Limits...
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.

Show MeSH

Related in: MedlinePlus

Detection of Py235 protein expression.(A) Indirect fluorescent antibody assay using mAb25.77 on 1%                            formaldehyde-fixed thin blood smears of WT or PY01365-KO schizonts. For                            each parasite line the first panel shows mAb 25.77 reactivity, the                            second shows mAb 25.77 reactivity and parasite nuclei labelled with                            DAPI, and the third shows the bright field image. (B) a dual labeling                            experiment of WT parasitized erythrocytes. In the first column of panels                            the antibodies used were rabbit anti-EBL, mAb 45B1 (AMA1) and mAb 48F8                            (RON4), each of which was detected by the appropriate                            fluorescence-labelled second antibody. In the second column of panels                            the same cells were stained with mAb 25.77, which was detected either                            with a fluorescence-labelled antibody (red) or was directly conjugated                            to a fluorescent dye (green). The third column of panels shows parasite                            nuclei labelled with DAPI, with the fourth column of panels showing the                            overlay of all three previous panels in the row (MERGE). The fifth                            column of panels shows the bright field images. (C) Immunoprecipitation                            analysis of WT (lanes 1, 3 and 5) and PY01365-KO (lanes 2, 4 and 6)                            parasite soluble proteins released into culture supernatant using mAbs                            25.77 (lanes 1 and 2), 25.37 (lanes 3 and 4) and normal mouse serum                            (lanes 5 and 6). The Py235 proteins detected by this analysis are shown.                            (D) Erythrocyte binding assay: detection of a single protein band eluted                            from erythrocytes and immunoprecipitated using mAbs 25.77 (lanes 1 and                            2) and 25.37 (lanes 3 and 4) and derived from WT (lanes 1 and 3) and KO                            (lanes 2 and 4) parasites, respectively. The proposed identities                            (Py235EBP-1 and Py235EBP-2) are indicated. (E) Alexa Fluor                            488-conjugated 25.77 (green) and Alexa Fluor 594-conjugated 25.37 (red)                            were used in a dual labeling experiment of WT parasitized erythrocytes.                            The third of four panels of an identical field shows parasite nuclei                            labelled with DAPI, with the fourth panel showing the overlay of all                            three previous panels (MERGE). Py235 proteins are recognised by both                            mAbs in each schizont.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3040676&req=5

ppat-1001288-g003: Detection of Py235 protein expression.(A) Indirect fluorescent antibody assay using mAb25.77 on 1% formaldehyde-fixed thin blood smears of WT or PY01365-KO schizonts. For each parasite line the first panel shows mAb 25.77 reactivity, the second shows mAb 25.77 reactivity and parasite nuclei labelled with DAPI, and the third shows the bright field image. (B) a dual labeling experiment of WT parasitized erythrocytes. In the first column of panels the antibodies used were rabbit anti-EBL, mAb 45B1 (AMA1) and mAb 48F8 (RON4), each of which was detected by the appropriate fluorescence-labelled second antibody. In the second column of panels the same cells were stained with mAb 25.77, which was detected either with a fluorescence-labelled antibody (red) or was directly conjugated to a fluorescent dye (green). The third column of panels shows parasite nuclei labelled with DAPI, with the fourth column of panels showing the overlay of all three previous panels in the row (MERGE). The fifth column of panels shows the bright field images. (C) Immunoprecipitation analysis of WT (lanes 1, 3 and 5) and PY01365-KO (lanes 2, 4 and 6) parasite soluble proteins released into culture supernatant using mAbs 25.77 (lanes 1 and 2), 25.37 (lanes 3 and 4) and normal mouse serum (lanes 5 and 6). The Py235 proteins detected by this analysis are shown. (D) Erythrocyte binding assay: detection of a single protein band eluted from erythrocytes and immunoprecipitated using mAbs 25.77 (lanes 1 and 2) and 25.37 (lanes 3 and 4) and derived from WT (lanes 1 and 3) and KO (lanes 2 and 4) parasites, respectively. The proposed identities (Py235EBP-1 and Py235EBP-2) are indicated. (E) Alexa Fluor 488-conjugated 25.77 (green) and Alexa Fluor 594-conjugated 25.37 (red) were used in a dual labeling experiment of WT parasitized erythrocytes. The third of four panels of an identical field shows parasite nuclei labelled with DAPI, with the fourth panel showing the overlay of all three previous panels (MERGE). Py235 proteins are recognised by both mAbs in each schizont.

Mentions: The mAb 25.77 had previously been used to identify Py235EBP-1, the product of the PY01365 gene. By IFA, this mAb gives a punctuate pattern of fluorescence in the WT parasite line (Figure 3A). Surprisingly, a similar pattern was also observed for the PY01365-KO parasite line, even though Py235ebp-1 is no longer being expressed. The pattern of reactivity (Figure 3B) was similar but not identical to that of antibodies specific for the micronemal protein, Apical Membrane Antigen 1 (AMA1) [39], the erythrocyte binding ligand protein (EBL), which has a dense granule location in this parasite line [40], and rhoptry neck protein 4 (RON 4) [41]. Furthermore, when proteins released into in vitro culture supernatant from radiolabeled WT and PY01365-KO parasitized erythrocytes were immunoprecipitated using mAbs 25.77 and 25.37 (Figure 3C), or bound to erythrocytes, eluted and then immunoprecipitated (Figure 3D), both mAbs recognized proteins of approximately 235 kDa showing that Py235 proteins were expressed by both WT and PY01365-KO parasite lines. Clearly the Py235 proteins now expressed by the KO parasite line, although at least in part different to those being expressed by the WT parasite, share common epitopes bound by the antibodies.


