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

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.

Figure 1. Influence of peptidase inhibitors on the cytopathogenic effect of the cell-free filtrate.

LMH monolayer was incubated with cell-free filtrate with and without different peptidase inhibitors. (A) Mean lesion scores, (B) cytotoxicity of LMH cells, as assessed by Promega CellTiter 96® aqueous solution at different time points. Cell-free filtrate was obtained after 24 h of incubation of 10⁷ axenically grown protozoa cells obtained from T. gallinae clone 5895-C1/06, passage18. Absorbance values for Promega CellTiter 96® aqueous solution were recorded at 490 nm using ELISA reader.

Figure 2. Demonstration of protease activity in cell-free filtrates from T. gallinae clone 5895-C1/06 by one-dimensional substrate SDS-PAGE.

A) Substrate (0.2% gelatin) 1-D SDS-PAGE (8%) with the low passage (49x) cell-free filtrate, B) conventional 1-D SDS PAGE (8%) with the low passage (49x) cell-free filtrate, C) Substrate (0.2% gelatin) 1-D SDS-PAGE (8%) with the high passage (130x) cell-free filtrate, and D) conventional 1-D SDS PAGE (8%) with the high passage (130x) cell-free filtrate.

Figure 3. Two-dimensional gel electrophoresis of the cell-free filtrate from clone 5895-C1/06, P53.

A) Substrate (0.2% gelatine) SDS-PAGE (8%) and B) conventional SDS-PAGE (8%) was used as the second dimension, respectively.

Figure 4. Comparison of the predicted amino acid sequences of T. gallinae cysteine peptidases.

Residues identical to consensus are shaded grey; * above a residue indicates a predicted start of mature protein; # above a residue indicates key active site; conserved cysteine residues involved in formation of disulphide bonds are labelled with.