SLI-activation of var genes in 3D7.

(A) Schematic for SLI strategy. HR: homology region; ATS: acidic terminal segment; NTS: N-terminal segment; 2A: T2A skip peptide; NEO-R: G418-resistance; hDHFR: human dihydrofolate reductase; arrows P1-4: primers for diagnostic PCR; X: desired var gene. (B, C) Activation of indicated PfEMP1. Scheme shows domain organisation. Agarose gels show PCR products confirming correct integration of the SLI plasmid. Product over 5′ integration junction (5’): P1+P2; 3’ integration junction (3’): P3+P4; original locus (ori): P1+P4; see (A) for primer positions, Table S5 for sequence; 3D7: parent; Int: integrant cell line. Fluorescence microscopy images show IFAs with indicated antibodies. Nuclei: Hoechst 33342; DIC: differential interference contrast; size bars 5 μm. (D) Western blot of trypsin cleavage assays with indicated parasites. Asterisks show protected PfEMP1 fragment. α-SBP1-N: control for integrity of host cell (breach of RBC membrane would result in a smaller SBP1 fragment). Marker in kDa. Replicates and full blots in Figure S4. (E, F) Pie charts with proportions of total var gene transcripts determined by qPCR of the indicated cell lines on G418 and after lifting G418. (G, H) Activation of PF3D7_0425800 in 3D7 or 3D7MEED. Scheme shows domain organisation. Agarose gels show PCR products confirming correct integration of the SLI plasmid as described in (A). Fluorescence microscopy images show IFAs as described in (B, C). Pie charts show proportions of total var gene transcripts of the indicated cell lines determined by RNAseq (normalized to TPM). (I) SuperPlot (121) showing percentage (log scale) of total var gene transcripts for non-activated var genes of the indicated cell line determined by RNAseq (normalized to TPM; small grey dots: individual var genes; large coloured dots: average of each replicate; bars: mean of averages of replicates with SD; n = 3 biological replicates; unpaired t-test; p-values indicated). See also Figure S1 and S4.

Clogging PTEX prevents PfEMP1 transfer into the host cell.

(A) Scheme: options for impact of WR-induced stabilisation of mDHFR folding on PfEMP1 export. Relevant domains of modified PfEMP1 indicated. Fluorescence microscopy images of IFAs with parasites of the indicated cell line + and – WR with the indicated antibodies. Nuclei: Hoechst 33342; DIC: differential interference contrast; size bars 5 μm. (B) Effect of blocking PTEX (+WR) with early (mal7 promoter) expressed SBP1-mDHFR-GFP on PfEMP1 export. Relevant expressed products are shown. Live cell images (top rows) and IFAs (bottom rows; as described in (A)) of parasites grown + and - WR. Graph: quantification of parasites with a PfEMP1 export phenotype + and - WR (4 biological replicates; dots: % cells per replicate; bars: mean of replicates with SD; n = 26 parasites per experiment and condition; +WR only parasites with an SBP1-mDHFR-GFP export phenotype were scored; unpaired t-test; p-values indicated). Scheme shows WR-dependent clogging of PTEX (right) or control (left); features explained in (A). (C) Effect of blocking PTEX with late (crt promoter) expressed SBP1-mDHFR-GFP-2A-KAHRP-mScarlet on PfEMP1 export. Relevant expressed products are shown. Live cell images (top rows) and IFAs (bottom rows, as described in (A)) + and - WR. Graph: quantification of parasites with PfEMP1 or REX1 export phenotype + and - WR (3 biological replicates; -WR, PfEMP1: n = 34, 76, 60; +WR, PfEMP1: n = 18, 48, 30; - WR, REX1: n = 18, 27, 35; +WR, REX1: n = 12, 31, 25; +WR, only parasites with an KAHRP-mScarlet (late PTEX block reporter) export phenotype were scored (dots: % cells per replicate; bars: mean of replicates with SD; unpaired t-test; p-values indicated). Scheme shows WR-dependent clogging of PTEX (right) or control (left); features explained in (A); note that due to late block, early expressed REX1 is in the host cell in both conditions. See also Figure S2.

