PROJECT SUMMARY In 2019, methicillin-resistant Staphylococcus aureus infections were the basis of nearly 400,000 hospitalizations per year with direct costs estimated at $1.7 billion. Furthermore, S. aureus is the leading cause of bacterial endocarditis. Understanding host-pathogen interactions that skew disease outcome will facilitate the design of efficacious therapeutic strategies. Neutrophils possess antimicrobial functions that are critical to the innate immune response to S. aureus. Preliminary data show that neutrophils lacking calprotectin (CP), a highly abundant immune protein expressed by neutrophils, exhibit altered mitochondrial homeostasis, where CP- deficient neutrophils produce more mitochondrial superoxide in response to S. aureus compared to wild-type neutrophils. As a result, CP-deficient neutrophils undergo elevated suicidal NETosis. In addition, increased suicidal NETosis correlates with CP-deficient mice having lower bacterial burdens specifically within the heart and increased survival during systemic S. aureus infection. This suggests that neutrophil and CP biology is unique within the heart. Aim 1 will identify why the heart offers a unique niche for CP biology during S. aureus infection. More specifically, we will address (i) whether cardiomyocyte function in CP-deficient mice is altered, thereby skewing the neutrophil response and (ii) identify factors driving CP secretion/retention by neutrophils during infection in the heart. These experiments are significant as they define the immunological and metabolic environment of the heart, compared to other sites of infection, and the implications this has on CP and neutrophil biology. Aim 2 will focus on the role of intracellular CP in altering mitochondrial homeostasis. We will specifically test the role of CP in regulating mitochondrial metabolism and metal homeostasis, and downstream impacts this has on neutrophil function. These experiments are significant because they identify CP as a molecular rheostat for neutrophil function by controlling mitochondrial homeostasis, which may be broadly applicable to other cells expressing CP during inflammation. This proposal provides critical scientific insights into the function of CP that may be especially efficacious as a biological target for treating S. aureus infections of the heart. In addition, the technological advancements achieved by this proposal will create a platform that can be applied to other inflammatory diseases.