The Plasmodium falciparum histone methyltransferase PfSET10 is dispensable for the regulation of antigenic variation and gene expression in blood-stage parasites

Abstract

The malaria parasite Plasmodium falciparum employs antigenic variation of the virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1) to escape adaptive immune responses during blood infection. Antigenic variation of PfEMP1 occurs through epigenetic switches in the mutually exclusive expression of individual members of the multi-copy var gene family. var genes are located in perinuclear clusters of transcriptionally inactive heterochromatin. Singular var gene activation is linked to locus repositioning into a dedicated zone at the nuclear periphery and deposition of histone 3 lysine 4 di-/trimethylation (H3K4me2/3) and H3K9 acetylation marks in the promoter region. While previous work identified the putative H3K4-specific methyltransferase PfSET10 as an essential enzyme and positive regulator of var gene expression, a recent study reported conflicting data. Here, we used iterative genome editing to engineer a conditional PfSET10 knockout line tailored to study the function of PfSET10 in var gene regulation. We demonstrate that PfSET10 is not required for mutually exclusive var gene expression and switching. We also show that PfSET10 is dispensable not only for asexual parasite proliferation but also for sexual conversion and gametocyte differentiation. Furthermore, comparative RNA-seq experiments revealed that PfSET10 plays no obvious role in regulating gene expression during asexual parasite development and gametocytogenesis. Interestingly, however, PfSET10 shows different subnuclear localization patterns in asexual and sexual stage parasites and female-specific expression in mature gametocytes. In summary, our work confirms in detail that PfSET10 is not involved in regulating var gene expression and is not required for blood-stage parasite viability, indicating PfSET10 may be important for life cycle progression in the mosquito vector or during liver stage development.IMPORTANCEThe malaria parasite Plasmodium falciparum infects hundreds of millions of people every year. To survive and proliferate in the human bloodstream, the parasites need to escape recognition by the host's immune system. To achieve this, P. falciparum can change the expression of surface antigens via a process called antigenic variation. This fascinating survival strategy is based on infrequent switches in the expression of single members of the var multigene family. Previous research reported conflicting results on the role of the epigenetic regulator PfSET10 in controlling mutually exclusive var gene expression and switching. Here, we unequivocally demonstrate that PfSET10 is neither required for antigenic variation nor the expression of any other proteins during blood-stage infection. This information is critical in directing our attention toward exploring alternative molecular mechanisms underlying the control of antigenic variation and investigating the function of PfSET10 in other life cycle stages.

Keywords: CRISPR-Cas9; PfEMP1; PfSET10; Plasmodium falciparum; RNA-seq; antigenic variation; gametocytes; histone methyltransferase; malaria; var genes.

Fig 1

PfSET10 is not essential for asexual parasite replication. (A) Live cell fluorescence microscopy of PfSET10-mNG expression in the 3D7/DiCre/SET10-mNG-iKO line during asexual parasite development. Numbers in brackets indicate the age range (hpi) of the synchronous parasite samples analyzed at six TPs during the IDC. DNA was stained with Hoechst. DIC, differential interference contrast. R, ring; T, trophozoite; S, schizont, seg., segmented; mNG, mNeonGreen. Representative images are shown. Scale bar, 5 µm. (B) Live cell fluorescence microscopy of PfSET10-mNG expression in the 3D7/DiCre/SET10-mNG-iKO line comparing control (DMSO) with PfSET10 KO (RAPA) parasites at 30–34 hpi. DNA was stained with Hoechst. DIC, differential interference contrast. Representative images are shown. Scale bar, 5 µm. (C) Parasite multiplication rates measured by flow cytometry of SYBR Green I-stained control (DMSO) and PfSET10 KO (RAPA) parasites over three consecutive generations. Parasitemia was measured at 20–28 hpi. The means ± s.d. (error bars) of three biological replicates are shown, with circles representing individual values. RAPA, rapamycin.

Fig 2

PfSET10 is not required for var gene switching. (A) Quantification of V08-2xTY-expressing cells in 3D7/DiCre/SET10-mNG-iKO/V08-2xTY^BSD parasites populations cultured in the absence (gray bar) or presence (white bar) of BSD-S-HCl pressure as assessed by IFAs using α-TY antibody at 32–40 hpi. The means ± s.d. (error bars) of three biological replicates are shown, with circles representing individual values. At least 150 individual iRBCs were counted for each condition and replicated. (B) Parasite multiplication rates were measured by flow cytometry of three SYBR Green I-stained clonal lines each obtained from DMSO-treated (gray bars) and RAPA-treated PfSET10 KO parasite populations (white bars) after the first (top) and second (bottom) invasion cycles (generations 1 and 2). Parasitemia was measured at 20–28 hpi. Values represent the results from a single challenge experiment. Clone IDs are indicated on the x-axis. RAPA, rapamycin.

