ABSTRACT/SUMMARY Necrotizing Fasciitis (NF) or “flesh-eating disease” is a rapidly progressing bacterial infection with severe necrosis of the dermis and underlying soft tissues. Treatment of NF requires systemic antibiotics and aggressive surgical debridement. Even with these treatments, NF has considerable morbidity and mortality. Thus, a better understanding of the pathophysiology of NF and identification of new treatment strategies to attenuate disease progression is required. Recent work has revealed that pro-inflammatory signals can increase or decrease cellular resistance to the cholesterol-dependent cytolysins (CDCs), key microbial toxins that permeabilize cells and destroy tissues. The induction of a CDC “resistant or sensitive state” for phagocytes was found to be dependent on the rapid reprogramming of cellular cholesterol homeostasis. Moreover, disrupting the ability of macrophages to reprogram their lipid metabolic state disrupts the induction of protective states by inflammatory signals. Thus, an inflammatory-lipid metabolic circuit in host cells serves as a determinant of the pathogenic potential of CDCs, a major virulence factor in necrotizing skin infections. In this application, we combine advanced methodologies (e.g., mass spectrometry, single-cell sequencing, and imaging) with genetic and pharmacologic models of lipid metabolism to understand if tissue lipid metabolism is a host factor that determines the pathogenic potential of CDCs and group A strep (GAS) infections. Specific Aim 1 will determine the molecular mechanism underlying how the CH25H-LXR metabolic axis mediates the protection of cells from CDC toxicity. Specifically, we will pursue our discovery that activation of the LXR signaling pathway profoundly protects phagocytes from CDC-mediated loss of membrane integrity. Combining lipidomics, transcriptomics, imaging, and functional assays with gain- and loss-of function models, we will molecularly dissect the lipid metabolic pathways necessary for LXR-mediated protection from CDC-mediated cytotoxicity. Specific aim 2 will focus on advancing our understanding of the cell types in the skin necessary and sufficient for LXR-induced protection from CDC tissue damage. We will apply advanced analytical techniques combined with mouse models of altered lipid metabolism to determine the cell types and lipid metabolic pathways involved in inducing a resistant state to CDCs in the skin. Specific Aim 3 determines which host lipid metabolism pathways are critical for resistance to localized or NF-like experimental GAS skin infection models. Our data shows that dysregulation of cholesterol metabolism potentiates CDC-mediated tissue damage but activating the LXR pathway induces a protective state. In this aim, we extend these exciting observations and mechanistically test if modulating lipid homeostasis in host tissues alters the pathogenesis of experimental NF models and may serve as an adjunct treatment. We expect that these studies...