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International Journal of Medical Microbiology 304 (2014) 121– 132

Contents lists available at ScienceDirect

International Journal of Medical Microbiology

jo ur nal homepage: www.elsev ier .co m/locate / i jmm

he phosphoproteome and its physiological dynamics intaphylococcus aureus

atrin Bäsell a, Andreas Ottoa, Sabryna Junkera, Daniela Zühlkea,erd-Martin Rappena, Sabrina Schmidta, Christian Hentschkera,oris Macekb, Knut Ohlsenc, Michael Heckera, Dörte Bechera,∗

Institute for Microbiology, Ernst Moritz Arndt University Greifswald, GermanyProteome Center, Interfaculty Institute for Cell Biology, University of Tübingen, GermanyInstitute for Molecular Infection Biology, Julius Maximillians University, Würzburg, Germany

r t i c l e i n f o

eywords:hosphopeptide enrichmenthosphoproteomero-Q diamondtaphylococcus aureusD-gelrginine/serine/threonine/tyrosinehosphorylation

a b s t r a c t

Phosphorylation events on proteins during growth and stress/starvation can represent crucial regulationprocesses inside the bacterial cell. Therefore, serine, threonine and tyrosine phosphorylation patternswere analyzed by two powerful complementary proteomic methods for the human pathogen Staphy-lococcus aureus. Using 2D-gel analysis with a phosphosensitive stain (Pro-Q Diamond) and gel-freetitanium dioxide based phosphopeptide enrichment, 103 putative phosphorylated proteins with suc-cessfully mapped 68 different phosphorylation sites were found in the soluble proteome of S. aureus.Additionally, in a proof of concept study, 8 proteins phosphorylated on arginine residues have been iden-tified. Most important for functional analyses of S. aureus, proteins related to pathogenicity and virulencewere found to be phosphorylated: the virulence regulator SarA, the potential antimicrobial target FbaA

and the elastin-binding protein EbpS. Besides newly identified phosphorylation sites we compared ourdataset with existing data from literature and subsequent experiments revealed additional phosphoryla-tion events on highly conserved localizations in FbaA. Differential analysis of phosphorylation signals onthe 2D-gels showed significant changes in phosphorylation under different physiological conditions for10 proteins. Among these, we were able to detect newly appearing signals for phosphorylated isoforms

itros

of FdaB and HchA under n

ntroduction

Continuously changing stress and starvation conditions inheir natural habitats have maintained an evolutionary pressureor bacteria toward the development of complex adaptationaletworks. For pathogenic bacteria, not only nutrient availabilitynd physico-chemical parameters of their environment, but alsoost defense mechanisms are of importance and elicit adaptivend immune evasion responses. Deeper insights into these adap-ive and evasive strategies are crucial for understanding microbial

hysiology in the host in general and pathogenicity in particularFlannagan et al., 2009).

Abbreviations: HCD, higher C-trap dissociation; MAHMA NONOate, 6-2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-hexanamine; SCX, strongation exchange chromatography; S/T/Y, serine/threonine/tyrosine; TE, Tris–EDTAethylenediaminetetraacetic acid) buffer.∗ Corresponding author.

E-mail address: dbecher@uni-greifswald.de (D. Becher).

438-4221/$ – see front matter © 2013 Elsevier GmbH. All rights reserved.ttp://dx.doi.org/10.1016/j.ijmm.2013.11.020

ative stress conditions.© 2013 Elsevier GmbH. All rights reserved.

Regulatory proteins and hence the regulatory networks areoften modulated by post translational modifications like phosphor-ylation and dephosphorylation events (Mijakovic and Macek, 2012;Ohlsen and Donat, 2010). Due to the negative charge, phosphory-lation may trigger protein activation, inactivation and degradationby reorganization of the protein structure (Johnson and Barford,1993). This modulation of protein activity is found in differentprocesses such as stress responses, central carbon metabolismand cell growth, as well as in virulence of bacterial pathogens(Eymann et al., 2007; Madec et al., 2002; Truong-Bolduc et al.,2008). Regarding pathogenic processes, phosphorylation eventsplay an important role in cell–cell interaction and adherence, inva-sion of bacterial infectors into host cells and changes in hostcellular structure and function (Chao et al., 2010; Schmidl et al.,2010).

In the last years knowledge on protein phosphorylation has beengained in large-scale studies for a wide range of bacteria rendering

insights into putative regulatory sites of post translational modi-fication including phosphorylation. Whilst the first studies merelyfocused on phosphorylation of histidine and aspartate residues intwo-component systems (Hoch, 2000) and of histidine residues

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laying a key role in regulation of the phosphotransferase sys-em (Deutscher et al., 2006), phosphoproteome studies targetinger/Thr/Tyr kinases (STYKs) and phosphatases were conducted notntil the post-genome era. More recently, it has been proven thatlso phosphorylation of arginine residues plays a role in biologicalegulation and is accessible by current analytical methods (Elsholzt al., 2012; Fuhrmann et al., 2009; Ge and Shan, 2011; Macek andijakovic, 2011; Pereira et al., 2011).Among virulent bacteria, Staphylococcus aureus is a major threat

s a human pathogen due to its dominant role in nosocomialnfections worldwide. Spreading antibiotic resistances and theneffectiveness of current antibiotic treatment strategies for S.ureus (Otto, 2010) urgently require global investigations on path-genicity and its regulation (Reyes et al., 2011; Young et al.,012).

Proteins that are regarded as major determinants of pathogen-city for S. aureus are found mainly to be secreted and/or cell-wallssociated (Bronner et al., 2004; Cheung et al., 2004). Here, expres-ion and targeting is closely coupled to growth phase and inducingtimuli. Accordingly, regulation of virulence is tightly controlled byifferent transcription factors leading to the activation or repres-ion of complete regulons (Cheung et al., 2008; Jelsbak et al., 2010;ekalanos, 1992).First studies on protein phosphorylation events involved in bac-

erial virulence focused on two component systems functioning aswitches for regulons specific for the effectors of pathogenicity.egulation of these systems is mainly characterized on molecu-

ar level by phosphorylation/dephosphorylation events on histidinend aspartate residues (Delaune et al., 2012; Dubrac and Msadek,004; Fournier and Hooper, 2000; Giraudo et al., 1999; Lina et al.,998; Yarwood et al., 2001).

Later, the role of S/T/Y-phosphorylation events in S. aureuseading to altered virulence gained attention. Here, an importantxample of phosphorylation-mediated regulation is the global reg-lator MgrA that is controlled by the eukaryotic-like protein kinaseknB. Post translational modification of MgrA is a key event in theontrol of expression of efflux pumps involved in drug resistanceTruong-Bolduc and Hooper, 2010; Truong-Bolduc et al., 2008).urthermore, PknB was shown to be important for the resistancegainst a large range of antibiotics and for the full expression ofirulence in S. aureus (Debarbouille et al., 2009).

