Clag9 is not essential for PfEMP1 surface expression in non-cytoadherent Plasmodium falciparum parasites with a chromosome 9 deletion

Abstract

Background: The expression of the clonally variant virulence factor PfEMP1 mediates the sequestration of Plasmodium falciparum infected erythrocytes in the host vasculature and contributes to chronic infection. Non-cytoadherent parasites with a chromosome 9 deletion lack clag9, a gene linked to cytoadhesion in previous studies. Here we present new clag9 data that challenge this view and show that surface the non-cytoadherence phenotype is linked to the expression of a non-functional PfEMP1.

Methodology/principal findings: Loss of adhesion in P. falciparum D10, a parasite line with a large chromosome 9 deletion, was investigated. Surface iodination analysis of non-cytoadherent D10 parasites and COS-7 surface expression of the CD36-binding PfEMP1 CIDR1α domain were performed and showed that these parasites express an unusual trypsin-resistant, non-functional PfEMP1 at the erythrocyte surface. However, the CIDR1α domain of this var gene expressed in COS-7 cells showed strong binding to CD36. Atomic Force Microscopy showed a slightly modified D10 knob morphology compared to adherent parasites. Trafficking of PfEMP1 and KAHRP remained functional in D10. We link the non-cytoadherence phenotype to a chromosome 9 breakage and healing event resulting in the loss of 25 subtelomeric genes including clag9. In contrast to previous studies, knockout of the clag9 gene from 3D7 did not interfere with parasite adhesion to CD36.

Conclusions/significance: Our data show the surface expression of non-functional PfEMP1 in D10 strongly indicating that genes other than clag9 deleted from chromosome 9 are involved in this virulence process possibly via post-translational modifications.

Figure 1. Binding assay on C32 melanoma cells.

A. P. falciparum D10 mature stages are unable to bind to expressed receptors of C32 melanoma cells (CD36≫ICAM-1≫CSA) compared to the positive control FCR3^CD36. B. The graph illustrates the number of bound PRBC per 100 C32 cells for FCR3^CD36 (black) and D10 (white). Results are the means ± standard deviation from five experiments. C. Microscopic images of Giemsa stained FCR3^CD36 and D10 on purified receptors. D. Quantification of binding assays for FCR3^CD36 (black) and D10 (white) on purified receptors spotted on plastic Petri dishes. Results are the means ± standard deviation from five experiments.

Figure 2. Analysis of P. falciparum D10.

A. Northern blot. Total RNA (10 µg) from P. falciparum D10 intra-erythrocytic stages hybridized with the D10 DBL1αdomain of PfEMP1 probe (lanes 1–2), the expected 8 kb transcript was detected. The same membrane was hybridized with the ATS probe to control for loading (lanes 3–4). R: Ring stages (6–12 h after invasion), S: Schizonts (34–36 h after invasion). Size markers are in kb. B–D. Effect of trypsin on PfEMP1 of FCR3^CD36 and D10 strains. B. Surface iodination. SDS-PAGE analysis of ¹²⁵I-labelled FCR3^CD36 (lanes 1–3) and D10 (lanes 4–6). C. Immunoprecipitation of FCR3^CD36 (lanes 1–3) and D10 (lanes 4–6) TX100_ins-SDS_sol trypsin-treated extracts with a pool of human immune sera. Arrowheads indicate the PfEMP1 antigen. Molecular weight markers are in kDa. D. Western blot. Mature FCR3^CD36 and D10 parasites were treated with 0 µg/ml (lanes 1 and 3), 10 µg/ml (lanes 2 and 4), or 100 µg/ml trypsin (lanes 3 and 6). TX100_ins-SDS_sol extracts were probed with goat anti-ATS and alkaline phosphatase-conjugated anti-goat and revealed with NBT/BCIP. The ATS antigen is shown by an arrow and an arrowhead indicates full- length PfEMP1. The same membrane was reacted with Hsp70 antibodies to control for loading. E. Surface immunofluorescence of FCR3^CD36 and D10. Plasmion-selected parasites were treated with trypsin prior to incubation with pooled human immune sera (PIAG) followed by goat anti-human IgG Alexa Fluor 488. Scale bars: 5 µm.

Figure 3. Localization of parasite exported proteins.

