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The RACK1 signaling scaffold protein selectively interacts with Yersinia pseudotuberculosis virulence function.

Thorslund SE, Edgren T, Pettersson J, Nordfelth R, Sellin ME, Ivanova E, Francis MS, Isaksson EL, Wolf-Watz H, Fällman M - PLoS ONE (2011)

Bottom Line: We show here that the virulence protein YopK has a role in orchestrating effector translocation necessary for productive antiphagocytosis.This resistance is not due to altered levels of translocated antiphagocytic effectors, and cells with reduced levels of RACK1 are still sensitive to the later occurring cytotoxic effect caused by the Yop effectors.Together, our data imply that the local event of Yersinia-mediated antiphagocytosis involves a step where YopK, by binding RACK1, ensures an immediate specific spatial delivery of antiphagocytic effectors leading to productive inhibition of phagocytosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Umeå University, Umeå, Sweden.

ABSTRACT
Many gram-negative bacteria use type III secretion systems to translocate effector proteins into host cells. These effectors interfere with cellular functions in a highly regulated manner resulting in effects that are beneficial for the bacteria. The pathogen Yersinia can resist phagocytosis by eukaryotic cells by translocating Yop effectors into the target cell cytoplasm. This is called antiphagocytosis, and constitutes an important virulence feature of this pathogen since it allows survival in immune cell rich lymphoid organs. We show here that the virulence protein YopK has a role in orchestrating effector translocation necessary for productive antiphagocytosis. We present data showing that YopK influences Yop effector translocation by modulating the ratio of the pore-forming proteins YopB and YopD in the target cell membrane. Further, we show that YopK that can interact with the translocators, is exposed inside target cells and binds to the eukaryotic signaling protein RACK1. This protein is engaged upon Y. pseudotuberculosis-mediated β1-integrin activation and localizes to phagocytic cups. Cells with downregulated RACK1 levels are protected from antiphagocytosis. This resistance is not due to altered levels of translocated antiphagocytic effectors, and cells with reduced levels of RACK1 are still sensitive to the later occurring cytotoxic effect caused by the Yop effectors. Further, a yopK mutant unable to bind RACK1 shows an avirulent phenotype during mouse infection, suggesting that RACK1 targeting by YopK is a requirement for virulence. Together, our data imply that the local event of Yersinia-mediated antiphagocytosis involves a step where YopK, by binding RACK1, ensures an immediate specific spatial delivery of antiphagocytic effectors leading to productive inhibition of phagocytosis.

