Surface Co-Expression of Two Different PfEMP1 Antigens on Single -Infected Erythrocytes Facilitates Binding to ICAM1 and PECAM1

When co-expressed on single IE, the PFD1253w and PF11_0008 PfEMP1 antigens mediate binding to two different endothelial receptors

Having established simultaneous expression of two species of PfEMP1 on the IE surface, we then analysed whether this dual expression affects the binding phenotype of the infected erythrocytes (Figure 6). 3D7_PFD1235w infected erythrocytes showed specific binding to CD54/ICAM1 transfected CHO cells (Figure 6A) and no binding to CHO-CD36 cells (Figure 6B) or wild type CHO cells (Figure 6C). The observed binding was specifically inhibited by anti-CD54/ICAM1 (15.2 and My13) antibodies (Figure 6A) which have been shown to inhibit binding of PfEMP1 to ICAM1/CD54 [44].

Erythrocytes infected with 3D7_{PF11_0008} did not bind CD54/ICAM1 (Figure 6A). The 3D7_{PFD1235w/PF11_0008} sub-line, which expressed both PFD1235w and PF11_0008, bound as strongly to CD54/ICAM1 as the PFD1235w infected cells which express only this ICAM1–binding antigen (Figure 6A). The 3D7_{PF11_0008} sub-line bound specifically to the un-stimulated HUVEC cells which constitutively express PECAM1/CD31 and von Willebrand factor. This binding was markedly reduced in the presence of antibodies to CD31/PECAM1 (Figure 6D), indicating that CD31/PECAM1 was important for PfEMP1-mediated binding in this assay. Cells expressing PFD1235w also showed very slight binding to un-stimulated HUVEC cells, probably explicable by a low level expression of CD54/ICAM1 on these cells as this binding was slightly reduced by CD54/ICAM1 antibodies (Figure 6D).

However cells expressing both PFD1235w and PF11_0008 bound strongly to the HUVEC cells, with binding levels 2–3 times higher than the binding of the cells expressing PF11_0008 only. This binding could only be partially inhibited by anti-CD31/PECAM1 and CD54/ICAM1 antibodies. This result indicates that cells co-expressing PFD1235w and PF11_0008 can bind both CD54/ICAM1 and CD31/PECAM1, whereas IE having only one of the two PfEMP1 species on the surface bind to one or another of the two receptors, but not both. Interestingly the binding of the 3D7_{PFD1235w/PF11_0008} sub-line to HUVEC cells (Figure 6D) was stronger than the binding of the 3D7_{PF11_0008} sub-line. This could reflect an additive effect of PfEMP1 co-expression.

Discussion

The complete P. falciparum 3D7 genome sequence and real-time quantitative RT-PCR enable accurate measurement of the relative amounts of var gene transcripts in intra-erythrocytic malaria parasite populations. To further analyse the relationship between var gene transcripts and the individual infected erythrocyte's PfEMP1 antigen surface expression and cytoadhesion phenotype, we have combined mRNA quantification and FISH analysis with FACS and single-cell live confocal microscopy using PfEMP1 antisera of demonstrated specificity in addition to in vitro adhesion assays, in antibody-selected, but otherwise genetically unmodified parasites. The primary intention was to test the ‘mutually exclusive expression’ hypothesis for the P. falciparum PfEMP1 mediated antigenic variation system.

A handful of studies have investigated this relationship between var transcripts and its eventual outcome in terms of surface PfEMP1 expression and adhesion. These have used parasites selected for rosetting [16], [45], adhesion to ICAM1 or CSA [15], [17], [19] or selected using the A4varICAM1 monoclonal antibody Bc6 [15], [46]. Data supporting exclusive antigen expression on IE has been obtained using ‘pan-reactive’ antibodies targeting the conserved Acidic Terminal Sequence of PfEMP1, which detected single PfEMP1-sized bands in immuno-precipitations and Western blots [16]. However, similar antibodies have also shown protein expression of several differently sized PfEMP1 bands in other experiments with adhesion-selected clonal populations [19] and immune sera-selected cultures are also known to express several species of PfEMP1 [39]. Additionally, a more recent study using a transfected A4 parasite line show surface co-expression of miniPfEMP1 protein with endogenous PfEMP1 [47].

Direct demonstration of PfEMP1 using gel electrophoretic methods is technically difficult due to the low amounts of PfEMP1 on parasitized erythrocytes and the fact that Western blot-based typing is rarely possible because typing sera usually recognise conformational epitopes and not denatured blotted PfEMP1. However, flow cytometry and confocal microscopy can detect native PfEMP1 conformations on live cells, at both cell and population level. We used this to analyse PfEMP1 surface expression on antibody-selected IE populations known to predominantly, but not exclusively, transcribe the PFD1235w and PF11_0008 var genes.

