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. 2007 May 1;9(5):1172–1190. doi: 10.1111/j.1462-5822.2006.00858.x

Global analysis of community-associated methicillin-resistant Staphylococcus aureus exoproteins reveals molecules produced in vitro and during infection

Christopher Burlak 1, Carl H Hammer 2, Mary-Ann Robinson 2, Adeline R Whitney 1, Martin J McGavin 3, Barry N Kreiswirth 4, Frank R DeLeo 1,*
PMCID: PMC2064037  PMID: 17217429

Abstract

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is a threat to human health worldwide. Although progress has been made, mechanisms of CA-MRSA pathogenesis are poorly understood and a comprehensive analysis of CA-MRSA exoproteins has not been conducted. To address that deficiency, we used proteomics to identify exoproteins made by MW2 (USA400) and LAC (USA300) during growth in vitro. Two hundred and fifty unique exoproteins were identified by 2-dimensional gel electrophoresis coupled with automated direct infusion-tandem mass spectrometry (ADI-MS/MS) analysis. Eleven known virulence-related exoproteins differed in abundance between the strains, including alpha-haemolysin (Hla), collagen adhesin (Cna), staphylokinase (Sak), coagulase (Coa), lipase (Lip), enterotoxin C3 (Sec3), enterotoxin Q (Seq), V8 protease (SspA) and cysteine protease (SspB). Mice infected with MW2 or LAC produced antibodies specific for known or putative virulence factors, such as autolysin (Atl), Cna, Ear, ferritin (Ftn), Lip, 1-phosphatidylinositol phosphodiesterase (Plc), Sak, Sec3 and SspB, indicating the exoproteins are made during infection in vivo. We used confocal microscopy to demonstrate aureolysin (Aur), Hla, SspA and SspB are produced following phagocytosis by human neutrophils, thereby linking exoprotein production in vitro with that during host–pathogen interaction. We conclude that the exoproteins identified herein likely account in part for the success of CA-MRSA as a human pathogen.

Introduction

Staphylococcus aureus causes a wide range of human diseases, including impetigo, cellulitis, food poisoning, toxic–shock syndrome, necrotizing pneumonia, endocarditis and sepsis (Lowy, 1998; Diekema et al., 2001). Decades of selective pressure with β-lactam antibiotics and close proximity of susceptible hosts have resulted in a high prevalence of methicillin-resistant S. aureus (MRSA) in hospitals worldwide (Chambers, 2001; Diekema et al., 2001). Although these factors logically explain the high incidence of hospital-associated MRSA infections, the molecular basis for the increased incidence and severity of community-acquired (or associated) MRSA (CA-MRSA) infections among healthy individuals remains incompletely defined (Chambers, 2001; 2005; McDougal et al., 2003; Fridkin et al., 2005; Miller et al., 2005; Zetola et al., 2005). Recent studies indicate strains that are the leading causes of CA-MRSA disease in the United States, represented by pulsed-field gel electrophoresis (PFGE) types USA300-0114 (McDougal et al., 2003; Fridkin et al., 2005; Kazakova et al., 2005; Diep et al., 2006) and USA400 (Centers for Disease Control and Prevention, 1999; Baba et al., 2002; McDougal et al., 2003; Adem et al., 2005), have enhanced virulence compared with leading causes of hospital infections (e.g. USA200) (Voyich et al., 2005). In addition to their distinct PFGE profiles (McDougal et al., 2003), these two CA-MRSA strains can be distinguished from one another and from other S. aureus strains on the basis of multilocus sequence typing (MLST or ST) and sequencing of the variable number tandem repeats in the staphylococcal protein A gene (spa); USA300-0114 is spa-type 1 (Shopsin et al., 1999) and multilocus sequence type ST8, while USA400 is ST1 (Enright et al., 2000). Both strains also have the type IV staphylococcal chromosomal cassette mec element, which is common among CA-MRSA but not typically found in hospital adapted nosocomial MRSA (Baba et al., 2002; Voyich et al., 2005; Diep et al., 2006). In addition, each strain has one or more common names, such as Los Angeles County clone (LAC) or FPR3757 (sequenced strain) used for USA300-0114 (Voyich et al., 2005; Diep et al., 2006), and MW2 for the prototype USA400 strain (Centers for Disease Control and Prevention, 1999; Baba et al., 2002). Enhanced virulence of USA300-0114 (referred to herein as either LAC or USA300) and USA400 (referred to herein as MW2) is linked in part to their ability to circumvent killing by neutrophils and cause host cell lysis (Voyich et al., 2005). It is likely that exoproteins (cell surface-associated and freely secreted proteins) produced by these strains are an important component of this enhanced virulence (Foster, 2005; Voyich et al., 2005; Diep et al., 2006).

