A family of cathepsin F cysteine proteases of Clonorchis sinensis is the major secreted proteins that are expressed in the intestine of the parasite

https://doi.org/10.1016/j.molbiopara.2009.11.006Get rights and content

Abstract

Cysteine proteases of helminth parasites play essential roles in parasite physiology as well as in a variety of important pathobiological processes. In this study, we identified a multigene family of cathepsin F cysteine proteases in Clonorchis sinensis (CsCFs). We identified a total of 12 CsCF genes through cDNA cloning using degenerate PCR primers followed by RACE. Sequence and phylogenetic analysis of the genes suggested they belonged to the cathepsin F-like enzyme family and further clustered into three different subfamilies. Enzymatic and proteomic analysis of C. sinensis excretory and secretory products (ESP) revealed that multiple isoforms of CsCF were the major proteins present in the ESP and the proteolytic activity of the ESP is mainly attributable to the enzymes. Comparative analysis of representative enzymes for each subfamily, CsCF-4, CsCF-6, and CsCF-11, showed that they share similar biochemical properties typical for cathepsin F-like enzymes, but significant differences were also identified. The enzymes were expressed throughout various developmental stages of the parasite and the transcripts increased gradually in accordance with the maturation of the parasite. Immunolocalization analysis of CsCFs showed that they were mainly localized in the intestine and intestinal contents of the parasite. These results collectively suggested that CsCFs, which are apparently synthesized in the epithelial cells lining the parasite intestine and secreted into the intestinal lumen of the parasite, might have a cooperative role for nutrient uptake in the parasite. Furthermore, they were eventually secreted into outside of the parasite and may perform additional functions for host–parasite interactions.

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.

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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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    Note: Nucleotide sequence data reported in this paper is available in the GenBank database under accession numbers DQ346207DQ346210 and DQ346212.

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