Transcriptomic analysis reveals reduced transcriptional activity in the malaria parasite Plasmodium cynomolgi during progression into dormancy
- Novartis Pharma AG, Europe
- Biomedical Primate Research Centre, The Netherlands
- Novartis Pharma AG, United States
- Mahidol University, Thailand
Figures
Figure 1 with 5 supplements
Comparison of transcriptional activity in schizonts (SZ) and hypnozoites (HZ) at day 6/7 and day 9.
Left panel: Distribution of average gene expression values in the schizont samples at day 6/7 (light blue; n = 3) and at day 9 (dark blue; n = 2), and distribution of average gene expression values in the hypnozoites samples at day 6/7 (light green; n = 4) and at day 9 (dark green; n = 2). FPKM, Fragments per kilobase of transcript per million mapped reads. Upper right panel: Venn diagrams show the overlap of genes expressed ≥1 FPKM and ≥10 FPKM, respectively, at day 6/7 and at day 9 in the schizont samples. Lower right panel: Venn diagrams show the overlap of genes expressed ≥1 FPKM and ≥10 FPKM, respectively, at day 6/7 and at day 9 in the hypnozoite samples.
Figure 1—figure supplement 1
Quality check of FACS-sorted parasite cells.
(A) Bar chart depicting the median fluorescence intensity (MFI) values of GFPhigh (schizonts) and GFPlow (hypnozoites) samples FACS-sorted at days 6, 9 and 10 post-sporozoite infection. MFI values are depicted as box whisker (min-max) plots. See Materials and methods for details. (B) FACS-purified GFPhigh and GFPlow fractions at day nine post sporozoite infection. GFPlow parasites were found both in- and outside hepatocytes after sorting (arrows). Day 9 GFPlow sorted parasite samples contained substantial amounts of uninfected hepatocytes. Scalebar 50 μm.
Figure 1—figure supplement 2
Correlation of gene expression values between biological replicates.
Scatter plot showing all pairwise log10 normalized CPM correlations between samples from (A) schizonts and (B) hypnozoites. The upper right part of the panel shows the value of the calculated Pearson correlation coefficients.
Figure 1—figure supplement 3
Possible contamination of day 10 hypnozoites with schizont derived transcripts.
(A) Venn diagrams show the overlap of genes expressed ≥3 FPKM at day 6/7 and 9 in the schizont samples (left, SZ) and in the hypnozoite samples (right, HZ), respectively. (B) Upper Table: Known merozoite-specific markers along with their mean expression levels (FPKM) in the schizont and hypnozoite samples at day 9 and day 10. Lower Table: Selected control genes along with their mean expression levels (FPKM) in the schizont and hypnozoite samples at day 9 and day 10. (C) RNA-FISH (RNAscope) of a hypnozoite and two liver schizonts at 10 days post-sporozoite infection. Staining of a hypnozoite (HZ) and a schizont (SZ2) probed with hsp70 (cy5). Staining was absent in fully mature schizonts (SZ1). Probing against msp1 (FITC) detected transcripts in schizonts only. Scalebar 10 μm.
Figure 1—figure supplement 4
Diagram showing mean gene expression values (FPKM) of schizogony markers in the schizont samples at day 6/7 and day 9.
https://doi.org/10.7554/eLife.41081.006
Figure 1—figure supplement 5
Expression levels of specific genes of interest.
(A) Table showing hypnozoite-specific genes previously identified in Voorberg-van der Wel et al., 2017 passing the cut-off of >2 fold change absolute value and 10% false discovery rate at day 6/7, along with their mean gene expression values (FPKM) in hypnozoites and schizonts at day 9. (B) Upper diagram: Mean gene expression values (FPKM) of 3 ApiAP2 factors in the hypnozoite samples at day 6/7 and day 9. Lower diagram: Mean gene expression values (FPKM) of known malaria drug targets (Voorberg-van der Wel et al., 2017) in the hypnozoite samples at day 6/7 and day 9.
