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的兼性细胞内细菌，感染并存活于巨噬细胞中。越来越多的证据表明，细胞内生长对哺乳动物鼠疫杆菌定植很重要。在进入巨噬细胞后，鼠疫杆菌停留在一个被称为含耶尔森菌液泡（YCV）的膜结合腔室中，但它不会被巨噬细胞杀死。此外，这种细菌还能有效地抑制YCV的酸化。我们假设鼠疫杆菌与特定宿主因子相互作用，以改变YCV的成熟和酸化。虽然已经确定了细胞内存活的重要细菌因素，但细菌在巨噬细胞内存活的分子机制尚未确定。为了确定这些因素/途径，我们建立了一种新的鼠疫杆菌生物报告器来监测鼠疫杆菌在巨噬细胞中的存活。利用这种生物报告器，我们开发了一种基于抑制rna的检测方法，以特异性地识别改变细胞内鼠疫杆菌的宿主因子

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.