Obligate intracellular bacteria in the order Rickettsiales cause emerging and potentially fatal infections. Orientia tsutsugamushi, an etiologic agent of scrub typhus, is one of the deadliest rickettsial pathogens to humans. Yet, it is severely understudied due, in part, to a lack of genetic manipulation tools. We have made important progress on this front by developing protocols for Tn5 transposon mutagenesis and allelic exchange in O. tsutsugamushi. We employed both approaches to insert a cassette that expresses mCherry and chloramphenicol acetyl transferase into the O. tsutsugamushi chromosome. Using chloramphenicol selection and enrichment via fluorescence activated cell sorting, we recovered a Tn5 mutant pool and an allelic exchange mutant in which we knocked out a virulence factor. The Tn5 mutant pool and knockout mutant can be maintained under antibiotic selection. In the R61 phase, we will further develop these genetic tools. In Aim 1, we will transform O. tsutsugamushi with Tn5 transposomes to generate an insertional mutant library that we will catalog. In Aim 2, we will complement this random mutagenesis approach by using allelic exchange to delete selected single-copy O. tsutsugamushi genes. For this purpose, we prioritized a set of virulence factors whose functions we previously defined to provide us with phenotypes to characterize in downstream R33 functional studies. While transposon and allelic exchange mutagenesis are exciting “firsts” for this organism, neither can be used to target essential genes in any other obligate intracellular bacterium. Therefore, in Aim 3 we will develop a riboswitch system that allows us to control protein expression in transgenic O. tsutsugamushi using a diffusible ligand. This approach can be used to block expression of any O. tsutsugamushi protein at any point during infection. Even if the protein is essential, the mutant can be maintained in the presence of inducer to ensure viability. To establish proof-of- principle for the riboswitch, we will target a gene that we have determined to be non-essential for viability. In the R33 phase, we will use the newly developed tools to study O. tsutsugamushi pathogenesis. Up to four allelic exchange mutants and the riboswitch mutant will be assayed alongside their wild-type counterparts for the ability to modulate host cell functions. The knockout mutants will also be assessed for pathogenesis in the mouse model. Up to two Tn5 mutants, prioritized based on the predicted functions of the genes that are disrupted, will be included in the in vitro and in vivo studies. Overall, we will establish the genetic basis of virulence in O. tsutsugamushi for the first time and provide valuable tools for the field. We envision that the proposed work will bolster the field by encouraging an influx of researchers to study scrub typhus.