In vitro effects of cysteine protease inhibitors on Trichomonas foetus–induced cytopathic changes in porcine intestinal epithelial cells

Abstract

OBJECTIVE To investigate the effects of specific cysteine protease (CP) inhibitors on cytopathic changes to porcine intestinal epithelial cells induced by Tritrichomonas foetus isolated from naturally infected cats. SAMPLE T foetus isolates from 4 naturally infected cats and nontransformed porcine intestinal epithelial cells. PROCEDURES T foetus isolates were treated with or without 0.1 to 1.0mM of the CP inhibitors antipain, cystatin, leupeptin, and chymostatin and the vinyl sulfone inhibitors WRR-483 and K11777. In-gel gelatin zymography was performed to evaluate the effects of these inhibitors on CP activity of T foetus isolates. Each treated or untreated isolate was also cocultured with monolayers of porcine intestinal epithelial cells for 24 hours, and cytopathic effects of T foetus were evaluated by light microscopy and crystal violet spectrophotometry. RESULTS Results of in-gel gelatin zymography suggested an ability of WRR-483, K11777, and cystatin to target specific zones of CP activity of the T foetus isolates. These inhibitors had no effect on T foetus growth, and the cytopathic changes to the intestinal epithelium induced by all 4 T foetus isolates were significantly inhibited. CONCLUSIONS AND CLINICAL RELEVANCE This study revealed that certain protease inhibitors were capable of inhibiting regions of CP activity (which has been suggested to cause intestinal cell damage in cats) in T foetus organisms and of ameliorating T foetus-induced cytopathic changes to porcine intestinal epithelium in vitro. Although additional research is needed, these inhibitors might be useful in the treatment of cats with trichomonosis.

Comparative membrane proteomic analysis of Tritrichomonas foetus isolates

“…Following such recognition, the parasite synthesizes cytotoxic molecules that assist in colonization of the epithelium. These molecules include neuraminidases 98,99 , enzymes with sialidase activity that cleave sialic acid from the vaginal epithelium, thus exposing α-D-Nacetylgalactosamine residues. Infection with T. foetus modifies the expression of glycoconjugates in the genital epithelium of infected females, both in cattle 100 and in a murine model 101,102 ; and recently we found that these changes may vary upon infection with different T. foetus isolates and with different degrees of pathogenicity 103 .…”

“…foetus, the presence of sialic acid on the surface of host cells is essential for its recognition and subsequent adhesion 98,99 . Following such recognition, the parasite synthesizes cytotoxic molecules that assist in colonization of the epithelium.…”

The conundrum of feline trichomonosis: the more we learn the ‘trickier’ it gets

“…25 Cysteine proteases appear to mediate both adhesion and cytotoxic effects on the intestinal epithelium, and are being explored as possible treatment targets to ameliorate the clinical signs of infection. 68,78,79 Large scale studies to define the transcriptome and proteome of Tritrichomonas species to identify virulence factors and to screen large libraries of drugs for effectiveness in killing Tritrichomonas species are actively ongoing by several teams of investigators. 68,80…”

“…25 Cysteine proteases appear to mediate both adhesion and cytotoxic effects on the intestinal epithelium, and are being explored as possible treatment targets to ameliorate the clinical signs of infection. 68,78,79…”