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Recruitment of EB1, a master regulator of microtubule dynamics, to the surface of the Theileria annulata schizont.

Woods KL, Theiler R, Mühlemann M, Segiser A, Huber S, Ansari HR, Pain A, Dobbelaere DA - PLoS Pathog. (2013)

Bottom Line: The latter does not egress to invade and transform other cells.Assuming the plus ends of growing MTs provide the first point of contact with the parasite, we focused on the complex protein machinery associated with these structures.Our findings provide important new insight into the mode of interaction between Theileria and the host cell cytoskeleton.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pathobiology, Department of Clinical Research and Veterinary Public Health, Vetsuisse Faculty, University of Bern, Bern, Switzerland.

ABSTRACT
The apicomplexan parasite Theileria annulata transforms infected host cells, inducing uncontrolled proliferation and clonal expansion of the parasitized cell population. Shortly after sporozoite entry into the target cell, the surrounding host cell membrane is dissolved and an array of host cell microtubules (MTs) surrounds the parasite, which develops into the transforming schizont. The latter does not egress to invade and transform other cells. Instead, it remains tethered to host cell MTs and, during mitosis and cytokinesis, engages the cell's astral and central spindle MTs to secure its distribution between the two daughter cells. The molecular mechanism by which the schizont recruits and stabilizes host cell MTs is not known. MT minus ends are mostly anchored in the MT organizing center, while the plus ends explore the cellular space, switching constantly between phases of growth and shrinkage (called dynamic instability). Assuming the plus ends of growing MTs provide the first point of contact with the parasite, we focused on the complex protein machinery associated with these structures. We now report how the schizont recruits end-binding protein 1 (EB1), a central component of the MT plus end protein interaction network and key regulator of host cell MT dynamics. Using a range of in vitro experiments, we demonstrate that T. annulata p104, a polymorphic antigen expressed on the schizont surface, functions as a genuine EB1-binding protein and can recruit EB1 in the absence of any other parasite proteins. Binding strictly depends on a consensus SxIP motif located in a highly disordered C-terminal region of p104. We further show that parasite interaction with host cell EB1 is cell cycle regulated. This is the first description of a pathogen-encoded protein to interact with EB1 via a bona-fide SxIP motif. Our findings provide important new insight into the mode of interaction between Theileria and the host cell cytoskeleton.

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The SKIP motif is required for p104 interaction with EB1.(A) COS-7 cell lysates were incubated with Halo-tagged p104-CT-V5, p104-NT-V5 and p104-CTSKNN-V5 linked to resin. The resin was washed with a high salt wash buffer and proteins eluted from the Halolink resin by cleavage with TEV protease. Eluates were subjected to immunoblot analysis using anti-V5 (to reveal p104-NT-V5, p104-CT-V5 and p104-CTSKNN-V5 in the eluates), anti-EB1 (to monitor EB1/p104 interaction) and anti-tubulin (as a control). (B). Lysate from TaC12 cells was subjected to pull-down using GST-EB1 or GST alone as a negative control. EB1 and its potential binding partner(s) was cleaved off GST-EB1 using precision protease and subjected to SDS-PAGE (10% gel) followed by immunoblot analysis using anti-p104 (1C12, upper panels) and anti-TaSP (middle panels). Control samples (right) were treated in the same manner. Ponceau staining (lower panels) shows cleaved-off EB1 (marked by asterisk). (C) Lysates of COS-7 cells expressing full-length EB1-GFP, EB1125–268-GFP and EB11–133-GFP were subjected to pull-down analysis using recombinant p104-CT-V5 and p104-CTSKNN-V5 as described above. EB1-GFP and EB1125–268-GFP interacted with p104-CT-V5. EB11–133-GFP failed to interact with p104-CT-V5, and p104-CTSKNN-V5 did not interact with any EB1 fragments. The lower panels represent immunoblots probed with anti-GFP to demonstrate the presence of EB1-GFP fragments in pull-down elutions. The top panel shows the same blot reprobed with anti-V5 to demonstrate the presence of bait proteins. EB1125–268-GFP is found as a doublet in transfected COS-7 cells, likely due to the presence of a second in-frame translational start codon. In all panels, 1% of the lysate (10 µl of 1 ml) was loaded as input; pull-down lanes represent 10% of eluted protein.
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ppat-1003346-g004: The SKIP motif is required for p104 interaction with EB1.(A) COS-7 cell lysates were incubated with Halo-tagged p104-CT-V5, p104-NT-V5 and p104-CTSKNN-V5 linked to resin. The resin was washed with a high salt wash buffer and proteins eluted from the Halolink resin by cleavage with TEV protease. Eluates were subjected to immunoblot analysis using anti-V5 (to reveal p104-NT-V5, p104-CT-V5 and p104-CTSKNN-V5 in the eluates), anti-EB1 (to monitor EB1/p104 interaction) and anti-tubulin (as a control). (B). Lysate from TaC12 cells was subjected to pull-down using GST-EB1 or GST alone as a negative control. EB1 and its potential binding partner(s) was cleaved off GST-EB1 using precision protease and subjected to SDS-PAGE (10% gel) followed by immunoblot analysis using anti-p104 (1C12, upper panels) and anti-TaSP (middle panels). Control samples (right) were treated in the same manner. Ponceau staining (lower panels) shows cleaved-off EB1 (marked by asterisk). (C) Lysates of COS-7 cells expressing full-length EB1-GFP, EB1125–268-GFP and EB11–133-GFP were subjected to pull-down analysis using recombinant p104-CT-V5 and p104-CTSKNN-V5 as described above. EB1-GFP and EB1125–268-GFP interacted with p104-CT-V5. EB11–133-GFP failed to interact with p104-CT-V5, and p104-CTSKNN-V5 did not interact with any EB1 fragments. The lower panels represent immunoblots probed with anti-GFP to demonstrate the presence of EB1-GFP fragments in pull-down elutions. The top panel shows the same blot reprobed with anti-V5 to demonstrate the presence of bait proteins. EB1125–268-GFP is found as a doublet in transfected COS-7 cells, likely due to the presence of a second in-frame translational start codon. In all panels, 1% of the lysate (10 µl of 1 ml) was loaded as input; pull-down lanes represent 10% of eluted protein.

