PROJECT SUMMARY Improved therapeutic approaches are needed for non-healing wounds, as they present a major challenge to the healthcare system by increasing treatment costs as well as rates of morbidity and mortality. The skin microbiome exists at the interface of all cutaneous wounds, but its potential as a novel therapeutic target remains untapped. Therefore, our long-term goal is to understand host-microbial interactions during wound healing, so we may leverage these mechanisms to identify wound healing treatments. To initiate this investigation, our lab previously performed a metagenomic analysis on wound samples from non-infected diabetic foot ulcers (DFUs) and unearthed a surprisingly prevalent wound inhabitant Alcaligenes faecalis. Very little is known about this species in the context of wounds, so we sought to investigate the effect of A. faecalis on wound healing. We observed the surprising finding that treating wounds with A. faecalis accelerates the rate of wound healing in vivo and activates a pro-epithelialization phenotype in keratinocytes. Thus, the central goal of this study is to identify the mechanism by which A. faecalis mediates accelerated wound healing. Pro- inflammatory cytokine signaling is necessary to promote re-epithelialization, and IL-6 in particular has been shown to induce activation of keratinocytes during healing. Therefore, I tested if A. faecalis induced a cytokine response in keratinocytes and found robust IL-6 production after treatment with A. faecalis conditioned media. A primary mechanism by which bacteria can modulate host responses is through production of secreted molecules. I found that sterile supernatant of A. faecalis, rather than bacterial-cell surface molecules, promotes keratinocyte migration and IL-6 production. Together, these findings lead to my hypothesis that A. faecalis produces secreted molecules that improve re-epithelialization by enhancing keratinocyte IL-6 signaling. Aim 1 will determine the host mechanisms of A. faecalis-induced accelerated healing through a combination of wound healing assays and transcriptional profiling. Aim 2 will identify the microbial genetic determinants of A. faecalis necessary to produce the pro-healing secreted molecule. I will use comparative genomics approach to identify genomic loci that segregate with a pro-wound healing phenotype. To complete this aim, I will leverage our collection of 44 A. faecalis clinical DFU isolates and perform a genomic multiple alignment. I will pair this genetic screen with a phenotypic screen using wound healing assays to determine which genomic locus segregates with the pro-healing phenotype. In conjunction with these experimental aims, I will also engage in a rigorous training plan at the University of Pennsylvania under the guidance of the MD/PhD Program and my PhD advisor. This training plan will foster my development as a future physician scientist through the following goals: strengthen independence as an investigator, ...