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Infect Immun. Oct 2004; 72(10): 6095–6105.
PMCID: PMC517558

Correct Promoter Control Is Needed for Trafficking of the Ring-Infected Erythrocyte Surface Antigen to the Host Cytosol in Transfected Malaria Parasites

Abstract

Following invasion of human erythrocytes, the malaria parasite, Plasmodium falciparum, exports proteins beyond the confines of its own plasma membrane to modify the properties of the host red cell membrane. These modifications are critical to the pathogenesis of malaria. Analysis of the P. falciparum genome sequence has identified a large number of molecules with putative atypical signal sequences. The signals remain poorly characterized; however, a number of molecules with these motifs localize to the host erythrocyte. To examine the role of these atypical signal sequences in the export of parasite proteins, we have generated transfected parasites expressing a chimeric protein comprising the N-terminal region of the P. falciparum ring-infected erythrocyte surface antigen (RESA) appended to green fluorescent protein (GFP). This N-terminal region contains a hydrophobic stretch of amino acids that is presumed to act as a noncanonical secretory signal sequence. Modulation of the timing of transgene expression demonstrates that trafficking of malaria proteins into the host erythrocyte is dependant on both the presence of an appropriate transport signal and the timing of expression. Transgene expression under the control of a trophozoite-specific promoter mistargets the chimeric molecule to the parasitophorous vacuole surrounding the parasite. However, expression of RESA-GFP in schizont stages, under the control of the RESA promoter, enables correct trafficking of a population of the chimeric protein to the host erythrocyte.

Due to the compartmentalization of eukaryotic cells, a sophisticated protein-trafficking system is needed to deliver proteins to their correct destinations. Proteins targeted to compartments other than the cytoplasm are synthesized with peptide motifs that act as a molecular address system (10). For example, in eukaryotes, a hydrophobic sequence located 3 to 17 amino acids from the N terminus can direct proteins across the endoplasmic reticulum (ER) membrane and into the secretory system (20, 26, 27, 36).

The malaria parasite, Plasmodium falciparum, spends part of its life cycle inside the enucleated erythrocytes of its human host, which are cells that lack their own machinery for protein synthesis and trafficking. As a consequence, an unusual and highly specialized secretory system is needed to target parasite proteins to their correct destinations. Indeed the parasite achieves the targeting of proteins not only to compartments within its own confines but to the parasitophorous vacuole (PV) in which it resides and onward to the PV membrane (PVM), the erythrocyte cytoplasm, and the host cell membrane (32, 34). Interestingly, the putative signal sequences of some exported parasite proteins are unusual in that they are recessed, with hydrophobic cores beginning 20 to 80 amino acids from the N terminus (24).

With the advent of transfection protocols for P. falciparum, it has become possible to begin an analysis of the signals that direct proteins to different compartments. For example, the signals that direct the exported protein-1 (Exp1) to the PV (2), the acyl carrier protein (ACP) to the apicoplast (38) and HRP2 (25), and the knob-associated histidine-rich protein (KAHRP) to the host cell cytosol (39) have been examined. For these studies, a series of constructs was designed in which gene fragments encoding the N-terminal regions of ACP, Exp1, HRP2, and KAHRP were appended to the reporter protein green fluorescent protein (GFP). These plasmids were then introduced into P. falciparum by using a stable transfection system, and the locations of the proteins were assessed by fluorescence microscopy. These studies showed that the leader sequence of ACP, the typical N-terminal signal sequence of Exp1 and HRP2, and the recessed signal sequence (hydrophobic core at residues 22 to 33) in KAHRP are all recognized by the ER signal recognition machinery. The signal sequences direct entry into the ER and default trafficking to the PV. These studies further showed that a bipartite signal in ACP mediates transit to the apicoplast and that downstream sequence elements in KAHRP and HRP2 are needed for the translocation of these proteins across the PVM and transfer to the host cell membrane (39).

In this work, we have examined the trafficking of a protein known as the ring-infected erythrocyte surface antigen (RESA). RESA is found in all field isolates of P. falciparum, suggesting that it facilitates survival of the parasite in vivo (29). The protein is produced in the final stages of schizont development and is stored in apical organelles, known as dense granules, within the individual merozoites (3). Following rupture and reinvasion of a new red blood cell by a merozoite, RESA is secreted into the newly formed PV and then transported, by an unknown mechanism, to the red cell membrane skeleton (13). The interaction of RESA with erythrocyte spectrin has been shown to stabilize the erythrocyte membrane (14, 17) and may be involved in repair of the host membrane following invasion.

