The Protein Expression of Streptococcus pyogenes Is Significantly

We show that the common human pathogen Streptococcus pyogenes rapidly remodel its cellular metabolism and virulence pathways when exposed to human ...
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The Protein Expression of Streptococcus pyogenes Is Significantly Influenced by Human Plasma Bjo1 rn P. Johansson,*,† Fredrik Levander,‡ Ulrich von Pawel-Rammingen,§ Tord Berggård,‡ Lars Bjo1 rck,† and Peter James‡ Department of Clinical Sciences, Lund University, Lund, Sweden, and Department of Electrical Measurements, Lund University, Lund, Sweden, and Department of Molecular Biology, Umeå University, Umeå, Sweden Received July 15, 2005

During the courser of infection, the common human pathogen Streptococcus pyogenes encounters plasma. We show that plasma causes S. pyogenes to rapidly remodel its cellular metabolism and virulence pathways. We also identified a variant of the major virulence factor, M1 protein, lacking 13 amino acids at the NH2-terminus in bacteria grown with plasma. The pronounced effect of plasma on protein expression, suggests this is an important adaptive mechanism with implications for S. pyogenes pathogenicity. Keywords: Streptococcus pyogenes • 2D-PAGE • plasma • M protein • post-translational modifications

Introduction Streptococcus pyogenes (group A streptococcus) is an important human pathogen causing superficial infections on epithelial barriers in the skin and upper respiratory tract. Since the late 1980s, an increase of the prevalence of invasive S. pyogenes infections causing severe clinical conditions such as necrotizing fasciitis, septicemia and streptococcal toxic shock syndrome, has attracted considerable attention and concern.1,2 The fatality rate for patients with these invasive infections is high, and postinfectious sequelae such as rheumatic fever also represents a significant clinical problem, especially in the developing world.2 During infection, S. pyogenes is exposed to human plasma. Superficial infectious sites with inflammation contain human plasma as a consequence of vascular leakage, and invasive strains can penetrate into the blood stream. Plasma is a rich growth medium, but it is also a reservoir for opsonizing antibodies, complement, and other components of the human immune system that S. pyogenes must evade in order to survive within the host. Research conducted over many years has shown that numerous interactions between plasma proteins and streptococcal proteins interfere with various host defense systems.3 Even though we are just beginning to understand the regulatory networks during S. pyogenes infections, it is well established that the expression of streptococcal genes is under tight transcriptional control. Several factors including temperature, pH, and ion-concentration, influence the protein * To whom correspondence should be addressed. Bjo¨rn P. Johansson, Clinical and Experimental Infectious Medicine, BMC B14, Tornava¨gen 10, SE-221 84 Lund, Sweden. Tel.: +46-46-2224489. Fax: +46-46-157756. E-mail: [email protected]. † Department of Clinical Sciences, Lund University. ‡ Department of Electrical Measurements, Lund University. § Department of Molecular Biology, Umeå University.

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expression.4-6 Numerous systems for transcriptional regulation of genes encoding virulence factors have been described in S. pyogenes. Among these is the multiple gene activator (Mga), which is stimulated during logarithmic growth by high levels of carbon dioxide or anaerobiosis.4,5 Mga regulates several genes involved in virulence, including the M protein, the complement inactivating enzyme C5a peptidase,7,8 and protein SIC,9 which inhibits the formation of membrane attack complexes and blocks the effect of antibacterial peptides.10 Rgg is a global regulatory factor that contributes to growth phase-dependent synthesis of proteins associated with secondary metabolism and thermal stress response in S. pyogenes.11,12 Rgg is also required for the expression of a number of secreted streptococcal virulence factors such as the proteinase SpeB, the M49 protein, and mitogenic factors 1 and 3.13-15 Moreover, the well studied streptococcal two-component gene regulatory system CovRS, negatively regulates the expression of several streptococcal virulence factors in both logarithmic and stationary growth phases.16,17 Understanding the mechanisms regulating the transcription of streptococcal genes is of great importance for comprehending the host-pathogen interplay during infection. However, in contrast to genomic research, investigations at the proteome level provide further insight into protein abundance and post-translational modifications. Previous studies on the S. pyogenes proteome have established that streptococci rapidly switch phenotype in response to the environment.4-6,14,18 However, the effect of plasma on protein expression in S. pyogenes has not previously been investigated. This is noteworthy, as the pathogen will encounter plasma during the course of infection (see above). Moreover, S. pyogenes expresses surface proteins with high and specific affinity for some of the most abundant human plasma proteins, such as albumin, fibrinogen, IgG, R2-macroglobulin, and kininogens.3 The present study examines the effects of human plasma on S. pyogenes protein expression, and presents a screen of the 10.1021/pr050217y CCC: $30.25

 2005 American Chemical Society

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Regulation of S. pyogenes Protein Expression

streptococcal proteome using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF). The results show a profound influence of plasma on protein expression, suggesting that this response is crucial for the adaptation of S. pyogenes to its human host.

Experimental Procedures Bacterial Strains and Growth Conditions. The S. pyogenes strain AP1 (serotype M1) originates from the World Health Organization Collaborating Center for References and Research on Streptococci, Institute of Hygiene and Epidemiology, Prague, Czech Republic. Triplicate cultures of AP1 were routinely grown in 50 mL Todd-Hewitt broth (TH) (Difco Laboratories, Detroit, Mich) in parallel with TH supplemented with 5% human plasma, at 37 °C in a 5% CO2-atmosphere. For standard measurements of S. pyogenes growth rate, quadruplicate cultures of AP1 were grown at the conditions stated above. The optical density at 620 nm and pH was measured at given intervals of 30 min until the bacterial cultures reached stationary growth phase. Protein Sample Preparation. Bacterial cultures in mid exponential growth phase (OD620 0.6) were harvested and immediately chilled on ice. Plasma proteins bound to streptococcal surfaces of bacteria grown in the presence of plasma were removed by acid glycine (pH 2.0), and the samples were subjected to extensive washing with ice cold PBS. The amount of bacteria in the different cultures was double checked by weighing the streptococcal pellets after centrifugation of the cultures. The bacteria were lysed in lysis buffer (8 M urea, 2% CHAPS, 1% DTT and 2% v/v Pharmalyte 4-7 or 6-11 respectively), by extensive vortexing with glass beads. Proteins were purified and precipitated using a 2D-clean up kit, (Amersham Biosciences, Uppsala, Sweden). In brief, protein samples, precipitant, and co-precipitant was mixed thoroughly and centrifugated down at 8000 × g for 10 min. The supernatant was carefully removed and the pellet was dispersed in prechilled wash buffer and wash additive. After 30 min of incubation at -20 °C the samples were centrifugated down and the pellet was solubilized in rehydration buffer (8M urea, 2% CHAPS, 0.01M DTT, 2% ampholytes, and bromphenol blue) and solubilized in rehydration buffer (8M urea, 2% CHAPS, 0.01M DTT, 2% ampholytes, and bromphenol blue). The protein concentration in the samples was measured using a protein micro assay (Bio-Rad) according to the manufacturers instructions. First Dimension. Immobilized pH gradient strips, nonlinear pH 4-7, 24 cm long (Amersham Biosciences), were rehydrated 12 h with the protein samples solubilized in rehydration buffer and isoelectric focusing was performed for 60 000 Vh with a maximum of 8000 V at an IPGphor (Amersham Biosciences). When analyzing streptococcal proteins within the basic pH range, 18 cm long, nonlinear 6-11 immobilized pH gradient strips were rehydrated in Destreak rehydration solution (Amersham Biosciences) for 12 h prior to sample entry by cup loading. The strips were subjected to iso-electric focusing for 35 000 Vh with a maximum of 8000 V at an IPGphor (Amersham Biosciences) under running conditions as previously described.19 Second Dimension, Staining and Visualization. After isoelectric focusing, the strips were equilibrated two times for 15 min in a buffer containing: Tris (50mM, pH 8.8) 6 M urea, 30% glycerol, 2% SDS, 0.002% bromphenol blue, and DTT (10 mg/

