A coordinated transcriptional switching network mediates antigenic variation of human malaria parasites
- Department of Microbiology and Immunology, Weill Cornell Medical College, United States
- Jill Roberts Center for Inflammatory Bowel Disease, Weill Cornell Medical College, United States
Figures
Figure 1 with 2 supplements
Detection of ‘single’ and ‘many’ var expression profiles in cultured parasite populations.
(A) Schematic representation of the single-many-single (SMS) model for var gene transcriptional switching. Each circle represents expression of an individual var gene and arrows represent switches …
Figure 1—figure supplement 1
Clone tree of wildtype 3D7 parasites.
Pie charts display the var expression profile for each subcloned population with each slice of the pie representing the expression level of a single var gene. Vertical and horizontal lines delineate …
Figure 1—figure supplement 2
Examination of var gene expression profiles of recent clones of NF54.
(A) Cloning by limiting dilution was performed using an uncloned, heterogenous population of the NF54 isolate. Eight individual clonal populations were generated and allowed to expand for 5 weeks, …
Figure 2
Detection of var gene minor transcript expression profiles in clonal populations overtime.
(A) Heatmap of var minor transcripts for clones 2–6. The clones were all derived from the same parent population as shown in Figure 1D. Pie charts show the initial var gene expression profiles above …
Figure 3
Convergence to var2csa expression in long-term cultures and targeted deletion of the var2csa locus.
(A) Changes in var expression over time in five clonal parasite populations, three ‘manys’ (clones 2–4) and two ‘singles’ (clones 5 and 6), over several months of continuous culture. Expression of va…
Figure 4
Alterations in var gene expression after deletion or disruption of var2csa.
(A) Changes in var expression profile over time in five clonal parasite populations containing deletions or disruption of the var2csa locus, including two in which the locus was disrupted by plasmid …
Figure 5 with 1 supplement
Parasites displaying ‘single’ vs. ‘many’ var expression profiles differ in replication cycle progression.
(A) Volcano plots showing differentially expressed genes derived from whole transcriptome comparisons of var2csa-mutated ‘single’ and ‘many’ parasite lines. Differentially expressed genes are shown …
Figure 5—figure supplement 1
Growth assays of parasites displaying the ‘single’ and ‘many’ var expression phenotypes.
Parasitemias were determined daily by flow cytometry and cultures were diluted by 10-fold whenever the parasitemia reached 0.5%. The daily parasitemia was multiplied by the exponential dilution …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Plasmodium falciparum) | var2csa | EuPathDB | PF3D7_1200600 | |
| Strain, strain background (P. falciparum) | NF54 | Delemarre and van der Kaay, 1979 (PMID:390409) | NF54 | |
| Strain, strain background (P. falciparum) | 3D7 | Walliker et al., 1987 (PMID:3299700) | 3D7 | |
| Transfected construct (P. falciparum) | pL6_eGFP | Ghorbal et al. (PMID:24880488) | CRISPR-targeting plasmid | |
| Transfected construct (P. falciparum) | pUF1_Cas9 | Ghorbal et al. (PMID:24880488) | CRISPR-targeting plasmid | |
| Software, algorithm | aligner HISAT2 | Kim et al., 2019 (PMID:31375807), Afgan et al., 2018 (PMID:29790989) | v.2.2.0 | |
| Software, algorithm | featureCounts | Liao et al., 2014 (PMID:24227677) | Package Rsubread v.2.0.1 | |
| Software, algorithm | DESeq2 | Love et al., 2014 (PMID:25516281) | v.3.14 | |
| Software, algorithm | EdgeR | Chen et al., 2016 (PMID:27508061), McCarthy et al., 2012 (PMID:22287627), Robinson et al., 2010 (PMID:19910308) | v.3.14 | |
| Software, algorithm | R | Rstudio (1.4.1717) | v.4.1.0 | |
| Software, algorithm | vegan | Community Ecology Package | v.2.5-7 | |
| Software, algorithm | PERMANOVA | Community Ecology Package | v.2.5-7 | |
| Software, algorithm | heatmap.2 | gplots | v.2.3.0 |
Additional files
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Supplementary file 1
List of differentially expressed genes when comparing var2csa-mutant ‘single’ lines to var2csa-mutant ‘many’ lines at ring stages.
