Scuticociliate proteinases may modulate turbot immune response by inducing apoptosis in pronephric leucocytes.
The role of proteinases of the histiophagous ciliate Philasterides dicentrarchi, purified by affinity chromatography in bacitracin-Sepharose, on apoptosis (programmed cell death) of turbot pronephric leucocytes (PL) was investigated. The results showed that more than 90% of proteinases purified by bacitracin-Sepharose were cysteine proteinases, which lacked significant caspase-3-like activity and generated three main gelatinolytic bands of molecular weights 36, 45 and 77 kDa as determined by gelatine-SDS-PAGE and immunoblot. Viability of PL cells after 24 h stimulation with P. dicentrarchi cysteine proteinases did not differ from that of non-stimulated cells. Apoptosis was confirmed by: (i) caspase activity, (ii) DNA fragmentation, and (iii) nucleus fragmentation. The caspase-3-like activity in PL incubated for 4h in the presence of 125, 250 and 500 microg/ml of proteinases increased in a dose-dependent fashion. The PL DNA was fragmented following 24-h exposure to P. dicentrarchi cysteine proteinases and characteristic DNA ladders consisting of multimers of approximately 180-200 pb were produced. Morphological changes, such as chromatin condensation and nucleus fragmentation, were observed under fluorescence microscopy after DAPI staining of the PL cells incubated with cysteine proteinase-incubated for 24 h. The results suggest that the pathogenic scuticociliate P. dicentrarchi may induce host leucocyte programmed cell death via the production of cysteine proteinases, as a mechanism of pathogenesis and evasion of the turbot innate immune response.
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Departamento de Microbiología y Parasitología, Laboratorio de Parasitología, Instituto de Investigación y Análisis Alimentarios, Universidad de Santiago de Compostela, C/ Constantino Candeira, s/n, 15782 Santiago de Compostela, Spain.
Castro
R
R
Lamas
J
J
Sanmartín
M L
ML
Santamarina
M T
MT
Leiro
J
J
D000818:Animals; D017209:Apoptosis / Q000276:immunology*; D053148:Caspase 3 / Q000096:biosynthesis / Q000276:immunology; D002467:Cell Nucleus / Q000276:immunology; D002470:Cell Survival / Q000276:immunology; D016798:Ciliophora / Q000276:immunology*; D016770:Ciliophora Infections / Q000276:immunology; D003546:Cysteine Endopeptidases / Q000276:immunology / Q000302:isolation & purification; D053938:DNA Fragmentation; D016054:DNA, Protozoan / Q000276:immunology; D004587:Electrophoresis, Agar Gel / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005393:Fish Diseases / Q000276:immunology; D005412:Flatfishes / Q000276:immunology*; D015151:Immunoblotting / Q000379:methods; D007668:Kidney / Q000166:cytology / Q000276:immunology; D007962:Leukocytes / Q000276:immunology*; D010447:Peptide Hydrolases / Q000276:immunology* / Q000302:isolation & purification; D011529:Protozoan Infections, Animal / Q000276:immunology
D016054:DNA, Protozoan; D010447:Peptide Hydrolases; D053148:Caspase 3; D003546:Cysteine Endopeptidases