mL). In the second step iodoacetamide (25 mg/mL) was used instead of DTT. The strips were layered onto 12% SDS-PAGE gels and electrophoresis was conducted with a constant electrical current of 10 W/gel, for 5 h. Gels were stained overnight in a 0.02% solution of the Coomassie Blue stain Phast Gel Blue R (Pharmacia Biotech, Uppsala, Sweden) in 30% methanol and 10% acetic acid, to visualize the separated protein spots. Matching and Analysis of the Protein Spots. Ettan progenesis software (Amersham Biosciences) was used for matching and analysis of the protein spot expression on gels. A reference gel was created by combining all of the spots from three +plasma gels and three -plasma gels into one image. Only spots present in all gels were used for normalization of spot volume and for comparison of spot intensity between gels. The reference gel was used for determination of existence and differences in spot volume between two sets of experiments. A factor of 2.5 times difference in the average spot volume between the two experimental conditions is reported as an upregulation/down-regulation of the protein level. Spot Picking, MALDI-TOF Mass Spectrometry and LC-MS/ MS. Automatic spot picking and protein digestion was performed in an Ettan Spot Handling Workstation (Amersham Biosciences). Individual protein spots were excised, digested with trypsin, and spotted on MALDI target plates. MALDI spectra were acquired in data-dependent mode on a Micromass M@ldi MALDI-TOF MS (Waters). MALDI spectrum processing and database searches were performed using the PIUMS software20 with batch filtering and differential settings as described previously.21 The molecular weight and pI of significantly matched proteins (p e 0.01) were compared with the corresponding spot position on the gels before being regarded as identified. Additionally, MS/MS analysis was performed on protein spots identified as M1 protein, using an atmospheric pressure MALDI (Masstech) Deca XP Plus ion trap (Thermo Electron, Kungens kurva, Sweden), with settings and data processing as described elsewhere.21 MS/MS database searches were performed with Mascot (www.matrixscience.com). For M1 protein isoform analysis, LC-MS/MS was performed on a Micromass Qtof Ultima (Waters, Sollentuna, Sweden) with a CapLC. The Swissprot-TrEMBL nonredundant databases and the S. pyogenes protein database were downloaded from ExPASy ftp-server (ftp.expasy.org).

Results Screening of S. pyogenes Protein Expression. The protein expression of strain AP1 grown in 5% human plasma in TH growth medium, approximately mimicking the amount of human plasma proteins in inflammatory exudates at the infectious site,22 was investigated in relation to growth in TH culture medium alone. Bacterial cultures were grown to exponential growth phase (OD620 0.6), and intracellular proteins were identified by 2D-electrophoresis and MALDI-TOF mass spectrometry. A total of 206 protein spots were identified, and 191 unique S. pyogenes proteins with a consistent expression in three separate experiments, were identified between the pI intervals of 4-7 and 6-11 (Figure 1 and Table 1). The protein expression was matched between different gels and analyzed for spot intensity. A factor of 2.5 times difference in average spot volume between the two experimental conditions is reported as an upregulation of the protein expression. In addition, numerous protein spots were identified as human plasma proteins that Journal of Proteome Research • Vol. 4, No. 6, 2005 2303

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Figure 1. Master gels of S. pyogenes protein expression during logarithmic growth phase S. pyogenes protein expression in the pI intervals of 4-7 (A and B) and 6-11 (C and D) was analyzed in Todd Hewitt medium (A and C), and Todd Hewitt medium supplemented with 5% human plasma (B and D). Proteins were separated by 2D-electrophoresis and identified by MALDI-TOF mass spectrometry. The streptococcal proteins identified by mass spectrometry are numbered and listed in Table 1. Proteins differentially expressed under the stated conditions are listed in Table 2.

had not been completely removed from the bacterial surface by washing in glycine buffer, pH 2.0. Growth Characteristics of AP1 Bacteria. The growth of strain AP1 cultured in TH or TH supplemented with 5% human plasma was monitored to examine the effect of human plasma on S. pyogenes growth. The optical density at 620 nm and the pH of the media was measured at given intervals of 30 min until the bacteria entered stationary growth phase. No difference in acidification of the media was observed, an important observation since the expression of several streptococcal virulence factors is influenced by altered pH. As seen in Figure 2a, AP1 entered logarithmic growth phase faster in TH supplemented with plasma than when grown in TH, plausibly as a result of the high nutritional value of human plasma. However, once having entered logarithmic growth phase, both set of cultures had equivalent growth rates as shown in Figure 2b. Hence, the increased expression of metabolic proteins observed in AP1 grown in human plasma does not influence the bacterial growth rate but is probably used for other biological processes such as biosynthesis of secreted proteins. Effect of Human Plasma on S. pyogenes Protein Expression. The expression of 39 protein spots representing 24 unique streptococcal proteins was significantly increased in bacteria exposed to human plasma (Table 2). Thirty-one spots were absent in bacteria grown in TH, whereas eight were expressed in both media but up-regulated by a factor of at least 2.5 times in plasma. In contrast, only two protein spots, one hypothetical protein and a septum placement protein involved in cell 2304