The ring-stages of antibody selected 3D7-derived lines showed polygenic var transcription with several abundant var mRNAs (Figure 4A1–C1, Figure 4A2, and Figure 4A3). Dynabeads sorted (Figure 4B2–D2), and FACS sorted lines (Figure 4B3) also showed polygenic var gene transcription. The real-time Q-PCR data are also supported by RNA-FISH and Northern blot analysis showing co-transcription of full-length PFD1235w and PF11_0008 var genes associated with single-cell nuclei of 3D7_{PFD1235w/PF11_0008} (Figure 5).

By contrast, the NF54_VAR2CSA sub-line had a single dominant gene, var2csa, whose transcript was 98% of total var mRNA (Figure 4D1). The dominant, exclusive var2csa transcription of repeatedly CSA selected parasites [41], [42], [48] is the best demonstration of ‘mutually exclusive’ var transcription. However, it is also to some extent a special case. The var2csa gene has its own promoter, upsE [10], and an unique upstream open reading frame involved in the control of var2csa expression [49] and thus its regulation has certain non-standard features [42]. Strictly monoallelic or unilocus expression of the var2csa gene has however, also been questioned by recent publications showing transcription of duplicated var2csa genes [50], which appears to be simultaneous in individual parasites [51]. However dual expression of both VAR2CSA variants on the erythrocyte surface has not been shown.

We observe neither monoallelic mRNA transcription, nor monomorphic antigen expression at the erythrocyte surface in individual 3D7_{PFD1235w/PF11_0008} IE. These cells clearly reacted with more than one specific anti-PfEMP1 antisera. Other selected sub-lines reacted exclusively with either antisera (Figure 1, Figure 2, Figure S1 and Figure S5). In our assays these antisera were highly specific for the immunizing antigen (Figure S1, S2, and S4). Our experiments appear to exclude the possibility that these antisera are non-specifically reacting with other surface expressed parasite antigens. We consider that these novel results indicate that, contrary to the current consensus, at least two different PfEMP1 antigens can in some circumstances be expressed on the surface of a single 3D7 IE.

A puzzling phenomenon associated with PfEMP1 surface staining with antigen and domain specific antisera remains. This is the observation that differentially labelled antisera raised against separate domains (Figure 1) or identical domains (unpublished data) of the same PfEMP1 protein consistently show non-colocalizing patterns of punctate surface fluorescence. This could be the result of some PfEMP1 topological phenomena, or more likely an artificial outcome of the experimental setup with cross-linking of primary via secondary species-specific antibodies [52].

Disregarding our flow cytometry and single-cell microscopy data the presence of several abundant full-length var mRNAs in the three antibody selected sub-lines (Figure 4A1–C1, Figure 4A2, Figure 4A3) could be explained by that fact that mRNA was extracted from in vitro cultured cell populations rather than individual cells. To exclude this possibility we did limiting dilution of the 3D7_{PFD1235w/PF11_0008} sub-line to obtain a clonal population of cells. From this experiment nine clones were obtained of which seven double positive clones show a slightly reduced complexity, but still multiplicity of var gene transcripts (compare Figure 4A4–C4 and Figure 4C1). Thus, the multiple var transcripts seen (Figure 4C1, Figure 4A2, and Figure 4A3) can not be explained by our use of a heterogeneous cell population.

At the genetic level, several DNA sequences required for appropriate var gene control have been reported e.g. the pairing of a 5′var promoter with an intronic promoter at the 3′end of the same var gene [24]. We therefore screened for potential confounding defects in our parasite lines and in clone 3. Using cross-intron primers for the abundantly transcribed var genes we found correct splicing of exon I to exon II. In addition, the real time Q-PCR for each abundant var gene transcript indicated similar transcript levels of several full-length var mRNAs in the early stage IE (Figure S6). Sequencing the intronic regions of the PFD1235w and PF11_0008 var genes located on two different chromosomes revealed that these were identical to the sequences available on PlasmoDB. Thus, constitutive transcription by single 3D7_{PFD1235w/PF11_0008} IE due to some defect in their respective intron sequences does not explain our dual surface expression data. In addition as shown by flow cytometry, two of the clones switched surface expression during culturing with a minor population still co-expressing PFD1235w and PF11_0008 and a majority expressing unidentified PfEMP1s (Figure S3). This strongly indicates a shut-down of PFD1235w and PF11_0008 PfEMP1 surface expression and further indicates constitutive transcription does not explain our dual surface staining data.

