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The requirement of aerobic organisms to control damage caused by reactive oxygen species has led to the evolution of the antioxidant systems. Peroxiredoxins are a large family of peroxidases which detoxify hydrogen peroxide at the expense of thiols. The parasitic nematode Haemonchus contortus contains two peroxiredoxins, HcPrx1 a mitochondrial protein and HcPrx2 a cytoplasmic protein. Although both peroxiredoxins contain the conserved eukaryotic motifs 'GGLG' and 'YF', identified as critical for hydrogen peroxide instability, both were stable to high concentrations of hydrogen peroxide, demonstrating different functions to their mammalian counterparts. H. contortus also contains two thioredoxin reductases and five different thioredoxin-like proteins. The activity of both peroxiredoxins was specific for the thioredoxin system; however, both could also be regenerated by the glutathione system when coupled to the nematode specific thioredoxin HcTrx5. Analysis of homologous genes in Caenorhabditis elegans showed that only CePrx2, which is secreted, was sensitive to the external oxidant hydrogen peroxide. However, both peroxiredoxins KO C. elegans were sensitive to intracellular free radicals and both peroxiredoxins protected DNA from free radical attack. The results demonstrate that the hydrogen peroxide detoxification and the antioxidant activity of the peroxiredoxins are separate activities that are independent of the 'GGLG' and 'YF' motifs., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Thioredoxin reductase (TrxR) is an evolutionary diverse enzyme. In higher eukaryotes, TrxR contains a selenocysteine in the active site which confers high activity and a broad substrate range. The parasitic nematode Haemonchus contortus contains two TrxRs, a cytoplasmic enzyme HcTrxR1 with a selenocysteine in the active site (Gly-Cys-SeCys-Gly), similar to the mammalian TrxR, and a mitochondrial enzyme HcTrxR2 with a Gly-Cyc-Cys-Gly active site which is unique to nematodes. Both enzymes were cloned and expressed and their activity compared to rat TrxR. Although the expressed HcTrxR1 contained a cysteine to replace the selenocycteine, it showed broad activity with thioredoxins from Escherichia coli, sheep, and H. contortus similar to the rat selenoenzyme. However, HcTrxR2 had high activity only with the mitochondrial H. contortus thioredoxin. HcTrxR2 was also active with the commonly used substrate 5,5'-dithiobis(2-nitrobenzoate) and sodium selenite and was inhibited by auranofin and the natural product compounds curcumin and theaflavin. This demonstrates that the non-seleno-enzyme HcTrxR2 is as active as the rat seleno-containing enzyme when reacting with the H. contortus thioredoxin.

The dependency of parasites on the cellular redox systems has led to their investigation as novel drug targets. Defence against oxidative damage is through the thioredoxin and glutathione systems. The classic thioredoxin is identified by the active site Cys-Gly-Pro-Cys (CGPC). Here we describe the identification of a unique thioredoxin in the parasitic nematode, Haemonchus contortus. This thioredoxin-related protein, termed HcTrx5, has an arginine in its active site (Cys-Arg-Ser-Cys; CRSC) that is not found in any other organism. Recombinant HcTrx5 was able to reduce the disulfide bond in insulin, and be regenerated by mammalian thioredoxin reductase with a K(m) 2.19+/-1.5 microM, similar to the classic thioredoxins. However, it was also able to reduce insulin when glutathione and glutathione reductase replaced the thioredoxin reductase. When coupled with H. contortus peroxiredoxin, HcTrx5 was active using either the thioredoxin reductase or the glutathione and glutathione reductase. HcTrx5 is expressed through the life cycle, with highest expression in the adult stage. The unique activity of this thioredoxin makes it a potential drug target for the control of this parasite.

Thioredoxins are a family of small proteins conserved through evolution, which are essential for the maintenance of cellular homeostasis. The "classic" thioredoxin, identified in most species, is a 12-kDa protein with a Cys-Pro-Gly-Cys (CPGC) active site. However, in nematodes a larger protein, 16 kDa, with a Cys-Pro-Pro-Cys (CPPC) active site was identified. We report that in the parasitic nematode Haemonchus contortus, both the 12-kDa (HcTrx1) and the 16-kDa (HcTrx3) species are expressed through the life cycle. However, the HcTrx3 is expressed at higher concentrations. Recombinant HcTrx1 and HcTrx3 were produced and both reduced insulin at a rate similar to that observed with ovine (host) and Escherichia coli thioredoxins and both were regenerated by a mammalian thioredoxin reductase, demonstrating that they have similar thioredoxin activity. Unlike mammalian thioredoxins, both proteins were able to reduce oxidised glutathione and hydrogen peroxide. This suggests essential roles for these proteins in response to oxidative stress and the host immune attack. Analysis of ivermectin-resistant H. contortus showed that expression of both genes were increased in a drug-resistant strain relative to a sensitive strain. Involvement in drug resistance identifies these thioredoxin proteins as potential drug targets for parasite control.