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The iron-regulated surface determinant protein B (IsdB) of Staphylococcus aureus is involved in the acquisition of iron from hemoglobin. Moreover, IsdB elicits an adaptive immune response in mice and humans. Here, we show that IsdB also has impact on innate immunity. IsdB induces the release of proinflammatory cytokines, including IL-6 and IL-1β, in innate immune cells of humans and mice. In silico analysis and thermophoresis show that IsdB directly binds to TLR4 with high affinity. TLR4 sensing was essential for the IsdB-mediated production of IL-6, IL-1β, and other cytokines as it was abolished by blocking of TLR4-MyD88-IRAK1/4-NF-κB signaling. The release of IL-1β additionally required activation of the NLRP3 inflammasome. In human monocytes infected with live S. aureus, IsdB was necessary for maximal IL-1β release. Our studies identify S. aureus IsdB as a novel pathogen-associated molecular pattern that triggers innate immune defense mechanisms.
The human antibody response to experimental colonization with Staphylococcus aureus NCTC8325-4
(2008)
The four main work packages and their most important results are briefly described as following. 1. Characterization of the extracellular proteome of S. aureus NCTC8325-4 Reference maps of the extracellular proteins of S. aureus NCTC8325-4 were produced at pH ranges 6-11 and 4-7. In total, 119 (pH 6-11) and 177 (pH 4-7) protein spots were identified, corresponding to 48 and 114 proteins, respectively. Among them were many well-known virulence factors such as alpha-hemolysin (Hla), beta-hemolysin Hlb, gamma-hemolysin subunits (HlgA-C), hyaluronate lyase (HysA) and staphylococcal superantigen-like protein 11 (Ssl11). We also detected various extracellular enzymes, which can cause tissue degradation and are involved in nutrient acquisition, for example, autolysin (Atl), glycerol ester hydrolase (Geh), lipase (Lip), thermonuclease (Nuc), several serine proteases SplA-F (SplA-F), V8 protease (SspA), cysteine protease (SspB), staphopain thiol proteinase (88195808, SspP). Many of these proteins probably also contribute to the virulence of S. aureus. 2. Optimization of a 2-D immunoblot (IB) method for the comprehensive investigation of IgG binding to S. aureus extracellular proteins (strain NCTC8325-4) The immune proteome of S. aureus NCTC8325-4 was revealed by probing 2-D blots of S. aureus extracellular proteins at the two pH ranges 6-11 and 4-7 with a pool of sera from 16 volunteers. IgG binding was detected with high sensitivity using a peroxidase-coupled secondary Ab in combination with an ECL-substrate. With application of the software package Delta2D, we could clearly define 66 immune reactive spots on the immunoblots (IBs) of pH range 6-11 and 38 spots on IBs of pH range 4-7. 72 of these 104 immune reactive spots could be identified by matching the IBs with the protein reference maps. These spots represented 36 identified proteins, many of which are known virulence factors, or they are involved in bacterial cell wall biosynthesis and degradation. Generally, the most abundant proteins were also highly immune reactive, but there was no strict correlation between protein abundance and immune reactivity. Some low abundance proteins, especially basic proteins, showed high immune reactivity on 2-D IBs, for example, Atl, 88195808 (SspP) and iron-regulated surface determinant protein A (IsdA). On the other hand, we observed proteins, which were present in large amounts but did not bind IgG such as peptidoglycan hydrolase (LytM) and a hypothetical protein 88193909 (SAOUHSC_00094). 3. Determination of the anti-staphylococcal Ab profiles of S. aureus carriers and noncarriers Comparing the serum IgG binding patterns of sera from the 16 individual volunteers, we observed pronounced heterogeneity in total IgG binding, spot patterns and spot intensities. Five spots were stronger in carriers than in noncarriers (P< 0.05, Mann-Whitney U test). These spots represent IgG binding to SspA, SspB, IsaA, and two hypothetical proteins. A principal component analysis based on differential IgG binding to these spots showed that the carriers were more closely related to each other than the noncarriers, but that they could not be clearly separated from the noncarriers. 