Tritrichomonas foetus: characterization of isolates and partial purification of a secreted cytotoxin

Abstract

Putative virulence factors including extracellular proteases, hemagglutinin, hemolysins, and soluble cytotoxins may play significant roles in the pathogenesis of trichomoniasis. The cytotoxicity, hemagglutinating, and hemolytic activity of Tritrichomonas foetus isolate ATCC #30003 and several field isolates were compared. All isolates were hemolytic toward mouse and bovine erythrocytes but not other tested species. The isolates varied significantly in hemagglutinating ability and cytotoxin production. A 40,000 Da soluble cytotoxin was partially purified and characterized. Chromatography separated cytotoxic activity from hemagglutinating and hemolytic activity but not from protease activity. However, protease assays indicated that protease activity was inversely correlated with cytotoxic activity. Characterization studies indicated that cytotoxic activity was destroyed by heat and acidic conditions but repeated freeze/thawing did not diminish activity. Target cell specificity assays showed Henle cells were twice as sensitive to the effects of the cytotoxin as Vero cells. These results suggest that T. foetus isolates vary in the production of virulence factors and produce a soluble relatively stable non-protease cytotoxic protein capable of killing cultured mammalian cells in vitro. Index Descriptors and Abbreviations: Trichomoniasis; Tritrichomonas foetus; cytotoxins; American Type Culture Collection (ATCC); concentrated parasite-conditioned medium (CPCM); cytotoxic unit (CU 50); hemagglutinating unit (HA 100); Dulbecco’s modified Eagle’s medium (DMEM); gel filtration chromatography (GF); hydrophobic interaction chromatography (HIC); ion exchange chromatography (IEC); optical density (OD); phosphate-buffered saline (PBS); serum-free RPMI-1640 medium (RPMI).