"Reverse Evolution" Approach to Identify Coxiella burnetii Strategies of Intracellular Survival

项目摘要 伯氏柯克斯体(Coxiella burnetii，CB)是自然界中的一种专性胞内细菌，是人类 急性Q热以及慢性病。一旦感染，CB就会沿着经细菌修饰的内吞胞体运输 途径，并建立了一个寄生液泡(PV)，它保留了许多成熟的特征 吞噬酶体。在宿主细胞内的整个感染周期中，CB面临着一系列的生理需求 以及酸化的细胞内小泡内生命所独有的压力。病原体能操控过多的寄主 通过细菌4型分泌系统(T4BSS)从PV内部的细胞路径和过程，以便 生存和复制。然而，无菌介质支持在细菌 它的许多适应细胞内生长所需的基因不再需要。删除和插入 突变体已经证明了一些CB宿主细胞特有的基因要求，然而，对 当细胞压力消除时，细菌基因的广度和/或它们的表达将发生变化 仍然是缺乏的。这是我们在理解CB如何颠覆宿主细胞过程中的一个根本差距 并将使人们对CB的生物学和发病机制有更广泛的了解。我们的长期目标是 研究旨在阐明CB在寄生过程中协调宿主细胞寄生的分子机制 感染。我们的假设是1-CB在最初的几代中改变了它的基因表达 从细胞内培养过渡到无菌培养，2-CB将逐渐获得和 从细胞内到无菌培养基经重复培养后积累DNA突变 传代，因为某些细菌基因/蛋白质不再需要成功的生长。我们会 通过1-识别早期和长期发生的CB基因表达变化来测试我们的假设 接种宿主细胞繁殖的CB的无菌培养液传代后，2-识别CB 接种无菌疫苗后在多代传代中发生/积累的基因突变/变化 在含有宿主细胞的生长培养基上繁殖CB。了解该病毒的致病机制 独特的病原体能在宿主细胞吞噬小体的恶劣环境中生存并引起疾病 使我们能够开发针对这种知之甚少的细菌的对策。

Project Summary Coxiella burnetii (Cb) is an obligate intracellular bacterium in nature and the causative agent of human acute Q fever as well as chronic disease. Upon infection, Cb is trafficked along a bacterially modified endocytic pathway and establishes a parasitophorous vacuole (PV) that retains many of the features of mature phagolysosomes. Throughout the infectious cycle within a host cell, Cb face an array of physiological needs and stresses unique to life within an acidified intracellular vesicle. The pathogen manipulates a plethora of host cell pathways and processes from within the PV via a bacterial Type 4 Secretion System (T4BSS) in order to survive and replicate. However, axenic media supports Cb replication in an environment where the bacterial genes required for many of its adaptations to intracellular growth are no longer needed. Deletion and insertion mutants have demonstrated some of Cb host cell specific gene requirements, however, an understanding of the breadth of the bacteria’s genes, and/or their expression, that will change when cellular stresses are removed is still lacking. This is a fundamental gap in our understanding of how Cb subverts host cell processes during infection and will lead to a broader understanding of Cb biology and pathogenesis. The long-term goal of our research is to elucidate molecular mechanisms employed by Cb to orchestrate parasitism of the host cell during infection. Our hypotheses are 1- Cb changes its gene expression within the first few passages upon transition from intracellular to axenic media growth, and 2- Cb will progressively acquire and accumulate DNA mutations upon transition from intracellular to axenic media growth after repetitive passages since certain bacterial genes/proteins are no longer required for successful growth. We will test our hypotheses by 1- Identifying Cb gene expression changes that occur during early and long term passages after inoculating axenic growth media with host cell propagated Cb, and 2- Identifying Cb genetic mutations/changes that occur/accumulate over multiple passages after inoculating axenic growth media with host cell propagated Cb. Understanding the virulence mechanisms employed by this unique pathogen to survive within the harsh environment of the host cell phagosome and cause disease will enable us to develop countermeasures to this poorly understood bacteria.