Investigating apicoplast membrane transporters of the malaria parasites Plasmodium berghei and P. falciparum
AuthorSayers, Claire Peta
AffiliationSchool of BioSciences
School of Botany
MetadataShow full item record
Document TypePhD thesis
Access StatusOpen Access
© 2017 Dr. Claire Peta Sayers
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.
Keywordsgenetic screen; malaria; Plasmodium; apicoplast; membrane transporter; knockout (KO); knockdown; localise; isopentenyl diphosphate (IPP)
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