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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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Analysis of Py235 gene family expression.(A) Quantitative real time RT-PCR (qPCR) analysis of genes that encode                            proteins recognized by the protective mAbs. Comparison of transcription                            levels of genes, expressed as number of copies transcribed per                                1×10−9 g DNA. qPCR was carried according to                            the MIQE guidelines. All qPCR reactions were set up in triplicates. The                            result of a representative experiment is shown. Transcripts from three                            PY235genes: PY01365, PY01185 and PY05995/PY03534 were examined from both                            WT and PY01365-KO parasites. (B) RNA-Seq coverage plot for PY01365 and                            flanking genes. The gene models are in light blue. The lines represent                            the coverage plots for the numbers of reads: the green line is for WT                            and the red line for PY01365-KO parasite lines, respectively. The                            knocked out gene PY01365 is not expressed, unlike two neighboring genes,                            PY00128 and PY01366 that are expressed at similar levels in both                            parasite lines. PY00129 is not expressed.
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ppat-1001288-g007: Analysis of Py235 gene family expression.(A) Quantitative real time RT-PCR (qPCR) analysis of genes that encode proteins recognized by the protective mAbs. Comparison of transcription levels of genes, expressed as number of copies transcribed per 1×10−9 g DNA. qPCR was carried according to the MIQE guidelines. All qPCR reactions were set up in triplicates. The result of a representative experiment is shown. Transcripts from three PY235genes: PY01365, PY01185 and PY05995/PY03534 were examined from both WT and PY01365-KO parasites. (B) RNA-Seq coverage plot for PY01365 and flanking genes. The gene models are in light blue. The lines represent the coverage plots for the numbers of reads: the green line is for WT and the red line for PY01365-KO parasite lines, respectively. The knocked out gene PY01365 is not expressed, unlike two neighboring genes, PY00128 and PY01366 that are expressed at similar levels in both parasite lines. PY00129 is not expressed.

Mentions: qPCR was carried out using primers specific to the genes of interest and to reference genes coding for PyEBL (PY04764), which is expressed at the same developmental stage as Py235 proteins, and the gene for the constitutively expressed protein Pyβ-tubulin (PY05711) (Table 2). Of the 3 genes in the Py235 family that were examined, PY01365 had the lowest transcription level followed by PY05995/PY03534, with PY01185 having the highest transcription level in the WT parasite line (Figure 7a).


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)

Analysis of Py235 gene family expression.(A) Quantitative real time RT-PCR (qPCR) analysis of genes that encode                            proteins recognized by the protective mAbs. Comparison of transcription                            levels of genes, expressed as number of copies transcribed per                                1×10−9 g DNA. qPCR was carried according to                            the MIQE guidelines. All qPCR reactions were set up in triplicates. The                            result of a representative experiment is shown. Transcripts from three                            PY235genes: PY01365, PY01185 and PY05995/PY03534 were examined from both                            WT and PY01365-KO parasites. (B) RNA-Seq coverage plot for PY01365 and                            flanking genes. The gene models are in light blue. The lines represent                            the coverage plots for the numbers of reads: the green line is for WT                            and the red line for PY01365-KO parasite lines, respectively. The                            knocked out gene PY01365 is not expressed, unlike two neighboring genes,                            PY00128 and PY01366 that are expressed at similar levels in both                            parasite lines. PY00129 is not expressed.
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Related In: Results  -  Collection

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

ppat-1001288-g007: Analysis of Py235 gene family expression.(A) Quantitative real time RT-PCR (qPCR) analysis of genes that encode proteins recognized by the protective mAbs. Comparison of transcription levels of genes, expressed as number of copies transcribed per 1×10−9 g DNA. qPCR was carried according to the MIQE guidelines. All qPCR reactions were set up in triplicates. The result of a representative experiment is shown. Transcripts from three PY235genes: PY01365, PY01185 and PY05995/PY03534 were examined from both WT and PY01365-KO parasites. (B) RNA-Seq coverage plot for PY01365 and flanking genes. The gene models are in light blue. The lines represent the coverage plots for the numbers of reads: the green line is for WT and the red line for PY01365-KO parasite lines, respectively. The knocked out gene PY01365 is not expressed, unlike two neighboring genes, PY00128 and PY01366 that are expressed at similar levels in both parasite lines. PY00129 is not expressed.
Mentions: qPCR was carried out using primers specific to the genes of interest and to reference genes coding for PyEBL (PY04764), which is expressed at the same developmental stage as Py235 proteins, and the gene for the constitutively expressed protein Pyβ-tubulin (PY05711) (Table 2). Of the 3 genes in the Py235 family that were examined, PY01365 had the lowest transcription level followed by PY05995/PY03534, with PY01185 having the highest transcription level in the WT parasite line (Figure 7a).

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