Streptococcus pyogenes - Journal of Bacteriology - American Society ...

JOURNAL OF BACTERIOLOGY, Mar. 2007, p. 1514–1522 0021-9193/07/$08.00⫹0 doi:10.1128/JB.01132-06 Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Vol. 189, No. 5

Proteomic Analysis and Identification of Streptococcus pyogenes Surface-Associated Proteins䌤 Anatoly Severin,1 Elliott Nickbarg,2† Joseph Wooters,2 Shakey A. Quazi,2 Yury V. Matsuka,1 Ellen Murphy,1 Ioannis K. Moutsatsos,2 Robert J. Zagursky,1 and Stephen B. Olmsted1* Wyeth Vaccine Research, Pearl River, New York,1 and Wyeth Research, Cambridge, Massachusetts2 Received 27 July 2006/Accepted 21 November 2006

Streptococcus pyogenes is a gram-positive human pathogen that causes a wide spectrum of disease, placing a significant burden on public health. Bacterial surface-associated proteins play crucial roles in host-pathogen interactions and pathogenesis and are important targets for the immune system. The identification of these proteins for vaccine development is an important goal of bacterial proteomics. Here we describe a method of proteolytic digestion of surface-exposed proteins to identify surface antigens of S. pyogenes. Peptides generated by trypsin digestion were analyzed by multidimensional tandem mass spectrometry. This approach allowed the identification of 79 proteins on the bacterial surface, including 14 proteins containing cell wall-anchoring motifs, 12 lipoproteins, 9 secreted proteins, 22 membrane-associated proteins, 1 bacteriophage-associated protein, and 21 proteins commonly identified as cytoplasmic. Thirty-three of these proteins have not been previously identified as cell surface associated in S. pyogenes. Several proteins were expressed in Escherichia coli, and the purified proteins were used to generate specific mouse antisera for use in a whole-cell enzymelinked immunosorbent assay. The immunoreactivity of specific antisera to some of these antigens confirmed their surface localization. The data reported here will provide guidance in the development of a novel vaccine to prevent infections caused by S. pyogenes. Streptococcus pyogenes, also known as group A Streptococcus, is a gram-positive bacterium that causes a wide spectrum of diseases ranging from mild localized infections, such as pharyngitis and impetigo, to severe invasive diseases, such as necrotizing fasciitis and streptococcal toxic shock-like syndrome. Invasive streptococcal disease is associated with high morbidity and mortality rates (37). S. pyogenes is also associated with a variety of autoimmune sequelae such as acute rheumatic fever, which after repeated episodes can result in rheumatic valvular heart disease, the most common cause of pediatric heart disease worldwide (11). In spite of the high mortality and substantial economic losses caused by these diseases, there is currently no licensed vaccine to prevent human S. pyogenes infections. For many years, efforts to develop a vaccine to protect against S. pyogenes infections were focused on the surfaceassociated M protein (19, 38), a major virulence factor of S. pyogenes. However, there are at least two significant limitations for using M protein as a vaccine antigen. First, the M protein contains a highly variable amino-terminal region that determines the S. pyogenes serotype. With over 150 different M serotypes identified, it is difficult to envision using the M protein as a broadly efficacious vaccine. Second, M protein elicits antibodies that are cross-reactive with human cardiac myosin and are associated with the development of acute rheumatic fever (10). To circumvent these issues, we have initiated an

alternative strategy to identify other proteins localized to the surface of S. pyogenes that could also serve as protective antigens. The availability of complete bacterial genomic sequences, as well as the powerful processing capabilities of bioinformatics, have begun to play an important role in the identification of protein vaccine candidates through genomics, transcriptional profiling, and proteomics (31, 32, 45, 62). Combining these technologies becomes an important first step in vaccine development. Recently, proteomic analyses of cell surface-associated proteins from Staphylococcus aureus (34), S. pyogenes (8), Staphylococcus epidermidis (53), Streptococcus agalactiae (20), Streptococcus pneumoniae (24), and Clostridium difficile (60) were reported. In these studies, the detection of cell surfaceassociated proteins was based on digestion of the peptidoglycan with lysozyme, mutanolysin M1, and/or lysostaphin in the presence of osmotic protective agents. The proteins external to the cytoplasmic membrane were released, separated by twodimensional (2D) gel electrophoresis, and digested, and the resulting peptides were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry or tandem mass spectrometry (MS/MS). By this approach, surprisingly few, if any, of the proteins covalently attached to the peptidoglycan through cell wall-anchoring motifs (29) were identified in those proteomic studies that included S. epidermidis (53), S. agalactiae (20), and S. pneumoniae (24). This is in contrast to genomic predictions of 10 such proteins covalently attached to the peptidoglycan in S. epidermidis, from 25 to 35 proteins (depending on the strain) in S. agalactiae, and from 14 to 16 proteins in S. pneumoniae (5, 9). Only 3 of 17 predicted peptidoglycan-attached proteins were found by proteomic analysis in S. pyogenes (8), and only 3 of 21 predicted peptidoglycan-attached proteins (49) were detected in S. aureus

* Corresponding author. Mailing address: Wyeth Vaccine Research, Bldg. 205/Room 225, 401 North Middletown Rd., Pearl River, NY 10965. Phone: (845) 602-7992. Fax: (845) 602-4350. E-mail: olmsteds @wyeth.com. † Present address: Schering Plough Research Institute, 840 Memorial Dr., Cambridge, MA 02139. 䌤 Published ahead of print on 1 December 2006. 1514

