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J. Biol. Chem., Vol. 278, Issue 28, 25977-25981, July 11, 2003

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PfSPATR, a Plasmodium falciparum Protein Containing an Altered Thrombospondin Type I Repeat Domain Is Expressed at Several Stages of the Parasite Life Cycle and Is the Target of Inhibitory Antibodies^*

Rana Chattopadhyay, Dharmendar Rathore , Hishasi Fujioka , Sanjai Kumar, Patricia de la Vega, David Haynes, Kathleen Moch, David Fryauff, Ruobing Wang, Daniel J. Carucci and Stephen L. Hoffman ¶

From the Malaria Program, Naval Medical Research Center, Silver Spring, Maryland 20910-7500, Laboratory of Malaria and Vector Research, NIAID, National Institutes of Health, Bethesda, Maryland 20892-0425, and the Institute of Pathology, Case Western Reserve University, Cleveland, Ohio 44106

Received for publication, January 27, 2003 , and in revised form, April 11, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The annotated sequence of chromosome 2 of Plasmodium falciparum was examined for genes encoding proteins that may be of interest for vaccine development. We describe here the characterization of a protein with an altered thrombospondin Type I repeat domain (PfSPATR) that is expressed in the sporozoite, asexual, and sexual erythrocytic stages of the parasite life cycle. Immunoelectron microscopy indicated that this protein was expressed on the surface of the sporozoites and around the rhoptries in the asexual erythrocytic stage. An Escherichia coli-produced recombinant form of the protein bound to HepG2 cells in a dose-dependent manner and antibodies raised against this protein blocked the invasion of sporozoites into a transformed hepatoma cell line. Sera from Ghanaian adults and from a volunteer who had been immunized with radiation-attenuated P. falciparum sporozoites specifically recognized the expression of this protein on transfected COS-7 cells. These data support the evaluation of this protein as a vaccine candidate.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Plasmodium parasites cause malaria and are transmitted by the bite of infected mosquitoes. Numerous candidate genes and strategies have been evaluated in an attempt to develop malaria vaccines (1–5), yet there is currently no licensed malaria vaccine, and it is unlikely that there will be one for at least a decade (6). The complex life cycle of the parasite with its distinct morphological and antigenic stages has been a major hurdle in developing such vaccines. It is anticipated that use of data from the recently completed genomic sequence of Plasmodium falciparum (7) will lead to an increased understanding of parasite biology that will eventually be translated into new drugs and vaccines for malaria (8).

Chromosome 2 was the first chromosome of P. falciparum to be sequenced, and initial analysis indicated that there were 209 genes on this chromosome (9). In an effort to discover additional protein candidates for vaccine development, we sought to characterize one of the genes from chromosome 2 of P. falciparum, which had been annotated as a putative secreted protein containing a thrombospondin Type I repeat (TSR)¹ domain (9). The TSR is an ancient eukaryotic domain (10) now known to be present in more than 300 different proteins (11), including surface antigens of pathogenic microorganisms (12). Numerous Plasmodium surface antigens have been shown to possess the TSR domain (13–15), and these proteins have been shown to be involved in ookinete and sporozoite motility, host cell attachment, and invasion (16–19), thus making them potentially good vaccine targets. In addition to a TSR domain, we found that the predicted protein also possessed a cysteine-rich signature that could represent a Type II EGF-like domain. The orthologue of this protein is present in the murine malaria parasite Plasmodium yoelii and named "secreted protein with altered thrombospondin repeat" or SPATR (20).

