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. 2022 May 30;13(1):3004.
doi: 10.1038/s41467-022-30605-z.

Stochastic expression of invasion genes in Plasmodium falciparum schizonts

Jaishree Tripathi  1 , Lei Zhu  2 , Sourav Nayak  2 , Michal Stoklasa  2 , Zbynek Bozdech  3
Affiliations

Stochastic expression of invasion genes in Plasmodium falciparum schizonts

Jaishree Tripathi et al. Nat Commun. .

Abstract

Genetically identical cells are known to exhibit differential phenotypes in the same environmental conditions. These phenotypic variants are linked to transcriptional stochasticity and have been shown to contribute towards adaptive flexibility of a wide range of unicellular organisms. Here, we investigate transcriptional heterogeneity and stochastic gene expression in Plasmodium falciparum by performing the quasilinear multiple annealing and looping based amplification cycles (MALBAC) based amplification and single cell RNA sequencing of blood stage schizonts. Our data reveals significant transcriptional variations in the schizont stage with a distinct group of highly variable invasion gene transcripts being identified. Moreover, the data reflects several diversification processes including putative developmental "checkpoint"; transcriptomically distinct parasite sub-populations and transcriptional switches in variable gene families (var, rifin, phist). Most of these features of transcriptional variability are preserved in isogenic parasite cell populations (albeit with a lesser amplitude) suggesting a role of epigenetic factors in cell-to-cell transcriptional variations in human malaria parasites. Lastly, we apply quantitative RT-PCR and RNA-FISH approach and confirm stochastic expression of key invasion genes, such as, msp1, msp3, msp7, eba181 and ama1 which represent prime candidates for invasion-blocking vaccines.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Highly reproducible MALBAC-based amplification of single cell transcriptomes from P. falciparum schizonts.
a A boxplot showing Pearson correlation coefficients for PfRNAdil (n = 30) amplified by SMARTseq2 (SSEQ2) or MALBAC (MAL). Asterisk denotes significant Wilcoxon rank sum test p-value (p < 2.2e-16). b Scatter plot depicting relationship between standard deviation (SD) and mean transcript expression (log2FPKM) across PfRNAdil replicates (n = 30) for the two techniques. c A scatter plot showing the probability of detection of transcripts (detected in atleast 50% PfRNAdil) across PfRNAdil replicates (n = 30) amplified by MALBAC or SMARTseq2. d A boxplot showing number of transcripts detected in 1-cell (isogenic (n = 97) and non-isogenic (n = 295)), 100-cells (n = 15) and PfRNAdil (n = 49) samples. e A frequency distribution plot of number of transcripts detected in 1-schizonts and 100-schizonts samples. µ denotes the mean number of transcripts expressed in the 1-cell and 100-cells group. f Correlation between averaged 1-cell and 100-cell transcriptomes. The range of averaged R2 values for individual 1-cell and 100-cell transcriptome correlations is shown as a boxplot (inset, median and standard deviation shown, n = 295 for 1-cell, n = 15 for 100-cells). g A violin plot depicting estimated age distribution of non-isogenic and isogenic schizonts. Source data are provided as a Source Data file. All box plots show centre line as median, box limits as upper and lower quartiles, whiskers as minimum to maximum values.
Fig. 2
Fig. 2. Schizont subpopulations with unique transcriptional profiles identified in non-isogenic P. falciparum parasites.
A multidimensional scaling (MDS) plot showing first two components (MDS1 and MDS2) depicting maximum differences between individual schizonts in a non-isogenic and b isogenic group. RSEC-based SCTS hierarchy is shown in right hand inset (for both (a, b)) with each SCTS represented by a different colour for non-isogenic schizonts. The two major age groups (36-40 HPI and >44 HPI) identified has been overlaid on the MDS plot. c A heatmap showing average expression of differentially expressed (DE) genes (mean normalised log2FPKM) in each SCTS of non-isogenic parasites. d A dot plot of various functional pathways enriched in non-isogenic parasite SCTS. The colour and size of each circle depicts p-value and mean log2FPKM value respectively with pathways of interest highlighted in grey. Source data are provided as a Source Data file.
Fig. 3
Fig. 3. Stochastic Gene Expression in P. falciparum Schizonts.
a A graph showing variability index score (VIS) of individual genes in non-isogenic schizonts (n = 271 1-cell) belonging to similar pseudotime interval (i.e. SCTS 1,4,5,6,7 (n = 118 1-cell) in inset) with key invasion genes labelled. Only genes expressed in more than 10 PfRNAdil were considered to avoid artificially inflated VIS. b A heatmap showing expression of HVGs (log2FPKM) in non-isogenic schizonts with genes of interest labelled. Higher and lower expression (log2FPKM) is shown in red and blue respectively. c A boxplot showing z-score calculated from Ct values for each gene transcript measured in single schizonts (n = 16 for rhop2, exp2; n = 38 for msp3, msp7, gap45) and PfRNAdil by qRT-PCR. Median Ct is shown as black line. Primer sequences are provided in Supplementary Data 6. Confocal microscopy images and line graphs showing transcript levels of d eba181, rh5, e msp1, msp3, msp7 and f ama1, in individual schizonts. Representative images of single 3D7MR4 schizonts stained for d eba181 (red) and rh5 (green), and, e msp1 (red), msp3 (green) and msp7 (purple) and f ama1 (green) transcripts using customised RNA probes are shown on the left. S6, S20, S24 and S6, S22, S23, S25 and S5, S6, S8 refers to the schizont order in the line graph for d–f respectively for which the image is shown. Parasite nuclei were stained with DAPI. Between 30 to 50 schizonts were imaged from one RNA-FISH experiment for each combination of probes. Line graphs on the right-hand side show the mean intensity quantified for each gene transcript in individual parasites using the ZEN 3.0 (blue edition ZEN lite) software. Source data are provided as a Source Data file. All box plots show centre line as median, box limits as upper and lower quartiles, whiskers as minimum to maximum values.
Fig. 4
Fig. 4. Transcriptional profile of variant surface antigen encoding genes.
Bubble plots depicting the frequency of expression and average expression (FPKM) for a var, c rifin and e phist genes in both non-isogenic and isogenic schizonts with same gene IDs highlighted in same colour in the two parasite groups. A distribution of number of b var, d rifin and f phist transcripts expressed per cell is shown as a line plot for non-isogenic and isogenic parasites. Different colours represent the distribution of transcripts expressed above 10 (black), 50 (red), 100 (green) or 200 (yellow) FPKM in individual schizonts. Source data are provided as a Source Data file.
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