Abstract
Trichomonas is an amitochondriate parasitic protozoon specialized for an anaerobic lifestyle. Nevertheless, it is exposed to oxygen and is able to cope with the resultant oxidative stress. In the absence of glutathione, cysteine has been thought to be the major antioxidant. We now report that the parasite contains thioredoxin reductase, which functions together with thioredoxin and thioredoxin peroxidase to detoxify potentially damaging oxidants. Thioredoxin reductase and thioredoxin also reduce cystine and so may play a role in maintaining the cellular cysteine levels. The importance of the thioredoxin system as one of the major antioxidant defense mechanisms in Trichomonas was confirmed by showing that the parasite responds to environmental changes resulting in increased oxidative stress by up-regulating thioredoxin and thioredoxin peroxidases levels. Sequence data indicate that the thioredoxin reductase of Trichomonas differs fundamentally in structure from that of its human host and thus may represent a useful drug target. The protein is generally similar to thioredoxin reductases present in other lower eukaryotes, all of which probably originated through horizontal gene transfer from a prokaryote. The phylogenetic signal in thioredoxin peroxidase is weak, but evidence from trees suggests that this gene has been subject to repeated horizontal gene transfers from different prokaryotes to different eukaryotes. The data are thus consistent with the complexity hypothesis that predicts that the evolution of simple pathways such as the thioredoxin cascade are likely to be affected by horizontal gene transfer between species.
Highlights
Trichomonas vaginalis is the protozoan parasite responsible for trichomoniasis in humans [1]
Analysis of the predicted amino acid sequences of the T. vaginalis thioredoxin reductase (TrxR) confirmed that it belongs to the L-TrxR group and is distinct from both the H-TrxR group and the AhpFs of bacteria
There are two small indels of 5 and 2 amino acids in the L-TrxRs of most other eukaryotes (including S. cerevisiae and E. histolytica), which are absent from the enzyme of T. vaginalis and the enzymes of two diplomonad parasites, G. lamblia and S. barkhanus, and of most prokaryotes
Summary
Growth and Harvesting of Parasites—A clonal cell line (G3) of T. vaginalis was routinely grown axenically in modified Diamond’s medium and harvested as previously described [17]. Cystine reductase activity of the TrxR/Trx couple was measured by monitoring the oxidation of NADPH at 340 nm in a reaction mixture comprising 0.1 M potassium phosphate, pH 7.0, 5 mM EDTA, 0.2 mM NADPH, 12.5 M rTrx, 44 pmol of rTrxR and varying concentrations of L-cystine (5–50 M). The Effects of Growth Conditions upon the Expression of TrxR, Trx, and TrxP in T. vaginalis—Parasites were grown in 25 ml of medium in tightly capped universal tubes with little gas phase, except for the “aerobic” cultures, which were grown in 500-ml tissue culture vessels with loose caps in a normally aspirated incubator. Cultures were initiated at 105 parasites/ml, and incubation was for l8 h at 37 °C, whereupon the parasites were harvested, washed, and stored as pellets at Ϫ70 °C until analysis [17]
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