Identification of host factors required for Yersinia pestis macrophage infection

描述（由申请人提供）：鼠疫是由鼠疫耶尔森菌引起的急性感染，鼠疫耶尔森菌是一种革兰氏阴性兼性细胞内细菌，感染巨噬细胞并存活。越来越多的证据表明，细胞内生长是重要的哺乳动物定居的Y。鼠疫进入巨噬细胞后，Y.鼠疫菌保留在一个被称为含耶尔森氏菌空泡（YCV）的膜结合区室中，但它不会被巨噬细胞杀死。此外，细菌主动抑制YCV的酸化。我们假设Y.鼠疫杆菌与特定的宿主因子相互作用，以改变YCV的成熟和酸化。虽然已经确定了对细胞内存活重要的细菌因子，但细菌在巨噬细胞内存活所使用的分子机制尚未确定。为了识别这些因素/途径，我们建立了一个新的Y。pestis bioreporter监测鼠疫菌。鼠疫菌在巨噬细胞中存活。使用这种生物报告，我们已经开发了一种抑制性RNA为基础的测定，以特异性地确定宿主因素，改变Y。胞内鼠疫菌 生存通过该检测，我们将进行全基因组筛选，以确定Y所需的宿主因子/途径。鼠疫菌在巨噬细胞中的存活（目的1）。使用计算机模拟和显微镜分析的组合，我们还将特异性地鉴定在我们的筛选中鉴定的改变吞噬体成熟以使Y受益的宿主因子。鼠疫（目标2）。这些研究将是第一个针对宿主细胞的研究，以了解Y。鼠疫菌在宿主细胞中生存。

DESCRIPTION (provided by applicant): Plague is an acute infection caused by Yersinia pestis, a Gram-negative, facultative intracellular bacterium that infects and survives in macrophages. Growing evidence suggests that intracellular growth is important for mammalian colonization by Y. pestis. Upon entry into macrophages, Y. pestis remains in a membrane-bound compartment called the Yersinia-containing vacuole (YCV), but it is not killed by the macrophage. Furthermore, the bacterium actively inhibits acidification of the YCV. We hypothesize that Y. pestis interacts with specific host factors in order to alter YCV maturation and acidification. While bacterial factors important for intracellular survival have been identifie, the molecular mechanisms used by the bacterium to survive within macrophages have not been defined. To identify these factors/pathways, we have built a novel Y. pestis bioreporter to monitor Y. pestis survival in macrophages. Using this bioreporter, we have developed an inhibitory RNA-based assay to specifically identify host factors that alter Y. pestis intracellular survival. With this assay we will perform a genome wide screen to identify host factors/pathways required for Y. pestis survival in macrophages (Aim 1). Using a combination of in silico and microscopy analysis, we will also specifically identify host factors identified in our screen that alter phagosome maturation to the benefit of Y. pestis (Aim 2). These studies will be the first to target the host cell to understand the mechanisms used by Y. pestis to survive in host cells.