Phosphoproteomic Analysis Reveals the Multiple Roles of

Nov 8, 2009 - Bacterial protein phosphorylation regulates virulence at gene levels;(19-21) for example, the eukaryotic-type Ser/Thr protein kinase Stk...
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Phosphoproteomic Analysis Reveals the Multiple Roles of Phosphorylation in Pathogenic Bacterium Streptococcus pneumoniae Xuesong Sun,†,# Feng Ge,†,# Chuan-Le Xiao,† Xing-Feng Yin,† Ruiguang Ge,‡ Liu-Hui Zhang,† and Qing-Yu He*,† Institute of Life and Health Engineering and National Engineering Research Center for Genetic Medicine, Jinan University, Guangzhou 510632, China, and The Laboratory of Integrative Biology, College of Life Sciences, Sun Yat-Sen University, Guangzhou 510006, China Received July 13, 2009

Recent phosphoproteomic characterizations of Bacillus subtilis, Escherichia coli, Lactococcus lactis, Pseudomonas putida, and Pseudomonas aeruginosa have suggested that protein phosphorylation on serine, threonine, and tyrosine residues is a major regulatory post-translational modification in bacteria. In this study, we carried out a global and site-specific phosphoproteomic analysis on the Gram-positive pathogenic bacterium Streptococcus pneumoniae. One hundred and two unique phosphopeptides and 163 phosphorylation sites with distributions of 47%/44%/9% for Ser/Thr/Tyr phosphorylations from 84 S. pneumoniae proteins were identified through the combined use of TiO2 enrichment and LC-MS/MS determination. The identified phosphoproteins were found to be involved in various biological processes including carbon/protein/nucleotide metabolisms, cell cycle and division regulation. A striking characteristic of S. pneumoniae phosphoproteome is the large number of multiple species-specific phosphorylated sites, indicating that high level of protein phosphorylation may play important roles in regulating many metabolic pathways and bacterial virulence. Keywords: Phosphoproteome / • Streptococcus pneumoniae / • Kinase

Introduction Protein phosphorylation is a key post-translational modification that is important for signal transduction and regulation.1,2 Eukaryotic cells rely on the specific covalent phosphorylation on the hydroxyl group of Ser/Thr/Tyr for signal transduction.3,4 Phosphorylation processes are catalyzed in a reversible fashion by specific protein kinases and phosphatases. In the process, protein kinases modify other proteins by chemically adding phosphate groups to amino acid residues of the proteins, whereas protein phosphatases remove the phosphate groups that have been attached by protein kinases. For many years, protein phosphorylation has been considered to exist exclusively in eukaryotes. The first observation demonstrating that protein phosphorylation also occurs in bacteria was reported in the late 1970s.5,6 Signal transduction in prokaryotes was previously thought to take place primarily through histidine kinases. The bacterial homologues of eukaryotic-type Ser/Thr kinases and phosphatases were later found to be necessary for cellular functions such as cell growth, differentiation, pathogenicity, heat shock response, and secondary metabolism.7,8 Recent phosphoproteomic studies on * To whom correspondence should be addressed: Qing-Yu He, Institute of Life and Health Engineering, Jinan University, Guangzhou 510632, China. Tel and fax: +86-20-85227039. E-mail: [email protected]. † Jinan University. # These authors contributed equally to this work. ‡ Sun Yat-Sen University. 10.1021/pr900612v

