Pneumococcal hydrogen peroxide regulates host cell kinase activity

Introduction Protein kinases are indispensable reversible molecular switches that adapt and control protein functions during cellular processes requiring rapid responses to internal and external events. Bacterial infections can affect kinase-mediated phosphorylation events, with consequences for both innate and adaptive immunity, through regulation of antigen presentation, pathogen recognition, cell invasiveness and phagocytosis. Streptococcus pneumoniae (Spn), a human respiratory tract pathogen and a major cause of community-acquired pneumoniae, affects phosphorylation-based signalling of several kinases, but the pneumococcal mediator(s) involved in this process remain elusive. In this study, we investigated the influence of pneumococcal H2O2 on the protein kinase activity of the human lung epithelial H441 cell line, a generally accepted model of alveolar epithelial cells. Methods We performed kinome analysis using PamGene microarray chips and protein analysis in Western blotting in H441 lung cells infected with Spn wild type (SpnWT) or with SpnΔlctOΔspxB -a deletion mutant strongly attenuated in H2O2 production- to assess the impact of pneumococcal hydrogen peroxide (H2O2) on global protein kinase activity profiles. Results Our kinome analysis provides direct evidence that kinase activity profiles in infected H441 cells significantly vary according to the levels of pneumococcal H2O2. A large number of kinases in H441 cells infected with SpnWT are significantly downregulated, whereas this no longer occurs in cells infected with the mutant SpnΔlctOΔspxB strain, which lacks H2O2. In particular, we describe for the first time H2O2-mediated downregulation of Protein kinase B (Akt1) and activation of lymphocyte-specific tyrosine protein kinase (Lck) via H2O2-mediated phosphorylation.


