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

细菌 CRISPR 干扰定义巨噬细胞对 B 族链球菌蛋白的反应

• 批准号: 10724607
• 负责人: David M Aronoff
• 金额: $ 25.29万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-15 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10724607
• 关键词: Adverse event; Affect; Alleles; Amniotic Fluid; Animal Model; Anti-Inflammatory Agents; Bacteria; Bacterial Genes; Binding; Binding Proteins; CRISPR interference; Candidate Disease Gene; Cell Wall; Cells; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Complement; Complement 5a; Complex; Confocal Microscopy; Dangerousness; Defect; Detection; Disease; Early treatment; Etiology; Fetal Development; Fetus; Gene Deletion; Gene Expression; Generations; Genes; Genetic; Goals; Growth; Health; Homeostasis; Human; Hyaluronidase; Immune; Immune Evasion; Immune response; Immunologic Surveillance; Immunophenotyping; Incubated; Infection; Inflammatory; Inflammatory Response; Innate Immune System; Interleukin-1 beta; Invaded; Lead; Libraries; Life; Macrophage; Macrophage Activation; Maternal-Fetal Exchange; Measurement; Mediating; Microbe; Modeling; Molecular Mimicry; Mothers; Mus; Natural Immunity; Newborn Infant; Outcome; Pathogenesis; Peptide Hydrolases; Phagocytes; Phagocytosis; Placenta; Play; Polysaccharides; Predisposition; Pregnancy; Pregnant Uterus; Pregnant Women; Premature Birth; Premature Labor; Prevention; Production; Protein Secretion; Proteins; Reactive Oxygen Species; Receptor Signaling; Role; Sialic Acids; Signal Transduction; Sorting; Streptococcal Infections; Streptococcus CAMP protein; Streptococcus Group B; Surface; TNF gene; Techniques; Testing; Therapeutic; Time; Tissues; United States; Vagina; Virulence; Western Blotting; Work; adverse pregnancy outcome; antimicrobial; candidate identification; capsule; clinically relevant; complement 4b-binding protein; complement system; cytokine; delivery complications; density; early onset; experimental study; fetal; high reward; high risk; in vivo; innovation; interest; intraamniotic infection; intrauterine infection; knock-down; microbial; mouse model; multimodality; mutant; neonatal sepsis; novel; novel therapeutics; novel vaccines; pathogen; post pregnancy; response; single-cell RNA sequencing; stillbirth; tool

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.

项目概要 B 族链球菌 (GBS) 是美国及世界各地子宫内感染的主要原因 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、新生儿败血症和全身性孕产妇疾病，这些都是 GBS 危及生命的原因之一。 严重宫内感染的一个常见病因是——在细菌性阴道定植者中——它具有 抑制和逃避胎儿和母体先天免疫监视的非凡能力 有效清除宫腔内的其他微生物。巨噬细胞是母胎的关键效应细胞。 先天免疫，在维持妊娠健康方面发挥重要作用，但无法消除 GBS 妊娠组织，为严重并发症奠定了基础。该提案的目的是检查， 详细信息，巨噬细胞和 GBS 细胞之间的分子相互作用，以发现基本机制 我们将使用 GBS 逃避和抑制妊娠巨噬细胞信号传导和细菌杀灭。 基于 CRISPR/Cas 的新型细菌基因抑制技术可系统地测试 GBS 菌株 缺乏特定的、高度保守的表面运输蛋白的敲低文库。 具有特定外化蛋白质缺陷的细胞将与离体人胎盘巨噬细胞共孵育，两者 母体和胎儿来源，检查它们对巨噬细胞细胞因子表达、吞噬​​作用和 我们将使用这些筛选来识别具有显着杀灭作用的新型 GBS 表面转运蛋白。 这些筛选的发现，包括几个对胎盘巨噬细胞免疫表型的影响。 已经进行了初步实验，将告知目标基因缺失 GBS 突变体的生成。 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 通过多重细胞因子分析、单细胞转录组学、免疫荧光详细了解巨噬细胞 共聚焦显微镜，以及临床相关 GBS 小鼠模型的体内结果检查 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疗法的外化蛋白质靶点 宫内感染。