Trypanosoma brucei pteridine reductase 1 is essential for survival in vitro and for virulence in mice

Natasha Sienkiewicz, Han B. Ong, Alan H. Fairlamb

    Research output: Contribution to journalArticle

    27 Citations (Scopus)

    Abstract

    Gene knockout and knockdown methods were used to examine essentiality of pteridine reductase (PTR1) in pterin metabolism in the African trypanosome. Attempts to generate PTR1 null mutants in bloodstream form Trypanosoma brucei proved unsuccessful; despite integration of drug selectable markers at the target locus, the gene for PTR1 was either retained at the same locus or elsewhere in the genome. However, RNA interference (RNAi) resulted in complete knockdown of endogenous protein after 48 h, followed by cell death after 4 days. This lethal phenotype was reversed by expression of enzymatically active Leishmania major PTR1 in RNAi lines (oeRNAi) or by addition of tetrahydrobiopterin to cultures. Loss of PTR1 was associated with gross morphological changes due to a defect in cytokinesis, resulting in cells with multiple nuclei and kinetoplasts, as well as multiple detached flagella. Electron microscopy also revealed increased numbers of glycosomes, while immunofluorescence microscopy showed increased and more diffuse staining for glycosomal matrix enzymes, indicative of mis-localisation to the cytosol. Mis-localisation was confirmed by digitonin fractionation experiments. RNAi cell lines were markedly less virulent than wild-type parasites in mice and virulence was restored in the oeRNAi line. Thus, PTR1 may be a drug target for human African trypanosomiasis.

    Original languageEnglish
    Pages (from-to)658-671
    Number of pages14
    JournalMolecular Microbiology
    Volume77
    Issue number3
    DOIs
    Publication statusPublished - Aug 2010

    Keywords

    • PARASITE LEISHMANIA-MAJOR
    • LIFE-CYCLE STAGES
    • RNA INTERFERENCE
    • TETRAHYDROBIOPTERIN BIOSYNTHESIS
    • METHOTREXATE RESISTANCE
    • AFRICAN TRYPANOSOMES
    • BLOOD-STREAM
    • DIHYDROPTERIDINE REDUCTASE
    • TRYPANOTHIONE REDUCTASE
    • DIHYDROFOLATE-REDUCTASE

    Cite this

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    title = "Trypanosoma brucei pteridine reductase 1 is essential for survival in vitro and for virulence in mice",
    abstract = "Gene knockout and knockdown methods were used to examine essentiality of pteridine reductase (PTR1) in pterin metabolism in the African trypanosome. Attempts to generate PTR1 null mutants in bloodstream form Trypanosoma brucei proved unsuccessful; despite integration of drug selectable markers at the target locus, the gene for PTR1 was either retained at the same locus or elsewhere in the genome. However, RNA interference (RNAi) resulted in complete knockdown of endogenous protein after 48 h, followed by cell death after 4 days. This lethal phenotype was reversed by expression of enzymatically active Leishmania major PTR1 in RNAi lines (oeRNAi) or by addition of tetrahydrobiopterin to cultures. Loss of PTR1 was associated with gross morphological changes due to a defect in cytokinesis, resulting in cells with multiple nuclei and kinetoplasts, as well as multiple detached flagella. Electron microscopy also revealed increased numbers of glycosomes, while immunofluorescence microscopy showed increased and more diffuse staining for glycosomal matrix enzymes, indicative of mis-localisation to the cytosol. Mis-localisation was confirmed by digitonin fractionation experiments. RNAi cell lines were markedly less virulent than wild-type parasites in mice and virulence was restored in the oeRNAi line. Thus, PTR1 may be a drug target for human African trypanosomiasis.",
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    author = "Natasha Sienkiewicz and Ong, {Han B.} and Fairlamb, {Alan H.}",
    year = "2010",
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    Trypanosoma brucei pteridine reductase 1 is essential for survival in vitro and for virulence in mice. / Sienkiewicz, Natasha; Ong, Han B.; Fairlamb, Alan H.

    In: Molecular Microbiology, Vol. 77, No. 3, 08.2010, p. 658-671.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - Trypanosoma brucei pteridine reductase 1 is essential for survival in vitro and for virulence in mice

    AU - Sienkiewicz, Natasha

    AU - Ong, Han B.

    AU - Fairlamb, Alan H.

    PY - 2010/8

    Y1 - 2010/8

    N2 - Gene knockout and knockdown methods were used to examine essentiality of pteridine reductase (PTR1) in pterin metabolism in the African trypanosome. Attempts to generate PTR1 null mutants in bloodstream form Trypanosoma brucei proved unsuccessful; despite integration of drug selectable markers at the target locus, the gene for PTR1 was either retained at the same locus or elsewhere in the genome. However, RNA interference (RNAi) resulted in complete knockdown of endogenous protein after 48 h, followed by cell death after 4 days. This lethal phenotype was reversed by expression of enzymatically active Leishmania major PTR1 in RNAi lines (oeRNAi) or by addition of tetrahydrobiopterin to cultures. Loss of PTR1 was associated with gross morphological changes due to a defect in cytokinesis, resulting in cells with multiple nuclei and kinetoplasts, as well as multiple detached flagella. Electron microscopy also revealed increased numbers of glycosomes, while immunofluorescence microscopy showed increased and more diffuse staining for glycosomal matrix enzymes, indicative of mis-localisation to the cytosol. Mis-localisation was confirmed by digitonin fractionation experiments. RNAi cell lines were markedly less virulent than wild-type parasites in mice and virulence was restored in the oeRNAi line. Thus, PTR1 may be a drug target for human African trypanosomiasis.

    AB - Gene knockout and knockdown methods were used to examine essentiality of pteridine reductase (PTR1) in pterin metabolism in the African trypanosome. Attempts to generate PTR1 null mutants in bloodstream form Trypanosoma brucei proved unsuccessful; despite integration of drug selectable markers at the target locus, the gene for PTR1 was either retained at the same locus or elsewhere in the genome. However, RNA interference (RNAi) resulted in complete knockdown of endogenous protein after 48 h, followed by cell death after 4 days. This lethal phenotype was reversed by expression of enzymatically active Leishmania major PTR1 in RNAi lines (oeRNAi) or by addition of tetrahydrobiopterin to cultures. Loss of PTR1 was associated with gross morphological changes due to a defect in cytokinesis, resulting in cells with multiple nuclei and kinetoplasts, as well as multiple detached flagella. Electron microscopy also revealed increased numbers of glycosomes, while immunofluorescence microscopy showed increased and more diffuse staining for glycosomal matrix enzymes, indicative of mis-localisation to the cytosol. Mis-localisation was confirmed by digitonin fractionation experiments. RNAi cell lines were markedly less virulent than wild-type parasites in mice and virulence was restored in the oeRNAi line. Thus, PTR1 may be a drug target for human African trypanosomiasis.

    KW - PARASITE LEISHMANIA-MAJOR

    KW - LIFE-CYCLE STAGES

    KW - RNA INTERFERENCE

    KW - TETRAHYDROBIOPTERIN BIOSYNTHESIS

    KW - METHOTREXATE RESISTANCE

    KW - AFRICAN TRYPANOSOMES

    KW - BLOOD-STREAM

    KW - DIHYDROPTERIDINE REDUCTASE

    KW - TRYPANOTHIONE REDUCTASE

    KW - DIHYDROFOLATE-REDUCTASE

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    SP - 658

    EP - 671

    JO - Molecular Microbiology

    JF - Molecular Microbiology

    SN - 0950-382X

    IS - 3

    ER -