In an effort to study the mode of trafficking of RESA, we prepared a construct comprising its putative recessed signal sequence fused to GFP and used the plasmid to transfect P. falciparum-infected erythrocytes. When the transgene was expressed under the control of the Hsp86 promoter, which drives expression mainly in trophozoite stage parasites, the chimeric protein was trafficked to the PV. However, when the RESA-GFP construct was expressed under the control of the putative RESA promoter, the timing of expression more closely resembled that of endogenous RESA, and at least part of the population of the chimeric molecules was correctly trafficked to the host erythrocyte. These data indicate that both sequence information and correct promoter control are needed for proper targeting of proteins in the malaria parasite.

MATERIALS AND METHODS

Transfection of P. falciparum with RESA constructs.

The sequence of RESA was obtained from PlasmoDB (http://www.plasmodb.org/plasmodb; gene locus:PFA0110w) and analyzed by using the SignalP algorithm (http://www.cbs.dtu.dk/services/SignalP-2.0/). The sequence encoding amino acid residues 1 to 117 of RESA from strain 3D7 (RESA1-351) was amplified by PCR from a plasmid preparation of a cDNA library (kindly donated by D. Kaslow, National Institutes of Health) (30) with primers 5′-AGATCTATGAGACCTTTTCATGCATATAC and 5′-CCTAGGTTTTTCGAAGGGTAAACCAAATAC (restriction enzyme sites are underlined). The resulting fragment was cloned upstream of the mut2 enhanced-GFP-coding region into the pHH2 vector (pHH2-RESA1-351-GFP) (37, 39). The insert was sequenced in both directions by fluorescent dideoxynucleotide termination. In a second plasmid construct (pRESA5′-RESA1-351-GFP), the Hsp86 promoter region driving the expression of the RESA1-351 fragment was replaced by an 875-bp noncoding region upstream of the RESA open reading frame on chromosome 1. This fragment was amplified by PCR from 3D7 strain genomic DNA with primers 5′-GAAGATCTGTTAGTCGTCTCTGTATTTTTG and 5′-GCATGAAAAGGTCTCATCTCGAGAATTATTTAGATA (restriction enzyme sites are underlined). P. falciparum-infected erythrocytes (strain 3D7) were transfected with the pHH2-RESA1-351-GFP or pRESA5′-RESA1-351-GFP plasmid and cultured in the presence of WR99210 (10 nM) as described earlier (15, 39).

Northern analysis.

Aliquots (30 ml, ~5% parasitemia) of tightly synchronized cultures (~4-h window) were sampled at 4-h intervals. RNA was prepared, separated on agarose gels, and transferred overnight to nylon membrane as described by Kyes et al. (23). Prehybridization was performed in Ultrahyb (Ambion) solution at 42°C for 1 h, and the α-[32P]dATP-labeled DNA probes (GFP or RESA open reading frame fragments) were hybridized to the RNA overnight at 42°C. The membranes were washed several times in 2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and exposed to Hyperfilm MP (Amersham) overnight at −80°C. The films were scanned and the images were analyzed with background correction and normalization by using NIH ImageJ (http://rsb.info.nih.gov/ij).

Subcellular fractionation and Western blot analysis of transfectants.

Transfectants were synchronized by sorbitol treatment, cultured to a parasitemia of 10% trophozoite stage parasites, and harvested by using Plasmagel as described by Heidrich et al. (21). For streptolysin O (SLO) lysis, 90 μl of toxin (~1,000 U; Sigma) in phosphate-buffered saline was mixed with 10 μl of 1 M dithiothreitol in phosphate-buffered saline, activated for 15 min at room temperature, and tested for the ability to lyse erythrocytes as described by Baumeister et al. (7). To release the erythrocyte contents, approximately 5 × 107 parasites were treated with 4 hemolytic units and separated into pellets and supernatants. To release the erythrocyte and PV contents, 5 × 107 parasites were subjected to lysis in the presence of 0.09% saponin (Sigma) and separated into soluble and particulate fractions by centrifugation (12). Equivalent fractional amounts of each of the samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (12.5% acrylamide) and Western analysis.

For immunoblot analyses, asynchronous or highly synchronous P. falciparum cultures were frozen, thawed, and resuspended in Laemmli buffer. Samples were subjected to SDS-PAGE (12.5% acrylamide) and subsequently transferred to a nitrocellulose membrane, probed with antiserum followed by horseradish peroxidase-conjugated secondary antibodies (Sigma), and developed with enhanced chemiluminescence reagent. Western blot membranes were probed with antibodies recognizing GFP (murine monoclonal; Roche), RESA (monoclonal antibody [MAb] 28/2) and P. falciparum S antigen (rabbit antiserum against strain 3D7) (both antibodies were obtained from Robin Anders, La Trobe University), and Hsp70 (rabbit antiserum) (9). The films were scanned and the images analyzed with background correction by using NIH ImageJ (http://rsb.info.nih.gov/ij).

Fluorescence microscopy.

