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

项目摘要 B族链球菌（GBS）是美国和世界各地子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、新生儿败血症和可能危及生命的全身性母体疾病。GBS之所以 严重子宫内感染的常见病因是，在细菌阴道定植者中， 抑制和逃避胎儿和母体先天免疫监视的特殊能力， 有效清除子宫腔内的其他微生物。巨噬细胞是母胎发育的关键效应子 先天免疫，并在维持妊娠期健康中发挥重要作用，但不能消除GBS， 妊娠组织，为严重的并发症做好准备。该提案的目的是审查，在 详细，巨噬细胞和GBS细胞之间的分子相互作用，以发现 GBS逃避和抑制妊娠巨噬细胞信号传导和细菌杀伤。我们将使用 基于CRISPR/Cas的新型细菌基因抑制技术，用于系统地测试来自 敲低文库缺乏特异性的、高度保守的、表面运输的蛋白。这些菌株 将与离体人胎盘巨噬细胞共孵育， 母体和胎儿来源的，以检查它们对巨噬细胞细胞因子表达、吞噬作用和 微生物杀灭我们将使用这些筛选来鉴定新的GBS表面运输蛋白， 对胎盘巨噬细胞免疫表型的影响。从这些屏幕上的发现，包括几个 已经在初步实验中，将告知靶基因缺失GBS突变体的产生。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。 巨噬细胞详细，通过多重细胞因子谱，单细胞转录组学，免疫荧光 共聚焦显微镜检查和临床相关GBS小鼠模型的体内结果检查 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性疾病的新疫苗或治疗剂的外部化蛋白质靶点 子宫内感染

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.