Generation of Orientia tsutsugamushi mutant strains for pathogenesis research

Scrub typhus is an emerging and life threating zoonosis caused by Orientia tsutsugamushi (Ot), a Gram-negative bacterium that replicates in macrophages and endothelial cells. Severe infection is linked to vascular dysfunction, multiorgan failure, and high mortality. Alarmingly, this infection has emerged to countries outside of the traditional “Tsutsugamushi triangle.” No vaccines are yet available, partially due to limited research in Ot biology, immunology, and pathogenesis, as genetic manipulation is not yet feasible for Ot strains. To address these knowledge gaps, we seek support for this R61/R33 project, in which we will apply random mutagenesis to generate mutants in Ot Karp strain, which is responsible for ~50% of infections in the world. In pilot studies with Rickettsia conorii, a member within the family Rickettsiaceae, we constructed a Himar1-mariner-based transposon plasmid (carrying a codon-optimized polycistronic construct encoding for antibiotic, fluorescent and luminescent markers) and tested its applicability for genome-wide mutagenesis. We also optimized electroporation protocols and transformed Ot Karp strain with a self-replicating plasmid and verified transformants for their expression of fluorescent proteins. These pilot studies establish the amicability of Ot for genetic manipulation and feasibility for application of Himar1-based mutagenesis for generation of a genome-wide mutant library for in-depth molecular investigation of Ot biology and genetics. Accordingly, in Aim 1 (R61 phase), we will apply a streamlined random mutagenesis approach to generate a genome-wide mutant library in Ot strain Karp and isolate clonal mutants by fluorescence-based live cell sorting and via plaque and focus forming assays. We will confirm the disrupted locus in each mutant by three independent approaches. A series of technical milestones have been developed to systematically guide the progression of the R61 phase and lay a solid foundation for the R33 phase of the project. Our objective is to generate a minimum of 50 Ot mutants which will be further classified and ranked based on the deleted gene, protein domain analysis, onservation of the disrupted gene among other Ot strains, and mutant growth kinetics in vitro. In Aim 2 (R33 phase), we will characterize novel Ot mutant strains for their infectivity in vitro and disease pathogenesis in our established mouse models. We will assess bacterial replication dynamics and immune responses elicited by Ot mutants during the infection of primary cell cultures in vitro. The genetic contributions to pathogenesis and bacterial dissemination by Ot mutants will be evaluated in vivo utilizing our highly susceptible CD-1 mouse model (i.v. route) and Infgr1 -/- C57BL/6 model (i.d. route) and by bioluminescence-based live imaging in the ABSL3. Overall, this is the first study to develop a genome-wide mutant library in Ot, a highly significant and valuable tool that will be shared among the research community. It will advance our understanding of Ot biology and pathogenesis and future development of therapeutics.