Outline of Experimental Design

<p>NF54 parasites were cloned by limiting dilution to create a clonal population (NF54/C3) that predominantly expressed PFD1005c. These parasites were transfected with the plasmid pV_bBB/IDH and the clone B12E3 containing a double crossover integration was isolated. After blasticidin selection, B12E3 exclusively expressed <i>bsd,</i> and all other <i>var</i> genes were transcriptionally silent. Drug pressure was then removed for two months during which time the <i>var</i> gene expression pattern became heterogenous. This heterogeneous B12E3 population was then re-cloned and clone DC-J isolated. DC-J predominantly expressed the <i>var</i> gene PFD1015c and had silenced the <i>bsd</i> gene. Growth of DC-J back under blasticidin pressure however results in reactivation of <i>bsd</i> expression and silencing of the rest of the <i>var</i> gene family, thus demonstrating the reversibility of the phenomenon.</p

Replacement of PfEMP1 Exon I with the Selectable Marker <i>bsd</i>

<div><p>(A) Schematic diagram showing integration of the <i>bsd</i> cassette from the construct pV_bBB/IDH into the NF54 <i>var</i> PFB1055c locus through double crossover recombination.</p><p>(B) Southern analysis was performed using HpaI digested DNA and probed for <i>bsd</i> and pUC18. The size of the DNA fragment is shown on the left. The absence of a 6.7-kb band when using a <i>bsd</i> probe and the absence of hybridization with plasmid backbone are indicative of a double crossover recombination event, resulting in replacement of exon I with the <i>bsd</i> cassette. This arrangement was confirmed by additional Southern blots and by sequencing across the sites of integration.</p></div

An Active <i>var</i> Promoter on an Episome Is Exclusively Expressed

<div><p>NF54/C3 parasites stably carrying the episome pVBB/IDH were grown under blasticidin pressure.</p><p>(A) Plasmid map of pVBB/IDH.</p><p>(B) Transcription levels were then measured by Q-RT-PCR and indicated that the parasites exclusively express <i>bsd</i> while all endogenous <i>var</i> genes are silent.</p></div

PfEMP1 Is Not Expressed in the Knock-Out Transgenic Lines

<p>Western blot analysis of whole cell extracts isolated from NF54 and the three transgenic lines B12E3, B15C2, and C7G12 growing under blasticidin pressure. Extract were probed with antibodies to either the conserved C terminus of PfEMP1 (α-ATS) or to the ER protein <i>Pf</i>39 (α <i>Pf</i>39). α-ATS signal appears only in NF45 parasites and not in any of the transgenic lines.</p

Integration of the <i>bsd</i> Expression Cassette into a Telomeric <i>var</i> Locus

<div><p>(A) Schematic diagram showing integration of the construct pV_bBB/IDH into the <i>var</i> PFL0020w locus through a single homologous recombination event within the <i>var</i> upstream region.</p><p>(B) Southern analysis was performed using BamHI-digested gDNA and hybridized with probes specific to <i>bsd</i> and pUC18. The linearized plasmid within the multiple copy concatameric insertion appears as a high intensity 6.7 kb band with both probes. The 6.1-kb <i>bsd</i> band and the ~13-kb pUC18 band correspond to fragments flanking the site of integration. This arrangement was confirmed by additional Southern blots and by sequencing across the sites of integration.</p><p>(C) Analysis of the level of transcription from each <i>var</i> gene in the genome shows that selection for <i>bsd</i> expression results in silencing of the entire gene family. The only “on” gene in parasites growing under drug pressure (top panel) is the locus expressing the <i>bsd</i> cassette. Analysis performed 10 wk after drug removal demonstrated that the culture has become transcriptionally heterogenous (bottom panel) with the majority of the genes upregulated, of which nine are expressed at levels greater than the control (relative copy number > 1).</p></div

Analysis of Levels of Transcription of the Entire <i>var</i> Family

<div><p>All values are presented as relative copy number to the housekeeping gene seryl-tRNA synthetase (PF07_0073).</p><p>Top panel: NF54 parasites were cloned by limiting dilution, and <i>var</i> gene expression was measured by Q-RT-PCR as soon as the culture reached the required parasitemia, approximately 6 wk after plating. The clone NF54/C3, which was used to generate all transgenic lines, was predominantly expressing <i>var</i> PFD1005c (located on Chromosome 4 internal cluster) while expression of the rest of the <i>var</i> family was virtually undetectable.</p><p>Second panel: The recombinant line B12E3 growing under blasticidin pressure only transcribed <i>bsd</i> (red), while transcription levels of the rest of the <i>var</i> family was close to zero (blue).</p><p>Central panel: Expression of additional <i>var</i> genes was easily observed after the parasites were grown for 10 wk without drug pressure. At this point the culture is transcriptionally heterogeneous and <i>bsd</i> is no longer the dominant gene. Five genes were expressed at levels equal to or greater than the control (copy number = 1).</p><p>Fourth panel: The transcription pattern of clone DC-J that was re-cloned from this culture represents a population that had switched away from <i>bsd</i> and now predominantly expresses <i>var</i> PFD1015c. Applying blasticidin to DC-J resulted in the selection of parasites that had switched back to exclusively expressing <i>bsd</i> (bottom panel).</p></div

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

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

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