 2010 American Chemical Society

classical model organisms, including the Gram-positive Bacillus subtilis (B. subtilis), Gram-negative Escherichia coli (E. coli), Pseudomonas putida (P. putida) and Pseudomonas aeruginosa (P. aeruginosa), and an industrial organism Lactococcus lactis (L. lactis), demonstrated that, in bacteria, phosphoproteins, especially the phosphorylated residues, are significantly more conserved than their nonphosphorylated counterparts.9-12 Some of the potential phosphorylated sites are highly conserved from Archaea to humans. Streptococcus pneumoniae (S. pneumoniae) is a Grampositive pathogenic bacterium, causing a number of human diseases including otitis, sinusitis, bacterial meningitis, sepsis, and pneumonia.13,14 Most affected by this organism are children, or individuals with immature/compromised immune systems, such as patients with diabetes or acquired immunodeficiency syndrome.15-17 Bacterial pathogens including S. pneumoniae have developed a variety of strategies to successfully infect hosts, including bacterial adherence to the host cells, pathogen uptake and invasion, and replication inside the host to lead to the final cell death.18 One strategy involves the protein phosphorylation at Ser/Thr/Tyr, resulting from the combinational actions of specific protein kinases and protein phosphatases from either the bacterial pathogens themselves or the host eukaryotic cells. Bacterial protein phosphorylation regulates virulence at gene levels;19-21 for example, the eukaryotic-type Ser/Thr protein kinase StkP works as a global regulator of gene expression in Journal of Proteome Research 2010, 9, 275–282 275 Published on Web 11/08/2009

research articles S. pneumoniae. Phosphorylation also controls the characteristics of bacterial cell surface via affecting the extracellular polysaccharide synthesis to guarantee optimal attachment to the host cells. This kind of regulation through phosphorylation modifications has been characterized in S. pneumoniae where CpsA, CpsB, CpsC and CpsD are components of a tyrosine phosphorylation regulatory system in the modulation of capsule synthesis in the bacterium.22 In addition, bacterial protein phosphorylation interferes with host phosphorylation-dependent signaling by exporting kinases and phosphatases, allowing the pathogens to survive in the host.23,24 Other than these reports about phosphorylation in specific proteins, the comprehensive phosphoproteome of Streptococcus species has not yet been globally characterized. In this study, we enzymatically digested the entire S. pneumoniae D39 proteome and enriched the phosphopeptides by using titanium dioxide (TiO2). The phosphopeptides were analyzed by a hybrid LTQ-Orbitrap mass spectrometer. Totally, 102 unique phosphopeptides from 85 proteins were identified with 163 phosphorylation sites: 77 on serine, 71 on threonine and 15 on tyrosine. The number of phosphorylation sites in S. pneumoniae is much larger than those in E. coli, B. subtilis and L. lactis despite the fact that S. pneumoniae has a genome size comparable to L. lactis and only half that of E. coli and B. subtilis. The identified phosphoproteins involved in several important biological processes were functionally categorized into an interaction map. This is the first time that an interaction network of phosphoproteins in bacteria was constructed, which may help us to better understand the significance of phosphorylation in key cellular mechanisms.

Materials and Methods Cell Culture and Protein Extraction. S. pneumoniae D39 was grown in Todd-Hewitt broth supplemented with 0.5% yeast extract (THY) in a controlled atmosphere chamber (37 °C, 5% CO2). Bacterial growth was monitored by the light absorbance at 600 nm using a Thermo model Evolutionary 300 spectrometer. At OD600 ∼0.6, cells were harvested by centrifugation at 5000 g for 20 min at 4 °C. The harvested cells were washed three times with prechilled PBS (10 mM, pH 7.4). Bacteria were resuspended in an appropriate volume of lysis buffer (15 mM Tris-HCl, 7 M urea, 2 M thio-urea, 1% DTT and 4% CHAPS, pH 8.0) with 5 mM phosphatase inhibitors including sodium fluoride, 2-glycerol phosphate, sodium vanadate, and sodium pyrophosphate. The mixture was freeze-thawed for three cycles and then sonicated 6 times each for 30 s. The lysate was centrifuged at 12 000 g for 10 min at 4 °C. The resulting supernatants were stored in aliquots at -80 °C until further use. Protein concentrations were determined with Bradford assay. Protein Digestion. Protein extracts (1 mg) were subjected to disulfide reduction with 10 mM of DTT (37 °C, 3 h) and alkylation with 20 mM of iodoacetamide (25 °C, 1 h in dark). Proteins were precipitated with 4 vol of ice-cold acetone, collected by centrifugation and washed with ethanol twice. The pellet was redissolved in 50 mM of ammonium bicarbonate and then digested with sequencing grade modified trypsin (1: 25 w/w) (Promega, Madison, WI) at 37 °C for 20 h. Digested peptides were dried in a vacuum centrifuge. Phosphopeptide Enrichment with TiO2 Resin. Phosphopeptides from digested peptides were enriched using the Phosphopeptide Enrichment TiO2 kit (Calbiochem, San Diego, CA) according to the manufacturer’s instruction with slight 276

