The macrophage Repairome

摘要 由成孔毒素(PFTs)破坏质膜是一种最常见和最古老的策略，由 细菌病原体感染它们的宿主并逃避宿主的免疫反应。李斯特菌溶血素O(LLO)是PFT 由食源性致病菌单核细胞增多性李斯特氏菌(Lm)产生。金黄色葡萄球菌是革兰氏阳性菌 对于李斯特菌病，这是一种严重的疾病，导致99%的人住院，尽管接受了治疗，但高达30%的死亡率。Lm是一种 感染包括巨噬细胞在内的大量细胞的兼性细胞内病原体。虽然LM生产 在众多的毒力因素中，LLO是唯一已知的在发病过程中不可或缺的因素。因此，LLO和 这种毒素靶向的宿主通路是开发治疗干预措施的有希望的靶点。 LIO在LM细胞内生命周期的所有阶段都会分泌，并与胆固醇结合以组装大的 跨膜孔复合体。长期以来，人们一直知道这种毒力因子会穿透LM的细胞膜。 含有吞噬小体，将LM释放到其复制的利基--胞浆中。最近证实，LLO 也穿孔宿主细胞质膜，这有助于细胞入侵和吞噬小体逃逸。 专门捕获和破坏微生物的单核/巨噬细胞进化出细胞保护 尽管存在LLO，但维持细胞稳态和存活的机制(称为巨噬细胞修复) 进攻。巨噬细胞如何修复其质膜并防止毒素攻击尚不完全清楚。这个 这个R03提案的目标是开发工具，使用无偏见的整体- 基因组筛查。我们实验室成功地开发了基于荧光的筛查方法来分析修复 暴露于LLO的细胞的机械。为了以公正的方式建立巨噬细胞修复系统，我们将 使用CRISPR/Cas9基因组编辑进行全基因组筛选。我们将生成一个CRISPR/Cas9库 在THP-1细胞(人单核细胞样细胞系)中筛选不能保持其完整性的细胞 在LLO暴露时，使用荧光激活细胞分选(FACS)可选择的表型(阳性筛选)。 事实上，细胞修复缺陷的受损细胞将是荧光的，而完整的细胞将排除 荧光染料。深度测序将产生维持巨噬细胞所需的候选基因清单 正直。我们将进行路径分析，选择最新颖和最有前途的路径，并验证 THP-1细胞中选择的通路。这些途径将在单核细胞增多性乳杆菌的背景下进行详细研究。 通过未来的R01资助感染。了解巨噬细胞用于中和细菌的机制 多孔性毒素有望促进新型抗菌治疗的发展，以缓解 传染病的负担。

Summary Plasma membrane disruption by pore-forming toxins (PFTs) is a most common and ancient strategy used by bacterial pathogens to infect their host and evade the host’s immune responses. Listeriolysin O (LLO) is a PFT produced by the foodborne pathogen Listeria monocytogenes (Lm). Lm is a Gram-positive bacterium responsible for listeriosis, a severe illness leading to 99% hospitalization and up to 30% fatality despite treatment. Lm is a facultative intracellular pathogen that infects a large array of cells including macrophages. Although Lm produces numerous virulence factors, LLO is uniquely known to be indispensable for pathogenesis. Therefore, LLO and the host pathways targeted by this toxin are promising targets for the development of therapeutic interventions. LLO is secreted at all stages of the Lm intracellular lifecycle and binds cholesterol to assemble a large transmembrane pore complex. This virulence factor has long been known to perforate the membrane of the Lm- containing phagosome to release Lm into its replicative niche, the cytosol. It was recently established that LLO also perforates the host cell plasma membrane, which facilitates cell invasion and phagosomal escape. Monocyte/Macrophages, which specialize in the capture and destruction of microbes, evolved cytoprotective mechanisms (referred to as the macrophage repairome) to maintain cell homeostasis and survival despite LLO attack. How macrophages repair their plasma membrane and prevent toxin attack is not fully understood. The goal of this R03 proposal is to develop tools to discover the “macrophage repairome” using unbiased whole- genome screening. Our lab successfully developed fluorescence-based screening methods to analyze the repair machineries of cells exposed to LLO. To establish the macrophage repairome in an unbiased fashion, we will perform a whole-genome screen using CRISPR/Cas9 genome editing. We will generate a CRISPR/Cas9 library in THP-1 cells (human monocyte-like cell line) and screen the library for cells unable to maintain their integrity upon LLO exposure using florescence-activated cell sorting (FACS)-selectable phenotype (positive screen). Indeed, damaged cells with deficient cell repair will be fluorescent, whereas intact cells will exclude the fluorescent dye. Deep sequencing will generate a list of candidate genes required for maintaining macrophage integrity. We will perform pathway analysis, select the most novel and promising pathways, and validate the selected pathways in THP-1 cells. These pathways will be studied in detail in the context of L. monocytogenes infection via future R01 funding. Understanding the mechanisms used by macrophages to counteract bacterial pore-forming toxins is expected to facilitate the development of novel antimicrobial treatments to alleviate the burden of infectious diseases.