Distinct Trafficking and Localization of STEVOR Proteins in Three Stages of the Plasmodium falciparum Life Cycle

Gametocyte culture and purification.The 3D7 strain of P. falciparum was cultured under standard conditions, and gametocytes were produced and harvested as previously described (24). To ensure stage specificity, gametocyte cultures were treated with 50 mM N-acetylglucosamine (Sigma) 5 to 8 days after the culture was initiated, to prevent further development of asexual-stage parasites (22). Early, middle, and late gametocyte preparations were taken after 6, 12, and 17 days of culture, respectively. The sexual-stage parasites were then harvested from the 30 to 45% and 45 to 54% interfaces of a Percoll (Sigma) gradient (8). The proportions of distinct developmental stages in these preparations were determined by using Giemsa-stained thin films prepared immediately after the Percoll harvest (Table 1).

Purification of asexual-stage parasites.Ring stage asexual parasites were harvested following treatment with 15% sorbitol (Sigma) to remove trophozoites and schizonts. Asexual cultures of 3D7 were enriched for late developmental stages by centrifugation through 60% Percoll in serum-free RPMI.

Sporozoite preparation. P. falciparum (uncloned line of the NF54 isolate) sporozoites were obtained from dissection of infected Anopheles stephensi mosquito salivary glands.

RNA isolation.Asexual and sexual parasites were harvested in TRI reagent (Sigma). RNA was stored in formamide by the procedure of Kyes et al. (18). RNA was ethanol precipitated and resuspended in nuclease-free water (Promega) for use in RT-PCR experiments.

RT-PCR amplification.RT-PCR amplification was performed with the Access RT-PCR System (Promega Corp., Southampton, United Kingdom) with specific primers designed to amplify pfs16 (CSo72 and CSo73; encoding the P. falciparum 16-kDa sexual-stage antigen), resa (CSo83 and CSo85; encoding ring-infected erythrocyte surface antigen), csp (Alloueche #1 and #2; encoding circumsporozoite protein), and the hypervariable loop of stevor transcripts (CSo75 and LM03: ACGTACGTACGTACGT), as previously described (1, 26, 27). The products were visualized on 1.5% agarose gels in Tris-borate EDTA buffer stained with ethidium bromide and photographed over a UV transilluminator.

Western blotting.Parasite proteins were extracted, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose as previously described (16). STEVOR proteins were detected by previously characterized mouse polyclonal sera (16) followed by horseradish peroxidase-linked secondary antibodies (Bio-Rad) and chemiluminescence (Pierce).

Antibodies and antisera.Rabbit and mouse antisera that recognize the STEVOR amino terminus (pepNPH, previously peptide 1) and carboxy terminus (pepIWL, peptide 3), and the MC protein PfSBP have been previously described (3, 16). Antisera to a third STEVOR peptide (EPMSTELEKELLETYE; pepEPM) were also used in the present study (16). Rabbit polyclonal antisera raised against recombinant Pfs16 (2) was a kind gift from David Baker. Mouse monoclonal antibody CT-1 to P. falciparum circumsporozoite protein was a kind gift of from G. Del Giudice (9).

Immunofluoresence of fixed parasite preparations.Parasites were washed three times in phosphate-buffered 0.9% saline (pH 7.4) (PBS)-0.1% bovine serum albumin (BSA) and diluted by 10⁴-fold. The parasite suspension was spotted on to each of 12 wells on Teflon-coated slides, dried in a laminar-flow hood and stored at −20°C. The slides were fixed in acetone on ice for 30 min immediately after removal from −20°C, dried at 22°C for 30 min, washed in PBS, and blocked in PBS-0.1% BSA for 30 min at 22°C in the dark. Primary antibody was applied diluted 1:100 (Pfs16) or 1:20 (STEVOR) in PBS-0.1% BSA-5mM sodium azide for 1 h, and then fluorescence-labeled secondary antibody diluted 1:400 was added for 30 min; the parasites were then given a final wash in PBS. This step was repeated for double-staining experiments. Following the final wash, the parasite nuclei were stained with TOTO-3 nucleic acid stain (Molecular Probes), mounted in Vectashield, and stored in the dark at 4°C until visualized using confocal microscopy (Zeiss Axioplan LSM510). The secondary antibodies used were goat anti-mouse immunoglobulin G (heavy plus light chains) [IgG (H +L)] conjugated to AlexaFluor 594 (red) and goat anti-rabbit IgG (H + L), conjugated to AlexaFluor 488 (green) (Cambridge Biosciences). Sporozoites were air dried on glass slides and fixed with cold methanol. The slides were blocked with 1% BSA for 30 min, and then polyclonal anti-STEVOR serum (diluted 1:100) or anti-CSP monoclonal antibody (diluted 1:1,000) was added. The preparation was incubated for 30 min with goat anti-mouse antibody coupled to fluorescein isothiocyanate (1 mg/ml) (Sigma).