Whether the simultaneous expression of more than one PfEMP1 occurs in single IE during natural infections is not known as we currently have a better understanding of malaria antigen variation in vitro than in vivo. In natural infections, P. falciparum sequesters in deep tissue prior to the onset of cell division and under physiological blood flow sequestration is mediated by avidity-dependent binding to multiple host-receptors, mimicking the process of leukocyte recruitment [53]–[55]. Antibodies to CD36 have been shown to reduce rolling and adhesion of IE, residual rolling being further inhibited by antibody to ICAM1 [55]. These receptors operate synergistically to mediate strong cytoadherence when coexpressed on endothelial cells [56].

PfEMP1 proteins have multiple domains [8] and most CIDR-α type domains bind CD36 [57], while some DBLβc2 domains bind ICAM1 [58]–[61] and some single PfEMP1 species have been shown to mediate multiple independent interactions with a diverse set of host receptors including CD31/PECAM-1, the blood group A antigen, normal nonimmune IgM, heparan sulfate–like glucosaminoglycan, and CD36 [62].

A study has suggested that the ability of parasites to bind to multiple receptors is correlated with disease severity [63]. In addition, several lines of evidence have implicated CD54/ICAM1 [64]–[67], CD31/PECAM1 [68] as well as PFD1235w and PF11_0008 [39], [69], [70] as having a role in severe disease. Interestingly, we found the PFD1235w and PF11_0008 surface co-expressed on the 3D7_{PFD1235w/PF11_0008} sub-line to mediate binding to CD54/ICAM1 and CD31/PECAM1, respectively (Figure 6). To our knowledge this is the first study to demonstrate dual receptor binding in malaria cytoadherence being mediated by two different PfEMP1 molecules on the surface of single IE. A potential mechanism for the two PfEMP1 interactions could involve a role for PFD1235w as a primary ligand for rolling and CD54/ICAM1 binding on the endothelium thus enabling further contacts with CD31/PECAM1 which could be mediated by PF11_0008, when both are present on the IE surface. Adhesion would then be improved through simultaneous binding to several receptors as indicated by our data in Figure 6. This process may be similar to that of lymphocyte rolling based on carbohydrate-receptor interactions [71].

If more than one PfEMP1 antigen is expressed on individual IE in in vivo infections it may be most advantageous during the immediately post-hepatocytic establishment phase. The first post-hepatocytic generation lacks epigenetic memory of var gene transcription and translation. Relaxed transcription [72] and translation may thus be initially unavoidable. It may also ensure the highest avidity binding interaction possible and rapid sequestration. Transcription of those var genes expressed by successfully sequestering survivors of the post-hepatocytic wave of infection would epigenetically mark these var genes for expression in their descendent population, whilst leaving their unexpressed and thus unmarked var genes to become silenced. Such an ‘early loose-tight late’ model for var gene transcription in blood stage infection is compatible with experiments demonstrating that transcription of a particular var gene promoter leads to the silencing of the other var genes in the repertoire [73].

Our var gene transcript level, PfEMP1 surface expression, and cytoadhesion data may be indicating that the primary role of the P. falciparum var genes is the sequestration reaction and that at least in the earliest phases of blood stage infection, escape of IE from the circulation to a sequestration site which ensures successful replication takes precedence over shielding the antigenic variation repertoire from immune surveillance. The tiny amounts of PfEMP1 present at very low parasitaemia are unlikely to be sufficient to promote protective seroconversion. As infections develop and variant-specific parasitaemia rises, the repertoire become protected by epigenetic silencing until finally a novel cytoadhesion phenotype expressing a new PfEMP1 variant outgrows the increasingly immunocompromised founder population and the process starts over.

In summary, we have shown protein expression of two different var genes on the membrane of single erythrocytes infected with P. falciparum 3D7 to facilitate cytoadhesion of the infected cells to two different human receptors. These observations contradicts the hypothesis of mutually exclusive PfEMP1 expression and at the same time offers an additional molecular explanation for how individual IE can mediate adhesion to multiple host receptors.

Materials and Methods

Malaria parasites and in vitro selection procedure

The P. falciparum isolate NF54 and the NF54 derived clone 3D7 were cultured in blood group 0 erythrocytes as previously described [74]. Cultures were routinely genotyped by PCR using primers targeting the polymorphic loci MSP2 and Glurp as described [75] and mycoplasma tested using the MycoAlert Mycoplasma Detection Kit (Lonza) following the manufactures instructions.

