Trichuris suis: thiol protease activity from adult worms.
Trichuris suis, the whipworm of swine, causes anemia, weight loss, anorexia, mucohemorrhagic diarrhea, and death in heavy infections. A zinc metalloprotease has been suggested to play a role in the severe enteric pathology associated with infection and the infiltration of opportunistic bacteria into deeper tissues in the swine colon. In this study, a thiol protease from gut extracts of adult T. suis and from excretory/secretory components (E/S) of adult worms was characterized using fluorogenic peptide substrates and protein substrate gels. The protease cleaved the fluorogenic substrate Z-Phe-Arg-AMC, and this cleavage was completely inhibited by the thiol protease inhibitors E-64, leupeptin, Z-Phe-Ala-CH2F, and Z-Phe-Arg-CH2F. Gelatin substrate gels and fluorescence assays using both the gut and the stichosome extracts and E/S revealed enhanced activity when 2 mM dithiothreitol or 5 mM cysteine was included in the incubation buffer, and optimal activity was seen over a pH range of 5.5 to 8.5. Incubation of gut extracts or E/S material with inhibitors of aspartic, serine, or metalloproteases had no effect on the cleavage of Z-Phe-Arg-AMC. Thiol protease activity was found in extracts of gut tissue but not in the extracts of stichocytes of adult worms. N-terminal amino acid sequencing of the protease revealed sequence homologies with cathepsin B-like thiol protease identified from parasitic and free-living nematodes.
Hill
D E
DE
USDA, ARS, LPSI, Parasite Biology and Epidemiology Laboratory, Beltsville, Maryland 20705, USA.
Sakanari
J A
JA
D000595:Amino Acid Sequence; D000818:Animals; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D005260:Female; D006863:Hydrogen-Ion Concentration; D008297:Male; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013552:Swine; D014258:Trichuris / Q000201:enzymology*
D015853:Cysteine Proteinase Inhibitors; D003546:Cysteine Endopeptidases