School of BioSciences - Theses
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ItemAn exploration of the impact of drug resistance mutations on the development of Plasmodium berghei throughout the parasite life cycle( 2021)Drug resistance in Plasmodium spp. significantly impedes malaria control and eradication efforts. Recent emergence of resistance to front line antimalarial treatments, such as artemisinin, means new strategies are needed to counter the spread of resistance and overcome the evolutionary agility of the malaria parasite. Malaria parasites undergo immense changes in metabolic activity between their vertebrate and mosquito life cycle stages. Consequently, drug selection for resistance during the vertebrate stage can have dire consequences for parasite fitness when it transitions to insect stages. We investigated drugs with targets likely to be under markedly different selection pressures between the two hosts to identify resistance mutations that could drive parasite failure in the mosquito, thereby blocking transmission. This works with the mitochondrion-encoded cytochrome b inhibitor atovaquone, where resistance is trapped in the host. We hypothesised that the same trap would hold true for drug targets encoded by the apicoplast, a parasite compartment homologous to the plastids of plants and algae that is very likely under differential selection between mammalian and insect life cycle stages. We selected for strains of the mouse malaria model Plasmodium berghei resistant to antimalarial drugs inhibiting apicoplast translation. We generated clonal strains resistant to azithromycin (PbAZMR) and clindamycin (PbCLINDR) but were unsuccessful in generating strains resistant to doxycycline. PbAZMR parasites carried mutations in the apicoplast-encoded 50S ribosomal protein L4, which also confer resistance to this class of drugs in P. falciparum, bacteria, and algae. PbCLINDR parasites had novel, independent mutations in an apicoplast translation pathway not previously linked to clindamycin resistance. Phenotype analysis of PbAZMR and PbCLINDR parasites across the life cycle showed that resistant parasites largely produce viable gametes and can infect mosquitoes. However, both PbAZMR and PbCLINDR parasites produced fewer midgut oocysts, and their oocysts developed aberrantly. Both PbAZMR and PbCLINDR parasites produced fewer salivary gland sporozoites, and—surprisingly—PbAZMR sporozoites lacked an apicoplast. Both PbAZMR and PbCLINDR parasites had severe impairments in their ability to transmit to naive mice, suggesting the resistance mutations had imposed cumulative fitness deficits in both mosquito and liver stages that reduced transmissibility. Our results suggest that the resistance trap initially discovered for mitochondrial gene targets could also work for apicoplast drug targets. Although apicoplast translation inhibitors are little used in therapy because the delayed death phenomenon (where parasite killing only occurs after a full asexual intraerythrocytic developmental cycle) makes them unsuitable for curing seriously ill patients, our demonstration that resistance to these drugs is poorly transmitted makes them of renewed interest as partners to protect fast killing frontline drugs. Combination therapies are better able to constrain resistance, and the safe, cheap, apicoplast inhibiting antimalarials could protect the efficacy of the primary compound and alleviate the spectre of combinatorial use selecting for multi-drug resistance, as has happened previously with sulfadoxine/pyrimethamine and artemisinin-based combination therapies.