Transcriptional changes in Plasmodium falciparum upon conditional knock down of mitochondrial ribosomal proteins RSM22 and L23

Abstract

The mitochondrion of malaria parasites is an attractive antimalarial drug target, which require mitoribosomes to translate genes encoded in the mitochondrial (mt) DNA. Plasmodium mitoribosomes are composed of highly fragmented ribosomal RNA (rRNA) encoded in the mtDNA. All mitoribosomal proteins (MRPs) and other assembly factors are encoded in the nuclear genome. Here, we have studied one putative assembly factor, RSM22 (Pf3D7_1027200) and one large subunit (LSU) MRP, L23 (Pf3D7_1239100) in Plasmodium falciparum. We show that both proteins localize to the mitochondrion. Conditional knock down (KD) of PfRSM22 or PfMRPL23 leads to reduced cytochrome bc1 complex activity and increased sensitivity to bc1 inhibitors such as atovaquone and ELQ-300. Using RNA sequencing as a tool, we reveal the transcriptomic changes of nuclear and mitochondrial genomes upon KD of these two proteins. In the early phase of KD, while most mt rRNAs and transcripts of putative MRPs were downregulated in the absence of PfRSM22, many mt rRNAs and several MRPs were upregulated after KD of PfMRPL23. The contrast effects in the early phase of KD likely suggests non-redundant roles of PfRSM22 and PfMRPL23 in the assembly of P. falciparum mitoribosomes. At the late time points of KD, loss of PfRSM22 and PfMRPL23 caused defects in many essential metabolic pathways and transcripts related to essential mitochondrial functions, leading to parasite death. In addition, we enlist mitochondrial proteins of unknown function that are likely novel Plasmodium MRPs based on their structural similarity to known MRPs as well as their expression profiles in KD parasites.

Fig 1. PfRSM22 and PfMRPL23 are localized to the mitochondrion and are essential for parasite survival.

(A) Images from fluorescence microscopy showing colocalization of PfRSM22-3HA (green) with Mitotracker (red). (B) Colocalization of PfMRPL23-3HA (green) with Mitotracker (red). In A and B, the parasite nucleus is stained using DAPI (blue). (C) Western blot showing expression of PfRSM22-3HA protein (62 KDa) in the presence or absence of 250 nM aTc over 6 days (3 IDCs). (D) Western blot showing expression of PfMRPL23-3HA protein (32 KDa) in the presence or absence of 250 nM aTc over 4 days (2 IDCs). Pf-Exp2 (33 KDa) was used as a loading control. Representative images of Giemsa-stained D10-PfRSM22-3HA parasites (E) and D10-PfMRPL23-3HA parasites (F) at the trophozoite stage in the presence (aTc ON) and absence of aTc (aTc OFF). Quantification of D10-PfRSM22-3HA parasite growth (G) and D10-PfMRPL23-3HA parasite growth (H) over 8 days in the presence and absence of aTc. Growth index was calculated by multiplication of parasitemia with splitting factors over the time course. Data shown here is mean ± SD of n = 3.

Fig 2. PfRSM22 and PfMRPL23 KD increased sensitivity to antimalarials targeting the parasite bc1 complex.

(A) The schematic represents key steps of growth inhibition assays measured by [³H] hypoxanthine incorporation. The diagram was created using BioRender.com. (B and C) Atovaquone hypersensitivity upon PfRSM22 and PfMRPL23 KD respectively. (D and E) ELQ-300 hypersensitivity upon PfRSM22 and PfMRPL23 KD respectively. Reduction in IC50 values and Hill Slope of the curve were reported for respective parasite lines. Data shown are the mean ± S.D. of triplicates from n = 3 independent experiments. (F and G) KD of PfRSM22 or PfMRPL23 did not show hypersensitivity to chloroquine.

Fig 3. Reduced cytochrome bc₁ complex activity upon PfRSM22 and PfMRPL23 KD.

