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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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RACK1 is not required for Yop translocation.(A) Translocation of ExoS resulting in modification of Ras in control and RACK1 RNAi HeLa cells. The cells were infected with a Y. pseudotuberculosis strain expressing ExoS [YPIII(pIB526), pTS103]. Modification of Ras was visualized by Western blot with anti-Ras antibodies. Detection of tubulin was used as loading control. (B) Translocation of YopE into control and RACK1 RNAi HeLa cells. Cells were infected with the indicated strains and then treated with proteinase K. Thereafter, the cells were lysed with digitonin or left untreated. The resulting supernatants were subjected to Western blot analysis using anti-YopE antisera. The translocation deficient yopD mutant was included as negative control and detection of Erk was used as loading control.
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pone-0016784-g004: RACK1 is not required for Yop translocation.(A) Translocation of ExoS resulting in modification of Ras in control and RACK1 RNAi HeLa cells. The cells were infected with a Y. pseudotuberculosis strain expressing ExoS [YPIII(pIB526), pTS103]. Modification of Ras was visualized by Western blot with anti-Ras antibodies. Detection of tubulin was used as loading control. (B) Translocation of YopE into control and RACK1 RNAi HeLa cells. Cells were infected with the indicated strains and then treated with proteinase K. Thereafter, the cells were lysed with digitonin or left untreated. The resulting supernatants were subjected to Western blot analysis using anti-YopE antisera. The translocation deficient yopD mutant was included as negative control and detection of Erk was used as loading control.

Mentions: To determine whether the level of RACK1 influences the amount of Yop effectors translocated, we employed two different methods to analyze T3SS-mediated translocation of effectors into eukaryotic cells: a translocation assay using the Pseudomonas aeruginosa effector ExoS [48] and the protease protection assay [49]. In the former technique, the ExoS-dependent ADP ribosylation of cytoplasmic proteins (e.g., Ras) allows sensitive detection of ExoS translocation into eukaryotic cells. We used the ExoS-producing Y. pseudotuberculosis strain YPIII(pIB526pTS103) (Table 1) to infect control and RACK1 RNAi HeLa cells. Lysates were analyzed by Western blot to detect modification of Ras. Both cell lines displayed comparable levels of Ras modification, indicating that similar amounts of ExoS were translocated into the different cells (Figure 4A). This agreed with the results of the protease protection assay where similar levels of the antiphagocytic effector YopE were translocated into the two cell types (Figure 4B). Collectively, this implies that RACK1 does not affect the level of Yop effector translocation per se. Consequently, the reduced antiphagocytic capacity of Y. pseudotuberculosis in contact with RACK1 RNAi cells cannot be explained by altered levels of antiphagocytic effectors inside these cells.


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)

RACK1 is not required for Yop translocation.(A) Translocation of ExoS resulting in modification of Ras in control and RACK1 RNAi HeLa cells. The cells were infected with a Y. pseudotuberculosis strain expressing ExoS [YPIII(pIB526), pTS103]. Modification of Ras was visualized by Western blot with anti-Ras antibodies. Detection of tubulin was used as loading control. (B) Translocation of YopE into control and RACK1 RNAi HeLa cells. Cells were infected with the indicated strains and then treated with proteinase K. Thereafter, the cells were lysed with digitonin or left untreated. The resulting supernatants were subjected to Western blot analysis using anti-YopE antisera. The translocation deficient yopD mutant was included as negative control and detection of Erk was used as loading control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3037380&req=5

pone-0016784-g004: RACK1 is not required for Yop translocation.(A) Translocation of ExoS resulting in modification of Ras in control and RACK1 RNAi HeLa cells. The cells were infected with a Y. pseudotuberculosis strain expressing ExoS [YPIII(pIB526), pTS103]. Modification of Ras was visualized by Western blot with anti-Ras antibodies. Detection of tubulin was used as loading control. (B) Translocation of YopE into control and RACK1 RNAi HeLa cells. Cells were infected with the indicated strains and then treated with proteinase K. Thereafter, the cells were lysed with digitonin or left untreated. The resulting supernatants were subjected to Western blot analysis using anti-YopE antisera. The translocation deficient yopD mutant was included as negative control and detection of Erk was used as loading control.
Mentions: To determine whether the level of RACK1 influences the amount of Yop effectors translocated, we employed two different methods to analyze T3SS-mediated translocation of effectors into eukaryotic cells: a translocation assay using the Pseudomonas aeruginosa effector ExoS [48] and the protease protection assay [49]. In the former technique, the ExoS-dependent ADP ribosylation of cytoplasmic proteins (e.g., Ras) allows sensitive detection of ExoS translocation into eukaryotic cells. We used the ExoS-producing Y. pseudotuberculosis strain YPIII(pIB526pTS103) (Table 1) to infect control and RACK1 RNAi HeLa cells. Lysates were analyzed by Western blot to detect modification of Ras. Both cell lines displayed comparable levels of Ras modification, indicating that similar amounts of ExoS were translocated into the different cells (Figure 4A). This agreed with the results of the protease protection assay where similar levels of the antiphagocytic effector YopE were translocated into the two cell types (Figure 4B). Collectively, this implies that RACK1 does not affect the level of Yop effector translocation per se. Consequently, the reduced antiphagocytic capacity of Y. pseudotuberculosis in contact with RACK1 RNAi cells cannot be explained by altered levels of antiphagocytic effectors inside these cells.

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