stevor and rif are Plasmodium falciparum multicopy gene families which potentially encode variant antigens

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Abstract

Several multicopy gene families have been described in Plasmodium falciparum, including the var genes that code for the variant surface antigen PfEMP1, the stevor family of subtelomeric open reading frames and the rif interspersed repetitive elements. This report documents the chromosomal location of stevor genes, their transcription and characteristics of the deduced protein. On 14 chromosomes, 34 stevor copies were identified from the Dd2 parasite line. Most are in subtelomeric regions within 50 kb of the telomere. stevor genes are located close to var genes and rif. All stevor genes sequenced had two exons: a short exon 1 encoding a start codon and a transmembrane domain; exon 2 encoding for the remainder of the ∼30 kDa protein and including two more transmembrane segments. A similar structure was found for copies of rif and its predicted protein. In both STEVOR and RIF proteins, a highly polymorphic region is predicted to be a loop on the outer side of the membrane. We propose that stevor and rif are members of a larger superfamily. The number of copies of stevor and rif, their location close to the var genes, their extreme polymorphism and the predicted structure of the proteins suggest that stevor and rif code for variant surface antigens.

Introduction

Malaria is still a major cause of mortality and morbidity in many areas of the world. Virulence factors for pathogenesis are not fully understood but one factor for Plasmodium falciparum infections is the cytoadherence of infected red cells to blood vessel walls. This is mediated by a family of highly polymorphic proteins named PfEMP1 for P. falciparum erythrocyte membrane protein 1 [1], [2], [3]. Different members of this protein family bind to different adhesion ligands. The proteins are also antigenically unique and switching of the individual PfEMP1 proteins during an infection is believed to be important for the maintenance of chronic infections [4], [5]. Approximately 50 var genes that code for PfEMP1 [6], [7], [8] are found in the parasite genome. They are located on most or all chromosomes [9], [10], [11]. Simultaneous transcription of multiple var genes from several chromosomes have been detected in a single ring-infected erythrocyte and only one of the transcribed var genes is selected to express PfEMP1 on the surface of the infected cell [12]. The expressed var gene switches in cloned lines and the copy expressed correlates with antigenic type and cytoadherence specificity [8]. Although domains of PfEMP1 bind ligands such as CD36 involved in cytoadherence [13], [14], other proteins such as sequestrin [15] may contribute to the binding. PfEMP1 is localized at electron dense protrusions of the infected red cell membrane. Several proteins are involved in knob formation and these proteins are important for cytoadherence [16].

Infected red cells also bind uninfected red cells to form rosettes. Domains of PfEMP1 bind to red cells and so PfEMP1 may also mediate rosetting [17], [18]. However, other small proteins (22–28 kDa), namely rosettins, have also been implicated [19]. Like PfEMP1, rosettins are antigenically polymorphic and may be the product of another multi-copy gene family.

Based on DNA hybridization patterns and the presence of an open reading frame in DNA fragments, three other multi-copy gene families have been identified in P. falciparum. The rif family of interspersed repetitive DNA was identified based on the isolation of a λgt11 clone a95 from a genomic library of P. falciparum [20]. Southern blots of this clone gave a complex banding pattern. The sequence of the clone contained a 1 kb open reading frame and a positive Northern blot showed that this sequence was transcribed in blood stage parasites. It was estimated that at least 11 copies are present per genome and it was suggested that this may correspond to a transposable element.

The first member of the second family was 7H8/6, a clone selected with monoclonal antibody 7H8 from a λgt11 P. falciparum genomic expression library derived from the Malayan camp line [21]. 7H8/6 hybridized to DNA isolated from all strains of P. falciparum tested. Similar to the rif clone, it gave distinctive multiple banding patterns [22] and has been used as a DNA finger printing probe to distinguish falciparum lines and field isolates [22], [23], [24]. As a multicopy gene, it is a sensitive target for the PCR-based measurement of low density parasitemia in humans [25]. Sequences which hybridize to 7H8/6 are located in the subtelomeric region of most chromosomes [22] and were called stevor for Subtelomeric, variable open reading frames [25].

The prototype member of the third family, Pf60 was also isolated from a λgt11 P. falciparum genomic expression library [24]. Pf60.1 was isolated with a polyclonal antibody raised against a 37 kDa protein from Babesia divergens and which reacts with 60 kDa protein located in the rhoptries of P. falciparum. It is estimated that there are ∼140 copies of the Pf60 family. Sequence homologies suggest this may be related to multicopy rhoptry proteins found in other Apicomplexia [24].

In this paper, we describe the genomic organization, chromosome location, DNA and cDNA repertoires, transcription of the stevor gene family and the predicted structure of the deduced proteins. We show that both stevor and rif genes have a similar two exon structure, potentially encode similar transmembrane proteins and the genes are co-localized in the subtelomeric regions, showing that in spite of little sequence homology, both are members of the same multi-copy superfamily.

Section snippets

Parasites

P. falciparum lines HB3, 3D7, D10, 10x, Palo Alto, Itg2, 7G8, T9-94, W/L, NF36, MS2, AE7, FCQ50, FCQ79, MAD71 and Z5 were used as described [22]. The P. falciparum cloned line W2 and its subclone, Dd2 were described by Oduola et al. [26] and Wellems et al. [27], respectively. The P. vivax sample was described previously [23].

YAC library and contigs

Yeast artificial chromosomes (YACs) from three libraries were used: (1) a YAC library prepared from the P. falciparum cloned line Dd2 [28]; (2) a YAC contig library of the

stevor and rif have two exons

Five stevor genes were sequenced from genomic DNA, one each from 3D7 YACs 515 and 872 (left and right arms of chromosome 3), two from D10 λgt10 genomic clones and one copy from Dd2. Further sequences are now available from chromosomes 2, 3, 4 and 12 from the malaria genome project.

All genomic sequences examined had a sequence consistent with a two exon structure (Fig. 1). The predicted intron had a high AT composition characteristic of P. falciparum, started with a GT and ended in a poly T

Discussion

The similarity of the stevor genes and the open reading frames within rif, the overall structure of the deduced proteins including some conserved cysteines and their arrangement of these DNA elements in to repetitive blocks of sequence containing both stevor and rif, shows that these two families are closely related.

The protein structure prediction routines strongly indicate that the STEVOR and RIF proteins are membrane bound. The structure of the RIF protein is predicted unambiguously by both

Acknowledgements

We are grateful for Drs Juan Cooper and Paul Horrocks for their assistance and helpful discussions, and Darrin Taylor for excellent technical assistance. We would like to thank Vikki Marshall for kindly supply the λgt10 library and Drs Ross L. Coppel, Jürgen Kun, Artur Scherf, Thomas E. Wellems, Ekkehard Werner, for supplying hybridization probes used in this work. This project was funded by the NH&MRC of Australia. Katja Fischer and Michael Lanzer are supported by Grants from the

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    Note: Nucleotide sequence data reported in this paper are available in the EMBL, GenBankTM and DDJB data bases. Accession numbers for 33 copies of the stevor hypervariable loop are AF065169 to AF065197 and AF065205 to AF065208; for six copies of full length stevor AF065198 to AF065201, AF065204 and AF065209; and for two copies of the varC′ sequences, AF065202 and AF065203.

    1

    Present address: Australian Army Malaria Institute, Gallipoli Barracks, Enoggera, Queensland 4052, Australia.

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