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Plasmodium berghei: plasmodium perforin-like protein 5 is required for mosquito midgut invasion in Anopheles stephensi.

Ecker A, Pinto SB, Baker KW, Kafatos FC, Sinden RE - Exp. Parasitol. (2007)

Bottom Line: In this study we show through phenotypic analysis of loss-of-function mutants that PPLP5 is equally essential for mosquito infection.Deltapplp5 ookinetes cannot invade midgut epithelial cells, but subsequent parasite development is rescued if the midgut is bypassed by injection of ookinetes into the hemocoel.The indistinguishable phenotypes of Deltapplp5 and Deltapplp3 ookinetes strongly suggest that these two proteins contribute to a common process.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell and Molecular Biology, Faculty of Natural Sciences, Imperial College London, Sir Alexander Fleming Building, Imperial College Road, London, UK. andrea.ecker@imperial.ac.uk <andrea.ecker@imperial.ac.uk>

ABSTRACT
During its life cycle the malarial parasite Plasmodium forms three invasive stages which have to invade different and specific cells for replication to ensue. Invasion is vital to parasite survival and consequently proteins responsible for invasion are considered to be candidate vaccine/drug targets. Plasmodium perforin-like proteins (PPLPs) have been implicated in invasion because they contain a predicted pore-forming domain. Ookinetes express three PPLPs, and one of them (PPLP3) has previously been shown to be essential for mosquito midgut invasion. In this study we show through phenotypic analysis of loss-of-function mutants that PPLP5 is equally essential for mosquito infection. Deltapplp5 ookinetes cannot invade midgut epithelial cells, but subsequent parasite development is rescued if the midgut is bypassed by injection of ookinetes into the hemocoel. The indistinguishable phenotypes of Deltapplp5 and Deltapplp3 ookinetes strongly suggest that these two proteins contribute to a common process.

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Related in: MedlinePlus

Generation of Δpplp5 parasites. Generation of constructs for targeted disruption of pplp5 by double homologous recombination were carried out as previously described (Dessens et al., 1999). Briefly, an upstream homology region of 469 bp was PCR amplified from Plasmodium berghei ANKA clone 2.34 genomic DNA using primers AE27A (5′-TTGGGCCCGTTGAATATGCATAGACAACATC-3′) and AE27B (5′-CCAAGCTTTCACAAATATAGGCTACTCTTGC-3′) and cloned into pBS-DHFR via ApaI and HindIII (restriction sites in bold). A downstream homology region of 570 bp was PCR amplified using primers AE27C (5′-TGAATTCTCATATTGAATAGGCCTTATATC-3′) and AE27D (5′-GGGGATCCTTTATCACTTCATATCCCAATAC-3′) and cloned into the plasmid with the upstream homology region via EcoRI and BamHI. The targeting cassette was released by ApaI and BamHI digestion. Parasite transfection using the Human T Cell Nucleofector Kit (amaxa), selection by pyrimethamine and dilution cloning were carried out as previously described (Waters et al., 1997; Janse et al., 2006). Diagnostic PCR (a) on genomic DNA from two independent Δpplp clones and control wt parasites. PCRs in lane 1 (27KO 5′-TTAGAATATTTTAAGCATTGGCTATC-3′ and 27WT 5′-CAAATGCCAACCAAATGCAC-3′), 3 (N-ter F and N-ter R) and 4 (MACPF-F 5′-TGAATTCGACCCATTTTTTATAAATATGTTGAA-3′ and MACPF-R 5′-TTCTCGAGTTAGCTAGAATAATATTCTAGAGCT-3′) are specific for the wt allele. The PCR in lane 2 is specific for integration of the gene targeting cassette (primers 27KO and 248 5′-GATGTGTTATGTGATTAATTCATACAC-3′). RT-PCR analysis (b) of pplp expression on total RNA isolated from purified in vitro cultivated ookinetes demonstrates absence of transcript in the Δpplp5 clones. pplp5 primers as in Fig. 1, p28F (5′-GCGAGATCTATGAATTTTAAATACAGTTTTATTTTTTTA-3′) and p28R (5′-GCGCCTAGCATTACTATCACGTAAATAACAAGTA-3′) amplify the pbs28 gene (642 bp).
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fig2: Generation of Δpplp5 parasites. Generation of constructs for targeted disruption of pplp5 by double homologous recombination were carried out as previously described (Dessens et al., 1999). Briefly, an upstream homology region of 469 bp was PCR amplified from Plasmodium berghei ANKA clone 2.34 genomic DNA using primers AE27A (5′-TTGGGCCCGTTGAATATGCATAGACAACATC-3′) and AE27B (5′-CCAAGCTTTCACAAATATAGGCTACTCTTGC-3′) and cloned into pBS-DHFR via ApaI and HindIII (restriction sites in bold). A downstream homology region of 570 bp was PCR amplified using primers AE27C (5′-TGAATTCTCATATTGAATAGGCCTTATATC-3′) and AE27D (5′-GGGGATCCTTTATCACTTCATATCCCAATAC-3′) and cloned into the plasmid with the upstream homology region via EcoRI and BamHI. The targeting cassette was released by ApaI and BamHI digestion. Parasite transfection using the Human T Cell Nucleofector Kit (amaxa), selection by pyrimethamine and dilution cloning were carried out as previously described (Waters et al., 1997; Janse et al., 2006). Diagnostic PCR (a) on genomic DNA from two independent Δpplp clones and control wt parasites. PCRs in lane 1 (27KO 5′-TTAGAATATTTTAAGCATTGGCTATC-3′ and 27WT 5′-CAAATGCCAACCAAATGCAC-3′), 3 (N-ter F and N-ter R) and 4 (MACPF-F 5′-TGAATTCGACCCATTTTTTATAAATATGTTGAA-3′ and MACPF-R 5′-TTCTCGAGTTAGCTAGAATAATATTCTAGAGCT-3′) are specific for the wt allele. The PCR in lane 2 is specific for integration of the gene targeting cassette (primers 27KO and 248 5′-GATGTGTTATGTGATTAATTCATACAC-3′). RT-PCR analysis (b) of pplp expression on total RNA isolated from purified in vitro cultivated ookinetes demonstrates absence of transcript in the Δpplp5 clones. pplp5 primers as in Fig. 1, p28F (5′-GCGAGATCTATGAATTTTAAATACAGTTTTATTTTTTTA-3′) and p28R (5′-GCGCCTAGCATTACTATCACGTAAATAACAAGTA-3′) amplify the pbs28 gene (642 bp).