As protein phosphorylation is assumed to play a major rolen regulation of infection-related processes, the present workddresses phosphorylation patterns and its changes under differenthysiological conditions in S. aureus COL. To achieve this goal, weombined complementary approaches and first visualized phos-horylation patterns on 2D-gels and determined phosphorylationites by mass spectrometry of excised and enzymatically digestedrotein spots. Second, we selectively enriched phosphopeptidesithout prior electrophoretical separation (gel-free) and analyzed

hem by mass spectrometry.Taken together, we could identify 108 phosphorylated proteins

ith 76 different phosphorylation sites on serine, threonine, tyro-ine and arginine residues. Furthermore, we followed changes inhosphorylation patterns on the 2D-gels for four different stressnd starvation conditions. Furthermore, we followed changesn phosphorylation patterns on the 2D-gels for four differenttress and starvation conditions. We report phosphorylation ofroteins functioning as virulence factors or being regarded asossible targets for antimicrobial therapy. Additionally, we pro-ose that these phosphorylation events are conserved withinrokaryotes. Most interestingly, our quantitative data suggest a

echanism of swift regulation of metabolic fluxes by protein

hosphorylation under nitrosative stress conditions to ensureupply with energy and to ensure detoxification of metabolic by-roducts.

cal Microbiology 304 (2014) 121– 132

Materials and methods

Bacterial strains and culture conditions

S. aureus COL (Shafer and Iandolo, 1979) was grown aerobicallyat 37 ◦C with vigorous shaking in synthetic medium (Gertz et al.,1999) with the following modifications: glycine and MOPS wereomitted; all amino acids were added to a final concentration of1 mM. Cultures for the gel-free approach targeting S/T/Y phospho-rylations were grown aerobically to an optical density of 1.5–2.0 at500 nm, which corresponds to the late exponential and transientgrowth phase. Cultures for the gel-free approach targeting argi-nine phosphorylations were grown aerobically in Luria Bertani (LB)medium to an optical density of 0.5 at 540 nm, which correspondsto the exponential growth phase.

Cell cultures were stressed at mid-log phase (OD500 = 0.5)and samples were taken at three distinct time points (beforetreatment, 15 min and 45 min after exposure to stress conditions).NO stress experiments were performed according to Hochgräfeet al. (Hochgräfe et al., 2008) by adding MAHMA NONOate[6-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-hexanamine; Sigma] serving as a NO donor to a final concentrationof 500 �M (Hochgräfe et al., 2008). Salt stress was imposed byaddition of 8% NaCl (w/v) to the culture. The thiol-specific oxidantdiamide was used at a concentration of 1 mM to trigger oxidativestress. Glucose deprivation was achieved by limiting glucoseconcentration to 0.05% (w/v) causing a growth arrest at an opticaldensity of 0.9. Here, samples were taken at mid-exponentialphase (OD500 = 0.5), at the transition from exponential growth tostationary phase and 3 h after transition. All experiments werecarried out in three biological replicates.

Sample preparation, 2D-PAGE, fluorescence staining, imageanalysis and quantitation

The bacterial cells were harvested on ice, centrifuged (10 min,8000 rpm, 4 ◦C) and washed three times with TE buffer. The cell pel-lets were resuspended in a buffer containing 8 M urea/2 M thioureaand 10 mM NaF (as phosphatase inhibitor) and disrupted usinga ribolyzer as described earlier (Becher et al., 2009). The solubleprotein fraction was obtained by removal of the cell debris by cen-trifugation and the protein concentration was determined usingRoti-Nanoquant (Roth, Karlsruhe, Germany). 2D-gel electrophore-sis, phospho-staining (Pro-Q diamond, Invitrogen, Carlsbad, CA),FlamingoTM fluorescent gel staining (Biorad, Hercules, CA)) andpicking of the 2D-gel spots were done according to Eymann et al.(2007). Image analysis, spot quantitation and determination ofputative phosphorylated spots were performed with DECODONDelta2D 4.0 software (Decodon GmbH, Greifswald, Germany;http://decodon.com).

Identification of proteins and phosphate containing peptides from2D-gel approach by MS

The manually picked gel pieces were digested and prepared formass spectrometric analysis according to Schmidl et al. (2010).LC–MS/MS analysis was carried out using a nanoACQUITY UPLCsystem (Waters, Milford, MA) coupled online to an LTQ Orbitrap XLmass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA).Concentration and desalting of peptide samples was achieved byusing a trap column (Symmetry C18, 5 �m, 180 �m inner diame-ter × 20 mm, Waters, Milford, MA) with subsequent elution onto

an analytical column (BEH130 C18, 1.7 �m, 100 �m inner diame-ter × 100 mm, Waters) by a binary gradient of buffer A (0.1% (v/v)acetic acid) and B (99.9% (v/v) acetonitrile, 0.1% (v/v) acetic acid)over a period of 45 min with a flow rate of 400 nl/min. The LTQ

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rbitrap XLTM was operated in data-dependent MS/MS mode.he full scan was recorded in the Orbitrap analyzer at resolution

= 60,000 with the lockmass option enabled (Olsen et al., 2005). Forhe five most intense precursor ions collision induced dissociationpectra in the LTQ analyzer were recorded. Multistage activationMSA) at −97.98, −48.99, −36.66 and −24.49 Thompson was usedo enhance fragmentation for putative phosphate-group containingons in all MS/MS events (Schroeder et al., 2004).

All acquired MS/MS spectra in .dta format were searchedgainst a target-decoy database (5236 entries) including all pro-ein sequences of S. aureus COL extracted from the National Centeror Biotechnology Information (NCBI) bacteria genomes in addi-ion to common contaminants and an appended set of the reversedequences created by BioworksBrowser 3.2 EF2 (Elias and Gygi,007) using Sorcerer-SEQUEST (ThermoFinnigan, San Jose, CA; ver-ion v.27, rev. 11). Peptide hits were filtered and consolidated toroteins with Scaffold2 (version Scaffold 2 02 03, Proteome Soft-are Inc., Portland, OR). Mass tolerance for peptide identification

n MS and MS/MS peaks were 10 ppm and 1 Da, respectively. Up towo missed tryptic cleavages were allowed. Methionine oxidationnd cysteine carbamidomethylation, as well as phosphorylation aterine, threonine or tyrosine were set as variable modifications.roteins were identified by at least two peptides with a probabilitycore higher than 99.9%, which was assigned by the Protein Prophetlgorithm (Nesvizhskii et al., 2003). Phosphorylated peptides whichave a peptide probability score higher than 95.0% as specified byhe Peptide Prophet algorithm (Keller et al., 2002) and fulfilled theollowing criteria were accepted: (1) only phosphopeptides with sixr more amino acids residues were allowed; (2) b- or y-ion cover-ge confirmed the phosphorylation site. These spectra are providedn the Supplemental material S2.