We have characterized its expression, localization, and function at different stages of the Plasmodium life cycle. We report that this protein is expressed at several stages of the life cycle, that it binds to hepatoma cells, and that antibodies to this protein inhibit P. falciparum sporozoite invasion of liver cells.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Reverse Transcription-PCR and Cloning—Total RNA was isolated from P. falciparum sporozoites, synchronized erythrocytic stages (ring, trophozoite, and schizont), and gametocytes using a High Pure RNA Isolation Kit (Roche Diagnostics). 2 µg of RNA from each of these stages was reverse transcribed using random hexamers and Superscript II RNase H^– reverse transcriptase (Invitrogen). 5 µl of the reverse transcribed product of each of the above stages was subjected to PCR using PfSPATR-specific primers. Primer design was based on the published sequence with GenBank^TM accession number AE001404 [GenBank] (9). The amplified products were cloned into TA Cloning vector (Invitrogen) and sequenced.

Recombinant Protein Expression and Purification—For protein expression, a 690-bp cDNA encoding the mature form of PfSPATR (without signal sequence) was cloned as a BamHI and EcoRI fragment into pGEX-3X (Amersham Biosciences), a GST-based Escherichia coli expression vector. The recombinant protein was expressed in BL21 Escherichia coli cells, and the expression was induced with 1 mM isopropyl-1-thio--D-galactopyranoside. The protein was purified on a glutathione-agarose column as per the manufacturer's instructions (Amersham Biosciences).

Generation of Anti-PfSPATR Serum in Mice—Outbred CD-1 mice were immunized intraperitoneally with 10 µg of the purified protein in Freund's complete adjuvant and boosted 3 and 6 weeks after the first immunization with 10 µg of protein in Freund's incomplete adjuvant. Sera were collected 12 days after the third dose. Anti-GST antibodies were depleted by passing the sera through an immobilized GST column (Pierce), which was confirmed by Western blot.

Immunofluorescence Assay—Spots of P. falciparum sporozoites and smears of erythrocytic stages and gametocytes were made on glass slides. Anti-PfSPATR serum at 1:20 to 1:6400 dilutions were added and incubated in a moist chamber at 37 °C. After 1 h, unbound material was removed by washing, and anti-mouse IgG-fluorescein isothiocyanate conjugate was added. Unbound conjugate was removed, and the slides were observed under UV light in a fluorescence microscope. Pre-immune mice sera were used as controls.

Immunoelectron Microscopy—Immunoelectron microscopy was carried out on sporozoites isolated from infected mosquito salivary glands and in vitro cultured blood stages of P. falciparum (Clone 3D7) using 1:40 anti-PfSPATR antiserum as described (21). Pre-immune sera were used as control.

Expression of PfSPATR on COS-7 Cells—DNA encoding the full-length open reading frame of PfSPATR was cloned in plasmid pRE4 (22), a mammalian expression vector, and the endotoxin-free plasmid was transfected into COS-7 cells using Lipofectin. Expression of PfSPATR was evaluated by immunofluorescence using murine anti-PfSPATR antibodies and human serum samples obtained from 1) naturally immune adult, lifelong residents of P. falciparum hyperendemic area in Ghana, 2) malaria-naive volunteers immunized with irradiated P. falciparum sporozoites, and 3) their controls exposed to the bite of uninfected mosquitoes (23). All sera were used at dilutions ranging from 1:50 to 1:400. The use of human serum samples for this experiment was approved by the institutional review board at the Naval Medical Research Center.

Cell Binding Assay—The hepatoma cell line HepG2 was used to evaluate the binding activity of PfSPATR. Cells were seeded in a 96-well plate a day before the start of the experiment. The next day the recombinant proteins, 0 to 1.00 µM, were added to the paraformaldehyde-fixed cells and incubated at 37 °C for 1 h. Unbound material was removed followed by the addition of murine anti-protein antibodies. After a 1-h incubation at 37 °C, alkaline phosphatase-conjugated goat anti-mouse antibody was added, and the bound protein was measured by a fluorescence-based assay using 4-methyllumbelliferyl phosphate as substrate (24).

