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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

RT-PCR analysis of pplp5 expression during mosquito development. Routine parasite maintenance in and mosquito infections from Theiler’s Original mice were carried out as previously described (Sinden et al., 2002). Plasmodium berghei ANKA 2.34 gametocytes (Gct) were harvested from mice treated for 2 days with sulfadiazine in the drinking water to decrease asexual parasitaemia, and purified by ammonium chloride lysis at 4 °C. Ookinetes (Okn) were cultured in vitro and purified using α-Pbs28 antibody (13.1) coupled to magnetic beads (Dynabead) as previously described (Siden-Kiamos et al., 2000; Sinden et al., 2002). Infected A. stephensi midguts were dissected on day 5 (d5) or day 10 (d10) of infection. Total RNA was isolated using TRIzol (Invitrogen), contaminant genomic DNA was removed by treatment with TURBO DNA-free™ (Ambion) and RNA was cleaned up using the RNeasy Mini Kit (Qiagen). Reverse transcription was performed on 1 μg of RNA using the TaqMan® Reverse Transcription Reagents with a mixture of Oligo-dT primers and Random Hexamers (Applied Biosystems) and the resulting cDNA was used in diagnostic PCRs. Primers N-ter F (5′-TGAATTCATGGGTGATCCACTATTTACT-3′) and N-ter R (5′-TTCTCGAGTTAAAACTTATAACTCTTATATTCATCATC-3′) amplify a 318 bp fragment of pplp5, and primers TubF (5′-CCAGATGGTCAAATGCCC-3′) and TubR (5′-CTGTGGTGATGGCCATGAAC-3′) a 432 bp fragment of the α-tubulin gene. + and − denote the presence or absence of RT.
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fig1: RT-PCR analysis of pplp5 expression during mosquito development. Routine parasite maintenance in and mosquito infections from Theiler’s Original mice were carried out as previously described (Sinden et al., 2002). Plasmodium berghei ANKA 2.34 gametocytes (Gct) were harvested from mice treated for 2 days with sulfadiazine in the drinking water to decrease asexual parasitaemia, and purified by ammonium chloride lysis at 4 °C. Ookinetes (Okn) were cultured in vitro and purified using α-Pbs28 antibody (13.1) coupled to magnetic beads (Dynabead) as previously described (Siden-Kiamos et al., 2000; Sinden et al., 2002). Infected A. stephensi midguts were dissected on day 5 (d5) or day 10 (d10) of infection. Total RNA was isolated using TRIzol (Invitrogen), contaminant genomic DNA was removed by treatment with TURBO DNA-free™ (Ambion) and RNA was cleaned up using the RNeasy Mini Kit (Qiagen). Reverse transcription was performed on 1 μg of RNA using the TaqMan® Reverse Transcription Reagents with a mixture of Oligo-dT primers and Random Hexamers (Applied Biosystems) and the resulting cDNA was used in diagnostic PCRs. Primers N-ter F (5′-TGAATTCATGGGTGATCCACTATTTACT-3′) and N-ter R (5′-TTCTCGAGTTAAAACTTATAACTCTTATATTCATCATC-3′) amplify a 318 bp fragment of pplp5, and primers TubF (5′-CCAGATGGTCAAATGCCC-3′) and TubR (5′-CTGTGGTGATGGCCATGAAC-3′) a 432 bp fragment of the α-tubulin gene. + and − denote the presence or absence of RT.

Mentions: Ookinete midgut invasion is a major population bottleneck in the malaria life cycle and proteins essential for invasion, such as PPLP3, may be prime targets for transmission blocking vaccines. Besides PPLP3, P. berghei ookinetes reportedly express PPLP4 (Hall et al., 2005; Raibaud et al., 2006), and we report here, for the first time, evidence for expression of PPLP5 in the ookinete. PPLP5 was detected by MudPIT in a surface enriched ookinete proteome (R.R. Stanway, unpublished data) and expression was confirmed by RT-PCR (Fig. 1) on cDNA prepared from P. berghei gametocytes and purified ookinetes. Interestingly pplp5 was also amplified from day 5 and day 10 oocyst cDNA, indicating that the gene may be expressed throughout parasite development in the mosquito. This is consistent with data from P. falciparum, where PPLP5 was detected by MS in gametocytes and sporozoites (Florens et al., 2002).


