Abstract
Proteasomes are intracellular complexes that control selective protein degradation in organisms ranging from Archaea to higher eukaryotes. These structures have multiple proteolytic activities that are required for cell differentiation, replication and maintaining cellular homeostasis. Here, we document the presence of the 20S proteasome in the protist parasite Tritrichomonas foetus. Complementary techniques, such as a combination of whole genome sequencing technologies, bioinformatics algorithms, cell fractionation and biochemistry and microscopy approaches were used to characterise the 20S proteasome of T. foetus. The 14 homologues of the typical eukaryotic proteasome subunits were identified in the T. foetus genome. Alignment analyses showed that the main regulatory and catalytic domains of the proteasome were conserved in the predicted amino acid sequences from T. foetus-proteasome subunits. Immunofluorescence assays using an anti-proteasome antibody revealed a labelling distributed throughout the cytosol as punctate cytoplasmic structures and in the perinuclear region. Electron microscopy of a T. foetus-proteasome-enriched fraction confirmed the presence of particles that resembled the typical eukaryotic 20S proteasome. Fluorogenic assays using specific peptidyl substrates detected presence of the three typical peptidase activities of eukaryotic proteasomes in T. foetus. As expected, these peptidase activities were inhibited by lactacystin, a well-known specific proteasome inhibitor, and were not affected by inhibitors of serine or cysteine proteases. During the transformation of T. foetus to endoflagellar form (EFF), also known as pseudocyst, we observed correlations between the EFF formation rates, increases in the proteasome activities and reduced levels of ubiquitin-protein conjugates. The growth, cell cycle and EFF transformation of T. foetus were inhibited after treatment with lactacystin in a dose-dependent manner. Lactacystin treatment also resulted in an accumulation of ubiquitinated proteins and caused increase in the amount of endoplasmic reticulum membranes in the parasite. Taken together, our results suggest that the ubiquitin-proteasome pathway is required for cell cycle and EFF transformation in T. foetus.
Highlights
The protist Tritrichomonas foetus (Excavata, Parabasalia) is an important pathogen that causes bovine and feline trichomonosis
PS parasites, those that exhibit a pear-shaped body with at least one visible external flagellum (S1A Fig), from axenic cultures maintained under standard conditions and endoflagellar form (EFF) under a temperature-based assay were taken [5, 7]
Populations that contained greater than 90% of parasites in either PS or EFF were used (S1 Fig)
Summary
The protist Tritrichomonas foetus (Excavata, Parabasalia) is an important pathogen that causes bovine and feline trichomonosis. Bovine trichomonosis is a venereal disease that leads to reproductive failure in infected herds, resulting in considerable economic burden in beef-producing areas where open range management and natural breeding are practiced [1]. In addition to its economic and veterinary importance, T. foetus is of interest from the perspective of cell biology. Similar to the related human pathogen Trichomonas vaginalis, T. foetus contains cell structures commonly found in eukaryotes, e.g. endoplasmic reticulum (ER) and Golgi complex. It contains unusual anaerobic energy-generating organelles called hydrogenosomes and a very peculiar cytoskeleton that includes a microtubular pelta-axostylar system, the costa, a large striated root, among others [3]. T. foetus has a crucial position in various schemes of eukaryotic evolution and presents a large genome, which makes it a fascinating model for evolutionary studies [4]
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