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division, were down-regulated. Several up-regulated spots are derived from a single protein. For example, adenylosuccinate synthetase (spots no. 73 and 85), a putative surface lipoprotein (spots no. 54 and 55) and two extracellular proteins, the M1 protein (spots no. 41, 72, 74, 87, 88, 89, 90, and 202) and C5a peptidase (spots no. 60-65) were all expressed in multiple forms in plasma environment in contrast to growth in TH. Expression and Post-translational Processing of Streptococcal Virulence Factors. Molecular mechanisms contributing to the pathogenicity of S. pyogenes have been characterized extensively, and it has become increasingly clear that the expression of genes involved in virulence is under tight transcriptional control. For instance, the well-characterized streptococcal virulence factors M1 protein and C5a peptidase, which are both regulated by the multiple gene activator (Mga),23-25 were identified as up-regulated in the presence of plasma. Interestingly, seven distinct protein spots absent in the gels from bacteria grown in culture medium alone were identified as the M1 protein and six spots were identified as C5a peptidase, indicating that S. pyogenes subject virulent proteins to post-translational modifications when exposed to human plasma (Figure 1, Figure 4, Table 2). LC-MS/MS analysis of the different M1 protein spots revealed that the M1 proteins present in spots no. 41, 87, 89, and 202 lacked a portion of the N-terminus (Figure 3). The peptide closest to the NH2-terminus found in these spots after trypsin digestion, AANNPAIQNIR, is only partially tryptic, suggesting that the protein is proteolytically processed. In contrary, protein spots

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Regulation of S. pyogenes Protein Expression Table 1. Summary of Identified Streptococcal Proteins Spots in Figure 1a spot no.

protein name

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Hypothetical protein Spy0593 Hypothetical protein Spy0684 Major cold-shock protein 30S ribosomal protein S6 Hypothetical protein Spy2114 10 kDa chaperonin Hypothetical protein Spy1875 50S ribosomal protein L7/L12 Putative thioredoxin Putative phosphotransferase system Hypothetical protein Spy0588 50S ribosomal protein L10 Hypothetical protein Spy1260 Putative alkyl hydroperoxidase Transcription elongation factor greA Hypothetical protein Spy1262 GrpE protein Triosephosphate isomerase Putative phosphoprotein phosphatase Adenylate kinase Putative Dtdp-4 keto-6-deoxyglucose3, 5-epimerase Hypothetical protein Spy0546 Putative purine nucleoside phosphorylase Putative uridine phosphorylase Putative purine nucleoside phosphorylase Fructose bisphosphate aldolase Putative branched-chain-amino acid aminotransferase Hypothetical UPF0082 protein Cell-division initiation protein DNA-directed RNA polymerase alpha chain Phosphoglycerate kinase Putative phosphopentomutase Enolase Putative ribosomal protein S1-like DNAbinding protein Putative dipeptidase Putative pyruvate kinase Chaperone protein dnaK Trigger factor Seryl tRNA synthetase Protein Gid homolog M protein type 1 Inosine-5’-monophosphatedehydrogenase Putattive beta-ketoacyl-ACP synthase II Putative integrase-phage associated Mannose-specific phospho-transferase system component IIAB 6-phosphofructokinase Putative o-acetylserine lyase Peptide deformylase Ribosome recycling factor Glutamyl-tRNA(Gln) amido-transferase subunit C Hypothetical protein Spy1277 Putative glutaredoxin Ribose-5-phosphate isomerase Putative surface lipoprotein Putative surface lipoprotein Hypothetical protein Spy0949 Putative 2-dehydropantoate 2-reductase Carbamoyl phosphate synthase small chain Putative cell-envelope proteinase C5a peptidase precursor C5a peptidase precursor C5a peptidase precursor C5a peptidase precursor C5a peptidase precursor C5a peptidase precursor Putative trans-2-enoyl ACP reductase II DNA gyrase subunit B Putative O-acetylserine lyase

22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68

Swiss-Prot ID/ TrEMBL Accession

pI/Mw (Da)

exp. value

quality

coverage (%)/ no. of peptides

Q9A0V8 Q9A0P1 CSPA_STRPY RS6_STRPY YL72_STRPY CH10_STRPY Q99Y43 RL7_STRPY Q99Y75 Q99Z65 Q9A0W2 RL10_STRPY Q99ZE7 Q99XR7 GREA_STRPY Q99ZE5 GRPE_STRPY TPIS_STRPY Q99YM9 KAD_STRPY Q9A046

5.61/8189 4.32/8643 5.16/7322 4.97/11081 4.88/10363 4.79/10331 4.69/9146 4.49/12255 4.39/11710 4.74/8951 4.78/16089 5.19/17434 4.65/17453 4.65/20483 4.67/17652 4.93/19943 4.68/22054 4.57/26486 4.60/27020 4.83/23699 5.07/22437

0.001 0.002 0 0 0.001 0 0 0 0.001 0.001 0 0 0.003 0 0 0 0 0 0 0 0.014

7.08 6.91 12.72 13.89 7.62 9.38 14.99 12.05 7.45 8.01 18.99 22.66 10.38 15.15 29.21 9.81 10.96 10.38 13.26 18.76 5.63

55/6 45/5 63/6 72/5 24/3 26/3 82/5 80/9 57/4 10/3 57/7 63/10 26/7 18/7 79/10 34/6 37/8 33/8 37/7 82/18 16/3

Q9A0Z1 Q9A082 Q99Y49 Q9A081 ALF_STRPY Q9A068

8.29/27869 4.98/28881 5.11/27828 5.15/26019 4.87/31077 4.90/37063

0.087 0 0.002 0 0 0

4.09 15.27 6.98 15.75 14.92 14.02

35/3 32/7 18/3 31/7 31/7 21/7

Y316_STRPY Q99YW2 RPOA_STRPY PGK_STRPY DEOB_STRPY ENO_STRPY Q9A066

4.49/25887 4.47/28933 4.90/34529 4.82/41998 4.80/44223 4.74/47225 4.90/43849

0.031 0 0 0 0.010 0 0.007

8.39 14.67 13.35 25.70 7.01 14.93 5.68

11/4 32/8 25/9 50/16 17/6 26/8 15/6

Q99ZU1 Q99ZD1 DNAK_STRPY TIG_STRPY SYS_STRPY GID_STRPY Q99XV0 IMDH_STRPY Q99YDL Q9A044 Q99YE6

4.87/51345 4.96/54535 4.62/64788 4.41/47118 5.12/48181 5.46/49424 6.46/54220 5.65/52676 5.37/43651 5.27/44179 5.19/35567

0 0 0 0 0 0 0 0 0 0.015 0

14.51 12.54 40.79 22.21 12.49 16.77 21.54 7.77 9.54 9.13 9.38

51/7 17/8 49/28 34/17 20/9 17/9 44/11 26/9 25/10 13/5 42/12

K6PF_STRPY Q99YN5 DEF_STRPY RRF_STRPY GATC_STRPY Q99ZD4 Q878 × 0 Q92IL8 Q877Y4 Q877Y4 Q9A033 PANE_STRPY CARA_STRPY Q9A180 SCA2_STRPY SCA2_STRPY SCA2_STRPY SCA2_STRPY SCA2_STRPY SCA2_STRPY Q99YD4 Q8RTZ5 Q8K6F3

