Intracellular Survival Determinants of Yersinia pestis

鼠疫耶尔森氏菌是一种兼性细胞内病原体，可以在巨噬细胞中存活和复制。虽然Y.鼠疫菌可能在鼠疫的发病过程中起关键作用，尤其是在鼠疫的早期阶段，但目前对鼠疫菌在这方面的作用知之甚少。鼠疫毒力阐明Y.鼠疫菌在巨噬细胞中的存活和复制，提出了以下具体目标。首先，遗传学方法将用于鉴定Y。巨噬细胞细胞内增殖所需的鼠疫基因（rip基因）。RIP基因编码的蛋白质将通过分子、细胞和免疫学技术进行研究，以揭示Y。鼠疫菌在巨噬细胞中增殖。第二，将进行DNA微阵列分析以表征全球 Y.鼠疫菌在巨噬细胞内外复制这些实验将确定在两种环境中差异表达的基因。有了这些信息在手，我们将有一个更好的理解Y。鼠疫适应其生理学以在巨噬细胞中存活和复制。第三，小鼠将通过气溶胶途径感染野生型Y。鼠疫菌或与AIM 1中产生的RIP突变体结合。肺、肝和脾中的细菌生长将作为时间的函数进行测量，以确定细胞内增殖如何促成毒力。细胞标记和显微镜技术将用于检测组织切片中的细胞内细菌，并鉴定含有细胞内细菌的宿主细胞类型。这些结合的方法将导致更好地理解Y。 这将有助于促进制定预防或治疗人类鼠疫的新战略。

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.