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YopK interacts with RACK1 and this interaction is important for virulence.(A) Co-immunoprecipitation of YopK and RACK1. Beads with YopK-FLAG or control beads were incubated with HeLa cell lysates. Co-immunoprecipitation of RACK1 was detected by Western blot with anti-RACK1 antibodies. Detection of YopK by anti-YopK antisera was performed as a control, and HeLa cell lysate (12.5% input) was loaded for comparison. (B) Co-precipitation of purified recombinant RACK1 and YopK. Recombinant GST and GST-RACK1 on beads were incubated with culture supernatants of a Y. pseudotuberculosis multiple yop mutant strain expressing YopK-FLAG in trans [YPIII(pIB29MEKBD, pAH11)]. Co-precipitation of YopK was detected by Western blot with anti-YopK antibodies. Detection of RACK1 by anti-RACK1 antisera was performed as a control, and the bacterial culture supernatant (12.5% input) was loaded for comparison. (C) Pairwise alanine scan of YopK using the yeast two-hybrid assay as a read out. Interaction between the YopK variants and RACK1 was determined as growth of yeast on selective plates. In the diagram, +++ indicates growth corresponding to that seen in strains with non-mutated YopK and RACK1, and – stands for no growth. The mutated sequence is indicated in green and the amino acids identified as important for the interaction are in red. (D) Real-time monitoring of Y. pseudotuberculosis infection using the IVIS® technique. Mice were orally infected with 5.2×108, 4.9×108, and 6.3×108 bacteria for the wild-type, yopK, and yopKAA strains respectively, and bioluminescent signals from the infected animals were plotted. Data are presented as photons/sec on the indicated days post infection, and the illustrated values represent mean ± sd for the five mice in each group. Data were compared by student's paired t-test, differences being considered significant at P<0.1. (E) Bioluminescent image of one representative mouse for each bacterial strain on days 3 and 13 post infection. The color bar presents the total number of emitted photons, with high and low bioluminescent signals indicated by red and blue, respectively.
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pone-0016784-g002: YopK interacts with RACK1 and this interaction is important for virulence.(A) Co-immunoprecipitation of YopK and RACK1. Beads with YopK-FLAG or control beads were incubated with HeLa cell lysates. Co-immunoprecipitation of RACK1 was detected by Western blot with anti-RACK1 antibodies. Detection of YopK by anti-YopK antisera was performed as a control, and HeLa cell lysate (12.5% input) was loaded for comparison. (B) Co-precipitation of purified recombinant RACK1 and YopK. Recombinant GST and GST-RACK1 on beads were incubated with culture supernatants of a Y. pseudotuberculosis multiple yop mutant strain expressing YopK-FLAG in trans [YPIII(pIB29MEKBD, pAH11)]. Co-precipitation of YopK was detected by Western blot with anti-YopK antibodies. Detection of RACK1 by anti-RACK1 antisera was performed as a control, and the bacterial culture supernatant (12.5% input) was loaded for comparison. (C) Pairwise alanine scan of YopK using the yeast two-hybrid assay as a read out. Interaction between the YopK variants and RACK1 was determined as growth of yeast on selective plates. In the diagram, +++ indicates growth corresponding to that seen in strains with non-mutated YopK and RACK1, and – stands for no growth. The mutated sequence is indicated in green and the amino acids identified as important for the interaction are in red. (D) Real-time monitoring of Y. pseudotuberculosis infection using the IVIS® technique. Mice were orally infected with 5.2×108, 4.9×108, and 6.3×108 bacteria for the wild-type, yopK, and yopKAA strains respectively, and bioluminescent signals from the infected animals were plotted. Data are presented as photons/sec on the indicated days post infection, and the illustrated values represent mean ± sd for the five mice in each group. Data were compared by student's paired t-test, differences being considered significant at P<0.1. (E) Bioluminescent image of one representative mouse for each bacterial strain on days 3 and 13 post infection. The color bar presents the total number of emitted photons, with high and low bioluminescent signals indicated by red and blue, respectively.

Mentions: Since YopK is indeed translocated, we next sought to identify a eukaryotic target protein by using YopK as bait in a yeast two-hybrid screen with a HeLa cell cDNA library. One of the positive clones identified encoded for the C-terminal portion (aa 71–317) of the scaffolding protein RACK1. This was particularly interesting since RACK1 interacts with the cytoplasmic domain of β-integrins [34] that serve as the receptors to which Y. pseudotuberculosis bind to establish contact with eukaryotic cells [36]. To verify that the YopK-RACK1 interaction observed in yeast also occurred in HeLa cells (our established eukaryotic cell model system), we performed pull down assays using an expression construct (YopK–FLAG fusion) and HeLa cell lysates. In short, secreted YopK protein was immobilized on beads, which were subsequently incubated with HeLa cell lysate. Western blot analysis of YopK-associated proteins revealed binding between RACK1 and YopK–FLAG (Figure 2A). This confirmed that YopK can interact with RACK1 in the eukaryotic cytosol, suggesting that this interaction is biologically relevant. To further explore the observed RACK1-YopK interaction, we performed additional pull down experiments, now using recombinant GST-RACK1 and supernatant from a multi-Yop mutant strain (unable to express YopE, YopH, YopM, YopB, YopD) expressing YopK. In brief, recombinant GST-RACK1 was immobilized on glutathione sepharose beads which were subsequently incubated with the Yersinia supernatant containing YopK. Western blot analysis of RACK1-associated proteins revealed binding between YopK and RACK1 (Figure 2B), thus indicating that the binding of YopK to RACK1 is direct, and not mediated by a bridging eukaryotic molecule.


