Multifaceted activity of listeriolysin O during host cell invasion by Listeria

描述（由申请方提供）：单核细胞增生李斯特菌是危及生命的食源性疾病嗜热链球菌病的病原体，也是细胞内寄生研究的模式生物。已经描述了细胞内病原体侵入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溶胶中的分泌装置，导致内化病原体的宿主细胞内吞机制的激活。我们发现了第三种入侵机制，即在宿主细胞穿孔时被L.单核细胞增多症，孔形成毒素溶血素O（LLO）。已知LLO介导包含新内化的细菌的内吞囊泡和在细胞到细胞传播期间形成的次级囊泡的破坏，导致L.单核细胞增多症进入细胞质，在那里它增殖。以前认为LLO仅在酸化的内体中有活性。然而，我们的实验室和其他人已经表明，由细胞外细菌释放的LLO有效地穿透宿主细胞，并在这种情况下发挥关键作用。特别是，我们发表的数据表明，LLO足以诱导L。单核细胞增多症内化到上皮细胞中。为了深入了解这种新的入侵途径，我们构建了一种LLO变体，该变体与宿主细胞结合并组装成前孔复合物，但不能形成孔。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，在由LLO损伤的细胞中，跨质膜的离子通量是细菌内化和存活途径的主要调节剂，其维持宿主细胞的完整性。本申请 将阐明宿主细胞对LLO诱导的离子通量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潜在的LLO依赖性细菌内化和细胞存活的信号事件。我们还将使用鼠模型测试LLO在体内细菌内化中的作用。目的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L。单核细胞增生内在化目的二是阐明LLO穿孔细胞的质膜修复机制，以及细胞膜修复与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。本申请中研究的新的侵入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宿主途径，可以针对细胞内病原体引起的广泛疾病进行治疗干预。

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.