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(34). The failure to detect most of the proteins covalently attached to peptidoglycan may be related to the low abundance of these proteins in cell walls and/or to their high hydrophobicity. It is challenging to obtain good separation of the highly hydrophobic proteins by isoelectrofocusing in the first dimension of a 2D gel (17). An alternate method has been used to directly digest proteins from purified cell wall preparations of Listeria without 2D gel separation. This analysis revealed 20 proteins containing the cell wall-anchoring motif from the 30 predicted to be present in the genome (6). A similar direct digestion approach detected 12 of 17 predicted peptidoglycan-attached proteins in S. pyogenes (50). In our study, we also employed a method of direct digestion of proteins from the bacterial surface to obtain a set of surfaceexposed proteins of S. pyogenes. Due to the known temporal regulation of protein expression in S. pyogenes, we analyzed cells harvested at both early- and late-exponential phases of growth to provide a more comprehensive set of surface-associated proteins. In addition to the proteomic approach, genomic analysis of the S. pyogenes genome was performed in silico with several algorithms designed to identify genes that encode surface-localized proteins. Using these approaches, we identified 79 proteins associated with the S. pyogenes surface. Fourteen of 17 predicted peptidoglycan-attached proteins were identified, as well as 12 of 27 predicted lipoproteins, 9 secreted proteins, 22 membrane proteins, 1 bacteriophageassociated protein, and 21 proteins more commonly identified as cytoplasmic. The identification of these surface-associated proteins has provided a valuable set of proteins that may be used in the development of a broadly efficacious vaccine to prevent S. pyogenes infections in humans. MATERIALS AND METHODS Bacteria, media, and reagents. The S. pyogenes M1 strain SF370 (ATCC 700294) was used in this study. The complete genomic sequence for this strain is available (16). Streptococci were grown in 3% Todd-Hewitt broth (Difco Laboratories, Detroit, MI) supplemented with 0.5% yeast extract (THY) at 37°C in a 5% CO2 atmosphere. Escherichia coli was cultured in HySoy medium containing 1% HySoy (Kerry Bio-Science, Norwich, NY), 0.5% yeast extract, 0.5% NaCl, and 90 mM sodium phosphate buffer (pH 7.2) supplemented with either 100 ␮g/ml ampicillin or 30 ␮g/ml kanamycin. Bioinformatics/gene mining. The genomic sequence was downloaded from the University of Oklahoma website (http://www.genome.ou.edu/strep.html), and open reading frames (ORFs) were determined with the established algorithms GLIMMER (13) and GeneMark (25). The initial annotation of the S. pyogenes ORFs was performed with the BLAST, version 2.0, gapped search algorithm, and BLASTP was used to identify homologous sequences (1). SignalP (36) was employed for identifying proteins destined for translocation across the cytoplasmic membrane. Putative membrane-associated proteins that contain at least one membrane-spanning domain were detected with the TopPred program (7). To predict protein localization in bacteria, the software PSORT (33) was used. The HMM Lipo algorithm was developed in-house to predict lipoproteins built on a hidden Markov model using a training data set of 131 biologically proven bacterial lipoproteins reported in the literature. Using 70 known proteins containing the LPXTG cell wall-anchoring motif, we also developed a hidden Markov model to predict proteins that are covalently attached to the peptidoglycan (35). Tryptic digestion of bacterial surface proteins. An overnight culture of S. pyogenes SF370 was diluted in 200 ml of fresh THY medium to an optical density at 600 nm (OD600) of 0.04 and was grown to early-exponential (OD600 of 0.35) and late-exponential (OD600 of 0.75) growth phases. Cells were harvested by centrifugation at 12,000 ⫻ g for 15 min and washed with 20 mM Tris-HCl (pH 7.6) containing 150 mM NaCl, followed by resuspension in 2 ml of the same buffer supplemented with 1 M D-arabinose, 10 mM CaCl2, and 80 ␮g of trypsin (Tpck treated) (Worthington, Lakewood, NJ). A control sample was treated the

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same way but without trypsin. After incubation of samples at 37°C for 4 h with gentle shaking, cells were removed by centrifugation at 20,000 ⫻ g for 15 min. The supernatants were sterilized by membrane filtration through a 0.22-␮m Millipore filter and were stored at ⫺20°C until use. Cryo field emission scanning electron microscopy (FE-SEM). Samples were prepared as described previously (39). Bacterial suspensions with or without trypsin treatment were centrifuged, washed twice, and resuspended to a concentration of 1 ⫻ 108 cells/ml in 10 mM phosphate-buffered saline (PBS) (pH 7.4). Each sample was placed on poly-L-lysine-coated glass coverslips following a gentle wash to remove excess bacteria. The coverslips were placed into fixative (2.0% glutaraldehyde, 0.1 M sodium cacodylate buffer containing 7.5% sucrose) for 30 min. The fixative was washed from samples twice with 0.1 M sodium cacodylate buffer for 10 min and then postfixed in 0.1 M sodium cacodylate containing 1% osmium tetroxide for 30 min. The samples were then washed twice with 0.1 M sodium cacodylate, dehydrated with ethanol, and partially rehydrated in 50% ethanol. The bacterial samples were then plunge frozen, partially freeze-dried, and sputter coated with chromium at ⫺95°C. The cells were cryo-viewed at ⫺95°C with a LEO 1550 field emission scanning electron microscope (Carl Zeiss MicroImaging, Inc., Thornwood, NY) operated at low accelerating voltages (1 to 1.5 keV) using a secondary electron detector for conventional topographical imaging and a high-resolution Robinson backscatter detector. Sample preparation for MS analysis. In order to reduce the complexity of the peptide mixture prior to MS/MS analysis, samples were separated off-line on a reversed-phase high-performance liquid chromatography (HPLC) Vydac C8 column (Grace Vydac Co., Hesperia, CA). Before separation, peptides were desalted on a Sephadex G-25 column (17 by 2.6 cm) at a flow rate of 2 ml/min and dried on a Savant SpeedVac lyophilizer (ThermoElectron Corp., Milford, MA). Lyophilized peptides were dissolved in 100 ␮l of 0.1% trifluoroacetic acid (TFA), insoluble material was removed by centrifugation at 20,000 ⫻ g for 10 min, and supernatant was applied on a 250- by 4.6-mm reversed-phase Vydac C8 HPLC column. Separation was performed with an Agilent 1100 series HPLC system. The material bound to the column was eluted at a flow rate of 1 ml/min with a linear gradient of acetonitrile (0 to 50%) in 0.1% TFA for 40 min at room temperature. The eluted peptides were detected by absorption at 206 nm, and 10 fractions, 5 ml each, were collected. These fractions were further separated with the on-line mode as described below. All fractions were lyophilized, dissolved in 50 ␮l of 0.1% formic acid, and stored at ⫺20°C. Peptide analysis by nano-LC-MS/MS. The peptide fractions were analyzed on a ThermoFinnigan LCQ DECA-XP quadruple ion trap mass spectrometer (ThermoFinnigan, San Jose, CA) equipped with a nano-electrospray interface. The nano-electrospray interface consisted of a microcapillary LC column (75/360 ␮m [inner diameter {ID}/outer diameter {OD}] by 10 cm) with direct infusion of analytes eluted from the column into the mass spectrometer. The microcapillary column was prepared according to a published procedure (2) with some modifications. Fused-silica capillary tubing (75/360 ␮m [ID/OD] by 50 cm) with silica frit (tip ID, 15 ␮m) (New Objective Inc., Woburn, MA) was packed with MAGIC C18AQ, 200-Å, 5-␮m reversed-phase beads (Michrom BioResources, Inc., Auburn, CA). Helium pressure (400 lb/in2) applied to the bomb containing a suspension of packing material in 50% acetonitrile–0.1% formic acid with constant stirring forced a steady flow of the beads into the capillary. The column was packed to a bed length of 10 cm, washed with 90% acetonitrile containing 0.1% formic acid, and conditioned with 5 pmol of angiotensin and one gradient run prior to use. A column flow rate of 250 nl/min was achieved by using a split of the mobile phase, containing 0.1% formic acid and the gradient acetonitrile concentration. In order to obtain the best distribution of peptides during the 75-min on-line LC run, three different gradients were employed to individual fractions depending on the acetonitrile concentration at which they eluted from the off-line C8 column. The fractions that eluted at the lowest acetonitrile concentration, fractions 2 and 3, were eluted from the on-line microcapillary column with a gradient of acetonitrile from 2% to 15% in 45 min, followed by a 15% to 90% acetonitrile gradient in 30 min. Fractions 4 to 6 were eluted with a 5% to 35% gradient in 45 min, followed by a 35% to 90% gradient in 30 min. The last fractions, 7 to 10, were eluted with a 30% to 65% gradient in 45 min, followed by a 65% to 90% gradient in 30 min. Peptide samples were analyzed in positive mode at a spray voltage of 1.6 kV and an ion-transferred capillary temperature of 145°C by a data-dependent acquisition method. The acquisition method included one MS scan (375 to 1,200 m/z) followed by MS/MS scans of the three most intense ions detected in the MS scan. The dynamic exclusion function (exclusion duration, 2 min; exclusion mass width, 0.75 to 1.75) was employed to increase the number of peptides that were analyzed. Database search and protein identification. Automated analyses of MS/MS data were performed with the commercially available software SEQUEST (Thermo-