Genes upregulated in ‘singles’ are denoted in blue while those upregulated in ‘manys’ are denoted in green. log2 FC = logFold Change, logCPM = log(normalized mean Count per Million), LR = likehood ratio, FDR = adjust p value.
- https://cdn.elifesciences.org/articles/83840/elife-83840-supp1-v2.xlsx
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Supplementary file 2
List of differentially expressed genes when comparing var2csa-mutant ‘single’ lines to var2csa-mutant ‘many’ lines in trophozoite stage.
Genes upregulated in ‘singles’ are denoted in blue while those upregulated in ‘manys’ are denoted in green. logCPM = log10(normalized mean Count per Million), LR = likelihood ratio, FDR = adjust p value.
- https://cdn.elifesciences.org/articles/83840/elife-83840-supp2-v2.xlsx
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Supplementary file 3
Annotation numbers and gene ontology terms for each of 562 genes upregulated in var2csa-mutant ‘single’ lines when compared to var2csa-mutant ‘many’ lines.
Also shown are expression values described for each gene across the 48-hr asexual replication cycle as established in the published dataset from Bartfai et al. These values were used to establish the approximate position within the standard asexual cycle for each sample.
- https://cdn.elifesciences.org/articles/83840/elife-83840-supp3-v2.xlsx
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Supplementary file 4
Annotation numbers and gene ontology terms for each of 49 genes upregulated in var2csa-mutant ‘many’ lines when compared to var2csa-mutant ‘single’ lines.
Also shown are expression values described for each gene across the 48-hr asexual replication cycle as established in the published dataset from Bartfai et al. These values were used to establish the approximate position within the standard asexual cycle for each sample.
- https://cdn.elifesciences.org/articles/83840/elife-83840-supp4-v2.xlsx
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Supplementary file 5
Detailed likelihood of each sample time point using the method described by Poran et al.
All time points refer to the 48-hr asexual replication cycle.
- https://cdn.elifesciences.org/articles/83840/elife-83840-supp5-v2.xlsx
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Supplementary file 6
Primers used during the study.
The top four primers were used for PCR amplification of specific homology blocks during the construction of the pL6-var2csa-promoter deletion plasmid. (A) Primers for plasmid pL6-var2csa-promoter-deletion and pLcas9 construction. (B) Additional quantitative real-time RT-PCR (qRT-PCR) primers for var transcription detection.
- https://cdn.elifesciences.org/articles/83840/elife-83840-supp6-v2.xlsx
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Supplementary file 7
Summary of the total number of reads and mapped reads for the transcriptome analysis of various ‘single’ and ‘many’ expression lines, including var2csa-mutant lines (ΔV2_1, V2dis3, ΔV2_7, V2dis1, V2dis2, ΔV2_5, and ΔV2_3) and wildtype lines (Subclone 3.1, WT_7, WT_rif2, and WT_rif1).
Analysis of ring stages is shown on the left and trophozoite stages are on the right.
- https://cdn.elifesciences.org/articles/83840/elife-83840-supp7-v2.xlsx
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Supplementary file 8
RNAseq dataset from Bártfai et al., 2010 used as the reference for estimating the replication cycle time point of each sample population using the method of Poran et al., 2017.
Transcription levels (FPKM) are shown for each gene through eight time points of the Plasmodium falciparum (3D7) asexual cycle.
- https://cdn.elifesciences.org/articles/83840/elife-83840-supp8-v2.xlsx
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Supplementary file 9
Microarray dataset of Plasmodium falciparum (HB3) gene expression from Bozdech et al., 2003 used as the reference to estimate the replication cycle time point of RNAseq samples using the method of Lemieux et al., 2009.
The form was kindly provided by Dr. Silvia Portugal.
- https://cdn.elifesciences.org/articles/83840/elife-83840-supp9-v2.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/83840/elife-83840-mdarchecklist1-v2.pdf
