Temporal Expression and Localization Patterns of Variant Surface Antigens in Clinical <em>Plasmodium falciparum</em> Isolates during Erythrocyte Schizogony

<div><p>Avoidance of antibody-mediated immune recognition allows parasites to establish chronic infections and enhances opportunities for transmission. The human malaria parasite <em>Plasmodium falciparum</em> possesses a number of multi-copy gene families, including <em>var</em>, <em>rif</em>, <em>stevor</em> and <em>pfmc-2tm,</em> which encode variant antigens believed to be expressed on the surfaces of infected erythrocytes. However, most studies of these antigens are based on <em>in vitro</em> analyses of culture-adapted isolates, most commonly the laboratory strain 3D7, and thus may not be representative of the unique challenges encountered by <em>P. falciparum</em> in the human host. To investigate the expression of the <em>var</em>, <em>rif-A</em>, <em>rif-B</em>, <em>stevor</em> and <em>pfmc-2tm</em> family genes under conditions that mimic more closely the natural course of infection, <em>ex vivo</em> clinical <em>P. falciparum</em> isolates were analyzed using a novel quantitative real-time PCR approach. Expression patterns in the clinical isolates at various time points during the first intraerythrocytic developmental cycle <em>in vitro</em> were compared to those of strain 3D7. In the clinical isolates, in contrast to strain 3D7, there was a peak of expression of the multi-copy gene families <em>rif-A</em>, <em>stevor</em> and <em>pfmc-2tm</em> at the young ring stage, in addition to the already known expression peak in trophozoites. Furthermore, most of the variant surface antigen families were overexpressed in the clinical isolates relative to 3D7, with the exception of the <em>pfmc-2tm</em> family, expression of which was higher in 3D7 parasites. Immunofluorescence analyses performed in parallel revealed two stage-dependent localization patterns of RIFIN, STEVOR and <em>Pf</em>MC-2TM. Proteins were exported into the infected erythrocyte at the young trophozoite stage, whereas they remained inside the parasite membrane during schizont stage and were subsequently observed in different compartments in the merozoite. These results reveal a complex pattern of expression of <em>P. falciparum</em> multi-copy gene families during clinical progression and are suggestive of diverse functional roles of the respective proteins.</p> </div

Localization of VSAs during the intraerythrocytic developmental cycle.

<p><b>A-C:</b> Representative immunofluorescence images of the indicated VSAs in different parasite developmental stages of clinical isolate #4 (<b>A</b>), 3D7 parasites (<b>B</b>), and free merozoites from isolate #1 (<b>C</b>). First row: Giemsa staining of the corresponding parasitic stage. Second row: Positive control serum obtained from a semi-immune patient. Third row: <i>Pf</i>EMP1-specific antibody, showing the presence of the protein in Maurer’s clefts over the entire time course (<b>A, B</b>). Third to eighth rows: 2TM proteins were exported into the host cell (12–36 hpi) during the trophozoite stage but remained inside the parasite in the schizont stage (48 hpi). Proteins of the RIFIN-A family frequently localized to Maurer’s clefts, particularly when using the α-RIF29n antiserum, and the erythrocyte membrane; STEVOR and <i>Pf</i>MC-2TM localized predominantly to the erythrocyte membrane (<b>A, B</b>). RIFIN and STEVOR proteins were also observed at the apical tip or at the merozoite membrane, respectively. Isolate #1 also exhibited <i>Pf</i>MC-2TM-specific fluorescence in free merozoites when using the α-P<i>f</i>MC-2TM-CT antiserum (<b>C</b>). All antibodies were visualized with Alexa488-conjugated secondary antibody (green), and nuclei were stained with DAPI (blue).</p

Validation of the degenerate primer pairs used in quantitative real-time PCR.