5.33/35748 5.43/33240 5.51/22862 5.68/20572 4.67/11069 4.86/12277 6.05/8208 6.82/15907 8.87/61261 8.87/61261 6.03/8695 4.84/33828 5.57/39757 6.11/181288 5.10/122032 5.10/122032 5.10/122032 5.10/122032 5.10/122032 5.10/122032 6.33/33859 5.22/33273 5.43/33212

0 0 0.046 0 0 0.003 0.015 0 0 0 0.006 0.037 0.088 0 0 0.001 0 0 0 0 0.001 0 0.006

15.49 28.35 11.39 16.01 15.60 8.40 8.08 20.80 17.99 17.99 13.66 6.98 5.77 11.26 14.14 7.32 13.01 28.75 9.88 12.51 7.53 13.5 8.05

45/15 46/13 18/6 40/6 77/7 26/3 72/4 53/4 13/5 13/5 30/3 14/3 13/3 8/10 12/11 7/8 10/10 18/16 4/5 9/10 25/5 13/5 12/5

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Table 1 (Continued) spot no.

69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 2306

protein name

Putative protease Hypothetical protein Spy0004 Hypothetical protein Spy1646 M Protein type 1 Adenylosuccinate synthetase M protein type 1 Putative transketolase Elongation factor G Putative ribosomal protein S1-like DNA-binding protein Glyceraldehyde 3-phosphate dehydrogenase Protein SIC Hypothetical protein Spy1262 Ribonuclease P protein component 50S ribosomal protein L7/L12 Putative ABC transporter Peptide deformylase Adenylosuccinate synthetase Glyceraldehyde 3-phosphate dehydrogenase M protein type 1 M protein type 1 M protein type 1 M protein type 1 Putative Dtdp-glucose-4, 6-dehydratase Hypothetical protein Spy1865 Probable DNA-directed RNA polymerase Probable DNA-directed RNA polymerase Elongation factor P Glycerol-3-phosphate dehydrogenase Putative acetoin dehydrogenase Putative phosphotransacetylase Mannose specific phospho-transferase 30S ribosomal protein S2 Ornithine carbamoyltransferase Hypothetical protein Spy0248 Putative seryl t-RNA synthetase Putative glyceraldehyde-3-phosphate dehydrogenase Hypothetical P protein UDP-glucose 6-dehydrogenase Heat shock protein Hypothetical protein Spy1343 Putative sugar ABC transporter Oligopeptide permease Putative preprotein translocase binding subunit Putative DNA-dependent RNA-polymerase subunit Putative endopeptidase Clp ATP-binding chain C Quenine tRNA-ribosyltransferase Hypothetical protein Spy0306 Hypothetical protein Spy0306 Heat shock protein Hypothetical protein Spy0924 Putative Maltose operone transcriptional repressor Oligotransport ATP-binding protein oppF GTP-binding ERA homolog Putative 5-keto-D-gluconate 5-reductase UTP glucose-1-phosphate uridylyltransferase 1 Hypothetical protein Thymidylate synthase Hypothetical protein Spy0326 Putative NAD(P)H-flavin oxidoreductase Uracil phosphoribosyl transferase Putative metaldependent transcriptional regulator Uracil phosphoribosyl transferase Orotate phosphoribosyltransferase 50S ribosomal protein L4 Putative l-serine dehydratase beta subunit Putative DNA/pantothenate metabolism flavoprotein Putative signal peptidase I Putative mutator protein 30S ribosomal protein S13 Hypothetical protein Spy1646 Putative holliday junction resolvase

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Swiss-Prot ID/ TrEMBL Accession

pI/Mw (Da)

exp. value

quality

coverage (%)/ no. of peptides

Q9A0W0 Q9A208 Q99YL4 Q99XV0 PURA_STRPY Q99XV0 Q99YJ3 EFG_STRPY Q9A066

5.07/34690 9.77/7625 6.36/12445 6.46/54220 5.45/47461 6.46/54220 4.98/77454 4.83/76397 4.90/43849

0.001 0 0.020 0 0 0 0.001 0.003 0

8.76 8.59 3.90 16.91 20.06 16.21 7.65 9.04 19.14

19/8 38/4 34/3 43/17 37/19 38/15 16/8 16/7 27/11

G3P_STRPY Q9JNH0 Q99ZE5 RNPA_STRPY RL7_STRPY Q9A1P7 DEF_STRPY PURA_STRPY G3P_STRPY Q99XV0 Q99XV0 Q99XV0 Q99XV0 Q9A045 Q99Y53 RPOZ_STRPY RPOZ_STRPY EFP_STRPY GPDA_STRPY Q99ZX7 Q99ZQ5 Q99YE6 RS2_STRPY OTCC_STRPY Q9A1J2 SYS_STRPY Q99Z67 Q9A0G5 UDG_STRPY Q99YC9 Q99Z89 Q99ZH5 Q9A1F8 Q99Y96 RPOB_STRPY Q99XR9 TGT_STRPY Q9A1F4 Q9A1F4 Q99YC9 RNZ_STRPY Q99ZC1 OPPF_STRPY ERA_STRPY Q9A0T1 HAC1_STRPY Q9ZB44 TYSY_STRPY Q9A1D9 Q9A120 UPP_STRPY Q9A158 UPP_STRPY PYRE_STRPY RL4_STRPY Q99XI6 Q99ZI0 Q99Y69 Q99YW5 RS13_STRPY Q99YL4 RUVX_STRPY

5.34/35811 4.93/19943 4.93/19943 10.22/13874 4.49/12255 5.45/36211 5.51/22862 5.45/47461 5.34/35811 6.46/54220 6.46/54220 6.46/54220 6.46/54220 5.41/38866 4.96/22111 5.53/11705 5.53/11705 4.85/20467 5.65/36680 4.86/35836 5.01/35824 5.19/35567 5.11/28269 5.19/37799 5.14/33867 5.12/48181 5.06/50368 6.90/43955 6.93/45569 6.76/40494 7.03/58395 6.27/55603 8.47/34896 5.67/94764 4.97/132790 6.42/90714 6.42/43054 6.39/41676 6.39/41676 6.76/40494 6.84/34429 6.72/38170 6.12/34715 6.11/34087 4.96/28183 6.41/33649 6.57/35728 6.72/32663 6.61/24988 6.51/25283 6.30/22825 5.99/24814 6.30/22825 6.43/22743 9.80/22125 6.70/24067 6.57/19443 6.76/22817 6.12/17460 10.52/13425 6.36/12445 6.84/15759