Introduction
The human genome encodes more than 535 protein kinases, which catalyse the phosphorylation of about 30% of all cellular proteins and function as reversible molecular switches, in order to regulate intracellular and extracellular signalling (1).These enzymes affect the functioning of the immune system and regulate transcription, metabolism, homeostasis, translation, cell cycle progression, differentiation, cytoskeletal rearrangement, apoptosis and intracellular communication (2,3).Based on the specificity for their downstream targets, protein kinases can be separated into different groups: 1) protein tyrosine kinases (PTKs), 2) serine/ threonine kinases (STKs), 3) dual specificity kinases, which phosphorylate both serine/threonine and tyrosine residues and 4) histidyl kinases, which transfer the phosphate group onto an aspartate residue (4).The majority of the human protein kinases -428-belong to the STKs, whereas 90 are PTKs (5).
Infections of mammalian cells with pathogenic bacteria can affect phosphorylation-based signalling (6).According to the WHO, infections with S. pneumoniae (Spn) are responsible for 1.6 million deaths worldwide each year, including around 700,000 deaths in children below 5 years of age (7).Spn is an encapsulated Grampositive pathogen that was found to reduce the activity of certain protein kinases such as Adenosine Monophosphate-activated Protein Kinase (AMPK-a) and Cyclin-dependent kinase (CDK) in infected mice (8).Spn-infection of THP-1 cells can reduce the activity of protein kinase B (a.k.a.Akt kinase), involved in phagocytosis (9).As such, the pathogen can modify the host's kinome to promote its survival.Spn possesses several virulence factors, including capsule and adhesion proteins (10), as well as the cholesterol-dependent cytolysin pneumolysin (Ply) and hydrogen peroxide (H 2 O 2 ).Endogenously generated H 2 O 2 is a by-product of the enzymes pyruvate oxidase (SpxB) and lactate oxidase (LctO) in Spn (11).Whereas SpxB converts pyruvate, inorganic phosphate and oxygen into acetyl phosphate, carbon dioxide (CO 2 ) and H 2 O 2 (12,13), LctO catalyses the formation of pyruvate and H 2 O 2 from L-lactate and oxygen (11,14).Whereas physiological H 2 O 2 levels in human plasma are in the range of 1 to 8 µM (15), Spn-derived H 2 O 2 can surpass concentrations of 1 mM in culture supernatants (16).The endogenous production of high levels of H 2 O 2 by Spn provides protection against other commensals like Staphylococcus aureus, Haemophilus influenzae, Moraxella catarrhalis and Neisseria meningitidis during colonisation of the nasopharynx (17).Moreover, pneumococcal-derived H 2 O 2 is considered as a virulence factor and induces DNA double-strand breaks and apoptosis in human alveolar epithelial cells and impairment of the alveolar-capillary barriers, both of which promote the spread of Spn in the host (18)(19)(20).Interestingly, deletion of SpxB decreases virulence of Spn in vivo (21).
To date, it is not known what pneumococcal virulence factor, if any, is the main mediator of Spn-induced kinase regulation in mammalian cells.In this study, we investigated the influence of pneumococcal H 2 O 2 on the protein kinase activity of the H441 human lung epithelial cell line, a generally accepted model of alveolar epithelial cells (22).The effects of H 2 O 2 released by Spn during infection on the (de-) activation status of the global kinome were investigated using a peptide-based kinase activity assay (PamStation platform), which enables identification of differentially activated kinases based on the phosphorylation of immobilized substrate peptides in infected lung cells (23).In our approach, we compared the kinomes of H441 cells infected with either Spn wild type (SpnWT) or SpnDlctODspxB mutant, the latter of which lacks LctO and SpxB and is therefore strongly attenuated in H 2 O 2 production (generates only 3% of SpnWT H 2 O 2 levels) (11).As we and others have previously shown that the SpnDlctODspxB mutant releases significantly lower amounts of the pore-forming toxin Ply, as compared to SpnWT (24,25), we also analyzed the kinome of cells infected with the Ply deletion mutant SpnDply, in order to exclude specific effects caused by reduced release of Ply in infected cells.
Our data demonstrate that kinase activity profiles in infected H441 cells significantly vary according to the levels of pneumococcal H 2 O 2 .Infection with SpnWT prominently downregulated several protein kinases of both the STK and PTK groups.These kinases, including protein kinase B (Akt), were significantly downregulated in SpnWT-infected cells, but not in SpnDlctODspxB-infected cells.
By contrast, Activity of Lymphocyte-specific tyrosine protein kinase (Lck) was significantly higher in cells infected with the SpnDlctODspxB mutant than in cells infected with the WT strain, in view of an H 2 O 2 -dependent increase in inhibitory phosphorylation of Lck at residue tyrosine 505.As a proof of principle, we demonstrate that the phosphorylation of both Akt (leading to its activation) and Lck (resulting in its inhibition) is affected by the addition of exogenous H 2 O 2 .

Infection assays and cell lysis
H441 cells were seeded in 10 cm cell culture plates at a density of 1.8×10 6 cells/plat and incubated at 37°C with 5% CO 2 for 48 h until 90% confluency.Spn cultures were grown on the day of infection in THB-Y medium for 3 h incubated at 37°C with 5% CO 2 to an OD 600nm of 0.40 and washed 2x with PBS and centrifuged for 4 min at 6,000 g.Spn pellets were resuspended in RPMI medium supplemented with 10% FCS.SpnWT or SpnDply or SpnDlctODspxB suspensions were adjusted to a multiplicity of infection (MOI) 45 in infection medium and added to H441 cells for 5 h.Then the cell culture supernatants were removed, and the remaining H441 cells were washed with 5 mL ice cold PBS.Finally, the cells were scraped down in 250 µl M-PER mammalian extract buffer (Thermo Scientific) containing protease inhibitor cocktail (Thermo Scientific) and Halt phosphatase inhibitor (Thermo Fisher Scientific).Next, samples were homogenised with a syringe and incubated for 1 h at 4°C followed by centrifugation for 15 min at 16,000 g at 4°C.The supernatants were aliquoted and frozen in liquid nitrogen and stored at -80°C.Individual aliquots were used for protein concentration determination (BCA assay), Western blot (Jess Simple Western) or peptide-based kinase activity (PamStation) measurement.

