TRANSPORT OF S-ADENOSYLMETHIONINE IN RICKETTSIA

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Members of the genus Rickettsia are the causative agents of human diseases that have had a significant impact on human history and that, despite the availability of effective treatment, continue to pose serious threats to human health. Epidemic and endemic typhus and rocky mountain spotted fever caused by R. prowazekii, R. typhi and R. rickettsii, respectively, are the most well known of the rickettsial diseases although numerous other spotted fevers have been recognized worldwide. All members of this genus are obligate intracellular parasites that can only grow within the cytoplasm (and occasionally the nucleus) of a eukaryotic host cell, unbounded by a vacuolar membrane. While restricted to this intracellular existence, the rickettsiae are capable of infecting an assortment of animal hosts ranging from arthropods to humans with arthropods serving as vectors for transmission of these bacteria to a variety of mammalian hosts. The ability to invade, grow within and eventually lyse eukaryotic cells is the basis for rickettsial pathogenicity and is dependent on mechanisms for exploiting this novel bacterial niche. The goal of this research is to identify and characterize the rickettsial transport system for S-adenosylmethionine (SAM), an essential component of rickettsial intracellular survival. This will be the first SAM transport system to be characterized in bacteria. In Specific Aim 1 the uptake parameters of this transport system will be completely characterized. For example, rickettsial SAM uptake will be defined kinetically and the substrate specificity and effect of inhibitors such as the SAM analog sinefungin determined. This bacterial transporter will then be compared to known SAM transporters found in mitochondria and yeast. Specific Aim 2 will compare and contrast SAM transport and synthesis in R. prowazekii and the very closely related R. typhi. Evidence suggests that gene meltdown is occurring in the rickettsial gene coding for methionine adenosyltransferase (MAT) and that these species may differ in their ability to synthesize and tranport SAM. The ability of R. typhi to synthesize and/or transport SAM will be determined and compared to R. prowazekii strains providing a unique model for evolution of the rickettsial minimal genome. Finally, in Specific Aim 3 we will identify the gene coding for the R. prowazekii SAM transporter and, building upon our expertise in rickettsial transporters, obtain functional expression in Escherichia coli. This is a critical first step in characterizing the structure and function of this transport system. Completion of these studies will contribute to our understanding of how this pathogen exploits the intracellular niche as well as provide insight into rickettsial evolution.