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J. BACTERIOL.

FIG. 1. Scanning electron microscopy of S. pyogenes cells. Late-exponential-phase bacterial cultures were left untreated (A) or treated with trypsin (see Materials and Methods) (B). Untreated S. pyogenes cells displayed large amounts of material on the surface. The cells treated with trypsin showed a significant reduction of surface-exposed material, as the cells appeared smooth and of smaller size. Bar, 300 nm.

Finnigan Corp.) by searching the entire NCBI database (15). Parameters for protein identification included a mass tolerance of 1.4 Da, a mass range of 700 to 3,500 Da, a maximum of two missed cleavages, and possible oxidation of methionine. The SEQUEST data from multiple samples were analyzed by using the SEQUESTonOracle software developed at Wyeth. This application collectively summarizes SEQUEST data derived from multiple samples. As a general approach, a minimum of two rank-one peptides with a cross-correlation factor (Xcorr) of 2 or higher are required for protein identification. Cloning, expression, and protein purification. Primer sets were designed for PCR amplification of desired genes based on the published genomic sequence of S. pyogenes SF370 (16). All PCRs used DNA from S. pyogenes SF370 as a template. PCR products were cloned into pET-28a(⫹) vector (Novagen, San Diego, CA) and transformed into E. coli BLR(DE3) by standard procedures. For protein expression, overnight cultures were diluted 1:25 into 3 liters of fresh HySoy medium and grown to an OD600 of 1.2. Protein expression was induced by the addition of 1 mM isopropyl-␤-D-thiogalactopyranoside. Recombinant proteins were isolated from the soluble fraction of E. coli and purified by standard chromatographic methods. Generation of polyclonal antisera. Swiss Webster mice (five per group) were immunized subcutaneously at weeks 0, 3, and 5 with 5 ␮g of purified protein prepared as described above, adjuvanted with 100 ␮g AlPO4 and 50 ␮g monophosphoryl lipid A, and then bled at week 6. Whole-cell ELISA. Detection of proteins on the bacterial surface was performed by whole-cell enzyme-linked immunosorbent assay (ELISA). Cultures grown in THY medium were harvested at late-exponential growth phase (OD600 of 0.8), centrifuged, and washed twice with PBS. The cells were diluted to a final OD600 of 0.2 in PBS, and 100 ␮l of cell suspension per well was dispensed into 96-well plates (Nalge Nunc, Rochester, NY). Cell suspensions were air dried and stored at 4°C until use. Specific polyclonal mouse sera and secondary goat anti-mouse immunoglobulin G alkaline phosphate-conjugated antibodies (Southern Biotechnology Associates Inc., Birmingham, AL) were diluted in PBS containing 0.05% Tween 20. Plates were developed by standard ELISA procedure using p-nitrophenyl phosphate (Sigma, St. Louis, MO) as a substrate. Antibody titers are expressed as the reciprocal of the dilution that gives an OD405 value of 0.1.