4. Does experimental colonization induce changes of the anti-staphylococcal Ab profiles? Finally, we tested whether symptom-free experimental colonization of the 16 volunteers with S. aureus NCTC8325-4 elicited an IgG response. When we compared sera obtained before colonization with those taken 4 weeks after the inoculation with the laboratory S. aureus strain, we did not observe major changes in the Ab patterns. We conclude that short- term colonization with a strain of low virulence does not suffice to induce an Ab production, which is comparable to that present already before the colonization. Thus, either long term high density colonization is required, or as we consider most likely, the adaptive immune response is primarily triggered by (minor) S. aureus infections. Taken together, in this work we have separated the soluble proteins from complex extracellular S. aureus protein extracts with good reproducibility, large coverage (pH 6-11 and 4-7) and high resolution. With application of an ECL substrate, our 2-D immunoblotting procedure resulted in the highly sensitive detection of IgG binding over a wide range of signal intensities. The most important finding with this technique was the pronounced variability of anti-staphylococcal Ab profiles in healthy adults. This could well explain differences in susceptibility to S. aureus infection and its complications. The Ab responses are presumably triggered by long-term colonization or, more likely, by minor infections with S. aureus, since experimental nasal colonization of healthy volunteers with a bacterial strain of low virulence did not induce impressive changes in the Ab profiles.
Understanding of the regulatory mechanisms controlling stress gene expression of S.aureus in response to environmental stress is very essential in studying its fitness and virulence. In this work, the changes in protein expression profiles as well as the gene transcription of S.aureus after heat exposure, osmotic stress and in response to the antibiotic puromycin were studied in order to provide detailed insights into the response of S.aureus to various kinds of environmental stress under in vitro conditions, namely: (1) to investigate the global response of S.aureus to heat stress conditions using transcriptomic and proteomic analyses. (2) to study the transcriptome and proteome of S.aureus in response to antibiotic substance puromycin. (3) to define the proteome signatures of S.aureus under NaCl stress condition. (4) to complete the proteome map of cytoplasmic proteins of S.aureus by identifying proteins exclusively synthesized during the exposure to stress. Firstly, the high resolution 2-D protein gel electrophoresis technique combined with MALDI-TOF-MS and a DNA array approach were used to investigate the cellular response of S.aureus to heat stress. A switch from normal growth temperature to high temperature condition revealed complex changes in the protein expression pattern as well as the genes expression profile. The effect of puromycin stress on S.aureus cells was analyzed, using a gel-based proteomic approach and transcriptomic analyses with DNA microarrays. We compared the protein synthesis pattern as well as the transcription data of S.aureus in response to puromycin stress with that in response to heat shock. The results demonstrated that both stress conditions induced specific, overlapping and general responses. Finally, the protein expression profile of S.aureus in response to NaCl stress was analyzed with 2D gel based proteomic approach. Our proteome analyses revealed the repression of the synthesis of many enzymes belong to different metabolism pathways . In summary, the signatures for stress or starvation stimuli can be used as diagnostic tools for the prediction of the mode of action of new antibiotics or for studying the physiological state of cells grown. Expression of the respective genes under in vivo conditions could provide some ideas on the environmental signals that specifically influence the survival of S.aureus within and outside the host.