Mentions: In an attempt to understand why the ookinete expresses more than one PPLP protein and to investigate their respective functions, we removed the entire coding region of pbpplp5 (PB000511.01.0) by double cross-over homologous recombination and integration of a modified Toxoplasma gondii dihydrofolate reductase/thymidylate synthase (dhfr/ts) gene cassette which confers resistance to the antimalarial drug pyrimethamine. Two independent transfections were carried out to generate two independent Δpplp5 clones, clone 1 and clone 2, which were characterised by diagnostic PCR (Fig. 2a). Successful gene disruption was further confirmed by our failure to amplify pplp5 mRNA from Δpplp5 ookinete cDNA (Fig. 2b).


Plasmodium berghei: plasmodium perforin-like protein 5 is required for mosquito midgut invasion in Anopheles stephensi.

Ecker A, Pinto SB, Baker KW, Kafatos FC, Sinden RE - Exp. Parasitol. (2007)

Generation of Δpplp5 parasites. Generation of constructs for targeted disruption of pplp5 by double homologous recombination were carried out as previously described (Dessens et al., 1999). Briefly, an upstream homology region of 469 bp was PCR amplified from Plasmodium berghei ANKA clone 2.34 genomic DNA using primers AE27A (5′-TTGGGCCCGTTGAATATGCATAGACAACATC-3′) and AE27B (5′-CCAAGCTTTCACAAATATAGGCTACTCTTGC-3′) and cloned into pBS-DHFR via ApaI and HindIII (restriction sites in bold). A downstream homology region of 570 bp was PCR amplified using primers AE27C (5′-TGAATTCTCATATTGAATAGGCCTTATATC-3′) and AE27D (5′-GGGGATCCTTTATCACTTCATATCCCAATAC-3′) and cloned into the plasmid with the upstream homology region via EcoRI and BamHI. The targeting cassette was released by ApaI and BamHI digestion. Parasite transfection using the Human T Cell Nucleofector Kit (amaxa), selection by pyrimethamine and dilution cloning were carried out as previously described (Waters et al., 1997; Janse et al., 2006). Diagnostic PCR (a) on genomic DNA from two independent Δpplp clones and control wt parasites. PCRs in lane 1 (27KO 5′-TTAGAATATTTTAAGCATTGGCTATC-3′ and 27WT 5′-CAAATGCCAACCAAATGCAC-3′), 3 (N-ter F and N-ter R) and 4 (MACPF-F 5′-TGAATTCGACCCATTTTTTATAAATATGTTGAA-3′ and MACPF-R 5′-TTCTCGAGTTAGCTAGAATAATATTCTAGAGCT-3′) are specific for the wt allele. The PCR in lane 2 is specific for integration of the gene targeting cassette (primers 27KO and 248 5′-GATGTGTTATGTGATTAATTCATACAC-3′). RT-PCR analysis (b) of pplp expression on total RNA isolated from purified in vitro cultivated ookinetes demonstrates absence of transcript in the Δpplp5 clones. pplp5 primers as in Fig. 1, p28F (5′-GCGAGATCTATGAATTTTAAATACAGTTTTATTTTTTTA-3′) and p28R (5′-GCGCCTAGCATTACTATCACGTAAATAACAAGTA-3′) amplify the pbs28 gene (642 bp).
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fig2: Generation of Δpplp5 parasites. Generation of constructs for targeted disruption of pplp5 by double homologous recombination were carried out as previously described (Dessens et al., 1999). Briefly, an upstream homology region of 469 bp was PCR amplified from Plasmodium berghei ANKA clone 2.34 genomic DNA using primers AE27A (5′-TTGGGCCCGTTGAATATGCATAGACAACATC-3′) and AE27B (5′-CCAAGCTTTCACAAATATAGGCTACTCTTGC-3′) and cloned into pBS-DHFR via ApaI and HindIII (restriction sites in bold). A downstream homology region of 570 bp was PCR amplified using primers AE27C (5′-TGAATTCTCATATTGAATAGGCCTTATATC-3′) and AE27D (5′-GGGGATCCTTTATCACTTCATATCCCAATAC-3′) and cloned into the plasmid with the upstream homology region via EcoRI and BamHI. The targeting cassette