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.
Highlights
Trichomonas gallinae, a flagellated protozoon is commonly found in the upper digestive tract of different bird species, including columbid, passerine, and psittacine birds as well as falconiformes [1,2,3,4,5,6], but can affect other organs depending on the virulence of the strain [7]
Influence of peptidase inhibitors on the cytopathogenic effect of cell-free filtrate from axenically grown T. gallinae
The results showed that the addition of either 1 mM Phenylmethylsulfonyl fluoride (PMSF), 270 mM E-64 or 135 mM TLCK to the cell-free filtrate of T. gallinae partially inhibited cytopathogenic effects induced by trichomonads (Figure 1)
Summary
Trichomonas gallinae, a flagellated protozoon is commonly found in the upper digestive tract of different bird species, including columbid, passerine, and psittacine birds as well as falconiformes [1,2,3,4,5,6], but can affect other organs depending on the virulence of the strain [7]. Extensive research performed on the analysis of the T. vaginalis culture media revealed the presence of various cysteine peptidases and other molecules that mediate cytotoxicity by damaging the target cell plasma membrane reviewed in Schwebke and Burgess [31]. Some of these cytotoxic molecules have perforin-like activity and create pores in erythrocyte membranes reviewed in Fiori et al [32]; whereas others are different lytic factors with phospholipase A2 activities to destroy nucleated cells and erythrocytes [33]
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