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Targeted disruption of py235ebp-1: invasion of erythrocytes by Plasmodium yoelii using an alternative Py235 erythrocyte binding protein.

Ogun SA, Tewari R, Otto TD, Howell SA, Knuepfer E, Cunningham DA, Xu Z, Pain A, Holder AA - PLoS Pathog. (2011)

Bottom Line: The PY01365, PY01185, and PY05995/PY03534 genes are part of a distinct subset of the py235 family.In P. falciparum, the RH protein genes are under epigenetic control and expression correlates with binding to distinct erythrocyte receptors and specific invasion pathways, whereas in P. yoelii YM all the genes are expressed and deletion of one does not result in upregulation of another.We propose that simultaneous expression of multiple Py235 ligands enables invasion of a wide range of host erythrocytes even in the presence of antibodies to one or more of the proteins and that this functional redundancy at the protein level gives the parasite phenotypic plasticity in the absence of differences in gene expression.

View Article: PubMed Central - PubMed

Affiliation: Division of Parasitology, MRC National Institute for Medical Research, London, UK. sogun@nimr.mrc.ac.uk

ABSTRACT
Plasmodium yoelii YM asexual blood stage parasites express multiple members of the py235 gene family, part of the super-family of genes including those coding for Plasmodium vivax reticulocyte binding proteins and Plasmodium falciparum RH proteins. We previously identified a Py235 erythrocyte binding protein (Py235EBP-1, encoded by the PY01365 gene) that is recognized by protective mAb 25.77. Proteins recognized by a second protective mAb 25.37 have been identified by mass spectrometry and are encoded by two genes, PY01185 and PY05995/PY03534. We deleted the PY01365 gene and examined the phenotype. The expression of the members of the py235 family in both the WT and gene deletion parasites was measured by quantitative RT-PCR and RNA-Seq. py235ebp-1 expression was undetectable in the knockout parasite, but transcription of other members of the family was essentially unaffected. The knockout parasites continued to react with mAb 25.77; and the 25.77-binding proteins in these parasites were the PY01185 and PY05995/PY03534 products. The PY01185 product was also identified as erythrocyte binding. There was no clear change in erythrocyte invasion profile suggesting that the PY01185 gene product (designated PY235EBP-2) is able to fulfill the role of EBP-1 by serving as an invasion ligand although the molecular details of its interaction with erythrocytes have not been examined. The PY01365, PY01185, and PY05995/PY03534 genes are part of a distinct subset of the py235 family. In P. falciparum, the RH protein genes are under epigenetic control and expression correlates with binding to distinct erythrocyte receptors and specific invasion pathways, whereas in P. yoelii YM all the genes are expressed and deletion of one does not result in upregulation of another. We propose that simultaneous expression of multiple Py235 ligands enables invasion of a wide range of host erythrocytes even in the presence of antibodies to one or more of the proteins and that this functional redundancy at the protein level gives the parasite phenotypic plasticity in the absence of differences in gene expression.

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Targeted disruption of PY01365 (py235ebp-1) gene in                                P. yoelii.(A) Schematic showing the targeting construct used for gene disruption by                            double homologous recombination into the PY01365 gene and the resultant                            targeted integrated locus. The positions of the DNA probes (A, B and C)                            used in Southern blot analysis are indicated below the WT chromosomal                            locus. (B) Southern blot analysis of P. yoelii genomic                            DNA from WT, transgenic parasites and cloned transgenic parasites,                            digested with Pci1 and Acc651. Probes used are a 921bp fragment of the                            Tg/DHFR sequence (lanes 1, 2, 8, 9 and 10) and Fragment B, (lanes 3 to                            7). The result of integration is seen at ∼3.8 Kb (lanes 2 and 4 to                            10); the unmodified band at ∼2.3Kb present in WT DNA was detected                            with Fragment B probe alone (lane 3). (C). Chromosomes from a cloned                            transgenic parasite line (lanes 1, 2, 5 and 6) or WT parasites (lanes 3,                            4, 7 and 8) were separated by pulse field gel electrophoresis and                            hybridized with Fragment C (lanes 1 to 4) and the Tg/DHFR probe (lanes 5                            to 8). The position of chromosomes 13/14 and the endogenous DHFR on                            chromosome 7 is indicated.
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ppat-1001288-g001: Targeted disruption of PY01365 (py235ebp-1) gene in P. yoelii.(A) Schematic showing the targeting construct used for gene disruption by double homologous recombination into the PY01365 gene and the resultant targeted integrated locus. The positions of the DNA probes (A, B and C) used in Southern blot analysis are indicated below the WT chromosomal locus. (B) Southern blot analysis of P. yoelii genomic DNA from WT, transgenic parasites and cloned transgenic parasites, digested with Pci1 and Acc651. Probes used are a 921bp fragment of the Tg/DHFR sequence (lanes 1, 2, 8, 9 and 10) and Fragment B, (lanes 3 to 7). The result of integration is seen at ∼3.8 Kb (lanes 2 and 4 to 10); the unmodified band at ∼2.3Kb present in WT DNA was detected with Fragment B probe alone (lane 3). (C). Chromosomes from a cloned transgenic parasite line (lanes 1, 2, 5 and 6) or WT parasites (lanes 3, 4, 7 and 8) were separated by pulse field gel electrophoresis and hybridized with Fragment C (lanes 1 to 4) and the Tg/DHFR probe (lanes 5 to 8). The position of chromosomes 13/14 and the endogenous DHFR on chromosome 7 is indicated.