Influenza A Virus (IAV), Staphylococcus aureus (staphylococci), and Streptococcus pneumoniae (pneumococci) are leading viral and bacterial causes of pneumonia. Dendritic cells (DCs) are present in the lower respiratory tract. They are characterized by low expression of co-stimulatory molecules, including CD80 and CD86 and high capacity of antigen uptake. Subsequently, DCs upregulate co-stimulatory signals and cytokine secretion to effectively induce T-cell priming. Here, we investigated these processes in response to bacterial and viral single as well as coinfections using human monocyte-derived (mo)DCs. Irrespective of single or coinfections, moDCs matured in response to IAV and/or staphylococcal infections, secreted a wide range of cytokines, and activated CD4+, CD8+ as well as double-negative T cells. In contrast, pneumococcal single and coinfections impaired moDC maturation, which was characterized by low expression of CD80 and CD86, downregulated expression of CD40, and a mild cytokine release resulting in abrogated CD4+ T-cell activation. These actions were attributed to the cholesterol-dependent cytotoxin pneumolysin (Ply). Infections with a ply-deficient mutant resulted in restored moDC maturation and exclusive CD4+ T-cell activation. These findings show that Ply has important immunomodulatory functions, supporting further investigations in specific modalities of Ply-DC interplay.
Staphylococcus aureus(S. aureus) is a pathobiont of humans as well as a multitude of animalspecies. The high prevalence of multi-resistant and more virulent strains ofS. aureusnecessitatesthe development of new prevention and treatment strategies forS. aureusinfection. Major advancestowards understanding the pathogenesis ofS. aureusdiseases have been made using conventionalmouse models, i.e., by infecting naïve laboratory mice with human-adaptedS. aureusstrains. However,the failure to transfer certain results obtained in these murine systems to humans highlights thelimitations of such models. Indeed, numerousS. aureusvaccine candidates showed promising resultsin conventional mouse models but failed to offer protection in human clinical trials. These limitationsarise not only from the widely discussed physiological differences between mice and humans, but alsofrom the lack of attention that is paid to the specific interactions ofS. aureuswith its respectivehost. For instance, animal-derivedS. aureuslineages show a high degree of host tropism and carry arepertoire of host-specific virulence and immune evasion factors. Mouse-adaptedS. aureusstrains,humanized mice, and microbiome-optimized mice are promising approaches to overcome theselimitations and could improve transferability of animal experiments to human trials in the future.
Staphylococcus aureus is a commensal that colonizes the skin and mucosa of 20-30% of the human population without leading to symptoms of diseases. However, it is also the most important cause of nosocomial infections. Those range from minor skin infections to life-threatening diseases such as pneumonia, endocarditis or septicaemia. Development of strains with resistance against many antibiotics complicates the situation further. The variety of strains with their various properties is one reason why no successful vaccine has been introduced to the market, yet. Therefore, efficient strategies for prevention and therapy of these dangerous infections are urgently needed. To accomplish these goals, the understanding of molecular interactions between host and pathogen is indispensable. Within this dissertation, several internalization experiments were performed aiming to investigate the interaction of S. aureus HG001 and human cell lines upon infection on the protein level. In order to obtain sufficient amounts of proteins for comprehensive physiological interpretations, it is necessary to enrich bacteria, secreted bacterial proteins or infected host cells upon internalization. In the framework of this thesis, bacteria which continuously produce green fluorescent protein (GFP) were employed. With that it was possible to sort bacteria from lysed host cells by flow cytometry or to separate host cells carrying bacteria after contact from those which did not. Subsequently, the proteins were proteolytically digested and peptides were analyzed by mass spectrometry in a gel-free proteomics approach. To allow such analyses also for staphylococci which do not produce GFP, such as clinical isolates, an additional protocol was developed. Prior to the infection, bacteria were labeled with fluorescent or para-magnetic nanoparticles. Afterwards bacteria could be separated from host cell debris by fluorescence-based cell sorting or with the help of a strong magnet. In order to cover also important secreted virulence factors of S. aureus HG001, phagosomes and engulfed bacteria and secreted proteins were isolated from infected host cells. Further steps of protocol optimization included improved bacterial cell counting by fluorescence-based flow cytometry, enhanced data analysis by combination of different search algorithms, and comprehensive functional annotation of proteins of the applied strain by sequence comparison with other strains and organisms. First, the proteome adaptation of internalized S. aureus