Development of genetic tools for overexpression and targeted mutagenesis of Orientia tsutsugamushi TPR proteins

Project Summary Orientia tsutsugamushi (O.t.) is the etiological agent of scrub typhus, a devastating disease with a high mortality rate that is transmitted by the bite of certain trombiculid mites or “chiggers”. Over one million individuals are infected annually, however, these statistics may be a gross underestimate of disease incidence as the “tsutsugamushi triangle”, where most cases are reported, encompasses large regions of jungle in rural subtropical environments with limited access to hospitals and diagnostic facilities. Beyond the tsutsugamushi triangle, recent reports have confirmed cases of the disease in South America, Africa, and the Middle East. There is no vaccine and antibiotic resistant strains have been reported. Without treatment, disease associated morbidities including hepatitis, renal failure, myocarditis, encephalitis, multiple organ failure, and death can occur. Despite its significant impact on global health, little is known of the molecular mechanisms the bacterium uses to infect and cause disease in humans. Specifically, the bacterial factors that promote host cell subversion and bacterial pathogenesis remain largely unknown. The key roadblock to a more detailed understanding of how O.t. causes disease lies in the inability to genetically manipulate the pathogen. Overcoming the genetic intractability of O.t. will remove the single largest barricade to extending our operational knowledge of the molecular mechanisms utilized by the bacterium to hijack host cells. Here we propose to develop the first functional system for genetic transformation of O.t. which will present a unique opportunity to overexpress epitope-tagged proteins in O.t. and generate the first site specific O.t. mutants. During the R61 phase, we will develop a shuttle vector and transformation method for use in O.t. (Aim 1) and adapt the TargeTron system to insertionally inactivate select TPR proteins (Aim 2). In the R33 phase, we will use these newly developed tools and mutants to determine whether select TPR are secreted proteins that promote host cell invasion or bacterial proliferation by perturbing the host cell cycle (Aim 3). Our proposed studies will generate the first tools and reagents to genetically alter O.t., a resource that will be invaluable to other Orientia researchers.