Fig 3

PfSET10 is not required for mutually exclusive var gene expression and switching. (A) Schematic explaining the sample collection workflow applied to quantify var gene transcripts by RNA-seq in DMSO control and RAPA-treated 3D7/DiCre/SET10-mNG-iKO/V08-2xTYBSD synchronous ring-stage parasites 2 and 10 generations after removal of BSD-S-HCl selection pressure. G, generation; R, ring stages; WT, DMSO-treated control parasites; KO, RAPA-treated PfSET10 KO parasites; hpi, hours post-invasion. (B) Heatmap showing normalized read counts (blue-red) and fold change in expression levels (orange-purple) of all var genes as quantified by RNA-seq of 3D7/DiCre/SET10-mNG-iKO/V08-2xTY^BSD ring-stage parasites (10–16 hpi). R-WT-G0, control parasites selected on BSD-S-HCl for v08-2xty expression. R-WT-G2 and R-WT-G10, DMSO-treated control parasites harvested 2 and 10 generations after release from BSD-S-HCl selection pressure, respectively. R-KO-G2 and R-KO-G10, RAPA-treated PfSET10 KO parasites harvested 2 and 10 generations after release from BSD-S-HCl selection pressure, respectively. Values represent the mean of three biological replicate RNA-seq experiments performed for each condition and TP. The dominant v08 gene PF3D7_1221000 has been highlighted. RAPA, rapamycin.

Fig 4

PfSET10 plays no major role in regulating gene expression in asexual blood-stage parasites. (A) Schematic explaining the sample collection workflow applied to quantify global gene expression by RNA-seq in synchronous DMSO control and RAPA-treated 3D7/DiCre/SET10-mNG-iKO/V08-2xTYBSD parasites at four time points during the IDC two generations after removal of BSD-S-HCl selection pressure. G, generation; R, ring stages; T, trophozoites; ES, early schizonts; LS, late schizonts; WT, DMSO-treated control parasites; KO, RAPA-treated PfSET10 KO parasites; hpi, hours post-invasion. (B) Principal component analysis plot of the triplicate transcriptomes determined by RNA-seq from synchronous 3D7/DiCre/SET10-mNG-iKO/V08-2xTY^BSD parasites at four TPs during the IDC, two generations after release from BSD-S-HCl selection pressure. R, ring stages (10–16 hpi); T, trophozoites (22–28 hpi); ES, early schizonts (30–36 hpi); LS, late schizonts (38–44 hpi). WT, DMSO-treated control parasites; KO, RAPA-treated PfSET10 KO parasites. G2, generation 2 after release from BSD-S-HCl selection pressure. RAPA, rapamycin.

Fig 5

PfSET10 shows female-specific expression in late-stage gametocytes. (A) Live cell fluorescence microscopy of NF54/DiCre/SET10-mNG-iKO gametocytes. PfSET10-mNG is visible from day 2 (stage I) onward as multiple diffuse signals overlap with the Hoechst-stained area. From day 8 (stage IV) onward, a PfSET10-mNG negative subpopulation emerges. DNA was stained with Hoechst, subpellicular microtubules were stained with SPY555-tubulin. DIC, differential interference contrast. Representative images are shown. Scale bar, 5 µm. (B) IFA overview image of NF54/DiCre/SET10-mNG-iKO stage V gametocytes (day 13) stained for the female-enriched marker Pfg377, PfSET10-mNG, and Hoechst. Representative images are shown. Scale bar, 5 µm. (C) Integrated Alexa Fluor 568 (Pfg377) and Alexa Fluor 488 (PfSET10-mNG) fluorescence signal intensity values resulting from automated image analysis of NF54/DiCre/SET10-mNG-iKO stage V gametocytes (day 13) stained with Hoechst, α-Pfg377/Alexa Fluor 568, and α-mNeonGreen/Alexa Fluor 488 antibodies. The scatter plot shows normalized integrated intensity values for Alexa Fluor 488 (PfSET10-mNG, y-axis) and Alexa Fluor 568 (Pfg377, x-axis) for 324 cells scored in a single experiment.