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)

Detection of Py235 protein expression.(A) Indirect fluorescent antibody assay using mAb25.77 on 1%                            formaldehyde-fixed thin blood smears of WT or PY01365-KO schizonts. For                            each parasite line the first panel shows mAb 25.77 reactivity, the                            second shows mAb 25.77 reactivity and parasite nuclei labelled with                            DAPI, and the third shows the bright field image. (B) a dual labeling                            experiment of WT parasitized erythrocytes. In the first column of panels                            the antibodies used were rabbit anti-EBL, mAb 45B1 (AMA1) and mAb 48F8                            (RON4), each of which was detected by the appropriate                            fluorescence-labelled second antibody. In the second column of panels                            the same cells were stained with mAb 25.77, which was detected either                            with a fluorescence-labelled antibody (red) or was directly conjugated                            to a fluorescent dye (green). The third column of panels shows parasite                            nuclei labelled with DAPI, with the fourth column of panels showing the                            overlay of all three previous panels in the row (MERGE). The fifth                            column of panels shows the bright field images. (C) Immunoprecipitation                            analysis of WT (lanes 1, 3 and 5) and PY01365-KO (lanes 2, 4 and 6)                            parasite soluble proteins released into culture supernatant using mAbs                            25.77 (lanes 1 and 2), 25.37 (lanes 3 and 4) and normal mouse serum                            (lanes 5 and 6). The Py235 proteins detected by this analysis are shown.                            (D) Erythrocyte binding assay: detection of a single protein band eluted                            from erythrocytes and immunoprecipitated using mAbs 25.77 (lanes 1 and                            2) and 25.37 (lanes 3 and 4) and derived from WT (lanes 1 and 3) and KO                            (lanes 2 and 4) parasites, respectively. The proposed identities                            (Py235EBP-1 and Py235EBP-2) are indicated. (E) Alexa Fluor                            488-conjugated 25.77 (green) and Alexa Fluor 594-conjugated 25.37 (red)                            were used in a dual labeling experiment of WT parasitized erythrocytes.                            The third of four panels of an identical field shows parasite nuclei                            labelled with DAPI, with the fourth panel showing the overlay of all                            three previous panels (MERGE). Py235 proteins are recognised by both                            mAbs in each schizont.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1001288-g003: Detection of Py235 protein expression.(A) Indirect fluorescent antibody assay using mAb25.77 on 1% formaldehyde-fixed thin blood smears of WT or PY01365-KO schizonts. For each parasite line the first panel shows mAb 25.77 reactivity, the second shows mAb 25.77 reactivity and parasite nuclei labelled with DAPI, and the third shows the bright field image. (B) a dual labeling experiment of WT parasitized erythrocytes. In the first column of panels the antibodies used were rabbit anti-EBL, mAb 45B1 (AMA1) and mAb 48F8 (RON4), each of which was detected by the appropriate fluorescence-labelled second antibody. In the second column of panels the same cells were stained with mAb 25.77, which was detected either with a fluorescence-labelled antibody (red) or was directly conjugated to a fluorescent dye (green). The third column of panels shows parasite nuclei labelled with DAPI, with the fourth column of panels showing the overlay of all three previous panels in the row (MERGE). The fifth column of panels shows the bright field images. (C) Immunoprecipitation analysis of WT (lanes 1, 3 and 5) and PY01365-KO (lanes 2, 4 and 6) parasite soluble proteins released into culture supernatant using mAbs 25.77 (lanes 1 and 2), 25.37 (lanes 3 and 4) and normal mouse serum (lanes 5 and 6). The Py235 proteins detected by this analysis are shown. (D) Erythrocyte binding assay: detection of a single protein band eluted from erythrocytes and immunoprecipitated using mAbs 25.77 (lanes 1 and 2) and 25.37 (lanes 3 and 4) and derived from WT (lanes 1 and 3) and KO (lanes 2 and 4) parasites, respectively. The proposed identities (Py235EBP-1 and Py235EBP-2) are indicated. (E) Alexa Fluor 488-conjugated 25.77 (green) and Alexa Fluor 594-conjugated 25.37 (red) were used in a dual labeling experiment of WT parasitized erythrocytes. The third of four panels of an identical field shows parasite nuclei labelled with DAPI, with the fourth panel showing the overlay of all three previous panels (MERGE). Py235 proteins are recognised by both mAbs in each schizont.
Mentions: The mAb 25.77 had previously been used to identify Py235EBP-1, the product of the PY01365 gene. By IFA, this mAb gives a punctuate pattern of fluorescence in the WT parasite line (Figure 3A). Surprisingly, a similar pattern was also observed for the PY01365-KO parasite line, even though Py235ebp-1 is no longer being expressed. The pattern of reactivity (Figure 3B) was similar but not identical to that of antibodies specific for the micronemal protein, Apical Membrane Antigen 1 (AMA1) [39], the erythrocyte binding ligand protein (EBL), which has a dense granule location in this parasite line [40], and rhoptry neck protein 4 (RON 4) [41]. Furthermore, when proteins released into in vitro culture supernatant from radiolabeled WT and PY01365-KO parasitized erythrocytes were immunoprecipitated using mAbs 25.77 and 25.37 (Figure 3C), or bound to erythrocytes, eluted and then immunoprecipitated (Figure 3D), both mAbs recognized proteins of approximately 235 kDa showing that Py235 proteins were expressed by both WT and PY01365-KO parasite lines. Clearly the Py235 proteins now expressed by the KO parasite line, although at least in part different to those being expressed by the WT parasite, share common epitopes bound by the antibodies.

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