The Amitochondriate Eukaryote Trichomonas vaginalis Contains a Divergent Thioredoxin-linked Peroxiredoxin Antioxidant System

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.

The Mercaptopyruvate Sulfurtransferase of Trichomonas vaginalis Links Cysteine Catabolism to the Production of Thioredoxin Persulfide

“…The parasite is not a strict anaerobe and is adapted to growth under microaerophilic conditions. T. vaginalis is exposed to oxygen in its environment, and mechanisms for defense against the resulting oxidative stress have been identified (27). Cysteine has been considered to be the major intracellular thiol (see Refs.9, 10, and 15); our data confirm this, because at ϳ600 M it comprised more than 70% of the total thiols detected.…”

“…A thioredoxin-reducing system consisting of 0.1 M rTvTrxR, 12.5 M rTvTrx, and 0.2 mM NADPH was incubated at 37°C, and the rate of NADPH oxidation was quantified by measuring absorbance at 340 nm. The low background rate in this assay is due to the NADPH oxidase activity of the rTvTrxR (27). The addition of 10 nM rTvMST had no effect, but the rate increased more than 10-fold when 0.3 mM 3-MP was subsequently added (Fig.…”

“…Reduced Thioredoxin Is a Sulfur Acceptor for rTvMST-The production of recombinant T. vaginalis thioredoxin reductase (rTvTrxR) and recombinant T. vaginalis thioredoxin (rTvTrx) was described previously (27). A thioredoxin-reducing system consisting of 0.1 M rTvTrxR, 12.5 M rTvTrx, and 0.2 mM NADPH was incubated at 37°C, and the rate of NADPH oxidation was quantified by measuring absorbance at 340 nm.…”

“…The rTvTrx(C38A) mutant was expressed and purified as described previously for rTvTrx (27). The purified protein was treated with DTT to reduce any dimers formed during expression or purification.…”

“…The rate of thioredoxin oxidation was determined by measuring the rate of oxidation of NADPH in 100 mM potassium phosphate, 200 M NADPH, 0.1 M rTvTrxR (27), 12.5 M rTvTrx (27), 0.3 mM 3-MP, 7.5 nM rTvMST, pH 7.0. The absorbance at 340 nm was measured for 5 min at 37°C, and the rate of NADPH oxidation was quantified using the extinction coefficient 6220 M Ϫ1 cm Ϫ1 .…”