el-free approach for mass spectrometric phosphopeptidedentification and determination of phosphorylation sites

For both S/T/Y and arginine detection of phosphopeptides0 mg protein of S. aureus were prepared for phosphopeptidenrichment by using strong cation exchange (SCX) chromatogra-hy and titanium dioxide beads according to Olsen and Macek2009). For the study targeting S/T/Y phosphorylated peptides, sam-les were separated and analyzed by LC–ESI-mass spectrometrysing a nanoACQUITY UPLC system (Waters, Milford, MA) withn analytical column (BEH130 C18, 1.7 �m, 100 �m inner diam-ter × 100 mm, Waters) coupled online to an LTQ Orbitrap XL masspectrometer (Thermo Fisher Scientific, Waltham, MA). Elutionas performed by a binary gradient of buffer A (0.1% (v/v) acetic

cid) and B (99.9% (v/v) acetonitrile, 0.1% (v/v) acetic acid) over aeriod of 80 or 90 min with a flow rate of 400 nl/min. The masspectrometric analysis followed as described above. For the studyargeting arginine phosphorylated peptides, samples were mea-ured online by LC–ESI–mass spectrometry using an Easy-nLCIIThermo Fisher Scientific, Waltham, MA) with self packed analyticalolumns (100 �m × 20 cm) containing C18 material (Phenomenex,schaffenburg, Germany) and coupled to a LTQ Orbitrap Velos

Thermo Fisher Scientific, Waltham, MA). An 80 min gradient, using.1% (v/v) acetic acid as buffer A and 99.9% (v/v) ACN with 0.1% (v/v)cetic acid as buffer B was used, applying a flow rate of 300 nl/min.ull Scan at a resolution of 60,000, recorded in the Orbitrap ana-yzer, was followed by the analysis of the 20 most intense precursorons (in the LTQ analyzer) according to data-dependent MS/MS

ode. Wideband activation and lockmass option was enabled

Olsen et al., 2005). Fragment spectra were recorded by enablingeutral loss option. Multistage activation (MSA) at −97.98, −48.99,36.66 and −24.49 Thompson was applied in all MS/MS events

o improve the fragmentation for putative phosphorylated ions

cal Microbiology 304 (2014) 121– 132 123

(Schroeder et al., 2004). For the HCD measurements, Full Scan at aresolution of 30,000 was carried out, enabling wideband activationand lockmass option. MS/MS Scans were recorded at a resolutionof 7500.

Raw data were processed using the MaxQuant software (version1.1.1.14 for serine, threonine or tyrosine phosphorylations, version1.2.2.7 for arginine phosphorylations). The peak lists were searchedusing Andromeda search engine (Max Planck Institute of Biochem-istry, Martinsried) against a database (2618 entries) composed of allprotein sequences of S. aureus COL extracted from the National Cen-ter for Biotechnology Information (NCBI) bacteria genomes (Coxand Mann, 2008). Randomizing the database and including con-taminants were selected in MaxQuant software.

The following criteria were used: Full tryptic specificity wasrequired; up to two missed cleavages were allowed and car-bamidomethylation was set as fixed modification. Oxidation (M)and phosphorylation (STYK) were set as variable modifications. Themaximum mass deviation in the database search was set to 6 ppmfor the precursor ion and 0.5 Da for fragment ions generated by CIDor 20 ppm for fragment ions generated by HCD. For data processingand phosphopeptide validation the MaxQuant software was used.Only peptides with six or more amino acids were accepted.

The false positive rate on protein and peptide level of thereported dataset is set to be <1%. Only phosphopeptides having aphosphorylation site probability higher than 0.75 were accepted forphosphorylation site determination. All peptides were additionallycurated with the criteria mentioned above. Annotated MS/MS spec-tra of all identified phosphopeptides are presented in Supplementalmaterial S3.

Results and discussion

For comprehensive mapping of protein phosphorylation in S.aureus COL under different physiological situations two comple-mentary workflows have been applied: a 2D-gel based approachaccording to Eymann et al. (Eymann et al., 2007) and a phosphopep-tide enrichment approach according to Olsen and Macek (2009).

The first method visualizes phosphorylated proteins on 2D-gelsand subsequently detects their identity by LC–MS/MS. The sec-ond approach is a phosphopeptide enrichment technique whichis specifically targeted to high throughput mass spectrometricidentification of phosphorylated peptides after strong cation chro-matography and subsequent titanium dioxide enrichment.

Gel-based analysis of the phosphoproteome of S. aureus COL

2D-gel based proteomics is a powerful method of separatingand visualizing complex protein mixtures on protein level. Besidesthe visualization of protein amount and its changes, sophisticatedstaining procedures have been established to specifically detectpost translational modifications like protein phosphorylations.

To distinguish between phosphorylated and non-phosphorylated protein species in our 2D-gel based analysis,we used two different stains for detecting either total proteinamount (FlamingoTM fluorescent gel stain) or putatively phos-phorylated protein species on serine, threonine and tyrosineresidues (the phosphate-specific stain Pro-Q diamond phospho-protein gel stain). As shown in a comparable study for Bacillussubtilis, the distributions in signal intensity for phospho-stain/totalprotein amount stain of proteins can be computed to ensurereliable differentiation between putatively phosphorylated and

non-phosphorylated proteins (Eymann et al., 2007). Protein spotsexhibiting a larger Pro-Q/Flamingo ratio lie outside of the standarddistribution and are therefore candidates for protein phosphor-ylation (false colored red in dual channel images). By using this

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24 K. Bäsell et al. / International Journal o

pproach, we were able to assign 73 putatively phosphorylatedrotein spots for the different physiological conditions of S. aureusOL.

Next, the gel-based approach was further refined by using a nar-ow pH range IPG strips (pH 4.5–5.5) to exploit its advantage foresolving the highest number of proteins. As expected, this resultedn an increased resolution of protein spots at a higher proteinxtract loading capacity and an improved rate of protein identifi-ation: additional 48 putatively phosphorylated protein spots (seeigs. 1 and 2) could be mapped on the 2D-gels compared to theclassical” (pH 4–7) 2D-gel method.