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)

RT-PCR analysis of pplp5 expression during mosquito development. Routine parasite maintenance in and mosquito infections from Theiler’s Original mice were carried out as previously described (Sinden et al., 2002). Plasmodium berghei ANKA 2.34 gametocytes (Gct) were harvested from mice treated for 2 days with sulfadiazine in the drinking water to decrease asexual parasitaemia, and purified by ammonium chloride lysis at 4 °C. Ookinetes (Okn) were cultured in vitro and purified using α-Pbs28 antibody (13.1) coupled to magnetic beads (Dynabead) as previously described (Siden-Kiamos et al., 2000; Sinden et al., 2002). Infected A. stephensi midguts were dissected on day 5 (d5) or day 10 (d10) of infection. Total RNA was isolated using TRIzol (Invitrogen), contaminant genomic DNA was removed by treatment with TURBO DNA-free™ (Ambion) and RNA was cleaned up using the RNeasy Mini Kit (Qiagen). Reverse transcription was performed on 1 μg of RNA using the TaqMan® Reverse Transcription Reagents with a mixture of Oligo-dT primers and Random Hexamers (Applied Biosystems) and the resulting cDNA was used in diagnostic PCRs. Primers N-ter F (5′-TGAATTCATGGGTGATCCACTATTTACT-3′) and N-ter R (5′-TTCTCGAGTTAAAACTTATAACTCTTATATTCATCATC-3′) amplify a 318 bp fragment of pplp5, and primers TubF (5′-CCAGATGGTCAAATGCCC-3′) and TubR (5′-CTGTGGTGATGGCCATGAAC-3′) a 432 bp fragment of the α-tubulin gene. + and − denote the presence or absence of RT.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC1916484&req=5

fig1: RT-PCR analysis of pplp5 expression during mosquito development. Routine parasite maintenance in and mosquito infections from Theiler’s Original mice were carried out as previously described (Sinden et al., 2002). Plasmodium berghei ANKA 2.34 gametocytes (Gct) were harvested from mice treated for 2 days with sulfadiazine in the drinking water to decrease asexual parasitaemia, and purified by ammonium chloride lysis at 4 °C. Ookinetes (Okn) were cultured in vitro and purified using α-Pbs28 antibody (13.1) coupled to magnetic beads (Dynabead) as previously described (Siden-Kiamos et al., 2000; Sinden et al., 2002). Infected A. stephensi midguts were dissected on day 5 (d5) or day 10 (d10) of infection. Total RNA was isolated using TRIzol (Invitrogen), contaminant genomic DNA was removed by treatment with TURBO DNA-free™ (Ambion) and RNA was cleaned up using the RNeasy Mini Kit (Qiagen). Reverse transcription was performed on 1 μg of RNA using the TaqMan® Reverse Transcription Reagents with a mixture of Oligo-dT primers and Random Hexamers (Applied Biosystems) and the resulting cDNA was used in diagnostic PCRs. Primers N-ter F (5′-TGAATTCATGGGTGATCCACTATTTACT-3′) and N-ter R (5′-TTCTCGAGTTAAAACTTATAACTCTTATATTCATCATC-3′) amplify a 318 bp fragment of pplp5, and primers TubF (5′-CCAGATGGTCAAATGCCC-3′) and TubR (5′-CTGTGGTGATGGCCATGAAC-3′) a 432 bp fragment of the α-tubulin gene. + and − denote the presence or absence of RT.
Mentions: Ookinete midgut invasion is a major population bottleneck in the malaria life cycle and proteins essential for invasion, such as PPLP3, may be prime targets for transmission blocking vaccines. Besides PPLP3, P. berghei ookinetes reportedly express PPLP4 (Hall et al., 2005; Raibaud et al., 2006), and we report here, for the first time, evidence for expression of PPLP5 in the ookinete. PPLP5 was detected by MudPIT in a surface enriched ookinete proteome (R.R. Stanway, unpublished data) and expression was confirmed by RT-PCR (Fig. 1) on cDNA prepared from P. berghei gametocytes and purified ookinetes. Interestingly pplp5 was also amplified from day 5 and day 10 oocyst cDNA, indicating that the gene may be expressed throughout parasite development in the mosquito. This is consistent with data from P. falciparum, where PPLP5 was detected by MS in gametocytes and sporozoites (Florens et al., 2002).

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