The Role of Glutathione Synthetase in Trypanothione Biosynthesis in Trypanosoma brucei

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Trypanosoma brucei is the causative agent of Human African trypanosomiasis, commonly called sleeping sickness, which is a debilitating disease for which treatment is not currently ideal. Trypanosome parasites differ from their human host by utilizing a novel cofactor termed trypanothione instead of glutathione for protection against reactive oxygen species. Trypanothione is formed by the conjugation of two molecules of glutathione to spermidine forming a link between the polyamine and thiol biosynthetic pathways. My work has investigated the enzyme annotated as glutathione synthetase (GS) in T. brucei to determine if it indeed catalyzes the synthesis of glutathione and if so, to define its kinetic parameters, decipher whether it has any role in the regulation of these pathways, and assess if it is essential for growth. To determine whether the putative TbGS gene was correctly identified through sequence homology, I cloned and expressed the T. brucei gene (TbGS) in Escherichia coli, and purified the recombinant protein. Using an ATP-coupled spectrophotometric assay, I was able to measure TbGS kinetic activity and determine that it was comparable to activities of other published GS homologs, indicating that this gene was correctly annotated. To investigate the physiological role of TbGS in T. brucei, I used genetic approaches to manipulate TbGS levels, first by RNAi, and then by employing conditional knockout models. RNAi was used to decrease protein levels; however, even though TbGS protein levels were depleted by more than eighty percent, there was no altered growth phenotype, and parasites did not have increased sensitivity to known inhibitors of the pathway. I then constructed a TbGS conditional double knockout (cDKO) parasite cell line that contained a tetracycline (tet) regulated episomal copy of TbGS. By removing tet from the media and stopping TbGS protein production, parasites entered growth arrest by day five, which correlated with depleted thiol pools. Parasites remained in growth arrest until day eight after which they resumed growth. This resumption of growth also correlated with the return of low levels of TbGS and thiol pools indicating that loss of trypanothione caused growth arrest. To evaluate if the loss of TbGS had any regulatory effect, levels of biosynthetic pathway proteins were assessed by western blot analysis. A three-fold increase was seen in γ-GCS levels as well as a decrease in AdoMetDC prozyme and ODC levels. Thus our studies have shown that not only is TbGS essential for parasite growth but have also uncovered cross regulation between the polyamine and thiol pathways.

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