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Casein kinase 1 isoform 2 is essential for bloodstream form Trypanosoma brucei.

Urbaniak MD - Mol. Biochem. Parasitol. (2009)

Bottom Line: Induction of RNA interference targeted against casein kinase 1 isoform 2 (TbCK1.2, Tb927.5.800) in bloodstream form Trypanosoma brucei in vitro results in rapid cessation of growth, gross morphological changes, multinucleation and ultimately cell death.A mutant of the highly homologous casein kinase 1 isoform 1 (Tb927.5.790) in bloodstream form T. brucei displays no growth or morphological phenotype in vitro.These data show that TbCK1.2 is an attractive target for anti-trypanosomal drug discovery.

View Article: PubMed Central - PubMed

Affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK. m.d.urbaniak@dundee.ac.uk

ABSTRACT
Induction of RNA interference targeted against casein kinase 1 isoform 2 (TbCK1.2, Tb927.5.800) in bloodstream form Trypanosoma brucei in vitro results in rapid cessation of growth, gross morphological changes, multinucleation and ultimately cell death. A mutant of the highly homologous casein kinase 1 isoform 1 (Tb927.5.790) in bloodstream form T. brucei displays no growth or morphological phenotype in vitro. A truncated form of TbCK1.2 expressed in Escherichia coli as a GST fusion produces catalytically active recombinant protein, facilitating screening for small molecule inhibitors. These data show that TbCK1.2 is an attractive target for anti-trypanosomal drug discovery.

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Growth and morphology of TbCK1.1 knockout and TbCK1.2 knockdown cells. (A) Growth of the TbCK1.1 double knockout (dKO) cell line compared to wild type (WT), inset shows the RT-PCR analysis of TbCK1.1 and TbCK1.2 mRNA levels; (B) growth of TbCK1.2 knockdown cells in the absence (−Tet) and presence (+Tet) of tetracycline, with RT-PCR inset; (C) Phase contrast and DAPI-stained microscopy of TbCK1.2 knockdown cells cultured in the absence (−Tet) and presence (+Tet) of tetracycline for 48 h, arrows indicate multinucleation. The ΔTbCK1.1::PAC/ΔTbCK1.1::HYG dKO cell line was created by homologous recombination. Knockdown of TbCK1.2 by tetracycline inducible RNAi was achieved with a TbCK1.2 specific fragment [15] PCR-amplified from genomic DNA (primers 5′-GACAGCGGCAATAATCC-3′ and 5′-CCACAACACCGCCAC-3′) and cloned into p2T7TAblue as described by Alibu et al. [13]. RT-PCR was performed using the Quick-Access RT-PCR system (Promega) using a common 5′-primer (5′-TGGCAGGGTTAAAGGC-3′) with two unique 3′-primers producing a 345 bp fragment for TbCK1.1 (5′-GACGGGATGTTCATC-3′) and a 320 bp fragment for TbCK1.2 (5′-TCGGTGTCATCACTC-3′). Microscopy was performed using cell fixed in 4% paraformaldehyde and stained with 2 μg/ml DAPI, with images acquired on a Zeiss Axiovert 200 M fluorescence microscope. Growth curves and microscopy images are representative examples of multiple experiments (n ≥ 3).
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fig1: Growth and morphology of TbCK1.1 knockout and TbCK1.2 knockdown cells. (A) Growth of the TbCK1.1 double knockout (dKO) cell line compared to wild type (WT), inset shows the RT-PCR analysis of TbCK1.1 and TbCK1.2 mRNA levels; (B) growth of TbCK1.2 knockdown cells in the absence (−Tet) and presence (+Tet) of tetracycline, with RT-PCR inset; (C) Phase contrast and DAPI-stained microscopy of TbCK1.2 knockdown cells cultured in the absence (−Tet) and presence (+Tet) of tetracycline for 48 h, arrows indicate multinucleation. The ΔTbCK1.1::PAC/ΔTbCK1.1::HYG dKO cell line was created by homologous recombination. Knockdown of TbCK1.2 by tetracycline inducible RNAi was achieved with a TbCK1.2 specific fragment [15] PCR-amplified from genomic DNA (primers 5′-GACAGCGGCAATAATCC-3′ and 5′-CCACAACACCGCCAC-3′) and cloned into p2T7TAblue as described by Alibu et al. [13]. RT-PCR was performed using the Quick-Access RT-PCR system (Promega) using a common 5′-primer (5′-TGGCAGGGTTAAAGGC-3′) with two unique 3′-primers producing a 345 bp fragment for TbCK1.1 (5′-GACGGGATGTTCATC-3′) and a 320 bp fragment for TbCK1.2 (5′-TCGGTGTCATCACTC-3′). Microscopy was performed using cell fixed in 4% paraformaldehyde and stained with 2 μg/ml DAPI, with images acquired on a Zeiss Axiovert 200 M fluorescence microscope. Growth curves and microscopy images are representative examples of multiple experiments (n ≥ 3).