RESULTS Tryptic digestion of bacterial surface-associated proteins. Here we describe a method for direct trypsin digestion of

surface-associated proteins from whole S. pyogenes cells. During this in situ digestion, special care was taken to prevent cell lysis so that the preparation would not be contaminated with cytoplasmic proteins. In the event that cell wall hydrolysis might occur during the 4-h incubation at 37°C, 1 M D-arabinose was included in the trypsin digestion mixture for protoplast stabilization. The integrity of the cells after trypsin treatment was confirmed by viable counts and cryo FE-SEM. Viable counts showed no difference in CFU among samples, and cryo FE-SEM confirmed the integrity of the cells (Fig. 1). Topographical examination at high magnification of untreated bacterial cells revealed large quantities of material on the surface of S. pyogenes. However, streptococcal cells treated with trypsin showed a reduction of visible surface material, as cells appeared smooth and their overall size was smaller. Most importantly, the bacterial cells in both samples appeared intact, and no damaged cells were visible in any of the fields examined. Proteins identified on the surface of S. pyogenes. A total of 79 surface-localized proteins were identified by proteomic analysis of S. pyogenes (Table 1). Of these proteins, 12 were detected only in cells grown to early-exponential phase, 17 were detected only in the cells at late-exponential phase, and 50 were observed in both growth phases. For cases in which a protein was identified in one growth phase but not in another, the sample in which the protein was not found was reanalyzed with SEQUESTonOracle parameters on the basis of the presence of a single peptide. This allowed a protein identification to be made, although with lower confidence. This approach was used only for comparison of the surface protein compositions of selected growth phase cultures. It did not affect the total number or confidence of identified proteins, as it was applied only when proteins had already been identified with

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TABLE 1. Cell surface-associated proteins of S. pyogenes SF370a

Gene

Proteins containing cell wall-anchoring motif SPy0128 SPy0130 SPy0269 SPy0416 SPy0747 SPy0843 SPy0872 SPy1357 SPy1972 SPy1983 SPy2009 SPy2010 SPy2018 Lipoproteins SPy0163 SPy0210 SPy0317 SPy0453 SPy0457 SPy1094 SPy1274 SPy1294 SPy1390 SPy1882 SPy2037

Secreted proteins SPy0019 SPy0167 SPy0292 SPy0469 SPy0714 SPy1037 SPy1733 SPy2039 SPy2066 Membrane-associated proteins SPy0010 SPy0041 SPy0097 SPy0127 SPy0613 SPy0818 SPy0836 SPy1154 SPy1315 SPy1370 SPy1544 SPy1649 SPy1686 SPy1697 SPy1798 SPy1892

Protein annotation

Molecular mass (Da)b

Early-exponential phase

Late-exponential phase

No. of peptides matched

Coverage (%)c


Coverage (%)c

Hypothetical protein Hypothetical protein Putative surface exclusion protein Putative cell envelope proteinase Conserved hypothetical protein Hypothetical protein Putative secreted 5⬘ nucleotidase Protein GRAB Putative pullulanase Collagen-like surface protein Hypothetical protein (fibronectin-binding protein) C5a peptidase precursor M protein type 1 Collagen-binding proteinf

37,188 24,204 94,697 181,288 99,837 111,504 73,214 22,836 128,866 36,2560 40,352 129,487 54,221 85,143

2 — 6 — 4 7 2 2 — 2 9 54 22 5

9 — 15 — 8 11 6 24 — 14 20 49 35 12

—d 3 3 5 4 6 — (1)e 2 — 5 58 20 7

— 14 8 6 8 12 — (8)e 3 — 16 46 33 10

Putative ABC transporter Hypothetical protein Conserved hypothetical protein Metal-binding protein of ABC transporter Putative cyclophilin-type protein Conserved hypothetical protein Putative amino acid ABC transporter, periplasmic amino acid-binding protein Putative maltose/maltodextrin-binding protein Putative protease maturation protein Putative acid phosphatase Conserved hypothetical, putative protease maturation protein Putative oligopeptide permeaseg

36,212 47,096 30,623 34,358 29,342 36,523 31,712

— 2 — — 4 7 (1)

— 9 — — 30 20 (3)

4 — 3 3 3 5 3

12 — 20 16 15 23 11

44,585 38,535 32,575 34,349

3 7 6 13

9 18 42 35

2 5 5 10

7 23 20 22

72,103

—

—

2

8

Putative secreted protein Streptolysin O precursor Penicillin-binding protein (D-alanyl-D-alanine carboxypeptidase) Putative 42-kDa protein Putative adhesion protein Conserved hypothetical protein Putative transcription regulator Cysteine proteinase, SpeB Putative dipeptidase

41,900 63,639 45,766

7 2 —

30 4 —

4 (1) 2

12 (2) 8

41,084 58,520 34,628 45,810 43,130 55,499

4 2 3 (1) — —

17 6 16 (2) — —

2 — 4 2 7 2

10 — 24 5 18 17

14,900 66,658 84,498 14,891 26,618 48,538 45,439 27,757 78,464 48,291 37,931 80,207 16,850 25,851 142,704

— 2 2 — (1) — 4 3 6 2 2 6 4 3 2

— 5 3 — (12) — 15 13 14 7 7 11 33 16 3

2 3 2 2 2 2 4 2 7 2 10 6 (1) 3 3

20 7 3 20 19 8 15 8 16 9 29 13 (15) 17 5

34,845

3

10

3

10

Putative cell division protein Putative acyltransferase Putative penicillin-binding protein 1b Putative signal peptidase I Putative triosephosphate isomerase Hypothetical protein Conserved hypothetical protein Hypothetical protein (sortase) Hypothetical protein Putative deacetylase Putative ornithine transcarbamylase Putative penicillin-binding protein 1A Hypothetical protein Hypothetical protein Hypothetical protein sharing similarity with several eukaryotic proteins Conserved hypothetical protein

Continued on following page

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J. BACTERIOL. TABLE 1—Continued

Gene

SPy1962 SPy2006 SPy2032 SPy2033 SPy2065 SPy2216 Cytoplasmic proteins (group 1)h SPy0274 SPy0611 SPy1073 SPy0731 SPy1262 SPy1282 SPy1371 SPy1372 SPy1547 SPy1738 SPy1881 SPy1889 SPy1896 SPy2070 SPy2072 Cytoplasmic proteins (group 2)i SPy0063 SPy0065 SPy0460 SPy0913 SPy1489 SPy1849 Phage-associated proteins SPy0688

Molecular mass (Da)b

Protein annotation

Early-exponential phase

Late-exponential phase


Coverage (%)c


Coverage (%)c

Putative prolyl-tRNA synthetase Hypothetical protein Putative ATP-binding cassette transporter-like protein Hypothetical protein Hypothetical protein Putative serine protease, DegP