PamStation kinome analysis
H441 cells infected with SpnWT or SpnDply or SpnDlctODspxB cultures were lysed as described in 2.3 and subjected to kinome profiling as described previously (26)(27)(28).For each condition, five experimental replicates were performed and non-infected cells were used as control group.For PTK analyses, 10 µg of the protein lysate (2 µg for STK, respectively) were dissolved in reaction buffer (proprietary information by the manufacturer) and added on the PamChip for analysis of tyrosine kinases (or serine/threonine, respectively).Peptide substrate phosphorylation was detected with secondary FITC-conjugated antibodies and monitored by a CCD camera (Evolve (Software Version), PamGene).All recorded images were further processed by bioinformatic software to derive a single numerical value reflecting the intensity of phosphorylation for each peptide and each sample (BioNavigator6, Version 6.3.67.0,PamGene).Individual values were corrected by logarithmical transformation and mathematical normalisation for the different replicates (x center = xmean[x]) before subsequent processing for database-assisted upstream kinase prediction.Kinases responsible for significant changes in peptide phosphorylation between two experimental conditions (e.g.non-infected control versus SpnWT), are described by two important parameters: The predicted differential kinase activity depicts the overall change of the peptide set that represents the group of substrates for the given kinase.In addition to this, the "mean specificity score" is stated, which is expressed as the negative log 10 p-value, where p < 0.05 refers to the statistical significance for the changes of the phosphorylation for the substrate peptide sets between the two experimental conditions.Therefore, kinases with a prediction of a highly differential kinase activity and a "mean specificity score" higher than 1.3 were considered as promising candidates for subsequent investigations.The step-wise comparisons of two distinct experimental groups facilitated the identification of Spn mutant specific peptide phosphorylation and the activation of responsible upstream kinases by Venn diagram analyses (BioVenn -a web application for the comparison and visualization of biological lists using area-proportional Venn diagrams (29).

Automated western immunoblotting with Jess ™ simple western
Jess Simple Western system (ProteinSimple, San Jose CA, USA) is an automated capillary-based size separation immunoassay.This technology was used to detect protein levels of kinases in lysates from H441 cells according to the manufacturer's instructions.For detection of Akt and Lck levels anti-p-Akt antibody (Cell Signaling 9271), anti-Akt antibody (Cell Signaling 9272), anti-Lck antibody (Cell Signaling) and anti-p-Lck antibody (Cell Signaling 2751) were used.Briefly, 2.4 µl protein extract was mixed with 0.6 µl fluorescent 5x master mix supplied 200 mM dithiothreitol (DTT) dissolved in sample buffer (Protein simple).All samples were denatured at 95°C for 5 min.Afterwards, the samples were spinned down and together with the size ladder (12-230 kDa) placed on ice until loading onto the plate.Once the plate is centrifuged (1,000 g, 5 min, room temperature) and placed into the instrument together with the cartridge, a fully automated process with standard instrument settings ensures separation of the samples followed by immobilization via UV light and chemiluminescent detection using primary and secondary horse-radish peroxidase antibodies.Images, i.e. light emission, are recorded by a CCD camera and analysis is performed by the Compass Simple Western software (version 4.1.0,Protein Simple).The results, i.e. protein expression reflected by band intensities, are displayed in traditional lane view as well as electropherograms, which allow for quantification by defining the respective area under the curve.

Statistical analyses
Statistical analyses were carried out using one-way ANOVA with GraphPad Prism software 5 (GraphPad Software, Inc., La Jolla, CA, USA).p < 0.05 was considered to indicate a statistically significant difference.