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Sun et al. modifications. Briefly, the tryptic digest was dried down, redissolved in 200 µL of TiO2 Phosphobind buffer containing 50 g/L of 2,5-dihydroxybenzoic acid and further mixed with 50 µL of TiO2 Phosphobind resins. After 30-min incubation, the supernatant was discarded, and TiO2 was washed three times with the washing buffer. After that, elution buffer was added twice to elute the phosphopeptides. The eluates were combined and dried using a Speed-Vac Concentrator and reconstituted in 5% acetonitrile (ACN)/0.1% of formic acid for LC-MS/MS analysis. All the buffers and the phosphoprotein purification resins were provided in the kit by the manufacturer. Peptide Analysis by the LC-MS/MS Approach. Online LCMS/MS was performed using a Finnigan Surveyor HPLC system with a 0.1 × 100 mm C18 reversed phase column (Michrom Bioresources, Auburn, CA) connected directly to a nanospray LTQ-Orbitrap mass spectrometer (Thermo Electron, San Jose, CA). Peptides were eluted with an increasing 0-35% (B/(A + B)) acetonitrile gradient (Buffer A, 0.1% formic acid and 5% ACN; Buffer B, 0.1% formic acid and 95% ACN) over the time of 90 min and monitored online with the mass spectrometer. The electrospray needle of the mass spectrometer was operated at 1.85 kV without sheath and auxiliary gas flow. Datadependent TOP5 method was used to generate fragment ions of individual peptides.25 The ion transfer tube was heated to 200 °C and ion fragmentation was achieved using 35% normalized collision energy for the MS2. Ion thresholds were set to 500 counts for MS2 and 120 counts for MS3. An activation q ) 0.25 and activation time of 30 ms was applied in MS2 acquisitions. For each cycle, one full MS scan in the Orbitrap at 1 × 106 AGC target was followed by 5 MS2 in the LTQ at 5000 AGC target on the five most intense ions. Selected ions were excluded from further analysis for 90 s. Maximum ion accumulation times were set at 500 ms for the full MS scan and 100 ms for the MS2 scans.26 To improve the fragmentation of phosphopeptides, an MS3 was triggered with similar accumulation times and AGC targets26 if a neutral loss peak at -98, -49, or -32.7 Da was detected in the MS2 and the peak belongs to the five most intense ions in the MS2 spectrum. Database Analysis and Manual Evaluation of Mass Spectra. For the peptide or protein identification, the forward and random NCBI S. pneumoniae D39 databases containing 1914 protein sequences were concatenated together. All the raw data files were processed using BioWorks 3.3.1 (Thermo Finnigan, San Jose, CA) and the derived peak lists were searched using the MASCOT search engine (Matrix Science, London, U.K.). The following search criteria were employed: full tryptic specificity required; two missed cleavages allowed; carbamidomethylation set as fixed modification, whereas oxidation (M), phospho (ST), and phospho (Y) considered as variable modifications. Initial mass deviation of precursor ions and fragment ions were allowed up to 10 ppm and 0.5 Da, respectively. This led to the identification of 7837 peptide sequences, including the phophorylated and nonphosphorylated peptides in samples. The phosphorylated peptides were then extracted by using MASCOT assignment. During the evaluation of the data, it was found that the peptide scores of the phosphopeptides assigned by MASCOT did not always correlate well with the quality of the spectra. Therefore, all fragmentation spectra of these potential phosphopeptides were manually verified using a method as described by Mann et al.9,10 The following validation criteria were used: (1) only peptides with more than 6 amino acid residues were considered; (2) Ser(P) and Thr(P) peptides must have a