68,703 92,649 46,608

2 3 6

8 5 21

— — 2

— — 7

14,569 64,062 42,760

4 — 7

34 — 25

5 2 7

37 9 29

Putative GAPDH Putative translation elongation factor Tu 50S ribosomal protein L7/L12 Putative enolase Conserved hypothetical protein Putative pyruvate kinase Putative NADP-dependent GAPDH Putative phosphoenolpyruvate:sugar phosphotransferase system enzyme Streptococcal antitumor protein Mannose-specific phosphotransferase system component IIAB Phosphoglycerate kinase Putative fructose-bisphosphate aldolase Transcription regulator; trigger factor, RopA Heat shock protein Heat shock protein

35,943 43,855 12,256 47,356 19,944 54,535 50,369 63,136

3 4 3 6 3 3 5 2

19 17 31 26 18 10 18 10

6 2 (1) 13 — 4 8 2

30 8 (10) 37 — 8 20 10

46,297 35,568

— —

— —

6 2

18 10

42,130 31,208 47,119 57,096 10,332

2 3 2 2 2

8 15 5 7 54

6 — (1) 4 —

22 — (3) 12 —

50S ribosomal protein L5 30S ribosomal protein S8 50S ribosomal protein L11 Putative ribosomal protein S1-like DNA-binding protein Putative histone-like DNA-binding protein Putative pyruvate formate-lyase

19,815 14,802 14,801 43,850

2 — 3 2

18 — 26 9

— 2 (1) (1)

— 20 (10) (4)

9,647 87,528

3 2

42 6

— 2

— 4

Putative major head protein; bacteriophage associated

29,190

—

—

2

15

a

Gene and protein annotations are according to the genomic sequence of S. pyogenes M1 strain SF370 (accession no. NC_002737.1). Theoretical values for protein precursors obtained from the NCBI database (http://www.ncbi.nlm.nih.gov). Percentage of protein sequence covered by the experimentally detected peptides. d —, not detected. e Only one peptide was detected, indicating that traces of the protein may be present. There was no change in the total number of proteins identified, as this protein was already identified with a high degree of confidence in one of the two growth conditions. f Protein is not annotated in S. pyogenes SF370. Gene coordinates in the SF370 genome (NC_002737.1) are 256,906 to 258,886. g Protein is not annotated in S. pyogenes SF370. Gene coordinates in the SF370 genome (NC_002737.1) are 116,794 to 119,064. h Cytoplasmic proteins previously detected on the bacterial surface. i Cytoplasmic proteins identified in this study to be on the surface S. pyogenes. b c

high confidence in one of the paired cultures. Nevertheless, when this approach was applied, additional precautions were taken to increase the confidence of the results. An Xcorr greater than 4 in at least two independent experiments was required for identification in these cases. Manual MS/MS spectrum inspections of these peptides revealed an almost perfect match between the experimental peptide sequence and the theoretical one. A genomic analysis that utilized various algorithms to predict protein secretion and cell surface localization was applied to the genomic sequence of the S. pyogenes strain SF370, and

only 58 of the 79 detected proteins were predicted to be surface localized. In this analysis, 17 proteins were predicted to be covalently attached to cell wall peptidoglycan via a sortasetargeted cell wall-anchoring motif. Proteomic analysis detected 14 of these proteins. Among them are M protein, C5a peptidase, a collagen-binding protein (not annotated in the SF370 genomic sequence), collagen-like protein SclA (SPy1983) (also known as Scl1) (47, 61), protein GRAB (for “protein G-related ␣-2-macroglobulin-binding” protein), pullulanase, a putative surface exclusion protein (SPy0269), a putative cell envelope proteinase (SPy0416), a putative secreted 5⬘ nucleotidase (SPy0872),

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a hypothetical protein (possible fibronectin-binding protein) (SPy2009), and four hypothetical proteins with unknown functions: SPy0128, SPy0130, SPy0747, and SPy0843. The remaining proteins containing a cell wall-anchoring motif—collagenlike protein SclB (SPy1054) (46, 56), a hypothetical protein (SPy1494), and a putative extracellular matrix-binding protein (SPy0737)—were not detected by proteomic analysis, suggesting that these proteins might not be expressed or are present in a very low quantities and/or are deeply imbedded in peptidoglycan under these experimental conditions. Twelve of the 79 experimentally detected surface-associated proteins were predicted to be lipoproteins, and nine others were predicted to be secreted based on having an N-terminal signal sequence and no transmembrane domain regions. The latter group contained true extracellular proteins, such as streptolysin O (SPy0167); streptococcal pyrogenic exotoxin B (SPy2039), also known as streptopain; and a putative secreted protein (SPy0019); as well as proteins known to be associated with the outer surface of the cytoplasmic membrane, such as penicillin-binding protein (D-alanyl-D-alanine carboxypeptidase) (SPy0292). Twenty-two proteins of the total observed were predicted to be membrane associated, according to the SignalP/TopPred algorithms. One bacteriophage-associated protein (SPy0688) was also detected. Proteomic analysis identified 15 proteins that have been shown to have dual localization in bacterial cells, both in cytoplasm and on the bacterial surface. Among these are the well-characterized surface-exposed proteins glyceraldehyde-3phosphate dehydrogenase (GAPDH) (42), enolase (41), streptococcal antitumor protein (arginine deiminase) (59), and elongation factor Tu (18). In addition to the cytoplasmic proteins found on the bacterial surface by others, our study revealed six new cytoplasmic proteins associated with the S. pyogenes envelope. These six include four ribosomal proteins— 50S-L5, 30S-S8, 50S-L11, and the S1-like DNA-binding protein—as well as a putative histone-like DNA-binding protein and pyruvate formate-lyase. To our knowledge, the association of these proteins with the bacterial surface has not been previously reported. Whole-cell ELISA. To confirm the proteomic observations, several of the 79 experimentally determined surface proteins were recombinantly expressed in E. coli. Mice were immunized with the purified recombinant proteins, the antisera from which were then used to assess the reactivity of the specific antibody to the surface of late-exponential-phase streptococcal cultures in a whole-cell ELISA. The ELISA titers for 16 representative sera that reflect the range in titers observed in this study are shown in Table 2. The emm and scpA genes, encoding M protein and C5a peptidase, respectively, were cloned as positive controls, as they are the most abundant streptococcal cell surface proteins. Consistent with this observation, their titers were the highest, at 859,394 and 471,344, respectively. The paired preimmune sera (week 0) were used as the negative controls for each protein, and all of them exhibited titers below 500. Titers fourfold higher than background were considered positive for surface localization. In general, whole-cell titers ranged from background to those shown for the M protein and the C5a peptidase. Elongation factor Tu demonstrated positive ELISA titers that confirmed its localization on the S. pyogenes surface (Table 2). In contrast, other proteins, such as SPy0097,

