Abstract
The phylum Apicomplexa contains a group of protozoa causing diseases in humans and livestock. Plasmodium spp., the causative agent of malaria, contains a mitochondrion that is very divergent from that of their hosts. The malarial mitochondrion is a clinically validated target for the antimalarial drug atovaquone, which specifically blocks the electron transfer activity of the bc1 complex of the mitochondrial electron transport chain (mtETC). Most mtETC proteins are nuclear-encoded and imported from the cytosol, but three key protein subunits are encoded in the Plasmodium mitochondrial genome: cyt b, COXI, and COXIII. They are translated inside the mitochondrion by mitochondrial ribosomes (mitoribosomes). Here, we characterize the function of one large mitoribosomal protein in Plasmodium falciparum, PfmRPL13. We found that PfmRPL13 localizes to the parasite mitochondrion and is refractory to genetic knockout. Ablation of PfmRPL13 using a conditional knockdown system (TetR-DOZI-aptamer) caused a series of adverse events in the parasite, including mtETC deficiency, loss of mitochondrial membrane potential (Δψm), and death. The PfmRPL13 knockdown parasite also became hypersensitive to proguanil, a drug proposed to target an alternative process for maintaining Δψm Surprisingly, transmission EM revealed that PfmRPL13 disruption also resulted in an unusually elongated and branched mitochondrion. The growth arrest of the knockdown parasite could be rescued with a second copy of PfmRPL13, but not by supplementation with decylubiquinone or addition of a yeast dihydroorotate dehydrogenase gene. In summary, we provide first and direct evidence that mitoribosomes are essential for malaria parasites to maintain the structural and functional integrity of the mitochondrion.
Highlights
The phylum Apicomplexa contains a group of protozoa causing diseases in humans and livestock
PfmRPL13 is essential in asexual blood stages of P. falciparum
The malarial mitochondrial electron transport chain (mtETC) has been the focus of many endeavors seeking novel antimalarial drugs [39], yet the structure and function of mitochondrial ribosomes, which translate critical protein subunits of the mtETC, remain entirely uncharacterized
Summary
PF3D7_0214200 (designated as PfmRPL13) is annotated as the putative mitochondrial ribosomal protein L13 in P. falciparum (www.plasmodb.org)3 [47]. When aTc was removed, the percentage of parasites with diffused staining in the PfmRPL13 knockdown parasites increased dramatically in the third and fourth cycles These data suggest that PfmRPL13 and, functional mitoribosomes were critical to maintain mitochondrial membrane potential and parasite health. The level of proguanil hypersensitivity in our PfmRPL13 knockdown parasites (55-fold) was comparable with that of the yDHODH line under atovaquone treatment (62-fold) These data suggest that PfmRPL13 genetic ablation caused a severe mtETC deficiency in a degree that was similar to that triggered by atovaquone inhibition. To gain an understanding of the mechanisms that led to parasite demise when PfmRPL13 was knocked down, we cantly after five cycles or longer without aTc (long term) These data suggested that, in the long term, yDHODH expression still failed to rescue mtETC defects resulting from PfmRPL13 ablation.
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