In Vitro Inhibition of Sporozoite Invasion (ISI) in HepG2 Cells—The ISI assay was performed as described (19). Briefly, 50,000 HepG2 cells were placed in each of the eight wells of tissue culture slides 2 days before the experiment. P. falciparum (NF54) sporozoites were isolated from mosquito salivary glands using a discontinuous gradient as described (25). 20,000 sporozoites were added to the cells along with anti-PfSPATR serum at a final dilution of 1:50 in the presence (20 or 10 µg/ml) or absence of PfSPATR protein. Anti-P. falciparum circumsporozoite protein (PfCSP) monoclonal antibody, NFS1, at a concentration of 10 µg/ml was added as a positive control (NFS1 monoclonal antibody was diluted 1:600 to achieve this concentration). After a 3-h incubation at 37 °C, the numbers of sporozoites that had invaded the hepatoma cells were counted. Percent inhibition was calculated by the following formula: [(mean number of invaded sporozoites in negative controls) – (mean number of invaded sporozoites in test) / (mean number of invaded sporozoites in negative controls)] x 100.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Selection, Cloning, and Expression of PfSPATR—Analysis of the published DNA sequence of chromosome 2 of P. falciparum identified a 1069-nucleotide sequence containing a two-exon gene encoding a 250-amino acid long putative secreted protein with an altered TSR domain (Fig. 1). Application of reverse transcriptase-PCR to assess its expression in sporozoites, infected erythrocytes (rings, trophozoites, schizonts, and gametocytes) revealed that the gene is transcribed in all the evaluated stages of the parasite life cycle (Fig. 2). The amplified fragment was 753 bp in length, and sequencing of the cDNA confirmed that the correct mRNA had been amplified (data not shown). To obtain recombinant PfSPATR protein, a 690-bp cDNA fragment encoding the mature protein was cloned in plasmid pGEX-3X, a GST-based E. coli expression vector. The construct was expressed in BL21 cells and induced with 1 mM isopropyl-1-thio--D-galactopyranoside for 3 h. Although the coding sequence had a high AT content, a characteristic feature of P. falciparum genes, we could detect the expression of the fusion protein on a Coomassie Blue-stained polyacrylamide gel (data not shown). The fusion protein was purified to homogeneity on a glutathione-agarose column (data not shown). Purified protein was used to immunize outbred CD-1 mice, and anti-PfSPATR serum was obtained.

View larger version (38K): [in this window] [in a new window]   FIG. 1. Schematic representation of PfSPATR gene and protein. The mature mRNA formed after removal of the intron region is 753 nucleotides in length and translates into a 250-amino acid polypeptide. The probable Type II EGF-like domain is boxed, whereas the altered thrombospondin domain is underlined. The first 21 amino acids represent the putative signal sequence and are shown in lowercase letters.

View larger version (52K): [in this window] [in a new window]   FIG. 2. Transcription of PfSPATR gene at different stages of the P. falciparum life cycle. The reaction was performed in the absence (–RT) and presence (+RT) of reverse transcriptase.

Localization of PfSPATR Protein in Different Parasite Stages—To determine whether the transcribed mRNA was associated with protein expression, we evaluated the cellular expression and localization of PfSPATR at different stages of the parasite life cycle. Immunofluorescence assays using anti-PfSPATR antibodies produced in mice demonstrated binding in all of the evaluated stages, viz. sporozoites, asexual erythrocytic stages, and gametocytes, suggesting that the protein is expressed in several stages of parasite life cycle (Fig. 3). The strongest reactivity was observed against sporozoites where the protein was detectable even at dilutions of 1:6400 of the antiserum. Sporozoites and asexual erythrocytic stages were further evaluated by immunoelectron microscopy to determine the specific location of PfSPATR. In longitudinal and cross-sections, PfSPATR was localized on the surface of sporozoites and was not detected in the intracellular organelles such as micronemes (Fig. 4, A and B). In asexual erythrocytic stages, PfSPATR was detectable around the rhoptries and to a lesser extent on the infected erythrocyte membrane (Fig. 4C). Western blot using anti-PfSPATR antiserum on sporozoite and blood-stage parasite lysates detected PfSPATR protein at its expected size of 30 kDa (data not shown).

