Mechanisms of Feline Tritrichomonas foetus Adherence to the Intestinal Epithelium and Novel Sites for Pharmacological Control

Background and Aims: Tritrichomonas foetus (TF) is a mucosal protozoan that parasitizes the feline ileum and colon resulting in chronic diarrhea. TF has a worldwide distribution with no effective therapies to treat infection. Thus, defining the cellular mechanisms of disease and developing novel treatment strategies are of considerable interest. The biochemical mechanisms by which TF causes diarrhea are largely unknown, however adhesion to the epithelium and elaboration of cellular proteases are considered to be key events in venereal trichomonad pathogenicity. Using a developed in vitro model of feline TF adhesion to intestinal epithelium, these studies explore the hypothesis that adherence is a key initiating event in TF pathogenicity and that this event is linked to its pathogenic effects and is a potential pharmacological target for prevention or amelioration of clinical disease. Methods: The effect of multiplicity of infection, viability of TF, binding competition, formalin fixation, cytoskeletal inhibitors, sialic acid and protease inhibitors on adherence of feline TF to IPEC-J2 monolayers was quantified by liquid scintillation counting and immunofluorescence. To evaluate for adhesion-dependent cytotoxicity, light microscopy and western blotting for cleaved cytokeratin 18 was performed following co-culture with whole cell trichomonads or their secretory products. Gelatin-SDS-PAGE was used to characterize the proteolytic profile of feline TF isolates. To assess the role of proteases as virulence factors in adhesion-dependent cytotoxicity, adhesion and cytotoxicity studies were performed in the presence or absence of protease inhibitors. Results: 3[H]thymidine and CFSE-labeled TF cytoadhered to IPEC-J2 monolayers in a dose and time-dependent manner with reproducible adhesion achieved by infection with 20x10 6 Tf for 6hrs. Clinical isolates of feline TF (n=5) demonstrated significantly greater adhesion to intestinal epithelium that Pentatrichomonas hominis , the latter of which is a presumably nonpathogenic trichomonad of domestic animals and people. Adhesion of TF required viable trophozoites and was dependent on specific receptor-ligand interactions. Treatment of TF with N-acetylneuraminic acid and cysteine protease inhibitors significantly blocked adherence. Protein patterns from TF isolates (n=4) were similar and revealed numerous protease activities belonging to both the serine and cysteine protease class. In contrast, feline Pentatrichomonas hominis, produced little protease activity compared to TF and had no detectable cysteine protease activity. TF stimulated IPEC-J2 cells to undergo apoptosis, an effect that was found to be both dependent on adhesion of the parasite to the intestinal epithelium and exposure to trichomonad cysteine proteases. Inhibition of cysteine protease activity significantly reduced adhesion and ameliorated cytopathogenicity. Conclusions: Our studies have resulted in the development a unique model that can be used for further study of TF cytopathogenicity. We demonstrate for the first time that TF adhere to the intestinal epithelium via specific receptor ligand interactions. Further, cytotoxicity is dependent on direct interaction of the epithelium and exposure to TF cell-associated cysteine proteases. These studies support a central role for cysteine protease activity in TF adhesion-dependent cytotoxicity of the intestinal epithelium and may suggest a novel molecular target for therapeutic intervention of intestinal trichomonosis.