The involvement of the cysteine proteases of Clonorchis sinensis metacercariae in excystment

Abstract

The effects of trypsin, bile, trypsin-bile, pepsin, dithiothreitol (DTT) and metacercarial excretory-secretory product (ESP) on the in vitro excystment of Clonorchis sinensis metacercariae were investigated. The majority of metacercariae excysted immediately in trypsin-bile in PBS solution, a process which was complete after 30 min of incubation. When incubated in metacercarial ESP in PBS, excystment was potentiated in the presence of 5 mM DTT, but was inhibited dose-dependently by a cysteine protease inhibitor, iodoacetic acid. Two active protease bands of 28 and 40 kDa were identified in the ESP of metacercariae by gelatin substrate SDS-PAGE. Scanning electron microscopy demonstrated that the larvae in solutions of DTT and ESP migrated through a small hole on the metacercarial wall, whereas larvae were liberated by entire wall disruption in trypsin solution. These results suggest that trypsin is a major extrinsic factor of the rapid excystment of C. sinensis metacercariae, and that endogenous cysteine proteases are also involved in metacercarial excystment.

Bile components and amino acids affect survival of the newly excysted juvenile Clonorchis sinensis in maintaining media

“…The mode of action of the bile is unclear, but is believed to induce nonspecific stimuli that cause muscular movement (Dixon 1966). In the case of C. sinensis, bile enhanced both metacercariae excystation and increased their motility (Li et al 2004b), observations confirmed in the present study. These findings are supportive of previous suggestions that bile is one of the major activating agents for intestinal parasites.…”

An update on metacercarial excystment of trematodes

“…The combination of extrinsic factors used in their in vitro experiments appears to be similar to what is provided by the definitive host for the successful in vivo excystation of M. turgidus. Li et al (2004) investigated the effects of trypsin, bile, trypsin-bile, pepsin, DTT, and metacercarial excretorysecretory products (ESP) on the in vitro excystment of Clonorchis sinensis metacercariae. The authors found that most of the metacercariae excysted immediately in a trypsin-bile phosphate-buffered saline (PBS) solution; excystation under these conditions was complete after 30 min of incubation.…”

Long-term storage of Clonorchis sinensis metacercariae in vitro

“…Endogenous cysteine proteinases of C. sinensis are known to help metacercariae excyst spontaneously during long-time refrigerated storage, and therefore, PBSI (PBS containing IAA) was expected promote viability during storage (Li et al 2004b). However, the authors found that PBSI was a poorer storage medium than PBSA or PBS, which suggests that the addition of IAA detrimentally affects metacercariae viability in storage.…”

“…Recently, ammonia produced in dead fish was found to be lethal to infecting metacercariae (Zhang et al 2003), and therefore, the isolation of metacercariae from fish improves their long-term survivals. In addition, endogenous cysteine proteinase was found to cause metacercariae to excyst spontaneously in cold saline (Li et al 2004b), suggesting that enhanced endogenous cysteine proteinase activity reduces metacercariae viability. Moreover, researches on animals require that metacercariae be maintained for longer than 60 days in the laboratory.…”