School of BioSciences - Theses
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ItemEvolution of drug-resistance genes in the asymptomatic Plasmodium falciparum reservoir of infection in Ghana( 2019)Ghana is one of the 11 countries in the world with the highest malaria burden. Like many other African countries, the majority of individuals of all ages harbour asymptomatic Plasmodium falciparum infections, which sustain malaria transmission. Yet these infections are largely undiagnosed and untreated. Chloroquine (CQ) was the main drug for treating clinical malaria in Africa until it was replaced with artemisinin-based combination therapies (ACTs) in the early 2000s due to treatment failures. At the same time, sulphadoxine-pyrimethamine (SP) was adopted for intermittent preventative treatment in pregnancy (IPTp). In order to inform future malaria control strategies in Ghana, I investigated the asymptomatic P. falciparum reservoir in Bongo District (BD), where malaria transmission is both high and seasonal. To evaluate the reservoir of asymptomatic P. falciparum infections including antimalarial drug-resistance markers in BD, a cross-sectional Pilot survey of ~700 participants (≥ 1 year) was undertaken at the end of the dry season in June 2012. Following the completion of this Pilot investigation a larger serial cross-sectional study (~2,000 participants) involving six seasonally timed surveys was completed between 2012 and 2016. This study was designed to evaluate the impact of indoor residual spraying with insecticides (IRS) on the prevalence and diversity of asymptomatic P. falciparum infections in BD before, during, and after the IRS intervention. At the end of the dry season in 2012 I showed that 38.3% of the population across all ages (1-85 year) carried asymptomatic P. falciparum infections. The majority (>70%) of these infections harboured CQ sensitive alleles (Pfcrt K76 and Pfmdr1 N86) and/or alleles associated with reduced response to SP (Pfdhfr I51R59N108/Pfdhps G437) and/or the ACT partner-drug, lumefantrine (Pfmdr1 N86F184). There was no evidence of selection of multilocus haplotypes (i.e. Pfcrt- Pfmdr1- Pfdhfr- Pfdhps) with predicted resistance to both CQ and SP, nor was there any evidence of artemisinin resistance based on Pfk13 genotyping. To further understand this rebound of CQ sensitivity in BD further analyses of the microsatellite loci flanking Pfcrt and Pfmdr1 indicated that the CQ sensitive alleles spread through the asymptomatic parasite reservoir via soft selective sweeps. They may have expanded from CQ sensitive lineages that survived CQ drug pressure, i.e. before Ghana switched to ACTs. Following the completion of the 3-rounds of IRS in BD, undertaken between 2013 and 2014, both the prevalence and multiplicity of asymptomatic P. falciparum infections among children (1-10 years) reduced significantly compared to the pre-IRS surveys. Interestingly, despite these reductions, parasite diversity as assessed by msp2 heterozygosity remained high and stable from the pre-IRS through to the post-IRS surveys. My findings suggest that the asymptomatic P. falciparum reservoir in BD poses a threat to malaria elimination and plays a role in the evolution of antimalarial resistance in Ghana. Therefore, strategies combining IRS with population-wide antimalarial treatments, potentially using ACTs with CQ, would have to be deployed and sustained in BD. Nonetheless, continuous monitoring of the molecular markers of resistance and for changes in the parasite diversity will be crucial to inform elimination strategies in Ghana and Africa.
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ItemInvestigating apicoplast membrane transporters of the malaria parasites Plasmodium berghei and P. falciparum( 2017)The malaria-causing parasite, genus Plasmodium, contains a unique non-photosynthetic plastid known as the apicoplast. The apicoplast is an essential organelle bound by four membranes. Although membrane transporters are attractive drug targets, only two transporters have been characterised in the apicoplast membranes. The aim of this thesis was to characterise membrane transporters of the apicoplast to ultimately identify novel drug targets to kill or perturb the malaria parasite. I selected 28 candidate genes and performed a genetic screen in P. berghei, which is amenable to medium throughput molecular genetics, to determine the blood stage essentiality and cellular localisation of these potential membrane transporters. My approach was successful, identifying eight blood stage essential genes in P. berghei, three of which are apicoplast-localised. I also found 20 blood stage dispensable P. berghei genes, four of which are apicoplast targeted. Interestingly, three apicoplast-localised candidates do not contain a canonical targeting signal, which led me to investigate novel apicoplast targeting mechanisms. Protein features required for the targeting of the only previously known leaderless apicoplast membrane transporter, PfoTPT, were dissected, and an N-terminal tyrosine was found to be necessary but not sufficient for apicoplast localisation. Similar conserved tyrosines were observed in all three novel leaderless apicoplast putative membrane transporters identified in this thesis, suggesting that this residue could be an important component of leaderless apicoplast targeting. Genes essential at the blood stage are potentially good drug targets because they are required in the medically important stage of malaria and perturbation can be assumed to kill the parasite. To further characterise my shortlisted essential apicoplast membrane transporter candidates, I switched my study organism to P. falciparum, which offers the best systems for the inducible knockdown of essential apicoplast genes. I created inducible ribozyme-mediated knockdown parasite lines for two P. falciparum putative membrane transporters shown to be essential in P. berghei, one of which produced ambiguous results. The advantage of doing inducible apicoplast knockdowns in P. falciparum cultures is that the loss of apicoplast-produced isopentenyl diphosphate (IPP) can be supplemented by adding IPP to the culture medium, thereby enabling lethal knockdowns to be maintained. I found that knockdown of PfDMT2, an essential apicoplast putative membrane transporter in P. berghei, was only viable when parasites were supplemented with IPP. Knockdown of PfDMT2 resulted in complete loss of the apicoplast, and these apicoplast-minus parasites were reliant on exogenous IPP to survive. PfDMT2 is therefore a crucial apicoplast membrane transporter and an excellent candidate for therapeutic intervention. Five novel apicoplast putative membrane transporters were identified, three of which are non-canonically targeted to the organelle. Additionally, two novel apicoplast putative membrane proteins were identified. This work has significantly contributed to the apicoplast transportome and will provide a platform for future studies to better understand apicoplast biology and whether it can be exploited to kill or perturb the malaria parasite.