In total, 121 putatively phosphorylated protein spots corre-ponding to 80 proteins were detected for S. aureus COL (seeigs. 1 and 2, and for further information see also Supporting infor-ation Tables S1-A and S1-B).One of the most important characteristics of 2D-gel based

roteomics is the separation and visualization of intact protein iso-orms, which are not detectable by other peptide based proteomics

ethods (shotgun proteomics). In our approach, 36 of the 80 phos-horylated proteins appeared as at least two isoforms of uniquelyatched proteins (see Fig. 2: e.g. Pyk, MetE, AhpF, RpsB).

To unambiguously determine the phosphorylation sites in the

el-based approach, we excised the protein spots of interest andubmitted the respective digests to high-accuracy LC–MS/MS.dditionally, we implemented a more targeted approach for the

ig. 1. Dual channel image of Flamingo-stained (total protein amount, green) and Pro-Q-ells (pH range 4–7). Phosphorylated proteins are labeled (see supporting information Tnumerated.

cal Microbiology 304 (2014) 121– 132

detection of phosphopeptides for the putatively phosphorylatedproteins without unambiguous identification of phosphopeptidesin the first place: in a second run, the digests of protein spotswere subjected to LC–MS/MS measurements with a parent masslist including all possible S/T/Y phosphorylation sites. Based on theexisting methodology for detection of putatively phosphorylatedproteins and optimized settings for the identification of phospho-sites we found 22 phosphorylated peptides from 19 proteins.

Altogether, in spite of the inherently limited quantities of phos-phorylated proteins in 2D-gels, we identified the phosphorylationsites for a quarter of all putatively phosphorylated proteins asdetermined by the phospho-specific Pro-Q diamond stain.

Gel-free analysis of the phosphoproteome of S. aureus COL

Apart from the 2D-gel based approach, a gel-free phospho-peptide centered workflow has succeeded markedly in bacterialphosphoproteomics since a first study on the phosphoproteome onB. subtilis by Macek et al. (2007). Here, the protein extract is at firstdigested with an appropriate protease and subjected to a combina-tion of strong ion exchange chromatography and TiO2 enrichment.

Starting with a high amount of protein extract (mg range), the low-abundant phosphopeptides in the digest are specifically retainedby TiO2 after an initial SCX separation taking advantage of theiracidic nature. With this approach, we were able to identify 49

stained (phosphorylated proteins, red) proteins of exponentially growing S. aureusable S1-A and S1-B). Multiple protein spots corresponding to the same protein are

K. Bäsell et al. / International Journal of Medical Microbiology 304 (2014) 121– 132 125

Fig. 2. Dual channel image of Flamingo-stained (total protein amount, green) and Pro-Q-stained (phosphorylated proteins, red) proteins of exponentially growing S. aureusc rting ip e to stH

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ells on a 2D-gel (pH range 4.5–5.5). Phosphorylated proteins are labeled (see supporotein are enumerated. Newly appearing phosphorylated protein spots in responschA-2, Pgm-3).

eptides with mapped phosphorylation sites belonging to 33 dif-erent proteins.

Only recently, Elsholz et al. have shown that phosphorylationn arginine residues exists in vivo and is functional in B. subtilisElsholz et al., 2012). In a proof of principle study we have investi-ated the existence of protein arginine phosphorylation in vivo in. aureus. We applied the standard gel-free and site-specific proto-ol (Olsen and Macek, 2009) with slight variations in timing of theorkflow to take into account the challenges of protein argininehosphorylation. This way, we could identify 8 phosphosites on 8ifferent proteins in S. aureus COL. These findings prove that argi-ine phosphorylations are experimentally accessible via gel-freehosphopoteome analyses even in other gram-positive bacteria.

omparison of gel-based vs. gel-free results

Altogether, the combination of gel-based and gel-freepproaches led to the identification of 108 phosphorylatedroteins (including the 80 putatively phosphorylated Pro-Q dia-ond stained proteins from the 2D-gel analysis) with 76 different

hosphorylation sites; among them we determined 38 serine, 19hreonine and 11 tyrosine and 8 arginine residues (see Table S1-And Figure S5-A).

The advantage of applying gel-based and gel-free workflows inlucidating post translational modifications is their complementar-ty with respect to the physico-chemical properties exploited forpecific enrichment and detection. As earlier studies have already

nformation Table S1-A and S1-B). Multiple protein spots corresponding to the sameress/starvation conditions are marked with a red circle and labeled in red (FdaB-2,

shown, the overlap of identified phosphorylation sites in gel-basedvs. gel-free studies is surprisingly limited (Eymann et al., 2007;Macek et al., 2007). Accordingly, we obtained an overlap of solelythree common annotated phosphosites on the same molecularentity in the present study. Besides, ten distinct phosphorylationsites found in the gel-free approach could be assigned to eightputatively phosphorylated proteins on the gel (MetE (3 phospho-sites), Pgm, Pyk, SACOL0427, SACOL0875, SACOL2173, SACOL2501,SodA2).

An explanation for this relatively small overlap may be found inthe different technical challenges of the two approaches. Despitethe specific staining of the post translational modification in theshifted protein spots in 2D-gel analysis, direct mass spectrometricevidence is often challenging. It is known that especially phosphategroups are both prone to irreversible adhesion on surfaces in sam-ple preparation and ion suppression effects in LC–MS/MS analyses(Mann et al., 2002). This leads to the caveat in gel-based phos-phoproteome analyses that, although biochemically stable during2D-gel analysis and phosphosensitive staining, further mapping ofphosphorylation sites in LC–MS/MS analyses is difficult withoutprior concentration steps as performed for the gel-free approach.Furthermore, the physico-chemical characteristics of the peptideswithin the analytical window of both methods are considerably

different, e.g. in pI. Most of the phosphorylated peptides detectedin the gel-based approach have a pI below 2.7 and are thereforeexcluded from the analytical window of the SCX approach. Themobile phase in the strong cation exchange chromatographic run is

1 f Medical Microbiology 304 (2014) 121– 132

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Fig. 3. Structure of the FBP-aldolase of M. tuberculosis including the substrate FBP.The surface colors are reflecting sequence homologies based on structure alignmentsof five different bacterial class II aldolases (M. tuberculosis, H. pylori, E. coli, Bacillusanthracis and Campylobacter jejuni). The scale from red to yellow relates from strongto weaker homology. Homologous putative phosphorylation sites are colored pink.

