Thiol metabolism in the parasitic nematode Haemonchus contortus

University of Technology, Sydney. Faculty of Science.Haemonchus contortus is an important parasitic nematode, both economically and pathologically. The emergence of widespread drug resistance requires new drug or vaccine targets to be identified. The requirement of aerobic organisms to control damage caused by reactive oxygen species and, the increased necessity of parasites to overcome the host immune response, has led to the investigation of antioxidant systems as potential targets. This work examines the thioredoxin antioxidant system in H. contortus, specifically the thioredoxin reductase and peroxiredoxin enzymes, to characterise their activity and determine if they are potential targets for parasite control. H. contortus contains two TrxRs, a cytoplasmic enzyme HcTrxRl with a selenocysteine in the active site, similar to the mammalian TrxR, and a mitochondrial enzyme HcTrxR2 with a nematode unique active site. HcTrxRl showed broad activity with thioredoxins from E. coli, sheep, and H. contortus while HcTrxR2 had high activity with only the mitochondrial H. contortus thioredoxin 1. Importantly, HcTrxRl was found to be more sensitive to the black tea inhibitor theaflavin than the selenocysteine containing mammalian TrxR, demonstrating the differences in the enzymes susceptibilities to inhibitors. To determine the function of the TrxR enzymes in nematodes, knockout (KO) strains of Caenorhabditis elegans were examined. TrxRl -/-KO worms were more sensitive to free radical attack and also to the anthelmintic ivermectin; while TrxR2 -/- KO eggs were highly sensitive to sodium hypochlorite. This demonstrates that inhibition of these enzymes would sensitise the nematodes to the host's immune attack. H. contortus contains two peroxiredoxins, the mitochondrial HcPrxl and the cytoplasmic HcPrx2. The activity of both peroxiredoxins was specific for the thioredoxin system; however, both peroxiredoxins were also able to be regenerated by the glutathione system when coupled to the nematode specific H. contortus thioredoxin 5. Both enzymes were stable to high concentrations of hydrogen peroxide which demonstrates different functions to their mammalian counterparts. A specific inhibitor of these peroxiredoxins was also identified which has minimal mammalian cytotoxicity. HcPrxl was found to be involved in drug resistance while HcPrx2 was found to be secreted and highly immunogenic. Analysis of homologous genes in C. elegans showed that both peroxiredoxin KO worms were sensitive to free radical attack; however, only the cytoplasmic CePrx2 KO C. elegans were sensitive to external oxidants. Overall, this work adds to the knowledge of H. contortus biology and identifies the enzymes of the thioredoxin system as potential drug or vaccine targets for parasite control