Parasitized erythrocytes expressing RESA1-117-GFP (>5% rings) were tightly synchronized by sorbitol treatment and sampled every 6 h. Live cells were mounted wet on a glass slide, covered by a glass coverslip, and imaged within 20 min at ambient temperature, using exposure settings that optimized the signal with a Zeiss Axioskop 2 microscope equipped with a PCO SensiCam (12-bit) camera and Axiovision 3 software. Captured images were processed by using Corel Draw and ImageJ software (http://rsb.info.nih.gov/ij). The contrast of the images was adjusted to visualize features of interest. For immunofluorescence analysis, methanol-fixed smears of asynchronous parasite cultures (strain 3D7) were probed with anti-RESA MAb (28/2) and rabbit anti-GFP (provided by Michael Ryan, La Trobe University) and subsequently incubated with secondary antibodies Alexa Fluor 568-conjugated anti-rabbit immunoglobulin G (IgG) (Molecular Probes) and fluorescein isothiocyanate-conjugated anti-mouse IgG (Dako). Semiquantitative image analysis of the 8-bit confocal images and estimation of Pearson's coefficients as a measure of overlap of the red and green channels were performed with the Image Correlator 3 and LUT importer plug-ins in the ImageJ software as described previously (1).

Electron microscopy.

An asynchronous culture of approximately 10% parasitemia was fixed with 1% glutaraldehyde in RPMI-HEPES (pH 7.2) on ice for 1 h, washed, and transferred into agarose blocks, which were dehydrated and embedded in LR Gold resin. Alternatively, cells were gently pelleted and 5-μl droplets of the cell pellet were sandwiched between brass freezer hats (type A; ProSciTech). The enclosed cell suspensions were subjected to high-pressure freezing with a Leica EM high-pressure freezer, the freezer hats were split apart, and the frozen cell suspensions were stored under liquid nitrogen. Frozen cell pellets were freeze-substituted in 0.1% uranyl acetate in acetone at −90°C for 72 h, and the temperature was raised to −50°C at 6°C per h. The cell pellets were scraped out of the freezer hats and rinsed in three 30-min changes of acetone. The samples were infiltrated with a graded series of HM20 low-temperature resin in acetone, consisting of 25% resin (8 h), 50% resin (overnight), 75% resin (8 h), and 100% resin (overnight). The infiltrated samples were placed in a fresh change of 100% resin in gelatin capsules, polymerized under UV light for 48 h at −50°C, and brought to room temperature at 6°C per h. The soft sample blocks were hardened under UV light for a further 24 h at room temperature. Thin sections were prepared and incubated with the anti-RESA MAb 28/2, followed by 10-nm-diameter gold-labeled goat anti-mouse IgG and/or rabbit anti-GFP, followed by anti-rabbit IgG conjugated to 15-nm-diameter gold particles. Sections were poststained with uranyl acetate and lead citrate to enhance contrast before examination in a JEOL JEM 2010HC transmission electron microscope. Semiquantitative analysis of the distribution of particles was done by counting particles of different sizes in the different compartments.

RESULTS

Amino acids 1 to 117 of RESA, when fused to GFP, allow secretion of the chimera in transfected P. falciparum.

The arrangement of the RESA gene and the encoded protein sequence (strain 3D7) are shown in Fig. Fig.1B.1B. Exon 1 of the RESA gene encodes the first 65 amino acids of the protein (Fig. 1B and C). Residues 52 to 64 are postulated to form the hydrophobic core of a recessed signal sequence, and the SignalP algorithm predicts a possible cleavage site after residue 69. The N-terminal region of the RESA sequence presumably contains the information to direct the protein into the secretory pathway and to the dense granules for storage. Following the eventual release of the dense-granule contents into the PV in the newly invaded ring stage parasite, additional information is presumably required to direct RESA across the PVM and to allow it to bind to spectrin in the erythrocyte membrane skeleton.

FIG. 1.
Organization of the RESA gene and transfection constructs. (A) Schematic diagram of the pHH2 and pRESA5′ constructs, showing insertion of RESA1-117 upstream of the GFP coding region. (B) Schematic diagram of the RESA gene. Exon 1 encodes residues ...

To study the signals involved in directing these trafficking events, we have generated a chimera comprising the coding sequence for the first 117 amino acids of RESA linked to the 5′ end of the coding region of mut2 enhanced GFP as a reporter molecule. The RESA1-351-GFP chimera was inserted into the transfection vector pHH2 (pHH2-RESA1-351-GFP), from which expression of the coding sequence for RESA1-117-GFP is driven by the Hsp86 promoter (40) (Fig. (Fig.1A).1A). In addition, we have generated a novel transfection vector, pRESA5′, in which 875 bp of sequence upstream from the P. falciparum 3D7 RESA-coding region on chromosome 1 (presumed to include the RESA promoter region) was used to replace the Hsp86 promoter region. For both pHH2-RESA1-351-GFP and pRESA5′-RESA1-351-GFP, the resultant GFP fusion proteins are expressed from a stably maintained episome within the transformed 3D7 P. falciparum blood stage parasites.