HG001 and the infected A549 host cells was investigated during the first hours of infection. It became clear, that the bacteria replicate inside the host during the first 6.5 h. After internalization the levels of bacterial enzymes involved in protein biosynthesis decreased. Furthermore, bacteria adapted their proteome to the harsh intracellular conditions such as oxygen limitation, cell wall stress, host defense in terms of oxidative stress, and nutrient limitation. After contact to S. aureus HG001, A549 cells produced increased amounts of cytokines (e.g. IL-8, IFN-γ) in comparison to non-treated A549 cells. In addition, activation of the immunoproteasome and hints of early apoptosis activity were observed. Afterwards, the response of S. aureus HG001 to internalization by A549, S9 or HEK 293 cells was compared on the proteome level. It was obvious, that the adaptation to stress and the reduced protein synthesis are conserved mechanisms. Host dependent differences were detected especially in the energy metabolism and the synthesis of some amino acids. Additionally, bacteria showed different intracellular replication patterns depending on the host cell line. A higher percentage of extracellular bacterial proteins was found in isolated phagosomes compared to the sorted samples. Selected low abundant virulence factors could be quantified at two points in time after infection with the help of the sensitive single reaction monitoring (SRM) method. Further, a heterogeneous mixture of several phagosomal maturation steps was present during the first 6.5 h after infection. Finally, the gel-free proteome analyses could be applied to investigate Bordetella pertussis, the cause of whooping cough, during iron limitation and after internalization, and the results were compared to the S. aureus HG001 data.
Proteolysis represents the final step in the life of a protein. It is one of the most important cellular processes assisted by chaperone systems and ensures an appropriate protein homeostasis. Protein degradation is essential for the removal of cytotoxic protein aggregates and mis-translated/mal-folded proteins, „unemployed“ and regulatory proteins to enable rapid cell adaptation to altering environmental conditions (Gottesman, 2003; Wiegert & Schumann, 2001; Parker, 1981; Stansfield et al., 1998; Drummond & Wilke, 2008; Goldberg, 1972; Gerth et al., 2008). The bacterial Clp (caseinolytic proteins) protease complexes are analogous to the eukaryotic 26S proteasome and consist of Hsp100/Clp proteins of the AAA+ superfamily and an associated barrel-like proteolytic chamber (e.g. ClpP). The Clp proteases seem to be responsible for the major protein turnover in low GC, Gram+ bacteria. The main goal of this thesis was to develop new methods and tools to investigate global proteolysis more precisely and to get a detailed understanding of protein degradation during starvation conditions and it´s regulation in low GC, Gram-positive bacteria. To analyse protein degradation under starvation conditions the well established glucose starvation model was used. In Bacillus subtilis it could be shown that approximately 200 proteins are selectively degraded in a glucose depletion induced stationary phase. Furthermore radioactive pulse-chase labelling experiments coupled with 2D-PAGE analysis revealed that mainly the ClpCP protease complex is involved in the degradation of proteins in the stationary growth phase. To investigate proteolysis in the human pathogen Staphylococcus aureus in the same way, a newly developed chemically defined medium was established suitable for radioactive pulse-chase labelling experiments under stable glucose starvation conditions. The degradation kinetics of individual 2D spots was significantly better resolved using 14C-BSA as an internal marker protein for the sample normalisation. A rather huge overlap was found within the functional protein classes that were degraded in B. subtilis and S. aureus the stationary phase. Among others, especially proteins involved in amino acid, nucleotide and cell wall biosynthesis were rapidly degraded, whereby not always the same and sometimes another enzymes from a biosynthetic chain were targeted for proteolysis. Despite the resolution power of the 2D-PAGE method, there are some drawbacks such as a limited "protein window" with regard to the molecular weight and isoelectric point, loss of low abundance proteins and a rather low reproducibility for time course experiments. Therefore a mass spectrometry based approach for the simultaneous detection of protein synthesis, accumulation and degradation was developed. This pulse-chase SILAC approach provides a very good reliability with a broad spectrum of proteins that can be analysed. Through the combination with ultracentrifugation even non-soluble and aggregated proteins could be analysed. Several hundred proteins were degraded in S. aureus during glucose starvation. Among them was the functional cluster of ribosomal proteins which is degraded in the early stationary phase. Furthermore proteins belonging to complexes were degraded with the same kinetic (e.g. NrdE, NrdF). In addition selective protein degradation took place according to functional categories (e.g., ribosomal proteins, biosynthetic, glycolytic enzymes) and