26 K. Bäsell et al. / International Journal o

djusted to a pH of approximately 2.7. Therefore, phosphorylatedeptides with a pI less than 2.7 are not fully protonated, resulting

n a zero or negative overall charge. As a result, these peptides areot retained by the SCX resin and are consecutively found in theowthrough of the SCX run as has been shown in earlier studiesBeausoleil et al., 2004; Eymann et al., 2007; Macek et al., 2007). Tonderline this situation, common identification of phosphosites,.g. in B. subtilis for distinct molecular entities was possible onlyhrough missed cleavage sites leading to a shift of the resultinghosphopeptides toward the analytical window of the respectiveethod (see Table S5-B; AroA, RsbRA, YbbT and YqhA) (Macek et al.,

007).Besides this exclusion based on the pI, other limitations in

ongruent identification in both approaches apply: Ten of the 33hosphoproteins identified in the gel-free approach would theo-etically be detectable in the pH range 4–7 but are not found inhe gel-based approach. For all other phosphoproteins it can betated that either the pI/molecular weight lies outside the gel ranger that the proteins comprise several transmembrane domains.hilst these proteins lie within reach for detection in the gel-freeorkflow as octylglycoside was used as detergent in the samplereparation process, in 2D-gel based analyses, membrane proteinsre mostly excluded due to their hydrophobic nature. Possiblexplanations for the proteins being theoretically detectable but notound are their low abundance and/or insufficient separation fromigh-abundant non-phosphorylated protein spots.

A unique feature of gel-free analyses has only been recently beeniscovered. According to the findings in B. subtilis, we were able toap 8 arginine phosphorylations in a proof of principle study for S.

ureus. Other than for S/T/Y phosphorylations, arginine phosphory-ations are not stable under the conditions the gel-based approachs performed and therefore targeting arginine phosphorylations byel-free approaches is truly complementary.

hosphorylation sites revisited: conserved phosphosites andodification of already characterized binding regions

S. aureus belongs to the highly relevant pathogens due to a mas-ive increase in resistance against a growing number of antibioticgents. Accordingly, phosphorylation events of proteins specific foracterial pathogens or proteins representing virulence factors aref high interest in a global phosphorylation study. Two groups ofroteins are in the focus of the current study: classical virulence fac-ors displayed at the surface or loosely attached to the bacterial cell

embrane as well as surface-located protein binding factors alsoound within the bacterial cell. Virulence factors that are secretedr only found in the membrane are not in the focus of the currenttudy and necessitate further fractionation schemes (Becher et al.,009).

For an overview of the cellular processes affected by pro-ein phosphorylation, we grouped the phosphorylated proteinsccording to their functional categories (JCVI cellular role cate-ory (Peterson et al., 2001)). Fifteen different functional classesould be distinguished, which confirmed the general importancef phosphorylation in cellular processes of S. aureus as alreadyemonstrated for other bacteria (Eymann et al., 2007; Macek et al.,008, 2007; Schmidl et al., 2010; Soufi et al., 2008a). A large groupmong the phosphorylated enzymes and transporters belongs tohe energy metabolism (Figure S5-C). This seems to reflect a gen-ral situation in many prokaryotic organisms. Despite a number ofhosphorylated proteins being found uniquely in a specific organ-

sm, with more datasets being accessible, the evidence for common

hosphorylation profiles in various prokaryotic species is growingMisra et al., 2011).

Two proteins involved in energy metabolism are most inter-sting in terms of conserved phosphorylation patterns in bacteria:

The structures were extracted from the RscB-PDB database (RCSB Protein Data Bank;http://www.rcsb.org/pdb/home/home.do) and were modified with Chimera 1.6.2.(UCSF Chimera package).

the glycolytic enzyme FbaA belonging to a subclass of isoenzymesunique to microorganisms (Fonvielle et al., 2004) and the glycolyticisomerase TpiA (Furuya and Ikeda, 2011).

Fructose-1,6-bisphosphate aldolase FbaA catalyzes the con-version of fructose-1,6-bisphosphate into dihydroxyacetone-phosphate and glyceraldehyde-3-phosphate. Two classes of thisenzyme have evolved: whilst class I is found in all kingdoms, classII exclusively occurs in microorganisms like bacteria and yeasts(Marsh and Lebherz, 1992). Due to the differing molecular modeof action, the class II aldolase is regarded as a promising new tar-get for antimicrobial therapy with intensive research on specificantagonists under way (Daher et al., 2010). We detected FbaA intwo Pro-Q diamond spots to be putatively phosphorylated witha phosphosite at T234. This is in agreement to a study givingexperimental evidence of the enzyme to be a substrate of a ser-ine/threonine kinase (SA1063, homologous to PknB) (Donat et al.,2009; Lomas-Lopez et al., 2007; Truong-Bolduc et al., 2008). Analignment of the homologous proteins from different bacteria withavailable phosphoprotein data (see Figure S5-D and Fig. 3) revealsthat three distinct serine and threonine residues in close proxim-ity to the annotated active site seem to be conserved (S50, S211and T234 of the staphylococcal FbaA). S50 has been determined asbeing phosphorylated in Listeria monocytogenes (Misra et al., 2011)and Streptococcus pneumoniae (Supplemental S5-A and S5-B). Forthe S/T211 residues, S211 in L. monocytogenes (Misra et al., 2011),S211 in S. pneumoniae (supplemental S5-A and S5-B) and T212 inB. subtilis (Macek et al., 2007) have been proven, and in the studyfrom Prisic et al. a phosphorylation event could not be specifiedfurther than to one of three different serines located in this region(S255, S257 or S260) (Prisic et al., 2010). Additionally, T234 has beenmapped as being modified in B. subtilis (T234 (Macek et al., 2007))and in the present study for S. aureus (Table S1-A). As judged fromthe theoretically accessible peptides from FbaA in S. aureus, it ismost probable that a possible phosphorylation at S50 is precludedfrom analysis due to peptidase specificity. To verify the remaining

hypothesized phosphorylation sites in FbaA, we changed the pro-tease for the enzymatic digest of the putatively phosphorylatedspot from trypsin to the V8 protease also known as GluC. Indeed,now we were able to map the two other additional, apparently

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ighly conserved phosphosites, namely S50 and T212. Althoughtill under examination, we believe that this pattern of phosphory-ation is highly conserved within the class II aldolases and that thisact will help to contribute to the development of new specific classI aldolase antagonists serving as antimicrobial agents in the future.

The second interesting enzyme from the group of “energyetabolism” is TpiA, a glycolytic enzyme with a putative role as

plasminogen binding factor (Furuya and Ikeda, 2011). TpiA haseen annotated to be post translationally modified in multiple stud-

es: Schmidl et al. (2010) and Voisin et al. (2007) found TpiA beingutatively phosphorylated in Mycoplasma pneumoniae and Campy-

obacter jejuni but did not determine any phosphosites. For B. subtilisnd S. aureus the serine residues S213 and S215 were mapped aseing modified ((Macek et al., 2007), Table S1-A). Here, the con-erved phosphosite is directly located at the triose binding siteFuruya and Ikeda, 2011) in a loop crucial for structural aspects innzymatic activity (Casteleijn et al., 2006). First of all, the vicin-ty of the phosphorylation site to the active site of the enzymemplies a regulatory role of the phosphorylation for glycolysis ineneral. The fact that TpiA may play a role in binding of eukary-tic glycoproteins apparently additionally opens up the questionf a potential regulatory role of this particular phosphorylationvent apart from the proper role in glycolysis. Here, future detailedocalization-specific phosphorylation analyses will possibly help tonderstand the intracellular function of the triose isomerase as wells its speculative role outside the cell.