Parasites expressing the GFP chimeric protein under the control of the Hsp86 promoter (RESA1-117-GFPHsp86) and the RESA promoter (RESA1-117-GFPRESA) were obtained 53 and 28 days after transfection, respectively. The transfected parasites were maintained in culture in the presence of a 10 nM concentration of the antifolate drug WR99210. They showed growth rates that were similar to those of each other and to those of untransfected parasites. This suggests that the expression of the GFP chimera did not confer a major growth disadvantage on the parasites.

Asynchronous parasite cultures of both transfectant lines were subjected to Western blotting and probed with an anti-RESA MAb (MAb 28/2) which recognizes the 3′ repeat region of full-length RESA (5). A rabbit antiserum against GFP was used to detect the transgene product. MAb 28/2 recognized a band of approximately 155 kDa (Fig. (Fig.2B,2B, upper panels), as reported previously (5, 17). When the same membrane was probed with antibodies recognizing GFP, no reactivity was observed in uninfected erythrocytes (not shown) or in the untransfected parental line, 3D7 (Fig. (Fig.2B,2B, lane a). In contrast, the RESA1-117-GFPHsp86 and RESA1-117-GFPRESA transfectants expressed proteins that were resolved as a doublet of approximately 32 and 31 kDa and a band of 27 kDa (Fig. (Fig.2B,2B, lanes b and c). The predicted size of the RESA1-117-GFP chimera is 40.3 kDa, which would be reduced to 32.2 kDa after cleavage of the predicted 69-amino-acid signal. Thus, it is likely that the 32-kDa band represents the initial processed form of RESA1-35-GFP in which the putative signal sequence has been removed, while the 31-kDa band results from further processing from the N-terminal end. This may occur in the ER or in a downstream compartment. In this context, it is interesting that endogenous RESA has been shown to migrate as a fine doublet under some conditions (17). The 27-kDa band presumably represents a further degradation product. A band of similar molecular mass has been reported previously (2, 39), and a direct comparison revealed that the RESA-GFP migrated at the same apparent molecular mass as the KAHRP-GFP degradation product that we have reported previously (reference 36 and data not shown). This product is thought to represent a processing event that digests the chimera back to the protease-resistant GFP core. It is presumed that this event occurs in the parasite food vacuole.

FIG. 2.
Transgene expression in parasites transfected with the RESA1-117-GFP constructs. (A) RNA was prepared from tightly synchronized cultures (~4-h window) collected at 4-h intervals, separated on agarose gels, and transferred to nylon membranes. The ...

Timing of expression of RESA1-117-GFPHsp and RESA1-117-GFPRESA.

To examine the temporal control of expression of the transgene under control of the different promoters, we undertook a Northern blot analysis of culture samples collected at different time intervals. We employed a probe that hybridizes only to the endogenous RESA gene and a probe that hybridizes to the GFP gene fragment in the transgene. We found maximal transcription of endogenous RESA in mature-stage parasites (40 to 48 h) (Fig. (Fig.2A),2A), in agreement with previous studies (11). By contrast, RESA1-117-GFPHsp86 was transcribed in ring and trophozoite stages, with a marked decrease in transcription in very-mature-stage parasites (Fig. (Fig.2A).2A). When expressed under the control of the endogenous promoter, the RESA-GFP chimera was maximally transcribed in late-stage parasites (44 h) but also in ring stage parasites (Fig. (Fig.2A2A).

Expression patterns were also examined at the protein level. Endogenous RESA has previously been shown to be present at high levels throughout the ring stage parasites but to decrease as the parasite matures, presumably due to proteolytic degradation (references 11 and 16 and data not shown). In this work, culture samples were collected at 4-h intervals and examined by Western blot analysis (Fig. (Fig.2C).2C). The endogenous housekeeping protein Hsp70 was used as a loading control (Fig. (Fig.2C,2C, upper panels). The level of expression of the transgene was significantly higher in RESA1-117-GFPHsp86 transfectants as judged by the required exposure times for the blots. A semiquantitative analysis of the level of RESA1-117-GFPHsp86 relative to Hsp70 revealed maximum expression of the transgene in mid-trophozoite stages (20 to 36 h postsynchronization). For example, the apparent transgene/Hsp70 ratio is 17-fold higher at 28 h than at 16 h. By contrast, expression of the transgene under the control of the putative RESA promoter was associated with high levels of the fusion protein in very-mature-stage parasites (44 to 48 h) and in the early ring stage (4 h postsynchronization) (Fig. (Fig.2C,2C, lower panels). The apparent transgene/Hsp70 ratio is 6.5-fold higher at 44 h and 8.8-fold higher at 4 h than at 16 h. It is of interest that a 43-kDa band was observed at 20 to 36 h after synchronization for the RESA1-117-GFPHsp86 transfectants and at 36 to 48 h for the RESA1-117-GFPRESA transfectants (Fig. (Fig.2C).2C). This species might represent the uncleaved protein, which is more readily visualized when it is first synthesized. Similarly, the 32/31-kDa species was present maximally at 24 to 32 h in the RESA1-117-GFPHsp86 transfectants and at 44 to 48 h and 4 h in RESA1-117-GFPRESA transfectants (Fig. (Fig.2C).2C). The level of the 27-kDa degradation product also varied with parasite stage.

