Cysteine proteinase activities in the fish pathogen Philasterides dicentrarchi (Ciliophora: Scuticociliatida).
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.
Paramá
A
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Laboratory of Parasitology, Institute of Food Investigation and Analysis, University of Santiago de Compostela, C/Constantino Candeira s.n., 15782 Santiago de Compostela, Spain.
Iglesias
R
R
Alvarez
M F
MF
Leiro
J
J
Ubeira
F M
FM
Sanmartín
M L
ML
D000818:Animals; D016798:Ciliophora / Q000201:enzymology*; D016770:Ciliophora Infections / Q000469:parasitology / Q000662:veterinary*; D024042:Collagen Type I / Q000378:metabolism; D003124:Colorimetry; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D005347:Fibroblasts / Q000469:parasitology; D005393:Fish Diseases / Q000469:parasitology*; D005412:Flatfishes; D005780:Gelatin / Q000378:metabolism; D006454:Hemoglobins / Q000378:metabolism
D024042:Collagen Type I; D015853:Cysteine Proteinase Inhibitors; D006454:Hemoglobins; D005780:Gelatin; D003546:Cysteine Endopeptidases