was released by ApaI and BamHI digestion. Parasite transfection using the Human T Cell Nucleofector Kit (amaxa), selection by pyrimethamine and dilution cloning were carried out as previously described (Waters et al., 1997; Janse et al., 2006). Diagnostic PCR (a) on genomic DNA from two independent Δpplp clones and control wt parasites. PCRs in lane 1 (27KO 5′-TTAGAATATTTTAAGCATTGGCTATC-3′ and 27WT 5′-CAAATGCCAACCAAATGCAC-3′), 3 (N-ter F and N-ter R) and 4 (MACPF-F 5′-TGAATTCGACCCATTTTTTATAAATATGTTGAA-3′ and MACPF-R 5′-TTCTCGAGTTAGCTAGAATAATATTCTAGAGCT-3′) are specific for the wt allele. The PCR in lane 2 is specific for integration of the gene targeting cassette (primers 27KO and 248 5′-GATGTGTTATGTGATTAATTCATACAC-3′). RT-PCR analysis (b) of pplp expression on total RNA isolated from purified in vitro cultivated ookinetes demonstrates absence of transcript in the Δpplp5 clones. pplp5 primers as in Fig. 1, p28F (5′-GCGAGATCTATGAATTTTAAATACAGTTTTATTTTTTTA-3′) and p28R (5′-GCGCCTAGCATTACTATCACGTAAATAACAAGTA-3′) amplify the pbs28 gene (642 bp).
Mentions: In an attempt to understand why the ookinete expresses more than one PPLP protein and to investigate their respective functions, we removed the entire coding region of pbpplp5 (PB000511.01.0) by double cross-over homologous recombination and integration of a modified Toxoplasma gondii dihydrofolate reductase/thymidylate synthase (dhfr/ts) gene cassette which confers resistance to the antimalarial drug pyrimethamine. Two independent transfections were carried out to generate two independent Δpplp5 clones, clone 1 and clone 2, which were characterised by diagnostic PCR (Fig. 2a). Successful gene disruption was further confirmed by our failure to amplify pplp5 mRNA from Δpplp5 ookinete cDNA (Fig. 2b).

Bottom Line: In this study we show through phenotypic analysis of loss-of-function mutants that PPLP5 is equally essential for mosquito infection.Deltapplp5 ookinetes cannot invade midgut epithelial cells, but subsequent parasite development is rescued if the midgut is bypassed by injection of ookinetes into the hemocoel.The indistinguishable phenotypes of Deltapplp5 and Deltapplp3 ookinetes strongly suggest that these two proteins contribute to a common process.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell and Molecular Biology, Faculty of Natural Sciences, Imperial College London, Sir Alexander Fleming Building, Imperial College Road, London, UK. andrea.ecker@imperial.ac.uk <andrea.ecker@imperial.ac.uk>

ABSTRACT
During its life cycle the malarial parasite Plasmodium forms three invasive stages which have to invade different and specific cells for replication to ensue. Invasion is vital to parasite survival and consequently proteins responsible for invasion are considered to be candidate vaccine/drug targets. Plasmodium perforin-like proteins (PPLPs) have been implicated in invasion because they contain a predicted pore-forming domain. Ookinetes express three PPLPs, and one of them (PPLP3) has previously been shown to be essential for mosquito midgut invasion. In this study we show through phenotypic analysis of loss-of-function mutants that PPLP5 is equally essential for mosquito infection. Deltapplp5 ookinetes cannot invade midgut epithelial cells, but subsequent parasite development is rescued if the midgut is bypassed by injection of ookinetes into the hemocoel. The indistinguishable phenotypes of Deltapplp5 and Deltapplp3 ookinetes strongly suggest that these two proteins contribute to a common process.

Show MeSH
Related in: MedlinePlus