IgG from rabbits immunized with DBL4γ or CIDR1α of 3D7 PFD1235w and CIDR2β of 3D7 PF11_0008 were used to obtain three different sub-lines of 3D7 parasites. 3D7_PFD1235w, originating from a previously selected line referred to as 3D7Dodowa1 [31] was obtained using sera targeting a PFD1235w-DBL4γ recombinant antigen. 3D7_{PF11_0008} originating from 3D7 was selected using sera targeting the CIDR2β of PF11_0008 and 3D7_{PFD1235w/PF11_0008} similarly originating from 3D7 was obtained using sera targeting a CIDR1α domain recombinant protein of PFD1235w. NF54_VAR2CSA kindly provided by Morten A. Nielsen was selected using rabbit antisera raised against a DBL5ε-6ε recombinant protein based on the var2csa gene. Briefly, the different rabbit sera were depleted on human uninfected erythrocytes type 0, incubated with gelatine purified trophozoite-stage 3D7/NF54 parasites for 30 min at 37°C, and unbound antibodies were removed by washing. Subsequently, IE were incubated with Protein A-coupled Dynabeads (Invitrogen) for 30 min at 37°C and bound IE were trapped using a magnet. Trapped IE were transferred to new culture flasks for continued in vitro culturing and the procedure was repeated until cultures stained positive by the selecting antisera in flow cytometry.

Dynabeads sorting of parasites

Gelatine purified 3D7_{PFD1235w/PF11_0008} IE was incubated for 30 min at 37°C with rabbit sera targeting (PFD1235w-DBL4γ, PFD1235w-CIDR1α, and PF11_0008-CIDR2β) and incubated with Protein A-coupled Dynabeads. Bound IE were trapped as described above, grown for less than one cycle to rings, following which RNA was purified, reverse transcribed and used for real-time quantitative PCR.

Flow cytometry sorting of parasites

MACS purified 3D7_{PFD1235w/PF11_0008} IEs were double stained with rat PFD1235w-DBL4γ and rabbit PF11_0008-CIDR2β antisera as described below and analysed on a FACSAria (Becton Dickinson). A total of 1×10⁶ double positive IE were collected and grown for less than one cycle to rings, following which RNA was purified, reverse transcribed and used for real-time quantitative PCR.

Limiting dilution of parasites

Late stage 3D7_{PFD1235w/PF11_0008} IE was obtained by MACS purification and cloned using a slightly modified version of the protocol described by Walliker and Beale [76]. In brief, 0.25 or 0.5 IE in a total volume of 100 µl RPMI 1640 (Lonza) with 1% hematocrit, 5 mg/ml Albumax II (Life Technologies, 0.18 mg/ml glutamine (Sigma-Aldrich), 0.05 mg/ml gentamicin (Gibco) and 10% Normal Human Serum were seeded into each well of flat bottomed 96-wells plates (NUNC). New media was added every second day and additional 1% hematocrit was added on day 5. A total of 64 wells were checked for the presence of IE by Giemsa staining on day 12 and16 yielding nine different clones. RNA was purified from all nine clones for real-time quantitative PCR analysis and the PFD1235w and PF11_0008 surface expression was similarly analysed by flow cytometry. One clone, clone 3 was selected for FISH and confocal microscopy analysis.

Protein expression

The DNA sequence encoding amino acid # 73-739 (DBL1α-CIDR1α: … CELDYRF…DTKTNPC), amino acid #473-817 (CIDR1α*: DYCQICP:… NGEPCTG), amino acid # 407-798 (CIDR1α: KDAKTDS…TLNGDIC), amino acid # 1239-1689 (DBL3β: CAETGGV… YATACDC), amino acid #1719-2255 (DBL4γ: PRDKTTG…LKGDKSL), amino acid #2258-2764 (DBL5δ: ACALKYG… SAKQKDC), and amino acid # 2242-3016 (DBL5δ-CIDR2β: CATVAKA…VTQPNIC) of 3D7 PFD1235w, amino acid #1553-1924 (CIDR2β: KKQEKLY…NVPANPC) and amino acid #1994-2378 (DBL4β: CNITKEH… HDDACAC) of 3D7 PF11_0008 and the DNA amplified by primers listed in Table S1 was cloned and expressed in a baculovirus system as described [39], [77]–[79]. DBL1x VAR2CSA protein was kindly provided by Ali Salanti and Madeleine Dahlbäck.