not to regulatory groups (e.g. CcpA, SigB regulon).The investigation of a clpP deletion mutant in S. aureus revealed a greater susceptibility to aggregation, where the cells try to counteract with the expression of chaperones like GroEL/ES, ClpB and DnaK. The renaturation process is very ATP consuming and only takes place in energy rich phases of growth (e.g. from exponential to transient growth phase). Protein aggregation was found enhanced in the stationary phase. Furthermore, a higher GTP level compared to the wild-type probably resulted in a stronger CodY mediated repression with a rather low level of amino acids in clpP mutant cell. In addition substances like glycerol, which thermodynamically stabilise proteins in refolding processes (Maeda et al., 1996; Feng & Yan, 2008), were found in higher levels compared to the wild-type. A strong response to reactive oxygen species was detected in the clpP mutant strain, which is probably due to ROS production during the early stages of protein aggregation. Altogether, different methods were used for investigation protein degradation at a proteome-wide scale. Hundreds of degradation candidates were identified by gel-based and gel-free approaches in S. aureus wild-type cells. “Unemployed” proteins (e.g. ribosomal proteins, biosynthetic enzymes) were degraded and proteins particularly required and synthesized in glucose-starved cells such as TCA cycle enzymes were stable in the stationary phase. Investigation of the clpP mutant strain supports a proposed model for the pleiotropic phenotype and provides a deeper insight in the fine-tuned protein quality control and the important role of ClpP during starving conditions.
Reversible posttranslational modifications play an important role during the regulation of many central processes in bacterial cells. Protein phosphorylation, in particular, can influence signal transduction processes and thus enables a distinct reaction of the cell to different stress and environmental conditions. In the case of the human pathogen Staphylococcus aureus, protein phosphorylation is involved in the adaptation to changing conditions during colonisation of human hosts. For this reason, the investigation of phosphorylations in S. aureus allows a better understanding of pathophysiology and virulence of this organism. Apart from stable phosphorylations at the amino acids serine, threonine and tyrosine, insights into energy-rich phosphorylations, for instance at arginine residues, gain more and more scientific attention. For this reason, one purpose of this study was the investigation of incidence and physiological relevance of this protein modification at a global scale. Firstly, the analysis of this modification was methodically optimised resulting in the identification of eight arginine phosphorylations in wild type cells of S. aureus COL. Secondly, the deletion mutant ΔptpB missing the gene that codes for an arginine phosphatase, was analysed. The characterisation of PtpB in vitro proved its activity and specificity towards arginine phosphorylations. This enabled the global analysis of the phosphoproteome with a focus on arginine phosphorylations. In addition to the optimisation of the phosphopeptide enrichment as part of the sample preparation, the data analysis process was adapted to the special challenges of energy-rich phosphorylations. Here, classical database search was extended by spectral library based analyses. In addition, synthetic peptides allow the generation of high quality mass spectra and the verification of database based evaluation strategies to ensure the quality of the spectral library. Next, S. aureus COL was cultivated under various conditions and several subcellular fractions were analysed with the aim to cover a broad part of the proteome. The combination of the spectra of synthetic peptides, the spectra of non-phosphorylated peptides from extensive cultivation experiments and the spectra of enriched phosphopeptides rendered the construction of a spectral library possible. This contained 2,270 proteins out of which 392 were found to be phosphorylated. A comparison of the database based analysis with spectral library based analysis showed the advantages of the latter when comparing the reproducibility of biological replicates. Thereby a permanent issue in phosphoproteomics was investigated. Hence, spectral libraries were used for the analysis of the phosphoproteome of S. aureus under control and stress conditions. 215 arginine phosphosites were identified within the mutant under control conditions and 117 under oxidative stress conditions. Oxidative stress was chosen because phenotypic characterisation of the mutant revealed that the most distinct growth changes in comparison with the wild type occurred after oxidative stress. These phenotypic changes were quantitatively approached in the last part of this work. Total proteome quantification of the wild type and mutant under control and stress conditions revealed an influence of the ptpB deletion on amino acid metabolism, oxidative stress response and virulence. The quantification of phosphopeptides by means of a combination of spectral library with Census based analysis finally confirmed the observations made during total proteome quantification.
