The Role of the Histone Methyltransferase PfSET10 in Antigenic Variation by Malaria Parasites: a Cautionary Tale

Abstract

The virulence of the malaria parasite Plasmodium falciparum is due in large part to its ability to avoid immune destruction through antigenic variation. This results from changes in expression within the multicopy var gene family that encodes the surface antigen P. falciparum erythrocyte protein one (PfEMP1). Understanding the mechanisms underlying this process has been a high-profile research focus for many years. The histone methyltransferase PfSET10 was previously identified as a key enzyme required both for parasite viability and for regulating var gene expression, thus making it a prominent target for developing antimalarial intervention strategies and the subject of considerable research focus. Here, however, we show that disruption of the gene encoding PfSET10 is not lethal and has no effect on var gene expression, in sharp contrast with previously published reports. The contradictory findings highlight the importance of reevaluating previous conclusions when new technologies become available and suggest the possibility of a previously unappreciated plasticity in epigenetic gene regulation in P. falciparumIMPORTANCE The identification of specific epigenetic regulatory proteins in infectious organisms has become a high-profile research topic and a focus for several drug development initiatives. However, studies that define specific roles for different epigenetic modifiers occasionally report differing results, and we similarly provide evidence regarding the histone methyltransferase PfSET10 that is in stark contrast with previously published results. We believe that the conflicting results, rather than suggesting erroneous conclusions, instead reflect the importance of revisiting previous conclusions using newly developed methodologies, as well as caution in interpreting seemingly contrary results in fields that are known to display considerable plasticity, for example metabolism and epigenetics.

Keywords: antigenic variation; chromatin modifications; epigenetic gene regulation; histone methyltransferase; malaria.

FIG 1

Analysis of PfSET10(−) asexual blood-stage parasites. (A) Schematic depicting PfSET10. The SET (blue box) and PHD zinc finger (green box) domains are indicated. aa, amino acid. (B) Schematic depicting the gene knockout (KO) strategy via single crossover recombination-based gene disruption using selection-linked integration-mediated targeted gene disruption (SLI-TGD). The vector pSLI-TGD was modified to contain a 900-bp sequence block (light red box) from near the 5′ end of the Pfset10 coding region (dark red box). The coding region was maintained in frame with a green fluorescent protein coding region (green box), a 2A “skip” peptide (gray box), and the Neo-R gene (blue) that provides resistance to the antibiotic G418. Medium containing G418 selects for integration into the locus and disruption of the Pfset10 coding region. Arrows indicate the position of primers 1 to 4 used to detect integration of the pSLI-TGD vector. Asterisks indicate a stop codon. GFP, green fluorescent protein; hDHFR; human dihydrofolate reductase for resistance to WR99210; NeoR, neomycin resistance; 2A, skip peptide. (C) Confirmation of vector integration for the PfSET10(−) parasites by diagnostic PCR using genomic DNA (gDNA) obtained from PfSET10(−) and the wild type (WT; P. falciparum strain NF54). 5′ Integration was detected using primers 1 and 4 (1,470 bp), and 3′ integration was detected using primers 2 and 3 (1,164 bp). Primers 3 and 4 were used to detect the presence of episomes (1,251 bp), and primers 1 and 2 were used for WT control (1,342 bp). (D) Confirmation of truncated PfSET10 tagged with GFP. Parasite lysates were subjected to Western blotting using polyclonal mouse anti-GFP (67 kDa). Lysates of WT and noninfected red blood cells (niRBC) were used as negative controls. Immunoblotting with mouse anti-Pf39 antiserum (39 kDa) served as a loading control. (E) Verification of GFP expression in the PfSET10(−) parasites. Live images of trophozoites (TZ) and schizonts (SZ) of the PfSET10(−) line detected GFP (green) associated with the parasite nuclei. The WT was used for a negative control. Nuclei were counterstained by Hoechst 33342 (blue). Bar, 5 μm. (F) Morphology of the PfSET10(−) asexual blood-stages. The morphology was compared via Giemsa staining of asexual blood stages of PfSET10(−) and the WT. TR, trophozoite; imSZ, immature schizont; mSZ, mature schizont. Bar, 5 μm. (G) Asexual blood stage replication of the PfSET10(−) line. Synchronized ring stage cultures of WT and PfSET10(−) with a starting parasitemia of 0.25% were maintained in cell culture medium, and the parasitemia was followed via Giemsa smears over a time period of 0 to 84 h. The experiment was performed in triplicate (mean ± standard deviation [SD]). (H) Steady-state Pfset10 mRNA levels of WT and two PfSET10(−) lines. qRT-PCR was used to detect expression levels in both rings and trophozoite-stage parasites. Expression levels are displayed relative to seryl-tRNA ligase. Results shown in panels C to H are representative of two to three independent biological replicates.

FIG 2

Assessment of var gene expression in WT and two PfSET10(−) lines. Steady-state mRNA levels for each var gene were determined using qRT-PCR and displayed relative to expression of seryl-tRNA ligase. RNA was extracted from each line at an initial time point (0 days, blue) and after 2 weeks of continuous culture (14 days, orange). Expression profiles for each knockout line (top and middle panels), as well as for wild-type parasites (bottom panel), are shown. The annotation number for each var gene is shown on the x axis of the bottom panel. Results are representative of three independent experiments.