Generation of antisera

All procedures complied with European or national regulations. Prior to immunization each animal was pre-bled and these sera were used as negative controls in the flow cytometry and confocal microscopy experiments. VAR2CSA DBL5ε-DBL6ε rat antisera, rabbit αDBL5ε-DBL6ε and mouse αDBL5ε antisera [80] were kindly provided by Ali Salanti and Madeleine Dahlbäck. PFD1235w-CIDR1α,PFD1235w-DBL4γ, and PF11_0008-CIDR2β rabbit, and PFD1235w-DBL1α-CIDR1α, PFD1235w-CIDR1α^*, PFD1235w-DBL3β, PFD1235w-DBL4γ, PFD1235w-DBL5δ, PFD1235w-DBL5δ-CIDR2β, and PF11_0008-DBL4β were raised by subcutaneous injection of 10–20 µg protein in complete Freund's adjuvant followed by several boosters of protein in incomplete Freund's adjuvant.

Ethics statement

All experiments including immunizations and bleeding of animals was approved by The Danish Animal Procedures Committee (“Dyreforsoegstilsynet”) as described in permit no. 2008/561-1498 and according to the guidelines described in act no. LBK 1306 (23/11/2007) and BEK 1273 (12/12/2005).

Affinity purification of antibodies

0.5 mg of PFD1235w-DBL4γ and PF11_0008-CIDR2β in each 1 ml were dialysed ON against coupling buffer and subsequently coupled to HiTrap NHS activated HP columns as described by the manufacturer (GE Healthcare). A pool of 3 ml PFD1235w-DBL4γ antisera from six rats and 6 ml PF11_0008-CIDR2β antisera from one rabbit were diluted 1∶1 in PBS and affinity purified on the HiTrap columns. Following elution in Glycin buffer (0.1 M, pH 2.8) antibodies were neutralised in Hepes buffer (1M, pH 8.0).

Depletion and blocking of antibodies

The affinity purified rabbit PFD1235w-DBL4γ and PF11_0008-CIDR2β antibodies were depleted for V5-His reactivity using a non-sense peptide (VLIM-tag) VLIMFNEQHKRGASTYWCPGKPIPNPLLGLD STRTGHHHHHH (Schafer-N) containing the V5-His epitope (underlined) and recombinant V5-HIS tagged VAR2CSA-DBL1x. The VLIM-tag peptide and VAR2CSA-DBL1x (both 1 mg/ml in PBS) were coupled to Epoxy M270 Dynabeads according to the manufacturer's instructions (Invitrogen). 200 µl of each of the two different species of affinity purified antibodies was incubated for 2 h at RT with 3 mg of coupled Dynabeads. Following this the supernatant containing the depleted antibody fraction was removed and re-incubated twice with freshly coupled Dynabeads. Antibodies bound to the Dynabeads were eluted in Glycin/HCl (0.1 M, pH 2.75) and neutralised in Tris buffer (1 M, pH 9.0). The reactivity of depleted and non-depleted antibody fractions was tested by ELISA (as described below) using PFD1235w-DBL4γ, PF11_0008-CIDR2β, VAR2CSA-DBL1x, and the VLIM-tag peptide as coating antigen. Additionally, the affinity purified and depleted PFD1235w-DBL4γ and PF11_0008-CIDR2β antibodies used for flow cytometry and confocal microscopy were tested by Luminex (as described below) on 49 different PfEMP1 recombinant proteins. Similarly rabbit antisera against PFD1235w-CIDR1α was depleted on recombinant V5-HIS-tagged VAR2CSA-DBL1x and tested by ELISA using PFD1235w-CIDR1α, PF08_0103-CIDR1α, PF11_0008-CIDR2β, VAR2CSA-DBL1x, and the VLIM-tag peptide as coating antigen.

Rat PFD1235w-DBL4γ, rabbit PFD1235w-CIDR1α or PF11_0008-CIDR2β antisera binding was blocked by incubation for 1 hr at 4°C using excess purified recombinant PFD1235w-DBL4γ, PFD1235w-CIDR1α, PF11_0008-CIDR2β, or PF08_0103-CIDR1α protein (3 µg/well containing 5 µl rat or 10 µl rabbit sera) and remaining cross-reactivity to parasite antigens expressed on the surface of infected erythrocytes was subsequently tested by flow cytometry as described below.