Secreted virulence proteins of S. aureus can be categorized based on proven or putative function. Cytolytic toxins, such as haemolysins (Hla, Hlb, HlgABC) and leukocidins (LukD/E and Panton–Valentine leukocidin, PVL), oligomerize to form pores on the cell surface (Walev et al., 1993; Bhakdi et al., 1998). Destruction of leucocytes (especially neutrophils), which can be mediated by these toxins, is likely a key component of CA-MRSA pathogenesis. For example, PVL has high specificity for granulocytes and is linked by epidemiology to CA-MRSA disease (Lina et al., 1999), although our recent studies indicate the toxin has a limited role in virulence (Voyich et al., 2006). Staphylococcal enterotoxins are secreted superantigens (SAg) that bind to major histocompatibility complex (MHC) class II proteins, resulting in CD4+ T-cell activation and immune modulation (Malchiodi et al., 1995; McCormick et al., 2001; Orwin et al., 2002; Llewelyn et al., 2004). S. aureus also secretes numerous proteases and lipases that degrade host components, and proteins that sequester antibody or inactivate antibiotics (Foster, 2005). As a step towards understanding the role played by S. aureus exoproteins in virulence, previous proteomics-based studies identified immunogenic proteins produced by strain COL (Vytvytska et al., 2002), evaluated the role played by S. aureus agr and sigB on secretion of virulence factors (Ziebandt et al., 2001), and tested the effects of linezolid on production of virulence factors (Bernardo et al., 2004). However, a comprehensive analysis of the exoproteins produced by CA-MRSA has not been conducted.

To that end, we used a proteomic approach to identify exoproteins of LAC and MW2 during growth in vitro and evaluated immunogenicity of the proteins using sera from mice infected with each strain. In addition, we used confocal microscopy to determine that selected exoproteins were produced within phagocytic vacuoles of human neutrophils following uptake, a phenomenon accompanied by host cell lysis.

Results

Resolution and identification of culture supernatant proteins produced by CA-MRSA strains

As an initial step towards gaining a comprehensive understanding of exoproteins made by the most prominent CA-MRSA strains, we resolved/identified proteins in MW2 (USA400) and LAC (USA300) culture supernatants using 2-dimensional gel electrophoresis (2-DGE) coupled with automated direct infusion-tandem mass spectrometry (ADI-MS/MS). Three hundred and fifty-three and 270 protein spots were resolved from MW2 and LAC culture media, respectively, at mid-exponential phase of growth (Fig. 1A–D). By comparison, 625 MW2 and 581 LAC proteins were resolved from culture supernatants at stationary phase of growth (Fig. 1B). Of the resolved exoproteins, 153 (60.2 ± 18%) from cultures at mid-exponential growth and 436 (67.9 ± 6.6%) from cultures at stationary growth matched between strains, indicating MW2 and LAC produce numerous proteins that co-migrate during 2-DGE (Fig. 2). We note that this analysis fails to account for variations of same or similar proteins with slightly different migration by 2-DGE. Therefore, our estimate of the degree of similarity between the two strains at the level of exoprotein (60% to 68%) is relatively conservative.

Fig. 1.

Fig. 1

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Distribution and function of proteins found in MW2 and LAC culture supernatants. A. MW2 and LAC were cultured to mid-exponential (Mid-exp.) or stationary phases of growth as indicated, at which point proteins were identified/resolved using proteomics as described in Experimental procedures. B. Numbers in yellow-shaded regions represent proteins identified in both MW2 and LAC supernatants at the indicated growth phase. Numbers in red- or blue-shaded regions indicate proteins identified in MW2 or LAC supernatants respectively. Results are derived from three separate experiments. C. Proteins identified by ADI-MS/MS were categorized based upon functional annotation. Numbers are the per cent of total proteins identified. D. Proteins identified at mid-exponential and stationary phases of growth were enumerated and categorized by functional annotation. Carb. Met., carbohydrate transport and metabolism; AA Met., amino acid transport and metabolism; Vir. & Def., virulence and defence mechanisms; Toxins & Hem., toxins and haemolysins; Stress Resp., stress response; Cell Div., cell division and maintenance; Prot. Synth., protein synthesis; Nuc. Biosynth., nucleotide biosynthesis; FA Biosynth., fatty acid biosynthesis; Trans. & Rep., transcription and replication.

Fig. 2.

Fig. 2

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2-DGE analyses of culture supernatant proteins produced by MW2 and LAC. Proteins from MW2 (green) and LAC (magenta) culture supernatants were analysed by proteomics. Left panel, proteins from cultures at mid-exponential (Mid-exp.) phase of growth. Right panel, proteins from cultures at stationary (Stat.) phase of growth. White areas are regions of overlap. Selected proteins are indicated. Images are representative of three separate experiments.

Using ADI-MS/MS and excluding protein isoforms and identifications from multiple gels, we identified 250 unique proteins from MW2 and LAC culture supernatants at the two phases of growth combined (98 at mid-exponential phase of growth and 228 at stationary phase) (Fig. 2 and Table 1). Proteins were separated into categories based on functional annotation to facilitate subsequent analyses (Fig. 1B and C, and Table 1).

Table 1.

MW2 and LAC culture supernatant proteins identified by ADI-MS/MS.