Effect of promoter control on localization of the RESA1-117-GFP chimera.

Fluorescence microscopy of live cells was used to examine the locations of the chimeras at different stages of growth (Fig. (Fig.3).3). It is important to note that the intensities of the images have been adjusted to optimize the fluorescence signal at each parasite stage. The RESA1-117-GFP chimera, when expressed under control of the Hsp86 promoter, is located largely in the PV that surrounds the parasite (Fig. (Fig.3,3, left panels). Some parasites showed a “necklace-of-beads” pattern around the periphery of the parasite (Fig. 3B and C), as has been reported previously for chimeras of N-terminal fragments of KAHRP and Exp1 with GFP (2, 39). Fluorescence photobleaching experiments were performed as described previously (2, 39) to examine the dynamics of the GFP chimera. These studies indicated that the chimera is present as a soluble protein within the PV (data not shown). However, the GFP fusion protein exhibited a lower-than-predicted diffusion rate and appeared to be restricted to particular subcompartments of the PV. Similar results have been reported for the KAHRP- and Exp1-GFP chimeras (2, 39).

FIG. 3.
Expression of the RESA1-117-GFP chimeric proteins at different stages of the intraerythrocytic life cycle of P. falciparum. The first image in each set represents the fluorescence signal from the GFP chimeric protein, the second is a bright-field or phase ...

In some cells the fluorescence was also observed in small compartments extending into the cytoplasm of the erythrocyte (Fig. 3D and E) and in a compartment within the parasite that appears to be the food vacuole (Fig. (Fig.3D).3D). It is important to note, however, that at the pH of the food vacuole (~pH 5), GFP fluorescence may be somewhat quenched (28). Schizont stage parasites displayed a segmented pattern, which indicates that the fusion protein surrounds the individual merozoites (Fig. (Fig.3E).3E). This again is consistent with trafficking of the protein to the PV. Upon lysis of the PVM, a “bunch-of-grapes” pattern around a fluorescent central remnant body remained, which points to the presence of a population of the chimera within the individual merozoites in addition to the PV-located population (Fig. (Fig.3F).3F). Also, a second remnant body was always observed in these stages (Fig. (Fig.3F3F).

These results suggest that the first 117 amino acids of the RESA protein are sufficient for entry into the secretory system and secretion from the parasite into the PV. However, when expressed under the control of the Hsp86 promoter, the RESA-GFP chimera is not able to access the secretory pathway that directs endogenous RESA into the erythrocyte cytosol.

When expressed under the control of the putative RESA promoter, the RESA1-117-GFP chimera is partially translocated into the host cell cytosol (Fig. 3G to L). Very-early-stage parasites retain the necklace-of-beads pattern around the periphery of the parasite (Fig. (Fig.3G).3G). However, as the parasites mature, they exhibit a dual-location pattern, with populations of the chimera located in both the parasite and the host cell cytosol (Fig. 3H to K). The level of RESA1-117-GFPRESA in the erythrocyte cytosol appears to be less than that in the parasite compartments, although it is important to remember that early-stage parasites occupy only a fraction of the volume of the erythrocyte. Nonetheless, it appears that RESA1-117-GFPRESA is less efficiently transported to the host cytosol than endogenous RESA (see Fig. Fig.5).5). The exported population appears to be diffusely distributed throughout the erythrocyte cytosol, rather than bound to the cytoskeleton as has been shown for endogenous RESA. This presumably reflects the absence of the spectrin-binding domain in the chimeric protein.

FIG. 5.
Immunofluorescence microscopy analysis of endogenous RESA and RESA1-117-GFP in transfected parasitized erythrocytes. Erythrocytes infected with RESA1-117-GFPHsp86 (A) or RESA1-117-GFPRESA (B) transfectants at the ring stage (top panels), trophozoite stage ...

Analysis of the intracellular location of the RESA1-117-GFP chimeras by subcellular fractionation.

