Programmed transcription of the var gene family, but not of stevor, in Plasmodium falciparum gametocytes

Abstract

The var genes encode Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) proteins, a set of highly diverse surface-expressed proteins that mediate adhesion of erythrocytes infected with asexual blood-stage parasites to host endothelium. Switching among expressed PfEMP1 variants in the course of a blood-stage infection is a key component of antigenic variation, and thus immune evasion, by the parasite. The majority of var loci are found in the subtelomeric regions of P. falciparum chromosomes associated with members of other multigene families, including stevor. Both PfEMP1 and STEVOR are expressed in gametocytes, the transmissible parasite stage, but the role of these proteins in the biology of sexual-stage parasites remains unknown. PfEMP1 may continue to mediate antigenic variation in gametocytes, which need to persist in the host for many days before reaching maturity. Using quantitative reverse transcription-PCR and Northern hybridization, we demonstrate that transcription of a defined subset of type C var loci occurs during gametocyte development in vitro. This transcriptional program occurs in gametocytes regardless of the var expression phenotype of their asexual progenitors and therefore is subject to regulatory processes distinct from those that manage antigenic variation in the asexual parasite. In contrast, the same stevor variants are transcribed in both gametocytes and their asexual progenitors. We also provide evidence that for both asexual parasites and gametocytes, var and stevor transcription patterns are not linked to each other.

FIG. 1.

Changes in var transcript abundance upon differentiation from asexual to sexual reproduction. (A) Transcription levels were measured by real-time PCR using a set of primers that amplify 58 var genes from clone 3D7. The relative abundance of transcript from each var gene is presented as a proportion of total var transcript abundance in the parasite population tested. An index ratio is given under each chart, indicating the var transcript abundance as a percentage of the estimated total var transcript abundance in ring-stage trophozoites, the stage with peak abundance. Numbered sectors of the pie charts correspond to the following genes: 1, PFD0625c; 2, PFE1640w; 3, PF07_0051; 4, PFD1015c; 5, PFL1955w; 6, PFL1960w; and 7, PF08_0103. (B) Transcript levels of PFD0625c, PFD1015c, and PF07_0051 normalized against levels of the control seryl-tRNA synthetase gene. Triplicate real-time measurements were performed for each cDNA, and the mean values are shown. Similar results were obtained when the experiment was repeated with an independent set of 3D7a gametocyte cultures (data not shown). Error bars represent 1 standard deviation. R, ring stages; T, trophozoite stages; G, gametocyte day 2, 4, or 9 cultures; gDNA, genomic DNA. CIDR1 and DBL2 are domains within PfEMP1 proteins.

FIG. 2.

Change in relative abundance of each var transcript upon differentiation from asexual to sexual reproduction of selected parasite lines with specific phenotypes. Transcript levels were measured by real-time PCR using a set of primers that amplify 58 var genes from clone 3D7. The relative abundance of transcript from each var gene is presented as a proportion of total var transcript abundance in the parasite population tested. Numbered sectors of the pie charts correspond to the following genes: 1, PFD0625c; 2, PFE1640w; 3, PF07_0051; 4, PFD1015c; 5, PFL1955w; 6, PFL1960w; 7, PF08_0103; 8, PF11_0008; 9, PF13_0003; 10, PFD1235w; 11, PF11_0007; and 12, PFF0010w. Transcript levels were measured in an unselected culture of clone 3D7a and in cultures of 3D7 which were either panned on trHBMEC (3D7-trHBMEC) or selected with IgG from semi-immune children (3D7-Dodowa1).

FIG. 3.

Different var types dominate transcript pools in asexual and sexual parasites. Transcript levels were measured by real-time PCR using a set of primers that amplify 58 var genes from clone 3D7. The relative abundance of transcript from each var gene is presented as a proportion of total var transcript abundance in the parasite population tested. Transcript levels were measured in an unselected culture of clone 3D7a and in cultures of 3D7 which were either panned on trHBMEC (3D7-trHBMEC) or selected with IgG from semi-immune children (3D7-Dodowa1). var genes are arranged according to the groups described by Lavstsen et al. (19).

FIG. 4.

Northern blots of ring-stage (R), trophozoite (T), and gametocyte (G) day 2, 4, or 9 cultures. M, 0.24- to 9.5-kb RNA marker. RNAs were size fractionated in either a 1% agarose gel (A) or a 0.8% agarose gel (D). Panels B and C are of the same gel.

FIG. 5.

Changes in stevor transcript upon differentiation from asexual to sexual reproduction. Transcript levels were measured by real-time PCR using a set of primers that amplify 37 stevor genes from clone 3D7. The relative abundance of transcripts from each stevor gene is presented as a proportion of total stevor transcript abundance in either trophozoites or the day 2 gametocyte population. Numbered sectors of the pie charts correspond to the following genes: 1, PFD0065w; 2, PFI0080w; 3, PF10_0395; and 4, PFF0850c.

FIG. 6.

Levels of stevor transcription in trophozoite-stage parasites either before or after selection on trHBMEC. Transcript levels were measured by real-time PCR using primers specific to each of 37 stevor genes and 58 var genes from clone 3D7. The relative abundance of transcripts from each stevor or var gene is presented as a proportion of total transcript abundance for the relevant gene family in the parasite population tested. Numbered sectors of the pie charts correspond to the following genes: 1, PFD0625c; 8, PF11_0008; 9, PF13_0003; 10, PFD1235w (var genes); 2, PFI0080w; 3, PF10_0395; 4, PFF0850c; and 5, PF11_0013 (stevor genes).