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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 an...
Autores principales: | , , , , , |
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Formato: | Texto |
Lenguaje: | English |
Publicado: |
Blackwell Publishing Ltd
2009
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2784880/ https://www.ncbi.nlm.nih.gov/pubmed/19558432 http://dx.doi.org/10.1111/j.1365-2958.2009.06761.x |
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author | Wyllie, Susan Oza, Sandra L Patterson, Stephen Spinks, Daniel Thompson, Stephen Fairlamb, Alan H |
author_facet | Wyllie, Susan Oza, Sandra L Patterson, Stephen Spinks, Daniel Thompson, Stephen Fairlamb, Alan H |
author_sort | Wyllie, Susan |
collection | PubMed |
description | 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. |
format | Text |
id | pubmed-2784880 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2009 |
publisher | Blackwell Publishing Ltd |
record_format | MEDLINE/PubMed |
spelling | pubmed-27848802009-12-08 Dissecting the essentiality of the bifunctional trypanothione synthetase-amidase in Trypanosoma brucei using chemical and genetic methods Wyllie, Susan Oza, Sandra L Patterson, Stephen Spinks, Daniel Thompson, Stephen Fairlamb, Alan H Mol Microbiol Research Articles 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. Blackwell Publishing Ltd 2009-11 2009-06-24 /pmc/articles/PMC2784880/ /pubmed/19558432 http://dx.doi.org/10.1111/j.1365-2958.2009.06761.x Text en Journal compilation © 2009 Blackwell Publishing http://creativecommons.org/licenses/by/2.5/ Re-use of this article is permitted in accordance with the Creative Commons Deed, Attribution 2.5, which does not permit commercial exploitation. |
spellingShingle | Research Articles Wyllie, Susan Oza, Sandra L Patterson, Stephen Spinks, Daniel Thompson, Stephen Fairlamb, Alan H Dissecting the essentiality of the bifunctional trypanothione synthetase-amidase in Trypanosoma brucei using chemical and genetic methods |
title | Dissecting the essentiality of the bifunctional trypanothione synthetase-amidase in Trypanosoma brucei using chemical and genetic methods |
title_full | Dissecting the essentiality of the bifunctional trypanothione synthetase-amidase in Trypanosoma brucei using chemical and genetic methods |
title_fullStr | Dissecting the essentiality of the bifunctional trypanothione synthetase-amidase in Trypanosoma brucei using chemical and genetic methods |
title_full_unstemmed | Dissecting the essentiality of the bifunctional trypanothione synthetase-amidase in Trypanosoma brucei using chemical and genetic methods |
title_short | Dissecting the essentiality of the bifunctional trypanothione synthetase-amidase in Trypanosoma brucei using chemical and genetic methods |
title_sort | dissecting the essentiality of the bifunctional trypanothione synthetase-amidase in trypanosoma brucei using chemical and genetic methods |
topic | Research Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2784880/ https://www.ncbi.nlm.nih.gov/pubmed/19558432 http://dx.doi.org/10.1111/j.1365-2958.2009.06761.x |
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