Multifaceted activity of listeriolysin O during host cell invasion by Listeria

李斯特氏菌入侵宿主细胞期间李斯特氏菌溶血素 O 的多方面活性

• 批准号: 8793094
• 负责人: Stephanie M M Seveau
• 金额: $ 37.63万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-02-01 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8793094
• 关键词: Accounting; Affect; Animal Model; Area; Bacteria; Binding; Cell Survival; Cell membrane; Cells; Cellular biology; Complex; Cytoplasm; Cytosol; Data; Development; Disease; Endocytic Vesicle; Endocytosis; Endosomes; Epithelial Cells; Event; F-Actin; Fluorescence; Generations; Goals; Health; Immune system; In Vitro; Infection; Knowledge; Laboratories; Lesion; Life; Listeria; Listeria monocytogenes; Listeria monocytogenes hlyA protein; Listeriosis; Mediating; Membrane; Modeling; Mus; Parasites; Pathway interactions; Peptides; Perforation; Play; Process; Proliferating; Proteins; Public Health; Publishing; Receptor Cell; Research; Role; Signal Pathway; Signal Transduction; Speed; Streptococcus pneumoniae plY protein; Surface; System; Testing; Therapeutic; Therapeutic Intervention; Toxin; Trypanosoma cruzi; Variant; Vesicle; Virulence Factors; Virus; Work; extracellular; fluorescence imaging; foodborne illness; foodborne pathogen; in vivo; injured; insight; intracellular parasitism; membrane model; neutralizing antibody; novel; novel therapeutics; pathogen; polymerization; porin; prophylactic; repaired; response; rho GTP-Binding Proteins; tool; uptake

DESCRIPTION (provided by applicant): Listeria monocytogenes is the etiologic agent of the life-threatening foodborne illness listeriosis and a model organism for the study of intracellular parasitism. Two major mechanisms of host cell invasion by intracellular pathogens have been described. Pathogens express surface invasins that specifically activate host cell receptors, or produce a secretion apparatus that injects effectors into the host cell cytosol, leading to the activation of the host cell endocytic machineries that internalize the pathogen. We discovered a third mechanism of invasion that is activated upon host cell perforation by the major virulence factor of L. monocytogenes, the pore-forming toxin listeriolysin O (LLO). LLO is known to mediate the disruption of the endocytic vesicle that contains newly internalized bacteria and the secondary vesicle formed during cell-to-cell spreading, leading to the release of L. monocytogenes into the cytoplasm, where it proliferates. It was previously thought that LLO was only active within acidified endosomes. However, our laboratory and others have shown that LLO released by extracellular bacteria efficiently perforates host cells and plays critical roles i this context. In particular, our published data demonstrate that LLO is sufficient to induce L. monocytogenes internalization into epithelial cells. To gain insight into this novel invasion pathway, we constructed a LLO variant that binds to host cells and assembles into a prepore complex, but is unable to form a pore. This unique tool allowed us to demonstrate that host cell perforation is required for LLO-induced bacterial internalization. Our recent data also show that in cells injured by LLO, ionic fluxes across the plasma membrane are master regulators of bacterial internalization and survival pathways that maintain host cell integrity. This application will elucidate host cell responses to ionic fluxes induced by LLO and how these responses control bacterial internalization and host cell survival. We will use state-of-the-art live-cell FRT fluorescence microcopy to identify and characterize signaling events underlying LLO-dependent bacterial internalization and cell survival. We will also test the role of LLO in bacterial internalization in vivo using the murine model. Aim 1 will elucidate signaling and endocytic pathways activated by LLO to induce F-actin-dependent L. monocytogenes internalization. Aim 2 will elucidate the mechanisms of plasma membrane repair in cells perforated by LLO and the cross-talk between membrane repair and host cell invasion in vitro and in vivo. Elucidating host cell responses to plasma membrane damage caused by pathogens and how these responses affect the course of infection are emerging areas of research. The novel invasion mechanism studied in this application is relevant to numerous pathogens as pore-forming toxins are common virulence factors of viruses as well as prokaryotic and eukaryotic intracellular parasites. This work is expected to identify host pathways that can be targeted for therapeutic interventions against a wide range of diseases caused by intracellular pathogens.

描述（由申请人提供）：单核细胞增生李斯特菌是危及生命的食源性疾病李斯特菌病的病原体，也是研究细胞内寄生的模型生物。已经描述了细胞内病原体入侵宿主细胞的两种主要机制。病原体表达特异性激活宿主细胞受体的表面侵袭素，或产生将效应物注入宿主细胞胞质的分泌装置，导致宿主细胞内吞机制的激活，从而内化病原体。我们发现了第三种入侵机制，该机制在宿主细胞穿孔时被单增李斯特菌的主要毒力因子——成孔毒素李斯特菌溶血素 O (LLO) 激活。众所周知，LLO 会介导内吞囊泡的破坏，内吞囊泡含有新内化的细菌，以及细胞间扩散过程中形成的次级囊泡，导致单核细胞增生利斯特氏菌释放到细胞质中，并在细胞质中增殖。此前人们认为 LLO 仅在酸化内体中具有活性。然而，我们的实验室和其他实验室已经证明，细胞外细菌释放的脂质连接寡糖能够有效地穿孔宿主细胞，并在此背景下发挥关键作用。特别是，我们发表的数据表明 LLO 足以诱导单核细胞增生利斯特氏菌内化到上皮细胞中。为了深入了解这种新的入侵途径，我们构建了一种 LLO 变体，它可以与宿主细胞结合并组装成前孔复合物，但无法形成孔。这种独特的工具使我们能够证明宿主细胞穿孔是 LLO 诱导的细菌内化所必需的。我们最近的数据还表明，在 LLO 损伤的细胞中，跨质膜的离子通量是细菌内化和维持宿主细胞完整性的生存途径的主要调节因子。这个应用程序 将阐明宿主细胞对 LLO 诱导的离子流的反应，以及这些反应如何控制细菌内化和宿主细胞存活。我们将使用最先进的活细胞 FRT 荧光显微镜来识别和表征 LLO 依赖性细菌内化和细胞存活的信号事件。我们还将使用小鼠模型测试 LLO 在体内细菌内化中的作用。目标 1 将阐明 LLO 激活的信号传导和内吞途径，以诱导 F-肌动蛋白依赖性单核细胞增生李斯特菌内化。目标 2 将阐明 LLO 穿孔细胞中质膜修复的机制，以及膜修复与体外和体内宿主细胞侵袭之间的相互作用。阐明宿主细胞对病原体引起的质膜损伤的反应以及这些反应如何影响感染过程是新兴的研究领域。本申请中研究的新型入侵机制与许多病原体相关，因为成孔毒素是病毒以及原核和真核细胞内寄生虫的常见毒力因子。这项工作预计将确定可针对细胞内病原体引起的多种疾病进行治疗干预的宿主途径。