Biochemistry and Pharmacology - Theses

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End date: 2022 × Start date: 2020 × Subject: Apicoplast × Subject: Mitochondria ×

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    Doxycycline has a dual mode of action against malaria parasites
    Crisafulli, Emily Marie ( 2021)
    Traditionally used as a broad-spectrum antibiotic, doxycycline is frequently used for malaria prophylaxis and treatment - the latter in combination with artemisinin derivatives. Its mechanism of action in Plasmodium spp. has not yet been fully elucidated, though there is substantial evidence that ribosomes in the apicoplast - a relict plastid - are the primary target, with doxycycline causing delayed death (a phenotype associated with inhibitors of apicoplast housekeeping). Inhibition of the apicoplast depletes isoprenoids, synthesised via a pathway housed in the organelle, perturbing the prenyl-dependent trafficking mechanism for haemoglobin uptake and trafficking. This same uptake of haemoglobin is required for activation of artemisinin derivatives. Here, we show that apicoplast-targeting antibiotics, such as doxycycline, reduce the abundance of the catalyst of artemisinin activation (free haem) in P. falciparum, likely through diminished haemoglobin digestion. We demonstrate antagonism between dihydroartemisinin and these antibiotics, likely because apicoplast inhibitors reduce artemisinin activation. Separately, we identify a secondary, more immediate target of doxycycline that exists at clinically relevant concentrations. We show that supplementation with the apicoplast-derived isoprenoid precursor, isopentenyl pyrophosphate, only rescues parasites from delayed death, demonstrating independence of the first cycle target from the relict plastid. Instead, we show that doxycycline depletes mitochondrial electron transport and selectively reduces the abundance of proteins encoded by the mitochondrion in the related apicomplexan parasite, Toxoplasma gondii, suggesting that inhibition of mitochondrial protein synthesis could underpin the immediate death phenotype caused by doxycycline. These data have potential clinical significance when considering the reliance on - and widespread use of - doxycycline and other apicoplast-targeting antibiotics in malaria endemic regions. They reinforce the strategic importance of rational choice of antimalarial combinations; and lay the groundwork for further exploration of the underlying mechanisms of drug resistance in Plasmodium parasites.