Intracellular Survival Determinants of Yersinia pestis

鼠疫的病原体鼠疫耶尔森氏菌是一种兼性的细胞内病原体，可以在巨噬细胞中生存和复制。虽然鼠疫杆菌的细胞内复制阶段可能在发病机制中发挥关键作用，特别是在鼠疫的早期阶段，但对鼠疫杆菌的这一方面的毒力知之甚少。为了阐明鼠疫杆菌在巨噬细胞中存活和复制的机制，提出了以下具体目标。首先，将使用遗传方法来鉴定巨噬细胞细胞内增殖所需的鼠疫耶尔森氏菌基因(RIP基因)。将使用分子、细胞和免疫学技术研究由RIP基因编码的蛋白质，以揭示鼠疫杆菌在巨噬细胞中增殖的潜在基础。其次，将进行DNA微阵列分析，以表征全球 鼠疫杆菌在巨噬细胞内外复制的基因表达谱。这些实验将识别在两种环境中差异表达的基因。有了这些信息，我们将更好地了解鼠疫杆菌如何适应它的生理来在巨噬细胞中生存和复制。第三，用野生型鼠疫杆菌或在Aim 1中产生的RIP突变体通过气溶胶途径实验感染小鼠。将测量细菌在肺、肝和脾中的生长随时间的变化，以确定细胞内增殖如何对毒力做出贡献。细胞标记和显微技术将用于检测组织切片中的细胞内细菌，并识别含有细胞内细菌的宿主细胞类型。这些结合起来的方法将使我们更好地理解Y。 鼠疫具有致病性，并将有助于推动制定新的战略，以预防或治疗人类人口中的鼠疫。

The agent of plague, Yersinia pestis, is a facultative intracellular pathogen that can survival and replicate in macrophages. Although the intracellular replication phase of Y. pestis is likely to play a key role in pathogenesis, especially during the early stages of plague, very little is known about this aspect of Y. pestis virulence. To elucidate the mechanism of Y. pestis survival and replication in macrophages, the following specific aims are proposed. First, genetic approaches will be used to identify Y. pestis genes that are required for intracellular proliferation in macrophages (rip genes). The proteins encoded by rip genes will be studied using molecular, cellular and immunological techniques to reveal the underlying basis for Y. pestis proliferation in macrophages. Second, DNA microarray analysis will be performed to characterize the global gene expression profile of Y. pestis replicating inside or outside of macrophages. These experiments will identify genes that are differentially expressed in the two environments. With this information in hand we will have a better understanding of how Y. pestis adapts its physiology to survive and replicate in macrophages. Third, mice will be experimentally infected by the aerosol route with wild-type Y. pestis or with rip mutants generated in aim 1. Bacterial growth in lungs, liver and spleen will be measured as a function of time to determine how intracellular proliferation contributes to virulence. Cell labeling and microscopic techniques will be used to detect intracellular bacteria in tissue sections and to identify host cell types that harbor intracellular bacteria. These combined approaches will lead to a better understanding of Y. pestis pathogenicity, and will help foster the development of new strategies to prevent or treat plague in the human population.