Molecular cloning and analysis of stage and tissue-specific expression of cathepsin B encoding genes from Fasciola gigantica☆
Introduction
Fasciola gigantica causes tropical fascioliasis in infected water buffaloes and cattle in Thailand and countries in the tropical region. Moreover, the parasite can also cross-infect human [1], which has been considered an important public health problem in many parts of the tropics. Control of the disease is so far carried out by treatment with antihelminthics, grazing management, and the application of molluscicides [2]. All of these preventive methods have limitations and transient effect. Vaccination is considered to be the ultimate, most cost-effective, and sustainable strategy. Many antigens have been tested as vaccine candidates in Fasciola spp., such as fatty acid binding proteins (FABP), glutathione S-transferase (GST), and cathepsin L proteases (CatL) [3], [4], [5], [6], [7], [8], because of their specific and important functions in the parasite’s survival. However, these vaccine candidates affect mainly the adult parasites which reside in the bile duct where lower concentrations of antibodies and immune effector cells are found. In addition, the adult parasites have already developed the full complements of immune evasion mechanisms. Therefore, the chance of damaging and killing parasites would be higher if the vaccine candidates can be directed at the newly excysted and juvenile stages.
In the parasitic life cycle, proteases have been deployed in some important tasks, which include tissue penetration, digestion of host tissue for nutrition, and evasion from the host immune responses [9], [10], [11]. Although the most characterized proteases in Fasciola spp. belong to the cathepsin L group of cysteine proteases [12], [13], Wilson et al. [14] have reported that cathepsin B proteases are the major proteases secreted by the newly excysted juvenile (NEJ) of F. hepatica. In a recent publication the cloning and expression of the major secreted cathepsin B protein from juvenile F. hepatica has been reported [15]. This protein could be detected in early stages of infection and the investigators suggested that the protease may help the juvenile parasite in penetration and migration through the host’s liver. However, cathepsin B gene fragments have also been amplified from adult F. hepatica RNA [16], but its function in adults has not been studied yet.
In the present study, we have cloned cDNAs of F. gigantica cathepsin B genes from three different stages (adult, newly excysted juveniles, and metacercariae) and analyzed the identity of their sequences. The genes have been characterized in respect to their copy number and differential expression in several developmental stages. The distribution of cathepsin B gene transcripts in F. gigantica tissues has been analyzed by RNA in situ hybridization using a cathepsin B antisense RNA probe.
Section snippets
Parasite specimens
F. gigantica metacercariae were obtained from experimentally infected snails Lymnaea ollula. To activate the excystment, the metacercariae were incubated in a solution containing 2% pepsin and 0.5% HCl at 37 °C for 45 min and then in a solution of 0.02 M sodium dithionite, 0.2% taurocholic acid, 1% NaHCO3, 0.8% NaCl, and 0.5% HCl at 37 °C for 45 min. The metacercariae were excysted in RPMI-1640 medium (Sigma Chemical Co., St. Louis, MO, USA) containing 10% normal sheep serum and 10 μg/ml gentamycin
Cloning and sequencing of the cDNAs encoding cathepsin B of F. gigantica
Screening of the cDNA libraries, using the generated F. gigantica cathepsin B fragments as probes, resulted in 270 positive clones out of 5×104 plaques from the metacercaria cDNA library, 100 positive clones out of 5×104 plaques from the NEJ cDNA library, and 12 positive clones out of 5×104 plaques in the adult cDNA library. Five of the ten selected clones from each library contained the complete coding sequences for cathepsin B genes. The clone isolated from the adult cDNA library (named
Discussion
In this study we have cloned three cDNAs encoding cathepsin B proteases from stage-specific libraries (adult, NEJ, and metacercaria) of F. gigantica and analyzed their stage- and tissue-specific expression by Northern hybridization, PCR techniques and RNA in situ hybridization. The three cathepsin B clones showed differences in length and nucleotide/amino acid sequences which imply that they are distinct members of the same gene family. Interestingly, FG cat-B2 showed 99% identity to F. hepatica
Acknowledgements
This research was supported by the Thailand Research Fund (Senior Research Fellowship to Prasert Sobhon, and Royal Golden Jubilee Ph.D. Scholarship to Krai Meemon).
References (36)
- et al.
Protection of cattle against Fasciola hepatica infection by vaccination with glutathione S-transferase
Vaccine
(1996) - et al.
Fasciola: kinetics and quality of humoral responses to fatty acid binding protein and cathepsin L following delivery as DNA vaccines in mice
Exp Parasitol
(2001) - et al.
Vaccination of sheep with purified cysteine proteinases of Fasciola hepatica decreases worm fecundity
Exp Parasitol
(1994) Nutritional adaptations to parasitism within the platyhelminthes
Int J Parasitol
(1997)- et al.
Thiol proteases released in vitro by Fasciola hepatica
Mol Biochem Parasitol
(1989) - et al.
Purification of a cathepsin L-like proteinase secreted by adult Fasciola hepatica
Mol Biochem Parasitol
(1993) - et al.
Fasciola hepatica: characterization and cloning of the major cathepsin B protease secreted by newly excysted juvenile liver fluke
Exp Parasitol
(1998) - et al.
Cloning of a protease gene family of Fasciola hepatica by the polymerase chain reaction
Mol Biochem Parasitol
(1994) - et al.
Molecular cloning and characterization of cathepsin L encoding genes from Fasciola gigantica
Parasitol Int
(2001) - et al.
Primary structures of Sm31/32 diagnostic proteins of Schistosoma mansoni and their identification as proteases
Mol Biochem Parasitol
(1989)
Human gastric adenocarcinoma cathepsin B: isolation and sequencing of full-length cDNAs and polymorphisms of the gene
Gene
Electron microscope studies of Fasciola hepatica XIII. Fine structure of newly excysted juvenile
Exp Parasitol
Fasciola hepatica: irradiation-induced alterations in carbohydrate and cathepsin-B protease expression in newly excysted juvenile liver fluke
Exp Parasitol
The shedding of the outer glycocalyx of juvenile Fasciola hepatica
Vet Parasitol
Schistosoma mansoni: eggshell formation is regulated by pH and calcium
Exp Parasitol
Electron microscope studies of Fasciola hepatica X. Egg formation
Exp Parasitol
Mosquito cathepsin B-like protease involved in embryonic degradation of vitellin is produced as a latent extraovarian precursor
J Biol Chem
Drosophila cathepsin B-like proteinase: a suggested role in yolk degradation
Arch Biochem Biophys
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Note: Nucleotide sequence data reported in this paper are available in the GenBank™, EMBL and DDBJ databases under the accession numbers AY227673- AY227675.