Diverse expression patterns of subgroups of the rif multigene family during Plasmodium falciparum gametocytogenesis

Abstract

Background: The maturation of Plasmodium falciparum gametocytes in the human host takes several days, during which the parasites need to efficiently evade the host immune system. Like asexual stage parasites, immature gametocytes can sequester at various sites in the human body, and only mature sexual stages are found in the circulation. Although the fundamental mechanisms of gametocyte immune evasion are still largely unknown, candidate molecules that may be involved include variant antigens encoded by multigene families in the P. falciparum genome, such as the PfEMP1, STEVOR and RIFIN proteins. While expression of the former two families in sexual stages has been investigated earlier, we report here RIFIN expression during gametocytogenesis.

Methodology/principal findings: Variants of two previously characterized RIFIN subfamilies (A- and B-type RIFINs) were found to be synthesized in gametocytes. Immunofluorescence experiments showed A-type RIFINs to be accumulated in a crescent-shaped pattern of discrete punctate structures at the infected erythrocyte membrane, while members of the B-type family were associated with the parasite. Transcription analysis demonstrated the existence of diverse transcriptional regulation patterns during sexual differentiation and indicated variant-specific regulation of B-type RIFINs, in contrast to group-specific regulation for A-type RIFINs. Phylogenetic analysis of 5'-upstream regions showed that the rif-gene family falls into five defined clusters, designated rups (rifupstream) A1, A2, AB, B and C. In trophozoites and early gametocytes, rif variants of the rupsA2-type were preferentially expressed.

Conclusions/significance: In this work we demonstrate the expression dynamics of the rif-gene family during sexual differentiation and present indications for subgroup specific regulation patterns. Therefore, our data provide a first foundation and point to new directions for future investigations of the potential role of RIFINs in gametocyte immune evasion.

Figure 1. Immunofluorescence analysis of RIFIN expression in asexual and sexual parasites.

A: Smears of trophozoite and gametocyte IE were analyzed for RIFIN localization by staining with rat anti-A_RIF40 and mouse anti-B_RIFΔNC antisera (green). Nuclei were stained with DAPI (blue). A-type RIFINs were visualized with anti-A_RIF40, while B-type RIFINs were detected with anti-B_RIFΔNC. B: Quantification of the proportion of RIFIN positive cells in trophozoites (T), immature gametocytes at day 6 (iG) and mature gametocytes at day 10 (mG) after induction of gametocytogenesis.

Figure 2. Western Blot Analysis of RIFIN expression during gametocytogenesis.

Total lysates from trophozoite and gametocyte IE at different time points during sexual development (stages shown representatively in the top panels) were analyzed using antisera generated against recombinant RIFIN proteins RIF40, RIFΔNC and RIF44. The antiserum against the gametocyte specific protein Pfg27 served as a stage control. Antibodies against the Maurer's clefts marker protein SBP1 and the soluble parasite protein phosphatase PP5 served as loading controls. Molecular size markers in kDa are indicated on the left.

Figure 3. Immunofluorescence colocalization in stage III gametocytes.

Methanol fixed smears of stage III gametocytes were incubated with antibodies against RIFIN and marker proteins. A: rat anti-A_RIF40 antibodies (green) and rabbit anti-Pfg27, mouse anti-CCP1, mouse anti-STEVOR or mouse anti-SBP1 (red). B: mouse anti-B_RIFΔNC (green) and rabbit anti-Pfg27 (red). Nuclei were stained with DAPI (blue).

Figure 4. RIFIN RNA expression during sexual development.

A: RT-PCR analysis of A- and B-type RIFINs in asexual and sexual stage parasites of the NF54 strain. RNA was harvested from 20–24 hour trophozoites (T) and at days 2, 4, 6, 8 and 10 of gametocytogenesis (D2–D10). RNA was reverse transcribed into cDNA (+) and PCR was performed with specific primers for A-RIFINs and B-RIFINs. RNA samples in which reverse transcriptase was omitted served as negative controls (−). Positive controls were performed on genomic DNA (g). B: RT-PCR analysis with specific primers detecting the A-type rif variant PFD1240w and the B-type rif variant PFI0025c. Stages are as in (A).

Figure 5. Changes in A-type rif gene transcription during sexual differentiation.

A-type rif RT-PCR products from 20–24 hour trophozoites (Troph) as well as day 2, 4, 6, 8 and 10 gametocytes (GD2–GD10) were cloned and single sequences analyzed. Frequencies are shown in percent of all clones. Blue arrows highlight downregulation, red arrows upregulation during sexual development. Three independent experiments are shown in black, light grey and dark grey.

Figure 6. Changes in B-type rif gene transcription during sexual differentiation.

B-type rif RT-PCR products from 20–24 hour trophozoites (Troph) as well as day 2, 4, 6, 8 and 10 gametocytes (GD2–GD10) were cloned and single sequences analyzed. Frequencies are shown in percent of all clones. Black arrows point out variants that are continuously detected, blue arrows highlight downregulation, red arrows upregulation during sexual development. Three independent experiments are shown in black, light grey and dark grey.

Figure 7. Definition of rups groups based on 5′-upstream sequences.

A: Distance tree of rif gene upstream regions (rups) from 3D7. The 5′-sequences containing 1500 bp upstream of each rif gene were aligned with ClustalW. Distance trees were generated using the p-distance/Neighbor-Joining method with pairwise deletion of gaps. The clusters rupsA1, rupsA2, rupsA/B and rupsB were verified by bootstrapping, and bootstrap values are indicated at the branches. The cluster rupsC could not be verified by bootstrapping. Star: RupsA2, rups A/B and rupsB variants with transcription orientation towards centromere, contrary to general transcription orientation. Hash: Upstream regions previously referred to as upsA-rif . B: Chromosomal orientation of variants belonging to each of the rups clusters.

Figure 8. Rups related A-type rif variant expression in asexual and sexual stages.

A: Proportion of A-type variants summarized by rups group in asexual and sexual stages. The mean and standard deviation of three independent experiments is shown. B: The frequencies of variants assigned to the rups groups in each stage were corrected for the frequency of variants of the respective rups group in the genome. A relative abundance value of 1 corresponds to the identical proportion as present in the 3D7 genome. A1: blue, A2: red, AB: yellow, C: green.