Semester

Summer

Date of Graduation

2004

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Microbiology, Immunology, and Cell Biology

Committee Chair

Herbert A. Thompson.

Abstract

Coxiella burnetii is a gram negative, obligate intracellular parasite that can only replicate in the acidified environment of a phagosome. Like other intracellular parasites, C. burnetii depends on the host cell for many of the substrates required for growth, such as amino acids. It had been observed that avirulent C. burnetii Nine Mile strain (Phase II) organisms in BHK-21 fibroblasts displayed improved growth when extracellular cytidine was introduced to infected cultures. This improved growth was not observed in wild type organisms in the same cell system. This observation, coupled with early enzyme activity studies, suggested that Phase II organisms had an inactive CTP synthase enzyme. To verify this hypothesis, nucleoside transport studies were performed in vitro in acidified media that determined that both wild type C. burnetii and the avirulent variant are permeable to nucleosides, but not nucleotides or nucleobases. Further, the CTP synthase gene from the wild type and avirulent organisms were sequenced and compared. The enzyme activity was examined by using both cell-free extacts and hexahistidine-tagged recombinant CTP synthase cloned from the wild type C. burnetii genome. It was found that the Phase I and Phase II CTP synthase genes were identical. Further, it was shown that the avirulent organism had a functional CTP synthase enzyme and that neither nucleoside transport nor CTP synthase activity were the explanations for the lower growth rate of Phase II organisms in BHK-21 fibroblasts. While the hypothesis was found to be invalid, enzyme inhibition experiments suggest the C. burnetii CTP synthase may be a potential target for drug development.

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