Thioredoxin reductase is essential for the survival of Plasmodium falciparum erythrocytic stages

The human malaria parasite Plasmodium falciparum poses an increasing threat to human health in the tropical regions of the world, and the validation and assessment of possible drug targets is required for the development of new antimalarials. It has been shown that the erythrocytic stages of the parasites, which are responsible for the pathology of the disease in humans, are under enhanced oxidative stress and are particularly vulnerable to exogenous challenges by reactive oxygen species. Therefore it is postulated that the disruption of the antioxidant and/or redox systems of the parasite is a feasible way to interfere with their development during erythrocytic schizogony. In order to test this suggestion thioredoxin reductase (TrxR), an enzyme heavily involved in maintenance of redox homeostasis and antioxidant defense, was knocked out in P. falciparum. It was impossible to generate parasites with a disrupted trxR gene suggesting that TrxR is essential for P. falciparum erythrocytic stages. Technical problems were excluded by transfecting a 3' replacement construct, which recombined correctly and transfectants did not show any phenotypic alterations. In order to prove that the trxR knockout was responsible for the lethal phenotype of the null mutants, a co-transfection with both the knockout construct and a construct containing the trxR coding region under the control of the calmodulin promoter was conducted. Despite the disruption of the trxR gene, parasites were viable. In a Southern blot analysis a complicated restriction pattern was obtained, but it was shown by pulse field gel electrophoresis and field inverse gel electrophoreses that only the trxR gene locus on chromosome 9 was targeted by the constructs. It was found that the co-transfected constructs form concatemeric structures prior to integration into the trxR gene locus, which is further supported by plasmid rescue followed by restriction analyses of the plasmids. Northern and Western blot analyses proved that the co-transfectants highly overexpress TrxR from the introduced gene. Our results demonstrate that TrxR is essential for the survival of the erythrocytic stages of P. falciparum.