<p>Primer pairs targeting the multi-copy gene families <i>var</i>, <i>rif-A</i>, <i>rif-B</i>, <i>stevor</i> and <i>pfmc-2tm</i> were designed to amplify a broad repertoire of different genes present in the 3D7 genome (first bar, 1), as indicated by <i>in silico</i> PCR results (second bar, 2). Experimental validation revealed similar numbers of genes amplified by the indicated primer pairs in every <i>P. falciparum</i> genotype relative to single-copy <i>fructose-bisphosphate aldolase</i> (RELATNO) (third to seventh bar, 3–7). <i>In silico</i> PCR results were experimentally confirmed for the <i>var</i> (red), <i>stevor</i> (green) and <i>pfmc-2tm</i> (yellow) primer pairs; however, the <i>rif-A</i> (dark blue) and <i>rif-B</i> (light blue) primer pairs only partially covered the genomic repertoire. Shown are mean values and standard deviations obtained by analysing two biological samples of 3D7 and <i>ex vivo</i> isolated gDNA of the clinical isolates in quadruplicates. 1: Number of genes present in the 3D7 genome (set as 100%); 2: number and percentage of genes amplified <i>in silico</i> using the one mismatch configuration (<a href="http://insilico.ehu.es" target="_blank">http://insilico.ehu.es</a>); 3–7: experimentally calculated RELATNOs of amplified genes of the indicated multi-copy gene families using gDNA from the 3D7 laboratory strain (3) (including percentages) and from clinical isolates #1 (4), #2 (5), #3 (6) and #4 (7).</p

Proportion of infected erythrocytes with VSAs exported to host cell compartments in clinical isolates and 3D7 at different developmental stages.

1<p>Time point not determined for isolate #1.</p>2<p>Time point of 44 h for isolate #4.</p><p>hpi, hours post-infection.</p

Immunoblot analysis of VSA abundance in the clinical isolate #5 and the 3D7 strain.

<p><b>A, B</b>: Isolate #5 (h: time of <i>in vitro</i> cultivation) and 3D7 (hpi: hours post infection) at successive developmental stages were harvested (<b>A</b>), and differences in VSA abundance in the membrane fraction were assessed by immunoblot using α-RIF40, α-RIF44, α-RIF50, α-STEVOR-mix, α-<i>Pf</i>MC-2TM-SC, and α-<i>Pf</i>MC-2TM-CT antisera (<b>B</b>). As expected, RIFIN and STEVOR were present at higher levels in the clinical isolate; in contrast, <i>Pf</i>MC-2TM proteins were quantitatively increased in 3D7 parasites. Differences were most obvious in pigmented parasite stages (trophozoites, schizonts, left), which also exhibited the highest levels of protein during intraerythrocyic development, but upon longer exposure (*), similar results were also observed for ring stage parasites (right). The luminal endoplasmic reticulum (ER) protein BiP (HSP70) served as a loading control.</p

Expression of multi-copy gene families in the 3D7 laboratory strain during erythrocyte schizogony.

<p>Expression profiles of the multi-copy gene families <i>var</i> (red line, squares), <i>rif-A</i> (dark blue line, triangles), <i>rif-B</i> (light blue, upside-down triangles), <i>stevor</i> (green line, diamonds), and <i>pfmc-2tm</i> (yellow line, dots) in long-term <i>in vitro</i> cultivated strain 3D7 during erythrocyte schizogony. In contrast to the clinical isolates, 3D7 parasites exhibited a slight increase of <i>rif-A</i>, <i>stevor</i> and <i>pfmc-2tm</i> expression during the ring stage, and the main peak of expression of the multi-copy gene families was observed in mid-age trophozoites. Transcription of the <i>var</i> gene family was restricted to ring stage parasites. Expression was normalized to the reference gene <i>fructose-bisphosphate aldolase</i> and is represented by either ΔCt (left chart) or relative expression (RELATEXP, right chart). 3D7 time course experiments were performed twice and two samples were analysed for each time point at least in duplicates. Graphically shown are mean values and standard deviations of all qPCR runs performed for the respective time points Parasite developmental age (hpi) and the corresponding parasitic stage (shown as Giemsa staining) are plotted on the x-axis.</p

Modification of <i>var</i> transcription by mosquito transmission.

<p>(A) The rate difference of the median expression for each individual <i>var</i> variant and the housekeeping controls shows the overexpression of the entire <i>var</i> gene family <i>in vivo</i> with exception of the <i>var2csa</i> gene PFL0030c in comparison to the parental Master Cell Bank (MCB) parasite line. Each point reflects the median for the volunteer samples at the day of patent infection (n = 18) divided by the median observed for the parasite generations 6, 8 and 21 from two vials of the MCB cell line (n = 6). Housekeeping genes used as controls, <i>var</i> gene names and groups are indicated. (B) Differences in gene expression on group level between the pre-mosquito MCB parasite lines and parasites recovered from the infected volunteers (VOL) at the day of patent infection determined by thick blood smear are displayed as group median with interquartile range (IQR). Significant differences in distributions between MCB and VOL series were tested via a Wilcoxon rank-sum test using a Bonferroni corrected significance level. The graph contains a scale brake at the y-axis to account for the huge variability in the gene expressions. Group affiliations are indicated above the graph. Red (A), orange (A, subfamily <i>var3</i>), dark red (A, subfamily <i>var1</i>), purple (B/A), blue (B), turquoise (B/C), green (C) and yellow (E).</p

<i>Var</i> transcription profiles of parasites recovered from infected volunteers at the day of first microscopically detectable parasitemia.