Stevor transcripts appear transiently in early gametocyte development.Transcripts of stevor, resa, pfs16, and csp were amplified by RT-PCR from total RNA isolated from P. falciparum asexual blood-stage parasites, 6-, 12- and 17-day gametocyte preparations, and salivary gland sporozoites (Fig. 1). The relevant abundance of each gametocyte developmental stage (I to V) at each of the three gametocyte time points is shown in Table 1. pfs16 transcripts were detected in all three gametocyte preparations, but stevor transcripts were detected in RNA only from early gametocytes. stevor transcripts were not detected in RNA from salivary gland sporozoites.

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FIG. 1.

RT-PCR of stevor in blood-stage parasite development and in sporozoites. Total RNA from each of the parasite life cycles stages shown was used as the template for RT-PCR amplification of transcripts of resa, pfs16, csp, and stevor. Ring, early trophozoite-stage asexual parasites; Tr/Schz, late trophozoite- and schizont-stage asexual parasites; G, gametocytes from 6-, 12-, or 17-day cultures; Spz, sporozoites from the salivary glands of infected mosquitoes; + and −, reactions in which avian myeloblastosis virus reverse transcriptase was present and absent, respectively. The 3D7A clone of P. falciparum was used throughout, except for the sporozoite material, which was derived from NF54, the parent isolate of 3D7A.

Transcripts of csp and resa were not only detected in RNA from sporozoites and asexual blood stages, respectively, as expected, but were also present throughout gametocyte development (Fig. 1). Although the circumsporozoite protein is regarded as a preerythrocytic antigen, the presence of csp transcripts in cultured blood-stage parasites is consistent with microarray data (4). We have previously shown that resa transcripts are not present in mature circulating gametocytes in Gambian malaria patients (27), and so the observed transcripts either represent residual resa expression in immature gametocytes or indicate that strict stage-specific transcriptional control of this gene is not occurring in 3D7 under culture conditions.

Anti-STEVOR sera detect proteins of identical size in gametocytes and trophozoites.Protein extracts of mid-stage gametocytes and of late trophozoites and schizonts both exhibited a protein band in the predicted STEVOR size range (30 to 40 kDa) that was detected by antisera raised to three different conserved STEVOR peptides (Fig. 2, upper arrow). An abundant polypeptide detected in all lanes at ∼30 kDa (Fig. 2, asterisk) was also detected by normal mouse serum, as were the higher-molecular-weight bands migrating more slowly than STEVOR. Thus, antibodies raised against three distinct STEVOR peptides and known to specifically react with STEVOR in asexual parasites (16, 17) give the same detection pattern in asexual- and sexual-stage parasites. We therefore conclude that full-length STEVOR polypeptides are expressed in gametocytes. Interestingly, a smaller protein band (∼25 kDa) that specifically reacts with each of the antisera was seen in gametocyte extracts only (Fig. 2, lower arrow). This band may correspond to truncated STEVOR polypeptides such as those predicted by our studies of stevor transcripts in gametocytes (26).

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FIG. 2.

Expression of STEVOR in asexual parasites and gametocytes. Parasite extracts were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and incubated with mouse antisera raised to STEVOR peptides pepNPH, pepEPM, and pepIWL or to normal mouse serum (ns). Secondary antibodies were as described in Materials and Methods. a, asexual parasite extract; g, gametocyte extract. Upper arrow, predicted STEVOR polypeptides of ∼40 kDa; lower arrow, additional polypeptides of ∼25 kDa; *, polypeptide present in normal mouse serum and recognised by all antisera.