The RACK1 signaling scaffold protein selectively interacts with Yersinia pseudotuberculosis virulence function.

Thorslund SE, Edgren T, Pettersson J, Nordfelth R, Sellin ME, Ivanova E, Francis MS, Isaksson EL, Wolf-Watz H, Fällman M - PLoS ONE (2011)

YopK interacts with RACK1 and this interaction is important for virulence.(A) Co-immunoprecipitation of YopK and RACK1. Beads with YopK-FLAG or control beads were incubated with HeLa cell lysates. Co-immunoprecipitation of RACK1 was detected by Western blot with anti-RACK1 antibodies. Detection of YopK by anti-YopK antisera was performed as a control, and HeLa cell lysate (12.5% input) was loaded for comparison. (B) Co-precipitation of purified recombinant RACK1 and YopK. Recombinant GST and GST-RACK1 on beads were incubated with culture supernatants of a Y. pseudotuberculosis multiple yop mutant strain expressing YopK-FLAG in trans [YPIII(pIB29MEKBD, pAH11)]. Co-precipitation of YopK was detected by Western blot with anti-YopK antibodies. Detection of RACK1 by anti-RACK1 antisera was performed as a control, and the bacterial culture supernatant (12.5% input) was loaded for comparison. (C) Pairwise alanine scan of YopK using the yeast two-hybrid assay as a read out. Interaction between the YopK variants and RACK1 was determined as growth of yeast on selective plates. In the diagram, +++ indicates growth corresponding to that seen in strains with non-mutated YopK and RACK1, and – stands for no growth. The mutated sequence is indicated in green and the amino acids identified as important for the interaction are in red. (D) Real-time monitoring of Y. pseudotuberculosis infection using the IVIS® technique. Mice were orally infected with 5.2×108, 4.9×108, and 6.3×108 bacteria for the wild-type, yopK, and yopKAA strains respectively, and bioluminescent signals from the infected animals were plotted. Data are presented as photons/sec on the indicated days post infection, and the illustrated values represent mean ± sd for the five mice in each group. Data were compared by student's paired t-test, differences being considered significant at P<0.1. (E) Bioluminescent image of one representative mouse for each bacterial strain on days 3 and 13 post infection. The color bar presents the total number of emitted photons, with high and low bioluminescent signals indicated by red and blue, respectively.
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Related In: Results  -  Collection

