Pathogenic bacteria require iron for their survival and virulence. The opportunistic pathogen Pseudomonasaeruginosa has multiple mechanisms by which it can acquire iron, including ferric and ferrous iron uptakesystems. However, within the host P. aeruginosa can adapt to utilize heme via the heme assimilation (has) andPseudomonas heme utilization (phu) systems. We have recently shown the OM receptor PhuR has a uniqueHis-Tyr coordination, which is an emerging motif in high affinity heme acquisition systems. 13C-heme isotopiclabeling studies combined with bacterial genetics suggest the PhuR receptor is the high capacity uptakereceptor, with the HasR receptor acting primarily as a sensor and regulator of heme utilization. Furthermore,we have shown the heme metabolite biliverdin IX? is a feedback regulator of the heme sensing system (has),as well as several virulence mechanisms including the pyochelin and Zn/Ni-pseudopaline uptake system, TypeIII secretion systems (ExoS and ExoT), and extracellular proteases (LasB). The goal of the proposal is tounderstand the regulation and molecular mechanism of heme acquisition in P. aeruginosa. Specifically, we willelucidate the heme-dependent regulatory elements controlling expression of the has system throughtranscriptional and translational fusion studies. Targeted transcriptional and post-transcriptional studies will becomplimented by global analysis through transcriptomic and and proteomic analyses. We will further definethe substrate specificity of the bis-His HasR and His-Tyr coordinated PhuR and their respective contributions toheme acquisition and regulation. Contributions of the Has and Phu systems to heme acquisition and virulencewithin the host will be tested in murine acute and chronic lung infection models. In addition, dual RNA-seq willbe performed to simultaneously determine the P. aeruginosa and murine host response to infection. MALDI-MSI will be used in combination with quantitative LC-MS methods to determine the spatial distribution hememetabolites (BVIX isomers) and host-pathogen biomarkers in PAO1 and heme utilization mutants. Completionof the studies will provide a molecular basis for P. aeruginosa adaption to heme utilization in the context of thehost-pathogen interaction.