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Figure 1. MS2 and MS3 spectra acquired in the LTQ mass spectrometer for a threonine-phosphorylated peptide (VVPEAPHETFLALDASTGQNALVQAK) from signal recognition particle-docking proteins FtsY. (A) As a specific signature of phosphorylation, pronounced neutral loss of phosphoric acid in the MS2 spectrum is indicated. (B) A MS3 spectrum, triggered by neutral loss of phosphoric acid in the MS2 spectrum. The identity of the phosphopeptide and the phosphorylated residue is shown in the panel.

pronounced neutral loss of phosphoric acid in the precursor ion and/or fragment ions or the neutral loss-dependent MS3 scan; (3) extensive coverage of b- and/or y-ion series was required; (4) proline-containing peptides must show pronounced cleavage N-terminally to the Pro residue; (5) all the other phosphopeptides without the above-mentioned typical fragmentation pattern were accepted when they were detected in two or more independent measurements or in two or more forms (e.g., with or without methionine oxidation, or as a complete and a missed cleavage form). Gene Ontology Network Generation and Visualization. Application of Gene Ontology (GO) database is required to submit the annotation files of S. pneumoniae D39 in a specified format.27 Each annotation row lists the GO term, the Uniport ID, and NCBI accession number. The bioinformatics toolbox of MATLAB was used to obtain one level of upward regulated GO terms of phosphoproteins and the interaction network was constructed by all GO terms.28,29 Each node was labeled by gene name, locus number or GO term number.

Results Phosphoproteome of Exponentially Growing S. pneumoniae. Here we used a label-free and gel-free method to carry out the global phosphoproteomic analysis of S. pneumoniae. In this approach, protein phosphorylation in pathogenic bacterium S. pneumoniae D39 was analyzed at the phosphorylation site level. One hundred and two unique phosphopeptides from 84 proteins were enriched with TiO2 resin from S. pneumoniae lysates and were further identified using a high-accuracy LTQOrbitrap mass spectrometer. Among the identified phosphopeptides, 163 phosphorylation sites were determined: 77 on serine, 71 on threonine and 15 on tyrosine. The low ratio of

tyrosine phosphoryaltion is not a surprise as this is always the case for the known phosphoproteomes, either from bacterial or human subjects. All the identified phosphopeptides with their protein NCBI accession numbers and gene names are listed in Supplementary Table S1. Relevant detailed information about the identified phosphopeptides is provided in the Supplementary Table S2. Figure 1 shows an example of the analysis of a phosphopeptide sequence and the assignment of the phosphorylation site. This triply charged peptide was detected in the MS2 spectrum at m/z 962.21. The MS2 spectrum of the peptide exhibited a clear loss of 98 Da, the signature cleavage of phosphoric acid molecule (H3PO4) from phosphorylated peptide ions (Figure 1A). To obtain the detailed information about the peptide backbone and the location of phosphorylation sites, a further MS3 analysis by the method of collision-induced dissociation was performed. MS3 spectrum identified the sequence as VVPEAPHETFLALDASTGQNALVQAK and the site of phosphorylation at Thr9 (Figure 1B). Classification of Phosphorylated Proteins. Information on the classes of the identified phosphoproteins was obtained using GO analysis and is presented in Table 1. Among the 84 identified S. pneumonaie phosphoproteins, 69 phosphoproteins were annotated for their functions in biological processes, and 39 for their respective cellular localizations. These phosphoproteins are present throughout the whole cell (Figure 2A), with around 10% phosphoproteins at the cell wall/membrane. Despite extensive and intensive literature searching, the localization information for around 50% of the identified phosphoproteins cannot be determined. The 84 phosphoproteins in S. pneumoniae are distributed widely as regards to their respective functions in biological processes (Figure 2B): 20% Journal of Proteome Research • Vol. 9, No. 1, 2010 277