STEVOR proteins are present throughout gametocyte development.To investigate the expression of STEVOR during gametocyte development, polyclonal mouse antisera raised against STEVOR (16) and rabbit antisera against Pfs16 (2) were applied to acetone-fixed staged gametocyte preparations (Fig. 3). Pfs16 is expressed throughout gametocyte development and is localized to the parasitophorous vacuole membrane (PVM) (2, 5). Gametocytes at 6 days showed perinuclear STEVOR localization (red) within the PVM, which is clearly delineated by Pfs16 staining (green) (Fig. 3, top row). In some 12-day gametocytes, the STEVOR signal was focused within the parasite; in others, it crossed the PVM or was external to the parasite, often clearly associated with the host erythrocyte membrane (Fig. 3, second row). A common pattern among 12-day gametocytes was for STEVOR to localize around the erythrocyte membrane but with an additional asymmetric focus of staining at the “waist” of the gametocyte. In this region, the erythrocyte membrane was closely juxtaposed with the PVM and parasite membrane, beneath the nucleus and the food vacuole with its characteristic pigment deposits (Fig. 3, third row, phase-contrast micrograph). In 17-day gametocytes, STEVOR antibodies bound in a punctate layer associated with, or directly underneath, the infected RBC membrane (Fig. 3, bottom row). These antibodies were raised against conserved amino acid sequences found at the amino and carboxy termini of most STEVOR (16) and are predicted to bind to cytoplasmic, rather than surface-exposed, domains of STEVOR. Thus, STEVOR proteins are present in gametocytes many days after the peak of transcription in early gametocytes.

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FIG. 3.

Expression profile of STEVOR in developing gametocytes. Parasites from staged gametocyte cultures were acetone fixed and coincubated with mouse antisera raised to STEVOR (red), rabbit antisera raised to Pfs16 (green), and nuclear stain TOTO-3 (blue). Secondary antibodies were as described in Materials and Methods. Labeled preparation were examined using either dark-field or phase-contrast confocal microscopy.

Colocalization studies with amino- and carboxy-terminal antisera.Previous studies of stevor transcription in P. falciaprum gametocytes suggest that some spliced transcripts encode truncated STEVOR polypeptides in which amino-terminal sequences are missing (26). To investigate this possibility, colocalization of antisera against STEVOR amino-terminal and carboxy-terminal peptides was investigated. All early, mid-stage, and mature gametocytes that were STEVOR positive reacted with antibodies directed to both the amino and carboxy termini (data not shown). We conclude that if truncated polypeptides are translated in gametocytes, as suggested by the results shown in Fig. 2, they occur in cells that also contain full-length STEVOR.

STEVOR polypeptides do not localize to MC in gametocytes.STEVOR polypeptides are located in MC in mature asexual parasites and remain associated with these vesicular structures in erythrocyte ghosts after schizont rupture (16). MC have previously been found in early gametocytes only (15). Consistent with this finding, antibodies to PfSBP, a marker for MC, recognized a small number of distinct, MC-like structures in 6-day gametocytes, but these were absent from 12- and 17-day gametocytes. We observed colocalization of STEVOR and PfSBP in schizonts, as previously described (16). However, STEVOR antibodies did not colocalize with antibodies against PfSBP in gametocytes (data not shown). Furthermore, trafficking of STEVOR protein occurred after MC had disappeared (Fig. 3). We conclude that although MC are present in early gametocytes, STEVOR polypeptides do not localize to MC in gametocytes and are trafficked to the erythrocyte membrane by an MC-independent process, consistent with the findings by Eksi et al. (10). Thus, the trafficking and localization of STEVOR is distinct in asexual and sexual parasites.

STEVOR polypeptides are expressed in salivary gland sporozoites.Antisera which recognize a conserved amino-terminal peptide found in the majority of STEVOR variants, PepNPH, recognize salivary gland sporozoites of NF54, the parent isolate of clone 3D7 (Fig. 4). In contrast to the surface labeling exhibited by CSP antisera, STEVOR appears to be organized in discrete foci within the sporozoites. This pattern is unlike that observed in either asexual parasites or gametocytes.

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FIG. 4.

Expression of STEVOR in salivary gland sporozoites. Methanol-fixed sporozoites were incubated with mouse sera raised against pepNPH (located in the N-terminal part of STEVOR) followed by a goat anti-mouse IgG coupled to fluorescein isothiocyanate.