0 0.001 0 0.040 0.001 0 0 0 0.001 0 0 0 0.001 0 0 0.001 0.002 0.043 0 0 0.005 0 0 0.001 0.007 0.001 0 0 0.002 0 0 0 0 0 0 0 0 0.002 0 0 0.087 0.007 0 0 0.009 0.001 0 0.007 0.003 0 0 0 0 0 0 0.043 0 0.099 0.001 0.001 0 0

14.05 10.83 9.81 6.02 7.28 15.70 12.75 20.06 9.00 16.73 11.58 21.02 9.30 18.79 13.68 6.71 6.20 7.02 10.40 9.51 8.64 18.90 17.50 10.15 8.37 9.66 18.54 15.13 6.24 17.3 32.56 18.12 11.19 39.15 13.80 10.21 25.19 6.20 9.16 23.89 3.09 7.05 9.64 16.18 6.90 7.47 13.77 5.95 5.94 9.09 13.10 8.73 31.97 8.64 7.16 4.47 11.14 3.92 6.81 6.57 21 24.28

17/7 22/6 23/4 32/6 71/6 21/6 52/7 37/19 19/8 62/13 28/16 41/17 13/14 12/13 52/9 60/6 63/5 14/3 13/6 20/6 11/5 41/12 40/9 9/7 16/7 15/9 17/9 21/8 17/4 33/9 27/15 28/14 14/9 36/30 24/15 12/9 19/8 15/4 22/6 22/9 35/3 22/9 11/5 23/8 18/3 28/6 23/8 17/4 24/4 24/5 26/6 18/4 45/12 34/5 12/6 13/5 32/10 14/4 27/4 20/3 42/7 44/8

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Regulation of S. pyogenes Protein Expression Table 1 (Continued) spot no.

protein name

Swiss-Prot ID/ TrEMBL Accession

pI/Mw (Da)

exp. value

quality

coverage (%)/ no. of peptides

140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206

ATP synthase epsilon chain (3R)-hyroxymyristoyl ACP dehydratase Putative proton translocating ATPase, delta subunit Methyltransferase GidB Hypothetical UPF0230 protein Spy1698 Putative acetyl-CoA carboxylase beta subunit Fatty acid/phospholipid synthesis protein plsX DNA-binding protein HU 50S ribosomal protein L29 50S ribosomal protein L30 50S ribosomal protein L33 30S ribosomal protein S15 30S ribosomal protein S16 30S ribosomal protein S10 30S ribosomal protein S19 Putative transcriptional regulator 50S ribosomal protein L14 50S ribosomal protein L19 30S ribosomal protein S11 50S ribosomal protein L17 30S ribosomal protein S7 50S ribosomal protein L13 50S ribosomal protein L6 Translation initiation factor IF-3 Hypothetical protein Spy1124 50S ribosomal protein L5 Putative dehydrofolate reductase Hypothetical protein Spy0305 50S ribosomal protein L9 30S ribosomal protein S5 30S ribosomal protein S8 50S ribosomal protein L21 50S ribosomal protein L11 50S ribosomal protein L23 50S ribosomal protein L31 type B Translation initiation factor IF-1 Hypothetical protein Spy1511 30S ribosomal protein S4 30S ribosomal protein S3 50S ribosomal protein L3 Hypothetical protein Spy0369 Putative cell-division ATP-binding protein Hypothetical protein Spy1161 50S ribosomal protein L1 Putative transcriptional regulator ATPase protein Probable inorganic polyphosphate-ATP/NAD-kinase Putative hemolysin Putative iron-sulfur cofactor synthesis protein Hypothetical protein Spy1533 Putative RNA-helicase Putative ATP-binding cassette transporter protein Putative glycosyl transferase Probable exodeoxyribonuclease VII large subunit Putative deoxyribodipyromidine photolyase Putative fibronectin-binding protein-like protein A Zink-binding protein adcA Putative RNA methyltransferase Nicotine adenine dinucleotide glycohydrolase Hypothetical protein Spy1781 Putative nucleolar protein 3-phosphoshikimate 1-carboxyvinyltransferase M protein type 1 Hypothetical protein Spy0316 60 kDa chaperonin Putative ABC transporter Hypothetical protein Spy1862

ATPE_STRPY FABZ_STRPY Q9A0J0 GIDB_STRPY YG98_STRPY Q99YE0 PLSX_STRPY DBH_STRPY RL29_STRPY Q9A1V6 RL33_STRPY Q99XZ0 RS16_STRPY RS10_STRPY RS19_STRPY Q99Y40 Q9A1W4 RL19_STRPY RS11_STRPY Q8P2Z2 RS7_STRPY Q99Y07 Q9A1V9 IF3_STRPY Q99ZQ9 RL5_STRPY Q8K7V9 Q9A1F5 RL9_STRPY RS5_STRPY RS8_STRPY Q9A0D6 RL11_STRPY Q9A1 × 2 R31B_STRPY IF1_STRPY Q99YW4 RS4_STRPY RS3_STRPY RL3_STRPY Q9A1B0 Q9A0S4 Q99ZM7 RL1_STRPY Q99YQ0 Q99XV7 PPNK_STRPY Q99YX6 Q9A0D9 Q99YU5 Q99Z69 Q99XU2 Q9A116 EX7L_STRPY Q99YX0 Q99ZY7 ADCA_STRPY YG06_STRPY Q9R304 Q99YB4 Q99ZG0 AROA_STRPY Q99XV0 Q9A1E6 CH60_STRPY Q9A1P7 YI62_STRPY

6.98/15553 6.91/15323 7.92/19902 7.77/26779 7.62/30873 7.70/31825 8.49/35496 9.00/9647 9.30/7962 10.12/6441 10.35/5912 10.27/10503 10.28/10251 9.92/11613 9.94/10622 9.57/14376 10.18/13061 11.10/13145 11.22/13370 9.81/14521 10.17/17679 9.91/16100 9.54/19432 9.83/20053 9.10/22051 9.34/19815 8.89/19372 8.69/22309 9.40/16512 9.79/17027 9.62/14802 9.76/11154 9.43/14801 9.62/10731 9.62/9854 8.06/8272 7.99/11561 9.98/23122 9.64/24133 10.13/22438 9.85/27007 9.52/26160 9.35/32205 9.34/24394 5.71/33445 8.91/29410 9.17/31376 8.80/30440 6.40/40279 8.86/41053 9.11/41023 9.11/46607 8.87/49891 8.92/50455 8.07/54892 8.25/63034 8.52/58520 9.32/50715 7.77/46526 8.60/51980 8.16/48897 8.78/46692 6.46/54220 4.49/25887 4.75/56964 5.45/36211 4.79/10975

