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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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PCR amplification of a py235 gene fragment spanning two contigs,                            PY05995 and PY03534.(A) A single PCR product of 392bp was obtained using a forward primer                            designed on DNA sequences from within the last 200 nucleotides of the                            3′ coding sequence of PY05995 and a reverse primer from within the                            first 200 nucleotides of the 5′ coding sequence of PY03534. (B)                            Alignment of part of the amino acid sequence translated from the                            directly sequenced PCR product with the relevant PY05995 and PY03534                            amino acid sequences (obtained from the NCBI database). The PCR product                            sequence aligned perfectly with that of the two contigs and filled a 24                            nucleotide gap coding for the eight amino acid residues indicated in                            red. (C) Confirmation of PY05995 and PY03534 concatenation by RNA-Seq.                            The two sequences were linked in the RNA-Seq data by 153 read pairs that                            mapped to the end of PY05995, and the beginning of PY03534. The graphs                            present the expression in the WT (green) and PY01365-KO (red) parasite                            lines. The two red diamonds joined by a line indicates the position of                            the qPCR primers.
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ppat-1001288-g005: PCR amplification of a py235 gene fragment spanning two contigs, PY05995 and PY03534.(A) A single PCR product of 392bp was obtained using a forward primer designed on DNA sequences from within the last 200 nucleotides of the 3′ coding sequence of PY05995 and a reverse primer from within the first 200 nucleotides of the 5′ coding sequence of PY03534. (B) Alignment of part of the amino acid sequence translated from the directly sequenced PCR product with the relevant PY05995 and PY03534 amino acid sequences (obtained from the NCBI database). The PCR product sequence aligned perfectly with that of the two contigs and filled a 24 nucleotide gap coding for the eight amino acid residues indicated in red. (C) Confirmation of PY05995 and PY03534 concatenation by RNA-Seq. The two sequences were linked in the RNA-Seq data by 153 read pairs that mapped to the end of PY05995, and the beginning of PY03534. The graphs present the expression in the WT (green) and PY01365-KO (red) parasite lines. The two red diamonds joined by a line indicates the position of the qPCR primers.

Mentions: The gene encoding the protein recognized by mAb 25.77 and expressed in WT parasites has previously been identified. A second protective mAb 25.37 also recognizes Py235 proteins and we wished to identify the protein(s) to which it binds. To identify the corresponding genes, peptide mass fingerprinting was carried out on the Py235 proteins affinity purified using mAb 25.37 from both schizonts (Figure 4A) and from culture supernatant of WT parasites maintained in vitro (Figure 4B) and fractionated by SDS-PAGE on a 5% gel. The peptides detected were derived from three contigs in the genome database; one contained a full length (8172bp) py235 gene sequence, PY01185, and the remaining two contained partial gene sequences (Table 1). Contig PY05995 is 2685bp in length and contains sequence that aligned with the 5′ end of other Py235 gene family members, while PY03534 (5478bp) aligned with the 3′ of other Py235 genes. To establish whether or not these two contigs are part of the same gene, primers designed to the 3′-sequence of PY05995 and to the 5′-sequence of PY03534 were used to amplify sequence from gDNA, and gave a single PCR product of the expected size, 392bp (Figure 5A). Sequence analysis and alignment with the gene sequences from the database showed perfect alignment (Figure 5B) and confirmed that the contigs were part of the same single full length Py235 gene, PY05995/PY03534. This conclusion was further confirmed by read pairs of the RNA-Seq data. 153 mates mapped to the end of PY03534 and the beginning of PY05995, and the entire gene could be assembled from the mapping reads (Figure 5C).


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)

PCR amplification of a py235 gene fragment spanning two contigs,                            PY05995 and PY03534.(A) A single PCR product of 392bp was obtained using a forward primer                            designed on DNA sequences from within the last 200 nucleotides of the                            3′ coding sequence of PY05995 and a reverse primer from within the                            first 200 nucleotides of the 5′ coding sequence of PY03534. (B)                            Alignment of part of the amino acid sequence translated from the                            directly sequenced PCR product with the relevant PY05995 and PY03534                            amino acid sequences (obtained from the NCBI database). The PCR product                            sequence aligned perfectly with that of the two contigs and filled a 24                            nucleotide gap coding for the eight amino acid residues indicated in                            red. (C) Confirmation of PY05995 and PY03534 concatenation by RNA-Seq.                            The two sequences were linked in the RNA-Seq data by 153 read pairs that                            mapped to the end of PY05995, and the beginning of PY03534. The graphs                            present the expression in the WT (green) and PY01365-KO (red) parasite                            lines. The two red diamonds joined by a line indicates the position of                            the qPCR primers.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040676&req=5

ppat-1001288-g005: PCR amplification of a py235 gene fragment spanning two contigs, PY05995 and PY03534.(A) A single PCR product of 392bp was obtained using a forward primer designed on DNA sequences from within the last 200 nucleotides of the 3′ coding sequence of PY05995 and a reverse primer from within the first 200 nucleotides of the 5′ coding sequence of PY03534. (B) Alignment of part of the amino acid sequence translated from the directly sequenced PCR product with the relevant PY05995 and PY03534 amino acid sequences (obtained from the NCBI database). The PCR product sequence aligned perfectly with that of the two contigs and filled a 24 nucleotide gap coding for the eight amino acid residues indicated in red. (C) Confirmation of PY05995 and PY03534 concatenation by RNA-Seq. The two sequences were linked in the RNA-Seq data by 153 read pairs that mapped to the end of PY05995, and the beginning of PY03534. The graphs present the expression in the WT (green) and PY01365-KO (red) parasite lines. The two red diamonds joined by a line indicates the position of the qPCR primers.
Mentions: The gene encoding the protein recognized by mAb 25.77 and expressed in WT parasites has previously been identified. A second protective mAb 25.37 also recognizes Py235 proteins and we wished to identify the protein(s) to which it binds. To identify the corresponding genes, peptide mass fingerprinting was carried out on the Py235 proteins affinity purified using mAb 25.37 from both schizonts (Figure 4A) and from culture supernatant of WT parasites maintained in vitro (Figure 4B) and fractionated by SDS-PAGE on a 5% gel. The peptides detected were derived from three contigs in the genome database; one contained a full length (8172bp) py235 gene sequence, PY01185, and the remaining two contained partial gene sequences (Table 1). Contig PY05995 is 2685bp in length and contains sequence that aligned with the 5′ end of other Py235 gene family members, while PY03534 (5478bp) aligned with the 3′ of other Py235 genes. To establish whether or not these two contigs are part of the same gene, primers designed to the 3′-sequence of PY05995 and to the 5′-sequence of PY03534 were used to amplify sequence from gDNA, and gave a single PCR product of the expected size, 392bp (Figure 5A). Sequence analysis and alignment with the gene sequences from the database showed perfect alignment (Figure 5B) and confirmed that the contigs were part of the same single full length Py235 gene, PY05995/PY03534. This conclusion was further confirmed by read pairs of the RNA-Seq data. 153 mates mapped to the end of PY03534 and the beginning of PY05995, and the entire gene could be assembled from the mapping reads (Figure 5C).

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