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Table 1. Cellular Functional Groups of the Identified Phosphoproteins from S. pneumoniae

pyruvate kinase phosphoglycerate kinase L-lactate dehydrogenase S-adenosylmethionine synthetase formate acetyltransferase protein jag ribosomal protein S1 chaperonin GroEL peptide chain release factor 1 ribosomal protein L15 ribosomal protein L3 ribosomal protein L14

Carbon Metabolism phosphoenolpyruvate carboxylase trans-2-enoyl-ACP reductase II sugar ABC transporter, ATP-binding protein maltose/maltodextrin ABC transporter PTS system, mannose-specific IIAB components Protein Metabolism ribosomal protein L1 chaperone protein DnaK chaperonin, 33 kDa translation elongation factor Tu translation initiation factor IF-1 initiation factor IF-2

Amino Acid and Peptide Metabolism oligopeptide ABC transporter, oligopeptide-binding protein cell wall-associated serine protease PrtA AmiA amino acid ABC transporter, amino acid-binding protein peptidyl-prolyl cis-trans isomerase aminopeptidase N peptidase, U32 family protein peptide deformylase Nucleic Acid Metabolism DNA-directed RNA polymerase, alpha subunit alanyl-tRNA synthetase DNA-directed RNA polymerase, beta subunit phenylalanyl-tRNA synthetase, alpha subunit DNA-directed RNA polymerase, beta’ subunit tyrosyl-tRNA synthetase galactose operon repressor isoleucyl-tRNA synthetase single-strand binding protein lysyl-tRNA synthetase adenylate kinase transcription-repair coupling factor hydrolase, TatD family protein catabolite control protein A ribonucleoside-diphosphate reductase, alpha subunit type II restriction endonuclease Cell Cycle and Cell Division cell division protein DivIVA serine/threonine protein kinase cell division protein FtsA folylpolyglutamate synthase signal recognition particle-docking protein FtsY Others L-lactate

dehydrogenase autolysin/N-acetylmuramoyl-L-alanine amidase oxidoreductase, pyridine nucleotide-disulfide, class I glycosyl transferase, group 1 family protein metallo-beta-lactamase superfamily protein domain choline binding protein C

endo-beta-N-acetylglucosaminidase UDP-N-acetylglucosamine pyrophosphorylase maltodextrin phosphorylase hydrolase, TatD family protein zinc metalloprotease ZmpB Unknown

conserved hypothetical protein lipoprotein

in nucleotide metabolism, 16% in protein metabolism, 12% in carbon metabolism, 8% in amino acid and peptide metabolism and 6% in cell cycle and cell division. The remaining 16 phosphoproteins have not been reported with relevance to their

pneumococcal histidine triad protein A precursor

specific functions in the cellular biological processes. Interestingly, several proteins including the alpha, beta and beta′ subunits of DNA-directed RNA polymerase, galactose operon repressor and catabolite control protein A involved in the

Figure 2. Distribution of the identified phosphoproteins of S. pneumoniae according to the cellular location (A) and biological function (B). 278

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Figure 3. Interaction network map of the identified phosphoproteins in S. pneumoniae D39. Purple represents phosphoproteins identified in this study; blue and gray are proteins which interacted with the phosphoproteins. Important nodes were highlighted in yellow.

transcriptional process were also found to be phosphorylated in S. pneumoniae. Interaction Network of Phosphoproteins. To better understand the regulatory mechanisms in S. pneumoniae, protein interaction network for the identified phosphoproteins was constructed by using a GO network generation tool. As shown in Figure 3, this phosphoprotein interaction map consisted of a large network covering 123 proteins and five subnetworks to include all upstream regulatory proteins and downstream targets. Alpha-ribonucleoside-diphosphate reductase (NrdE), L-lactate dehydrogenase (Ldh) and lysyl-tRNA synthetase (LysS) were found to be the partial nodes with high connectivity, with both NrdE and LysS being the downstream targets of Ldh. LysS interacts closely with four translational factors, translation initiation factor IF-1 (InfA), ribosomal protein L3 (RplC), ribosomal protein L1 (RplA) and signal recognition particledocking protein (FtsY). Ldh possibly mediates the glycolysis to provide energy for the subsequent translational processes in S. pneumoniae and thus indirectly regulates protein synthesis. In addition, several enzymes involved in tRNA aminoacylation, including LysS, alpha-phenylalanyl-tRNA synthetase (PheS), tyrosyl-tRNA synthetase (TyrS), alanyl-tRNA synthetase (Alas),