0.002 0 0.022 0.001 0 0.002 0 0.002 0.001 0.001 0.001 0 0 0 0 0 0.001 0 0 0.001 0.001 0 0 0 0.030 0 0.007 0.004 0 0 0 0.002 0 0 0 0.003 0 0 0 0 0 0 0.003 0.004 0 0 0.001 0 0 0 0 0 0 0.020 0 0 0 0 0 0 0.007 0.004 0 0 0 0 0.006

6.44 15.66 5.83 7.41 8.57 7.36 11.57 6.11 7.27 7.82 6.82 8.91 7.93 8.31 17.14 14.98 7.22 12.34 11.20 11.87 6.59 8.34 18.27 8.52 3.48 16.93 5.68 5.19 18.12 13.14 9.39 7.26 13.28 8.44 14.66 5.7 15.09 22.36 13.95 14.52 20.52 14.38 5.78 5.98 14.02 8.54 7.59 8.52 7.77 28.91 14.91 11.27 22.01 3.89 10.80 8.26 11.55 29.10 17.68 11.55 5.8 11.51 19.54 30.24 29.59 21.54 6.09

21/3 23/4 10/2 16/5 41/6 16/4 23/6 30/3 29/3 43/3 36/3 37/5 50/5 39/4 57/9 41/8 32/6 54/8 46/7 59/7 29/5 54/6 37/7 26/4 23/3 54/8 15/6 9/2 43/7 26/3 9/2 37/3 22/3 56/5 50/5 25/3 56/6 39/11 45/12 38/9 33/8 34/8 12/4 13/4 22/7 16/4 20/4 20/6 8/3 54/18 12/6 22/9 22/10 10/4 15/7 9/5 18/8 23/8 23/7 19/9 9/3 22/11 34/9 45/10 46/18 44/11 34/4

a Significantly matched proteins by peptide mass fingerprinting (p E 0.01) are included in the Table. The identifier of each protein is the primary accession number in the ExPASy protein database. Several proteins were expressed in multiple forms, with similar Mw and pI on the gels.

88 and 90 contained the fully tryptic peptide EVIEDLAANNPAIQNIR and no trace of the partially tryptic peptide. The

LC-MS/MS spectra of all six protein digests were scanned for the two peptides, and only one of the forms was present in Journal of Proteome Research • Vol. 4, No. 6, 2005 2307

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Figure 2. Growth rate of the AP1 strain in Todd Hewitt growth medium or Todd Hewitt supplemented with 5% human plasma. (A) Quadruplicate cultures of AP1 bacteria were grown in Todd Hewitt medium (b) or Todd Hewitt supplemented with 5% human plasma (O), at 37 °C in 5% CO2- 20% O2 atmosphere. The optical density at 620 nm and the pH were measured at given intervals of 30 min until the bacterial cultures entered stationary growth phase. Having entered logarithmic growth, both set of cultures had equivalent growth rates under both experimental setups, as indicated by the trend lines in (B). Results are given as mean values ( SD.

any sample. No other post-translational modification was found in the analyzed spots. In addition, a putative cell-envelope proteinase (Spot no. 59) was also increasingly expressed in bacteria grown in plasma. Although the protein has high sequence homologies to C5a peptidase, further characterization of the protein is needed to elucidate whether it plays an active role in S. pyogenes pathogenicity. Up-Regulated Metabolic Proteins. Gel analysis revealed that exposure to human plasma affects the transcription of several metabolic and cell-maintenance genes. Seven proteins involved in protein metabolism (Spot nos. 49, 57, 68, 76, 84, 93, 94, and 101, Tables 1 and 2), three proteins involved in glycolytic pathways (Spot nos. 86, 91, and 104, Tables 1 and 2), and one protein important for phospholipid biosynthesis (Spot no. 96, Tables 1 and 2), were increasingly expressed in plasma environment. Furthermore, two ribosomal proteins (Spot nos. 77 and 100, Table 1 and 2), and two proteins involved in DNA and RNA synthesis were also significantly up-regulated (Spot nos. 67 and 73, Table 1 and 2). Up-Regulated Transport Proteins. Two putative transport system proteins were identified as up-regulated in bacteria grown in plasma (See Table 2). First, a putative sugar ABC transporter belonging to the ABC transporter family, and, second, a putative surface lipoprotein belonging to the extracellular solute-binding protein family 5. ABC transporters are involved in active transport of solutes across the cytoplasmic membrane, whereas members of the solute-binding protein family 5 do not play a basic role in the transport process per se, but probably serve as receptors to trigger translocation of the solute across the membrane. The putative surface lipoprotein appeared in two spots, possibly as a result of posttranslational modification. Up-Regulated Hypothetical Proteins. Hypothetical proteins are open reading frames encoding putative proteins without any significant homologies to proteins with known functions. A total of 35 proteins, annotated as hypothetical proteins, were identified in this study to be expressed in vitro (Table 1). Three of these were up-regulated in response to plasma, and one of 2308

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them, spot no. 102, (Spy0248) contains a K homology domain, often found in RNA-binding proteins, such as ribosomal proteins and transcription factors.26,27 Putative Streptococcal Proteins Homologous to Bacterial Virulence Factors. Screening the AP1 proteome identified two putative proteins, equivalently expressed under both growth conditions that might contribute to S. pyogenes virulence. First, a 63 kDa putative fibronectin-binding protein (Spot no. 195) with 71% sequence homology to FbpA in S. gordonii and 96% identity to Fbp54 in S. pyogenes (http://www.ncbi.nlm.nih.gov/ BLAST), and, second, a putative hemolysin (Spot no. 187) with 74% identity to Hemolysin A in S. agalactiae. Moreover, two putative streptococcal proteinases, both absent in a TH environment but expressed during growth in human plasma, were also recognized as potential virulence factors. First, a putative protease belonging to the MEROPS peptidase family U32 (Spot no. 69), where the type example is a collagenase, and, second, a putative cell-envelope proteinase (Spot no. 59) with high sequence homologies to C5a peptidase. SignalP predictions (http://www.cbs.dtu.dk/services/SignalP) of the putative virulence factors revealed that only the cell-envelope proteinase contains a signal sequence in the NH2-terminal part and a LPXTG motif in the C-terminal part, making it unclear whether the putative hemolysin, putative proteinase and the putative fibronectin binding protein are secreted or not. However, a novel category of extracellular proteins lacking apparent signal sequences has been described.28-31

