Cloning and cross-species comparison of the thrombospondin-related anonymous protein (TRAP) gene from Plasmodium knowlesi, Plasmodium vivax and Plasmodium gallinaceum1

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Abstract

To examine the structure of the Plasmodium sporozoite micronemal protein, thrombospondin-related anonymous protein (TRAP) we have isolated TRAP genes from three species of Plasmodium: P. gallinaceum (PgTRAP), P. knowlesi (PkTRAP) and P. vivax (PvTRAP). Thus it is now possible to compare the TRAP gene from a total of six species of Plasmodium. The overall structure of TRAP is conserved in all species; specifically, an amino-terminal A-domain similar to magnesium-binding domains of mammalian integrins; a thrombospondin-like sulfatide-binding domain similar to region II in Plasmodium circumsporozoite protein; an acidic asparagine/proline-rich repeat region; a trans-membrane domain and a short acidic cytoplasmic region with a highly conserved carboxy terminus. The overall structure of TRAP from P. gallinaceum and P. falciparum (PfTRAP) is conserved and phylogenetic analysis suggests a monophyletic relationship of avian P. gallinaceum and human P. falciparum. Comparison of the amino acid sequences of the A-domain of PgTRAP and PfTRAP indicates a more rapid divergence of this domain with respect to the rest of the protein in these two species. The structural differences of PgTRAP and PfTRAP may relate to the distinct invasion pathways, macrophage and endothelial cell invasion of P. gallinaceum sporozoites versus hepatocyte invasion of P. falciparum.

Introduction

The progression of Plasmodium through its lifecycle in the vertebrate host and insect vector requires a bewildering sequence of recognition, adhesion and invasion events of host cells and tissues. To accomplish these interactions the parasite possesses a multitude of cell surface receptors that are often, but not always, discretely stage-specific in expression. The adhesion molecule TRAP (thrombospondin-related anonymous protein; termed sporozoite surface protein 2 (SSP2) in P. yoelii) initially was identified in the asexual erythrocytic stage [1]but has been largely studied as a sporozoite microneme or cell-surface protein 2, 3, 4. TRAP is proposed to contribute, along with circumsporozoite protein (CSP), to hepatocyte recognition and/or invasion 2, 5, 6, 7. TRAP-specific antisera blocks in vitro invasion of hepatocytes by sporozoites and bacterial-produced recombinant TRAP has been shown to bind to hepatocytes and heparin sulfate proteoglycans. TRAP shows promise as a vaccine component—individuals living in malaria endemic areas exhibit an age-dependent humoral immune response against TRAP [7]; in a mouse model vaccination with PyTRAP/SSP2 plus CSP of P. yoelii confers complete protection against sporozoite challenge [8]; and cytotoxic CD8+T-cells have been shown to confer protection via recognition of PyTRAP/SSP2 [9]or PfTRAP [10].

Although a ligand for TRAP has not yet been reported, the character of its multi-domain structure and microneme/membrane surface localization suggests a role in adhesion. The amino terminal portion of TRAP contains an approximately 200 amino acid long domain that is similar to a magnesium-binding `A-domain', described as a ligand-binding domain in a subset of mammalian α- and β-integrins 11, 12, 13. Crystal structures of A-domains from two α-integrins indicate several regions involved in coordination of a magnesium ion 12, 13, including a DXSXS motif that is conserved near the amino terminus of the mature TRAP protein. A-domains are also found in plasma proteins, such as von Willebrand factor and extracellular matrix proteins such as collagens (reviewed in Ref. [12]). In the Apicomplexa, A-domains have been identified in an Eimeria tenella microneme protein, Etp100 [14]and as multiple tandemly arrayed copies in a recently described CSP/TRAP-related protein, CTRP, in P. falciparum [15]. A putative role in adhesion has not yet been determined for Etp100 or CTRP.

