Dissecting the essentiality of the bifunctional trypanothione synthetase-amidase in Trypanosoma brucei using chemical and genetic methods

Susan Wyllie, Sandra L. Oza, Stephen Patterson, Daniel Spinks, Stephen Thompson, Alan H. Fairlamb (Lead / Corresponding author)

    Research output: Contribution to journalArticle

    53 Citations (Scopus)

    Abstract

    P>The bifunctional trypanothione synthetase-amidase (TRYS) comprises two structurally distinct catalytic domains for synthesis and hydrolysis of trypanothione (N1,N8-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.

    Original languageEnglish
    Pages (from-to)529-540
    Number of pages12
    JournalMolecular Microbiology
    Volume74
    Issue number3
    DOIs
    Publication statusPublished - Nov 2009

    Keywords

    • SLEEPING SICKNESS PARASITE
    • BLOOD-STREAM FORMS
    • AFRICAN TRYPANOSOMES
    • CRITHIDIA-FASCICULATA
    • ALPHA-DIFLUOROMETHYLORNITHINE
    • LEISHMANIA-DONOVANI
    • S-TRANSFERASE
    • DRUG TARGET
    • REDUCTASE
    • GLUTATHIONE

    Cite this

    @article{87fa83671fa74a2f83563392f3944681,
    title = "Dissecting the essentiality of the bifunctional trypanothione synthetase-amidase in Trypanosoma brucei using chemical and genetic methods",
    abstract = "P>The bifunctional trypanothione synthetase-amidase (TRYS) comprises two structurally distinct catalytic domains for synthesis and hydrolysis of trypanothione (N1,N8-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.",
    keywords = "SLEEPING SICKNESS PARASITE, BLOOD-STREAM FORMS, AFRICAN TRYPANOSOMES, CRITHIDIA-FASCICULATA, ALPHA-DIFLUOROMETHYLORNITHINE, LEISHMANIA-DONOVANI, S-TRANSFERASE, DRUG TARGET, REDUCTASE, GLUTATHIONE",
    author = "Susan Wyllie and Oza, {Sandra L.} and Stephen Patterson and Daniel Spinks and Stephen Thompson and Fairlamb, {Alan H.}",
    year = "2009",
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    TY - JOUR

    T1 - Dissecting the essentiality of the bifunctional trypanothione synthetase-amidase in Trypanosoma brucei using chemical and genetic methods

    AU - Wyllie, Susan

    AU - Oza, Sandra L.

    AU - Patterson, Stephen

    AU - Spinks, Daniel

    AU - Thompson, Stephen

    AU - Fairlamb, Alan H.

    PY - 2009/11

    Y1 - 2009/11

    N2 - P>The bifunctional trypanothione synthetase-amidase (TRYS) comprises two structurally distinct catalytic domains for synthesis and hydrolysis of trypanothione (N1,N8-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.

    AB - P>The bifunctional trypanothione synthetase-amidase (TRYS) comprises two structurally distinct catalytic domains for synthesis and hydrolysis of trypanothione (N1,N8-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.

    KW - SLEEPING SICKNESS PARASITE

    KW - BLOOD-STREAM FORMS

    KW - AFRICAN TRYPANOSOMES

    KW - CRITHIDIA-FASCICULATA

    KW - ALPHA-DIFLUOROMETHYLORNITHINE

    KW - LEISHMANIA-DONOVANI

    KW - S-TRANSFERASE

    KW - DRUG TARGET

    KW - REDUCTASE

    KW - GLUTATHIONE

    UR - http://www.scopus.com/inward/record.url?scp=70350381791&partnerID=8YFLogxK

    U2 - 10.1111/j.1365-2958.2009.06761.x

    DO - 10.1111/j.1365-2958.2009.06761.x

    M3 - Article

    VL - 74

    SP - 529

    EP - 540

    JO - Molecular Microbiology

    JF - Molecular Microbiology

    SN - 0950-382X

    IS - 3

    ER -