Mentions: Disruption of the py235ebp-1 (PY01365) by insertion of the DHFR cassette by double homologous recombination (Figure 1A) was carried out. Southern blot analysis of digested gDNA from transfected parasites selected with pyrimethamine identified a single band of the expected size in this population, when the filter was probed with a fragment of DHFR/TS (Figure 1B, lane 2). In contrast, hybridization with the probe that binds to the 3′ coding region of py235ebp-1 (Fragment B), detected DNA in both the wild type (WT) (2.3Kb) and the KO (3.8Kb) parasite lines as expected (Figure 1B, lanes 3 and 4). Four individual clones (1 to 3 shown) derived from the population of parasites gave a similar result with both fragment B (Figure 1B, lanes 5 to 7) and the DHFR/TS probe (Figure 1B, lanes 8 to 10), clearly showing that the PY01365 gene had been disrupted. Further evidence that the PY01365 gene had been deleted from the P. yoelii genome was obtained by chromosome analysis (Figure 1C). Hybridization with the probe that binds to a 5′ coding sequence of PY01365 (Fragment C), detected a signal only in the WT parasite lanes, (Figure 1C, lanes 3 and 4). Hybridization with a probe that binds the 3′ UTR of DHFR/TS detected both the modified py235ebp-1 locus (chromosome 13/14) in the KO parasite line (Figure 1C, lanes 5 and 6) and the endogenous dhfr locus (chromosome 7) in both KO and WT parasites (Figure 1C, lanes 5 to 8). Hybridization of a chromosome blot with the Fragment B probe identified a band in all the lanes as expected (data not shown).


Targeted disruption of py235ebp-1: invasion of erythrocytes by Plasmodium yoelii using an alternative Py235 erythrocyte binding protein.

Ogun SA, Tewari R, Otto TD, Howell SA, Knuepfer E, Cunningham DA, Xu Z, Pain A, Holder AA - PLoS Pathog. (2011)

Targeted disruption of PY01365 (py235ebp-1) gene in                                P. yoelii.(A) Schematic showing the targeting construct used for gene disruption by                            double homologous recombination into the PY01365 gene and the resultant                            targeted integrated locus. The positions of the DNA probes (A, B and C)                            used in Southern blot analysis are indicated below the WT chromosomal                            locus. (B) Southern blot analysis of P. yoelii genomic                            DNA from WT, transgenic parasites and cloned transgenic parasites,                            digested with Pci1 and Acc651. Probes used are a 921bp fragment of the                            Tg/DHFR sequence (lanes 1, 2, 8, 9 and 10) and Fragment B, (lanes 3 to                            7). The result of integration is seen at ∼3.8 Kb (lanes 2 and 4 to                            10); the unmodified band at ∼2.3Kb present in WT DNA was detected                            with Fragment B probe alone (lane 3). (C). Chromosomes from a cloned                            transgenic parasite line (lanes 1, 2, 5 and 6) or WT parasites (lanes 3,                            4, 7 and 8) were separated by pulse field gel electrophoresis and                            hybridized with Fragment C (lanes 1 to 4) and the Tg/DHFR probe (lanes 5                            to 8). The position of chromosomes 13/14 and the endogenous DHFR on                            chromosome 7 is indicated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3040676&req=5

