Host Responses to the Pore-Forming Toxin Listeriolysin O

宿主对成孔毒素李斯特菌溶血素 O 的反应

• 批准号: 10376220
• 负责人: Amal O Amer
• 金额: $ 67.17万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10376220
• 关键词: Adjuvant; Antigen Presentation; Antigen-Presenting Cells; Antigens; Bacteria; Binding; Calcium; Calcium Channel; Cancer Vaccines; Cathepsins; Cell Death; Cell Survival; Cell membrane; Cell physiology; Cell surface; Cells; Cholesterol; Communicable Diseases; Complex; Consequentialism; Cytolysins; Cytoskeletal Proteins; Cytosol; Data; Dendritic Cells; Development; Disease; Epithelial Cells; Event; Excision; Exocytosis; Exposure to; Family; Fluorescence Microscopy; Genes; Goals; Gram-Positive Bacteria; Histocompatibility Antigens Class II; Homeostasis; Hospitalization; Image; Immune; Immune response; Immunity; In Vitro; Infection; Inflammasome; Inflammatory; Injury; Innate Immune Response; Knowledge; Laboratories; Length; Libraries; Life; Life Cycle Stages; Listeria; Listeria monocytogenes; Listeria monocytogenes hlyA protein; Listeriosis; Lysosomes; Mediating; Membrane; Microscopy; Mind; Modeling; Myopathy; Neurodegenerative Disorders; Pathogenesis; Pathology; Pathway interactions; Perforation; Phagosomes; Plasma Cells; Play; Process; Property; Protein Kinase C; Proteins; Proteomics; Publishing; Recombinants; Reporting; Resolution; Role; Signal Transduction; Small Interfering RNA; Speed; Structure-Activity Relationship; T cell response; T-Lymphocyte; Testing; Therapeutic Intervention; Time; Toxin; Toxoids; Vaccines; Vacuole; Variant; Virulence; Virulence Factors; Work; anti-cancer; antimicrobial; base; cathepsin K; cell type; foodborne illness; foodborne pathogen; in vivo; macrophage; molecular assembly/self assembly; novel; novel therapeutic intervention; pathogen; premature; rational design; recruit; repaired; response; screening; spatiotemporal; trafficking; vaccine development; voltage

Summary Listeria monocytogenes is a facultative intracellular pathogen responsible for the life-threatening disease listeriosis. Although Lm produces numerous virulence factors, the secreted pore-forming toxin LLO is indispensable for pathogenesis. LLO is secreted at all stages of the Listeria intracellular life cycle and binds to cholesterol to form transmembrane pores. This virulence factor perforates the membrane of the Listeria- containing endocytic vacuoles to release the bacterium into its replicative niche, the cytosol. It was recently established that LLO also perforates the host cell plasma membrane, which promotes host cell invasion. It remains to elucidate how infected cells maintain viability despite perforation of their plasma membranes and how this low-grade perforation impacts the course of Listeria infection. The work performed in Aim 1 will establish novel mechanisms that maintain viability of infected cells despite perforation of their plasma membranes by LLO. Preliminary studies, via screening of a large siRNA library, led to the identification of novel families of host proteins that were not previously known to repair the plasma membrane of toxin-perforated cells. The Aim 1 studies will establish the mechanisms of action of these novel proteins. Specifically, they will determine the role plasma membrane depolarization plays in organizing calcium-dependent lysosome exocytosis, leading to the release of cytoprotective cathepsins on the cell surface. Aim 1 will also establish a new role for the septins, a family of cytoskeletal proteins, in plasma membrane repair. These studies will employ high-speed and super- resolution microscopy to analyze the molecular assemblies that orchestrate plasma membrane repair. The Aim 2 studies will establish the impact of plasma membrane perforation by LLO on Listeria intracellular survival and the innate immune response of antigen-presenting cells. We showed that transient plasma membrane perforation by LLO triggers Ca2+ influx-dependent activation of conventional PKCs on the endosomal network, a signaling event that is critical for Listeria phagosome escape. Aim 2 will identify the PKCs effectors by SILAC- based quantitative proteomic approach and how they contribute to the release of Listeria into the cytosol. Plasma membrane perforation also causes K+ efflux, which is known to activate the NLRP3 inflammasome. Aim 2 will dissect the role of low-grade plasma membrane perforation in the maturation of antigen presenting cells to enhance T cell immunity, in vitro and in vivo. This work is expected to broadly impact the development of vaccines and novel therapeutic strategies for a wide range of diseases in which pore-forming toxins are employed by pathogens.

总结 单核细胞增生李斯特菌是一种兼性胞内致病菌， 紫斑病。虽然Lm产生许多毒力因子，但分泌的成孔毒素LLO是 是发病机制不可或缺的。LLO在李斯特菌细胞内生命周期的所有阶段分泌，并结合至 胆固醇以形成跨膜孔。这种毒力因子穿透李斯特菌的细胞膜- 含有内吞空泡以将细菌释放到其复制小生境，即细胞质中。最近有 已经确定LLO也穿透宿主细胞质膜，这促进宿主细胞侵入。它 仍然需要阐明感染细胞如何在质膜穿孔的情况下保持活力，以及如何 这种低度穿孔影响李斯特菌感染的过程。在目标1中进行的工作将建立 新的机制，保持感染细胞的活力，尽管他们的质膜穿孔的LLO。 初步研究，通过筛选一个大的siRNA文库，导致鉴定新的家庭的主机 这些蛋白质以前不知道能修复毒素穿孔细胞的质膜。目标1 研究将建立这些新蛋白质的作用机制。具体来说，他们将决定 质膜去极化在组织钙依赖性溶酶体胞吐作用中起作用，导致 在细胞表面释放细胞保护性组织蛋白酶。目标1还将为septins确立一个新的角色， 细胞骨架蛋白家族，在质膜修复中。这些研究将采用高速和超- 分辨率显微镜来分析编排质膜修复的分子组装。目的 2项研究将确定LLO造成的质膜穿孔对李斯特菌细胞内存活的影响， 抗原呈递细胞的先天免疫反应。我们发现瞬时质膜 LLO穿孔触发内体网络上常规PKC的Ca 2+内流依赖性激活， 这是李斯特菌吞噬体逃逸的关键信号事件。目标2将通过SILAC鉴定PKC效应物- 基于定量蛋白质组学的方法，以及它们如何有助于李斯特菌释放到胞质溶胶中。血浆 膜穿孔还引起K+流出，已知K+流出激活NLRP 3炎性体。目标2将 剖析低度质膜穿孔在抗原呈递细胞成熟中的作用， 增强T细胞免疫，在体外和体内。这项工作预计将广泛影响疫苗的开发 以及用于多种疾病的新的治疗策略， 病原体