Identification of a conserved var gene in different Plasmodium falciparum strains

Abstract

Background: The multicopy var gene family of Plasmodium falciparum is of crucial importance for pathogenesis and antigenic variation. So far only var2csa, the var gene responsible for placental malaria, was found to be highly conserved among all P. falciparum strains. Here, a new conserved 3D7 var gene (PF3D7_0617400) is identified in several field isolates.

Methods: DNA sequencing, transcriptional analysis, Cluster of Differentiation (CD) 36-receptor binding, indirect immunofluorescence with PF3D7_0617400-antibodies and quantification of surface reactivity against semi-immune sera were used to characterize an NF54 clone and a Gabonese field isolate clone (MOA C3) transcribing the gene. A population of 714 whole genome sequenced parasites was analysed to characterize the conservation of the locus in African and Asian isolates. The genetic diversity of two var2csa fragments was compared with the genetic diversity of 57 microsatellites fragments in field isolates.

Results: PFGA01_060022400 was identified in a Gabonese parasite isolate (MOA) from a chronic infection and found to be 99% identical with PF3D7_0617400 of the 3D7 genome strain. Transcriptional analysis and immunofluorescence showed expression of the gene in an NF54 and a MOA clone but CD36 binding assays and surface reactivity to semi-immune sera differed markedly in the two clones. Long-read Pacific bioscience whole genome sequencing showed that PFGA01_060022400 is located in the internal cluster of chromosome 6. The full length PFGA01_060022400 was detected in 36 of 714 P. falciparum isolates and 500 bp fragments were identified in more than 100 isolates. var2csa was in parts highly conserved (H_e = 0) but in other parts as variable (H_e = 0.86) as the 57 microsatellites markers (H_e = 0.8).

Conclusions: Individual var gene sequences exhibit conservation in the global parasite population suggesting that purifying selection may limit overall genetic diversity of some var genes. Notably, field and laboratory isolates expressing the same var gene exhibit markedly different phenotypes.

Keywords: Genetic diversity; Malaria; Microsatellites; PfEMP1; Recombination; VSA; rifin; stevor; var genes.

Fig. 1

Screenshot of ACT program comparing the assembled sequence of PF3D7_0617400 of the field isolate clone MOA C3 with the reference strain 3D7. The upper part of the figure depicts the 3D7 reference sequence. The lower part shows the MOA C3 sequence. Blue and yellow = sequence identical. White: missing sequence (insertion). For orientation, the sequence next to the insertion that differs between MOAC3 and 3D7 is coloured in yellow. The additional small white areas represent homopolymer runs. Visual examination of direct alignments of the homopolymer runs revealed no differences in these areas (data not shown)

Fig. 2

a Gel electrophoresis after PCR with 6 specific PF3D7_0617400 primers (primer pairs 1,2,3,4,5,6 and a 100 bp marker) on MOA C3 cDNA reveals transcription of the entire exon 1 sequence. b qRT-PCR with P_C3 and two other MOA DBL primers (encoding for the dominant var genes in the other clones generated in the same limiting dilution experiment) [19] shows that PF3D7_0617400 is transcribed as the dominant var gene in MOA-C3. c qRT-PCR with NF54 specific primer set on cDNA of NF54 A3 [22] shows continued dominant transcription of PF3D7_0617400. Transcription is quantified in relative copy numbers of the housekeeping gene arginyl tRNA synthetase in MOA C3 and NFA3

Fig. 3

CD36 binding capacity of a NF45 A3 before CD36 selection and after one and two rounds of selection and b MOA C3 before CD36 selection as well as after one to four rounds of selection. c Mean fluorescence intensity (MFI) of FACS analysis with semi-immune sera from the MOA individual [20] on NF54 A3 and MOA C3 after CD36 selection

Fig. 4

Immunofluorescence with a specific antibody against the CIDRa2.1 of PF3D7_0617400 shows expression in MOA C3 and NF54 A3 but not in the field isolate Δ MOA D2: a MOA C3, b NF54 A3, c Δ MOA D2 (propagated without blasticidin pressure). The first images displays light microscopy, the second image identifies parasitized RBCs by Hoechst DNA staining (blue) and the third image displays IFA with mouse anti- CIDRa2.1-PF3D7_0617400 antibody and GFP. Note the anti- CIDRa2.1 antibodies signal in MOA C3 and NF54 A3. The D2 DBL expressed in Δ MOA D2 [20] is not detected by the antibody

Fig. 5

PF3D7_0617400 and var2csa in field isolates. a Gel electrophoresis of PCR fragments of PF3D7_0617400 on all field isolates and controls. PCR fragments of PF3D7_0617400 amplified (primer pair P0F + P0.1R) on two field isolated (5798, MOA C3) and on the NF54 clones 3D7 and E5 (positive controls). NC = negative control. The gel was digitally rearranged to match the gel shown in b. b Gel electrophoresis of PCR fragments of var2csa on all field isolates and controls. PCR fragments of var2csa amplified by one primer pair (10F and 75R, [5]) on all field isolates and on the NF54 clones 3D7 and E5 (positive controls). NC = negative control

Fig. 6

a ACT program comparative analysis of the central cluster of chromosome 6 in the 3D7 genome strain, MOA C3 (PFGA01) (middle part of figure) and 5798 (PFTG01) (lower part of figure), note that the stevor and rifin genes located downstream of PF3D7_0617400 are also conserved in the 3 strains. b Amino acid alignment depicting the amino acid sequence difference between 3D7 and the two field isolates

Fig. 7

Exon 1 conservation of PF3D7_0617400, PFGA01_060022400, PFDd2_070015900 and PFD3D7_0412400 in a global population of parasites. The amount of sharing of 4 var genes among a population of 714 parasites from Africa and Asia [15]. Blue parts correspond to fragments > 2000 bp, red parts correspond to fragments > 500 bp. The y axis shows the number of isolates carrying the respective fragments. Note that the second part of exon I is conserved to a higher degree than the first part. The small insertion that marks the difference between PF3D7_0617400 and PFGA01_060022400 can be appreciated. Note that PFDd2_070015900 is conserved along the entire exon1, consistent with a location within the pfcrt genetic sweep. PFD3D7_0412400 is shown as an example of a nonconserved UpsC 3D7 var gene. Exon 2 sequences were excluded from the analysis

Fig. 8

Microsatellite position and genetic diversity a 14 chromosomes of P. falciparum with positions of var genes indicated in red and positions of microsatellites indicated in green. b Observed H_e of microsatellite loci on all 14 chromosomes of P. falciparum across all field isolates. 0=no diversity, 1= max. diversity

Fig. 9

Scatterplot showing the H_e of the 57 microsatellites versus the two H_e of fragment (I) and (II) of var2csa. Note that the H_e of the fragment I of var2csa is 0 and thus highly conserved. In contrast the H_e of fragment II is 0,8 and thus in the same range as the H_e of the MS markers