Dissecting the essentiality of the bifunctional trypanothione synthetase-amidase in Trypanosoma brucei using chemical and genetic methods.
The bifunctional trypanothione synthetase-amidase (TRYS) comprises two structurally distinct catalytic domains for synthesis and hydrolysis of trypanothione (N(1),N(8)-bis(glutathionyl)spermidine). This unique dithiol plays a pivotal role in thiol-redox homeostasis and in defence against chemical and oxidative stress in trypanosomatids. A tetracycline-dependent conditional double knockout of TRYS (cDKO) was generated in bloodstream Trypanosoma brucei. Culture of cDKO parasites without tetracycline induction resulted in loss of trypanothione and accumulation of glutathione, followed by growth inhibition and cell lysis after 6 days. In the absence of inducer, cDKO cells were unable to infect mice, confirming that this enzyme is essential for virulence in vivo as well as in vitro. To establish whether both enzymatic functions were essential, an amidase-dead mutant cDKO line was generated. In the presence of inducer, this line showed decreased growth in vitro and decreased virulence in vivo, indicating that the amidase function is not absolutely required for viability. The druggability of TRYS was assessed using a potent small molecule inhibitor developed in our laboratory. Growth inhibition correlated in rank order cDKO, single KO, wild-type and overexpressing lines and produced the predicted biochemical phenotype. The synthetase function of TRYS is thus unequivocally validated as a drug target by both chemical and genetic methods.
Wyllie
Susan
S
Division of Biological Chemistry and Drug Discovery, Wellcome Trust Biocentre, College of Life Sciences, University of Dundee, Dundee, DD1 5EH, Scotland, UK.
Oza
Sandra L
SL
Patterson
Stephen
S
Spinks
Daniel
D
Thompson
Stephen
S
Fairlamb
Alan H
AH
D019732:Amide Synthases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D004791:Enzyme Inhibitors / Q000494:pharmacology; D055786:Gene Knockout Techniques; D005978:Glutathione / Q000031:analogs & derivatives / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018384:Oxidative Stress; D011073:Polyamines / Q000378:metabolism; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism; D013095:Spermidine / Q000031:analogs & derivatives / Q000378:metabolism; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014344:Trypanocidal Agents / Q000494:pharmacology; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D014349:Trypanosoma cruzi / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D014353:Trypanosomiasis, African / Q000469:parasitology
D004791:Enzyme Inhibitors; D011073:Polyamines; D015800:Protozoan Proteins; D013438:Sulfhydryl Compounds; D014344:Trypanocidal Agents; C012333:glutathionylspermidine; C044809:trypanothione; D000581:Amidohydrolases; C019665:amidase; D019732:Amide Synthases; C064407:trypanothione synthetase; D005978:Glutathione; D013095:Spermidine