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A WD40-repeat protein unique to malaria parasites associates with adhesion protein complexes and is crucial for blood stage progeny.

von Bohl A, Kuehn A, Simon N, Ngongang VN, Spehr M, Baumeister S, Przyborski JM, Fischer R, Pradel G - Malar. J. (2015)

Bottom Line: In gametocytes, sexual precursor cells mediating parasite transmission to the mosquito vector, plasma membrane-associated proteins primarily belong to the PfCCp and 6-cys families with roles in fertilization.In mature schizonts, the protein localizes underneath the merozoite micronemes and interacts with PfAMA1, while in gametocytes PfWLP1 primarily accumulates underneath the plasma membrane and associates with PfCCp1 and Pfs230.Reverse genetics failed to disrupt the pfwlp1 gene, while haemagglutinin-tagging was feasible, suggesting a crucial function for PfWLP1 during blood stage replication.

View Article: PubMed Central - PubMed

Affiliation: Division of Cellular and Applied Infection Biology, Institute for Biology II, RWTH Aachen University, Worringerweg 1, 52074, Aachen, Germany. andreas.von.bohl@rwth-aachen.de.

ABSTRACT

Background: During development in human erythrocytes, Plasmodium falciparum parasites display a remarkable number of adhesive proteins on their plasma membrane. In the invasive merozoites, these include members of the PfMSP1 and PfAMA1/RON complexes, which facilitate contact between merozoites and red blood cells. In gametocytes, sexual precursor cells mediating parasite transmission to the mosquito vector, plasma membrane-associated proteins primarily belong to the PfCCp and 6-cys families with roles in fertilization. This study describes a newly identified WD40-repeat protein unique to Plasmodium species that associates with adhesion protein complexes of both merozoites and gametocytes.

Methods: The WD40-repeat protein-like protein PfWLP1 was identified via co-immunoprecipitation assays followed by mass spectrometry and characterized using biochemical and immunohistochemistry methods. Reverse genetics were employed for functional analysis.

Results: PfWLP1 is expressed both in schizonts and gametocytes. In mature schizonts, the protein localizes underneath the merozoite micronemes and interacts with PfAMA1, while in gametocytes PfWLP1 primarily accumulates underneath the plasma membrane and associates with PfCCp1 and Pfs230. Reverse genetics failed to disrupt the pfwlp1 gene, while haemagglutinin-tagging was feasible, suggesting a crucial function for PfWLP1 during blood stage replication.

Conclusions: This is the first report on a plasmodial WD40-repeat protein associating with cell adhesion proteins. Since WD40 domains are known to mediate protein-protein contact by serving as a rigid scaffold for protein interactions, the presented data suggest that PfWLP1 supports the stability of adhesion protein complexes of the plasmodial blood stages.

No MeSH data available.


Related in: MedlinePlus

Expression of PfWLP1-HA in asexual blood stages and gametocytes. a Detection of PfWLP1-HA in parasite lysates. Western blot analysis of lysates from ring stages (Ring), trophozoites (Troph), schizonts (Schiz), immature gametocytes (imGc), mature gametocytes (mGc) and activated gametocytes (aGc) of line PfWLP1-HA, using rabbit anti-HA antibody, detected PfWLP1-HA migrating at the expected size of ~108 kDa. Lysates of WT asexual blood stages (WT ABs) and gametocytes (WT Gc) as well as of non-infected erythrocytes (Ec) was used as negative controls. Equal loading of lanes was confirmed by blotting with mouse antisera against Pf39 (~39 kDa). bPfWLP1 expression in the PfWLP1-HA asexual blood stages. PfWLP1-HA was immunolabelled with anti-HA antisera (green); the asexual blood stages were visualized by Evans Blue counterstaining (red). The parasite nuclei were highlighted by Hoechst nuclear stain (blue). cPfWLP1 expression in the PfWLP1-HA non-activated and activated gametocytes. PfWLP1-HA was immunolabelled with anti-HA antisera (green). The sexual stages were visualized with antisera against Pfs230 (red); nuclei were stained with Hoechst (blue). Bar 5 µm. Data are representative of two to five independent experiments
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Fig4: Expression of PfWLP1-HA in asexual blood stages and gametocytes. a Detection of PfWLP1-HA in parasite lysates. Western blot analysis of lysates from ring stages (Ring), trophozoites (Troph), schizonts (Schiz), immature gametocytes (imGc), mature gametocytes (mGc) and activated gametocytes (aGc) of line PfWLP1-HA, using rabbit anti-HA antibody, detected PfWLP1-HA migrating at the expected size of ~108 kDa. Lysates of WT asexual blood stages (WT ABs) and gametocytes (WT Gc) as well as of non-infected erythrocytes (Ec) was used as negative controls. Equal loading of lanes was confirmed by blotting with mouse antisera against Pf39 (~39 kDa). bPfWLP1 expression in the PfWLP1-HA asexual blood stages. PfWLP1-HA was immunolabelled with anti-HA antisera (green); the asexual blood stages were visualized by Evans Blue counterstaining (red). The parasite nuclei were highlighted by Hoechst nuclear stain (blue). cPfWLP1 expression in the PfWLP1-HA non-activated and activated gametocytes. PfWLP1-HA was immunolabelled with anti-HA antisera (green). The sexual stages were visualized with antisera against Pfs230 (red); nuclei were stained with Hoechst (blue). Bar 5 µm. Data are representative of two to five independent experiments