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TABLE 2. Detection of proteins on the surface of S. pyogenes strain SF370 by whole-cell ELISAa Gene

Protein

SPy0097 SPy0130 SPy0416 SPy0453 SPy0457 SPy0611 SPy0843 SPy1154 SPy1274

Putative penicillin-binding protein 1b Hypothetical protein Putative cell envelope proteinase Metal-binding protein of ABC transporter Putative cyclophilin-type protein Putative translation elongation factor Tu Hypothetical protein Hypothetical protein (sortase) Putative amino acid ABC transporter periplasmic amino acid-binding protein Putative penicillin-binding protein 1A Putative transcription regulator Putative acid phosphatase Conserved hypothetical protein Collagen-like surface protein C5a peptidase precursor M protein serotype 1

SPy1649 SPy1733 SPy1882 SPy1892 SPy1983 SPy2010 SPy2018

Antibody titer

1,486 24,669 52,422 5,355 8,214 4,007 220,289 4,861 1,716 1,277 3,555 2,000 1,539 5,412 471,344 859,394

a Swiss Webster mice were immunized three times with 5 ␮g of purified proteins, and antisera were collected at week 6. S. pyogenes cultured in THY was harvested at an OD600 of 0.8 and used to determine reactivity of the sera on the bacterial surface by whole-cell ELISA with secondary goat anti-mouse immunoglobulin G alkaline phosphate-conjugated antibodies.

SPy1274, SPy1649, and SPy1892 were identified by proteomics but fell below the threshold for antibody reactivity, suggesting that these proteins are either in low abundance, not accessible to antibody binding, or both. Taken together, the antibody titers confirmed the surface localizations of 12 out of the 16 proteins tested in the whole-cell ELISA. DISCUSSION In this report, a method has been employed to determine the protein composition of the surface of S. pyogenes strain SF370. Bacterial cells harvested at both early- and late-exponential growth phases were treated in situ with trypsin to release peptide fragments from surface-associated proteins. Following chromatographic separation in the first dimension on a C8 reversed-phase HPLC column off-line to reduce the complexity of the peptide mixture, the samples were analyzed on a C18 reversed-phase LC microcolumn in-line with direct infusion of peptides into a mass spectrometer (nano-LC-MS/MS). Special precautions were taken during the trypsin digestion of the whole cells to prevent the contamination of surfaceassociated proteins with proteins that are normally confined to the intracellular compartment. Even though the cell wall peptidoglycan does not contain trypsin-sensitive bonds that would result in cell lysis during the digestion procedure, it was possible that endogenous cell wall autolytic enzymes might cause cell wall hydrolysis during the 4-h incubation at 37°C. For this reason, the trypsin digestion was performed in the presence of 1 M D-arabinose, which ensured that even if cell wall hydrolysis occurred, it would cause the formation of protoplasts instead of cell lysis, and the protoplasts could then be removed by centrifugation. While this study does not address the mechanism by which proteins localize to the cell surface, several lines of evidence suggested that no measurable lysis occurred during the treat-

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ment of the cells with trypsin. Firstly, electron microscopic examination of the cells after treatment with trypsin did not reveal any broken or damaged cells; secondly, viable counts showed no reduction in CFU after trypsin treatment; and thirdly, neither lactate dehydrogenase nor pyruvate oxidase, the most abundant intracellular proteins (57), were detected among the cytoplasmic proteins associated with the cell surface. Collectively, these data indicate that detection of cytoplasmic proteins on the surface of S. pyogenes was not due to contamination as a result of cell lysis during sample preparation. A total of 79 surface-associated proteins were identified on the S. pyogenes surface by this proteomic approach (Table 1). By a bioinformatic analysis, 58 of these proteins were predicted to be surface localized and were grouped into four categories: proteins covalently attached to peptidoglycan through the cell wall-anchoring motif (14 proteins), lipoproteins (12 proteins), membrane-associated proteins (22 proteins), and secreted proteins (9 proteins). In addition to these, 21 cytoplasmic proteins were also identified on the bacterial surface. Our proteomic analysis revealed 12 of the predicted 15 LPXTG-containing proteins and an additional two proteins with different cell wall-anchoring motifs. The hypothetical protein SPy0128 contains the EVPTG-anchoring motif, and a collagen-binding protein, Cpa, contains a VVPTG motif and is not annotated in the SF370 genome sequence. These cell wallanchoring motifs were recently reported in S. pyogenes, and it was suggested that they could serve as substrates for SrtC sortase (3). Proteins covalently attached to the cell wall, such as the well-characterized M protein, C5a peptidase, and protein GRAB, play an important role in S. pyogenes virulence by either modulating the host immune response or promoting the adherence to host cell factors (for a review, see reference 11). There are limited data on the role of other proteins containing cell wall-anchoring motifs in bacterial virulence and hostpathogen interactions. An M1 strain of S. pyogenes with genetically inactivated sclA demonstrated a significant reduction of adherence to human A549 epithelial cells (26). Recently, the interaction of this protein with the ␣2␤1 integrin and induction of host cell signaling was reported (21). Pullulanase (PulA), another LPXTG-containing protein, has also demonstrated glycoprotein-binding activity (22). A mutant deficient in PulA expression showed significantly reduced binding to thyroglobulin, fetuin, asialofetuin, and mucin. Lastly, analysis of convalescent-phase sera from patients with invasive infections, noninvasive soft tissue infections, pharyngitis, and rheumatic fever indicated that the cell wall-attached proteins SPy0747, SPy0843, SPy0872, and SPy1970 were expressed in vivo (48). Differential surface expression of some proteins was detected in the two growth phases studied (Table 1). For example, SPy0128, SPy0872, and SPy1983 were expressed only in the early-exponential growth phase, while the expression of SPy0130, SPy0416, and SPy1972 was observed during lateexponential growth. These data may reflect the regulation of expression of these proteins during bacterial growth, although the significance of this regulation is not yet understood. The differential expression of collagen-like protein SclA observed in this study is consistent with a previously published report showing that sclA was transcribed in mid-log phase but not in the late-log phase of growth (26). Additionally, our data sug-

