Quantitative analysis of growth in a streamlined obligate intracellular pathogen

立克次体属斑点热群（SFG）成员是专性胞内病原体 引起人类斑疹热的疾病，从轻微到危及生命。像所有的物种一样 立克次氏体，SFG细菌经历了基因组精简，并依赖于真核宿主， 几十种必需代谢物值得注意的是，还原进化导致了基因组中因子的丢失或改变。 预测影响肽聚糖细胞壁代谢的代谢和形态发生途径。肽聚糖 代谢对于生长和生存力是必不可少的，因此是有效的抗生素靶标。的影响 与游离立克次体相比，立克次体生长和分裂机制的基因组精简 亲戚，不清楚。这种知识上的差距持续存在，部分原因是我们缺乏研究增长的工具 有足够的分辨率来获得一个机械的理解。我们假设 代谢和形态发生途径的还原进化需要已知机制的多样化 以支持细胞内生长。在这项提案中，我们的目标是开发和应用定量分析的方法， 巴氏立克次体在真核宿主细胞中的生长和细胞壁代谢。R. parkeri是一种SFG生物， 导致相对轻微的疾病。因此，它是一种BSL 2病原体，已被研究为代表， 了解SFG发病机制。通过本文的工作，我们将拓展R.帕克里调查 它的细胞内生长机制在aim1中，我们将开发和应用成像方法， 分析个体的细胞形态、亚细胞蛋白分布、细胞周期状态和生长动力学 R.在真核宿主细胞的细胞质中存在Parkeri。在目标2中，我们将确定和的化学成分 R.肽聚糖细胞壁代谢的空间模式parkeri在宿主细胞质中生长。完成 提出的目标将提供基础知识的生长动力学和机制的PG 蜱传播的专性细胞内人类病原体的代谢，并建立广泛适用的定量 研究真核宿主细胞内细菌生长的方法。最终，信息和方法 从这个项目中得出的结论将为限制立克次体的预防和治疗策略的发展提供信息。 疾病

Members of the Spotted Fever Group (SFG) of the bacterial genus Rickettsia are obligate intracellular pathogens that cause spotted fever disease in humans ranging from mild to life-threatening. Like all species in the order Rickettsiales, SFG bacteria have undergone genome streamlining and are dependent on a eukaryotic host for dozens of essential metabolites. Notably, reductive evolution has led to loss or modification of factors in metabolic and morphogenetic pathways predicted to impact peptidoglycan cell wall metabolism. Peptidoglycan metabolism is essential for growth and viability and is therefore an effective antibiotic target. The impact of genome streamlining on the mechanisms of growth and division of the Rickettsiales, as compared to their free- living relatives, is not clear. This gap in knowledge persists, in part, because we lack tools to investigate growth of intracellular bacteria with sufficient resolution to derive a mechanistic understanding. We hypothesize that reductive evolution of metabolic and morphogenetic pathways necessitates diversification of known mechanisms to support intracellular growth. In this proposal, we aim to develop and apply methods to quantitatively analyze growth and cell wall metabolism of Rickettsia parkeri in eukaryotic host cells. R. parkeri is a SFG organism that causes relatively mild disease. As such, it is a BSL2 pathogen that has been studied as a representative to understand SFG pathogenesis. Through the proposed work, we will expand the study of R. parkeri to investigate its intracellular growth mechanisms. In aim 1, we will develop and apply imaging methods to quantitatively analyze the cell shape of, subcellular protein distribution in, cell cycle status of, and kinetics of growth of individual R. parkeri in the cytoplasm of eukaryotic host cells. In aim 2, we will determine the chemical composition of and spatial patterning of peptidoglycan cell wall metabolism of R. parkeri growing in the host cytoplasm. Completion of the proposed aims will provide foundational knowledge on the growth kinetics and mechanisms of PG metabolism of a tick-borne, obligate intracellular human pathogen and establish broadly applicable quantitative approaches for studying growth of bacteria within a eukaryotic host cell. Ultimately, the information and methods derived from this project will inform development of preventive and therapeutic strategies for limiting Rickettsial disease.