View larger version (49K): [in this window] [in a new window]   FIG. 3. Immunofluorescence assay using anti-PfSPATR sera from mice immunized with the recombinant protein to detect PfSPATR protein expression in different stages of P. falciparum. A, sporozoite (1:6400). B, trophozoite (1:640). C, asexual erythrocytic stage schizont (1:640). D, merozoites escaping from a late stage schizont (1:640). E, gametocyte (1:640). Dilutions of antisera used are in parentheses.

View larger version (108K): [in this window] [in a new window]   FIG. 4. Localization of PfSPATR in P. falciparum by immunoelectron microscopy. A, longitudinal section of sporozoite. B, cross-section of sporozoite. C, cross-section of an infected erythrocyte containing a schizont. R, rhoptry; Mz, merozoites; FV, food vacuole; Hz, hemozoin pigments; E, erythrocyte membrane; Mn, micronemes.

Reactivity of Sera from Malaria Endemic Regions with PfSPATR on COS-7 Cells—Having established expression, we investigated whether sera from individuals exposed to P. falciparum parasites recognized PfSPATR. Employing a plasmid expressing PfSPATR to transiently transfect COS-7 cells, it was found that the protein was expressed on the surface of the cells and was readily recognized by the anti-PfSPATR serum (Fig. 5A) but not by the pre-immune serum (Fig. 5B). Sera from a malaria-naive volunteer who had been immunized with radiation-attenuated P. falciparum sporozoites (Fig. 5C) and five clinically immune adults (Fig. 5, E–I) from a region of Ghana with intense P. falciparum-malaria transmission recognized the PfSPATR expressed on COS-7 cells. However, serum from a volunteer who was also immunized with irradiated sporozoites but had low anti-sporozoite antibodies (Fig. 5D) as characterized by immunofluorescence assay and sera from two nonimmune adults (Fig. 5, J and K) did not recognize PfSPATR expression.

View larger version (71K): [in this window] [in a new window]   FIG. 5. Recognition of PfSPATR expression on COS-7 cells. A, serum from mice immunized with PfSPATR-recombinant protein. B, serum taken prior to immunization with PfSPATR-recombinant protein. C, serum from a volunteer immunized with radiation-attenuated P. falciparum sporozoites who had high levels of anti-sporozoite antibodies. D, serum from a volunteer who had been immunized with irradiated P. falciparum sporozoites whose serum was negative for anti-sporozoite antibodies. E–I, sera from semi-immune adults from Ghana and Africa. J–K, sera from nonimmune adults from the United States.

Biological Function of PfSPATR—As PfSPATR showed strong surface localization on sporozoites, we investigated its possible involvement in cell-cell interaction by evaluating its potential for binding to the human hepatocyte cell line, HepG2. The protein demonstrated potent binding to HepG2 cells that was dose-dependent (Fig. 6) and comparable with that of Pf-CSP. In contrast, serum albumin used as a negative control showed no binding (data not shown). This result suggested that PfSPATR could function as another parasite ligand involved in the interaction of sporozoites with liver cells and that a receptor for PfSPATR was present on HepG2 cells.

View larger version (15K): [in this window] [in a new window]   FIG. 6. Binding of PfSPATR protein to hepatoma cells. Binding activity of PfSPATR (filled circles) and recombinant PfCSP (open circles) was evaluated on HepG2 cells in a fluorescence-based assay.

In Vitro Inhibition of Sporozoite Invasion—As PfSPATR efficiently bound HepG2 cells, we investigated the ability of antibodies against PfSPATR to prevent sporozoite invasion of human liver cells. Anti-PfSPATR serum at a final dilution of 1:50 inhibited sporozoite invasion by more than 80%. Nonimmune control serum showed no inhibition suggesting that the inhibitory property of anti-PfSPATR serum was specific. The inhibitory activity was comparable with that of an anti-PfCSP monoclonal antibody that prevented invasion by more than 90% at 10 µg/ml. This invasion inhibition was antigen-specific as shown by the addition of free recombinant protein in the assay, which completely reversed the inhibition (Fig. 7).

