Molecular cloning and analysis of stage and tissue-specific expression of cathepsin B encoding genes from Fasciola gigantica

https://doi.org/10.1016/j.molbiopara.2004.02.010Get rights and content

Abstract

The transcriptional products of Fasciola gigantica genes encoding cathepsin B proteases were cloned from adult, newly excysted juvenile (NEJ), and metacercarial stages. The obtained cDNAs were named FG cat-B1, FG cat-B2, and FG cat-B3. The deduced amino acid sequences of the encoded proteases have identities ranging from 64 to 79%. Sequence comparison with homologous proteins showed that all functional important residues formerly described for cathepsin B are conserved. Southern analysis confirmed the presence of a family of related cathepsin B genes in the genome of F. gigantica. Northern analysis revealed a common transcript size of 1400 nucleotides with abundant cathepsin B transcripts detected in metacercarial and NEJ stages. Cathepsin B transcripts were located by RNA in situ hybridization in the caecal epithelial cells, in cells underlining the proximal part of the digestive tract, and in the tegumental cells underlining the surface tegument. Furthermore, transcripts were detected in the tissues of the reproductive system including cells of prostate, Mehlis, and vitelline glands, testis, and eggs. Stage-specific gene expression was investigated by RT-PCR using gene-specific primers and hybridization with a labeled cathepsin B probe. FG cat-B1 transcripts were detected in all stages, whereas FG cat-B2 and FG cat-B3 transcripts were expressed in metacercariae, NEJ, and juvenile parasites only. The switching off of the cat-B2 and cat-B3 genes during the maturation of the parasites implicates that these enzymes may be involved in digesting host tissues during penetration and migration to the liver, whereas cat-B1 present in all stages may perform general digestive function.

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).

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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.

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