Abstract
The mitochondrion of malaria parasites contains several clinically validated drug targets. Within Plasmodium spp., the causative agents of malaria, the mitochondrial DNA (mtDNA) is only 6 kb long, being the smallest mitochondrial genome among all eukaryotes. The mtDNA encodes only three proteins of the mitochondrial electron transport chain and ∼27 small, fragmented rRNA genes having lengths of 22-195 nucleotides. The rRNA fragments are thought to form a mitochondrial ribosome (mitoribosome), together with ribosomal proteins imported from the cytosol. The mitoribosome of Plasmodium falciparum is essential for maintenance of the mitochondrial membrane potential and parasite viability. However, the role of the mitoribosome in sustaining the metabolic status of the parasite mitochondrion remains unclear. The small ribosomal subunit in P. falciparum has 14 annotated mitoribosomal proteins, and employing a CRISPR/Cas9-based conditional knockdown tool, here we verified the location and tested the essentiality of three candidates (PfmtRPS12, PfmtRPS17, and PfmtRPS18). Using immuno-EM, we provide evidence that the P. falciparum mitoribosome is closely associated with the mitochondrial inner membrane. Upon knockdown of the mitoribosome, parasites became hypersensitive to inhibitors targeting mitochondrial Complex III (bc1), dihydroorotate dehydrogenase (DHOD), and the F1F0-ATP synthase complex. Furthermore, the mitoribosome knockdown blocked the pyrimidine biosynthesis pathway and reduced the cellular pool of pyrimidine nucleotides. These results suggest that disruption of the P. falciparum mitoribosome compromises the metabolic capacity of the mitochondrion, rendering the parasite hypersensitive to a panel of inhibitors that target mitochondrial functions.
Highlights
The mitochondrion of malaria parasites contains several clinically validated drug targets
The SSU of the Plasmodium mitoribosome is composed of 14 annotated proteins, at least 12 fragmented ribosomal RNA (rRNA), and likely a number of additional ribosomal proteins that are not identifiable via currently available bioinformatics approaches
Based on the assembly map of the well-studied bacterial 30S ribosome [39] and the 6-kb sequence of Plasmodium mitochondrial DNA (mtDNA) [17], we predicted relative positions of 12 mitoribosomal rRNA fragments in 5Ј to 3Ј directions according to their homology to different domains of the 16S rRNA molecule (Fig. 1)
Summary
In Plasmodium spp., there are a total of 41 annotated mitoribosomal proteins [8], most of which are universally conserved proteins present in all ribosomes (PlasmoDB, RRID: SCR_013331). These results provide strong evidence that genetic ablation of the Plasmodium mitoribosome sensitizes the parasite to antimalarial drugs targeting the mtETC and Complex V This synthetic lethality only occurred in the PfmtRPS12 line, in which a genetic knockdown caused a significant fitness cost but did not severely affect parasite growth. In the data described above, we observed that genetic knockdown of SSU proteins in the Plasmodium mitoribosome resulted in parasite growth arrest (Fig. 4, PfmtRPS12/ PfmtRPS17), reduction of bc activity (Fig. 5, all lines), and synthetic lethality with antimalarials targeting the mtETC (Fig. 6, PfmtRPS12). Our data show knockdown of the Plasmodium mitoribosome largely recapitulates the effect of atovaquone on inhibiting pyrimidine biosynthesis
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