A family of cathepsin F cysteine proteases of Clonorchis sinensis is the major secreted proteins that are expressed in the intestine of the parasite☆
Graphical abstract
Localizations of CsCF-4, CsCF-6, and CsCF-11. (A) Sections of Clonorchis sinensis adult worms were probed with rat anti-CsCF-4 and mouse anti-CsCF-6 followed by FITC-conjugated anti-rat IgG and TRITC-conjugated anti-mouse IgG. The slide was observed with a confocal laser scanning microscope (10× magnification). (B) Sections of C. sinensis adult worms were probed with mouse anti-CsCF-6 and rat anti-CsCF-11 followed by FITC-conjugated anti-rat IgG and TRITC-conjugated anti-mouse IgG. The slide was observed with a confocal laser scanning microscope (100× magnification). I, intestine; L, intestinal lumen; U, uterus; SR, seminal receptacle. Scale bar indicated 100 μm.
Introduction
Cysteine proteases of trematode parasites play essential roles in parasite physiology such as protein processing and turnover of parasite proteins as well as in various aspects of their pathogenicity such as facilitation of parasite penetration or invasion into host tissue, hydrolysis of host proteins for nutrient uptake, and modulation of the host immune system [1], [2], [3], [4], [5], [6], [7], [8]. Comprehensive studies have been done to characterize the biochemical properties and pathophysiological roles of cysteine proteases of trematode parasites and their essential roles in parasite survival or growth make them attractive targets for vaccines or chemotherapeutic agents [1], [9], [10], [11], [12].
Clonorchis sinensis is a causative agent of clonorchiasis in humans. It is prevalent in Far East Asian countries, e.g. China, Korea, Japan and Taiwan, and is estimated to infect about 35 million people worldwide [13]. Clonorchiasis is usually acquired by the ingestion of raw or under cooked fresh water fish that are infected with C. sinensis metacercariae. The excysted metacercariae in the host's duodenum migrate through the ampulla of Vater into the bile duct where they mature into adult worms. Most pathologic manifestations of clonorchiasis are mainly caused by inflammation and intermittent obstruction of the biliary ducts, but chronic and severe infections might be complicated by cholangitis, cholelithiasis, and cholangiectasis and even by cholangiocarcinoma [14], [15], [16].
Cysteine proteases of C. sinensis are likely to be developmentally regulated and are thought to participate in essential biological processes such as stage transition, immune evasion and nutrient uptake from the host [17], [18], [19], [20], [21], [22]. Several genes encoding C. sinensis cysteine proteases have been identified and partially characterized [19], [20], [22], [23], [24]. Recent studies that have shown cysteine proteases make up a large proportion of the total transcriptome of C. sinensis [25], [26] have also illustrated their biological importance for parasite physiology and survival in the host. However, the nature and the biological functions of these enzymes are poorly understood.
Here, we describe a multigene family of cathepsin F cysteine proteases of C. sinensis (CsCFs). Our findings revealed that at least 12 genes encoding cathepsin F proteins, which clustered into three separate groups based on their amino acid sequences, were identified in C. sinensis. We also characterized representatives of each group of protease biochemically and analyzed their expression during the developmental stages of the parasite. Our results demonstrated that CsCFs, which are synthesized in the intestinal epithelium and secreted into the intestinal lumen of the parasite, are major secreted proteins of C. sinensis. These enzymes might play important roles in nutrient uptake by C. sinensis as well as other key functions in parasite–host interactions.
Section snippets
Parasites
The metacercariae of C. sinensis were collected from naturally infected Pseudorasbora parva obtained in Korea. Sprague–Dawley rats were infected orally with 100 metacercariae each. Two-week-old juvenile worms were harvested from the livers of rats 2 weeks after experimental infection with the metacercariae. The 4-, 6-, and 9-week-old adult worms were also collected from the livers of rats 4, 6 or 9 weeks after experimental infection, respectively [22]. The worms were washed 5 times with cold
Identification and cloning of a multigene family of CsCFs
Following PCR with degenerate primers and sequencing analysis of 240 randomly selected clones from 3 developmental stages of C. sinensis, we obtained a total of 12 different partial gene sequences putatively encoding cysteine proteases of C. sinensis. Megablast nucleotide blast search of the NCBI database revealed that 6 (namely CsCF-1 to CsCF-6) of the sequences were identified previously reported, but the other 6 sequences (CsCF-7 to CsCF-12) were novel sequences which were different from all
Discussion
In this study, we identified a multigene family of CsCFs and characterized the biochemical and functional properties of the representative enzymes for each subfamily. As like in other parasitic helminthes, cysteine proteases have been considered to be one of the most abundantly expressed protease families in C. sinensis and they are likely to be developmentally regulated throughout various developmental stages of the parasite [18]. In search of new cysteine proteases in C. sinensis, we
Acknowledgement
This study was supported by a grant of the Korea Centers for Disease Control and Prevention (2008-E00272-00).
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