Barbara C. Lockwood

The use of a highly sensitive electrophoretic method to compare the proteinases of trichomonads

A highly sensitive electrophoretic method involving gelatin-containing polyacrylamide gels has been used to analyse trichomonad proteinases. Multiple forms, optimally active at pH 5-6, were present in all four species examined, but the species could be distinguished from one another by both quantitative and qualitative differences. The intestinal parasites, Trichomitus batrachorum and Pentatrichomonas hominis, had lower specific activities than the urogenital parasites, Trichomonas vaginalis and Tritrichomonas foetus, and, in the case of P. hominis, there were fewer enzyme forms. The high activity proteinases of Tritrichomonas foetus had low apparent molecular weights (less than 25 kDa), while the predominant enzymes of Trichomonas vaginalis were of high apparent molecular weight (68-110 kDa). Distinct differences were also observed between the proteinase patterns of various isolates of T. vaginalis. All of the enzymes were stimulated by dithiothreitol, suggesting that they were cysteine proteinases. This was confirmed for the T. vaginalis and Tritrichomonas foetus proteinases from their inhibition by antipain, leupeptin, TLCK and iodoacetic acid. The method allows the detection of proteinases in samples of Trichomonas vaginalis containing as few as 10(4) cells or as little as 1 microgram protein. It was also possible to detect proteinase activity released into the medium. For both T. vaginalis and Tritrichomonas foetus, the extracellular enzymes present during early log phase were qualitatively different from the intracellular proteinases, although the latter were present in samples of media obtained from later cultures (cell densities greater than 1 X 10(5) parasites ml-1). The results show the potential of this technique for detecting proteinases in trichomonad samples in studies aimed at determining proteinase function in pathogenesis and host-parasite relationships.

Trichomonas vaginalis, Tritrichomonas foetus, and Trichomitus batrachorum: Comparative proteolytic activity

At least four proteolytic activities were detected in the lysates of each of Trichomonas vaginalis, Tritrichomonas foetus, and Trichomitus batrachorum. These were HPAase, a dithiothreitol-dependent activity on hide powder azure; AZCase, a dithiothreitol-dependent activity on azocasein; and two distinct activities towards peptide nitroanilide derivatives--one was optimally active at pH 7 and stimulated by dithiothreitol; the other had no dithiothreitol requirement and was highly active at pH 5. HPAase and AZCase were active over a broad pH range. Overall, with respect to these four activities, T. batrachorum and T. vaginalis were quite similar. In contrast, T. vaginalis and T. foetus differed from one another in several respects, notably the level of HPAase activity and the properties of the dithiothreitol-independent activity. Multiple bands of proteinase activity were demonstrated with each species after electrophoresis of parasite extracts on polyacrylamide gels containing denatured haemoglobin. They appeared optimally at acid pH and in the presence of dithiothreitol. The proteinase band patterns of T. foetus were similarly complex (at least six bands), whereas T. batrachorum gave a much simpler pattern (three bands). The sensitivities to proteinase inhibitors suggested that all the activities were due to cysteine proteinases. The results show that there are some similarities in the proteolytic activities of all three trichomonad species, and that the two parasites of the urinogenital tracts of mammals possess additional features in common.

The release of hydrolases from Trichomonas vaginalis and Tritrichomonas foetus.

Trichomonas vaginalis and Tritrichomonas foetus were found to release large amounts of beta-N-acetylglucosaminidase (EC 3.2.1.30), alpha-mannosidase (EC 3.2.1.24), beta-glucosidase (EC 3.2.1.21), acid phosphatase (EC 3.1.3.2) and proteinases during axenic growth in vitro. The enzymes were released continually throughout the growth phase, with the extracellular activity being of the same order as that within the cells. There was differential release of proteinases from Trichomonas vaginalis. The subcellular localization of the hydrolases was determined by differential and isopycnic centrifugation. The intracellular enzymes were shown to be mostly located within particle populations. Centrifugation on Percoll gradients allowed the separation of sub-populations of the particles in T. vaginalis; two distinct sub-populations were apparent with equilibrium densities in 20% (v/v) Percoll of 1.035 and 1.050 g cm-3 respectively. The higher density particles were rich in the hydrolases released most abundantly, suggesting a possible link between enzyme release and these organelles. Distinct subpopulations of hydrolase-containing particles were not detected in Tritrichomonas foetus. The results demonstrate that hydrolytic enzyme release represents a major activity during trichomonad growth.

