Project Summary Group B Streptococcus (GBS) is a major cause of intrauterine infections in the United States and around the world. These infections commonly lead to serious adverse pregnancy outcomes including stillbirth, preterm labor, neonatal sepsis, and systemic maternal disease, which can be life-threatening. One reason that GBS is such a common etiology of serious intrauterine infection is that—among bacterial vaginal colonizers—it has exceptional abilities to suppress and evade fetal and maternal innate immune surveillance that is highly effective at clearing other microbes from the intrauterine cavity. Macrophages are key effectors of maternofetal innate immunity, and serve important roles in maintaining gestational health, yet can fail to eliminate GBS from pregnancy tissues, setting the stage for serious complications. The goal of this proposal is to examine, in detail, molecular interactions between macrophages and GBS cells to discover basic mechanisms of GBS evasion and suppression of gestational macrophage signaling and bacterial killing. We will use novel CRISPR/Cas-based bacterial gene suppression techniques to systematically test GBS strains from knockdown libraries that are deficient in specific, highly conserved, surface trafficked proteins. These strains with specific externalized protein defects will be coincubated with ex vivo human placental macrophages, both maternal and fetal-derived, to examine their effects on macrophage cytokine expression, phagocytosis, and microbial killing. We will use these screens to identify novel GBS surface trafficked proteins with significant effects on placental macrophage immunophenotypes. Discoveries from these screens, including several already made in preliminary experiments, will inform generation of targeted gene deletion GBS mutants. These mutants and appropriate complemented controls will then be used to characterize effects on placental macrophages in detail, through multiplex cytokine profiling, single-cell transcriptomics, immunofluorescent confocal microscopy, and examination of in vivo outcomes from a clinically relevant mouse model of GBS intrauterine infection. Our two proposed aims will use innovative, multimodal approaches to identify GBS externalized protein targets for new vaccines or therapeutics for prevention and early treatment of dangerous intrauterine infections.