Identification of a conserved <i>var </i>gene in different <i>Plasmodium falciparum</i> strains

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 (He = 0) but in other parts as variable (He = 0.86) as the 57 microsatellites markers (He = 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

In vitro variant surface antigen expression in Plasmodium falciparum parasites from a semi-immune individual is not correlated with var gene transcription

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is considered to be the main variant surface antigen (VSA) of Plasmodium falciparum and is mainly localized on electron-dense knobs in the membrane of the infected erythrocyte. Switches in PfEMP1 expression provide the basis for antigenic variation and are thought to be critical for parasite persistence during chronic infections. Recently, strain transcending anti-PfEMP1 immunity has been shown to develop early in life, challenging the role of PfEMP1 in antigenic variation during chronic infections. In this work we investigate how P. falciparum achieves persistence during a chronic asymptomatic infection. The infected individual (MOA) was parasitemic for 42 days and multilocus vargene genotyping showed persistence of the same parasite population throughout the infection. Parasites from the beginning of the infection were adapted to tissue culture and cloned by limiting dilution. Flow cytometry using convalescent serum detected a variable surface recognition signal on isogenic clonal parasites. Quantitative real-time PCR with a field isolate specific vargene primer set showed that the surface recognition signal was not correlated with transcription of individual vargenes. Strain transcending anti-PfEMP1 immunity of the convalescent serum was demonstrated with CD36 selected and PfEMP1 knock-down NF54 clones. In contrast, knock-down of PfEMP1 did not have an effect on the antibody recognition signal in MOA clones. Trypsinisation of the membrane surface proteins abolished the surface recognition signal and immune electron microscopy revealed that antibodies from the convalescent serum bound to membrane areas without knobs and with knobs. Together the data indicate that PfEMP1 is not the main variable surface antigen during a chronic infection and suggest a role for trypsin sensitive non-PfEMP1 VSAs for parasite persistence in chronic infections

No correlation of FACS and <i>var</i> signals in MOA bulk and clones at >65 generations.

<p>(A) The MOA bulk culture exhibits low <i>var</i> gene transcription signals, but a high MFI (201). (B) Clone D2 exhibits the highest MFI (489) in the entire population and high <i>var</i> gene transcription signals for transcripts P_D2 and D2_18. (C) and (D) Clones D5 and C3 display low <i>var</i> gene transcription signals but show MFIs in the medium range (MFI of 63.67 and 139.67 respectively).</p

Flow cytometry signals with day 70 serum on MOA bulk and MOA clones are variable.

<p>(A) Mean fluorescence intensities (MFI) obtained with MOA serum of day 70 followed by staining with a secondary antibody attached to FITC shows variable surface recognition signals ranging from low (< 80 MFI) to medium (81–160 MFI) and high (> 160 MFI) in MOA bulk and in the MOA clones. Error bars reflect the mean error of at least three independent experiments (Student´s t-test: MOA bulk vs. D5: p = 0.008, A1 vs. H6: p = 0.0003, D2 vs. D5: p = 0.0001, D2 vs. H6: p = 0.008, D5 vs. A1: p = 0.0002). (B) and (C) show transmission and scanning electron microscopy graphs of two representative clones with a highly significant difference in surface recognition signal (MOA D2 and MOA D5) and the presence of knobs (arrows) in equal numbers. Knobs were quantified per TEM cut of each clone.</p

MOA <i>in vivo</i> DBL transcripts and corresponding <i>in vivo</i> DNA DBLs.

<p>MOA <i>in vivo</i> DBL transcripts and corresponding <i>in vivo</i> DNA DBLs.</p

No correlation of FACS signal and switching or transcription strength at 35 generations after cloning.

