Receptor-Mediated Endocytosis of Legionella Pneumophila

描述（改编自申请人摘要）：军团菌的能力 嗜肺菌（Lpn）引起肺炎是依赖于细胞内复制 在肺吞噬细胞和上皮细胞内。在环境中， 细菌在细胞内与变形虫一起繁殖的地方无处不在。 原生动物内的入侵和细胞内复制是导致 Lpn在环境中的扩增和传播， 传播和感染人类。我们最近发现， 蠕虫状哈特曼氏菌的Lpn是由细菌介导的 连接到b2整联蛋白样半乳糖/N-乙酰半乳糖胺凝集素上， 原生动物表面细菌对凝集素的附着与 诱导原生动物基因表达和酪氨酸去磷酸化 凝集素和几种细胞骨架蛋白，包括肌动蛋白、粘着斑 激酶、桩蛋白和黏着斑蛋白。我们的数据表明， 原生动物的宿主。透射电子显微镜 表明，这些细菌诱导的细胞过程的操纵， 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 有些细菌是通过内吞作用而内化的，但有些细菌是通过卷曲吞噬作用而内化的。我们 初步数据表明，Lpn的进入机制是新的，这可能 有助于其随后的细胞内命运。我们的假设是 凝集素是一种参与Lpn摄取的原生动物受体， 从细胞骨架中分离出来我们的具体目标 是，1）克隆凝集素编码基因，并检查其调节， 表达; 2）克隆和表征结合所述细菌配体的细菌配体， 凝集素受体和配体-受体相互作用的模式; 3）确定 凝集素受体的细胞分布及其随后的命运 内化;以及4）评估受体与受体的相互作用 细胞骨架从拟议的研究中得出的结果将揭示新的 细胞内病原体摄取的范例，并将有助于我们 了解分子靶向进入内部“受保护的空泡” 真核细胞我们建议的研究也可能有助于设计未来的 预防策略，以控制Lpn的扩增和传播， 水生环境是细菌传播的唯一来源， 人类我们的研究可能揭示Lpn的潜在致病进化， 更进化的哺乳动物细胞，并可能有助于理解 分枝杆菌和衣原体入侵原生动物。

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.