Bacterial CRISPR interference to define macrophage responses to group B Streptococcus proteins

项目摘要 B组链球菌(GBS)是美国和世界各地宫内感染的主要原因 世界。这些感染通常会导致严重的不良妊娠结局，包括死产、早产 分娩、新生儿败血症和系统性母体疾病，这些都可能危及生命。GBS之所以成为 这种严重宫内感染的常见病因是--在细菌性阴道定植者中--它具有 具有特殊的能力来抑制和躲避胎儿和孕妇的天然免疫监视 有效地清除宫腔内的其他微生物。巨噬细胞是母婴发育的关键效应细胞 先天免疫，并在维持妊娠健康方面发挥重要作用，但不能从 妊娠组织，为严重并发症埋下伏笔。这项提案的目标是审查，在 巨噬细胞与GBS细胞的分子相互作用以揭示其基本机制 GBS逃避和抑制妊娠巨噬细胞信号和细菌杀伤。我们将使用 基于CRISPR/Cas的细菌基因抑制新技术用于系统检测GBS菌株 敲除文库缺乏特定的、高度保守的、表面运输的蛋白质。这些菌株 带有特定外化蛋白缺陷的细胞将与体外培养的人胎盘巨噬细胞共同孵育，两者 以检测它们对巨噬细胞细胞因子表达、吞噬功能和 微生物杀戮。我们将使用这些筛选来鉴定新的GBS表面交易蛋白，这些蛋白具有显著的 胎盘巨噬细胞免疫表型的影响。这些屏幕上的发现，包括几个 已经在初步实验中做出，将通知生成靶向基因缺失的GBS突变体。这些 然后将使用突变体和适当的补充对照来表征对胎盘的影响 巨噬细胞的细节，通过多重细胞因子图谱，单细胞转录，免疫荧光 共聚焦显微镜和临床相关的GBS小鼠模型体内结果的检查 宫内感染。我们提出的两个目标将使用创新的多模式方法来识别GBS 用于预防和早期治疗危险疾病的新疫苗或治疗药物的外化蛋白质靶标 宫内感染。

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.