Activated PfEMP1s in IT4 are functional and cytoadherent.

(A, B) Activation of indicated PfEMP1. Scheme shows domain organisation. Agarose gel shows PCR products confirming correct integration of the SLI plasmid as described in Fig. 1A, Table S5 for sequence; IT4: parent; Int: integrant cell line. Fluorescence microscopy images show IFAs with indicated antibodies. Nuclei: Hoechst 33342; DIC: differential interference contrast; size bars 5 μm. (C) Pie charts show proportions of total var gene transcripts of the indicated cell lines determined by RNAseq (normalized to TPM). (D) Western blot of trypsin cleavage assays with indicated parasites. Asterisks show protected PfEMP1 fragment. α-SBP1-N: control for integrity of host. Marker in kDa. Replicates and full blots in Figure S4. (E, F) SuperPlots showing binding assays of indicated cell lines against decorin or CSA-expressing HBEC-5i cells (3 biological replicates with 15 fields of view/experiment and condition; bars: mean of averages of replicates with SD; unpaired t-test; p-values are indicated). Small grey dots: bound iE/field of view, extrapolated to mm2. Larger coloured dots: average of bound iE/mm2/replicate. Same color indicates experiment conducted in parallel. iE: infected erythrocytes. (G) SuperPlot of binding assays of indicated cell lines against CHO cells expressing GFP, CD36 or ICAM-1 (3 biological replicates with 15 field of views/experiment and condition; bars: mean of averages of replicates with SD; unpaired t-test; p-values are indicated). Small grey dots: bound iE/field of view, extrapolated to mm2. Larger coloured dots: average bound iE/mm2/replicate. iE: infected erythrocytes.

Activation of further PfEMP1s with different binding properties in IT4.

(A, B, C) Activation of indicated PfEMP1. Scheme shows domain organisation. Agarose gel shows PCR products confirming correct integration of the SLI plasmid as described in Fig. 1A, Table S5 for sequence; IT4: parent; Int: integrant cell line. Fluorescence microscopy images show IFAs with indicated antibodies. Nuclei: Hoechst 33342; DIC: differential interference contrast; size bars 5 μm. Pie charts show proportions of total var gene transcripts of the indicated cell lines determined by RNAseq (normalized to TPM). (D) Western blot of trypsin cleavage assays with indicated parasites. Asterisks show protected PfEMP1 fragment. α-SBP1-N: control for integrity of host cell. Marker in kDa. Replicates and full blots in Figure S4. (E) SuperPlot of binding assays of indicated cell lines against CHO cells expressing GFP, CD36 or ICAM-1 (3 biological replicates with 15 fields of view/experiment and condition; bars: mean of averages of replicates with SD; unpaired t-test; p-values are indicated). Small grey dots: bound iE/field of view, extrapolated to mm2. Larger coloured dots: average of bound iE/mm2/replicate. iE: infected erythrocytes. (F) Western blot of trypsin cleavage assay as described in (D) and pie chart showing proportions of total var gene transcripts determined by RNAseq (normalized to TPM) of IT4var19-HAendo parasites after five rounds of panning on EPCR. See also Figure S1. (G) SuperPlot of binding assays of indicated cell lines against CHO cells expressing GFP, CD36, ICAM-1 or EPCR as described in (E). iE: infected erythrocytes. (I) Volcano plot showing differential expression analysis (DeSeq2) of EPCR-panned against unpanned IT4var19-HAendo parasites.

Second endogenous modification with SLI2 in a SLI var gene cell line.

