Malaria is among the greatest disease threats to global public health. As part of the effort to combat the disease, the development of new anti-malarials remains imperative. The causative agent of malaria is a parasitic pathogen of the genus Plasmodium which is transmitted to humans through the bite of an anopheline mosquito. The parasite contains a relic, non-photosynthetic plastid (called the apicoplast) of red algal origin that was acquired by an ancestral form through a secondary endosymbiotic event. The apicoplast supports critical biosynthetic processes in the parasite that are divergent from the equivalent host pathway, providing substantial opportunity for the identification of possible drug targets. The Plasmodium DNA gyrase is a eubacterial type II topoisomerase that is expected to function within the apicoplast, with a principal role in regulating the topological transitions of the 35 kb circular apicoplast genome. In this study, the anti-bacterial ciprofloxacin was used to probe the cellular functions of DNA gyrase. For further characterization of enzyme functions, transgenic parasites were generated to develop a system for conditional gene regulation. Results from this study indicated that targeting DNA gyrase with ciprofloxacin inhibited the replication of apicoplast DNA and the accumulation of apicoplast RNA transcripts, and blocked the production of apicoplast ribosomal RNA. The apicoplast nucleoid appeared reduced in size and did not segregate normally, and the apicoplast displayed abnormal morphology. In contrast, mitochondrial DNA replication, transcription, and segregation were unaffected. The DNA gyrase appeared to associate with the apicoplast DNA at non-specific loci, implicating numerous functional roles for the enzyme. Results from the conditional gene regulation studies indicated that the Tet-transactivator system has potential applicability for controlling endogenous gene expression in P. falciparum. Artificial promoters were successfully targeted to the pfpm4 genomic locus, and piggyBac insertion of transactivator (TaTi2) expression cassettes resulted in stable TaTi2 expression. In washout studies, the temporal pattern of pfpm4 RNA expression closely followed the expression of tati2. In the induced state of target transgene expression the steady-state TaTi2 levels decreased, a phenomenon that will be further investigated.
|Advisor:||Dame, John B.|
|School:||University of Florida|
|School Location:||United States -- Florida|
|Source:||DAI-B 70/11, Dissertation Abstracts International|
|Keywords:||DNA gyrase, Gene regulation, Malaria, Plasmodium, Ribosomal RNA|
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