Receptor-Mediated Endocytosis of Legionella Pneumophila

描述(摘自申请者的摘要)：军团菌的能力 嗜肺性肺炎(LPN)引起肺炎依赖于细胞内复制 在肺吞噬细胞和上皮细胞内。在环境中， 细菌无处不在，它们与阿米巴一起在细胞内繁殖。 原生动物的入侵和细胞内复制在 LPN在环境和环境中的扩增和传播 对人类的传播和传染性。我们最近已经表明， 原生动物蠕形哈特曼氏菌的LPN是由细菌介导的 将b2整合素样半乳糖/N-乙酰半乳糖胺凝集素附着在 原生动物的表面。细菌对凝集素的附着与 原生动物基因表达的诱导和酪氨酸去磷酸化 凝集素与肌动蛋白、粘着斑等几种细胞骨架蛋白 激酶、帕西林和纽蛋白。我们的数据显示细胞骨架在 原生动物寄宿在细菌附着上。透射电子显微镜 表明这些细菌诱导的对细胞过程的操纵 原生动物宿主与大多数细菌的进入有关 通过类似于受体介导的杯状内陷 内吞作用，但有些细菌是通过卷曲吞噬作用内化的。我们的 初步数据表明，LPN的进入机制是新的，这可能 导致其随后在细胞内的命运。我们的假设是 凝集素是一种参与摄取LPN的原生动物受体，是一种解离的 在细菌附着和入侵时从细胞骨架上。我们的具体目标 目的：1)克隆凝集素编码基因，并研究其对凝集素的调节作用 表达；2)克隆和鉴定与之结合的细菌配体 凝集素受体与配体-受体相互作用方式；3)测定 凝集素受体在细胞内的分布及其随后的命运 内化；以及4)评估受体与 细胞骨架。为拟议研究得出的结果将揭示新的 细胞内病原体吸收的范例，并将有助于我们的 对分子靶向内部“受保护液泡”的理解 真核细胞。我们提出的研究也可能有助于未来的设计 控制LPN在人群中扩增和传播的预防策略 水生环境，这是细菌传播到 人类。我们的研究可能揭示LPN潜在的致病进化以侵袭 更进化的哺乳动物细胞，可能有助于理解 分支杆菌和衣原体对原生动物的入侵。

Description (Adapted from applicant's abstract): The ability of Legionella pneumophila (Lpn) to cause pneumonia is dependent on intracellular replication within pulmonary phagocytic and epithelial cells. In the environment, the bacteria are ubiquitous where they multiply intracellularly with amoebae. Invasion and intracellular replication within protozoa play major factors in the amplification and dissemination of Lpn in the environment and in transmission and infectivity to humans. We have recently shown that uptake of Lpn by the protozoan Hartmannella vermiformis is mediated by bacterial attachment to a b2 integrin-like Galactose/N-acetyl-galactosamine lectin on the protozoan surface. Bacterial attachment to the lectin is associated with induction of protozoan gene expression and with tyrosine dephosphorylation of the lectin and several cytoskeletal proteins including actin, focal adhesion kinase, paxillin, and vinculin. Our data suggested a cytoskeletal disruption in the protozoan host upon bacterial attachment. Transmission electron microscopy showed that these bacterial-induced manipulations of cell processes in the protozoan host are associated with entry of the majority of the bacteria through a cup shape-like invagination that resemble receptor-mediated endocytosis, but some bacteria are internalized by coiling phagocytosis. Our preliminary data suggest that the mechanism of entry of Lpn is novel, which may contribute to its subsequent intracellular fate. Our hypothesis is that the lectin is a protozoan receptor involved in uptake of Lpn, and is dissociated from the cytoskeleton upon bacterial attachment and invasion. Our specific aims are, 1) to clone the lectin encoding gene and examine its regulation of expression; 2) to clone and characterize the bacterial ligand that binds the lectin receptor and the mode of ligand-receptor interaction; 3) to determine cellular distribution of the lectin receptor and its subsequent fate after internalization; and 4) to evaluate the interaction of the receptor with the cytoskeleton. The results derived for the proposed studies will uncover new paradigms of uptake of intracellular pathogens and will contribute to our understanding of targeting of molecules into a "protected vacuole" inside eukaryotic cells. Our proposed studies may also facilitate the design of future preventive strategies to control the amplification and spread of Lpn in the aquatic environment, which is the only source of bacterial transmission to humans. Our studies may uncover potential pathogenic evolution of Lpn to invade the more evolved mammalian cells, and may contribute to the understanding of invasion of protozoa by Mycobacterium and Chlamydia.