Characterization of a cysteine protease from Tritrichomonas foetus that induces host-cell apoptosis

Tritrichomonas foetus is a serious veterinary pathogen, causing bovine trichomoniasis and affecting cattle herds world-wide, resulting in inflammation of the genital tract, infertility and huge economic losses. The parasite secretes a cysteine protease (CP8), which induces cytotoxicity and apoptosis in bovine vaginal and uterine epithelial cells. Mallinson et al. [D.J. Mallinson, J. Livingstone, K.M. Appleton, S.J. Lees, G.H. Coombs, M.J. North, Microbiology 1995, 141 (12) 3077-3085.] originally reported a partial DNA sequence of T foetus CP8 based on PCR cloning of T.foetus genomic DNA. Here we report the biochemical properties of the CP8 enzyme. Kinetic properties and the substrate specificity profile of T foetus CP8 were studied using positional scanning synthetic combinatorial libraries and Michaelis-Menten kinetic analysis of three synthetic fluorogenic substrates. The preferred substrate Z-Leu-Arg-MCA prevented host-cell death/apoptosis induced by CP8. In addition, the DNA sequence was completed by 3' and 5' rapid amplification of cDNA ends (RACE) and the full-length amino acid sequence was obtained. (C) 2008 Elsevier Inc. All rights reserved.