Project Summary Staphylococcus aureus is a Gram-positive pathogen that causes a wide range of superficial and invasive infections. An essential component of S. aureus infectivity and pathogenicity is the cell wall. Comprised of a thick layer of peptidoglycan (PG), the cell wall not only ensures cell viability but also provides protection against external stressors. Given its vital role in bacterial cell survival, PG is a major target of clinically relevant antibiotics. Apart from its role as a protective barrier, S. aureus PG serves as a pathogen-associated molecular pattern that promotes inflammation during infection. However, the full extent of pattern recognition receptors responsible for sensing S. aureus PG and the molecular features required for recognition are not fully explored. Nevertheless, we know that host-PG interactions can stimulate robust inflammation and production of the critical pro- inflammatory cytokine, IL-1β. Indeed, the prior literature argues that the degree of PG recognition by immune cells can shift the nature and duration of the IL-1β response, potentially leading to either infection clearance or inflammatory pathology and persistence. S. aureus PG is composed of repeating disaccharide subunits that are highly crosslinked via peptide cross-bridges. These glycans are remodeled by four hydrolases known as glucosaminidases. Recently, our lab surveyed glucosaminidase mutants to determine how PG remodeling might drive innate immunity. Our findings highlighted that a single enzyme, SagB, was required for S. aureus-mediated induction of IL-1β by bone marrow-derived macrophages. Notably, a ΔsagB mutant failed to stimulate the production of IL-1β while leaving other pro-inflammatory cytokines unaffected. Purified PG isolated from WT S. aureus was sufficient to induce macrophage production of IL-1β, whereas PG from a ΔsagB mutant did not. Furthermore, a ΔsagB mutant elicited reduced IL-1β in infected skin along with decreased inflammatory pathology. In systemic infections, the ΔsagB mutant displayed attenuated virulence. Lastly, we discovered that the SagB-mediated IL-1β response was independent of the NLRP3 inflammasome and caspase-1/11 activation, suggesting the requirement of other caspases or proteases in PG-mediated IL-1β maturation. Based on these findings, we hypothesize that SagB processes PG to generate specific glycans crucial for IL-1β maturation via an NLRP3-independent process (Aim 1) and that SagB-processed PG elicits IL-1β and promotes inflammatory pathology in vivo (Aim 2). Aim 1 will (i) determine the minimal PG component required to induce IL-1β from macrophages, (ii) interrogate the cellular pathway PG activates to produce IL-1β, and (iii) assess the localization of PG using cellular and immunological approaches. Aim 2 will use both systemic and skin and soft tissue infection models to determine if SagB is required to promote inflammation and if SagB-dependent IL-1β production is required to stimulate these re...