PROJECT SUMMARY Orientia tsutsugamushi (Ot) is in an obligate intracellular vector-borne Rickettsiales bacterium that causes the life-threatening human disease scrub typhus, estimated to affect at least one million people per year globally. Scrub typhus is known to be endemic in many parts of Asia, including India and China, but recent reports have demonstrated the presence of Ot bacteria in Latin America, the Middle East and Africa, suggesting a more global distribution. Whilst Ot infections can be treated with tetracyclines, azithromycin and chloramphenicol, the bacterium is intrinsically resistant to several classes of front-line antibiotics, and misdiagnoses frequently result in ineffective therapy and life-threatening complications. Clinical presentation of scrub typhus resembles other tropical infections, and currently available diagnostic tests all suffer serious limitations. Recent reports of antibiotic resistant strains of Ot have resulted in an urgent need for improved diagnostic and treatment options, as well as effective vaccines. Our understanding of the fundamental biology of Ot is limited compared with equivalent human pathogens. This is partly because it is technically difficult to work with – as an obligate intracellular BSL3 level pathogen – and partly because it has been historically under recognised. In the current project we address questions of metabolic quiescence and latent infection in Ot. Metabolic quiescence describes a state in which cells switch off all metabolic processes including protein synthesis and cell division. Bacteria which enter into this state are often resistant to antibiotics. We have recently found that Ot enters into a metabolically quiescent state as a part of its infection cycle. In Aims 1 and 2 we will use a range of experimental techniques to understand the mechanisms Ot uses to enter into this state and exit from it, as well as the environmental triggers that Ot senses and responds to. This knowledge is essential in understanding the fundamental biology of this important human pathogen, and these studies will also provide a new model system for studying bacterial quiescence. Ot is a particularly good system in which to study signals and mechanisms of quiescence because it has a highly reduced (simplified) genome and because it can easily be synchronised between different states. Metabolic quiescence is frequently involved in establishing latent infection. This describes a state in which patients have recovered from acute infection but retain a number of non-replicating pathogens in their bodies, primed for potential relapse of disease. There is evidence that Ot can cause latent infections in animals and humans, but the evidence and details are weak. This is an important question as it will guide patient care and is also a prerequisite for the development of accurate and safe models for vaccine development. In Aim 3 we will link our studies on metabolic quiescence to latent infection, by using both...