Abstract Diabetic foot ulcers (DFUs) and diabetic foot infections (DFI) are one of the most challenging complications of diabetes due to high morbidity and associated mortality and precede the majority of non-traumatic lower limb amputations in the adult population. Diabetic foot microbiome of intact skin, prior to the onset of the ulcer, is characterized by the low level of Staphylococcus epidermidis (SE) and high levels of Staphylococcus aureus (SA). Persistent level of bacteria in ulcer tissue, resulting in prolonged and deregulated inflammation is one of the leading causes of lower leg amputations in patients suffering from DFUs. To gain greater insight into relationship between innate immune responses and wound healing outcomes, we propose to study how commensal microorganism SE prevents intracellular accumulation of SA and accelerates the diabetic wound healing process. The long term goal of this project is to prevent DFI by understanding the mechanism and developing new therapeutic strategies targeting cutaneous intracellular pathogens in patients with DFUs. We have already shown that downregulation of an innate-antimicrobial protein P-2 in keratinocytes and gamma delta (GD) T cells results in accumulation of intracellular MRSA in DFUs, contributing to persistent unresolved inflammation. Furthermore, loss of P-2 in murine models is associated with both, lower antimicrobial activity and accumulation of intracellular MRSA, and impaired epithelialization. Importantly, we have shown that killing of intracellular MRSA is enhanced in skin after exposure to SE. Based on robust preliminary data we postulate that SE colonization modifies diabetic skin and wound environment to prevent intracellular infections by pathogenic SA. Our hypothesis is that intracellular bacteria modify expression and function of P-2 to affect bacterial clearance and inflammatory response, directly impacting healing in DFU. The objective of this project is to determine mechanism by which SE prevents intracellular infections by pathogenic SA in a diabetic mouse and human wounds. We hypothesized that SE colonization modifies wound environment in order to prevent or resolve persistent bacterial wound infections. To test our hypothesis, we will evaluate SE-mediated induction of P-2 in professional and non-professional phagocytes during acute diabetic and non-diabetic wound healing process in vivo, using multiple animal and human models and samples obtained from DFU patients. We will also identify gene expression signatures and pathways in these cells that are differentially regulated in “low intracellular SA non-healing” vs “high intracellular SA healing” DFU (Aim 1). We will characterize SE protective mechanisms against intracellular MRSA in diabetic wound infections (Aim 2). Our findings will provide important new knowledge regarding the role and mechanisms by which commensal SE may prevent persistent diabetic wound infections. Targeting intracellular bacteria niche to accele...