(A) Schematic presentation of the Plasmodium mtETC. Complex III (bc₁ complex) activity was measured while complex II and complex IV were inhibited using malonate and KCN respectively. Modified from our previous publication [10]. Created with BioRender.com. Cyt c reduction was measured at 550 nm representing functionality of bc₁ complex in enriched and solubilized mitochondria from PfRSM22 KD (B) and PfMRPL23 KD (C). The bc₁ enzymatic activity of the knockdown parasites in PfRSM22 (orange) and PfMRPL23 (blue) was normalized to their respective aTc ON controls. Kruskal-Wallis test in n = 3 experiments. ***, p < 0.0001; **, p < 0.001.

Fig 4. Transcriptomic changes in the early phase after KD of PfRSM22 and PfMRPL23.

Volcano plots of differentially regulated transcripts (p adj <0.05) on day 2 post KD in the PfRSM22 KD line (A) and in the PfMRPL23 line (B). Each black dot in the Volcano plots represents a gene regulated at least by 2-fold (log2 fold ≥ 1 and ≤ -1). Each grey dot indicates a gene differentially regulated less than 2-fold (log2 fold <1 and > -1). (C) Significantly downregulated KEGG functional pathways based on the number of genes (gene counts) following the KD of PfRSM22 or PfMRPL23 on day 2. (D) Significantly upregulated KEGG functional pathways based on the number of genes (gene counts) following the KD of PfRSM22 or PfMRPL23 on day 2.

Fig 5. Early effects of PfRSM22 and PfMRPL23 KD on transcripts of mitoribosomal components.

(A) Log2 fold change of putative PfMRPs on day 2 KD of PfRSM22 (orange) or PfMRPL23 (blue). (B) Log2 fold change of mt rRNA fragments on day 2 KD of PfRSM22 (orange) or PfMRPL23 (blue). The positions of mt rRNA transcripts are shown in S5 Fig.

Fig 6. Transcriptomic changes in the late phase KD of PfRSM22 and PfMRPL23.

Volcano plots of differentially expressed transcripts in the PfRSM22 KD parasite line on day 6 (A) and in the PfMRPL23 KD parasite line on day 4 (B) post aTc removal. Each black dot in the volcano plots represents a gene differentially regulated (log2 fold change ≥ 1 and ≤ -1). Each grey dot represents a gene differentially regulated with log2 fold change <1 and >-1. (C) KEGG functional pathways based on the number of genes (gene counts) that belong to a pathway downregulated due to PfRSM22 KD on day 6 and PfMRPL23 KD on day 4. (D) KEGG functional pathways upregulated based on the number of genes (gene counts) on day 6 of PfRSM22 KD and on day 4 of PfMRPL23 KD.

Fig 7. Genes regulated in common after KD of PfRSM22 and PfMRPL23 in the late phase.

(A) Venn diagrams depict the relationship of downregulated gene transcripts in the PfRSM22 KD line on day 6 (orange) and in the PfMRPL23 KD line on day 4 (blue) to the 517 mitochondrial related transcripts (green). Transcripts downregulated ≥ 2-fold are included in this analysis. (B) Heat map of commonly downregulated (68 genes) and upregulated genes (2 genes) between PfRSM22 KD and PfMRPL23 KD at the late phase. (C) Venn diagrams depict the relationship of upregulated gene transcripts in the PfRSM22 KD line on day 6 (orange) and in the PfMRPL23 KD line on day 4 (blue) to the 517 mitochondrial related transcripts (green). Transcripts upregulated ≥ 2-fold are included in this analysis.

Fig 8. Late effects of PfRSM22 and PfMRPL23 KDs on transcripts of mitoribosomal components.

(A) Log2 fold change of putative PfMRPs on day 6 after PfRSM22 KD (orange) and on day 4 after PfMRPL23 KD (blue). (B) Log2 fold change of mt rRNA fragments on day 6 after PfRSM22 KD (orange) and on day 4 after PfMRPL23 KD (blue).