Mentions: Parasite preparations for indirect immunofluorescence assays (IFAs) included mixed asexual blood stages and mixed gametocyte cultures as well as activated gametocyte-cultures at 15 min post-activation of WT strain NF54 or line PfWLP-1-HA. Cell monolayers were air-dried on glass slides and subsequently fixed with 4 % w/v paraformaldehyde/PBS (pH 7.4) for 10 min at RT. For membrane permeabilization and blocking of non-specific binding sites, preparations were permeabilized with 0.1 % v/v Triton X-100/125 mM glycine (Carl Roth)/PBS at RT for 30 min, followed by blocking with 3 % w/v BSA/PBS for 1 h. To liberate live gametocytes from the enveloping RBC and parasitophorous vacuole membranes, gametocytes were treated with 0.05 % w/v saponin/medium for 3 min at 37 °C prior fixation, as described in Simon et al. [18]. In these cases, no further membrane permeabilization step was employed. Preparations were then incubated with polyclonal mouse antisera specific for Pfs230, PfCCp1, PfCCp4, and PfWLP1 or with rabbit antisera directed against the HA-tag or PfGAP50 at 37 °C for 2 h. NMS was used for negative control. Binding of primary antibody was detected by incubating the preparations with polyclonal Alexa Fluor 488-conjugated goat anti-mouse antibodies (Invitrogen Molecular Probes) at RT for 1 h. The different parasite stages or cell structures were either detected by double-labelling with stage-specific antibodies, i.e., polyclonal rabbit antisera directed against PfMSP1, PfAMA1, PfEXP1, Pfs230, PfGAP45, or alpha-tubulin, followed by incubation with polyclonal Alexa Fluor 594- or Alexa Fluor 633-conjugated goat anti-rabbit antibodies (Invitrogen Molecular Probes), or the cells were counterstained with 0.05 % w/v Evans Blue (Sigma-Aldrich)/PBS for 1 min at RT. Antisera dilutions of 1:50 to 1:1000 were used. The parasite nuclei were highlighted by incubating the specimens with Hoechst nuclear stain 33342 (Invitrogen Molecular Probes) for 1 min at RT. Labelled specimens were examined by confocal laser scanning microscopy using a Zeiss LSM 510 microscope (Additional file 3) or a Leica TCS SP5 DM6000 CFS microscope, equipped with a 20× 1.0 NA water immersion objective (Additional file 4B). Dyes were excited using the 488 nm line of an argon laser and a 633 nm HeNe laser. Otherwise the labelled specimens were investigated using an Olympus BX41 fluorescence microscope in combination with a ProgRes Speed XT5 camera (Fig. 2a, b), or using a Leica DM5500 B fluorescence microscope in combination with a Leica DFC365 FX camera (Figs. 2a–c, 3a–c, 4b, c, 5a; Additional files 5, 6, 7A). Digital images were processed using Adobe Photoshop CS software. PfWLP1 expression was quantified by determining the percentage of immune-labelled gametocytes (n = 100) in three individual experiments. Labelling of non-permeabilized and permeabilized gametocytes (n = 50) was determined in triplicate.Fig. 2