Flow cytometry

Parasite cultures were repeatedly antibody selected as described above and grown for 3–5 cycles prior to doing flow cytometry. Single colour flow cytometry surface staining was done with minor modifications as described [74]. In brief, IE were purified on a magnet-activated cell sorting column (MACS) and 2×10⁵ ethidium bromide-labelled IE were incubated for 30 min at 4°C in 5 µl rat, 10 µl rabbit sera, or 15 µl affinity purified and tag-depleted antibody depleted of anti-human erythrocyte antibodies and then incubated for 30 min at 4°C with FITC-conjugated goat-anti-rat IgG (1∶150, Zymed) or FITC-conjugated goat-anti-rabbit IgG (1∶200, Vector Laboratories). Prior to doing two colour flow cytometry surface staining the purity of the MACS-purified IE was verified by running a small sample of ethidium bromide-labelled IE on a flow cytometer. Sub-line MACS preparations with ≥90% IE were used for two colour flow cytometry. In the case of clone 3 MACS preparations with 75–80% IE were used. In brief, 2×10⁵ MACS-purified unlabelled IE were incubated with 5 µl rat and 10 µl rabbit sera depleted of anti-human erythrocyte antibodies, followed by incubation with FITC-conjugated goat-anti-rat IgG (1∶150, Zymed) and Alexa Fluor 610-R-PE-conjugated goat-anti-rabbit IgG (1∶200, Molecular Probes). Samples were analysed on a Cytomics FC 500 MPL flow cytometer (Beckman Coulter) and data analysed using WinList version 6.0 (Verity Software House Inc.). IE stained with one surface colour was gated based on the ethidium bromide staining to exclude uninfected erythrocytes. Two colour stained non-ethidium bromide-labelled IE were gated based on forward and side scatter values since ethidium bromide could not be used due to overlapping spectra with the Alexa Fluor 610-R-PE-conjugated anti-rabbit used. For removal of surface PfEMP1 and analysis of antisera cross-reactivity with trypsin resistant surface IE antigens expressed on late-stage trophozoites and schizonts IE were treated with 1 g/l porcine trypsin and 0.2 g/l EDTA solution in Hanks balanced salt solution (Sigma-Aldrich) for 10 min at 37°C. The reaction was stopped by adding 10% foetal calf serum (FCS) and cells washed three times in PBS plus 2% FCS. Controls were incubated similarly in PBS plus 2% FCS or in a trypsin/EDTA solution containing 10% FCS.

Confocal microscopy

Laser scanning confocal microscopy was performed on all samples analysed by flow cytometry in order to observe the staining patterns on individual IE. MACS purified unlabelled infected cells from the same batches of parasites tested by flow cytometry were incubated with individual antibodies or in combination with the PFD1235w, PF11_0008, and VAR2CSA antibodies as previously described [81]. Briefly, 1 µl packed IE were washed in 1% BSA in PBS (BSA/PBS) and the pellet was incubated in 100 µl BSA/PBS and 3 µl of the respective antibodies for 30 minutes at 4°C. The IE were washed three times in BSA/PBS. The IE stained with primary antibody were then incubated with secondary antibodies, either Alexa 488 anti-rat IgG (Invitrogen), Alexa 568 anti-rabbit and/or 568 Alexa anti-mouse IgG (Invitrogen) and DAPI (3 µl of a 3 µg/ml solution) for 30 minutes at 4°C. The IE were washed three times and visualised as live, unfixed cells using a Nikon TE 2000-E confocal Nikon microscope with 60x oil immersion objective lens (DIC). The images were processed using Adobe Photoshop software and displayed with the 5 µm scale bar calculated by the EZ-C1 software.

ELISA and luminex

Affinity purified and depleted rat and rabbit antibodies were tested by ELISA. Wells of Maxisorp plates (Nunc, Roskilde, Denmark) were coated with 1 µg/ml of testing antigen in Glycin/HCl buffer (0.1 M, pH 2.75), antibodies were diluted 1∶100 in blocking buffer (PBS, 0.5 M NaCl, 1% Triton-X-100, 1% BSA, pH 7.2), and plates were washed and developed as described previously [77]. In addition, the affinity purified and depleted antibodies were tested in the BioPlex¹⁰⁰ System (BioRad) as previously described [40]. Briefly, 0.1 mg Baculovirus produced proteins and the VLIM-tag peptide were individually coupled to 1.25•10⁷ Luminex xMAP technology microsphere beads (Ramcon). Plex 1 and 2 contained 6 and 45 different proteins, respectively. Three protein domains were coupled twice in plex 2 and two were identical to domains in plex 1 (Table S1). Prior to multiplexing, protein coupling was verified by incubating beads (1∶333) with mouse anti-V5 antibody (1∶10,000, Invitrogen) followed by biotinylated anti-mouse IgG (1∶500, DakoCytomation). The biotinylated antibody was detected using PE-streptavidin (1∶500, Sigma) and diluents used were PBS/TB (PBS, 0.05% (v/v) Tween-20, 0.1% (w/v) BSA, pH 7.4). Multiplexed beads (1∶333) were incubated with rat or rabbit antibodies (1∶1000) followed by incubation with biotinylated anti-rat IgG (Sigma) or anti-rabbit IgG antibody (The Binding Site) diluted 1∶1000. Detection was done with PE-streptavidine 1∶100 or 1∶1000 for rat and rabbit antibodies, respectively. Beads were resuspended in 100 µl diluents and a minimum of 100 beads from each set of multiplexed beads were analyzed to yield the mean fluorescence intensity (MFI). Each affinity purified and depleted antibody was analyzed in duplicates, the average MFIs and standard deviation were calculated based on three independently repeated experiments.

