Multifaceted activity of listeriolysin O during host cell invasion by Listeria

描述(申请人提供)：单核细胞增多性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式生物。细胞内病原体入侵宿主细胞的两种主要机制已被描述。病原体表达特定激活宿主细胞受体的表面入侵蛋白，或产生一种分泌装置，将效应物注入宿主细胞胞浆，导致宿主细胞内化病原体的内吞机制激活。我们发现了第三种入侵机制，该机制是在宿主细胞穿孔时由单核细胞增生性李斯特菌的主要毒力因子--致孔毒素李斯特溶素O(LLO)激活的。众所周知，LIO可以破坏含有新近内化细菌的内吞小泡和在细胞间传播过程中形成的次生小泡，导致单核细胞增多性乳杆菌释放到细胞质中，在那里增殖。以前认为LLO只在酸化的内吞体内有活性。然而，我们的实验室和其他人已经证明，由胞外细菌释放的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足以诱导单核细胞增多性乳杆菌内化到上皮细胞。为了深入了解这一新的入侵途径，我们构建了一个LLO变体，它与宿主细胞结合并组装成前孔复合体，但不能形成孔。这一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，穿过质膜的离子通量是维持宿主细胞完整性的细菌内化和生存途径的主要调节因素。此应用程序 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的内化和宿主细胞的生存。我们将使用最先进的活细胞FRT荧光显微镜来识别和表征依赖LLO的细菌内化和细胞生存的信号事件。我们还将使用小鼠模型测试LLO在体内细菌内化中的作用。目的1阐明LLO激活的信号和内吞途径诱导F-肌动蛋白依赖的L-单核细胞增多症的内化。目的2阐明LLO穿孔细胞质膜修复的机制，以及细胞膜修复与宿主细胞体内外侵袭的相互作用。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。本应用中研究的新的入侵机制与许多病原体有关，因为致孔毒素是病毒以及原核和真核胞内寄生虫的常见毒力因子。这项工作有望确定可作为针对由细胞内病原体引起的广泛疾病的治疗干预的宿主途径。

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.