ϕSa3 prophage as a molecular regulatory switch of Staphylococcus aureus β-toxin production

The goal of this proposal is to understand the dynamics of phage – bacterium – mammalian host interaction shaping Staphylococcus aureus fitness and adaptation to new environments. S. aureus is a leading cause of life-threatening diseases like pneumonia, infective endocarditis (IE), and septicemia. b-toxin is a sphingomye- linase known to increase the severity of these infections. However, the b-toxin hlb gene is disrupted by the ϕSa3 family of prophages in up to 96% of S. aureus human isolates. Yet, b-toxin-producing variants often arise during infections and are more virulent in experimental IE and pneumonia in rabbits. Little is known about the underlying mechanisms driving ϕSa3 mobilization and b-toxin production. Our recent studies demonstrated that b-toxin production is controlled by phage excision, where ϕSa3 functions as a phage-regulatory switch (phage-RS) that allows hlb expression in response to host signals. Here, we addressed the uncharacterized role of S. aureus prophages as molecular regulatory switches and b-toxin’s novel role in dysregulation of vascular regeneration. In turn, these studies will provide critical insight into S. aureus use of prophages for host adaptation and fitness, redefining the phage-bacterium interaction and b-toxin’s role in pathogenesis. ϕSa3 excision and integration or cell lysis are mediated by the timing and expression levels of phage genes, that in turn dictate phage dynamics. Yet, S. aureus sensing and responding to specific environments can differentially define the ϕSa3 dynamics as a result of the regulatory networks of the specific clonal lineage. Aim 1 will seek to elucidate the temporal ϕSa3 dynamics driving ‘active lysogeny’ and b-toxin production in S. aureus lineages of medical importance and under conditions encountered in the mammalian host. Several phage-RSs have now been described in various microbial species. Although incompletely understood, in all these examples phage excision leads to the alteration or regulation of critical processes. However, the mechanism controlling phage-RSs and their interaction with the native regulatory system of the microbial host have not been elucidated. Aim 2 will directly address this and will determine whether regulators of hlb expression trigger ϕSa3 excision to allow the timely production of b-toxin and/or alter phage dynamics. β-toxin is a sphingomyelinase (SMase). SMases hydrolyze sphingomyelin in eukaryotic membranes into bioactive sphingolipids widely recognized as essential signaling molecules that promote health or disease. Yet, the vascular responses specific to β-toxin SMase activity and its link to β-toxin’s anti-regenerative effects remain speculative. Aim 3 will elucidate the underlying molecular responses driving β-toxin’s vascular effects. In particular, the physiological context and sphingolipid metabolites associated with those responses. Project Number: 1R01AI182111-01A1 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Wilmara Salgado Pabon | Institution: UNIVERSITY OF WISCONSIN-MADISON, MADISON, WI | Award Amount: $560,178 | Activity Code: R01 | Study Section: Bacterial Virulence Study Section [BV] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R01AI18211101A1