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Publication type: Journal Article
Document type: Full Paper

Year: 2012

Author(s): Amin, A; Nöbauer, K; Patzl, M; Berger, E; Hess, M; Bilic, I

Title: Cysteine peptidases, secreted by Trichomonas gallinae, are involved in the cytopathogenic effects on a permanent chicken liver cell culture.

Source: PLoS One. 2012; 7(5):e37417



Authors Vetmeduni Vienna:

Bilic Ivana
Hess Michael
Nöbauer Katharina
Patzl Martina

Vetmed Research Units
University Clinic for Poultry and Fish Medicine, Clinical Unit of Poultry Medicine
Institute of Immunology
VetCore


Abstract:
Trichomonas gallinae, the aetiological agent of avian trichomonosis, was shown to secrete soluble factors involved in cytopathogenic effect on a permanent chicken liver (LMH) cell culture. The present study focused on the characterization of these molecules. The addition of specific peptidase inhibitors to the cell-free filtrate partially inhibited the monolayer destruction, which implied the presence of peptidases in the filtrate and their involvement in the cytopathogenic effect. One-dimensional substrate (gelatin) SDS-PAGE confirmed the proteolytic character of the filtrate by demonstrating the proteolytic activity within the molecular weight range from 38 to 110 kDa. In addition, the proteolytic activity was specifically inhibited by addition of TLCK and E-64 cysteine peptidase inhibitors implying their cysteine peptidase nature. Furthermore, variations in the intensity and the number of proteolytic bands were observed between cell-free filtrates of low and high passages of the same T. gallinae clonal culture. Two-dimensional substrate gel electrophoresis of concentrated T. gallinae cell-free filtrate identified at least six proteolytic spots. The mass spectrometric analysis of spots from 2-D gels identified the presence of at least two different Clan CA, family C1, cathepsin L-like cysteine peptidases in the cell-free filtrate of T. gallinae. In parallel, a PCR approach using degenerated primers based on the conserved amino acid sequence region of cysteine peptidases from Trichomonas vaginalis identified the coding sequences for four different Clan CA, family C1, cathepsin L-like cysteine peptidases. Finally, this is the first report analyzing molecules secreted by T. gallinae and demonstrating the ubiquity of peptidases secreted by this protozoon.

Keywords Pubmed: Animals
Cells, Cultured
Chickens
Cysteine Endopeptidases/genetics
Cysteine Endopeptidases/metabolism*
Cysteine Proteinase Inhibitors/pharmacology*
DNA Primers/genetics
Electrophoresis, Gel, Two-Dimensional
Electrophoresis, Polyacrylamide Gel
Hepatocytes/metabolism*
Leucine/analogs & derivatives
Mass Spectrometry
Tosyllysine Chloromethyl Ketone
Trichomonas/enzymology*


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