Dr. Mary Weber, Associate Professor in Microbiology and Immunology, has been awarded a 3-year National Institutes of Health/National Institute of Allergy and Infectious Diseases R61: Genetic Tools for Understanding Rickettsial and Related Infections Research Grant titled: “Development of genetic tools for overexpression and targeted mutagenesis of Orientia tsutsugamushi TPR proteins.” The major goal of this project is to examine Orientia tsutsugamushi (O.t.) which is an obligate intracellular pathogen and the etiological agent of scrub typhus, a potentially fatal disease that afflicts over one million people each year. The molecular details of how this important emerging pathogen co-opts the host are largely unknown, predominately due to a lack of tools for genetic manipulation of this significant human pathogen. The purpose of this research is to develop genetic tools for use in O.t., which will serve as the first crucial step towards identifying bacterial and host factors that are necessary for O.t. pathogenesis. This project involves collaboration with Dr. Jeanne Salje, Assistant Professor, at the University of Cambridge.

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 mostcases are reported, encompasses large regions of jungle in rural subtropical environments with limited access to hospitalsand diagnostic facilities. Beyond the tsutsugamushi triangle, recent reports have confirmed cases of the disease in SouthAmerica, Africa, and the Middle East. There is no vaccine and antibiotic resistant strains have been reported. Withouttreatment, 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 thebacterium uses to infect and cause disease in humans. Specifically, the bacterial factors that promote host cell subversionand bacterial pathogenesis remain largely unknown. The key roadblock to a more detailed understanding of how O.t. causesdisease lies in the inability to genetically manipulate the pathogen. Overcoming the genetic intractability of O.t. will removethe single largest barricade to extending our operational knowledge of the molecular mechanisms utilized by the bacteriumto hijack host cells. Here we propose to develop the first functional system for genetic transformation of O.t. which willpresent 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 theTargeTron system to insertionally inactivate select TPR proteins (Aim 2). In the R33 phase, we will use these newlydeveloped tools and mutants to determine whether select TPR are secreted proteins that promote host cell invasion orbacterial proliferation by perturbing the host cell cycle (Aim 3). Our proposed studies will generate the first tools andreagents to genetically alter O.t., a resource that will be invaluable to other Orientia researchers.