RNA extraction and cDNA synthesis

Antibody selected IE were grown for 3–5 cycles and erythrocytes infected by trophozoite/schizont-stage parasites (20–48 h post invasion) from in vitro cultures were MACS purified and used for flow cytometry, confocal microscopy and for re-invasion of uninfected erythrocytes in new cultures flasks. IE used for RNA extraction were harvested when re-invaded parasites were at the ring-stage as confirmed by microscopy of Giemsa stained thin smears.

Total RNA was prepared using Trizol (Invitrogen) as recommended by the manufacturers and treated with DNase1 (Invitrogen) for 15 min at 37°C. Absence of DNA in RNA samples was confirmed by stable base fluorescence after 40 cycles of real-time PCR with seryl-tRNA synthetase and fructose-bisphosphate aldolase primers as previously described [41]. Superscript II was used to reverse transcribe DNA-free RNA primed with random hexamer primers (Invitrogen) at 25°C for 10 min and 42 C for 50 min followed by 70°C for 15 min.

Quantitative real-time PCR

Quantitative real-time PCR was performed using a Rotorgene thermal cycler system (Corbett Research), Quanti-Tect SYBR Green PCR Master Mix (QIAGEN), and real-time PCR-optimized and gene-specific primers (0.5 µM) for each of the full-length var genes in the P. falciparum 3D7 genome and to the endogenous control genes seryl-tRNA synthetase and fructose-bisphosphate aldolase. Quantification was done using Rotorgene software version 6.0. mRNA transcript copy numbers were calculated for each var transcript using primer pair specific standard curves generated from real-time Q-PCR measurements of 10 fold genomic DNA dilutions as described [43]. The proportions of individual var gene transcripts relative to the total copy number of all var transcripts or to the total number of endogenous control genes was subsequently calculated for comparison of var transcript profiles within and between samples and depicted in pie-charts.

FISH analysis

RNA-FISH was done on ring stage parasites of 3D7_PFD1235w, 3D7_{PF11_0008}, and 3D7_{PFD1235w/PF11_0008} IE. PCR products were amplified using 3D7 genomic DNA and PFD1235w (fw: 5′-GGGATCCGACACGTCGAGACAGAGG-′3; rv: 5′-GGAGAAGCTTTGCAGCGGACTTCACA-′3) and PF11_0008 (fw: 5′-GGGATCCTAGTTATTTGACGCACCAGC-′3 and 5′-CGTGAATTCGGTGGCTGTACCTTCCC-3′) specific primers containing restriction sites (underlined). The PFD1235w and PF11_0008 PCR products were subsequently cloned into the pSPT19 or pSPT18 vector (Roche Applied Science), respectively and sequenced on a 3130 Genetic Analyzer (AppliedBiosystems). Digoxigenin (DIG)- and biotin-labelled antisense RNA probes were generated using a DIG RNA Labelling Kit and Biotin RNA Labelling Mix (Roche Applied Science). The var2CSA anti-sense probe described previously [50] was included as a negative control. The specificity of the RNA probes was confirmed by standard Northern blotting analysis according to the DIG Application Manual found at http://www.roche-applied-science.com/PROD_INF/MANUALS/DIG_MAN/dig_toc.htm and [39].

The RNA-FISH slides were prepared according to a standard FISH protocol with minor modifications [82], [83]. Following hybridization over-night at 48°C, single stained anti-sense RNA were detected by α-DIG HRP conjugated Ab or α-biotin HRP conjugated Ab and the dual stained anti-sense RNA were detected with both antibodies. The signal was further detected using the TSA Plus Fluorscence Palette System (PerkinElmer) with FITC and/or Cyanine 3, according to the protocol as described [83]. As control, slides were treated with RNAse (Sigma-Aldrich) at a concentration of 100 µg/ml for 30 min at 37°C prior to hybridization with the probes. Slides were washed, mounted with anti-fade reagent containing DAPI (Invitrogen) and images captured using a Nikon TE 2000-E confocal microscope as described above. The positivity of a total number of 100 IE was scored in each experiment.