Thiol or sulfhydryl groups are highly reactive functional groups in cellular systems. Molecules carrying thiol groups are mostly derivatives of the amino acid cysteine and are grouped as low molecular weight (LMW)-thiols: coenzyme A (CoA), glutathione (GSH) or bacillithiol (BSH). LMW-thiols can help in the maintenance of the reduced cellular environment as so called redox-buffers. Additionally, they act as co-factors in enzyme reactions or help in the detoxification of reactive oxygen or nitrogen species, electrophilic compounds or thiophilic metalloids (arsenite, tellurite). In proteins from different organisms cysteine is underrepresented compared to other amino acids, but still overtakes diverse roles. It is an important determinant in the tertiary and quaternary structure of proteins. The nucleophilic character of the thiol or thiolate group, respectively, makes cysteine the catalytically active amino acids of different enzymes. As a precursor cysteine participates in the formation of Fe-S clusters and coordinates different co-factors like heme, iron or zinc. The main goal of this study was the investigation of the different cellular thiol pools, now defined as the thiolome. The thiolome is the entity of the cellular thiol pools, i.e. LMW-thiols and protein thiols, and the dynamics between these pools. In Bacillus subtilis and Staphylococcus aureus mixed disulfides between protein thiols and free LMW-thiols, so called S-thiolations, were identified in different proteins in response to the thiol specific reagent diamide. Some of these S-thiolations were located at catalytically active cysteine residues. Subsequent analysis of metabolites supports this: the S-thiolation of the cobalamine-independent methionine-synthase MetE led to a decrease of the cellular methionine content. Additionally, the conversion of threonine to different branched-chain amino acids (BCAAs) was disrupted by the S-thiolation of the branched-chain amino acid aminotransferase YwaA, thereby probably inducing the synthesis of ppGpp, the alarmon of the stringent response. In addition to the identification of S-thiolations a technique was established which allowed the discrimination between intra- and intermolecular disulfides. The non-reducing/ reducing diagonal gel electrophoresis was applied to B. subtilis and S. aureus and confirmed known existing disulfide bonds, e.g. in alkyl hydroperoxide reductase AhpC or the thiol peroxidase Tpx. In response to diamide an increase of specific disulfide bonds in different proteins was observed. The analysis of the LMW-thiol content by an HPLC-approach allowed the observation of the dynamics of the thiolome. In response to diamide the reduced LMW-thiol content decreased by 75%, reduced protein thiols by 60%. Collaborations with other working groups allowed the identification of BSH in this approach. Additionally, an unknown thiol was found that is likely a derivative of BSH. Screening of the LMW-thiol content of different S. aureus-strains under various growth conditions revealed that strains 8325-4 and SH1000 lack BSH. The lack of BSH was attributed to an 8 bp-duplication in the bshC-gene that encodes the last enzyme of the BSH-synthesis. BSH-production was restored by transducing plasmid-borne functional BshC from strain Newman into strains 8325-4 and SH1000. The reconstitution of the BSH-synthesis aided in the resistance to the antibiotic fosfomycin but did not increase the resistance to different oxidants (diamide, sodium hypochlorite, hydrogen peroxide). The production of BSH had also positive effects on the survival of S. aureus inside human bronchial epithelial cells and murine macrophages in phagocytosis assays. Additionally, a GSH-uptake was observed into S. aureus which has before been known as a GSH-free bacterium. Taken together, this thesis provides the first insights into both, the LMW-thiol- and protein thiol pool of low GC, Gram-positive bacteria under different conditions. A plethora of different methodologies was used to describe the thiolome. The bacterial thiolome is a sophisticated system which is tightly regulated, but also flexible enough to not rely on determined molecules like BSH. The influences of the thiolome are not restricted to its own system and regulation, but also affect different branches of cellular physiology like the metabolism of BCAAs.
