败血症对宿主反应的影响。单细胞蛋白质组学方法(CITE-SEQ) 将致力于捕捉侵袭性专业吞噬细胞的异质性/极化 聚磷酸盐。多聚磷酸盐将在小鼠和人类的脓毒症样本中进行测量。(2)至 表征多聚磷酸盐对培养的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。如果是这样的话，我们会研究 多聚磷酸盐抑制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(氧磷酸盐，糖酵解)。此外，亲和纯化与无标记相结合 蛋白质组学将致力于鉴定巨噬细胞中新的多聚磷酸靶向蛋白；以更好地 了解多聚磷酸盐在脓毒症中如何干扰吞噬细胞反应的机制。(3)在灵芝中 小鼠，用缺乏聚磷酸盐的大肠杆菌突变体(Δppk)单克隆，我们将研究细菌如何- 衍生聚磷酸盐形成单一微生物CLP脓毒症前后的先天免疫。腹膜和腹膜 将对肠粘膜巨噬细胞的功能、转录组可塑性进行表征和比较 和免疫代谢表型。这项研究项目将为尚未探索的活动提供新的见解 细菌多聚磷酸盐在脓毒症宿主-病原体相互作用的网络中的作用，并最终可能 治疗逆转不良适应性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.