Mentions: In order to investigate the essentiality of TbCK1.1 for the survival of bloodstream form T. brucei the haploid TbCK1.1 genes were replaced with drug resistance genes by homologous recombination. Approximately 500 bp of the 5′- and 3′-UTR sequences immediately adjacent to TbCK1.1 were PCR amplified from genomic DNA using primers that allowed the two products to be knitted together in a second PCR to create a restriction enzyme site between the UTRs, allowing a drug resistance gene to be inserted [12]. The puromycin acetyltransferase (PAC) and hygromycin phosphotransferase (HYG) drug resistance genes were inserted between the UTRs and the resulting constructs used sequentially to replace both alleles of TbCK1.1 generating a ΔTbCK1.1::PAC/ΔTbCK1.1::HYG double knockout (dKO) cell line. Reverse transcriptase-PCR (RT-PCR) confirmed the absence of TbCK1.1 mRNA and revealed that the TbCK1.2 mRNA level was not significantly upregulated in response to the loss of TbCK1.1. The TbCK1.1 dKO cell line had normal morphology (not shown) and its growth was unaltered compare to the wild type (Fig. 1A), demonstrating that TbCK1.1 is not essential in vitro.


Casein kinase 1 isoform 2 is essential for bloodstream form Trypanosoma brucei.

Urbaniak MD - Mol. Biochem. Parasitol. (2009)