<p>(A) Clone J 1 transcribes the <i>in vivo</i> transcript d0_37 and additionally DBL D2_18 but exhibits a low surface signal (MFI of 71). (B) and (C) Transcription of d0_37 and DBL D7_33 in Clone H4 is associated with a high surface signal (MFI of 223.67), but exclusive transcription of DBL D7_33 in clone B10 has a medium surface signal (MFI 109). (D) The exclusive transcription of D2_69 in clone E10 (at the highest individual transcription signal of all clones) is associated with low surface signal (MFI of 55.33). (E) Clone C4 Transcription of D5_101 and C3_36 at close to identical copy numbers. The clone has a medium MFI of 84.</p

FACS signal with MOA day 70 serum of the laboratory strain E5 increases after CD36 selection.

<p>(A) <i>var</i> gene transcription profile of the laboratory clone E5 prior to CD36 receptor binding selection. Transcription was quantified with an E5-specific primer set (x-axis) (n = 3, standard errors are given). (B) E5 <i>var</i> gene transcription after 3 consecutive rounds of selection for binding to the CD36+ receptor. (C) Surface antigen recognition measured by flow cytometry with serum of MOA d70 shows a stronger signal for the CD36-selected E5 lab strain (standard errors given, n = 3).</p

No correlation of <i>var</i> gene transcription and flow cytometry signals in clonal parasite populations.

<p>(A) Left panel: <i>var</i> gene transcription profile of the clone B5 transcribing the MOA <i>in vivo</i> transcript d0_37 with the highest transcription signal. The transcription signal is quantified as relative copy number (RCN) of the housekeeping gene arginyl-tRNA synthetase (PFL0900 c) (n = 3, standard errors are given) on the y-axis. The 36 primer pairs are depicted on the x-axis. The 6 primer pairs quantifying <i>in vivo</i> transcripts are underlined. Right panel: The corresponding dot plot after incubation with MOA serum of day 70. DNA signals by staining with Hoechst-33342 are depicted on the x-axis ("Pacific Blue-A"), the antibody recognition signal is depicted at the y-axis ("FITC-A"). Uninfected red blood cells are shown in area Q3, infected erythrocytes with both strong (upper cloud) and weak (lower cloud) signals accumulate in Q4. (B) and (C) <i>var</i> gene transcription profiles of clones MOA E8 and MOA G3 transcribing d0_37 at lower transcription signals but exhibiting higher surface signals than clone B5. (D) Heat map correlating <i>var</i> gene transcription and flow cytometry signal in 19 MOA clones and the MOA bulk culture. The FACS signal is quantified by the left bar with a colour code ranging from black (high: MFI > 160) to dark grey (medium: MFI 81–159) to light grey (weak: MFI < 80). All MOA clones and MOA bulk are sorted according to their corresponding flow cytometry signal and depicted on the y-axis. The MOA specific primer set for 36 <i>var</i> loci is listed on the x-axis. <i>var</i> gene transcription is colour coded as indicated by the bar on the right. <i>var</i> genes marked in red are those with the highest transcription signal and <i>var</i> genes in white are those with very low (<3%) or no transcription. Note that clones transcribing d0_37 and T0_36 are evenly distributed from high to low flow cytometry signals.</p

The MOA D2 surface recognition signal is trypsin sensitive.

<p>(A) Surface recognition signal with day 70 sera before and after trypsinisation of CD36-selected ΔE5E2 (left panel) and of MOA D2 (right panel). Both ΔE5E2 and MOA D2 were incubated with MOA serum of day 70 and labelled with a secondary FITC antibody for detection in flow cytometry. Trypsinisation resulted in a significant decrease of the antibody recognition signal (standard errors are given, p = 0.05 for ΔE5E2 (n = 4) and p = 0.003 (n = 3) for D2) in both cell lines. (B) Immuno-TEM after MOA day 70 serum labelling on CD36 selected ΔE5E2 parasites and MOA D2 parasites. A secondary, 12 nm gold-labelled anti-human IgG antibody was added. In ΔE5E2 and MOA D2, gold particles (marked with arrows) could be detected on membrane areas with knobs as well as on the membrane areas without knobs.</p