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.