isoleucyl-tRNA synthetase (Lies), form a small compact network with aspartate-ammonia ligase (AsnA) in their core node. Bacterial Phosphoproteomes. The genome size of S. pneumoniae is similar to that of L. lactis and around one-half of those from both E. coli and B. subtilis, whereas the number of phosphorylation proteins (84) in S. pneumoniae is larger than those in E. coli (79), B. subtilis (78) and L. lactis (63) (Table 2). Only 56 and 57 unique phosphopeptides were identified in P. putida and P. aeruginosa, respectively, using Ti-HAMMOC and Zr-HAMMOC.12 The high phosphorylation level is a particular feature of S. pneumoniae as its number of phosphorylated sites is around 2-fold of those from other bacteria with known phosphoproteomes. Similar to L. lactis, S. pneumoniae has almost equal phosphorylation sites on serine and threonine; whereas for other bacteria, about 70% of the phosphorylation sites were identified on threonine. Another notable characteristic of the S. pneumoniae phosphoproteome is the high percentage of proteins with multiple phosphorylation sites: among the 84 identified phosphoproteins, 22 (∼26%) contained three or more phosphorylation sites. The only protein present in all the known bacterial phosphoproteomes is phosphoglycerate kinase (Table 3). The phosJournal of Proteome Research • Vol. 9, No. 1, 2010 279

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Table 2. Phosphoproteomic Comparison between S. pneumoniae and Other Bacteria

S. pneumoniae E. coli B. subtilis L. lactis

genome size (ORFs)

no. of phosphoproteins

no. of phosphopeptides

no. of phospho- sites

pS (%)

pT (%)

pY (%)

ref.

∼2069 ∼4300 ∼4100 ∼2600

84 79 78 63

102 105 103 102

163 81 78 79

47.2 67.9 69.2 46.5

43.8 23.5 20.5 50.6

9 8.6 10.3 2.7

This work 9 10 11

Table 3. Putative Phosphoproteins Conserved among S. pneumoniae, E. coli, B. subtilis, and L. lactis gene name

S. pneumoniae

E. coli

B. subtilis

L. lactis

pgk adk dnaK gatA lysS metK pheS infA glnA ldh rplA rplO tyrS

+ + + + + + + + + + + + +

+ + + + + + +

+

+

+ + + + + +

phoproteome of S. pneumoniae has another six, five and one proteins in common with those in E. coli, L. lactis and B. subtilis, respectively. The distribution of the functional classes for the identified phosphoproteins in S. pneumoniae is different from the other three published bacterial phosphoproteomes: a majority of S. pneumoniae phosphoproteins are involved in nucleotide and protein metabolism, whereas phosphoproteins involved in carbon metabolism are the major components of the phosphoproteomes in B. subtilis and L. lactis. The higher occurrence of phosphorylation in proteins involved in the regulation of gene expression in S. pneumoniae appears to be closely correlated with the higher virulence of S. pneumoniae, when compared with the other two bacteria. Other studies also reported that serine/threonine/tyrosine phophorylation can control bacterial virulence in the level of gene expression.21 It is therefore possible that the phosphorylation level of the protein expression regulators is a particular feature in comparing the virulence of different bacterial pathogens. Protein Kinase Target Motif in S. pneumoniae. To investigate the motif specificities between eukaryotic and prokaryotic kinases, the identified phosphorylated sites in S. pneumoniae were searched against the target sequences of eukaryotic protein kinases through SCANSITE (http://scansite.mit.edu) with high, medium, and low stringency.30 Table 4 lists the phosphopeptides that were identified in this study and predicted to be associated with a eukaryotic kinase binding motif. Ninety-one Ser/Thr-phosphorylated peptides and 3 Tyr-phosphorylated peptides matched the target motifs for eukaryotic kinases at low stringency, while only 2 peptides (ATTIKIATVNRSGSE and AGNIKNLSVKELKQG) matched at high stringency (Supplementary Table S3). The database searching results exhibited the substantial contribution from PKC-type kinases to the protein phosphorylation of S. pneumoniae. Statistics of Amino Acids around Phosphorylated Sites. The types of amino acids at -6 to +6 positions centering around the phophorylated amino acid were analyzed. Tyrosine phosphorylation by specific kinases is a key regulatory process in bacteria.19 We found here that the neighbors of the phosphoTyr were significantly rich in Gly, especially on positions -6, -3, -1, and +2. Leucine was presented with the highest 280

