Quantitative analysis of growth in a streamlined obligate intracellular pathogen

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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.