Discussion The pathogenesis of S. pyogenes is highly complex and different strains of S. pyogenes show considerable genetic variation.2 Consequently, the choice of strain(s) to be studied can always be debated. There are however several reasons why we selected the AP1 strain for the present investigation. It represents one of the best-characterized S. pyogenes strains. In particular, its plasma protein-binding properties have been studied in detail.32-34 Moreover, AP1 is of the M1 serotype, the serotype most frequently associated with severe invasive infections.2,35 Apart from the M1 protein which is expressed by all strains of the M1 serotype, AP1 bacteria also express protein H, an M-like surface protein with affinity for three plasma proteins; albumin, IgG, and fibronectin.33,34 Many strains of the M1 serotype do not have sph, the gene encoding protein H.36 Interestingly, many M1 strains that carry sph, express the gene only when injected into mice, but not when grown in vitro.37 In contrast to the vast majority of S. pyogenes strains, AP1 is naturally virulent to mice.38 Theoretically, protein H could contribute to this property, which may prove to be valuable in future studies on protein expression during experimental S. pyogenes infection in mice. S. pyogenes alters its protein expression in response to environmental changes.4,5,39 Nevertheless, most experiments with S. pyogenes have been conducted in culture media that have few similarities to the in vivo conditions the bacteria encounter during infection. For instance, S. pyogenes is constantly exposed to human plasma during superficial infections, as a consequence of the local inflammatory response, but the effect of human plasma on S. pyogenes protein expression is not known. We have therefore constructed a proteome map for nonsecreted proteins of S. pyogenes in standard laboratory culture medium TH, and TH supplemented with human plasma. Previous studies on the S. pyogenes proteome have mostly examined proteins released into the growth me-

research articles

Regulation of S. pyogenes Protein Expression Table 2. Differentially Expressed Streptococcal Proteinsa functional categories

cell-division DNA and RNA synthesis Glycolytic pathways hypothetical proteins

phospholipid biosynthesis protein metabolism

Ribosomal Transport Virulence Other

protein name by plasma

spot no. value

regulation/no. of peptides

Q99YW2 PURA_STRPY PURA_STRPY Q8RTZ5 G3P_STRPY Q99Z67 Q9A045 Y316_STRPY Q9A180 Q9A0W0 Q9A208 Q9A1J2 GPDA_STRPY RRF_STRPY PANE_STRPY Q8K6F3 EFG_STRPY DEF_STRPY RPOZ_STRPY RPOZ_STRPY OTCC_STRPY Q9A066 RS2_STRPY Q877Y4 Q877Y4 Q9A1P7 Q99XV0 Q99XV0 SCA2_STRPY GID_STRPY

29 73 85 67 86 104 91 28 59 69 70 102 96 49 57 68 76 84 93 94 101 77 100 54 55 83 41 72, 74, 87-90, 202 60-65 40

down new new new new new new down new new up new new up new new up new up up new new up new new new up new new up

expectation

0 0 0 0 0.001 0 0 0.031 0 0.001 0 0.007 0 0 0.037 0.006 0.003 0 0.001 0.002 0.001 0 0 0 0 0 0 0-0.001 0-0.001 0

coverage (%)

32/8 37/19 37/19 13/5 19/8 17/9 12/13 11/4 8/10 19.8 38/4 16/7 13/6 40/6 14/3 12/5 16/7 52/7 60/6 63/5 9/7 27/11 40/9 13/5 13/5 21/6 44/11 13-62/13-171 4-18/5-162 17/9

a All S. pyogenes proteins that were up- or down-regulated during growth in human plasma by a factor of at least 2.5, are listed and classified into categories based on their biological functions. Most proteins were up-regulated (Up) or newly synthesized (new), whereas only two proteins were down-regulated (Down).

dium6,18,40 whereas the experimental design of the present study identifies changes in the intracellular pool of proteins in response to plasma. This map permits consistent identification of proteins, as the coordinates of the protein spots are highly reproducible on high-resolution 2-DE gels. The bacteria were harvested during logarithmic growth. Although, it is not known that growth phases in vitro directly reflect those of an infection in vivo, it seems likely that bacteria are multiplying when the infection is being established in a tissue. Logarithmic growth phase is also connected with the expression of several surface-associated and secreted molecules essential for colonization of host tissues.41,42 The acidification of the growth media, cell concentration, and growth rate, were continuously measured to confirm that differences in protein expression between the two experimental setups were not due to such parameters. Although the bacteria entered logarithmic growth faster in culture medium supplemented with plasma, the generation time was the same in both cultures at the time for harvesting. Most of the spots identified are proteins important for cell maintenance or involved in various metabolic pathways. In addition, two surface-associated virulence factors (M1 protein and the C5a peptidase) and some proteins of unknown function were also identified. Extracellular proteins of Gram-positive bacteria are rapidly processed and exported after synthesis.43 Thus, only proteins present in sufficient concentrations at the time of harvesting are identified on the gels, explaining why several proteins known to be transcribed during logarithmic growth could not be detected. Several metabolic proteins were up-regulated in S. pyogenes in response to human plasma. A majority of these proteins is associated with protein metabolism and biosynthesis. Among

these are, for instance, two ribosomal proteins catalyzing mRNA-directed protein synthesis in S. pyogenes, and elongation factor G. Moreover, a ribosome recycling factor that dissociates ribosomes from mRNA after translation, as well as a peptide deformylase removing the formyl group of newly synthesized proteins, were also up-regulated. The enhanced expression of ribosomal proteins and proteins involved in protein biosynthesis demonstrates a general increase of protein synthesis in plasma environment. Plasma also significantly up-regulated glycolytic enzymes. Among these is the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) which plays an important role in glycolysis and gluconeogenesis. GAPDH has also been found at the bacterial surface where it binds plasminogen, indicating a possible role in S. pyogenes virulence.28,29,31 Furthermore, a protein homologous to GAPDH was also identified following growth in THplasma, but not in bacteria grown in TH without plasma. Only two streptococcal proteins were down-regulated in bacteria grown in plasma. First, a hypothetical protein of approximately 26 kDa with unknown function (Spot no. 28), and, second, a septum placement protein involved in cell division (Spot no. 29). The effect of septum placement proteins in S. pyogenes is unclear, but it has been reported that an inactivation of a homologous protein in Bacillus subtilis produces a minicell phenotype.44 However, microscopical analysis of S. pyogenes did not reveal any morphological differences between bacteria grown in TH and TH supplemented with plasma (data not shown). The M1 protein and the C5a peptidase, both well-characterized surface-attached virulence factors of S. pyogenes, were significantly up-regulated in response to plasma, and appeared in multiple forms. The M1 protein from bacteria grown in TH Journal of Proteome Research • Vol. 4, No. 6, 2005 2309

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Figure 4. Up-regulation and modification of M1 protein and C5a peptidase in plasma environment. AP1 bacteria expressed six distinct C5a peptidase spots when grown in Todd Hewitt supplemented with plasma (A), but showed no C5a peptidase expression in Todd Hewitt (B). Also, M1 protein was up-regulated (spot no. 41), and gave rise to several spots when grown in Todd Hewitt supplemented with plasma (C), as compared to Todd Hewitt alone (D).

