Feline Tritrichomonas foetus adhere to intestinal epithelium by receptor–ligand-dependent mechanisms

Abstract

Tritrichomonas foetus (TF) is a protozoan that infects the feline ileum and colon resulting in chronic diarrhea. Up to 30% of young purebred cats are infected with TF and the infection is recognized as pandemic. Only a single drug, characterized by a narrow margin of safety and emerging development of resistance, is effective for treatment. While the venereal pathogenicity of bovine TF is attributed to adherence to uterovaginal epithelium, the pathogenesis of diarrhea in feline TF infection is unknown. The aim of this study was to establish an in vitro model of feline TF adhesion to intestinal epithelium. Confluent monolayers of porcine intestinal epithelial cells (IPEC-J2) were infected with axenic cultures of feline TF that were labeled with [3H] thymidine or CFSE and harvested at log-phase. The effect of multiplicity and duration of infection, viability of TF, binding competition, formalin fixation and cytoskeletal inhibitors on adherence of feline TF to IPEC-J2 monolayers was quantified by liquid scintillation counting and immunofluorescence. [3H] thymidine and CFSE-labeled TF reproducibly adhered to IPEC-J2 monolayers. Clinical isolates of feline TF adhered to the intestinal epithelium in significantly greater numbers than Pentatrichomonas hominis, the latter of which is a presumably nonpathogenic trichomonad. Adhesion of TF required viable trophozoites but was independent of cytoskeletal activity. Based on saturation and competition binding experiments, adherence of feline TF to the epithelium occurred via specific receptor-ligand interactions. The developed model provides a valuable resource for assessing pathogenic mechanisms of feline TF and developing novel pharmacologic therapies for blocking the adhesion of feline TF to the intestinal epithelium.

Cysteine Protease Activity of Feline Tritrichomonas foetus Promotes Adhesion-Dependent Cytotoxicity to Intestinal Epithelial Cells

“…IPEC-J2 cells were grown in a coculture media, which included advanced Dulbecco's modified eagle medium-nutrient mixture F-12 (DMEM-F12) supplemented with 5 g/ml each of insulin, transferrin, and selenium, 5 ng/ml epidermal growth factor (EGF), 100 IU/ml penicillin, 100 mg/ml streptomycin, and 5% fetal bovine serum, and incubated at 37°C in 5% CO 2 Trichomonads. Isolation and culture of feline T. foetus and Pentatrichomonas hominis isolates were performed as previously described (15). Trichomonads were harvested in mid-to late-logarithmic phase by centrifugation at 250 ϫ g and washed twice in Hanks' balanced salt solution (HBSS).…”

“…Gels were then incubated in dH 2 Labeling of trichomonads. Labeling of trichomonads was performed as previously described (15). Briefly, trichomonads were harvested in the late logarithmic phase of growth and inoculated into modified Diamond's media containing 4 Ci/ml [ 3 H]thymidine (17 Ci/mmol) (American Radiolabeled Chemicals).…”

“…Adhesion assays were performed as described previously (15). For coculture adhesion assays, 20 ϫ 10 6 [ 3 H] thymidine-labeled T. foetus cells were inoculated into the apical media of confluent, polarized (transepithelial electrical resistance, Ն2,000 ⍀ · 4.67 cm 2 ) IPEC-J2 epithelial monolayers seeded onto polycarbonate inserts.…”

“…Using a coculture assay approach, we have previously demonstrated that feline T. foetus adheres to intestinal epithelial monolayers in vitro by kinetics that suggest a specific interaction of T. foetus with the epithelium (15). In venereal trichomonosis, adherence of trichomonads to the urogenital epithelium and elaboration of proteases are recognized as central events in mediating host cellular pathogenicity (16)(17)(18).…”

Mechanisms ofTritrichomonas foetusPathogenicity in Cats with Insights from Venereal Trichomonosis

“…In panel C , 3 H‐thymidine‐labeled trichomonads demonstrate saturable binding when added in increasing numbers to the apical media of monolayers of porcine intestinal epithelial cells. Figure 5B and C reproduced from47 with written permission.…”