Malaria, a parasitic disease caused by five species of the protozoan parasite Plasmodium, still kills an estimated 0.6 million people each year, almost all in the third world African countries. With renewed emphasis on global eradication of malaria, genome-based discovery of novel anti-transmission candidates has been identified as one of the priority research areas for the immediate future. The aim of this study was to exploit the benefits of a combination of classical forward/reverse genetics approaches, flow cytometry and high throughput whole genome sequencing to examine the molecular basis of gametocytogenesis in the rodent malaria parasite, P. berghei. Plasmodium is known to spontaneously generate gametocyte non-producing (GNP) mutants if asexually maintained for a long time. Using a sex-specific fluorescently labelled P. berghei parental line, ten parallel isogenic lineages were asexually maintained in mice by repeated mechanical passage for a year. Three, out of the potential ten lines, developed the GNP phenotype at the end of the study. The three GNP and their isogenic parental lines were sequenced using the Illumina platform and the sequence variations analysed. It was discovered that one single gene, a transcription factor with an AP2 domain (PBANKA_143750), was uniquely mutated in all the three de novo GNP mutants and also in the two pre-existing GNP mutants. The gene, called AP2-G, was thus implicated in regulating a switch associated with commitment to gametocytogenesis. Further conclusive evidence was generated using targeted AP2-G knockout studies (producing the GNP phenotype) and complementation studies in the AP2-G mutants (restoring the WT phenotype). AP2-G was also shown to recognize and bind to a conserved DNA motif in the selected gene promoters in a sequence-specific manner. Inhibition of this interaction by a synthetic customized polyamide compound, ISS-15, was also demonstrated in vitro. Collectively, the work done in the thesis (together with simultaneous independent evidence of involvement of the P. falciparum orthologue of AP2-G in gametocytogenesis) established AP2-G as the critical regulator of the commitment to gametocytogenesis in the form of a molecular switch.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:622062 |
| Date | January 2014 |
| Creators | Sinha, Abhinav |
| Publisher | University of Glasgow |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://theses.gla.ac.uk/5580/ |
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