The macrophage Repairome

总结 由成孔毒素（PFT）引起的质膜破坏是细胞毒性最常见和最古老的策略， 细菌病原体感染宿主并逃避宿主的免疫反应。李斯特菌溶血素O（LLO）是一种PFT 由食源性病原体单核细胞增生李斯特菌（Lm）产生。Lm是一种革兰氏阳性菌 对于肺炎，一种严重的疾病，导致99%的住院治疗和高达30%的死亡率，尽管治疗。Lm是 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 在众多的毒力因子中，LLO是唯一已知的致病机制所不可或缺的。因此，LLO和 这种毒素所靶向的宿主途径是开发治疗干预的有希望的靶点。 LLO在Lm细胞内生命周期的所有阶段都被分泌，并结合胆固醇以组装大的 跨膜孔复合体。这种毒力因子长期以来一直被认为是破坏Lm的膜， 含有吞噬体以将Lm释放到其复制龛（细胞质）中。最近确定， 也可穿透宿主细胞质膜，促进细胞侵入和吞噬体逃逸。 单核细胞/巨噬细胞，专门捕获和破坏微生物， 巨噬细胞修复蛋白（称为巨噬细胞修复蛋白）维持细胞稳态和存活，尽管LLO 攻击巨噬细胞如何修复其质膜并防止毒素攻击尚未完全了解。的 这个R 03提案的目标是开发工具来发现“巨噬细胞修复”使用无偏见的整体， 基因组筛选我们的实验室成功地开发了基于荧光的筛选方法来分析修复 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组，我们将 使用CRISPR/Cas9基因组编辑进行全基因组筛选。我们将构建一个CRISPR/Cas9文库， 在THP-1细胞（人单核细胞样细胞系）中，并筛选文库中不能保持其完整性的细胞 在LLO暴露后，使用荧光激活细胞分选（FACS）可选择的表型（阳性筛选）。 事实上，具有缺陷细胞修复的受损细胞将是荧光的，而完整细胞将排除荧光。 荧光染料。深度测序将生成维持巨噬细胞所需的候选基因列表 完整我们将进行途径分析，选择最新颖和最有前途的途径，并验证 THP-1细胞中的选择途径。这些途径将在L.单核 通过未来的R 01资金感染。了解巨噬细胞抵抗细菌的机制 成孔毒素有望促进新型抗微生物治疗的发展， 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.