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Table 4. SCANSITE Prediction for Eukaryotic Protein Kinases and Binding Motifs at High Stringency (0.2%), Medium Stringency (1.0%), and Low Stringency (5.0%) within Identified Phosphorylated Sites of S. pneumoniae D39 kinase

kinase group type

high

mid

low

PKC_delta PKC_mu DNA_PK Cam_Kin2 PKC_epsilon Akt_Kin ATM_Kin PKA_Kin Erk1_Kin Casn_Kin1 PKC_zeta Crk_SH2 GSK3_Kin PDK1_Bind PLCg_CSH2 Casn_Kin2 Clk2_Kin p38_Kin 1433_m1 Abl_SH2 Itk_Kin Nck_SH2 PKC_common

Baso_ST_kin Baso_ST_kin DNA_dam_kin Baso_ST_kin Baso_ST_kin Baso_ST_kin DNA_dam_kin Baso_ST_kin Pro_ST_kin Acid_ST_kin Baso_ST_kin SH2 Acid_ST_kin Kin_bind SH2 Acid_ST_kin Baso_ST_kin Pro_ST_kin pST_bind SH2 Y_kin SH2 Baso_ST_kin

1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

3 0 3 2 2 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0

6 7 2 5 2 12 7 5 3 2 2 3 3 3 3 2 2 2 1 1 1 1 1

frequency in the nearby sequences of phospho-Ser, especially on positions -1, +1, and +2, and Val was the second. Among the seven amino acids showing high occurrence frequencies surrounding the phosphorylated Ser/Tyr/Thr, six amino acids are hydrophobic residues including Pro, Val, Leu, Ile, Ala, and Phe (Figure 4), suggesting that hydrophobic residues are critical for protein phosphorylation by sequence-specific kinases in S. pneumoniae.

Discussion Signaling via Ser/Thr/Tyr phosphorylation is implicated in the regulation of bacterial virulence.18 Phosphorylation in bacterial proteins can interfere with eukaryotic signal transduction, rendering the host more prone to be infected.31 Recently, an increasing number of bacterial phosphoproteomes, from E. coli, B. subtilis, L. lactis, P. putida, and P. aeruginosa, was reported with the technical developments in phosphopeptide enrichment and mass spectrometric analysis.9-12 However, these are either typical model bacteria or industrial bacterium. In the current study, we focused on a pathogenic bacterium S. pneumoniae, using TiO2 enrichment and high-accuracy LTQ-Orbitrap MS analysis to systematically characterize its phosphoproteome for better understanding of the phosphorylation regulation in the bacterium. The present work demonstrated the first trial to uncover the phosphoproteomic interaction network in bacteria, which enclosed all upstream regulatory proteins and downstream targets in S. pneumoniae. The interaction network indicated that phosphorylation in S. pneumoniae regulates many impor-