Both examples of classic enzymes of the central carbonetabolism with implicated roles in bacterial virulence or puta-

ively serving as antimicrobial targets underline that the molecularharacterization of these enzymes, e.g. in crystallization studies,eeds further refinement with respect to potential phosphorylationvents that should also be taken into account in calculation of elec-ron densities. Here, especially the prediction of binding pocketsnd potential binding structures without knowledge of phosphor-lation events will lead to biased results (Daher et al., 2010).

We also mapped two distinct phosphosites in the pleiotropic vir-lence regulator SarA: S106 and on S109 (Table S1-A, S1-B). It wasound earlier that SarA is a target of the kinases PknB and SA0077n vitro and in vivo, leaving the phosphorylation site unidentifiedDidier et al., 2010). Although the potential site of post translational

odification that was postulated due to functional reasons differsrom the sites that have now been mapped (Didier et al., 2010),ur findings correlate to the phosphosites of MgrA. This regulatorf the SarA family is phosphorylated by PknB at residues S110 and113 (Truong-Bolduc and Hooper, 2010). The fact that both homol-gous proteins with regulator function combined with DNA bindingapacity are phosphorylated in the same region suggests that thesehosphorylations are important for the regulatory events as wasreviously shown for MgrA (Truong-Bolduc and Hooper, 2010).

Interestingly, Sun et al. have determined recently phosphory-ation occurring in the SarA/MgrA family (SarA, MgrA, and SarZ)n conserved cysteine residues (Sun et al., 2012). It remains to beroven if the cysteine phosphorylation is the main PTM event onhese proteins, or if at least for SarA either PTM on serines 106 and09 and the proposed cysteine are of regulatory importance in vivo.

A genuine membrane-bound protein found in our study by gel-ree methods is the elastin binding protein EbpS (SACOL1522).lthough studies on the elastin binding function have proven toe doubtful (Park et al., 1999; Roche et al., 2004), involvement inirulence has again been suggested according to a global networknalysis (Overton et al., 2011). This protein has two extracellularomains, a conserved LysM domain functioning in peptidoglycan

inding and an N-terminal extracellular stretch that was originallyroposed to exert an elastin binding function. It is striking that threehosphosites are located in the latter extracellular loop at residues2, T64 and S128. We think, with our data at hand, this will add a

cal Microbiology 304 (2014) 121– 132 127

new aspect in potential target binding, underlining the importanceof post translational modifications for virulence. Consequently,future studies on pathogen–host interaction should generally con-sider these modifications in developing binding assays or mappingspecific binding sites.

Determination of protein phosphorylation under stress andstarvation

Protein phosphorylation is a possibility for regulatory finetuning of the bacterial metabolism. Therefore, changes in phos-phorylation patterns in response to stress and starvation conditionsmay be directly linked to regulatory processes taking place insidethe bacterial cell. Our gel-based approach supports a quan-titative estimation of changes in the phosphorylation eventsfor putatively phosphorylated proteins by differential gel imageanalysis. In order to decipher changes, newly appearing phospho-rylated protein species and proteins with significantly increased ordecreased ratios of phosphorylation intensity signals (log 2 Pro-Q-signalstress/starvation/Pro-Q-signalcontrol>1 and <−1) were consideredto be significantly altered.

Pathogenic bacteria are generally exposed to changing environ-mental conditions including variations in pH, changing nutrientsupply, dehydration and host cell defense mechanisms. To reflectthese conditions we applied glucose starvation, salt stress, diamidestress and NO stress to cultures of growing S. aureus COL. Accord-ingly, we determined the relative changes in phosphorylationpatterns for ten putatively phosphorylated proteins on the 2D-gels(see Table 1).

Contrary to the expected, the quantitative results for the oxida-tive stress and the osmotic stress were not leading to new insightsinto potential regulations on the phosphorylation level. It is knownthat especially for stress caused by reactive oxygen species insidethe cell, post translational modifications of active sites at cys-teine residues are protecting the cells from irreversible oxidation.Apparently it seems that for both physiological situations other reg-ulatory processes are determinants for a swift cellular reaction tocounteract the detrimental effects of the stress or besides that, inthese cases our analytical window for examination of quantitativechanges on 2D-gels is not sufficient for evaluation of stress specificchanges in the phosphorylation pattern.

Whilst we found numerically the most changes in phosphor-ylation patterns in glucose starved cells (DeoB-1, DeoB-3, Pgm-3,SACOL1588, Pyk-4 and SACOL1483), exposure to nitrosative stressdid not only alter the levels, but led to new phosphorylations inFdaB and HchA (see Fig. 4). NO is regarded as a potent agent of theinnate immune response of the host being faced with pathogenicbacteria. Therefore, protection against NO is essential for survivalof S. aureus inside its host (Lewis et al., 1995; Nalwaya and Deen,2005). With NO leading to inhibition of aerobic respiration, it hasbeen shown earlier that cellular processes belonging to anaerobicmetabolism are induced, and enhanced glycolytic activity could bedetermined (Borisov et al., 2004; Hochgräfe et al., 2008).

S. aureus possesses a multilevel answer to nitrosative stress:first, to sustain energy charge and redox homeostasis, the bacteriashift to fermentation with high rates of l- and d-lactate secretion.Inactivation of the active sites of pyruvate dehydrogenase (PDH)and pyruvate formate-lyase (PFL) prevents pyruvate from beingintroduced into the tricarboxylic acid cycle (TCA) with concomi-tant depletion of the acetyl-CoA level (Richardson et al., 2008).Second, the cell heavily induces the protein Hmp which is regardedas a NO scavenging enzyme (Muntel et al., 2012; Richardson et al.,

2008). Third, the general protection of the free thiols is maintainedby three different thiol redox buffers: cysteine, coenzyme A andBacillithiol (Hochgräfe et al., 2008). These protective systems notonly allow the cell to survive the nitrosative damage induced by

128 K. Bäsell et al. / International Journal of Medical Microbiology 304 (2014) 121– 132

Table 1Quantitation results for Pro-Q Diamond signal intensities of phosphorylated proteins. The table contains only those protein spots revealing altered signal intensities with anaverage change of ±1 in the log 2 range. The bar charts refer to the left axes showing absolute % V values and the orange circles reflect the average log 2 ratio of the trans/statvs. control (glucose starvation) or the 15′/45′ vs. control sample (nitrosative stress, oxidative stress and osmotic stress). The quantitation was conducted with the softwareDelta 2D (Decodon GmbH, Greifswald, Germany).