<p>(A) Heat map showing the individual <i>var</i> gene expression for all volunteer samples taken when parasites were present in the thick blood smear ranked by mean expression. To correct for individual differences in the overall <i>var</i> expression levels, the expression for each <i>var</i> gene was normalized against the total <i>var</i> expression in each sample. The color scale indicates the relative expression levels with red representing values above the median, blue representing values below the median, and white representing median. Grey means not detected. The number of sporozoites (200, 800, 2500 and 3200) and mode of injection (iv = intravenous, id = intradermal) used for each volunteer are indicated below. (B) The distribution of the relative gene expression per <i>var</i> gene and control genes is shown in a dot plot for all volunteer samples at the day of patent infection defined as parasites present in the thick blood smear (n = 18). Each point represents a <i>var</i> gene expression value relative to the normalizing gene <i>sbp1</i> observed per volunteer sample and the median expression per <i>var</i> gene is marked. Housekeeping genes used as controls, <i>var</i> gene names and groups are indicated. (C) Proportion of <i>var</i> gene expression by group across all volunteers at the day of patent infection. For comparison, genomic proportion of each <i>var</i> gene group is indicated after the color code. (D) Comparison of the expression levels between subtelomeric and centromeric <i>var</i> gene variants. The box plot shows the distribution of transcript levels for each individual <i>var</i> gene relative to <i>sbp1</i> according to the chromosomal localization of the genes for all 18 volunteer samples at the day of patent infection. Gene expression varied significantly between both gene sets (Wilcoxon rank-sum test, p<0.0001) with median expression of 232 (IQR: 87–510) for telomeric <i>var</i> genes and 40 (IQR: 18–94) for centromeric <i>var</i> genes. (E) The heat map of pairwise Spearman’s rank correlation coefficients (R) between expression profiles illustrates the positive correlation between all 18 volunteer samples at the day of patent infection. Volunteer samples were ranked by the sum of their correlation coefficients. The color scale indicates the correlation coefficient in the range from 0 to 1. With exception of isolate 25.1 versus the isolates 02.1 (p = 0.0015), 25.3 (p = 0.0029) and 32.4 (p = 0.0030) all expressions correlated at a significance level below 0.001.</p

<i>Var</i> transcription profiles of parasites from infected volunteers 1–2 days before parasites were microscopically detectable.

<p>(A) Heat map showing the individual <i>var</i> gene expression profiles for samples obtained from volunteers one or two days before parasites were present in the thick blood smear. To correct for individual differences in the overall <i>var</i> expression levels, the expression for each <i>var</i> gene was normalized against the total <i>var</i> expression in each sample. Expression is ranked by mean expression obtained from “day of patent infection” samples (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005538#ppat.1005538.g001" target="_blank">Fig 1A</a>). The color scale indicates the relative expression levels with red representing values above the median, blue representing values below the median, and white representing median. Grey means not detected. (B) Relative gene expression is shown in a dot plot for the volunteer samples one time point before thick blood smear positivity (n = 8). Gene IDs of <i>var</i> genes and controls are indicated on the x-axis, <i>var</i> gene groups are indicated above the graph. (C) The distribution of <i>var</i> transcripts according to <i>var</i> group affiliation in the volunteer samples 1–2 days before parasites were detected in the thick blood smear is displayed by summarization of the total <i>var</i> gene expression and calculation of the proportion for each <i>var</i> group. The genomic proportion of each <i>var</i> gene group is indicated after the color code. (D) The pairwise Spearman’s rank correlation heat map demonstrates a positive correlation between the expression profiles on the day of first microscopically detectable parasitemia and 1–2 days before in the same volunteer and between volunteer samples.</p

Overview of volunteer characteristics infected with PfSPZ Challenge.

<p>Overview of volunteer characteristics infected with PfSPZ Challenge.</p