ppat-1001288-g001: Targeted disruption of PY01365 (py235ebp-1) gene in P. yoelii.(A) Schematic showing the targeting construct used for gene disruption by double homologous recombination into the PY01365 gene and the resultant targeted integrated locus. The positions of the DNA probes (A, B and C) used in Southern blot analysis are indicated below the WT chromosomal locus. (B) Southern blot analysis of P. yoelii genomic DNA from WT, transgenic parasites and cloned transgenic parasites, digested with Pci1 and Acc651. Probes used are a 921bp fragment of the Tg/DHFR sequence (lanes 1, 2, 8, 9 and 10) and Fragment B, (lanes 3 to 7). The result of integration is seen at ∼3.8 Kb (lanes 2 and 4 to 10); the unmodified band at ∼2.3Kb present in WT DNA was detected with Fragment B probe alone (lane 3). (C). Chromosomes from a cloned transgenic parasite line (lanes 1, 2, 5 and 6) or WT parasites (lanes 3, 4, 7 and 8) were separated by pulse field gel electrophoresis and hybridized with Fragment C (lanes 1 to 4) and the Tg/DHFR probe (lanes 5 to 8). The position of chromosomes 13/14 and the endogenous DHFR on chromosome 7 is indicated.
Mentions: Disruption of the py235ebp-1 (PY01365) by insertion of the DHFR cassette by double homologous recombination (Figure 1A) was carried out. Southern blot analysis of digested gDNA from transfected parasites selected with pyrimethamine identified a single band of the expected size in this population, when the filter was probed with a fragment of DHFR/TS (Figure 1B, lane 2). In contrast, hybridization with the probe that binds to the 3′ coding region of py235ebp-1 (Fragment B), detected DNA in both the wild type (WT) (2.3Kb) and the KO (3.8Kb) parasite lines as expected (Figure 1B, lanes 3 and 4). Four individual clones (1 to 3 shown) derived from the population of parasites gave a similar result with both fragment B (Figure 1B, lanes 5 to 7) and the DHFR/TS probe (Figure 1B, lanes 8 to 10), clearly showing that the PY01365 gene had been disrupted. Further evidence that the PY01365 gene had been deleted from the P. yoelii genome was obtained by chromosome analysis (Figure 1C). Hybridization with the probe that binds to a 5′ coding sequence of PY01365 (Fragment C), detected a signal only in the WT parasite lanes, (Figure 1C, lanes 3 and 4). Hybridization with a probe that binds the 3′ UTR of DHFR/TS detected both the modified py235ebp-1 locus (chromosome 13/14) in the KO parasite line (Figure 1C, lanes 5 and 6) and the endogenous dhfr locus (chromosome 7) in both KO and WT parasites (Figure 1C, lanes 5 to 8). Hybridization of a chromosome blot with the Fragment B probe identified a band in all the lanes as expected (data not shown).

Bottom Line: The PY01365, PY01185, and PY05995/PY03534 genes are part of a distinct subset of the py235 family.In P. falciparum, the RH protein genes are under epigenetic control and expression correlates with binding to distinct erythrocyte receptors and specific invasion pathways, whereas in P. yoelii YM all the genes are expressed and deletion of one does not result in upregulation of another.We propose that simultaneous expression of multiple Py235 ligands enables invasion of a wide range of host erythrocytes even in the presence of antibodies to one or more of the proteins and that this functional redundancy at the protein level gives the parasite phenotypic plasticity in the absence of differences in gene expression.

View Article: PubMed Central - PubMed

Affiliation: Division of Parasitology, MRC National Institute for Medical Research, London, UK. sogun@nimr.mrc.ac.uk

ABSTRACT
Plasmodium yoelii YM asexual blood stage parasites express multiple members of the py235 gene family, part of the super-family of genes including those coding for Plasmodium vivax reticulocyte binding proteins and Plasmodium falciparum RH proteins. We previously identified a Py235 erythrocyte binding protein (Py235EBP-1, encoded by the PY01365 gene) that is recognized by protective mAb 25.77. Proteins recognized by a second protective mAb 25.37 have been identified by mass spectrometry and are encoded by two genes, PY01185 and PY05995/PY03534. We deleted the PY01365 gene and examined the phenotype. The expression of the members of the py235 family in both the WT and gene deletion parasites was measured by quantitative RT-PCR and RNA-Seq. py235ebp-1 expression was undetectable in the knockout parasite, but transcription of other members of the family was essentially unaffected. The knockout parasites continued to react with mAb 25.77; and the 25.77-binding proteins in these parasites were the PY01185 and PY05995/PY03534 products. The PY01185 product was also identified as erythrocyte binding. There was no clear change in erythrocyte invasion profile suggesting that the PY01185 gene product (designated PY235EBP-2) is able to fulfill the role of EBP-1 by serving as an invasion ligand although the molecular details of its interaction with erythrocytes have not been examined. The PY01365, PY01185, and PY05995/PY03534 genes are part of a distinct subset of the py235 family. In P. falciparum, the RH protein genes are under epigenetic control and expression correlates with binding to distinct erythrocyte receptors and specific invasion pathways, whereas in P. yoelii YM all the genes are expressed and deletion of one does not result in upregulation of another. We propose that simultaneous expression of multiple Py235 ligands enables invasion of a wide range of host erythrocytes even in the presence of antibodies to one or more of the proteins and that this functional redundancy at the protein level gives the parasite phenotypic plasticity in the absence of differences in gene expression.

Show MeSH
Related in: MedlinePlus