Proteases in pathogenic and nonpathogenic haemoflagellates, Cryptobia spp. (Sarcomastigophora:Kinetoplastida), of fishes

Abstract

Proteases were detected in Cryptobia salmositica (pathogenic and nonpathogenic vaccine strains), C. bullocki, and C. catostorni using azocasein and hide powder azure as substrates. Maximum activity occurred in acidic pH and the pathogenic strain of C, salmositica had the highest activity.Cysteine protease was found in pathogenic and nonpathogenic Cryptobia spp., but metallo-protease was only present in the pathogenic strain of C. salmositica. Five enzyrnatic bands were detected in the pathogenic C. salmositica using haemoglobin-SDS-PAGE: four of these were cysteine proteases (49,60, 66 and 97 kDa) and the other was a metallo-protease (200 kDa). The pathogenic C. salmositica lost the metallo-protease after 10 mo of in vitro culture. We suggest that the metallo-protease of the pathogenic C. salmositica is related to pathogenicity of the parasite.

Purified metallo-protease from the pathogenic haemoflagellate Cryptobia salmositica and its in vitro proteolytic activities

“…infected fish are not anaemic) after 1 yr of serial in vitro cultivation (Woo & Li 1990). We (Zuo & Woo 1997a) also found a significant loss of the metallo-protease ifi the parasite after 10 mo of continuous in vitro culture and suggested that the decrease in metallo-protease is related to the loss of virulence. In the present study we purified the metallo-protease (200 kDa) from the pathogenic C. salrnositica and confirmed its proteolytic activities.…”

“…The proteolytic activity in the parasite lysate or in the purified fractions was detected using azocasein (AZC) as the substrate as described previously (Zuo & Woo 1997a). Briefly, 100 p1 of the sample was incubated with 0.5 m1 of AZC (10 mg ml-l) and 0.5 m1 phosphate buffer (0.1 M, pH 6.0) at 37°C for 2 h. 0.2 m1 of 50% trichloroacetic acid (TCA) was then added to terminate the reaction and the tube left to stand at 4°C for 30 min.…”

“…Fractions (2 m1 per tube) were collected. Fractions with proteolytic activity were detected as described above and the metallo-protease and cysteine protease were identified by using protease inhibitors, l , lo-phenanthroline and E-64, respectively (Zuo & Woo 1997a).…”

“…The haemoglobin was removed by centrifugation and the membrane pellet was washed 3 times with PBS. The membrane pellet was then added to PBS containing 0.2% (v/v) Triton X-100 and membrane proteins were extracted as described previously (Zuo & Woo 1996). To determine the degradation of membrane protein by the metalloprotease, 300 1-11 of the membrane protein sample (0.5 m g ml-' in PBS, pH 7.2) was added to 100 p1 of the purified metallo-protease (0.1 mg ml-' in PBS, pH 7.2) and 100 p1 of PBS containing 20 mM CaC1,.…”

“…The pathogenic Cryptobia salmositica has both cysteine and metallo-proteases, but the parasite has lost its metallo-protease in the avirulent strain (Zuo & Woo 1997a). Also, the avirulent strain of C. salmositica (see Woo & Li 1990) and the nonpathogenic C. catostomi (see Bower & Woo 1977) of white suckers Catostomus commersoni have only cysteine protease and their 'Addressee for correspondence.…”