Doxycycline is a tetracycline-class antibiotic used for malarial prophylaxis and as a partner drug with quinine for treating malaria. Doxycycline’s antimalarial mechanism of action has widely been accepted as a translation inhibitor, specifically blocking the prokaryotic 70S ribosomes of the Plasmodium apicoplast. At low concentrations (<5 μM) doxycycline exhibits a delayed death phenotype, typical of inhibitors of apicoplast housekeeping processes. In this process parasites are morphologically normal in their first growth cycle after treatment, dying only after one round of replication.
At higher concentrations (≥10 μM) doxycycline has schizonticidal activity in the first cycle via an unknown and apicoplast-independent mechanism. In other eukaryotes, doxycycline also inhibits mitochondrial translation. The mitochondrial genome of Plasmodium falciparum encodes only 3 proteins, each of them subunits of complexes III or IV of the electron transport chain. To investigate whether mitochondrial translation is a target at high doxycycline concentrations, we assessed protein abundance in drug treated parasites using mass spectrometry. Assessing steady state protein levels along with protein turnover using isotope-labelled amino acids we directly detected apicoplast encoded proteins for the first time and showed that these proteins decrease in abundance following doxycycline treatment. We detected no differences in mitochondrial translation via this method, although background mitochondrial protein abundance was already very low.
As an alternative assay for mitochondrial protein translation, we assayed the oxygen consumption of the electron transport chain for the mitochondrial encoded complexes. We detected severe perturbations to parasites’ oxidative phosphorylation using a Seahorse assay. The reduced oxygen consumption rate following doxycycline treatment strongly suggests that Plasmodium mitochondrial translation is indeed a target of doxycycline. This disrupted electron transport chain phenotype was also replicated in Seahorse assays of the related protozoan parasite Toxoplasma gondii.
These studies confirm that the apicoplast ribosome is the major target in Plasmodium for antibiotics targeting protein translation, but indicates that at higher doxycycline concentrations, mitochondrial translation and thus mitochondrial electron transport are also impacted. This reveals a second target for these drugs in malaria treatment and insight into their strange dynamics of killing.