ENDOGENOUS CYSTEINE PROTEASE

To infect definitive or paratenic hosts, metacercariae of Paragonimus westermani should excyst in the host intestine. Optimum conditions for the excystment have been known to be pH 8-9 and a temperature of 40 C. Under these conditions, excystment of P. westermani metacercariae was accelerated in the presence of I mM dithiothreitol (DTT). The DTT acceleration was antagonized dose-dependently by cysteine protease inhibitors of L-trans-epoxysuccinylleucylamido(4-guanidino)butane (E- 64, 2-20 MM) or leupeptin (0.1-1 mM), suggesting that certain cysteine proteases of the metacercaria are involved in excystment. Protease activities were detected in excretory-secretory products (ESP) of newly excysted metacercariae. Two distinct proteases were purified by DEAE anion-exchange chromatography of the ESP. While a 27-kDa protease exhibited endodipeptidolytic activity at pH 5-8.5 and remained stable at neutral pH for 3 days, the 28-kDa enzyme was stable at pH 5-7.5, with lower activity at pH 8.5. Both proteases hydrolyzed collagen, fibronectin, and myosin within I hr at pH 8. These results suggest that cysteine proteases secreted by P. westermani metacercariae modulate excystment. Different trematode species utilize different extrinsic and pos- sibly intrinsic factors in triggering excystment of metacercariae. As for Paragonimus westermani, which is the most important species causing endemic paragonimiasis in east and southeast Asia, the extrinsic factors favoring excystment of metacercariae include, among others, a pH of 8-8.5, a temperature of 40 C, and bile salts. Under these in vitro conditions, excystment of the metacercariae reached about 50% after a 4-hr incubation. Bile salt is, however, unlikely to be an absolute requirement because of its inconsistent effects. In contrast, in vivo experi- ments show that the excystment of the metacercariae is com- pleted within 30 min in a rat's intestine. The excystment mech- anism for P. westermani, as for most other trematodes, remains poorly understood (Yokogawa, 1965). In studies with Fasciola hepatica, excystment of metacercar- iae was stimulated by a combination of factors that included a temperature of 39 C, an alkaline pH, CO2, and bile salts. In addition, low redox potentials were shown to be important (Dix- on, 1966). These results suggest that excystment of F. hepatica metacercariae may produce a secretion that contains an un- known factor responding to low redox potential (Dixon, 1966; Smyth and Halton, 1983; Sommerville and Rogers, 1987). With P. westermani, we hypothesized that excretory-secretory prod- ucts (ESP) from metacercariae, activated by specific stimuli, promote excystment. In the present in vitro study, we found that a reductant, dithiothreitol (DTT) and cysteine protease in- hibitors affected the excystment. Subsequently, 2 purified pro- teases in metacercarial ESP were characterized as cysteine pro- teases.