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A multi-omics approach identifies key hubs associated with cell type-specific responses of airway epithelial cells to staphylococcal alpha-toxin.

Richter E, Harms M, Ventz K, Gierok P, Chilukoti RK, Hildebrandt JP, Mostertz J, Hochgräfe F - PLoS ONE (2015)

Bottom Line: Phosphoproteomics revealed a substantial impact on phosphorylation-dependent signaling in both cell models and highlights alterations in signaling pathways associated with cell-cell and cell-matrix contacts as well as the actin cytoskeleton as key features of early rHla-induced effects.Along comparable changes in down-stream activity of major protein kinases significant differences between both models were found upon rHla-treatment including activation of the epidermal growth factor receptor EGFR and mitogen-activated protein kinases MAPK1/3 signaling in S9 and repression in 16HBE14o- cells.Strikingly, inhibition of the EGFR sensitized S9 cells to Hla indicating that the cellular capacity of activation of the EGFR is a major protective determinant against Hla-mediated cytotoxic effects.

View Article: PubMed Central - PubMed

Affiliation: Competence Center Functional Genomics, Junior Research Group Pathoproteomics, University of Greifswald, 17489, Greifswald, Germany.

ABSTRACT
Responsiveness of cells to alpha-toxin (Hla) from Staphylococcus aureus appears to occur in a cell-type dependent manner. Here, we compare two human bronchial epithelial cell lines, i.e. Hla-susceptible 16HBE14o- and Hla-resistant S9 cells, by a quantitative multi-omics strategy for a better understanding of Hla-induced cellular programs. Phosphoproteomics revealed a substantial impact on phosphorylation-dependent signaling in both cell models and highlights alterations in signaling pathways associated with cell-cell and cell-matrix contacts as well as the actin cytoskeleton as key features of early rHla-induced effects. Along comparable changes in down-stream activity of major protein kinases significant differences between both models were found upon rHla-treatment including activation of the epidermal growth factor receptor EGFR and mitogen-activated protein kinases MAPK1/3 signaling in S9 and repression in 16HBE14o- cells. System-wide transcript and protein expression profiling indicate induction of an immediate early response in either model. In addition, EGFR and MAPK1/3-mediated changes in gene expression suggest cellular recovery and survival in S9 cells but cell death in 16HBE14o- cells. Strikingly, inhibition of the EGFR sensitized S9 cells to Hla indicating that the cellular capacity of activation of the EGFR is a major protective determinant against Hla-mediated cytotoxic effects.

No MeSH data available.


Related in: MedlinePlus

Involvement of surface proteins in Hla mediated cytotoxicity.A. Western blot analysis of ADAM10 expression in 16HBE14o- and S9 cells. Signals corresponding to mature and processed ADAM10 are indicated. B. Quantification of surface E-cadherin (left panel) and ADAM10 (right panel) in 16HBE14o- (red), A549 (blue) and S9 (orange) cells as analyzed by flow cytometry. Corresponding isotype controls are indicated as dashed shapes. C. Flow cytometry analysis of surface expression of E-cadherin (left panels) and ADAM10 (right panels) in 16HBE14o- (upper panels) and S9 (lower panels) cells after 2 h under control conditions (blue) or following 2 h treatment with rHla (red). The corresponding isotype control is indicated by a dashed line. MFI: mean fluorescence intensity.
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pone.0122089.g006: Involvement of surface proteins in Hla mediated cytotoxicity.A. Western blot analysis of ADAM10 expression in 16HBE14o- and S9 cells. Signals corresponding to mature and processed ADAM10 are indicated. B. Quantification of surface E-cadherin (left panel) and ADAM10 (right panel) in 16HBE14o- (red), A549 (blue) and S9 (orange) cells as analyzed by flow cytometry. Corresponding isotype controls are indicated as dashed shapes. C. Flow cytometry analysis of surface expression of E-cadherin (left panels) and ADAM10 (right panels) in 16HBE14o- (upper panels) and S9 (lower panels) cells after 2 h under control conditions (blue) or following 2 h treatment with rHla (red). The corresponding isotype control is indicated by a dashed line. MFI: mean fluorescence intensity.

Mentions: Primarily based on experiments with human A549 alveolar cells, ADAM10 has recently been identified as a proteinaceous membrane-receptor for Hla which is involved in pore formation and mediation of toxic effects [9]. We therefore compared the level of ADAM10 in 16HBE14o-, S9 cells and A549 cells. Western blot analysis revealed a higher expression of ADAM10 in whole cell lysates of 16HBE14o- cells compared to S9 cells (Fig. 6A). Similar results were obtained for surface-exposed ADAM10 as analyzed by flow cytometry. S9 cells possess only 25% and A549 cells 80% of the surface-accessible ADAM10 content compared to 16HBE14o- cells (Fig. 6B, right panel). To characterize the role of ADAM10 in rHla-mediated responses in S9 and 16HBE14o- cells in more detail, we manipulated the level of ADAM10 by siRNA-mediated knockdown and studied the influence on cell viability and activation of specific kinases during rHla-treatment (S3 and S4 Figs.). Treatment with siRNAs resulted in efficient ADAM10 reduction in either cell line (S3A Fig.). Upon reduction of ADAM10 protein levels we observed a marked reversal of the deleterious effect of 6 h rHla-treatment on general metabolic condition of S9 cells (S3B Fig.).