View larger version (29K): [in this window] [in a new window]   FIG. 7. In vitro inhibition of sporozoite invasion into HepG2 cells by anti-PfSPATR serum. The final dilution of anti-PfSPATR serum and control antisera used was 1:50, and the NFS1 monoclonal antibody against PfCSP was diluted 1:600 to give a final concentration of 10 µg/ml. The numbers of P. falciparum sporozoites invading liver cells were counted in the presence of anti-PfSPATR serum alone or with the recombinant-PfSPATR protein (20 or 10 µg/ml) used as a competitor to neutralize the inhibitory affect of anti-PfSPATR antibodies.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
We investigated a protein of P. falciparum that is encoded by a gene located on chromosome 2 of the parasite (9). Based on protein sequence homology, the protein had been suggested to possess an altered TSR domain toward the carboxyl terminus (20). The central motif present in most TSR-family proteins is WSXW (where "X" can be any amino acid) followed by CSXTCG (26). As the CSXTCG motif is absent in the SPATR sequence, it has been referred to as an "altered thrombospondin domain" (20). In Plasmodium, TSR-containing genes show synteny linkage conservation between different species (27). Along with the altered TSR domain, the protein has a 15-amino acid region (⁷²NSRNCWCPRGYILCS⁸⁶) that has characteristics suggestive of a Type II EGF-like signature sequence. The BLOCKS protein domain data base and search tool (www.blocks.fhcrc.org) that predicted this characteristic indicated that the PfSPATR sequence was not typical of the Type II EGF signature sequence found in a diverse range of proteins (28). Interestingly, no other protein domain data base and search tool predicted this Type II EGF-like signature. Although the precise roles of the TSR and potential EGF domains are as yet unclear, these domains are located in the extracellular region of membrane-bound proteins or in proteins known to be secreted (29). Although TSR (13–15) or EGF domains (30–32) are individually present in a number of Plasmodium proteins, no Plasmodium antigen has been reported in which these two domains are predicted to be present together.

We found that the PfSPATR gene is transcribed during the sporozoite and the major erythrocytic stages of the parasite life cycle (Fig. 2). Sporozoites for our studies were produced in mosquitoes, and the erythrocytic stage parasites were produced in cultures of erythrocytes, so there could not have been any cross-contamination. The expressed sequence matched perfectly with the predicted gene structure of the protein, which also verified the predicted exon-intron boundaries of the gene (Fig. 1). Only a few Plasmodium proteins have been reported to be expressed during multiple stages of the parasite life cycle (33).

We evaluated the cellular expression of PfSPATR by immunofluorescence assay and by immunoelectron microscopy and found that the protein is present in the sporozoite, asexual erythrocytic, and gametocyte stages of the life cycle (Figs. 3 and 4). In sporozoites it was exclusively located on the surface, whereas in asexual blood stages it was present around the rhoptries of merozoites and on the membrane of infected erythrocytes (Fig. 4). The presence of this protein on the surface of the parasite in different stages led us to investigate whether this protein was recognized by the host immune system in P. falciparum-infected individuals. The PfSPATR protein that was expressed on the cell surface of transfected COS-7 cells was recognized by sera from naturally infected clinically immune adult Africans, indicating that this protein was recognized by the host immune system (Fig. 5). The fact that the serum from an individual immunized with irradiated sporozoites recognized the protein corroborates the expression and immunogenicity of the protein during the early pre-erythrocytic stage of the parasite infection in humans. Serum from a volunteer with low levels of anti-sporozoite antibodies and control sera from two nonimmune donors did not recognize PfS-PATR expression on COS-7 cells indicating that this reaction was specific.