Growth and morphology of TbCK1.1 knockout and TbCK1.2 knockdown cells. (A) Growth of the TbCK1.1 double knockout (dKO) cell line compared to wild type (WT), inset shows the RT-PCR analysis of TbCK1.1 and TbCK1.2 mRNA levels; (B) growth of TbCK1.2 knockdown cells in the absence (−Tet) and presence (+Tet) of tetracycline, with RT-PCR inset; (C) Phase contrast and DAPI-stained microscopy of TbCK1.2 knockdown cells cultured in the absence (−Tet) and presence (+Tet) of tetracycline for 48 h, arrows indicate multinucleation. The ΔTbCK1.1::PAC/ΔTbCK1.1::HYG dKO cell line was created by homologous recombination. Knockdown of TbCK1.2 by tetracycline inducible RNAi was achieved with a TbCK1.2 specific fragment [15] PCR-amplified from genomic DNA (primers 5′-GACAGCGGCAATAATCC-3′ and 5′-CCACAACACCGCCAC-3′) and cloned into p2T7TAblue as described by Alibu et al. [13]. RT-PCR was performed using the Quick-Access RT-PCR system (Promega) using a common 5′-primer (5′-TGGCAGGGTTAAAGGC-3′) with two unique 3′-primers producing a 345 bp fragment for TbCK1.1 (5′-GACGGGATGTTCATC-3′) and a 320 bp fragment for TbCK1.2 (5′-TCGGTGTCATCACTC-3′). Microscopy was performed using cell fixed in 4% paraformaldehyde and stained with 2 μg/ml DAPI, with images acquired on a Zeiss Axiovert 200 M fluorescence microscope. Growth curves and microscopy images are representative examples of multiple experiments (n ≥ 3).
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fig1: Growth and morphology of TbCK1.1 knockout and TbCK1.2 knockdown cells. (A) Growth of the TbCK1.1 double knockout (dKO) cell line compared to wild type (WT), inset shows the RT-PCR analysis of TbCK1.1 and TbCK1.2 mRNA levels; (B) growth of TbCK1.2 knockdown cells in the absence (−Tet) and presence (+Tet) of tetracycline, with RT-PCR inset; (C) Phase contrast and DAPI-stained microscopy of TbCK1.2 knockdown cells cultured in the absence (−Tet) and presence (+Tet) of tetracycline for 48 h, arrows indicate multinucleation. The ΔTbCK1.1::PAC/ΔTbCK1.1::HYG dKO cell line was created by homologous recombination. Knockdown of TbCK1.2 by tetracycline inducible RNAi was achieved with a TbCK1.2 specific fragment [15] PCR-amplified from genomic DNA (primers 5′-GACAGCGGCAATAATCC-3′ and 5′-CCACAACACCGCCAC-3′) and cloned into p2T7TAblue as described by Alibu et al. [13]. RT-PCR was performed using the Quick-Access RT-PCR system (Promega) using a common 5′-primer (5′-TGGCAGGGTTAAAGGC-3′) with two unique 3′-primers producing a 345 bp fragment for TbCK1.1 (5′-GACGGGATGTTCATC-3′) and a 320 bp fragment for TbCK1.2 (5′-TCGGTGTCATCACTC-3′). Microscopy was performed using cell fixed in 4% paraformaldehyde and stained with 2 μg/ml DAPI, with images acquired on a Zeiss Axiovert 200 M fluorescence microscope. Growth curves and microscopy images are representative examples of multiple experiments (n ≥ 3).
Mentions: In order to investigate the essentiality of TbCK1.1 for the survival of bloodstream form T. brucei the haploid TbCK1.1 genes were replaced with drug resistance genes by homologous recombination. Approximately 500 bp of the 5′- and 3′-UTR sequences immediately adjacent to TbCK1.1 were PCR amplified from genomic DNA using primers that allowed the two products to be knitted together in a second PCR to create a restriction enzyme site between the UTRs, allowing a drug resistance gene to be inserted [12]. The puromycin acetyltransferase (PAC) and hygromycin phosphotransferase (HYG) drug resistance genes were inserted between the UTRs and the resulting constructs used sequentially to replace both alleles of TbCK1.1 generating a ΔTbCK1.1::PAC/ΔTbCK1.1::HYG double knockout (dKO) cell line. Reverse transcriptase-PCR (RT-PCR) confirmed the absence of TbCK1.1 mRNA and revealed that the TbCK1.2 mRNA level was not significantly upregulated in response to the loss of TbCK1.1. The TbCK1.1 dKO cell line had normal morphology (not shown) and its growth was unaltered compare to the wild type (Fig. 1A), demonstrating that TbCK1.1 is not essential in vitro.

Bottom Line: Induction of RNA interference targeted against casein kinase 1 isoform 2 (TbCK1.2, Tb927.5.800) in bloodstream form Trypanosoma brucei in vitro results in rapid cessation of growth, gross morphological changes, multinucleation and ultimately cell death.A mutant of the highly homologous casein kinase 1 isoform 1 (Tb927.5.790) in bloodstream form T. brucei displays no growth or morphological phenotype in vitro.These data show that TbCK1.2 is an attractive target for anti-trypanosomal drug discovery.

View Article: PubMed Central - PubMed

Affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK. m.d.urbaniak@dundee.ac.uk

ABSTRACT
Induction of RNA interference targeted against casein kinase 1 isoform 2 (TbCK1.2, Tb927.5.800) in bloodstream form Trypanosoma brucei in vitro results in rapid cessation of growth, gross morphological changes, multinucleation and ultimately cell death. A mutant of the highly homologous casein kinase 1 isoform 1 (Tb927.5.790) in bloodstream form T. brucei displays no growth or morphological phenotype in vitro. A truncated form of TbCK1.2 expressed in Escherichia coli as a GST fusion produces catalytically active recombinant protein, facilitating screening for small molecule inhibitors. These data show that TbCK1.2 is an attractive target for anti-trypanosomal drug discovery.

Show MeSH
Related in: MedlinePlus