Mentions: We next carried out pull-down experiments to confirm that T. annulata p104 is a functional EB1 binding partner. Recombinant Halo-tagged p104 fragments containing a C-terminal V5 tag were produced in E. coli and used for pull-down analysis of lysates prepared from COS-7 cells. Recombinant p104-CT migrates more slowly in SDS-PAGE than predicted (70 kDa rather than the predicted 46 kDa), while the N-terminal fragment has an apparent molecular weight as expected of 55 kDa (Figure 4A and 1C). Endogenous EB1 was detected after pull-down with recombinant p104-CT, but not with p104-NT. Mutation of the SKIP domain to SKNN (p104-CTSKNN) abolished the interaction between EB1 and p104-CT, supporting our observations made by IFM that p104 can interact with EB1, and that this interaction is mediated via the SKIP motif. Conversely, in pull-down assays performed on lysates of T. annulata-infected TaC12 cells, GST-tagged EB1 produced in E. coli bound parasite-derived p104 (Figure 4B), but not TaSP, another major schizont surface protein [7].


Recruitment of EB1, a master regulator of microtubule dynamics, to the surface of the Theileria annulata schizont.

Woods KL, Theiler R, Mühlemann M, Segiser A, Huber S, Ansari HR, Pain A, Dobbelaere DA - PLoS Pathog. (2013)

The SKIP motif is required for p104 interaction with EB1.(A) COS-7 cell lysates were incubated with Halo-tagged p104-CT-V5, p104-NT-V5 and p104-CTSKNN-V5 linked to resin. The resin was washed with a high salt wash buffer and proteins eluted from the Halolink resin by cleavage with TEV protease. Eluates were subjected to immunoblot analysis using anti-V5 (to reveal p104-NT-V5, p104-CT-V5 and p104-CTSKNN-V5 in the eluates), anti-EB1 (to monitor EB1/p104 interaction) and anti-tubulin (as a control). (B). Lysate from TaC12 cells was subjected to pull-down using GST-EB1 or GST alone as a negative control. EB1 and its potential binding partner(s) was cleaved off GST-EB1 using precision protease and subjected to SDS-PAGE (10% gel) followed by immunoblot analysis using anti-p104 (1C12, upper panels) and anti-TaSP (middle panels). Control samples (right) were treated in the same manner. Ponceau staining (lower panels) shows cleaved-off EB1 (marked by asterisk). (C) Lysates of COS-7 cells expressing full-length EB1-GFP, EB1125–268-GFP and EB11–133-GFP were subjected to pull-down analysis using recombinant p104-CT-V5 and p104-CTSKNN-V5 as described above. EB1-GFP and EB1125–268-GFP interacted with p104-CT-V5. EB11–133-GFP failed to interact with p104-CT-V5, and p104-CTSKNN-V5 did not interact with any EB1 fragments. The lower panels represent immunoblots probed with anti-GFP to demonstrate the presence of EB1-GFP fragments in pull-down elutions. The top panel shows the same blot reprobed with anti-V5 to demonstrate the presence of bait proteins. EB1125–268-GFP is found as a doublet in transfected COS-7 cells, likely due to the presence of a second in-frame translational start codon. In all panels, 1% of the lysate (10 µl of 1 ml) was loaded as input; pull-down lanes represent 10% of eluted protein.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3649978&req=5

