Fig 1.
Generation of parasite lines conditionally expressing MPPα.
(A) Schematic of MPPα locus modification using CRISPR-Cas9. Vertical arrow indicates position of sgRNA binding. Also indicated are the 5’ and 3’ homologous regions (HR), 5’ HR coming from MPPα CDS and the 3’ HR coming from 3’UTR. (B) Using primers indicated by PCR1, amplification of the forward and reverse primers from the endogenous locus shows that the locus is disrupted in the MPPα transfected line. Using primers indicated for PCR2, amplification of the forward primer from the endogenous locus and the reverse primer from the HA tag present in the plasmid determines correct integration of the donor DNA into the parasite genome. (C) Representative immunofluorescence images of a parasite conditionally expressing MPPα, showing its mitochondrial localization.
Fig 2.
MPPα is essential for parasite viability.
(A) Western blot analysis using anti-HA and anti-HSP60 antibodies. (B) Quantification of Western blot normalized to intensities of anti-HSP60. Pixel intensities were measured via Image J. (C) Assessment of growth of MPPα (+) and MPPα(-) parasites. Growth assay data was conducted in triplicate by flow cytometry using SYBR green stained parasites. Graphpad Prism was used for statistical analysis and plotting of parasitemia.
Fig 3.
Mitochondrial membrane potential is disrupted in knockdown parasites.
(A) Live cell microscopy images of MPPα (+) and MPPα (–) parasites. Mitotracker staining (red) revealed tubular morphology in MPPα(+) parasites but was diffused in varying number of MPPα(-) parasites starting as early as Day 2 following the knockdown (B) Quantitation of morphological profiles of 30 parasites at each time points was assessed, showing gradual increase in parasites showing diffuse Mitotracker staining resulting from disruption of mitochondrial membrane potential.
Fig 4.
Knockdown of MPPα expression causes hypersensitivity to proguanil.
(A) Parasite growth inhibition assessed by 3H-hypoxanthine incorporation following proguanil treatment. For MPPα- parasites, aTc was removed for 48 h followed by 3H-hypoxanthine addition for 72hr. MPPα(+) parasites were also grown for 72hr followed by 3H-hypoxanthine addition. (B) EC50 values for DSM1, atovaquone, PA21A092, and proguanil in MPPα+ and MPPα- parasites. While the EC50 values for Complex III, DHOD and PfATP4 inhibitors were similar for MPPα(+) and MPPα(-) parasites, MPPα(-) parasites were almost 200-fold hypersensitive to proguanil. (C) Parasite growth inhibition assessed by 3H-hypoxanthine incorporation following proguanil treatment and addition of 25 μM decyl-ubiquinone where indicated. For MPPα(-) parasites, aTc was removed for 48 h followed by 3H-hypoxanthine addition for 72hr.
Fig 5.
Proteomic analysis of MPPα immunoprecipitation (A) Volcano plot of enriched proteins with a log2-fold change of label free quantification (LFQ) intensity and p-values of < 0.05 found in immunoprecipitated MPPα samples.
The red dots represent proteins present in Complex III. The purple dots represent proteins that are predicted to be targeted to the mitochondria. The blue dots represent the other proteins that were significantly enriched in the immunoprecipitated samples. (B) Pie chart depicting the number of proteins that fall into the categories of Complex III, likely mitochondrially targeted, and other enriched proteins.