Pneumococcal H 2 O 2 downregulates kinase activity in Spn-infected H441 cells
In order to investigate whether Spn-derived H 2 O 2 affects protein kinase activity in lung cells, we infected H441 lung cells with SpnWT or with a deletion mutant strongly impaired in H 2 O 2 production, due to the lack of pyruvate oxidase (SpxB) and lactate oxidase (LctO): SpnDlctODspxB (11).Additionally, a pneumolysin (Ply) deletion mutant (SpnDply) was tested, to exclude effects caused by the decreased release of Ply observed in the SpnDlctODspxB mutant (24,25).Non-infected vehicle-treated or Spn-infected H441 cells were lysed for protein isolation five hours (h) post treatment/ infection, respectively.Kinome profiling of lysates from infected or non-infected cells was performed on microarray chips using the PamStation platform to analyze kinase activities of protein tyrosine kinases (PTKs) and serine/threonine kinases (STKs) (Figures 1 A, B).PamGene microarray chips contain designed substrate peptides, representing kinase phosphorylation sites for PTKs (196 different peptides in total) or STKs (144 different peptides in total) (30).
The differential pattern of kinase-mediated peptidephosphorylation in protein extracts of H441 cells infected with different Spn strains (SpnWT, SpnDlctODspxB or SpnDply) vs. noninfected cells from five biological experiments and their respective mathematical mean values are visualized as heat maps in Based on the determined phospho-peptide signatures comparing two experimental groups, a bioinformatics-based analysis of upstream kinases was performed, allowing for a prediction of kinase activity that is differentially regulated between the depicted conditions.Phylogenetic mapping of topranked dysregulated kinases (specificity score > 1.3) in H441 cells infected with SpnDlctODspxB vs. SpnWT revealed a kinase cluster that was not downregulated in SpnDlctODspxB:   Protein kinase B (Akt1) belongs to significantly upregulated kinases in SpnDlctODspxB (Figure 3A).Akt1 is involved in cell cycle progression, cell growth, cell survival and apoptosis and is part of the highly conserved phosphatidylinositol-3 kinase (PI3K)/ Akt/mammalian target of rapamycin (mTOR) signaling pathway (26,27).The phosphorylation of immobilized peptides that serve as substrates for Akt1 in the PamStation assays are depicted in Figure 4.The relative intensities of peptide phosphorylation that serve as Akt1 substrates demonstrates significant downregulation of profiles in cells infected with SpnWT and SpnDply (H 2 O 2 producers) (Figure 4).In contrast, the peptide phosphorylation profiles of cells infected with SpnDlctODspxB and non-infected cells show a significantly higher activity of Akt1, as compared to cells infected with SpnWT (Figure 4).
In order to confirm the effect of endogenously generated pneumococcal H 2 O 2 on Akt kinase activity, we performed Western blot analysis using specific antibodies against total and phospho-Akt to measure their ratio in H441 lung cells infected with SpnWT, SpnDply and SpnDlctODspxB.The results show that infection of H441 cells with SpnWT and SpnDply decreases both Akt total protein expression and phosphorylation, assessed as the p-Akt/Akt ratio (Figures 5A, B).Akt levels and p-Akt/Akt ratio in SpnDlctODspxB-infected cells as well as in non-infected cells are significantly higher than in SpnWT-infected cells (Figures 5A, B).The reduced p-Akt/Akt ratio in SpnWT-infected cells, as compared to infection with SpnDlctODspxB is consistent with the predicted activation of Akt in H441 lung cells detected by PamStation kinome profiling data (Figures 3A, 4).Reduction of p-Akt/Akt ratio in H441 cells after infection with H 2 O 2 generating strains (SpnWT and SpnDply), but not SpnDlctODspxB strongly suggests an effect of H 2 O 2 on phosphorylation of Akt.