(A) Schematic for SLI2 strategy for second genome modification in SLI cell line with activated var gene. HR: homology region; ATS: acidic terminal segment; NTS: NTS domain; 2A: T2A skip peptide; NEO-R: neomycin-resistance gene; yDHODH: yeast dihydroorotate dehydrogenase; BSD-R: BSD-resistance gene, arrows P1-8 primers for diagnostic PCR; X: desired var gene; PTP1: PfEMP1 transport protein 1. (B) Agarose gel shows PCR products confirming correct integration of the SLI2 plasmid and perpetuation of the SLI plasmid integration. SLI2 integration: product over 5′ integration junction (5’): P5 + P8; over 3’ integration junction (3’): P7 + P6; original locus (ori): P5 + P6; SLI integration PCRs as described in Fig. 1A; IT4: parent; Int: integrant cell line; primers in Table S5. (C) Fluorescence microscopy images of live IT4var1-HAendo+PTP1TGD-GFP parasites. (D) Fluorescence microscopy images of IFAs with indicated antibodies. Nuclei: Hoechst 33342; DIC: differential interference contrast; size bars 5 μm. Nuclei: Hoechst 33342; DIC: differential interference contrast; size bars 5 μm. (E) Western blot of trypsin cleavage assays with IT4var1-HAendo+PTP1-TGD parasites. μ-SBP1-N: control for integrity of host cell. Marker in kDa. Replicates and full blots in Figure S4. (F) SuperPlot of binding assays of indicated cell lines against CHO cells expressing GFP, CD36 or ICAM-1 (3 biological replicates with 15 fields of view/experiment and condition; bars: mean of averages of replicates with SD; unpaired t-test; p-values are indicated). Small grey dots: bound iE/field of view, extrapolated to mm2. Larger coloured dots: average of bound iE/mm2/replicate. iE: infected erythrocytes.

Proxiome of PfEMP1 from living parasites.

(A) Schematic of the three different 3xHA-tagged BirA*-IT4var1 fusion constructs and the position respective to the membrane in the fusion constructs reaching the host cell surface. (B, C, D) Confirmation of the activation and modification of the indicated IT4var1-BirA* fusions. Fluorescence microscopy images show IFAs with indicated antibodies or streptavidin. Nuclei: Hoechst 33342; DIC: differential interference contrast; size bars 5 μm. Pie charts show proportions of total var gene transcripts of the indicated cell lines determined by RNAseq (normalized to TPM). (E) Western blot of trypsin cleavage assays with indicated parasites. Asterisks show protected PfEMP1 fragment. α-SBP1-N: control for integrity of host cell. Marker in kDa. Replicates and full blots in Figure S4. (F) SuperPlot of binding assays of indicated cell lines against CHO cells expressing GFP, CD36 or ICAM-1 (3 biological replicates with 15 fields of view/experiment and condition; bars: mean of averages of replicates with SD; unpaired t-test; p-values are indicated). Small grey dots: bound iE/field of view, extrapolated to mm2. Larger coloured dots: average of bound iE/mm2/replicate. iE: infected erythrocytes. (G) Western blot of extracts of the indicated cell lines after incubation with biotin for 24 hours. Streptavidin probes biotinylated proteins; α-aldolase is the loading control. (H, I, J) Volcano plots showing enrichment of biotinylated proteins extracted with SDS from the indicated cell lines compared to IT4 wildtype parasites (24 hours growth with biotin). Only the quadrant with positive enrichment is shown, full plots in Figure S5 and further comparisons in Figure S6.

New proteins needed for PfEMP1 cytoadherence function.

(A) Domain schematic of candidates selected for analysis (Ty1-tagging and disruption (TGD) using SLI2) in IT4var1-BirA*Pos1endo. (B) SuperPlot of binding assays of the indicated cell lines against CHO cells expressing GFP, CD36 or ICAM-1 (3 or 4 (control and PTEF-TGD) biological replicates with 15 fields of view/experiment and condition; bars: mean of averages of replicates with SD; unpaired t-test; p-values are indicated). Small grey dots: bound iE/field of view, extrapolated to mm2. Larger coloured dots: average of bound iE/mm2/replicate. iE: infected erythrocytes. (C) Western blot of trypsin cleavage assays with indicated parasites. Asterisks show protected PfEMP1 fragment. α-SBP1-N: control for integrity of host cell. Marker in kDa. Replicates and full blots in Figure S4. See also Figure S7.