Adjacent to the A-domain in TRAP is a cysteine-rich region containing a motif (WSPCSVTCG in P. falciparum TRAP) that is highly similar to region II in CSP 16, 17, thrombospondin 18, 19, properdin [20], and is present in multiple tandem copies in Etp100 [14]and CTRP [15]. Evidence from a number of experiments with TRAP and CSP recombinant constructs and peptides suggest that this region is involved in binding to hepatocytes, specifically, to host sulfated glycoconjugates 5, 6, 21, 22, 23. A third extracellular five domain is an acidic asparagine/proline-rich region that is proposed to be a structural β-sheet `stalk' of the receptor and has been suggested to be involved in an as yet poorly understood immune evasion mechanism 24, 25. Finally, TRAP possesses a single trans-membrane region and a short (30–40 aa) cytoplasmic region with an acidic carboxy-terminus that is highly conserved across Plasmodium species. The carboxy-terminus of TRAP shares with CTRP the terminal amino acids WN preceded by a short stretch of acidic aa (PEENEWN in P. falciparum TRAP).

TRAP expression in asexual erythrocytic stages is not as well documented as microneme/cell surface-localized expression in sporozoites and has not been firmly established, due to the lack of protein expression data [2]. Recently, however, it has been shown that anti-peptide sera recognizing the CSP-like region II of TRAP inhibits asexual-stage merozoite invasion of erythrocytes [26]. Conclusive demonstration that TRAP is expressed in different Plasmodium stages would suggest that it is involved in functional events other than in specific cellular recognition of hepatocytes. The attractiveness of TRAP as a vaccine component would be enhanced if vaccination with it affects multiple stages of the malaria life cycle. To study the structure of TRAP and to provide reagents for studies of its function and potential for a vaccine we have isolated the TRAP gene from P. knowlesi (PkTRAP); P. vivax (PvTRAP); and P. gallinaceum (PgTRAP). Cloning of these genes was based upon sequence similarity of previously cloned P. falciparum (PfTRAP) and P. yoelii (PyTRAP/SSP2) TRAP genes 1, 2, 3.

Section snippets

Parasite strains and genomic DNA isolation

Genomic DNA (gDNA) of P. vivax was purified, as described previously [27], from Rhesus blood infected with the Sal-I strain [28]. A second lot of P. vivax gDNA (Sal-I) was purified using a QIAamp Tissue Kit (Qiagen). P. knowlesi gDNA was derived from parasites enriched from the blood of Rhesus monkeys infected with strain H/clone A and was kindly provided by Diana Hudson-Taylor (LPD, NIAID, NIH). A second lot of P. knowlesi gDNA was purified using a QIAamp Tissue Kit from similarly infected

Cross-species sequence comparison of Plasmodium TRAPs

Comparison of PfTRAP and PyTRAP/SSP2 nucleotide sequences suggested three conserved regions for the cloning of TRAP from other Plasmodium species by PCR using degenerate primer pairs (Fig. 1). Amplification of PvTRAP and PkTRAP was achieved using oligonucleotide primers corresponding to a region within the mature protein near the signal peptide cleavage site (DEIKYSEEVC) and to a region at the carboxy terminus (PE(E/D)N(D/E)WN). Near full-length coding regions of TRAP genes from P. vivax and P.

Discussion

To understand the function of TRAP in the Plasmodium lifecycle it is desirable to take advantages of the attributes of `model' systems of different Plasmodium species. In addition, to aid in determining the utility of TRAP as a recombinant protein vaccine or DNA vaccine it will be useful to study more than one Plasmodium model. For these reasons and to characterize the structure of TRAP through a cross-species comparison, we have cloned TRAP from three additional species of Plasmodium: P.

Acknowledgements

We thank Bill Collins for generously providing P. vivax parasites and Andre Laughinghouse and Joseph S.B. Vinetz for providing P. gallinaceum ookinetes. The authors thank Jessica Kissinger and Detlef Leipe for performing the phylogenetic analysis. We are grateful to Kathryn Robson for allowing use of the PbTRAP sequence prior to publication and for informative discussions. We would also like to thank Tom McCutchan and Louis Miller for sharing information on P. gallinaceum CSP sequence and for

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    Note: Nucleotide sequence data reported in this paper have been submitted to GenBank™ with the accession numbers PgTRAP: U64899; PkTRAP: U64900; and PvTRAP: U64901.

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