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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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Identification using mass spectrometry analysis of proteins from the                            PY01365-KO parasite line, which are recognized by mAb 25.77.Py235 proteins were purified from a detergent-lysate of PY01365-KO                            parasitized erythrocytes or the corresponding culture supernatant by                            affinity chromatography on mAb 25.77. Fractions of purified protein                            eluted sequentially from the affinity column were resolved by SDS-PAGE                            on a 5% polyacrylamide gel. The protein bands were excised,                            reduced and alkylated and digested with trypsin, and the peptides                            analyzed by MALDI-ToF mass spectrometry. The peptide mass fingerprints                            were used to identify the genes encoded by the purified proteins. (A)                            Proteins purified from schizonts: band1, PY01185; band 2, PY01185; band                            3, PY03534. (B) Proteins purified from culture supernatant: Band 1,                            PY01185; band 2, PY03534. Essentially two Py235 contigs were identified                            from both the schizont and supernatant preparations.
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ppat-1001288-g006: Identification using mass spectrometry analysis of proteins from the PY01365-KO parasite line, which are recognized by mAb 25.77.Py235 proteins were purified from a detergent-lysate of PY01365-KO parasitized erythrocytes or the corresponding culture supernatant by affinity chromatography on mAb 25.77. Fractions of purified protein eluted sequentially from the affinity column were resolved by SDS-PAGE on a 5% polyacrylamide gel. The protein bands were excised, reduced and alkylated and digested with trypsin, and the peptides analyzed by MALDI-ToF mass spectrometry. The peptide mass fingerprints were used to identify the genes encoded by the purified proteins. (A) Proteins purified from schizonts: band1, PY01185; band 2, PY01185; band 3, PY03534. (B) Proteins purified from culture supernatant: Band 1, PY01185; band 2, PY03534. Essentially two Py235 contigs were identified from both the schizont and supernatant preparations.

Mentions: We were interested to identify the Py235 proteins expressed by the PY01365-KO parasite line that were recognised by mAb 25.77 in the absence of Py235EBP-1. Py235 proteins affinity purified using mAb 25.77 from both detergent-solubilised parasite preparations (Figure 6A) and culture supernatant (Figure 6B) from the PY01365-KO parasite line were fractionated on a 5% SDS-PAGE gel and processed for mass spectrometry analysis. MASCOT searches using the peptides and the NCBI database gave significant matches to three Py235 contigs, PY01185, PY05995 and PY03534 (Table 2), mirroring the results obtained with mAb 25.37 and WT parasites. The results suggest that mAb 25.77 has a higher affinity for PY01365, than PY01185 and PY05995/PY03534 protein products, since in the absence of PY01365 mAb 25.77 was able now to detect the other proteins. While mass spectrometry analysis of proteins affinity purified from WT parasites using mAb 25.77 routinely clearly identified PY01365 (Py235EBP-1) (Table 2), peptides derived from PY01185, PY05995 and PY03534 were also present in small amounts (data not shown). Due to the high number of unique peptides required for positive identification of these large proteins, the few unique peptides obtained for PY01185, PY05995 and PY03534 was insufficient.


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)

Identification using mass spectrometry analysis of proteins from the                            PY01365-KO parasite line, which are recognized by mAb 25.77.Py235 proteins were purified from a detergent-lysate of PY01365-KO                            parasitized erythrocytes or the corresponding culture supernatant by                            affinity chromatography on mAb 25.77. Fractions of purified protein                            eluted sequentially from the affinity column were resolved by SDS-PAGE                            on a 5% polyacrylamide gel. The protein bands were excised,                            reduced and alkylated and digested with trypsin, and the peptides                            analyzed by MALDI-ToF mass spectrometry. The peptide mass fingerprints                            were used to identify the genes encoded by the purified proteins. (A)                            Proteins purified from schizonts: band1, PY01185; band 2, PY01185; band                            3, PY03534. (B) Proteins purified from culture supernatant: Band 1,                            PY01185; band 2, PY03534. Essentially two Py235 contigs were identified                            from both the schizont and supernatant preparations.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040676&req=5

ppat-1001288-g006: Identification using mass spectrometry analysis of proteins from the PY01365-KO parasite line, which are recognized by mAb 25.77.Py235 proteins were purified from a detergent-lysate of PY01365-KO parasitized erythrocytes or the corresponding culture supernatant by affinity chromatography on mAb 25.77. Fractions of purified protein eluted sequentially from the affinity column were resolved by SDS-PAGE on a 5% polyacrylamide gel. The protein bands were excised, reduced and alkylated and digested with trypsin, and the peptides analyzed by MALDI-ToF mass spectrometry. The peptide mass fingerprints were used to identify the genes encoded by the purified proteins. (A) Proteins purified from schizonts: band1, PY01185; band 2, PY01185; band 3, PY03534. (B) Proteins purified from culture supernatant: Band 1, PY01185; band 2, PY03534. Essentially two Py235 contigs were identified from both the schizont and supernatant preparations.
Mentions: We were interested to identify the Py235 proteins expressed by the PY01365-KO parasite line that were recognised by mAb 25.77 in the absence of Py235EBP-1. Py235 proteins affinity purified using mAb 25.77 from both detergent-solubilised parasite preparations (Figure 6A) and culture supernatant (Figure 6B) from the PY01365-KO parasite line were fractionated on a 5% SDS-PAGE gel and processed for mass spectrometry analysis. MASCOT searches using the peptides and the NCBI database gave significant matches to three Py235 contigs, PY01185, PY05995 and PY03534 (Table 2), mirroring the results obtained with mAb 25.37 and WT parasites. The results suggest that mAb 25.77 has a higher affinity for PY01365, than PY01185 and PY05995/PY03534 protein products, since in the absence of PY01365 mAb 25.77 was able now to detect the other proteins. While mass spectrometry analysis of proteins affinity purified from WT parasites using mAb 25.77 routinely clearly identified PY01365 (Py235EBP-1) (Table 2), peptides derived from PY01185, PY05995 and PY03534 were also present in small amounts (data not shown). Due to the high number of unique peptides required for positive identification of these large proteins, the few unique peptides obtained for PY01185, PY05995 and PY03534 was insufficient.

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