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pone-0016784-g002: YopK interacts with RACK1 and this interaction is important for virulence.(A) Co-immunoprecipitation of YopK and RACK1. Beads with YopK-FLAG or control beads were incubated with HeLa cell lysates. Co-immunoprecipitation of RACK1 was detected by Western blot with anti-RACK1 antibodies. Detection of YopK by anti-YopK antisera was performed as a control, and HeLa cell lysate (12.5% input) was loaded for comparison. (B) Co-precipitation of purified recombinant RACK1 and YopK. Recombinant GST and GST-RACK1 on beads were incubated with culture supernatants of a Y. pseudotuberculosis multiple yop mutant strain expressing YopK-FLAG in trans [YPIII(pIB29MEKBD, pAH11)]. Co-precipitation of YopK was detected by Western blot with anti-YopK antibodies. Detection of RACK1 by anti-RACK1 antisera was performed as a control, and the bacterial culture supernatant (12.5% input) was loaded for comparison. (C) Pairwise alanine scan of YopK using the yeast two-hybrid assay as a read out. Interaction between the YopK variants and RACK1 was determined as growth of yeast on selective plates. In the diagram, +++ indicates growth corresponding to that seen in strains with non-mutated YopK and RACK1, and – stands for no growth. The mutated sequence is indicated in green and the amino acids identified as important for the interaction are in red. (D) Real-time monitoring of Y. pseudotuberculosis infection using the IVIS® technique. Mice were orally infected with 5.2×108, 4.9×108, and 6.3×108 bacteria for the wild-type, yopK, and yopKAA strains respectively, and bioluminescent signals from the infected animals were plotted. Data are presented as photons/sec on the indicated days post infection, and the illustrated values represent mean ± sd for the five mice in each group. Data were compared by student's paired t-test, differences being considered significant at P<0.1. (E) Bioluminescent image of one representative mouse for each bacterial strain on days 3 and 13 post infection. The color bar presents the total number of emitted photons, with high and low bioluminescent signals indicated by red and blue, respectively.
Mentions: Since YopK is indeed translocated, we next sought to identify a eukaryotic target protein by using YopK as bait in a yeast two-hybrid screen with a HeLa cell cDNA library. One of the positive clones identified encoded for the C-terminal portion (aa 71–317) of the scaffolding protein RACK1. This was particularly interesting since RACK1 interacts with the cytoplasmic domain of β-integrins [34] that serve as the receptors to which Y. pseudotuberculosis bind to establish contact with eukaryotic cells [36]. To verify that the YopK-RACK1 interaction observed in yeast also occurred in HeLa cells (our established eukaryotic cell model system), we performed pull down assays using an expression construct (YopK–FLAG fusion) and HeLa cell lysates. In short, secreted YopK protein was immobilized on beads, which were subsequently incubated with HeLa cell lysate. Western blot analysis of YopK-associated proteins revealed binding between RACK1 and YopK–FLAG (Figure 2A). This confirmed that YopK can interact with RACK1 in the eukaryotic cytosol, suggesting that this interaction is biologically relevant. To further explore the observed RACK1-YopK interaction, we performed additional pull down experiments, now using recombinant GST-RACK1 and supernatant from a multi-Yop mutant strain (unable to express YopE, YopH, YopM, YopB, YopD) expressing YopK. In brief, recombinant GST-RACK1 was immobilized on glutathione sepharose beads which were subsequently incubated with the Yersinia supernatant containing YopK. Western blot analysis of RACK1-associated proteins revealed binding between YopK and RACK1 (Figure 2B), thus indicating that the binding of YopK to RACK1 is direct, and not mediated by a bridging eukaryotic molecule.

Bottom Line: We show here that the virulence protein YopK has a role in orchestrating effector translocation necessary for productive antiphagocytosis.This resistance is not due to altered levels of translocated antiphagocytic effectors, and cells with reduced levels of RACK1 are still sensitive to the later occurring cytotoxic effect caused by the Yop effectors.Together, our data imply that the local event of Yersinia-mediated antiphagocytosis involves a step where YopK, by binding RACK1, ensures an immediate specific spatial delivery of antiphagocytic effectors leading to productive inhibition of phagocytosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Umeå University, Umeå, Sweden.

ABSTRACT
Many gram-negative bacteria use type III secretion systems to translocate effector proteins into host cells. These effectors interfere with cellular functions in a highly regulated manner resulting in effects that are beneficial for the bacteria. The pathogen Yersinia can resist phagocytosis by eukaryotic cells by translocating Yop effectors into the target cell cytoplasm. This is called antiphagocytosis, and constitutes an important virulence feature of this pathogen since it allows survival in immune cell rich lymphoid organs. We show here that the virulence protein YopK has a role in orchestrating effector translocation necessary for productive antiphagocytosis. We present data showing that YopK influences Yop effector translocation by modulating the ratio of the pore-forming proteins YopB and YopD in the target cell membrane. Further, we show that YopK that can interact with the translocators, is exposed inside target cells and binds to the eukaryotic signaling protein RACK1. This protein is engaged upon Y. pseudotuberculosis-mediated β1-integrin activation and localizes to phagocytic cups. Cells with downregulated RACK1 levels are protected from antiphagocytosis. This resistance is not due to altered levels of translocated antiphagocytic effectors, and cells with reduced levels of RACK1 are still sensitive to the later occurring cytotoxic effect caused by the Yop effectors. Further, a yopK mutant unable to bind RACK1 shows an avirulent phenotype during mouse infection, suggesting that RACK1 targeting by YopK is a requirement for virulence. Together, our data imply that the local event of Yersinia-mediated antiphagocytosis involves a step where YopK, by binding RACK1, ensures an immediate specific spatial delivery of antiphagocytic effectors leading to productive inhibition of phagocytosis.

Show MeSH
Related in: MedlinePlus