Figure 2 with 1 supplement
Pathway analysis of the malaria liver stages.
(A) Heat map representing expression of Plasmodium pathways in schizonts and hypnozoites at days 6/7 and 9. A total of 257 biological pathways annotated in P. falciparum were assigned to P. cynomolgi through orthology (see Materials and methods in Voorberg-van der Wel et al., 2017). Pathway expression is shown with a color gradient from white (where the fraction of genes detected above 1 FPKM is 0%) to red (where this fraction is 100%). (B) Overall percentage of active pathways in schizonts and hypnozoites at days 6/7 and 9.
Figure 2—figure supplement 1
Expression levels of selected pathways of interest.
Heat map of pathways clustered into higher-level groups of similar functions (http://mpmp.huji.ac.il/; April, 2018) in (A) Schizonts and (B) Hypnozoites at days 6/7 and 9. For each group the mean expression was calculated. Expression is shown with a color gradient from white (where the fraction of genes detected above 1 FPKM is 0%) to red (where this fraction is 100%). (C) Selected pathways, which show high expression levels in hypnozoites at days 6/7 and 9.
Figure 3 with 1 supplement
Validation of genes involved in the export and transport of parasite proteins.
(A) Immunofluorescent staining patterns of UIS4 (Alexa-594), EXP-1 (Alexa-594) and HSP70 (FITC) in day 9 P. cynomolgi hypnozoites (HZ) and liver schizonts (SZ). Scalebar 25 μm. (B) EXP-1 is expressed in P. vivax hypnozoites (HZ) and liver schizonts (SZ) at day 8 post sporozoite infection. Colocalization of EXP-1 and UIS4 suggests that EXP-1 is expressed on the PVM, in agreement with other Plasmodium species. DNA was localized with DAPI. Scalebar 5 μm (HZ) and 15 μm (SZ). (C) RNA-FISH staining of Alba1 (cy3) and PTEX150 (cy3) in day 10 P. cynomolgi hypnozoites (Hz) and liver schizonts (Sz). Scalebar 10 μm.
Figure 3—figure supplement 1
Validation of selected genes.
(A) Diagram showing mean gene expression values (FPKM) of UIS4 and EXP-1 in hypnozoites at day 6/7 and day 9. (B) Immunofluorescent staining patterns of UIS4 (Alexa), EXP-1 (Alexa) and HSP70 (FITC) in P. cynomolgi liver stage parasites at different times post sporozoite infection. Scalebar 25 μm. (C) Diagram showing mean gene expression values (FPKM) of genes coding for Alba1-4 and genes coding for components of the translocon PTEX in hypnozoites day 6/7 and day 9, respectively.
Additional files
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Supplementary file 1
P.cynomolgi samples used for RNA-seq, related to Figure 1.
- https://doi.org/10.7554/eLife.41081.012
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Supplementary file 2
Genes with expression vaues ≥ 3 FPKM in hypnozoite samples at day 9 or day 10, related to Figure 1—figure supplement 3B.
- https://doi.org/10.7554/eLife.41081.013
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Supplementary file 3
Genes with expression vaues ≥10 FPKM in hypnozoite samples at day 9 and/or day 10, related to Figure 1.
- https://doi.org/10.7554/eLife.41081.014
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Supplementary file 4
Expression of Plasmodium pathways in schizont and hypnozoite samples at day 9 and day 10.
The table indicates if pathways are significantly up- or down-regulated over time.
- https://doi.org/10.7554/eLife.41081.015
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Supplementary file 5
Gene expression values (FPKM) generated by RNA-seq of hypnozoite (Hz) and liver schizont (Sz) samples, related to Figure 1.
- https://doi.org/10.7554/eLife.41081.016
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Transparent reporting form
- https://doi.org/10.7554/eLife.41081.017
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Transcriptomic analysis reveals reduced transcriptional activity in the malaria parasite Plasmodium cynomolgi during progression into dormancy
eLife 7:e41081.
https://doi.org/10.7554/eLife.41081
