RNA interference identifies two hydroperoxide metabolizing enzymes that are essential to the bloodstream form of the african trypanosome.
Detoxification of hydroperoxides in trypanosomes is mediated by a series of linked redox pathways that are dependent on the parasite-specific thiol trypanothione for reducing equivalents. These pathways are characterized by differences in subcellular location, electron transport molecules, and substrate specificity. To determine the functional significance of the enzymes involved, we have used a tetracycline-inducible RNA interference system to down-regulate expression of each of the corresponding transcripts in bloodstream form Trypanosoma brucei. We have identified two peroxidases, a cytosolic peroxiredoxin (TbCPX) and a member of the non-selenium glutathione-dependent peroxidase family (TbGPXI), that appear to be essential for the viability of this clinically relevant stage of the parasite life cycle. The addition of tetracycline to the cultures resulted in a major reduction in mRNA levels and enzyme activity, a dramatic fall in growth rate, and significant cell death. Furthermore, within 20 h of adding tetracycline, cells in which the cytosolic peroxiredoxin transcript was targeted were found to be 16-fold more susceptible to killing by exogenous hydrogen peroxide. We also observed that knockdown of the tryparedoxin TbT-PNI, a thioredoxin-like protein that facilitates electron transport to both TbCPX and TbGPXI, resulted in a reduction in growth rate. These experiments therefore identify redox pathways that are essential for oxidative defense in T. brucei and validate the corresponding peroxidases as targets for drug design.
Wilkinson
Shane R
SR
Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, United Kingdom. shane.wilkinson@lshtm.ac.uk
Horn
David
D
Prathalingam
S Radhika
SR
Kelly
John M
JM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D006861:Hydrogen Peroxide / Q000378:metabolism*; D008969:Molecular Sequence Data; D034622:RNA Interference; D017386:Sequence Homology, Amino Acid; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
D017931:DNA Primers; D006861:Hydrogen Peroxide