Bacterial infections represent an increasing threat in human health and hospital- acquired infections meanwhile account for 99,000 deaths every year in the United States (Ventola, 2015). Live-threating bacterial infections will certainly emerge to an even more serious concern in future, essentially by accelerated development of antibiotic resistance. Only recently, the discovery of plasmid-encoded mcr-1, that confers resistance against colistin, marks the point where this highly transmissible resistance mechanism is now reported for every so far developed antibiotic (Liu et al., 2016). Staphylococcus aureus is a Gram-positive bacterium and well-known for its ability to quickly acquire resistance toward antibiotics either by chromosomal mutations and/or horizontal gene transfer (Pantosti et al., 2007). Although approximately 30% of the population is colonized with S. aureus (Kluytmans et al., 1997), it can transform to an invasive pathogen that causes a wide range of severe infections including pneumonia. The success of S. aureus as opportunistic pathogen can be attributed to combinations of several beneficial properties and capabilities including the expression of an arsenal of virulence factors (Archer, 1998), intracellular persistence (Garzoni & Kelley, 2009) and subversion of host cell defense mechanisms (Schnaith et al., 2007). The airway epithelium is the first line of defense against bacterial pathogens by forming a relative impermeable physical barrier composed of epithelial cells that are linked by tight junctions, desmosomes and adherence junctions (Davies & Garrod, 1997). Additionally, the airway epithelium mediates the detection of bacterial pathogens via toll-like receptors (TLRs) that recognize a variety of bacterial molecular patterns such as lipopolysaccharide (LPS), peptidoglycan and flaggelin (Sha et al., 2012). This interaction is transduced via protein phosphorylations into the cell in order to promote adaptation to the infection by initiation of the adaptive and innate immune defense. Although few insights where obtained of the signaling host responses towards staphylococcal infections (Agerer et al., 2003; 2005; Ellington et al., 2001), a comprehensive description of the host signaling network is largely missing. Thus, this dissertation thesis focuses on the decipherment of phosphorylation-mediated signaling responses towards S. aureus infections in non- professional and professional phagocytes by mass spectrometry-based phosphoproteomic techniques. The results of this thesis are summarized in the four chapters. Chapter I introduces to recent advances in the development of methodologies applied in the field of phosphoproteomics, including quantification strategies, peptide fractionation techniques and phosphopeptide enrichment methods applied for the system-wide characterization of protein phosphorylations by mass spectrometry. Additionally, publications reporting phosphorylation-based host signaling responses towards bacterial pathogens or their molecular patterns that applied mass spectrometry-based phosphoproteomics are discussed. In chapter II, the responses of the human bronchial epithelial cell lines 16HBE14o- and S9 following challenge with staphylococcal alpha- toxin at the level of proteome and phosphoproteome are summarized. General and cell type-specific signaling events are highlighted and evidences linking the activity of the epidermal growth factor receptor (EGFR) with differences in tolerance toward alpha-toxin are provided. Chapter III describes the modulation of the host signaling network of 16HBE14o- airway epithelial cells triggered by infection with S. aureus including temporal dissection of signaling events. Several protein kinases were identified as important signaling hubs mediating the host response. Targeted pharmaceutical inhibition of these kinases was probed and resulted in reduction of intracellular bacterial load. Chapter IV describes the rearrangement of the kinome by the differentiation of THP-1 monocytes to macrophage-like cells by application of quantitative kinomics. This approach identified the kinase MAP3K7 (TAK1) as key mediator of bacterial clearance, chemokine secretion and the differentiation process itself.