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Phosphoproteome of Streptococcus pneumoniae

eron repressor was identified to carry three phosphorylated sites. PTS permease also showed to be phosphorylated; this protein controls the activity of transcription activators and antiterminators by phophorylating them in response to the availability of the respective substrate.33 A key enzyme involved in sugar transport, mannose-specific phosphotransferase system enzyme IIAB component, was also found to be phosphorylated on tyrosine. With this regard, protein phosphorylation seems to play a critical role in the transcription in pathogenic Streptococcus species. Although a large number of bacterial phosphoproteins have been identified so far, there was less effort to investigate the motif specificities between eukaryotic and prokaryotic kinases. In this work, the identified phosphorylated sites in S. pneumoniae were searched against the motif database of eukaryotic protein kinases with SCANSITE. With high stringency, PKC kinase was found to be the only kinase matched with eukaryotic kinase motif. This is not surprising as previous works on E. coli and B. subtilis also revealed large differential substrate specificity for bacterial and eukaryotic Ser/Thr/Tyr kinases.9,10 However, the statistics of amino acids surrounding the phosphorylated center indicated that phosphorylation in S. pneumoniae prefers hydrophobic environments (Figure 4). Higher proportion of multiple phosphorylation sites existed in the phosphoproteome of the pathogenic S. pneumoniae as compared to those in other model bacteria.9-12 Although different phosphopeptide enrichment conditions may contribute to the difference in phosphorylation detection, the commercially available TiO2 enrichment kit has been shown to have a good reproducibility in many other experiments. Higher occurrence of multiple phosphorylation in bacterial proteins has been noted in bacteria under stress or overloading proteolytic conditions.34 Several studies have established that Ser/ Thr/Tyr phosphorylation can control bacterial virulence at the gene level.22,35-38 These observations support the notion that active bacterial phosphorylation is closely related to virulence mechanisms including adhesion to the host, stimulation and regulation of pathogenic functions and biochemical warfare, scrambling the host signaling cascades and impairing its defense mechanisms.21 To survive and infect host, the pathogen S. pneumoniae has to develop an active metabolic mechanism regulated by phosphorylation-mediated regulatory switches. Figure 4. Statistics of the types of amino acids at -6 to +6 positions centering around the phophorylated amino acid.

tant biological processes such as carbon metabolism, protein synthesis and tRNA synthesis. Phosphoglycerate kinase (PGK) was found to be an important node in this network. Functioning as a key enzyme for ATP generation in glycolytic pathway (Table 3), PGK was identified in all the four bacterial phosphoproteomes, S. pneumoniae, E. coli, B. subtilis, and L. lactis. However, PGK phosphorylation was found on Tyr residue in S. pneumoniae, while it happened on Ser and Thr in B. subtilis and E. coli, and on Ser in L. lactis.9-11 This specificity may implicate the special role of PGK phosphorylation in the regulation of S. pneumoniae virulence, as bacterial tyrosine phosphorylation has been proposed to be closely related to the virulence mechanisms of many pathogens.32 In this work, many important proteins involved in transcription and its regulation were identified to be phosphorylated in Streptococcus species. For example, three subunits of RNA polymerase that directly control the production of RNA were found phosphorylated. A transcription regulator-galactose op-

Conclusions In this paper, we globally characterized the phosphoproteome from a pathogenic bacterium S. pneumonaie. The functional classification and interaction network of the identified phosphoproteins indicated that phosphorylation regulates many important processes including carbon metabolism, protein biosynthesis and tRNA synthesis. Compared to other bacteria with known phosphoproteomes, S. pneumoniae has a bigger size of phosphoproteome and higher proportion of multiple phosphorylation sites. These results imply that the high phosphorylation level may be closely related to bacterial virulence and pathogenesis. It will be interesting to investigate the Ser/Thr/Tyr phosphorylation as a potential avenue to depress the bacterial growth.

Acknowledgment. This work was partially supported by the 2007 Chang-Jiang Scholars Program, “211” Projects, National Natural Science Foundation of China (20871057, to Q.-Y.H.; 20801061, to R.G.) and Talents Start-up Foundation of Jinan University (JNU 51208047, to X.S.; JNU 51207040, to F.G.). Journal of Proteome Research • Vol. 9, No. 1, 2010 281

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