Analysis of the proteinases of Trypanosoma brucei.

A method comprising enzyme separation by SDS-PAGE and subsequent use of peptidyl aminomethylcoumarins as substrates has been used to study proteinases of the protozoan parasite Trypanosoma brucei. The application of this method has allowed investigation of the substrate specificities of individual proteinases in cell lysates without the need for enzyme purification. The results show that T. brucei contains a group of cysteine proteinases, probably four in number, with substrate and inhibitor specificities similar to those of cathepsin L. A second group of proteinases, larger enzymes with significantly different substrate specificities and sensitivity to inhibitors, was also detected. Peptidyl diazomethanes inhibited the cysteine proteinases and also parasite growth, offering promise that peculiarities in the substrate specificity of trypanosomal cysteine proteinases could be exploited by compounds of this type.

Polyamine biosynthesis in trichomonads.

Trichomonas vaginalis, Tritrichomonas foetus and Trichomitus batrachorum grown in modified Diamonds medium all had high concentrations of putrescine and lower concentrations of spermidine and spermine. Ornithine decarboxylase (ODC; EC 4.1.1.17) was detectable in all three species although at significantly different levels. Trichomonas vaginalis had the highest activity (typically around 1.85 nmol min-1 (mg protein)-1), Trichomitus batrachorum the lowest (0.11 nmol min-1 (mg protein)-1). The Trichomonas vaginalis ODC had an apparent Mr of 230 000 and was severely inhibited by alpha-difluoromethylornithine (DFMO). S-Adenosyl-methionine decarboxylase (EC 4.1.1.50) could not be detected in T. batrachorum but was present in the other two species. Arginine decarboxylase was apparently absent from all three. All three trichomonad species were able to accumulate spermidine and putrescine from the medium. When T. vaginalis was grown in the presence of DFMO (4 mM), which had little effect on parasite growth, ODC activity was reduced by over 99% and the polyamine content was altered; putrescine concentrations were decreased, those of spermidine and spermine remained the same or were raised. DFMO-treated cells accumulated more exogenous putrescine than untreated control cells. The results suggest that the lack of effect of DFMO on T. vaginalis in culture was due to the parasite being able to accumulate polyamines from the growth medium. It appears, therefore, that testing DFMO and similar compounds in axenic trichomonad cultures may well not give a true indication of their effectiveness in vivo where sources of exogenous polyamines may not be available.

Uptake of amino acids by the parasitic, flagellated protist Trichomonas vaginalis

HPLC techniques have been applied to study amino acid uptake and release by Trichomonas vaginalis under a variety of conditions. Studies on the growth of T. vaginalis in complex media and the survival of the parasite in simple media, with and without amino acids and/or maltose, have shown that the growth or survival of T. vaginalis is better in the presence of maltose than when it is absent, and that greater amounts of amino acids are consumed by T. vaginalis in the absence of maltose. The results are consistent with several amino acids, notably arginine, threonine, leucine and methionine, being used by T. vaginalis as energy substrates. T. vaginalis released alanine and glycine into the culture media, the excretion being greater in the presence of maltose. These studies have provided new data on the uptake and release of amino acids by T. vaginalis and pave the way for detailed analysis of key enzymes and the regulation of the pathways involved.

Proteinase activity in rumen ciliate protozoa

Azocasein-degrading proteinase activity was detected in all rumen ciliate protozoa that were examined from four entodiniomorphid and two holotrich genera. All of the activities were optimal in the range pH 4.0-5.0 and were inhibited by cysteine proteinase inhibitors, notably leupeptin. The inhibition profiles and extent of inhibition observed with the different groups of inhibitors were organism-specific. Gelatin-SDS-polyacrylamide gel electrophoresis of protozoal lysates revealed multiple forms of the proteinases in the species examined. The number of enzymes detected, their molecular masses, the level of activity and inhibitor susceptibility was genus-dependent. The proteinase profiles of the two holotrich species differed and inter-species differences were also apparent among species of the genus Entodinium. The characteristics and molecular size distribution of rumen bacterial proteinases were different to the protozoal proteinases. Low levels of proteinase activity, of apparently bacterial origin, were detected by gelatin-SDS-PAGE analysis of cell-free rumen liquor.