A WD40-repeat protein unique to malaria parasites associates with adhesion protein complexes and is crucial for blood stage progeny.

von Bohl A, Kuehn A, Simon N, Ngongang VN, Spehr M, Baumeister S, Przyborski JM, Fischer R, Pradel G - Malar. J. (2015)

Expression of PfWLP1-HA in asexual blood stages and gametocytes. a Detection of PfWLP1-HA in parasite lysates. Western blot analysis of lysates from ring stages (Ring), trophozoites (Troph), schizonts (Schiz), immature gametocytes (imGc), mature gametocytes (mGc) and activated gametocytes (aGc) of line PfWLP1-HA, using rabbit anti-HA antibody, detected PfWLP1-HA migrating at the expected size of ~108 kDa. Lysates of WT asexual blood stages (WT ABs) and gametocytes (WT Gc) as well as of non-infected erythrocytes (Ec) was used as negative controls. Equal loading of lanes was confirmed by blotting with mouse antisera against Pf39 (~39 kDa). bPfWLP1 expression in the PfWLP1-HA asexual blood stages. PfWLP1-HA was immunolabelled with anti-HA antisera (green); the asexual blood stages were visualized by Evans Blue counterstaining (red). The parasite nuclei were highlighted by Hoechst nuclear stain (blue). cPfWLP1 expression in the PfWLP1-HA non-activated and activated gametocytes. PfWLP1-HA was immunolabelled with anti-HA antisera (green). The sexual stages were visualized with antisera against Pfs230 (red); nuclei were stained with Hoechst (blue). Bar 5 µm. Data are representative of two to five independent experiments
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Related In: Results  -  Collection

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Fig4: Expression of PfWLP1-HA in asexual blood stages and gametocytes. a Detection of PfWLP1-HA in parasite lysates. Western blot analysis of lysates from ring stages (Ring), trophozoites (Troph), schizonts (Schiz), immature gametocytes (imGc), mature gametocytes (mGc) and activated gametocytes (aGc) of line PfWLP1-HA, using rabbit anti-HA antibody, detected PfWLP1-HA migrating at the expected size of ~108 kDa. Lysates of WT asexual blood stages (WT ABs) and gametocytes (WT Gc) as well as of non-infected erythrocytes (Ec) was used as negative controls. Equal loading of lanes was confirmed by blotting with mouse antisera against Pf39 (~39 kDa). bPfWLP1 expression in the PfWLP1-HA asexual blood stages. PfWLP1-HA was immunolabelled with anti-HA antisera (green); the asexual blood stages were visualized by Evans Blue counterstaining (red). The parasite nuclei were highlighted by Hoechst nuclear stain (blue). cPfWLP1 expression in the PfWLP1-HA non-activated and activated gametocytes. PfWLP1-HA was immunolabelled with anti-HA antisera (green). The sexual stages were visualized with antisera against Pfs230 (red); nuclei were stained with Hoechst (blue). Bar 5 µm. Data are representative of two to five independent experiments
Mentions: Parasite preparations for indirect immunofluorescence assays (IFAs) included mixed asexual blood stages and mixed gametocyte cultures as well as activated gametocyte-cultures at 15 min post-activation of WT strain NF54 or line PfWLP-1-HA. Cell monolayers were air-dried on glass slides and subsequently fixed with 4 % w/v paraformaldehyde/PBS (pH 7.4) for 10 min at RT. For membrane permeabilization and blocking of non-specific binding sites, preparations were permeabilized with 0.1 % v/v Triton X-100/125 mM glycine (Carl Roth)/PBS at RT for 30 min, followed by blocking with 3 % w/v BSA/PBS for 1 h. To liberate live gametocytes from the enveloping RBC and parasitophorous vacuole membranes, gametocytes were treated with 0.05 % w/v saponin/medium for 3 min at 37 °C prior fixation, as described in Simon et al. [18]. In these cases, no further membrane permeabilization step was employed. Preparations were then incubated with polyclonal mouse antisera specific for Pfs230, PfCCp1, PfCCp4, and PfWLP1 or with rabbit antisera directed against the HA-tag or PfGAP50 at 37 °C for 2 h. NMS was used for negative control. Binding of primary antibody was detected by incubating the preparations with polyclonal Alexa Fluor 488-conjugated goat anti-mouse antibodies (Invitrogen Molecular Probes) at RT for 1 h. The different parasite stages or cell structures were either detected by double-labelling with stage-specific antibodies, i.e., polyclonal rabbit antisera directed against PfMSP1, PfAMA1, PfEXP1, Pfs230, PfGAP45, or alpha-tubulin, followed by incubation with polyclonal Alexa Fluor 594- or Alexa Fluor 633-conjugated goat anti-rabbit antibodies (Invitrogen Molecular Probes), or the cells were counterstained with 0.05 % w/v Evans Blue (Sigma-Aldrich)/PBS for 1 min at RT. Antisera dilutions of 1:50 to 1:1000 were used. The parasite nuclei were highlighted by incubating the specimens with Hoechst nuclear stain 33342 (Invitrogen Molecular Probes) for 1 min at RT. Labelled specimens were examined by confocal laser scanning microscopy using a Zeiss LSM 510 microscope (Additional file 3) or a Leica TCS SP5 DM6000 CFS microscope, equipped with a 20× 1.0 NA water immersion objective (Additional file 4B). Dyes were excited using the 488 nm line of an argon laser and a 633 nm HeNe laser. Otherwise the labelled specimens were investigated using an Olympus BX41 fluorescence microscope in combination with a ProgRes Speed XT5 camera (Fig. 2a, b), or using a Leica DM5500 B fluorescence microscope in combination with a Leica DFC365 FX camera (Figs. 2a–c, 3a–c, 4b, c, 5a; Additional files 5, 6, 7A). Digital images were processed using Adobe Photoshop CS software. PfWLP1 expression was quantified by determining the percentage of immune-labelled gametocytes (n = 100) in three individual experiments. Labelling of non-permeabilized and permeabilized gametocytes (n = 50) was determined in triplicate.Fig. 2