Enhanced inhibitory Lck phosphorylation in the absence of H 2 O 2
The lymphocyte-specific tyrosine protein kinase (Lck) plays a role in cell proliferation and apoptosis of H441 cells, used in this study (31).Lck catalytic activity was reported to be induced by H 2 O 2 (32,33).Phosphorylation of Tyr-505, located near the carboxyl terminus of Lck, was reported to induce inhibition of Lck, due to stabilisation of a biologically inactive conformation (34).The consequences of H 2 O 2 production by SpnWT and SpnDply on the phosphorylation intensities of immobilized peptides that serve as substrates for Lck in PamStation assays are illustrated in Figure 6.The results show similar phosphorylation patterns between cells infected  Intensity of phosphorylation of distinct peptides that serve as substrates for kinase in PamStation assays.Peptide ID with sequence information together with the respective phospho-site, i.e.Ser or Thr residues, are given.The color-coded heat map displays the relative intensitiy of phosphorylation after normalization of the log-transformed raw data.For all four experimental groups, i.e. non-infected, SpnWT, SpnDply, and SpnDlctODspxB, the respective sets of five replicates each were used to calculate the final value, i.e. mathematical mean.with the H 2 O 2 -producing strains SpnWT and On the other hand, non-infected or SpnDlctODspxB-infected cells indicate phosphorylation patterns similar to one another but very different from SpnWT-infected cells (Figure 6).In order to analyse the effect of pneumococcal H 2 O 2 on Lck activity in H441 cells infected with SpnWT, SpnDply and SpnDlctODspxB, Lck and p-Lck levels were measured using anti-Lck antibody and anti-phospho Tyr505-Lck antibody to detect alterations in the phosphorylation of Lck during infection.The results show inactivation of Lck in H441 cells infected with SpnDlctODspxB and non-infected cells, as determined by increased phosphorylation of Lck at tyrosine 505 accompanied by a comparable high p-Lck/Lck ratio (Figures 7A, B).Interestingly, H 2 O 2 -generating strains (SpnWT and SpnDply) seem to maintain or even induce the Lck activity indicated by decreased phosphorylation of Lck at tyrosine 505 leading to a lower p-Lck/Lck ratio.These finding suggest H 2 O 2 -mediated activation of Lck.

Addition of external H 2 O 2 modulates kinase activity of Lck and Akt
In order to substantiate that the detected differences in Lck and Akt activities in SpnWT-infected vs. SpnDlctODspxB-infected H441 cells are predominantly due to pneumococcal H 2 O 2 (Figures 5, 7), we performed infection experiments in the presence of exogenously added H 2 O 2 (1 mM) in cells infected with SpnDlctODspxB.As such, we found that the infection with SpnWT as well as addition of exogenous H 2 O 2 to cells infected with SpnDlctODspxB significantly reduced the levels of p-Akt and non-phosphorylated Akt as compared to SpnDlctODspxB-infected cells (Figures 8A-C).Levels of Lck phosphorylation were reduced in H441 cells infected with SpnWT or with SpnDlctODspxB after additional H 2 O 2 treatment (Figures 8B-D).The reduction of Lck and Akt phosphorylation levels triggered by exogenous H 2 O 2 treatment of SpnDlctODspxBinfected cells was similar to what was observed following SpnWT infection, indicating a direct impact of pneumococcal-derived H 2 O 2 on the phosphorylation and activity of Akt and Lck.

