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Cysteine proteinase activities in the fish pathogen Philasterides dicentrarchi (Ciliophora: Scuticociliatida)

Published online by Cambridge University Press:  06 May 2004

A. PARAMÁ
Affiliation:
Laboratory of Parasitology, Institute of Food Investigation and Analysis, University of Santiago de Compostela, C/Constantino Candeira s.n., 15782 Santiago de Compostela, Spain
R. IGLESIAS
Affiliation:
Laboratory of Parasitology, Faculty of Sciences, University of Vigo, Lagoas-Marcosende s/n, 36200 Vigo, Spain
M. F. ÁLVAREZ
Affiliation:
Laboratory of Parasitology, Institute of Food Investigation and Analysis, University of Santiago de Compostela, C/Constantino Candeira s.n., 15782 Santiago de Compostela, Spain
J. LEIRO
Affiliation:
Laboratory of Parasitology, Institute of Food Investigation and Analysis, University of Santiago de Compostela, C/Constantino Candeira s.n., 15782 Santiago de Compostela, Spain
F. M. UBEIRA
Affiliation:
Laboratory of Parasitology, Institute of Food Investigation and Analysis, University of Santiago de Compostela, C/Constantino Candeira s.n., 15782 Santiago de Compostela, Spain
M. L. SANMARTÍN
Affiliation:
Laboratory of Parasitology, Institute of Food Investigation and Analysis, University of Santiago de Compostela, C/Constantino Candeira s.n., 15782 Santiago de Compostela, Spain

Abstract

This study investigated protease activities in a crude extract and in vitro excretion/secretion (E/S) products of Philasterides dicentrarchi, a ciliate fish parasite causing economically significant losses in aquaculture. Gelatin/SDS–PAGE analysis (pH 4, reducing conditions) detected 7 bands with gelatinolytic activity (approximate molecular weights 30–63 kDa) in the crude extract. The banding pattern observed in analysis of E/S products was practically identical, except for 1 low-molecular-weight band detected in the crude extract but not in the E/S products. In assays with synthetic peptide p-nitroanilide substrates, the crude extract hydrolysed substrates characteristic of cysteine proteases, namely Z-Arg-Arg pNA, Bz-Phe-Val-Arg pNA and Z-Phe-Arg pNA. These activities were strongly inhibited by the cysteine protease inhibitor E-64 and by Ac-Leu-Val-Lys aldehyde, a potent inhibitor of cysteine proteases of the cathepsin B protease subfamily. The proteases present in the crude extract degraded both type-I collagen and haemoglobin in vitro, consistent with roles in tissue invasion and nutrition respectively. Again, E-64 completely (collagen) or markedly (haemoglobin) inhibited this degradation. Finally, the histolytic activity of the ciliate in turbot fibroblast monolayers was strongly reduced in the presence of E-64, confirming the importance of secreted cysteine proteinases in the biology of Philasterides dicentrarchi.

Type
Research Article
Copyright
2004 Cambridge University Press

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