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J. Biol. Chem., Vol. 277, Issue 29, 25970-25975, July 19, 2002
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From the 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.
Thioredoxin Reductase Is Essential for the Survival of
Plasmodium falciparum Erythrocytic Stages*
§,¶
,,, and**
Bernhard Nocht Institute for Tropical
Medicine, Department of Biochemical Parasitology,
20359 Hamburg, Germany, ¶ The Walter and Eliza Hall Institute of
Medical Research, P. O. Royal Melbourne Hospital,
3050 Melbourne, Victoria, Australia, and ** University of
Dundee, School of Life Sciences,
Dundee DD1 5EH, Scotland, United Kingdom
*
This work was supported in part by the Deutsche
Forschungsgemeinschaft Grants DFG MU837/1-1 and DFG WA395/14-4 and the
Wellcome Trust.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Recipient of an Emmy-Nöther-Fellowship.
Wellcome Trust Senior Fellow. To whom correspondence should be
addressed: University of Dundee, School of Life Sciences, Division of
Biological Chemistry and Molecular Microbiology, MSI/WTB Complex, Dundee DD1 5EH, Scotland, UK. Tel.: 44-1382-345760; Fax: 44-1382- 345764; E-mail: s.muller@dundee.ac.uk.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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