Discussion
Protein kinase-mediated phosphorylation represents the most common post-translational protein modification in signalling processes.Kinase phosphorylation events triggered by bacterial infections can have consequences for the immune response by regulation of antigen presentation, pathogen recognition, cell invasiveness and phagocytosis (6,9) also foster intracellular Spn survival by oxidative inactivation of the host's lysosomal cysteine cathepsins (35).H O 2 has the capacity to modulate protein kinase activity either directly by oxidation of kinase regulatory cysteine and/or methionine residues or indirectly by oxidation of protein tyrosine phosphatases (PTPs) (36)(37)(38).In agreement with our results showing a significant downregulation of Akt activity following infection by SpnWT, but not by mutant SpnDlctODspxB, oxidation of regulatory cysteine residues by H 2 O 2 was shown to inactivate Akt kinase and Fibroblast growth factor receptor (FGFR) kinase (39,40).H 2 O 2 is also able to indirectly modulate activity of certain tyrosine kinases -such as focal adhesion kinase (FAK)-through inhibition of tyrosine phosphatase (38,41).In this regard, oxidation may be similar to phosphorylation, having the capacity to both inhibit and stimulate kinase activity, depending on the site of modification (42).
In this study, we analysed the impact of Spn-derived H 2 O 2 on kinase activity and identified differentially regulated kinases in H441 lung cells infected with SpnWT, SpnDlctODspxB or SpnDply as compared to non-infected cells.The deletion of the two main H 2 O 2 producing enzymes SpxB and LctO in Spn decreases the H 2 O 2 levels to 3% of that measured in SpnWT (11).Our results show that infection of H441 cells with SpnWT significantly downregulated a large number of kinases.Kinase activity profiles of SpnDlctODspxBvs.SpnWT-infected cells were significantly different and revealed higher activity of several kinases in the absence of H 2 O 2 generation in SpnDlctODspxB, including Akt, Lmr1, FGFR1, FLT3, Brk, CK1, AMPKa1, PKG1, PKG2, PKAa and PRKX (Figure 3A).Further kinases (Lck, FRK, Src Fyn, BLK, Hck, Yes, CDK1, CDK2, ERK1, ERK2, ERK5, p38-d, p38-g, AlphaK1 and SgK307) were found to be moderately dysregulated in SpnDlctODspxB-vs.non-infected cells (Figure 3B), likely due to H 2 O 2 -independent effects.The similar intensities of peptide phosphorylation in cells infected with SpnWT and SpnDply suggest that the previously described reduction of Ply release in the SpnDlctODspxB mutant is not the main cause for the distinct kinome profile of SpnDlctODspxB-infected cells (Figures 2A-D), thus substantiating the important role of pneumococcal H 2 O 2 (24,25).We selected two of the top ranked significantly dysregulated kinases identified during infection with SpnWT -Akt kinase and the Src family kinase Lck-for further Western blotting analysis.
Our results show that infection of H441 cells with the H 2 O 2producing strain SpnWT decreases Akt protein expression as well as Akt phosphorylation, assessed as the p-Akt/Akt ratio in H441 cells.In contrast, Akt protein levels and p-Akt/Akt ratio in SpnDlctODspxB infected cells as well as non-infected cells are significantly higher (Figures 5A, B).The reduced phosphorylation of Akt during infection of H441 cells with SpnWT that we observed correlates well with the findings previously reported with Spn infection in the A549 epithelial cell line, which also FIGURE 6 Intensity of phosphorylation of distinct peptides that serve as substrates for the Lck kinase in PamStation assays.Peptide ID with sequence information together with the respective phospho-site, i.e.Tyr residues, are given.The color-coded heat map displays the relative intensity of phosphorylation after normalization of the log-transformed raw data.For all four experimental groups, i.e. non-infected, SpnWT, SpnDply, and SpnDlctODspxB, the respective sets of five replicates each, were used to calculate the final value, i.e. mathematical mean.(43).Noteworthy, we demonstrate that external addition of H 2 O 2 to cells infected SpnDlctODspxB decreases the phospho-Akt and total Akt levels comparable to cells infected with SpnWT (Figure 8A).These results suggest that pneumococcal-derived H 2 O 2 reduces the total Akt and p-Akt levels.
The Src family kinase Lck was reported to be inactivated by phosphorylation of inhibitory tyrosine residue (Tyr-505) in the carboxyl-terminal region (32,34).Lck phosphorylation at Tyr-505 stabilizes Lck in a biologically inactive conformation, as it associates intramolecularly with the domain in the amino-terminal half of the protein (33) In summary, we provide direct evidence that pneumococcalderived H 2 O 2 has the capacity to modify host kinase activity in the course of Spn infection.We describe for the first time pneumococcal H 2 O 2 -specific downregulation of the activity of Akt kinase and activation of Lck.The exact mechanisms and signal pathways responsible for these effects require further study.