Glucose starvationDeoB-1 %V control (co) %V

transient%V stationary log 2-ratio: %V

transient/%V colog 2-ratio: %Vstationary/%Vco

Biol. replicate 1 0.24 0.26 0.44 0.12 0.88Biol. replicate 2 0.12 0.04 0.66 −1.49 2.44Biol. replicate 3 0.24 0.61 0.84 1.36 1.83Average 0.20 0.30 0.64 0.00 1.71stdev 0.07 0.28 0.20

DeoB-3 %V control (co) %Vtransient

%V stationary log 2-ratio: %Vtransient/%V co

log 2-ratio: %Vstationary/%Vco

Biol. replicate 1 0.25 0.31 0.54 0.29 1.10Biol. replicate 2 0.23 0.04 0.57 −2.74 1.28Biol. replicate 3 0.25 0.77 0.52 1.61 1.06Average 0.25 0.37 0.54 -0.28 1.15stdev 0.01 0.37 0.02

Pgm-3 %V control (co) %Vtransient

%V stationary log 2-ratio: %Vtransient/%V co

log 2-ratio: %Vstationary/%Vco

Biol. replicate 1 0.14 0.21 0.25 0.62 0.85Biol. replicate 2 0.12 0.05 0.34 -1.40 1.49Biol. replicate 3 0.14 0.18 0.46 0.35 1.72Average 0.13 0.14 0.35 -0.14 1.35stdev 0.01 0.09 0.10

SACOL1588 %V control (co) %V transient %Vstationary

log 2-ratio: %Vtransient/%V co

log 2-ratio: %Vstationary/%Vco

Biol. replicate 1 0.13 0.15 0.26 0.25 1.04Biol. replicate 2 0.17 0.08 0.36 −1.17 1.05Biol. replicate 3 0.13 0.28 0.32 1.14 1.33Average 0.14 0.17 0.31 0.07 1.14stdev 0.03 0.10 0.05

Pyk-4 %V control (co) %Vtransient

%V stationary log 2-ratio: %Vtransient/%V co

log 2-ratio: %Vstationary/%Vco

Biol. replicate 1 0.91 0.80 0.41 -1.15 -0.18Biol. replicate 2 1.05 0.02 0.06 -5.45 -4.18Biol. replicate 3 0.91 0.69 0.41 -0.40 -1.17Average 0.96 0.51 0.29 -2.33 -1.84stdev 0.08 0.42 0.20

SACOL1483 %V control (co) %V transient %Vstationary

log 2-ratio: %Vtransient/%V co

log 2-ratio: %Vstationary/%Vco

Biol. replicate 1 0.25 0.12 0.10 -1.11 -1.34Biol. replicate 2 0.15 0.02 0.06 -2.63 -1.36Biol. replicate 3 0.25 0.05 0.06 -2.31 -2.03Average 0.22 0.06 0.07 -2.02 -1.58stdev 0.06 0.05 0.02

K. Bäsell et al. / International Journal of Medical Microbiology 304 (2014) 121– 132 129

Table 1 (Continued )

Nitrosative stressFdaB-2 %V control (co) %V 15 min

NO%V 45 min NO log 2-ratio: %V

15 min/%V colog 2-ratio: %V45 min/%V co

Biol. replicate 1 0.38 2.30 2.26 2.62 2.59Biol. replicate 2 0.33 2.01 1.69 2.60 2.34Biol. replicate 3 0.37 2.11 2.06 2.50 2.48Average 0.36 2.14 2.00 2.57 2.47stdev 0.02 0.15 0.29

HchA %V control (co) %V 15 minNO

%V 45 min NO log 2-ratio: %V15 min/%V co

log 2-ratio: %V45 min/%V co

Biol. replicate 1 0.30 1.04 1.21 1.78 2.00Biol. replicate 2 0.19 0.60 0.37 1.63 0.95Biol. replicate 3 0.39 1.23 1.06 1.67 1.45Average 0.29 0.96 0.88 1.69 1.47stdev 0.10 0.32 0.45

oxidative stressPyk-4 %V control (co) %V 15 min

diamide%V 45 mindiamide

log 2-ratio: %V15 min/%V co

log 2-ratio: %V45 min/%V co

Biol. replicate 1 1.67 3.07 2.07 0.88 0.31Biol. replicate 2 1.28 2.79 2.49 1.12 0.96Biol. replicate 3 1.28 2.96 3.59 1.21 1.49Average 1.41 2.94 2.71 1.07 0.92stdev 0.22 0.14 0.78

Osmotic stressDeoB-1 %V control (co) %V 15 min

NaCl%V 45 min NaCl log 2-ratio: %V

15 min/%V colog 2-ratio: %V45 min/%V co

Biol. replicate 1 0.09 0.13 0.29 0.45 1.62Biol. replicate 2 0.09 nd. 0.26 nd. 1.49Biol. replicate 3 0.11 0.17 0.23 0.45 1.07Average 0.10 0.15 0.26 0.45 1.39stdev 0.01 0.03 0.03

Fig. 4. Significant changes of the Pro-Q signal intensities of FdaB-2 and HchA-2 during nitrosative stress. The bar charts display signal intensities in volume percentage (%V)for each protein spot, each replicate and each time point, respectively. The assigned region on the 2D-gel image (Pro-Q diamond (red) vs. Flamingo fluorescent stain (green))for the corresponding spot is shown on top of the single bar chart representations. Volume percentage values in the bar chart for control spots are not to be followed visuallyon the dual channel image. The calculated values are based on the spot detection algorithm implemented in Delta2D taking into account gray scale values below the limit ofdetection in quantification workflows to avoid calculation on spots without signal (division by zero).

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O but also, unlike other pathogenic bacteria, to replicate duringitrosative stress (Richardson et al., 2008).

A consequence of the change toward lactate fermentation ishe fact that the cell solely relies on the conversion of phospho-ylated sugars to pyruvate with subsequent conversion to lactate.. aureus possesses two different fructose-bisphosphate aldolaseshat function in the conversion of C6 phosphosugars to C3 metabo-ites, FbaA and FdaB. The Pro-Q diamond signal of the putativelyhosphorylated FdaB is already present to a certain amount dur-

ng exponential growth, but after the onset of NO stress a newutatively phosphorylated Pro-Q diamond signal appears (FdaB-, see Fig. 4). It may be speculated that similar to the situation

n Borrelia burgdorferi (Bourret et al., 2011), the class II fructose-ldolase FbaA is prone to nitrosative damage in S. aureus. As theonversion of fructose-1,6-bisphosphate into dihydroxyacetone-hosphate (DHAP) and glyceraldehyde-3-phosphate is essential in

actate fermentation, most probably the NO insensitive isoenzymedaB, a class I aldolase, is compensating for the assumed loss ofunction.