A multi-omics approach identifies key hubs associated with cell type-specific responses of airway epithelial cells to staphylococcal alpha-toxin.

Richter E, Harms M, Ventz K, Gierok P, Chilukoti RK, Hildebrandt JP, Mostertz J, Hochgräfe F - PLoS ONE (2015)

Involvement of surface proteins in Hla mediated cytotoxicity.A. Western blot analysis of ADAM10 expression in 16HBE14o- and S9 cells. Signals corresponding to mature and processed ADAM10 are indicated. B. Quantification of surface E-cadherin (left panel) and ADAM10 (right panel) in 16HBE14o- (red), A549 (blue) and S9 (orange) cells as analyzed by flow cytometry. Corresponding isotype controls are indicated as dashed shapes. C. Flow cytometry analysis of surface expression of E-cadherin (left panels) and ADAM10 (right panels) in 16HBE14o- (upper panels) and S9 (lower panels) cells after 2 h under control conditions (blue) or following 2 h treatment with rHla (red). The corresponding isotype control is indicated by a dashed line. MFI: mean fluorescence intensity.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4376684&req=5

pone.0122089.g006: Involvement of surface proteins in Hla mediated cytotoxicity.A. Western blot analysis of ADAM10 expression in 16HBE14o- and S9 cells. Signals corresponding to mature and processed ADAM10 are indicated. B. Quantification of surface E-cadherin (left panel) and ADAM10 (right panel) in 16HBE14o- (red), A549 (blue) and S9 (orange) cells as analyzed by flow cytometry. Corresponding isotype controls are indicated as dashed shapes. C. Flow cytometry analysis of surface expression of E-cadherin (left panels) and ADAM10 (right panels) in 16HBE14o- (upper panels) and S9 (lower panels) cells after 2 h under control conditions (blue) or following 2 h treatment with rHla (red). The corresponding isotype control is indicated by a dashed line. MFI: mean fluorescence intensity.
Mentions: Primarily based on experiments with human A549 alveolar cells, ADAM10 has recently been identified as a proteinaceous membrane-receptor for Hla which is involved in pore formation and mediation of toxic effects [9]. We therefore compared the level of ADAM10 in 16HBE14o-, S9 cells and A549 cells. Western blot analysis revealed a higher expression of ADAM10 in whole cell lysates of 16HBE14o- cells compared to S9 cells (Fig. 6A). Similar results were obtained for surface-exposed ADAM10 as analyzed by flow cytometry. S9 cells possess only 25% and A549 cells 80% of the surface-accessible ADAM10 content compared to 16HBE14o- cells (Fig. 6B, right panel). To characterize the role of ADAM10 in rHla-mediated responses in S9 and 16HBE14o- cells in more detail, we manipulated the level of ADAM10 by siRNA-mediated knockdown and studied the influence on cell viability and activation of specific kinases during rHla-treatment (S3 and S4 Figs.). Treatment with siRNAs resulted in efficient ADAM10 reduction in either cell line (S3A Fig.). Upon reduction of ADAM10 protein levels we observed a marked reversal of the deleterious effect of 6 h rHla-treatment on general metabolic condition of S9 cells (S3B Fig.).

Bottom Line: Phosphoproteomics revealed a substantial impact on phosphorylation-dependent signaling in both cell models and highlights alterations in signaling pathways associated with cell-cell and cell-matrix contacts as well as the actin cytoskeleton as key features of early rHla-induced effects.Along comparable changes in down-stream activity of major protein kinases significant differences between both models were found upon rHla-treatment including activation of the epidermal growth factor receptor EGFR and mitogen-activated protein kinases MAPK1/3 signaling in S9 and repression in 16HBE14o- cells.Strikingly, inhibition of the EGFR sensitized S9 cells to Hla indicating that the cellular capacity of activation of the EGFR is a major protective determinant against Hla-mediated cytotoxic effects.

View Article: PubMed Central - PubMed

Affiliation: Competence Center Functional Genomics, Junior Research Group Pathoproteomics, University of Greifswald, 17489, Greifswald, Germany.

ABSTRACT
Responsiveness of cells to alpha-toxin (Hla) from Staphylococcus aureus appears to occur in a cell-type dependent manner. Here, we compare two human bronchial epithelial cell lines, i.e. Hla-susceptible 16HBE14o- and Hla-resistant S9 cells, by a quantitative multi-omics strategy for a better understanding of Hla-induced cellular programs. Phosphoproteomics revealed a substantial impact on phosphorylation-dependent signaling in both cell models and highlights alterations in signaling pathways associated with cell-cell and cell-matrix contacts as well as the actin cytoskeleton as key features of early rHla-induced effects. Along comparable changes in down-stream activity of major protein kinases significant differences between both models were found upon rHla-treatment including activation of the epidermal growth factor receptor EGFR and mitogen-activated protein kinases MAPK1/3 signaling in S9 and repression in 16HBE14o- cells. System-wide transcript and protein expression profiling indicate induction of an immediate early response in either model. In addition, EGFR and MAPK1/3-mediated changes in gene expression suggest cellular recovery and survival in S9 cells but cell death in 16HBE14o- cells. Strikingly, inhibition of the EGFR sensitized S9 cells to Hla indicating that the cellular capacity of activation of the EGFR is a major protective determinant against Hla-mediated cytotoxic effects.

No MeSH data available.


Related in: MedlinePlus