Quantitative Proteomics Reveals the Dynamics of Protein Phosphorylation in Human Bronchial Epithelial Cells during Internalization, Phagosomal Escape, and Intracellular Replication of <i>Staphylococcus aureus</i>

Internalization of <i>Staphylococcus aureus</i> by nonprofessional phagocytic cells is a major suspected cause of persistent and difficult-to-treat infections, including pneumonia. In this study, we established an infection model with 16HBE14o- human bronchial epithelial cells and demonstrated internalization, escape from phagosomal clearance, and intracellular replication of <i>S. aureus</i> HG001 within the first 4 h postinfection. We used quantitative phosphoproteomics to identify characteristic signaling networks in the host at different infection stages. Although we found only minor changes in protein abundance, the infection was accompanied by highly dynamic alterations in phosphorylation events primarily in proteins that are associated with pathways of cytoskeleton dynamics, cell–cell and cell–matrix contacts, vesicle trafficking, autophagy, and GTPase signaling. Analyses of host protein kinases by kinase-substrate mapping, active regulatory site immunoblotting, and prediction algorithms highlighted known and novel host kinases with putative critical roles in <i>S. aureus</i> infection-accompanied signaling including FAK, PKA, PKC, and CDK. Targeted pharmacological inhibition of these kinases resulted in a significant reduction of intracellular <i>S. aureus</i> cells. The current study constitutes a valuable resource for better understanding the infection-relevant molecular pathomechanisms of airway cells and for developing novel host-centric anti-infective strategies for treating <i>S. aureus</i> infections

Initiation of Dopaminergic Differentiation of Nurr1^- Mesencephalic Precursor Cells Depends on Activation of Multiple Mitogen-Activated Protein Kinase Pathways

Interleukin-1 (IL-1) plays a pivotal role in terminal dopaminergic differentiation of midbrain-derived neural precursor cells already committed to the mesencephalic dopaminergic phenotype (named mdNPCs for mesencephalic dopaminergic neural precursor cells). Here we characterized the molecular events in long-term expanded rat nuclear receptor related-1^- (Nurr1^-) mdNPCs in response to IL-1β during their terminal dopaminergic specification. We showed that IL-1β induced a rapid induction of mRNA of dopaminergic key fate-determining transcription factors, such as Nurr1 and Pitx3, and a subsequent increase of tyrosine hydroxylase protein as an early marker for dopaminergic neurons in vitro. These effects of IL-1β were specific for mdNPCs and were not observed in striatal neural precursor cells (NPCs). Surprisingly, IL-1β did not activate the NF-κB pathway or the transcription factor activating protein 1 (AP-1), but inhibition of nuclear translocation of NF-κB by SN50 facilitated IL-1β-induced Nurr1 expression and dopaminergic differentiation of mdNPCs. Incubation of mdNPCs with IL-1β led to a rapid phosphorylation of ERK1/2 and p38 mitogen-activated protein (MAP) kinases within 1 to 3 hours, whereas Jun kinase was not phosphorylated in response to IL-1β. Consistently, inhibition of the ERK1/2 pathway or p38 MAP kinase blocked Nurr1 upregulation and further dopaminergic specification of mdNPCs, but not differentiation into MAP2ab^+ neurons. IL-1 receptor antagonist did not block early dopaminergic differentiation events, suggesting that the effects of IL-1β are not mediated through activation of IL-1 receptor type I. Our results indicate that induction of terminal dopaminergic specification of Nurr1^- mdNPCs by IL-1β depends on activation of the ERK1/2 and p38 MAP kinase pathway

Cell survival and general metabolic fitness of the human bronchial epithelial cells 16HBE14o- and S9 after treatment with 2,000 ng/ml rHla.

<p>A. Cell counts of 16HBE14o- and S9 cells 2 h, 6 h and 24 h post rHla-treatment (black bars) compared to mock treated cells (white bars). Data represent mean ± SEM (n = 3). B. General metabolic condition of rHla-treated 16HBE14o- (diamonds) and S9 (triangles) cells as indicated by a resazurin-based assay. Measurements after rHla-treatment are normalized to the initial reading before rHla addition. Data represent mean ± SEM (n = 3).</p

rHla-induced EGFR activation mediates resistance of S9 cells towards Hla.

<p>A. Cell counts of rHla-treated S9 cells co-treated with or without the EGFR-selective inhibitor tyrphostin AG1478. Graphs represent means ± SEM (n = 3). B. Quantification of EGFR surface expression in 16HBE14o- (red), A549 (blue) and S9 (orange) epithelial cells as analyzed by flow cytometry. The corresponding isotype control is indicated by a dashed line. MFI: mean fluorescence intensity.</p

DNA-microarray-based transcription profiles of 16HBE14o- and S9 cells under influence of rHla.

<p>A. Volcano plots of mRNA expression differences under control conditions and after treatment with rHla in 16HBE14o- and S9 as a function of statistical significance (ANOVA post hoc p≤0.05) and intensity ratio at the level of probe-sets. Probe sets indicating significant higher expression after rHla-treatment are indicated in ruby, probe sets indicating higher expression under control conditions are in cyan, and probe sets with no significant expression differences are in gray. Numbers indicate the number of gene-specific mRNAs increased (ruby) and decreased (cyan) after assigning probe-sets to genes. B. Expression level changes for selected immediate early response genes after rHla treatment of 16HBE14o- (open circles) and S9 (filled circles) cells (***p<0.001; **p<0.01; *p<0.05). C. Top five cellular functions affected after rHla-treatment in the category molecular functions for 16HBE14o- (black bars) and S9 (gray bars) as predicted by IPA Downstream Effects Analysis based on differentially expressed genes. D. Functional trends after rHla-treatment in all categories as predicted by IPA Downstream Effects Analysis based on direction of change of differentially expressed genes (16HBE14o- upper panel, S9 lower panel). The word cloud depicts the frequency of terms by font size and predicted increase in functional activity is shown in ruby and decrease in activity is shown in cyan.</p

Involvement of surface proteins in Hla mediated cytotoxicity.

<p><b>A.</b> 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.</p

Functional annotation analysis of proteins with altered phosphorylation in rHla-treated 16HBE14o- and S9 cells.

<p>A. KEGG pathways with statistically noticeable number of proteins with altered phosphorylation following rHla-treatment for 2 h. The dotted vertical line indicates the cut-off for significant enrichment (Benjamini-Hochberg corrected p-value ≤0.01). Numbers next to the horizontal bars provide the number of assigned proteins. B. Matrix for cross-comparison of recurrent proteins with altered phosphorylation in both cell types between KEGG pathways from panel A. Numbers greater than the half of the maximum possible are highlighted. C. Ranking of phosphoproteins with altered phosphorylation that appear in at least three enriched KEGG pathways. D. Network visualization of proteins from the enriched KEGG pathways that have been identified with altered phosphorylation in both cell lines. Grey lines indicate experimentally validated protein-protein-interactions and red arrows kinase-substrate relationship taken from STRING and PhosphoSitePlus, respectively. rHla-induced changes are indicated in ruby (increased p-sites); cyan (decreased) or purple (both) for kinases (diamond shape) and substrates (circles) for 16HBE14o- cells (left half of shape) and S9 cells (right).</p