Identification of host factors required for Yersinia pestis macrophage infection

描述(申请人提供)：鼠疫是一种由鼠疫耶尔森氏菌引起的急性感染，是一种革兰氏阴性的兼性细胞内细菌，感染巨噬细胞并存活。越来越多的证据表明，细胞内生长对鼠疫杆菌在哺乳动物中的定植很重要。进入巨噬细胞后，鼠疫杆菌仍然留在一个被称为含耶尔森氏菌的液泡(YCV)的膜结合的隔间里，但它不会被巨噬细胞杀死。此外，该细菌还能有效地抑制YCV的酸化。我们假设鼠疫耶尔森氏菌与特定的宿主因子相互作用，以改变YCV的成熟和酸化。虽然细菌对细胞内生存的重要因素已经被识别，但细菌在巨噬细胞内生存所使用的分子机制还没有被确定。为了确定这些因素/途径，我们构建了一种新型的鼠疫耶尔森氏菌生物报告程序来监测鼠疫杆菌在巨噬细胞中的存活。使用这种生物报告程序，我们已经开发了一种基于抑制RNA的分析方法，以特异性地识别改变鼠疫杆菌细胞内的宿主因素。 生死存亡。通过这项试验，我们将进行全基因组筛选，以确定鼠疫杆菌在巨噬细胞中生存所需的宿主因子/途径(目标1)。结合电子显微镜和显微镜分析，我们还将专门识别在我们的筛查中发现的有利于鼠疫杆菌的改变吞噬小体成熟的宿主因素(目标2)。这些研究将是第一次以宿主细胞为目标，以了解鼠疫杆菌在宿主细胞中生存的机制。

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.