The expression of PfSPATR protein on the sporozoite surface and its recognition by the host immune system in infected individuals led us to investigate its biological role in the parasite life cycle. Other known Plasmodium antigens with similar properties have been shown to be involved in cell-cell interactions (16–18). We hypothesized a role for this protein in the binding of sporozoites to liver cells, a property known to be associated with other sporozoite proteins possessing a TSR domain. PfSPATR bound to human liver cells, and its binding was comparable with that of PfCSP, the predominant sporozoite surface protein (Fig. 6). The binding of PfSPATR appeared to be specific and is presumed to be involved in the sporozoite invasion of liver cells as evidenced by inhibition of invasion by anti-PfSPATR antibodies and reversal of inhibition by the addition of recombinant PfSPATR protein (Fig. 7). It will be interesting to determine whether there are anti-PfSPATR antibodies that block the invasion-inhibiting activity of anti-PfSPATR antibodies as has been demonstrated for MSP-19 (34).

The presence of antibodies that partially inhibit the sporozoite invasion of hepatocytes does not indicate that an individual will be protected against P. falciparum infection. If a mosquito injects 20 sporozoites and 19 of them are inhibited from invading hepatocytes by antibodies to PfSPATR or against any other sporozoite protein, the subject will not be protected against developing P. falciparum infection, because within 1 week a single successfully invaded sporozoite can give rise to 10,000 merozoites each of which can invade erythrocytes. Immunization of volunteers with a number of PfCSP-recombinant protein vaccines has elicited antibodies to sporozoites that successfully inhibit sporozoite invasion of hepatoma cells in vitro but fail to protect the volunteers against challenge (35). Nonetheless, we know from passive transfer studies in mice and monkeys (36) that antibodies against sporozoites can completely protect against sporozoite challenge. In those cases, the invasion-inhibitory activity was generally >95%. We are currently investigating the potential of anti-SPATR antibodies to protect against infection in the P. yoelii rodent malaria model.

The SPATR protein is present in multiple Plasmodium species. It is present in the transcriptome of P. yoelii sporozoites (20), and we have also identified its orthologue in Plasmodium knowlesi and Plasmodium vivax species.² The presence of this protein in human, simian, and rodent malaria parasite species suggests that the protein plays an important role in the biology of the parasite. Numerous efforts are currently under way to develop an effective vaccine against malaria (37). The complex life cycle of the parasite with distinct sets of antigens expressed during various stages of development has made vaccine design and development a major challenge to malaria researchers. We have described herein a molecule that holds potential for investigation as a malaria vaccine candidate. Its multistage expression by sporozoites, asexual erythrocytic forms, and gametocytes, along with its possible role in liver cell invasion, suggests that PfSPATR could be a valuable new vaccine component.

	FOOTNOTES

 
^* This work was supported by Naval Medical Research and Development Command Work Unit 6000.RAD1.F.A0309. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Sanaria, 308 Argosy Drive, Gaithersburg, MD 20878. Tel.: 240-299-3178; Fax: 301-990-6370; E-mail: SLHoffman{at}sanaria.com.

¹ The abbreviations used are: TSR, thrombospondin Type I repeat; PfCSP, Plasmodium falciparum circumsporozoite protein; PfSPATR, Plasmodium falciparum-secreted protein with altered thrombospondin repeat; EGF, epidermal growth factor; GST, glutathione S-transferase.

² R. Chattopadhyay, unpublished results.

	ACKNOWLEDGMENTS

 
We thank Dr. Gary H. Cohen and Dr. Roselyn J. Eisenberg, University of Pennsylvania, Philadelphia for providing the pRE4 plasmid, DL6 and ID3 monoclonal antibodies used in expression of PfSPATR in COS-7 cells work and Dr. Yupin Charoenvit of the Malaria Program, United States Naval Medical Research Center, Silver Spring, MD for providing sera from irradiated sporozoite trial volunteers. We also acknowledge Dr. Stefan H. I. Kappe, Department of Pathology, New York University School of Medicine, New York and Dr. Mani Subramanian, Human Genome Sciences, Rockville, MD for their critical comments and suggestions.

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