Generation and characterization of a large-scale transposon mutant library of Rickettsia parkeri

The order Rickettsiales includes arthropod-associated, obligate intracellular bacteria that cause diseases in humans ranging from relatively mild to potentially fatal. A mechanistic understanding of growth and pathogenesis of rickettsial pathogens would potentiate therapeutic strategies to control rickettsial disease. However, because of their obligate intracellular lifestyle and consequent challenges in culturing and genetically manipulating these species, our understanding of fundamental aspects of rickettsial biology is limited. Within the Rickettsia genus, the Spotted Fever Group (SFG) includes tick-borne human pathogens that cause diseases ranging from mild to life-threatening. Among the SFG bacteria, R. parkeri causes a relatively mild disease and presents a tractable model for probing the biology of this group. In the past, research leveraging a relatively small collection of transposon mutants of R. parkeri has yielded important insights into Rickettsia pathogenesis and host interactions. Here, we propose to expand the genetic toolkit available to study R. parkeri, generating a large-scale transposon mutant library of R. parkeri in the R61 phase of this project and using it to gain mechanistic insights into rickettsial growth and division in the R33 phase. In Aim 1, we will develop and validate plasmid constructs for (1) generating transposon mutants of R. parkeri with desired characteristics and (2) expressing genes of interest with and without tags in R. parkeri. In Aim 2, we will generate, map, and organize a library of ~2000 transposon mutants of R. parkeri. In the R33 phase, we will perform global analysis of growth kinetics and cell morphology of all mutants in the transposon library in Aim 3. In Aim 4, we will leverage insights from morphology screening to identify and characterize candidate factors that are important for peptidoglycan cell wall hydrolysis during R. parkeri cell division. Completion of this project will generate important genetic resources for the study of all aspects of R. parkeri biology and will provide foundational knowledge about growth and cell division in an important tick-borne, obligate intracellular pathogen that may aid in the design of new antibacterial therapeutic approaches.