Bottom Line: In gametocytes, sexual precursor cells mediating parasite transmission to the mosquito vector, plasma membrane-associated proteins primarily belong to the PfCCp and 6-cys families with roles in fertilization.In mature schizonts, the protein localizes underneath the merozoite micronemes and interacts with PfAMA1, while in gametocytes PfWLP1 primarily accumulates underneath the plasma membrane and associates with PfCCp1 and Pfs230.Reverse genetics failed to disrupt the pfwlp1 gene, while haemagglutinin-tagging was feasible, suggesting a crucial function for PfWLP1 during blood stage replication.

View Article: PubMed Central - PubMed

Affiliation: Division of Cellular and Applied Infection Biology, Institute for Biology II, RWTH Aachen University, Worringerweg 1, 52074, Aachen, Germany. andreas.von.bohl@rwth-aachen.de.

ABSTRACT

Background: During development in human erythrocytes, Plasmodium falciparum parasites display a remarkable number of adhesive proteins on their plasma membrane. In the invasive merozoites, these include members of the PfMSP1 and PfAMA1/RON complexes, which facilitate contact between merozoites and red blood cells. In gametocytes, sexual precursor cells mediating parasite transmission to the mosquito vector, plasma membrane-associated proteins primarily belong to the PfCCp and 6-cys families with roles in fertilization. This study describes a newly identified WD40-repeat protein unique to Plasmodium species that associates with adhesion protein complexes of both merozoites and gametocytes.

Methods: The WD40-repeat protein-like protein PfWLP1 was identified via co-immunoprecipitation assays followed by mass spectrometry and characterized using biochemical and immunohistochemistry methods. Reverse genetics were employed for functional analysis.

Results: PfWLP1 is expressed both in schizonts and gametocytes. In mature schizonts, the protein localizes underneath the merozoite micronemes and interacts with PfAMA1, while in gametocytes PfWLP1 primarily accumulates underneath the plasma membrane and associates with PfCCp1 and Pfs230. Reverse genetics failed to disrupt the pfwlp1 gene, while haemagglutinin-tagging was feasible, suggesting a crucial function for PfWLP1 during blood stage replication.

Conclusions: This is the first report on a plasmodial WD40-repeat protein associating with cell adhesion proteins. Since WD40 domains are known to mediate protein-protein contact by serving as a rigid scaffold for protein interactions, the presented data suggest that PfWLP1 supports the stability of adhesion protein complexes of the plasmodial blood stages.

No MeSH data available.


Related in: MedlinePlus