Project Summary Staphylococci are ubiquitous bacterial residents of human skin and major causes of antibiotic-resistant infections. Of the ~40 skin-associated species, S. aureus and S. epidermidis have the greatest pathogenic potential: S. aureus is the leading cause of skin and soft tissue infections and S. epidermidis is the most common cause of infections associated with indwelling medical devices. Compounding the problem, S. epidermidis strains harbor a reservoir of genes that enhance fitness/virulence (e.g. genes that encode toxins and antibiotic resistance) which can be horizontally transferred to S. aureus. In light of these facts, a thorough understanding of the mechanisms that regulate horizontal gene transfer between these species would be an invaluable asset in neutralizing or stemming the flow of these factors at the source. In this context, staphylococcal phages (i.e. viruses) and the immune systems targeted against them have profound impacts on staphylococcal survival and pathogenesis. For instance, lysogenic phages can enhance pathogenic potential by transferring pathogenicity islands from one strain to another and carrying virulence factors that integrate along with the phage genome into the host. In contrast, strictly lytic phages can kill the bacterial host within minutes and are being used as alternative therapeutics to combat antibiotic-resistant infections. Bacterial immune systems target lytic and lysogenic phages alike, and can therefore counter these opposing effects. As it stands, we are only just beginning to understand these dynamics and identify the specific immune systems that staphylococci employ, and alarmingly, almost nothing is known about how these systems are horizontally spread. These knowledge gaps continue to undermine our ability to implement effective therapeutics and improve overall healthcare outcomes. The long-term objective of this R01 project is to gain a comprehensive understanding of the anti-phage immune systems in staphylococci and the pathways by which they spread. Towards this goal, this research uses S. epidermidis and a collection of diverse phages as model organisms to achieve three specific aims: Aim 1 will identify and characterize new anti-phage defenses in a suite of S. epidermidis clinical isolates using genetics, biochemical, and bioinformatics approaches. Aim 2 will determine the major genetic and environmental factors that drive mobilization of these defenses using molecular and genetic approaches. Aim 3 seeks to determine the global impacts of the molecular machinery that mediate defense mobilization using high-throughput genetics approaches. By revealing new insights into mechanisms of anti-phage defenses in staphylococci and the pathways by which they spread, the proposed work will enable the development of more effective approaches for not only combatting the spread of resistance to antibiotics but also saving the burgeoning phage therapeutics from a similar fate. This work will a...