J. BACTERIOL.

gesting differential expression of SPy0872 and SPy1972 are also in agreement with the data on the expression of these proteins in an M1 strain of S. pyogenes (48). Antibody reactivity, as determined by whole-cell ELISA, confirmed surface localization of 12 of the 16 proteins shown in Table 2. Compared to the positive controls M protein and C5a peptidase, whole-cell antibody titers ranged from background to ⬎800,000. These differences most likely reflect the abundance of the protein on the bacterial surface, the length and amino acid composition of the polypeptide chain, and the number of accessible epitopes for antibody binding. The hypothetical protein SPy0843, the putative cell envelope proteinase SPy0416, and the hypothetical protein SPy0130 showed comparatively high antibody titers, of 220,289, 52,422 and 24,669, respectively. These data suggest that the cells produce sufficient amounts of these proteins on the surface and that these proteins could be considered potential vaccine candidates. Both the hypothetical protein SPy0843 and the putative cell envelope proteinase (SPy0416) have been shown to be protective in a mouse challenge model following active immunization with the respective protein (48, 50). Twenty-one cytoplasmic proteins were also detected on the surface of S. pyogenes, 15 of which were observed on the surface of gram-positive bacteria in earlier studies by proteomic analysis. For some, functional analyses have shown them to be physiologically relevant. The best-characterized proteins in this category are enolase, GAPDH, and elongation factor Tu. Surface-associated enolase was shown to be the major plasminogen-binding protein in S. pyogenes (14, 41) and S. pneumoniae (4) and is involved in invasion and adherence of bacteria to human pharyngeal cells (44). The multifunctional surface protein GAPDH of S. pyogenes binds plasminogen, fibronectin, and myosin (42, 58), possesses ADP-ribosylating activities (40), and regulates host cell signaling (43). Recently, it was shown that GAPDH recognizes the pharyngeal membrane-bound urokinase plasminogen activator receptor CD87, which contributes to bacterial adherence and plays a significant role in pathogenesis (23). The translation elongation factor Tu has also been shown to be a surface-associated protein in S. pyogenes (8, 50) and other gram-positive bacteria (18, 53, 55, 57). Little is known about the possible functions of surface-associated elongation factors on the bacterial surface. Elongation factor Tu of Lactobacillus johnsonii is involved in the attachment of this pathogen to human intestinal cells and mucins (18), while elongation factor Tu of Mycobacterium pneumoniae binds fibronectin, which mediates the attachment of the pathogen to host cells (12). Several other surface-associated cytoplasmic proteins reported in this study were also detected on the bacterial surface in previous proteomic analyses. Phosphoglycerate kinase and fructose bisphosphate aldolase were reported on the surface of S. pneumoniae (24), S. pyogenes (8, 50), Clostridium pneumoniae (12), and S. oralis (57). Ribosomal protein L7/L12 was detected on the surface of S. oralis (57), S. aureus (30), and S. pyogenes (8). Heat shock protein was observed on the surface of C. pneumoniae (30), S. agalactiae (20, 30), Listeria monocytogenes (54), and Clostridium difficile (60). Pyruvate kinase was also reported as a surface-associated protein in S. pyogenes (8), C. difficile (60), and L. monocytogenes (54). The detection of the cytoplasmic protein RopA on the S.

VOL. 189, 2007


pyogenes surface reported here and previously (8) is intriguing. S. pyogenes secretes the cysteine proteinase SpeB exclusively through the ExPortal, a single unique membrane microdomain containing Sec translocons (51, 52). Trigger factor, RopA, a ribosome-associated chaperone with a peptidyl-prolyl cis-trans isomerase activity, is essential for the secretion and maturation of SpeB (27, 28). The deletion of the central region of the trigger factor results in a normal level of proteinase secretion, but the secreted proteinase exhibited a defect in its maturation. HtrA (DegP) is another protein involved in the maturation of the secreted SpeB and is a part of the ExPortal (52). We hypothesize that the trigger factor, RopA, might serve as a component of the ExPortal in order to participate in the maturation of SpeB. Our proteomic data reported here favor this hypothesis. Both RopA and DegP were found to be on the surface of S. pyogenes (Table 1). In spite of a growing list of cytoplasmic proteins identified on the bacterial surface, the mechanisms of their surface localization and attachment to the bacterial envelope remain unclear. These proteins do not contain signal peptides that direct proteins into secretory pathways and do not possess any known cell wall-anchoring motifs. Other, specifically designed experiments are needed to elucidate the mechanism(s) of delivery and attachment of these proteins to the bacterial surface. During the preparation of the manuscript, Rodrı´guezOrtega et al. (50) published a data set of S. pyogenes SF370 surface-associated proteins in which a total of 72 proteins were identified by a similar approach of direct proteolytic digestion of proteins from the bacterial surface. A comparison of these recently published data with the data reported here revealed substantial differences in the sets of detected proteins. Only 33 proteins were common to both studies. Although the cause of the disparity in protein identification is not clear, it is possible that differences in the algorithms utilized for protein identification are partially responsible. As was noted in the paper published by Rodrı´guez-Ortega et al., some proteins were identified with a low degree of confidence by using only one peptide for their identification. A majority of those proteins (28 of 35) were not detected in our study. In contrast, for our analysis we required a minimum of two peptides for confident protein identification. Secondly, in the study of Rodrı´guezOrtega et al., protein identification was based on a search against just the S. pyogenes SF370 database, which contained 1,819 entries (50). Our protein identification was performed with the entire NCBI nonredundant database consisting of 3,292,837 proteins. Searching peptides against a large database reduces the potential rate of false-positive hits. It also allows identification of proteins in multiple strains of the same species for which genome sequences have been deposited. As reported here, two identified proteins (a putative collagen-binding protein and a putative oligopeptide permease) are not annotated in the S. pyogenes SF370 genome, and yet the DNA sequences encoding these proteins are present. Coordinates for these two proteins in the S. pyogenes SF370 genome were identified by a tBLASTn analysis. Finally, we analyzed both early- and lateexponential phase cultures to capture a more comprehensive set of surface-associated proteins. As a result, this study reports 33 additional proteins that were not identified in the previously published analyses. Nevertheless, our data complement the data published previously (50), and together they

