Functional elucidation of lactate dehydrogenase in Toxoplasma gondii by double-stranded RNA gene suppression mediation.
Date of Award
Chemistry and Biochemistry
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Toxoplasma gondii is an intracellular parasite of humans and other mammals which can differentiate between an active and a dormant form. Our primary interest was to modulate gene expression in T. gondii in order to investigate a putative gene function. The capability of double-stranded RNA to down-regulate gene expression has been demonstrated in various organisms but has yet to be demonstrated in intracellular organisms such as T. gondii. Therefore, the effect of in vitro synthesized double-stranded RNA was first investigated for the down-regulation effect of the homologous gene in T. gondii. Three non-essential marker genes which encode the green fluorescent protein, uracil phosphoribosyl transferase and hypoxanthine-xanthine-guanine phosphoribosyltransferase were used in the study. Double-stranded RNA was efficiently electroporated into the parasites and specifically lowered the expression of the homologous marker gene. The down-regulation effects can be observed for three successive propagations of the parasites, suggesting the potential use of double-stranded RNA in T. gondii. Subsequently, the down-regulation effect of double-stranded RNA was further characterized in transgenic T. gondii expressing double-stranded RNA. We constructed a plasmid coding for double-stranded RNA homologous to the uracil phosphoribosyl transferase gene. A stable parasite line with down-regulated uracil phosphoribosyl transferase expression was generated and shown to have lowered levels of the corresponding transcript as compared to wild type parasites. The steady state level of the expressed double-stranded RNA was quantified and correlated to that of the homologous mRNA in order to estimate the copy number of double-stranded RNA which can effectively exhibit the down-regulation effect. Most importantly, the double stranded RNA was used for functional elucidation of lactate dehydrogenase which has long been hypothesized to be essential for parasite metabolism and differentiation. We generated stable transgenic parasite lines in which the expression of lactate dehydrogenase was knocked-down by double-stranded RNA. The differentiation processes of these parasite lines were impaired. In vivo studies in a murine model system revealed that these parasite lines were unable to establish a chronic infection. This study was the first to demonstrate that lactate dehydrogenase expression is essential for parasite differentiation.Dept. of Chemistry and Biochemistry. Paper copy at Leddy Library: Theses & Major Papers - Basement, West Bldg. / Call Number: Thesis2005 .A43. Source: Dissertation Abstracts International, Volume: 66-11, Section: B, page: 5953. Thesis (Ph.D.)--University of Windsor (Canada), 2005.
Al-Anouti, Fatme., "Functional elucidation of lactate dehydrogenase in Toxoplasma gondii by double-stranded RNA gene suppression mediation." (2005). Electronic Theses and Dissertations. 2172.