Cathepsin F Cysteine Protease of the Human Liver Fluke, Opisthorchis viverrini

Abstract

BackgroundThe liver fluke Opisthorchis viverrini is classified as a class I carcinogen due to the association between cholangiocarcinoma and chronic O. viverrini infection. During its feeding activity within the bile duct, the parasite secretes several cathepsin F cysteine proteases that may induce or contribute to the pathologies associated with hepatobiliary abnormalities.Methodology/Principal FindingsHere, we describe the cDNA, gene organization, phylogenetic relationships, immunolocalization, and functional characterization of the cathepsin F cysteine protease gene, here termed Ov-cf-1, from O. viverrini. The full length mRNA of 1020 nucleotides (nt) encoded a 326 amino acid zymogen consisting of a predicted signal peptide (18 amino acids, aa), prosegment (95 aa), and mature protease (213 aa). BLAST analysis using the Ov-CF-1 protein as the query revealed that the protease shared identity with cathepsin F-like cysteine proteases of other trematodes, including Clonorchis sinensis (81%), Paragonimus westermani (58%), Schistosoma mansoni and S. japonicum (52%), and with vertebrate cathepsin F (51%). Transcripts encoding the protease were detected in all developmental stages that parasitize the mammalian host. The Ov-cf-1 gene, of ∼3 kb in length, included seven exons interrupted by six introns; the exons ranged from 69 to 267 bp in length, the introns from 43 to 1,060 bp. The six intron/exon boundaries of Ov-cf-1 were conserved with intron/exon boundaries in the human cathepsin F gene, although the gene structure of human cathepsin F is more complex. Unlike Ov-CF-1, human cathepsin F zymogen includes a cystatin domain in the prosegment region. Phylogenetic analysis revealed that the fluke, human, and other cathepsin Fs branched together in a clade discrete from the cathepsin L cysteine proteases. A recombinant Ov-CF-1 zymogen that displayed low-level activity was expressed in the yeast Pichia pastoris. Although the recombinant protease did not autocatalytically process and activate to a mature enzyme, trans-processing by Fasciola hepatica cathepsin L cleaved the prosegment of Ov-CF-1, releasing a mature cathepsin F with activity against the peptide Z-Phe-Arg-NHMec >50 times that of the zymogen. Immunocytochemistry using antibodies raised against the recombinant enzyme showed that Ov-CF-1 is expressed in the gut of the mature hermaphroditic fluke and also in the reproductive structures, including vitelline glands, egg, and testis. Ov-CF-1 was detected in bile duct epithelial cells surrounding the flukes several weeks after infection of hamsters with O. viverrini and, in addition, had accumulated in the secondary (small) bile ducts where flukes cannot reach due to their large size.Conclusions/SignificanceA cathepsin F cysteine protease of the human liver fluke O. viverrini has been characterized at the gene and protein level. Secretion of this protease may contribute to the hepatobiliary abnormalities, including cholangiocarcinogenesis, observed in individuals infected with this parasite.

Excretory/secretory products of the carcinogenic liver fluke are endocytosed by human cholangiocytes and drive cell proliferation and IL6 production

“…Sripa and Kaewkes (2000a) observed an intense inflammatory response in areas of the biliary epithelium where parasite antigens were present, particularly those tissues in contact with the fluke. Moreover, numerous studies have provided evidence, albeit with low-resolution microscopy, that fluke antigens are detected inside cholangiocytes lining the biliary epithelium in O. viverrini- infected hamsters (Sripa and Kaewkes, 2000a; Pinlaor et al, 2009; Smout et al, 2009). Until now, the mechanism by which liver fluke antigens are internalised by cholangiocytes remained elusive.…”

Functional Genes and Proteins of Clonorchis sinensis

“…The fact that cysteine proteases are secreted into ES products suggests that act as digestive agents in the intestine. A recent study on cathepsin F of O. viverrini revealed enzyme in epithelial cells lining the bile ducts of infected animals, which suggested to possibility that it may stimulate biliary epithelium inflammation and proliferation and promote cholangiocarcinogenesis [57]. Hence, the extracorporeal roles of the cysteine proteases of C. sinensis warrant further investigation.…”

“…Genomic and proteomic analysis of several major global helminth parasites have revealed that parasite-derived proteases are key virulence factors [44][45][46][47][48][49][50][51][52]. The proteases of helminth parasites play numerous indispensable roles in parasite physiology, such as, in protein processing and the turnover of parasite proteins, and in various pathogenic aspects, such as, the facilitation of parasite penetration or invasion into host tissue, the hydrolysis of host proteins for nutrient uptake, and host immune system modulation [46,[53][54][55][56][57][58][59]. Accordingly, their activities are essential for parasite survival and growth, which suggests that they are attractive targets for vaccines or chemotherapeutic agents [53,[60][61][62][63].…”

Molecular cloning of a multidomain cysteine protease and protease inhibitor precursor gene from the tobacco hornworm (Manduca sexta) and functional expression of the cathepsin F-like cysteine protease domain

“…However, it has been predicted that the N-terminal region of human procathepsin F has a cystatin-like fold (Nägler et al, 1999). Cathepsin F from the human liver fluke, Opisthorchis viverrini, lacks a region corresponding to the cystatin-like domain (Pinlaor et al, 2009). …”