Molecular cloning of genes encoding oncosphere proteins reveals conservation of modular protein structure in cestode antigens☆
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.
References (28)
- et al.
Hydatidosis/cysticercosis: immune mechanisms and immunization against infection
Adv. Parasitol.
(1982) - et al.
Sequence analysis of a gene family encoding Taenia ovis vaccine antigens expressed during embryogenesis of eggs
Mol. Biochem. Parasitol.
(1997) - et al.
Identification and cDNA cloning of two novel low molecular weight host-protective antigens from Taenia ovis oncospheres
Int. J. Parasitol.
(1996) - et al.
Taenia saginata vaccination against cysticercosis in cattle with recombinant oncosphere antigens
Exp. Parasitol.
(1996) - et al.
A Taenia solium oncosphere protein homologous to host-protective Taenia ovis and Taenia saginata 18 kDa antigens
Int. J. Parasitol.
(1998) - et al.
Alternative splicing and sequence diversity of transcripts from the oncosphere stage of Taenia solium with homology to the 45W antigen of Taenia ovis
Mol. Biochem. Parasitol.
(2001) - et al.
A gene family expressing a host-protective antigen of Echinococcus granulosus
Mol. Biochem. Parasitol.
(2001) - et al.
Characterization of the gene family encoding a host-protective antigen of the tapeworm Taenia ovis
Mol. Biochem. Parasitol.
(1995) - et al.
Isolation and characterisation of nucleic acids from the hydatid organisms, Echinococcus spp. (Cestoda)
Mol. Biochem. Parasitol.
(1985) Detection of specific sequences among DNA fragments separated by gel electrophoresis
J. Mol. Biol.
(1975)