Molecular cloning of genes encoding oncosphere proteins reveals conservation of modular protein structure in cestode antigens

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Abstract

Recombinant oncosphere antigens have been found to be remarkably effective when used as vaccines against cysticercosis and hydatid disease. Comparison of the structural features of these proteins and their associated genes suggest common features between antigens. Here molecular cloning is used to complete comparisons of Taenia solium, Taenia saginata, Taenia ovis, Echinococcus granulosus and Echinococcus multilocularis oncosphere antigens and genes. The exon/intron structure of genes cloned from T. solium and T. ovis genomic DNA (tsol16 and to16, respectively) in this study was found to be highly conserved. Two closely related tsol16 genes were cloned from the T. solium genome. Their corresponding transcripts were cloned from T. solium oncospheres and a comparison of their deduced amino-acid sequence with that of the protein encoded by to16 indicates that these proteins are the most highly conserved oncosphere proteins identified so far. Cloning of another gene from T. solium (designated tsol18) and comparison with the homologous gene of T. saginata (tsa18) also revealed substantial conservation of gene structure. Comparisons of the genes cloned in this study with genes encoding oncosphere antigens from other taeniid cestodes identified striking conservation of exon structure. The highly conserved regions of the genes encode a putative secretory signal and fibronectin type III domain in each of the oncosphere proteins. The location of exon boundaries in relation to protein features identifies a clear modular structure among all members of these oncosphere antigens. Identification of structural conservation of genes encoding antigenic proteins across several taeniid species suggests that the encoded proteins play important roles in host infection and parasite survival.

Introduction

The taeniid cestodes are parasites in which the adult occurs as a tapeworm and the metacestode (larval stage) is commonly a cysticercus or hydatid cyst. Taenia species show a high degree of host specificity. Taenia solium infects humans as an adult tapeworm and occurs as a larval cysticercus in pigs. The larval stage can also infect humans and often results in the serious condition known as neurocysticercosis, which is a major cause of neurological disease worldwide [1]. The oncosphere or early larval life cycle stage of the taeniid cestodes has been shown to be a rich source of antigens capable of stimulating protective immune responses in the intermediate host against infection with the larval stage (reviewed by Rickard and Williams [2]). On this basis, several host-protective recombinant antigens have been cloned from the oncosphere stage of taeniid cestodes. The 45W antigen cloned from Taenia ovis oncospheres is able to be used as a vaccine to prevent cysticercosis infection in sheep [3], and the gene encoding it has subsequently been shown to be a member of a multi-gene family in T. ovis [4]. TO16 and TO18 are two other recombinant antigens that have been independently identified from T. ovis oncospheres [5] and have also been shown to protect sheep against cysticercosis. Molecular cloning of cDNA derived from Taenia saginata oncosphere mRNA has identified homologues of TO45W and TO18, designated TSA9 and TSA18, respectively, that have been shown to prevent cysticercosis in cattle [6]. The T. solium cDNA homologues of some of these T. ovis/T. saginata transcripts have been characterised [7], [8].

Identification and molecular cloning of the eg95 cDNA from Echinococcus granulosus oncospheres has also resulted in development of a very effective hydatid vaccine in sheep [9] and recent studies have shown that eg95 belongs to a family of closely related genes in E. granulosus [10]. A homologue of eg95 has been cloned from Echinococcus multilocularis, designated em95, which has been shown to encode an antigen that prevents infection of immunised mice with the larval stage of E. multilocularis [11].

The proteins encoded by the oncosphere mRNAs, referred to above, all contain at least one fibronectin type III (FnIII) domain [12] but detailed comparisons identifying common genomic structural features between the orthologous genes from different species of Taenia/Echinococcus have not been conducted. This is because molecular cloning of genes from chromosomal DNA has thus, far limited comparisons to the members of gene families of to45W, eg95 and tso45. This study describes the molecular cloning and analysis of genes and transcripts from T. solium and provides detailed comparisons between the various genes that encode taeniid host-protective antigens. The T. solium genes/transcripts described herein were isolated to enable identification of putative vaccine antigens to prevent transmission of T. solium cysticercosis. Characterisation of genes encoding taeniid antigens was completed by cloning of the genes encoding TSA9 of T. saginata and TO16 of T. ovis.

Section snippets

Isolation of parasite nucleic acids

Tapeworm specimens were obtained and genomic DNA extracted as described previously [13], [14]. Total cellular RNA was extracted [15] using TRIZOL (Life Technologies) from oncospheres which had been hatched from eggs, activated in vitro [16] and purified by density gradient centrifugation [17].

Southern blot hybridisation

Restriction-digested genomic DNA was separated by electrophoresis in TAE buffer, transferred [18] onto positively charged nylon membranes (Roche Diagnostics), dried, UV cross-linked (Stratalinker,

Results and discussion

Southern blot and hybridisation probing of EcoRI-digested T. ovis and T. solium genomic DNA identified two DNA fragments in each sample that hybridised with the to16 cDNA probe (Fig. 1A). T. solium genomic DNA produced restriction fragments of 2.37 and 0.46 kb (Fig. lA, lane 2) while T. ovis produced 4.5 and 3.2 kb fragments (Fig. lA, lane 3). Initial DNA sequence comparisons between the tsol16 and the to16 genes showed that the 0.46 kb T. solium restriction fragment identified in the Southern

Acknowledgements

This work was funded with research grants from the National Health and Medical Research Council of Australia.

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Note: Nucleotide sequence data reported in this paper are available in the Genbank™, EMBL and DDBJ databases under accession numbers AY147841AY147845.

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