School of BioSciences - Theses
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ItemDrug targets in the apicoplast of malaria parasites( 2017)Human malaria is caused by six species of Plasmodium, and despite considerable effort, it remains as one of the deadliest of infectious diseases. African children under the age of five are the most vulnerable population. Drugs are our primary means of malaria control, but parasite drug resistance constantly erodes their efficacy. Thus, the search for new drugs and novel drug targets continues to be a high priority. Plasmodium has a plant-like plastid organelle known as the apicoplast. The apicoplast is no longer photosynthetic, but like plant plastids, it is derived from endosymbiotic bacteria and performs crucial metabolic functions. Apicoplast housekeeping and metabolic pathways are similar to those of prokaryotic bacteria, which offers distinct selectivity for inhibition and hence killing of the parasite. Knowledge of the apicoplast contents and functions are mostly derived from bioinformatics analysis of Plasmodium genome data. Many antibiotics, presumed to target the apicoplast, can kill the parasite, and several are already in use to treat malarial patients or in clinical trials but their actual targets have not yet been verified. In Chapter 2, I attempted to isolate the apicoplast (using flow cytometry from a parasite line in which the apicoplast is green fluorescent tagged) and define the apicoplast proteome using mass spectrometry. In total, I identified 732 proteins from isolated sub-cellular structures, which included some known and/or predicted apicoplast proteins but also many non-apicoplast proteins. I also found numerous mitochondrial proteins in my apicoplast fraction and confirmed that mitochondrial DNA was present in the sorted material. Mitochondria and apicoplasts are physically linked and exchange metabolites in Plasmodium, and this attachment appeared to survive the flow cytometry sorting, bringing mitochondria along with the apicoplasts. Although the apicoplast isolation and mass spectrometry protocol showed some promise, more work will be required to generate a stringent and robust apicoplast proteome. In Chapter 3, I used isopentenyl pyrophosphate (IPP) supplementation in conjunction with a battery of apicoplast viability assays to validate whether or not 22 presumed apicoplast targeting drugs do indeed have their primary target in the apicoplast. I confirmed a primary apicoplast target for nine antibiotics, all of which cause so-called delayed death drugs whereby they kill parasites at least 10 times more efficiently during the 2nd life cycle of drug application. These nine antibiotics appear to impact apicoplast housekeeping machineries on the basis of apicoplast degradation during drug treatment. IPP supplementation also rescued parasites from two IPP biosynthesis pathway inhibitors, but these drugs did not result in apicoplasts degradation. Moreover, these drugs caused immediate death making them potentially better suited to therapy for severe malaria, whereas the delayed death drugs would appear more suited to prophylaxis or use as partners to fast acting drugs. IPP supplementation assays confirmed that apicoplast fatty acid biosynthesis and photosynthesis are not valid drug targets in the red blood cell phase of the parasite. One drug, actinonin (which in bacteria abrogates a post-translational modification process), emerged as unique among those having primary targets in the apicoplast. Actinonin caused immediate death and apicoplast degradation. This unique combination suggested that it has a deadly impact on apicoplast biogenesis but does not target the housekeeping pathways perturbed by the nine delayed death antibiotics, which likely impact DNA replication and protein translation. In Chapter 4, I identified the likely target of actinonin through selection of drug resistance and genotyping. Taking my lead from published work on the related parasite Toxoplasma gondii showing that a putative apicoplast protease, TgFtsH1, is the target of actinonin, I was able to show by direct sequencing of select genes that actinonin likely targets an orthologue, PfFtsH1, on the basis of a point mutation in this gene. My genotyping also indicated that actinonin likely does not target post-translational modification of apicoplast synthesised proteins since these genes remained wild-type in the resistant line. My data provide the best evidence yet that actinonin targets PfFtsH1, and make localisation and determination of its role in the apicoplast a high priority to better understand how this unique drug lead works. In Chapter 5, I investigated the activity of verapamil, a drug used in human medicine but also known to be antimalarial. I showed that verapamil causes delayed death, killing parasites at drastically lower concentrations in the 2nd red blood cell cycle. Based on the strong pattern of delayed death observed for nine delayed death antibiotics characterised in Chapter 3 on apicoplast housekeeping, I hypothesised that verapamil would also inhibit apicoplast housekeeping. However, IPP supplementation assays showed that verapamil death cannot be rescued with IPP supplementation and does not detectably perturb the apicoplast, which refuted my initial hypothesis. I selected for verapamil resistance in P. falciparum and genotyped the resistant parasites but was not able to identify any mutation(s) definitely associated with resistance. This data leads to a new working hypothesis that verapamil has a target outside the apicoplast, perhaps impacting the cytosolic utilisation of apicoplast synthesised IPP to cause delayed death. In Chapter 6, I present a recap of my findings and some directions as to where I think this work could eventually lead and what types of investigations should be the highest priority in pursuing apicoplast drug targets to further combat malaria.