Given the difficulty of quantitating the level of GFP in the erythrocyte cytosol and parasite compartments from fluorescence micrographs, we have employed selective permeabilization protocols to determine the levels of RESA-GFP in the different compartments. The transfected parasites were subjected to treatment with 0.09% saponin, which disrupts the host cell membrane and the PV membrane, or to SLO treatment, which permeabilizes only the host cell membrane (Fig. (Fig.4).4). As expected, treatment of the transfected parasitized erythrocytes with saponin released most of the PV-located S antigen (Fig. (Fig.4,4, top panels), while the intraparasitic component, ER-located calcium-binding protein (PfERC), remained associated with the pellet fraction (Fig. (Fig.4,4, middle panels). Under these conditions, both the RESA1-117-GFPHsp86 (Fig. (Fig.4A,4A, bottom panel) and the RESA1-117-GFPRESA (Fig. (Fig.4B,4B, bottom panel) chimeras were largely released into the supernatant, indicating a PV or erythrocyte cytosol location (note that the protein released in the RESA1-117-GFPHsp86 cells represents the 32-kDa species, while that in the RESA1-117-GFPRESA cells is the 31-kDa species). Treatment of the transfectants with 4 hemolytic units of SLO released more than 97% of the hemoglobin (data not shown) but not the S antigen (Fig. (Fig.4,4, top panels) or PfERC (Fig. (Fig.4,4, middle panels). Under these conditions, the RESA1-117-GFPHsp86 remained in the pellet (Fig. (Fig.4A,4A, bottom panel), while RESA1-117-GFPRESA was partly released (Fig. (Fig.4B,4B, bottom panel). This indicates the export of a part of the population (~30%) of the RESA1-117-GFPRESA chimera into the erythrocyte cytosol, while RESA1-117-GFPHsp86 remains trapped in the PV. Interestingly, the population of the chimera that is released by SLO into the erythrocyte cytosol in the RESA1-117-GFPRESA transfectants has a molecular mass of 31 kDa, while the population that is trapped in the parasite comprises mainly the 32- and 27-kDa species.

FIG. 4.
Subfractionation of the RESA1-117-GFP transfectants. Transfectants were grown to trophozoite stage after synchronization, Plasmagel purified (approximately 1 × 107 RESA1-117-GFPHsp86 and 5 × 107 RESA1-117-GFPRESA transfectants), solubilized ...

RESA1-117-GFPHsp86 and RESA1-117-GFPRESA transfectants show different localization patterns in immunofluorescence assays.

In order to compare the subcellular locations of the RESA1-117-GFP chimeras in the two transgenic parasite lines with that of endogenous RESA, we performed immunofluorescence microscopy with an anti-RESA MAb (which recognizes only the endogenous protein) and an anti-GFP antiserum (Fig. (Fig.5).5). In early-ring stage parasites, the anti-RESA antibody revealed some endogenous RESA associated with the PV (Fig. (Fig.5A,5A, top panel), which appeared to be relocated to the erythrocyte cytosol as the parasite matured (Fig. (Fig.5A5A middle panel, and B, top two panels). In schizont stage parasites, the antibody against endogenous RESA revealed a punctate fluorescence pattern that is consistent with labeling of dense granules in individual merozoites (Fig. (Fig.5,5, bottom panels). In ring stage RESA1-117-GFPHsp86 transfectants, the anti-GFP antiserum recognized punctate compartments within the PV, in accordance with the subcompartments seen in live cells (necklace of beads). Analysis of green (RESA) and red (GFP) pixel overlap in a series of five images of ring and early-trophozoite stage-infected erythrocytes yielded a Pearson's coefficient of 0.28. By contrast, in the RESA1-117-GFPRESA transfectants, the anti-GFP antiserum recognized the chimera within both the parasite and erythrocyte compartments, with significant overlap of labeling of endogenous RESA (green) and the chimera (red) in the host cell compartment (Fig. (Fig.5B,5B, top and middle panels) (the average Pearson's coefficient for red and green overlap for 5 cells is 0.57). In the schizont stage in both transfectant lines, the GFP chimera showed partial overlap with endogenous RESA (Fig. (Fig.5,5, lower panels), although it is clear that the transgene product is also present in additional compartments. The limited resolution of the fluorescence images precludes accurate quantitation of the degree of overlap within the merozoites. Nonetheless, the immunofluorescence data are consistent with the suggestion that while some of the population of chimeric proteins in the RESA1-117-GFPRESA transfectants is correctly trafficked to the erythrocyte cytosol, some appears to be mistargeted and accumulates within the parasite or PV.

Ultrastructural analysis of the RESA1-117-GFPHsp86 and RESA1-117-GFPRESA transfectants.