A. Double-labelling of synchronous air-dried D10 (top row) and FCR3^CD36 (bottom row) with guinea pig anti-PfEMP1 antibodies (1∶500) and purified rabbit anti-Pf332 IgG (1∶200) revealed with a mixture of goat anti-guinea pig Alexa Fluor 488 (1∶500) and Alexa Fluor 594-conjugated anti-rabbit IgG (1∶1000). B. Labelling of PfEMP1 (1∶500, green) and knobs on the PRBC surface with monoclonal anti-KAHRP (mAb89, 1∶800, red), followed by anti-guinea pig Alexa Fluor 488 and anti-mouse Alexa Fluor 594. Scale bars: 5 µm.

Figure 4. AFM analysis of erythrocytes infected by P. falciparum FCR3^CD36 and D10.

A. Representative amplitude images of infected red blood cells. Insets show Giemsa staining of the analysed PRBC. B. Height images were used to measure knob homogeneity, number, diameter and height on infected erythrocytes. Knob homogeneity and number were analyzed using mean ± standard deviation (5≤N cells≤15). Knob diameter and height were analyzed using mean ± standard error of the mean (5≤N cells≤15). Scale bars: 1 µm.

Figure 5. Expression and binding of D10^var1 CIDR1α minimal domain.

A. Diagram of D10^var1. The different domains are shown, including the location of the PfEMP1 CIDR1α minimal domain (M2) used for expression by COS-7 cells. The region spanning the intron between exons 1 and 2 of D10^var1 was sequenced from cDNA. B. Beads bound to COS-7 cells expressing M2 on the surface. C. Quantification of binding on COS-7 cells. The CIDR1α minimal domain of the D10^var1 PfEMP1 binds equally well to COS-7 cells as that of the Malayan Camp (MC^varCD36, positive control). In contrast, there is no significant binding of the FCR3^var1CSA M2 domain. pDisplay control represents COS-7 cells transfected with pDisplay not containing the insert.

Figure 6. Generation and characterization of clag9 KO.

A. Subtelomeric chromosome 9 deletion in D10. Schematic representation of the genes missing from the right arm of chromosome 9, as compared to 3D7. The break point open reading frame (BPORF) is present but none of the 25 genes between the BPORF and the telemores, including clag9 (red), were detected by PCR (data not shown). Each block corresponds to a single gene, clag9 and BPORF are larger for emphasis and the size of blocks does not illustrate gene size. The genes shown in yellow have been functionally identified. The identity of the remaining genes has not yet been elucidated. B. Plasmid pCC1 exon 1+8 knockout leading to the disruption of the clag9 gene by allelic exchange with insertion of the hDHFR selection marker. Primers (arrows) used for verification of KO and hDHFR integration, the restriction sites, and Southern blot expected fragment sizes are shown. C. PCR analysis of 3D7 (lanes 1–4), the clag9 E5KO (lanes 5–8), and F4KO (lanes 9–12). Lanes 1, 5, and 9: 5′UTR-F - exon 1-R; lanes 2, 6, and 10: 5′UTR-F- hDHFR 5′ out; lanes 3, 7, and 11: hDHFR 3′ out – exon 8-RC3′; lanes 4, 8, and 12: exon 2-F - exon 6-R. D–F. Southern blot of clag9 knockout. Genomic DNA digested by EcoRI (lanes 1 and 4), SpeI (lanes 2 and 5) or EcoRI+SpeI (lanes 3 and 6) separated on 0.8% agarose gel, transferred onto Hybond N+ and hybridized with different probes. D. Exon 1, present in both 3D7 and the clag9 E5KO. E. Hybridization of Exon 6 only with 3D7. F. Integration of the hDHFR selectable marker in the E5KO.

Figure 7. Immunofluorescence and binding assays of clag9 E5KO.

A. Labelling of CLAG9 in 3D7 and E5KO. Note there is labelling of E5KO rhoptries with mAb7H8/50 but not with anti-CLAG9 specific sera due to gene disruption. Scale bars = 5 µm. B. Trafficking of PfEMP1 and KAHRP by E5KO; labelling was identical to 3D7. Scale bars = 5 µm. C. Chemiluminescent western blot analysis of normal red blood cells (NRBC), E5KO and 3D7 parasite extracts reacted with anti-CLAG9 antibodies. D. Binding of clag9 E5KO on C32 melanoma cells. E. Binding assay of 3D7 and E5KO PRBC on purified CD36 receptor. Error bars indicate the standard error of the mean from 4 experiments.