Figure 2 (
Figures 2A, B for PTK, Figures 2C, D for STK).The figure depicts relative changes in phosphorylation of the highly dysregulated peptides on the PTK PamChips (182 peptides in total after quality control) and STK PamChips (92 peptides in total after quality control) representing kinase substrate phospho-sites.Peptide phosphorylation patterns for PTKs as well as STKs in non-infected H441 cells and those infected with SpnDlctODspxB indicate more similarities between each other as compared to the very different patterns of non-infected controls on the one hand and cells infected with SpnWT or SpnDply on the other hand, the latter two of which generate high levels of H 2 O 2 .Interestingly, infections with SpnWT and SpnDply show a similar pattern, indicating that the lack of Ply does not greatly affect the kinome (Figures 2A-D).The kinases (both STK and PTK) are mainly downregulated in SpnWTand SpnDply-infected cells, as compared to SpnDlctODspxB-infected cells.Taken together, these results demonstrate an important effect of Spn-derived H 2 O 2 on human kinase activity in H441 cells.The full data sets representing the underlying numerical values of the phosphorylation intensities are listed in Supplementary Tables S1, S2 (Supplementary material).

1 (
FIGURE 1 (A) Schematic representation of Spn infection: H441 lung cells were separately infected with either SpnWT, SpnDlctODspxB or SpnDply for 5 (h) Noninfected vehicle-treated H441 cells were used as control.(B) Kinome profiling of Spn-infected H441 lung cells using PamStation assay.Cell lysates were placed on protein tyrosine kinase (PTK) and serine/threonine kinase (STK) PamStation Chips.Every Chip contains four arrays covered with covalently bound peptides.Activated kinases present in the cell lysates phosphorylate peptides on the Chip.Phosphorylation of PTK or STK substrate peptides was detected using anti-phosphorylation antibodies and secondary FITC-conjugated antibodies.Created with BioRender.

2
FIGURE 2 Intensities of peptide phosphorylation induced by differential kinase activities in non-infected H441 cells and H441 cells infected with SpnWT, SpnDlctODspxB or SpnDply.The heat maps illustrate the intensities of peptide phosphorylation of five independent experiments.Relative intensities of peptide phosphorylation of protein tyrosine kinases (PTKs) (A) and serine/threonine kinases (STKs) (C) are depicted.(B, D) represent the means of all determined relative intensities of PTK and STK peptide phosphorylation.

FIGURE 5
FIGURE 5Spn-derived H 2 O 2 reduces p-Akt/Akt ratio in H441 cells.(A) Protein levels of total Akt and phosphorylated Akt were analysed by Western blot after infection of H441 cells with SpnWT, SpnDply and SpnDlctODspxB.Five independent infections were performed and lysates were analysed with antip-Akt, anti-Akt and anti-Vincullin antibody.(B) Quantification of p-Akt/Akt ratios following determination of protein levels of phosphorylated Akt and total Akt (p-Akt/Akt) by Western blot.Significant differences are denoted with asterisks (p < 0.05 = *; p < 0.001 = ***).n.s., not significant.
. Our results indicate that infection of H441 cells with the H 2 O 2 -deficient SpnDlctODspxB strain, but not with H 2 O 2producing strains SpnWT and SpnDply, increases phosphorylation of Lck at Tyr-505 (Figures 7A, B).These results suggest an H 2 O 2dependent activation of Lck in the presence of pneumococcalderived H 2 O 2 , albeit in an indirect manner, i.e. through inhibition of inhibitory phosphorylation.The impact of H 2 O 2 on Lck was substantiated, as also demonstrated for Akt, by addition of exogenous H 2 O 2 to SpnDlctODspxB-infected cells.The addition of H 2 O 2 to SpnDlctODspxB-infected cells led to a reduction in Lck phosphorylation, similar as what was observed upon infection with SpnWT (Figures 8B-D).These results suggest that reduced Lck phosphorylation at Tyr-505 depends on H 2 O 2 .