The intracellular location of RESA1-117-GFP chimeras was further assessed by immunoelectron microscopy of samples of parasitized erythrocytes by using antibodies recognizing RESA and GFP (Fig. (Fig.6).6). In ring- to early-trophozoite stage parasites, endogenous RESA is located mainly at the erythrocyte membrane (Fig. (Fig.6).6). Indeed, an analysis of sections from three different cells revealed that 78% of the 10-nm-diameter particles were in the erythrocyte cytosol or closely associated with the erythrocyte membrane. Gold particles were also occasionally observed associated with the PV (Fig. (Fig.6A,6A, panel b; Fig. Fig.6B,6B, panel a) (7% of 10-nm-diameter particles) and with structures in the parasite cytosol (Fig. (Fig.6A,6A, panel c; Fig. Fig.6B,6B, panel a) (15% of 10-nm-diameter particles). These finding are in agreement with previous reports (3, 13) and lead to the suggestion that the PV is a transit compartment for RESA en route to the host cell membrane. When RESA1-117-GFPHsp86 transfectants were examined in dual-labeling protocols, gold particles were almost completely restricted to the parasite cytosol (65% of 15-nm-diameter particles) and the PV (Fig. (Fig.6A,6A, panels a and c) (30% of 15-nm-diameter particles). By contrast, an analysis of sections from tranfectants in which the RESA1-117-GFPRESA was expressed under the control of the RESA promoter revealed gold particles in the host cell cytosol (70% of 15-nm-diameter particles) as well as in the parasite cytosol (20% of 15-nm-diameter particles) and in the PV (10% of 15-nm-diameter particles) (Fig. (Fig.6B).6B). The exported population of RESA1-117-GFPRESA appears to be free in the host cell cytosol, while endogenous RESA is located predominantly at the erythrocyte membrane.

FIG. 6.
Transmission electron microscopy and immunogold labeling of RESA1-117-GFP transfectants. Sections were probed with rabbit anti-GFP, followed by 15-nm-diameter gold-conjugated anti-rabbit IgG and/or 28/2 anti-RESA MAb, followed by 10-nm-diameter gold-conjugated ...

DISCUSSION

Proteins destined for extraparasitic locations in malaria parasite-infected erythrocytes are thought to transit through the ER and the PV prior to transfer to the erythrocyte cytosol (6, 39); however, the nature of the polypeptide secretory signals that direct proteins to their correct destinations is currently the subject of some debate (2, 12, 39). An analysis of the sequences of exported parasite proteins reveals some rather unusual motifs. Proteins destined for sites in the ER, the parasite plasma membrane, the PV, or the PVM appear to have a “classical” hydrophobic N-terminal signal sequence, i.e., a stretch of 10 to 15 hydrophobic amino acids commencing 3 to 17 amino acids from the N terminus (35). By contrast, many proteins that are directed past the PVM to the erythrocyte cytosol have a hydrophobic stretch of amino acids that is recessed (20 to 80 amino acids) from the N terminus (4). These exported proteins include proteins expressed in trophozoite stage parasites, such as KAHRP and the mature parasite-infected erythrocyte surface antigen, as well as RESA, which is synthesized in schizonts and trafficked to the erythrocyte membrane shortly after reinvasion.

The release of the complete P. falciparum genome sequence (18) and the advent of transfection of malaria parasites has led to attempts to dissect plasmodial trafficking signals. Work from Waller et al. (38), Wickham et al. (39), Adisa et al. (2), and Lopez-Estrano et al. (25) indicates that the noncanonical signal sequence of KAHRP and the classical signal sequences of ACP, Exp1, and HRP2 can direct default transport to the PV but that additional signal information is needed for export beyond this point. It has been suggested that basic amino acids in the region downstream from the hydrophobic region might play a role in trafficking of proteins across the PV (25). However, it is not clear whether proteins such as KAHRP and HRP2, which are secreted directly after synthesis, use the same export pathway as proteins such as RESA, which transit via a storage granule.

In this work, we have examined the trafficking of RESA by preparing two chimeric gene constructs encoding an N-terminal fragment of RESA linked to the reporter protein GFP. The two constructs differed only in their promoter regions. The HH2 plasmid contains 848 bp of the Hsp86 5′ untranslated region. This construct has previously been used successfully for studies of trophozoite stage-expressed proteins (2, 39). In addition, we replaced the Hsp86 5′ untranslated region with 875 bp of the RESA 5′ untranslated region. This construct was used in an effort to obtain correct timing of expression of the RESA transgene. Both plasmids were used to prepare stably transfected P. falciparum.

The timing of transcription of the transgene under control of the two promoters was examined by Northern and Western blot analyses. As expected, RESA1-117-GFPHsp86 was maximally expressed in trophozoite stage parasites. The expression pattern for RESA1-117-GFPRESA more closely represented that of endogenous RESA in that there were increased levels of the fusion protein in schizont and early ring stages; however, in contrast to endogenous RESA, the transgene was also expressed throughout the ring stages. There are various reasons why the timing of expression of the chimeric protein might not be identical to that for endogenous RESA. Two possibilities are that expression from the RESA promoter in this episomal plasmid context is somewhat leaky or that additional promoter elements upstream of the cloned region are needed for very tightly controlled timing of expression.

