PROJECT SUMMARY/ABSTRACT: Antibiotic resistant bacterial infections are an immediate public threat to the United States and global healthcare systems. We must gain a deeper understanding of the innate immune system’s response to bacterial pathogens to facilitate development of host-directed therapeutic approaches to combat bacterial infection. Many clinically relevant bacterial pathogens harbor a flagellum comprised of the structural protein flagellin, which is recognized by the extracellular Toll-like receptor 5 (TLR5) and the intracellular neuronal apoptosis inhibitory protein 5 (NAIP5) sensor. NAIP5 sensing of flagellin results in the formation of a multiprotein inflammasome complex containing the NLR caspase recruitment domain-containing protein 4 (NLRC4) which activates caspase-1, triggering an inflammatory cell death called pyroptosis. In the course of infection, a number of pathogens including Salmonella enterica serovar Typhimurium (S. Tm) deliver flagellin into the host cytosol, leading to activation of the NAIP5-NLRC4 inflammasome. Multiple studies suggest that in mice, NAIP6 also senses flagellin. Why there are two distinct sensors for cytosolic flagellin, as well as the biological circumstances under which NAIP6 might sense flagellin is unknown. Intriguingly, bacterial flagellins from many clinically relevant bacteria that do not activate NAIP5, lack a conserved arginine at the C terminus. Moreover, truncated S. Tm flagellin with a stop codon inserted in the place of conserved three amino acids in its D0 domain (termed D0STOP) abrogates recognition of flagellin by the host cell. These observations suggest that the C terminus of flagellin is essential for NAIP5 sensing and that bacterial pathogens may escape NAIP5 by altering this domain. My preliminary findings reveal that TLR2 priming of murine bone marrow derived macrophages (BMDMs) leads to activation of a NLRC4-dependent response to D0STOP flagellin when delivered using the heterologous Type III secretion system of Yersinia pseudotuberculosis (Yp). Altogether, these findings and my preliminary data provoke the conceptually novel hypothesis that TLR2 signaling licenses NAIP6-dependent flagellin sensing to overcome bacterial evasion of NAIP5. In Aim 1, I plan to determine how TLR2 licenses NAIP6- NLRC4 inflammasome activation in TLR2 primed BMDMs and test if NAIP6 is sufficient to recognize flagellins that evade NAIP5 sensing using Yp as a delivery system and a retroviral expression vector system to reconstitute the NAIP5/6-NLRC4 inflammasome in 293T cells. In Aim 2, I will assess the contribution of NAIP6 flagellin detection in eliciting protective host responses using Yp as a delivery system in Naip5-/- and Naip1-6D/D mice. The scientific goal of this fellowship is to uncover a new mechanism for innate detection of flagellin and understand why mice harbor two highly similar cytosolic sensors that detect flagellin. Another goal is to advance my training as a scientist to propel a ...