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...