Trypanosomes lacking trypanothione reductase are avirulent and show increased sensitivity to oxidative stress.
In Kinetoplastida, trypanothione and trypanothione reductase (TRYR) provide an intracellular reducing environment, substituting for the glutathione-glutathione reductase system found in most other organisms. To investigate the physiological role of TRYR in Trypanosoma brucei, we generated cells containing just one trypanothione reductase gene, TRYR, which was under the control of a tetracycline-inducible promoter. This enabled us to regulate TRYR activity in the cells from less than 1% to 400% of wild-type levels by adjusting the concentration of added tetracycline. In normal growth medium (which contains reducing agents), trypanosomes containing less than 10% of wild-type enzyme activity were unable to grow, although the levels of reduced trypanothione and total thiols remained constant. In media lacking reducing agents, hypersensitivity towards hydrogen peroxide (EC50 = 3.5 microM) was observed compared with the wild type (EC50 = 223 microM). The depletion of TRYR had no effect on susceptibility to melarsen oxide. The infectivity and virulence of the parasites in mice was dependent upon tetracycline-regulated TRYR activity: if the trypanosomes were injected into mice in the absence of tetracycline, no infection was detectable; and when tetracycline was withdrawn from previously infected animals, the parasitaemia was suppressed.
Krieger
S
S
Zentrum für Molekulare Biologie der Universität Heidelberg, Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany.
Schwarz
W
W
Ariyanayagam
M R
MR
Fairlamb
A H
AH
Krauth-Siegel
R L
RL
Clayton
C
C
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000981:Antiprotozoal Agents / Q000494:pharmacology; D001152:Arsenicals / Q000494:pharmacology; D002453:Cell Cycle / Q000235:genetics; D002455:Cell Division / Q000187:drug effects / Q000235:genetics; D004318:Doxycycline / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D009154:Mutation; D009247:NADH, NADPH Oxidoreductases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D018384:Oxidative Stress; D011401:Promoter Regions, Genetic; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013752:Tetracycline / Q000494:pharmacology; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000472:pathogenicity*; D014353:Trypanosomiasis, African / Q000188:drug therapy; D014774:Virulence
D000900:Anti-Bacterial Agents; D000981:Antiprotozoal Agents; D001152:Arsenicals; D004791:Enzyme Inhibitors; D013438:Sulfhydryl Compounds; C044719:melarsen oxide; D009247:NADH, NADPH Oxidoreductases; C049608:trypanothione reductase; D013752:Tetracycline; D004318:Doxycycline