In ring stage parasites, the RESA-GFP chimera was present in a necklace-of-beads pattern as has been reported previously for KAHRP- and Exp1-GFP chimeras (2, 39). Photobleaching analysis of GFP chimeras in the PV indicates that the individual “beads” or subcompartments are not fully interconnected (reference 2 and data not shown). Moreover Spielmann et al. (31) have recently provided evidence for distinct locations for two PV-associated proteins, and Behari and Haldar (8) have previously reported proteins that localized to subcompartments of the PVM. These compartments may represent specialized subdomains of the PV where proteins destined for forward transit are separated from PV-resident proteins.

Western blot analysis indicated that the RESA1-117-GFP was processed to a doublet with an apparent molecular mass of 32 and 31 kDa, presumably by removal of the N-terminal segment by a signal peptidase in the ER and further processing within the secretory system. A 27-kDa species was also observed for both chimeras. Previous studies have suggested that the 27-kDa species represents a proteolytically resistant GFP core and may result from GFP chimera that is mistargeted to the food vacuole (2, 38, 39).

Live cells were examined by fluorescence microscopy to determine the subcellular locations of the GFP reporter in the two transfectants. When expressed under the control of the Hsp86 promoter, RESA1-117-GFP is present in the PV and in limited extensions of the PV as well as in a compartment within the parasite cytoplasm that appears to be the food vacuole. There is no detectable release of GFP into the erythrocyte cytosol. When expressed under the control of the putative RESA promoter, a subpopulation of the GFP chimera is exported into the erythrocyte cytosol. The data obtained with live cells were confirmed by using immunofluorescence and immunoelectron microscopy. These techniques also showed that the PV is an intermediate compartment in the secretory process for both endogenous RESA and RESA1-117-GFPRESA.

Selective permeabilization studies have previously been used to examine the subcellular locations of parasite proteins (2, 7, 12). We used these protocols in an attempt to obtain a semiquantitative estimate of the level of RESA1-117-GFPRESA export. These data indicated that ~30% of the total population of the chimera is released into the host cell cytosol. The RESA1-117-GFPRESA molecules in the erythrocyte cytosol exhibit a diffuse fluorescence pattern, which indicates that the protein is present as a soluble protein in this compartment. By contrast, endogenous RESA associates with spectrin molecules within the membrane skeleton (17). Previous studies have mapped the spectrin-binding domain of RESA to a 48-amino-acid region located between the two blocks of repetitive amino acid sequence, which is not present in the chimeric proteins. Therefore, the chimera generated in these studies would not be expected to bind to spectrin.

The RESA1-117-GFP chimera apparently is trafficked to the erythrocyte cytosol only if the timing of expression is correct. Given that there are no coding sequence differences between the two RESA constructs examined in this study (and that the PV appears to be an intermediate depot in RESA trafficking), the question of how the parasite differentiates between the two constructs arises. It is likely that proteins that are synthesized in late schizogony, such as RESA, rely on a “just-in-time” sorting strategy. That is, they may need to be synthesized at the precise time when the secretory organelles for which they are destined (i.e., the dense granules) are being formed. Passage of the RESA1-117-GFP chimera through the dense granules may deliver the protein to a specialized region of the PV from which forward transit can occur. Alternatively, passage through the dense granules could allow a critical proteolytic processing event to occur, and it is interesting that the chimera released into the erythrocyte cytosol in RESA1-117-GFPRESA is preferentially the 31-kDa species. This processing event may be an important step in correct delivery of the chimera. It is interesting that precise timing of expression of the P. falciparum apical membrane protein has also been shown to be critical for its correct trafficking to an apical compartment (22, 33). This compartment has been recently shown to be the micronemes (19). Thus, a strict timing mechanism also appears to operate for delivery of other schizont stage proteins. Clearly, further analysis of these complex pathways is needed, and an increased understanding of the routes for protein trafficking in parasitized erythrocytes may point to novel targets for inhibiting parasite development.

Acknowledgments

This work was supported by the National Health and Medical Research Council, Australia. M.R. was supported by a fellowship from the Deutscher Akademischer Austauschdienst (DAAD). M.W. was supported by an Australian Postgraduate Research Award.

We thank Nick Klonis, Michael Ryan, and Robin Anders (La Trobe University) for providing antibodies and for useful discussions. We thank Denise Fernando, Simon Crawford, and Rob Glaisher for assistance with the electron microscopy protocols.

Notes

Editor: W. A. Petri, Jr.

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