Quantitative analysis of growth in a streamlined obligate intracellular pathogen

立克次体细菌属斑点热群(SFG)的成员是专性胞内病原体 这会导致人类患上从轻微到危及生命的斑点热病。像该目中的所有物种一样 Rickettsiales，SFG细菌经历了基因组流线化，并依赖于真核宿主 几十种必需的代谢物。值得注意的是，简约进化导致了因子的丢失或修改 预计代谢和形态发生途径会影响肽聚糖细胞壁的代谢。肽多糖 新陈代谢对生长和生存至关重要，因此是有效的抗生素靶点。网络的影响 关于立克次体生长和分裂机制的基因组简化，与它们的游离态相比 活着的亲属，目前还不清楚。这种知识差距之所以持续存在，部分原因是我们缺乏研究增长的工具 以足够的分辨率对细胞内细菌进行分析，从而得出机理上的理解。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。在这项建议中，我们的目标是开发和应用方法来定量分析 帕氏立克次体在真核宿主细胞中的生长和细胞壁代谢。帕克氏杆菌是一种SFG生物体 会导致相对轻微的疾病。因此，它是一种BSL2病原体，已被研究为代表 了解SFG的发病机制。通过所提出的工作，我们将扩大对帕克氏杆菌的研究，以探讨 它的细胞内生长机制。在目标1中，我们将开发和应用成像方法来定量地 分析个体的细胞形态、亚细胞内蛋白质分布、细胞周期状态和生长动力学 真核宿主细胞胞质中的帕氏杆菌。在目标2中，我们将确定和的化学成分 生长在寄主细胞质中的帕氏杆菌肽多聚糖细胞壁代谢的空间格局。完成 将为PG的生长动力学和机理提供基础知识 壁虱传播的专性细胞内病原体的代谢和建立广泛适用的定量 研究细菌在真核宿主细胞内生长的方法。归根结底，信息和方法 该项目将为制定限制立克次体的预防和治疗策略提供信息 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.