Bacterial cell wall remodeling, innate immunity, and pathogenesis

Project Summary Infections caused by Staphylococcus aureus remain one of the most significant burdens to human health with ~750,000 associated deaths worldwide reported in 2019; a third of which were directly attributed to antimicrobial resistance. Many therapeutics that target peptidoglycan synthesis are ineffective against S. aureus on account of these resistance adaptations. In the absence of effective antimicrobials, the host depends on rapid detection by the immune system to provide protection against S. aureus infection. However, this response must be calibrated to balance the beneficial outcomes of inflammation on infection clearance with the pathological consequences of excessive inflammation that include tissue damage and compromised infection resolution. A portion of the host inflammatory response against S. aureus is incited by peptidoglycan, yet we lack a molecular understanding of how peptidoglycan recognition shapes the balance between immune-mediated clearance of microbes and inflammatory pathology. One of the primary cytokines required for the host inflammatory response to S. aureus is IL-1β, yet we also do not know how S. aureus calibrates IL-1β responses to persist during infection. We recently tested if S. aureus peptidoglycan modifying enzymes known as glucosaminidases shape innate immune responses to S. aureus and identified the staphylococcal N-acetyl glucosaminidase B, SagB, as a central mediator IL-1β maturation by immune cells. SagB appears to promote IL-1β production from innate cells by processing peptidoglycan to shortened lengths that are normally required to establish cell wall rigidity and bacterial cell size. SagB-mediated IL-1β production was independent of canonical pattern recognition receptors that recognize Gram positive peptidoglycan as well as the NLRP3 inflammasome. Instead, infection of macrophages with a ∆sagB mutant led to reduced caspase-1/11-independent cleavage of pro-IL-1β and its accumulation in the macrophage cytosol. Furthermore, SagB was required for virulence in systemic infection and promoted IL-1β production and inflammatory pathology in a skin and soft tissue infection model. Our supporting data suggests that SagB-mediated processing of peptidoglycan can offset the balance between beneficial and pathological inflammation through a previously undescribed mechanism of IL-1β maturation. We hypothesize that SagB processes peptidoglycan to a biologically active short glycan required for host induction of IL-1β. Furthermore, we hypothesize SagB-mediated IL-1β production by immune cells occurs via a non- canonical pathway that drives maturation and possibly release and promotes inflammatory pathology and infection in vivo. Aim 1 will determine how and where SagB-modified peptidoglycan products are recognized by innate immune cells and determine how peptidoglycan cross-linking and glycan length drive recognition. Aim 2 will investigate the mechanism of SagB-dependent IL-1β maturation and release. Aim 3 will establish how SagB promotes infection during local and disseminated infection and will ascertain how peptidoglycan synthesis enzymes or cell wall targeting antibiotics interface with SagB to promote infection.