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

基本信息

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
项目类别：
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

项目摘要

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.

项目摘要 B组链球菌（GBS）是美国及其周围的乳肽内感染的主要原因世界。这些感染通常会导致严重的不良怀孕结局，包括死产，早产劳动，新生儿败血症和全身性材料疾病，可能会威胁生命。 GBS是这种严重插入式感染的这种常见病因是（在阴道殖民地细菌中）抑制和逃避胎儿和孕产妇先天免疫监视的特殊能力有效地从插入腔中清除其他微生物。巨噬细胞是含母元的关键作用先天免疫，并在维持妊娠健康方面发挥重要作用，但可能无法消除GB 怀孕组织，为严重的并发症奠定了基础。该提议的目的是检查细节，巨噬细胞和GBS细胞之间的分子相互作用，以发现基本机制 GBS逃避和抑制妊娠巨噬细胞信号传导和杀死细菌。我们将使用新型CRISPR/CAS基细菌基因抑制技术从系统地测试了GBS菌株缺乏特定，高度组成的表面运输蛋白的敲低文库。这些菌株与特定的外部化蛋白质缺陷一起将与离体人体占地巨噬细胞相一致孕产妇和胎儿衍生，以检查它们对巨噬细胞因子表达，吞噬作用和微生物杀死。我们将使用这些屏幕来识别具有重要意义的新型GBS表面运输蛋白对胎盘巨噬细胞免疫表型的影响。从这些屏幕中发现，包括几个已经在初步实验中进行的，将为靶向基因缺失GBS突变体的产生提供信息。这些然后将使用突变体和适当的完整控件来表征当地的影响巨噬细胞通过多重细胞因子谱分析，单细胞转录组学，免疫荧光。共聚焦显微镜以及从GBS的临床相关小鼠模型中检查体内结局插入感染。我们提出的两个目标将使用创新的多式联运方法来识别GBS 用于预防和早期治疗危险的新疫苗或治疗的外部蛋白质靶标插入感染。