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provide the most comprehensive set available of S. pyogenes surface-associated proteins for future vaccine development to prevent human S. pyogenes disease. ACKNOWLEDGMENTS We thank Bret Sellman and Alex Ruzin for critical review of the manuscript and David Russell for assistance and development of the HMM algorithms. REFERENCES 1. Altschul, S., T. Madden, A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389–3402. 2. Arnott, D. P., W. J. Henzel, and J. T. Stuits. 1995. Identification of proteins from two-dimensional electrophoresis gels by peptide mass fingerprinting, p. 226–243. In A. P. Snyder (ed.), Biochemical and biotechnological applications of electrospray ionization mass spectrometry. American Chemical Society, Washington, DC. 3. Barnett, T. C., A. R. Patel, and J. R. Scott. 2004. A novel sortase, SrtC2, from Streptococcus pyogenes anchors a surface protein containing a QVPTGV motif to the cell wall. J. Bacteriol. 186:5865–5875. 4. Bergmann, S., D. Wild, O. Diekmann, R. Frank, D. Bracht, G. S. Chhatwal, and S. Hammerschmidt. 2003. Identification of a novel plasmin(ogen)-binding motif in surface displayed ␣-enolase of Streptococcus pneumoniae. Mol. Microbiol. 49:411–423. 5. Boekhorst, J., M. W. H. J. de Been, M. Kleerebezem, and R. J. Siezen. 2005. Genome-wide detection and analysis of cell wall-bound proteins with LPxTG-like sorting motifs. J. Bacteriol. 187:4928–4934. 6. Calvo, E., M. Graciela Pucciarelli, H. Bierne, P. Cossart, J. P. Albar, and F. Garcı´a-del Portillo. 2005. Analysis of the Listeria cell wall proteome by two-dimensional nanoliquid chromatography coupled to mass spectrometry. Proteomics 5:433–443. 7. Claros, M. G., and G. von Heijne. 1994. TopPred II: an improved software for membrane protein structure predictions. Comput. Appl. Biosci. 10:685– 686. 8. Cole, J. N., R. D. Ramirez, B. J. Currie, S. J. Cordwell, S. P. Djordjevic, and M. J. Walker. 2005. Surface analyses and immune reactivities of major cell wall-associated proteins of group A Streptococcus. Infect. Immun. 73:3137– 3146. 9. Comfort, D., and R. T. Clubb. 2004. A comparative genome analysis identifies distinct sorting pathways in gram-positive bacteria. Infect. Immun. 72:2710–2722. 10. Cunningham, M. W. 2003. Autoimmunity and molecular mimicry in the pathogenesis of post-streptococcal heart disease. Front. Biosci. 8:s533–s543. 11. Cunningham, M. W. 2000. Pathogenesis of group A streptococcal infections. Clin. Microbiol. Rev. 13:470–511. 12. Dallo, S. F., T. R. Kannan, M. W. Blaylock, and J. B. Baseman. 2002. Elongation factor Tu and E1 b subunit of pyruvate dehydrogenase complex act as fibronectin binding proteins in Mycoplasma pneumoniae. Mol. Microbiol. 46:1041–1051. 13. Delcher, A., D. Harmon, S. Kasif, O. White, and S. Salzberg. 1999. Improved microbial gene identification with GLIMMER. Nucleic Acids Res. 27:4636– 4641. 14. Derbise, A., Y. P. Song, S. Parikh, V. A. Fischetti, and V. Pancholi. 2004. Role of the C-terminal lysine residues of streptococcal surface enolase in Glu- and Lys-plasminogen-binding activities of group A streptococci. Infect. Immun. 72:94–105. 15. Eng, J. K., A. L. McCormack, I. Yates, and R. John. 1994. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass Spectrom. 5:976–989. 16. Ferretti, J. J., W. M. McShan, D. Ajdic, D. J. Savic, G. Savic, K. Lyon, C. Primeaux, S. Sezate, A. N. Suvorov, S. Kenton, H. S. Lai, S. P. Lin, Y. Qian, H. G. Jia, F. Z. Najar, Q. Ren, H. Zhu, L. Song, J. White, X. Yuan, S. W. Clifton, B. A. Roe, and R. McLaughlin. 2001. Complete genome sequence of an M1 strain of Streptococcus pyogenes. Proc. Natl. Acad. Sci. USA 98:4658– 4663. 17. Fountoulakis, M., and B. Taracs. 2001. Effect of strong detergents and chaotropes on the detection of proteins in two-dimensional gels. Electrophoresis 22:1593–1602. 18. Granato, D., G. E. Bergonzelli, R. D. Pridmore, L. Marvin, M. Rouvet, and I. E. Corthesy-Theulaz. 2004. Cell surface-associated elongation factor Tu mediates the attachment of Lactobacillus johnsonii NCC533 (La1) to human intestinal cells and mucins. Infect. Immun. 72:2160–2169. 19. Hu, M. C., M. A. Walls, S. D. Stroop, M. A. Reddish, B. Beall, and J. B. Dale. 2002. Immunogenicity of a 26-valent group A streptococcal vaccine. Infect. Immun. 70:2171–2177. 20. Hughes, M. J. G., J. C. Moore, J. D. Lane, R. Wilson, P. K. Pribul, Z. N. Younes, R. J. Dobson, P. Everest, A. J. Reason, J. M. Redfern, F. M. Greer, T. Paxton, M. Panico, H. R. Morris, R. G. Feldman, and J. D. Santangelo.

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