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

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

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

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

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

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