Elucidating the evolution of Coxiella to uncover critical metabolic pathways

项目总结/摘要 贝氏柯克斯体的致病性取决于其在宿主细胞内溶酶体来源的敌对空泡中生长的能力。 人体细胞然而，代谢过程的关键C。伯内特氏菌细胞内生长在很大程度上是未知的。 这种知识的缺乏阻碍了对其基本生物学和发病机制的理解， 开发更好的治疗药物。长期目标是了解Coxiella独特生理学的分子细节，并将这些知识应用于开发新的治疗策略。本申请的目的是确定代谢途径，是至关重要的C。Burnetii的细胞内生长。的 根据初步数据提出的中心假设是C.贝氏体是从蜱虫进化而来的 通过水平基因转移（HGT）获得关键代谢基因，从而与相关的祖先相关联。一种新的进化基因组学方法将被用来确定代谢途径是至关重要的C。贝氏内 细胞生长这项研究的基本原理是，一旦代谢过程对C。伯内特氏菌的细胞内生长被鉴定，阻断这些途径的药理学试剂可以被开发出来 更有效地治疗慢性感染。该项目的目标将通过三个具体的 目的：（1）确定C.贝氏体与蜱相关柯克斯体的区别工作 假设是C.贝氏体的细胞内生理学不会出现在无毒的蜱虫中， 相关Coxiella来自喙鸟蜱的一种密切相关的柯克斯体的基因组将被测序，并与C。Burnetii的基因组(2)定义代谢途径是至关重要的C。伯内特氏 细胞内生长工作假设是通过HGT获得的基因在C.因为它们对病原体的生理至关重要。系统发育方法将用于识别 HGT衍生基因及其功能将使用RNA-seq和遗传工具进行验证。(3)作为服务的证明 原理，确定了血红素生物合成对C. Burnetii的细胞内生长。工作 假设血红素的生物合成对柯克斯体的生长至关重要。血红素生成和细胞内生长 将测定血红素途径缺陷菌株的浓度。这项研究是创新的，因为它（a）使用了一种新的方法 克服了目前在全基因组范围内研究柯克斯体的局限性，以及（B）基于一种新的 人类病原体通过大规模HGT从蜱虫共生体进化而来的概念。拟议的项目意义重大，因为它将（a）揭示对病原体细胞内生长至关重要的代谢途径， (b)确定新的治疗靶点，例如，HemA和HemL对于C. Burnetii，但不存在于人类细胞中，（c）提供了一种用于鉴定参与 宿主适应，这可以广泛应用于其他病原体，如弗朗西斯和立克次体属。 其中无毒的蜱共生体可以与有毒的人类特异性菌株进行比较。

PROJECT SUMMARY/ABSTRACT Coxiella burnetii's pathogenicity depends on its ability to grow in a lysosome-derived hostile vacuole within human cells. However, metabolic processes critical to C. burnetii's intracellular growth are largely unknown. This lack of knowledge has prevented both the understanding of its basic biology and pathogenesis, and the development of better therapeutic agents. The long-term goal is to understand the molecular details of Coxiella's distinctive physiology, and to apply this knowledge to developing novel therapeutic strategies. The objective of this application is to identify metabolic pathways that are vital to C. burnetii's intracellular growth. The central hypothesis, which was formulated based on preliminary data, is that C. burnetii evolved from a tick- associated ancestor by acquiring critical metabolic genes through horizontal gene transfer (HGT). A novel evolutionary genomics approach will be used to identify metabolic pathways that are critical to C. burnetii's intra- cellular growth. The rationale for the proposed research is that once metabolic processes important to C. burnetii's intracellular growth are identified, pharmacological agents that block these pathways could be developed to treat chronic infections more effectively. The objective of this project will be accomplished by three specific aims: (1) Identify metabolic pathways that distinguish C. burnetii from tick-associated Coxiella. The working hypothesis is that genes critical to C. burnetii's intracellular physiology will not be present in avirulent tick- associated Coxiella. The genome of a closely related Coxiella from the tick Ornithodoros rostratus will be sequenced and compared to C. burnetii's genome. (2) Define metabolic pathways that are critical to C. burnetii's intracellular growth. The working hypothesis is that genes acquired via HGT are being maintained in C. burnetii because they are critical to the pathogen's physiology. Phylogenetic approaches will be used to identify HGT-derived genes, and their functions will be validated using RNA-seq and genetic tools. (3) As a proof of principle, determine the importance of heme biosynthesis to C. burnetii's intracellular growth. The working hypothesis is that heme biosynthesis is crucial to Coxiella's growth. Heme production and intracellular growth of heme pathway-deficient strains will be assayed. This study is innovative because it (a) uses a novel approach that overcomes the current limitations in studying Coxiella at a genome-wide scale, and (b) is based on a novel concept that the human pathogen evolved from a tick symbiont via massive HGT. The proposed project is significant because it will (a) uncover metabolic pathways that are critical to the pathogen's intracellular growth, (b) identify new therapeutic targets, for example, HemA and HemL are essential for heme biosynthesis in C. burnetii but are not present in humans cells, (c) provide a model approach for identifying genes involved in host adaptation, which could be applied broadly to other pathogens such as Francisella and Rickettsia spp. where avirulent tick symbionts could be compared to virulent human-specialized strains.