ppat-1003346-g004: The SKIP motif is required for p104 interaction with EB1.(A) COS-7 cell lysates were incubated with Halo-tagged p104-CT-V5, p104-NT-V5 and p104-CTSKNN-V5 linked to resin. The resin was washed with a high salt wash buffer and proteins eluted from the Halolink resin by cleavage with TEV protease. Eluates were subjected to immunoblot analysis using anti-V5 (to reveal p104-NT-V5, p104-CT-V5 and p104-CTSKNN-V5 in the eluates), anti-EB1 (to monitor EB1/p104 interaction) and anti-tubulin (as a control). (B). Lysate from TaC12 cells was subjected to pull-down using GST-EB1 or GST alone as a negative control. EB1 and its potential binding partner(s) was cleaved off GST-EB1 using precision protease and subjected to SDS-PAGE (10% gel) followed by immunoblot analysis using anti-p104 (1C12, upper panels) and anti-TaSP (middle panels). Control samples (right) were treated in the same manner. Ponceau staining (lower panels) shows cleaved-off EB1 (marked by asterisk). (C) Lysates of COS-7 cells expressing full-length EB1-GFP, EB1125–268-GFP and EB11–133-GFP were subjected to pull-down analysis using recombinant p104-CT-V5 and p104-CTSKNN-V5 as described above. EB1-GFP and EB1125–268-GFP interacted with p104-CT-V5. EB11–133-GFP failed to interact with p104-CT-V5, and p104-CTSKNN-V5 did not interact with any EB1 fragments. The lower panels represent immunoblots probed with anti-GFP to demonstrate the presence of EB1-GFP fragments in pull-down elutions. The top panel shows the same blot reprobed with anti-V5 to demonstrate the presence of bait proteins. EB1125–268-GFP is found as a doublet in transfected COS-7 cells, likely due to the presence of a second in-frame translational start codon. In all panels, 1% of the lysate (10 µl of 1 ml) was loaded as input; pull-down lanes represent 10% of eluted protein.
Mentions: We next carried out pull-down experiments to confirm that T. annulata p104 is a functional EB1 binding partner. Recombinant Halo-tagged p104 fragments containing a C-terminal V5 tag were produced in E. coli and used for pull-down analysis of lysates prepared from COS-7 cells. Recombinant p104-CT migrates more slowly in SDS-PAGE than predicted (70 kDa rather than the predicted 46 kDa), while the N-terminal fragment has an apparent molecular weight as expected of 55 kDa (Figure 4A and 1C). Endogenous EB1 was detected after pull-down with recombinant p104-CT, but not with p104-NT. Mutation of the SKIP domain to SKNN (p104-CTSKNN) abolished the interaction between EB1 and p104-CT, supporting our observations made by IFM that p104 can interact with EB1, and that this interaction is mediated via the SKIP motif. Conversely, in pull-down assays performed on lysates of T. annulata-infected TaC12 cells, GST-tagged EB1 produced in E. coli bound parasite-derived p104 (Figure 4B), but not TaSP, another major schizont surface protein [7].

Bottom Line: The latter does not egress to invade and transform other cells.Assuming the plus ends of growing MTs provide the first point of contact with the parasite, we focused on the complex protein machinery associated with these structures.Our findings provide important new insight into the mode of interaction between Theileria and the host cell cytoskeleton.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pathobiology, Department of Clinical Research and Veterinary Public Health, Vetsuisse Faculty, University of Bern, Bern, Switzerland.

ABSTRACT
The apicomplexan parasite Theileria annulata transforms infected host cells, inducing uncontrolled proliferation and clonal expansion of the parasitized cell population. Shortly after sporozoite entry into the target cell, the surrounding host cell membrane is dissolved and an array of host cell microtubules (MTs) surrounds the parasite, which develops into the transforming schizont. The latter does not egress to invade and transform other cells. Instead, it remains tethered to host cell MTs and, during mitosis and cytokinesis, engages the cell's astral and central spindle MTs to secure its distribution between the two daughter cells. The molecular mechanism by which the schizont recruits and stabilizes host cell MTs is not known. MT minus ends are mostly anchored in the MT organizing center, while the plus ends explore the cellular space, switching constantly between phases of growth and shrinkage (called dynamic instability). Assuming the plus ends of growing MTs provide the first point of contact with the parasite, we focused on the complex protein machinery associated with these structures. We now report how the schizont recruits end-binding protein 1 (EB1), a central component of the MT plus end protein interaction network and key regulator of host cell MT dynamics. Using a range of in vitro experiments, we demonstrate that T. annulata p104, a polymorphic antigen expressed on the schizont surface, functions as a genuine EB1-binding protein and can recruit EB1 in the absence of any other parasite proteins. Binding strictly depends on a consensus SxIP motif located in a highly disordered C-terminal region of p104. We further show that parasite interaction with host cell EB1 is cell cycle regulated. This is the first description of a pathogen-encoded protein to interact with EB1 via a bona-fide SxIP motif. Our findings provide important new insight into the mode of interaction between Theileria and the host cell cytoskeleton.

Show MeSH
Related in: MedlinePlus