Figure 3. LC-MS/MS spectra identified two forms of M1 protein in strain AP1 grown in plasma. Six distinct protein spots identified as M1 protein by MALDI-TOF MS were analyzed for posttranslational modifications by LC-MS/MS. Two forms of M1 protein were identified in AP1 bacteria during mid exponential growth phase in plasma, an intact native form with a fully tryptic peptide closest to the NH2-terminus, EVIEDLAANNPAIQNIR, (A) and a processed form, AANNPAIQNIR, lacking the 13 NH2terminal amino acids (B).

supplemented with plasma was identified in eight distinct protein spots, compared to a single spot in bacteria grown in TH. LC-MS/MS analysis identified two distinct M1 protein forms, a mature nonprocessed M1 protein, and a variant lacking the NH2-terminal 13 amino acids. This processing should be due to proteolytic cleavage in plasma environment during logarithmic growth, and the precise mechanism responsible for the cleavage of this important virulence factor is currently under investigation. Previous studies have suggested that the M1 protein can be subjected to phosphorylation and/or processing when anchored to the bacterial surface.45-49 The loss of the NH2terminal 24 amino acids alters M1 nonimmune binding to immunoglobulin G, resulting in a less virulent phenotype.45-47 It has also been reported that genetically distinct subpopulations can occur within the same M type, presumably due to alterations during DNA replication.50,51 Such events result in M proteins of different sizes and partly changed amino acid sequences in the hyper-variable region, thus allowing daughter cells to avoid antibody recognition during infection. However, the modification of M1 protein reported in this study has not been reported before. It represents a novel mechanism to alter 2310

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the hyper-variable NH2-terminal region of the M1 protein, which may play a role in S. pyogenes pathogenicity. The cell wall-anchored subtilisin-like serine protease C5a peptidase prevents opsonization and interferes with chemotactic recruitment of phagocytic cells.7,52 C5a peptidase is produced with an NH2-terminal 31 amino acid residue signal peptide, responsible for exporting the protein across the membrane. C5a peptidase expression in strain AP1 is completely repressed in TH growth medium, whereas six distinct protein spots are identified as C5a peptidase in bacteria grown in TH- plasma. Heterogeneity has been described for the C5a peptidase gene between different S. pyogenes strains,53 but not within the same strain. Neither have post-translational modifications of the C5a peptidase been reported previously. Recently, Graham et al.39 reported that S. pyogenes rapidly remodels its cellular metabolism and virulence pathways when inoculated in human whole blood. Although blood and plasma are two distinct environments that S. pyogenes encounters during different stages of infection, it emphasizes our findings that the bacteria rapidly responds to molecular signals in human plasma by altering its metabolism and accumulate mgaregulated proteins when exposed to plasma. The results also underline the power of S. pyogenes adaptation to its human host, and provide a starting point for future investigations of the processing and modification of important virulence determinants.

Conclusion The common and important bacterial pathogen Streptococcus pyogenes coordinately regulates its protein expression in order to survive in its human host. Here we show that S. pyogenes significantly alters its protein expression when exposed to human plasma when compared to growth in standard

research articles

Regulation of S. pyogenes Protein Expression

laboratory culture medium. By using two-dimensional-gel electrophoresis and MALDI-TOF mass spectrometry, 206 protein spots were identified as 191 unique streptococcal proteins. Twenty-four of the 191 proteins were up-regulated at least 2.5fold in bacteria grown in plasma, whereas two were downregulated. The production of proteins involved in protein biosynthesis, carbohydrate metabolism, DNA/RNA synthesis, and phospholipid biosynthesis, was generally increased in plasma. Interestingly, the M1 protein and C5a peptidase, two major streptococcal virulence factors, were significantly upregulated and identified in multiple forms in bacteria grown in plasma. LC-MS/MS analysis of the M1 protein identified two distinct forms, a native form and a previously uncharacterized variant lacking the 13 NH2-terminal amino acid residues. As a consequence of the local inflammatory response in infected tissues, and during invasive infections, S. pyogenes will encounter plasma. The pronounced effect of plasma on protein expression, suggests that this represents an important adaptive mechanism with implications for S. pyogenes pathogenicity. Thus, the map of intracellular proteins from S. pyogenes grown in the classical TH medium, or TH supplemented with 5% human plasma presented here, should facilitate future studies of the protein expression of this significant human pathogen, during various environmental conditions.

Acknowledgment. We thank Ms Ulrika Brynnel and Dr Anna-Karin Påhlman for excellent technical assistance and expert help. This work was supported by the Swedish Research Council (projects 7480, 15401), the Foundations of Kock and O ¨ sterlund, and Hansa Medical AB. References (1) Kaplan, E. L. Eur. J. Clin. Microbiol. Infect. Dis. 1991, 10, 55-57. (2) Cunningham, M. W. Clin. Microbiol. Rev. 2000, 13, 470-511. (3) Navarre, W. W.; Schneewind, O. Microbiol. Mol. Biol. Rev. 1999, 63, 174-229. (4) Podbielski, A.; Peterson, J. A.; Cleary, P. Mol. Microbiol. 1992, 6, 2253-2265. (5) Caparon, M. G.; Geist, R. T.; Perez-Casal, J.; Scott, J. R. J. Bacteriol. 1992, 174, 5693-5701. (6) Nakamura, T.; Hasegawa, T.; Torii, K.; Hasegawa, Y.; et al. Arch. Microbiol. 2004, 181, 74-81. (7) Wexler, D. E.; Chenoweth, D. E.; Cleary, P. P. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 8144-8148. (8) Chen, C.; Bormann, N.; Cleary, P. P. Mol. Gen. Genet. 1993, 241, 685-693. (9) Åkesson, P.; Sjo¨holm, A. G.; Bjo¨rck, L. J. Biol. Chem. 1996, 271, 1081-1088. (10) Frick, I. M.; Åkesson, P.; Rasmussen, M.; Schmidtchen, A.; et al. J. Biol. Chem. 2003, 278, 16561-16566. (11) Chaussee, M. A.; Callegari, E. A.; Chaussee, M. S. J. Bacteriol. 2004, 186, 7091-7099. (12) Chaussee, M. S.; Somerville, G. A.; Reitzer, L.; Musser, J. M. J. Bacteriol. 2003, 185, 6016-6024. (13) Chaussee, M. S.; Sylva, G. L.; Sturdevant, D. E.; Smoot, L. M.; et al. Infect. Immun. 2002, 70, 762-770. (14) Chaussee, M. S.; Watson, R. O.; Smoot, J. C.; Musser, J. M. Infect. Immun. 2001, 69, 822-831. (15) Chaussee, M. S.; Ajdic, D.; Ferretti, J. J. Infect. Immun. 1999, 67, 1715-1722. (16) Graham, M. R.; Smoot, L. M.; Migliaccio, C. A.; Virtaneva, K.; et al. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 13855-13860.

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