Parasitology



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


A. PARAMÁ a1, R. IGLESIAS a2, M. F. ÁLVAREZ a1, J. LEIRO a1, F. M. UBEIRA a1 and M. L. SANMARTÍN a1c1
a1 Laboratory of Parasitology, Institute of Food Investigation and Analysis, University of Santiago de Compostela, C/Constantino Candeira s.n., 15782 Santiago de Compostela, Spain
a2 Laboratory of Parasitology, Faculty of Sciences, University of Vigo, Lagoas-Marcosende s/n, 36200 Vigo, Spain

Article author query
parama a   [PubMed][Google Scholar] 
iglesias r   [PubMed][Google Scholar] 
alvarez m   [PubMed][Google Scholar] 
leiro j   [PubMed][Google Scholar] 
ubeira f   [PubMed][Google Scholar] 
sanmartin m   [PubMed][Google Scholar] 

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.

(Received March 25 2003)
(Revised October 20 2003)
(Accepted October 23 2003)


Key Words: Philasterides dicentrarchi; fish pathogen; scuticociliatosis; ciliate; cysteine protease; histolytic activity.

Correspondence:
c1 Instituto de Investigación y Análisis Alimentarios, Universidad de Santiago de Compostela, C/Constantino Candeira s.n., 15782 Santiago de Compostela, Spain. Tel: +34 981 563100, ext. 16031, 14893. Fax: +34 981 547171. E-mail: mpduran@usc.es


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