Intron real-time PCR and sequencing of intron regions

RNA was isolated from the 3D7_PFD1235w, 3D7_{PF11_0008}, and 3D7_{PFD1235w/PF11_0008} sub-lines and cDNA generated as described above. Real-time quantitative PCR was done using primers amplifying the intron spanning region and parts of exon 1 of the var genes listed in Table S2 as well as the seryl-tRNA synthetase and fructose bisphosphate aldolase [41]. In addition, PCR products were amplified from cDNA using TaKaRA La Taq (Lonza) and the intron spanning primers of PFD1235w and PF11_0008 and the var genes listed in Table S2. The PCR products were sequenced on a 3130 Genetic Analyzer using the same set of primers. For comparison genomic DNA was purified from a batch culture of 3D7_{PFD1235w/PF11_0008} as well as from clone 3 and the intron spanning region of PFD1235w and PF11_0008 was sequenced.

Adhesion assays

Adhesion assays were done as described previously [84]. In brief, ring stage IE were cultured over night in hypoxanthine-free RPMI 1640 (Lonza) with 10% hypoxanthine-free Albumax (Invitrogen) and 8.75 MBq ³H-hypoxanthine (Amersham) per ml packed erythrocytes. 96-well flat bottomed plates (Nunc) were coated with 1% gelatine and HUVEC cells (PromoCell) grown in endothelial cell growth medium (Promocell) to maximum 6^th passage were seeded at 550,000 cells per well and grown to become confluent. CHO cells (CHO-wild type, CHO-CD36, CHO-CD54/ICAM1 from ATCC) were grown in RPMI 1640 with 1% added glutamine and 10% foetal calf serum (Lonza) and similarly seeded at 550,000 cells per well. On the day of the assay, ³H-labelled parasite cultures were enriched for late-stages by gelatine floatation and 50 µl of labelled trophozoites (12,000 counts per minute) were added in duplicates or triplicates to the 96-well plates containing HUVEC or CHO cells. The plates were left to incubate on a rocking table for 2 hrs at 37°C. Monoclonal mouse anti-CD54/ICAM1 My13 (10 µg/ml, Invitrogen), anti-CD54/ICAM1 15.2 (20 µg/ml, AbD Serotec), anti-CD36 (10 µg/ml, R&D) and anti-CD31/PECAM-1 9G11 (20 µg/ml, R&D Systems) was added in anti-adhesion assays. Unbound IE were removed using a washing robot (Biomek 2000, Beckman Coulter) and cells binding IE were harvested onto filter paper (Unifilter-96, GF/C, PerkinElmer) using a Filtermate Harvester (PerkinElmer). Following addition of 50 µl/well of scintillation liquid (Microscint-20, PerkinElmer) counting was done on a Topcount NXT (Perkin-Elmer). The ³H max-values were determined as the counts per minute of 50 µl of parasite added to one cell-free well and harvested directly onto a filter paper. To standardise, all values obtained was given as a ratio compared to the max value obtained in the particular assay. The percentage binding of the different 3D7 sub-lines were calculated relative to the binding of the 3D7_PFD1235w parasite line adhering to CD54/ICAM1 (Figure 6A–C) or the 3D7_{PF11_0008} parasite line adhering to HUVEC (Figure 6D) (e.g. binding of these equalling a 100%).

Accession numbers

Nucleotide sequence and protein data reported in this paper are available from http://plasmodb.org/plasmo/under accession numbers (accession numbers of data available from NCBI are shown in brackets): MAL6P1.1/PFF1595c (XP_966307), MAL6P1.314 (XP_965999), MAL6P1.316/PFF0010w (XP_965997), MAL6P1.4/PFF1580c (XP_966305), PF07_0049 (XP_001349031), PF07_0050 (XP_001349033), PF07_0051 (XP_001349034), PF07_0073 (XP_001349080) (seryl-tRNA synthetase), PF08_0103 (XP_001349434), PF08_0140 (XP_001349512), PF08_0141 (XP_001349513), PF11_0008 (XP_001347692), PF11_0521 (XP_001348176), PF13_0003 (XP_001349740), PFA0015c (XP_001350938), PFC0005w (XP_001351080), PFD0005w (XP_001351319.), PFD0020c (XP_001351321), PFD0615c (XP_001351435), PFD0625c (XP_001351437), PFD1235w (XP_001351561), PFE1640w (XP_001351876), PFI1820w (XP_001352240), PFL0005w (XP_001350410), PFL0020w (XP_001350413), PFL0030c (XP_001350415) (VAR2CSA), PFL1955w (XP_001350797), and PFL2665c (XP_001350935).

Supporting Information

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