The conversion of metabolites in the glycolytic pathway toward-lactate may be regarded as a type of overflow metabolismLandmann et al., 2011). This comes with severe implications forhe cell: it is anticipated that an imbalance between fluxes of thepper and the lower part of the glycolytic pathway may result in theccumulation of phosphorylated glycolytic intermediates. In thisituation, the excess of DHAP poses a severe threat to the organ-sm: DHAP is easily enzymatically or non-enzymatically convertednto methylglyoxal which is cytotoxic and must be removed by alyoxalase (Cooper, 1984; Ferguson et al., 1998; Landmann et al.,011).

Accordingly, we see HchA, a protein that is described both with ahaperone function (Hsp31) and a glyoxalase III function indepen-ent from glutathione, that markedly changes in Pro-Q diamondignal with a second Pro-Q diamond spot appearing during NO-tress in our study (HchA-2, see Fig. 4). In a study on nitrosativetress in S. aureus it was shown earlier that HchA shifts toward

more acidic pH on a 2D-gel due to an anticipated modificationn peroxide stressed cells after preincubation with NO (Hochgräfet al., 2008). Lomas-Lopez et al. have shown that HchA is phos-horylated on a tyrosine residue in S. aureus growing in minimaledium (Lomas-Lopez et al., 2007). Combined with our data, this

uggests that the enzyme is indeed already phosphorylated inrowing cells and additionally, after the onset of NO stress, aew phosphorylation signal according to Pro-Q diamond stainingmerges on the 2D-gel.

In the close relative B. subtilis, it has been observed recentlyhat methylglyoxal synthases function as alternative ways ofirecting C3 sugar phosphates toward lactate in overflow sit-ations being stringently regulated depending on the type ofarbon source by an interacting regulating protein Crh/Crh-PLandmann et al., 2011). As Crh and the methylglyoxal synthasere to date not found in S. aureus, we believe that HchA func-ions as a detoxification system for non-enzymatically formed

ethylglyoxal. Consequently, we hypothesize that phosphoryla-ion events are either responsible or an indication for a swiftegulation in the basic carbon metabolism and eventually for detox-fication systems of methylglyoxal in NO-stressed staphylococcalells.

oncluding remarks

With respect to the elucidation of regulatory processes inathogenic bacteria, our knowledge based on transcriptome androteome data as well as on detailed biochemical characterizationas been expanded considerably during the last years. However,

cal Microbiology 304 (2014) 121– 132

the large-scale characterization of post translational modificationsstill lags behind. Since 2007, the number of studies on bacterialphosphoproteomics based on gel-free approaches has increasedsignificantly and first studies aim at comparing the publisheddatasets (Macek and Mijakovic, 2011). The goal for such a com-parison is the discovery of phosphorylation patterns for conservedmetabolic pathways or specific functions that may be affected bythis post translational modification (Soufi et al., 2008b).

Consequently, protein–protein interaction networks of phos-phorylated proteins have been computed for S. pneumonia andHelicobacter pylori (Ge et al., 2011; Misra et al., 2011), and Misraet al. as well as Sun et al. have compared both conserved sites ofribosomal and glycolytic proteins and phosphoproteins in generalbetween phosphoproteome datasets (Misra et al., 2011; Sun et al.,2012).

The key question in our study was the presumed link of phos-phorylation with processes of bacterial metabolism, virulence andhost-interaction in the human pathogen S. aureus. We pursuedtwo different strategies. The first comprised a comprehensivemapping of phosphosites by gel-based and gel-free phospho-targeted approaches. The second was a quantitative trackingof putatively phosphorylated proteins by differential image gelanalysis.

Altogether, we could identify 108 phosphorylated proteins with76 different phosphorylation sites. The complementarity of thegel-based and the gel-free method is discussed with emphasison specificities of each method resulting in almost disjunctivephosphopeptide datasets. Despite the fact that we distinguish 15different functional classes according to JCVI, a key aspect of phos-phorylation events is the energy metabolism. It could be shown thatFbaA, a class II aldolase and intensively investigated potential targetfor antimicrobial agents, is phosphorylated at distinct sites appar-ently conserved within various pathogens. Phosphorylation sitescould be mapped accurately for TpiA, a glycolytic enzyme addi-tionally found to be able to bind to eukaryotic glycoproteins andfurthermore for EbpS, a membrane protein with annotated extra-cellular binding activities that has been found to be phosphorylatedon one of the extracellular loops. Consequently, we strongly suggestconsideration of phosphorylation events in detailed biochemicalbinding assays in the development of new antimicrobial targetsand the molecular characterization of adhesion factors.

A key finding was the variation in phosphorylation patterns inresponse to nitrosative stress. Upon NO stress, for two enzymes ofthe lower part of glycolysis new, putatively phosphorylated Pro-Q spots appear. We speculate that this might be a swift way ofmodulating metabolic pathways compensating for enzyme inacti-vation and coping with a metabolic overflow situation leading to anincreased level of cytotoxic, non-enzymatically formed metabolicby-products.

Altogether, this comprehensive phosphoproteome dataset pro-vides an excellent base for further investigations on regulatorymechanisms involved in pathogenicity/virulence. In the light ofour findings on arginine phosphorylations that goes together withthe respective work in B. subtilis (Elsholz et al., 2012), cysteinephosphorylations that play a role in the regulation of virulencein S. aureus (Sun et al., 2012) together with findings on crosstalkbetween phosphorylation and acetylation events in pathogenbacteria (Soufi et al., 2012; van Noort et al., 2012), further stud-ies targeting not only the S/T/Y-phosphorylations on a global scaleare needed.

Conflict of interest

The authors have declared no conflict of interest.

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cknowledgements

This work was supported by grants from the Deutscheorschungsgemeinschaft (SFB/TR34 and HE1887/8-1) and theundesministerium für Bildung und Forschung (0315828A andIK-FunGene). We thank Sebastian Grund and Jürgen Bartel forxcellent technical assistance and Martin Zander for support in cul-ivation of Staphylococcus aureus cells and sample preparation. Weould like to thank Gottfried Palm for helpful hints on aspects of

tructural biology. We thank Holger Kock for critical reading of theanuscript.

ppendix A. Supplementary data

Supplementary data associated with this article can beound, in the online version, at http://dx.doi.org/10.1016/j.ijmm.013.11.020.

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