Multifaceted activity of listeriolysin O during host cell invasion by Listeria

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，也是研究细胞内寄生的模式生物。细胞内病原体侵袭宿主细胞的两个主要机制已经被描述。病原体表达表面入侵蛋白，特异性激活宿主细胞受体，或产生分泌装置，将效应器注射到宿主细胞细胞质中，导致宿主细胞内吞机制的激活，使病原体内化。我们发现了第三种入侵机制，它在宿主细胞穿孔时被单核细胞增生乳杆菌的主要毒力因子激活，即成孔毒素李斯特菌溶素O （LLO）。已知LLO可以介导含有新内化细菌的内吞囊泡和细胞间扩散过程中形成的次级囊泡的破坏，导致单核增生乳杆菌释放到细胞质中，并在那里增殖。以前认为LLO只在酸化的核内体中有活性。然而，我们的实验室和其他人已经表明，细胞外细菌释放的LLO可以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足以诱导单核增生乳杆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，它与宿主细胞结合并组装成预孔复合物，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低密度脂蛋白诱导的细菌内化所必需的。我们最近的数据还表明，在被LLO损伤的细胞中，通过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剂。这个应用程序

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.