Protein name (Entrez protein name)a Entrez proteina MWE pIE MP SC% MS/MS MOWSE score
Carbohydrate transport and metabolism (61)
 Acetate kinase (AckA)C1,D1 13701506 44029 5.7 10 31 226
 Acetoin reductase (ButA)D2 49482369 27199 5.0 3 15 104
 Aconitate hydratase (AcnA)B1,C1,D1 49241672 98850 4.9 24 34 878
 Adenylate kinase (Adk)B1,D2 49484445 23959 4.8 6 29 245
 Adenylosuccinate synthetase, putative (PurA)C1 49482270 47522 5.1 4 12 146
 Alcohol dehydrogenase (Adh)B5,D6 14246373 36039 5.5 13 45 558
 Alcohol dehydrogenase, zinc-containing (AdhE)D1 87162223 36244 5.3 4 13 93
 Aldehyde dehydrogenase (AldA1)B1,D1 49242475 51936 5.1 7 19 179
 ATP synthase beta chain (AtpD)A1,C2,D1 49484327 51368 4.7 4 11 101
 ATP synthase alpha chain (AtpA)D1 49484329 54550 4.9 4 9 82
 Citrate synthase II (GltA)B2,C1,D2 49483937 42566 5.4 14 50 507
 CoA synthetase protein, putative (FadE)B1,C1,D1 49483408 42060 4.9 9 23 256
 Deoxyribose-phosphate aldolase (Dra)B1 14247910 23327 4.7 3 17 56
 Dihydrolipoamide acetyltransferase: subunit E2 (PdhC)A2,C2,D2 581570 46411 4.9 16 46 817
 Dihydrolipoamide dehydrogenase: subunit E3 (PdhC)A2,B2,C1,D2 48874 49421 5.0 16 46 803
 Phosphoglycerate mutase (Gpm)B2 14246544 56419 4.7 12 29 590
 Enolase (Eno)A2,B3,C2,D4 6015099 47088 4.5 18 54 1257
 Methylenetetrahydrofolate dehydrogenase (FolD)B2,D1 49244345 30824 5.4 13 68 859
 Formate tetrahydrofolate ligase (Fhs)B1,C1,D1 83288210 59876 5.8 16 44 759
 Formate acetyltransferase (PflB)D2 49482458 84808 5.3 6 11 193
 Fructose bisphosphate aldolase class I (Fba)A1,B4,C2,D6 88196553 33034 4.9 14 57 683
 D-Fructose 6-phosphate amidotransferase (GlmS)D1 49484376 65839 4.9 6 15 151
 6-Phospho-beta-galactosidase (LacG)A1 644835 116131 5.3 1 1 42
 Glucose 6-phosphate isomerase A (GpiA)A1,B1,C1,D1 49244181 49777 4.8 20 53 1008
 Glucose 6-phosphate 1-dehydrogenase (Zwf)D1 49483755 56929 5.3 7 15 227
 Glyceraldehyde 3-phosphate dehydrogenase 1 (Gap)A2,B5,C3,D5 49244087 36258 4.9 13 47 770
 Glyceraldehyde 3-phosphate dehydrogenase subunit B (GapB)D1 38195941 36899 6.0 4 14 106
 Glyceraldehyde 3-phosphate dehydrogenase subunit C (GapC)C1,D1 38195943 36227 4.9 3 10 65
 Glycerate dehydrogenase (MW2224)B1,D2 14248079 34681 5.1 10 41 377
 Glycine cleavage system H protein (GcvH)A1,C1,D2 49241194 14072 4.0 2 30 194
 Glycine C-acetyltransferase, similar to (MW0505)D1 14246318 41426 5.3 2 5 58
 Hexulose 6-phosphate synthase, putative (SgaH)B1,D1 87160968 22404 4.6 12 81 656
 Hydrolase (HAD superfamily) (MW0575)B1,D1 49241218 27962 4.5 4 16 67
 Indole-3-pyruvate decarboxylase, putative (IpdC)D1 14245955 60490 5.1 1 2 59
 Isocitrate dehydrogenase (Idh)B1,D1 49244963 46408 4.9 4 10 129
 L-Lactate dehydrogenase 1 (Ddh)B3,D4 57286685 34562 5.0 10 34 368
 Malate:quinone oxidoreductase (Mqo2)A1,B1,C1,D1 21205698 55892 6.2 7 17 301
 Mannitol-1-phosphate 5-dehydrogenase (MtlD)B3,D1 49242510 40801 5.0 10 36 357
 2-C-Methyl-D-erythritol 4-phosphate cytidylyltransferase (IspDF)B1 49482491 26640 5.4 3 12 77
 NAD-dependent dehydrogenase, putative (MW2068)B2 49245380 24057 5.0 15 81 643
 Oxoglutarate dehydrogenase (Ogdh)B1,D1 21204471 105289 5.4 14 22 401
 Phosphate acetyltransferase, putative (Pta)A1,B4,C1,D5 22212856 20383 4.8 8 44 523
 Phosphoenolpyruvate carboxykinase (PckA)B1,D1 49484033 59370 5.7 20 44 678
 Phosphogluconate dehydrogenase (Pgd)A1,C1,D1 14247282 51751 5.1 5 15 122
 Phosphopentomutase, putative (Drm)A1,B1,C2,D2 14246544 56419 4.7 14 36 672
 Phosphoenolpyruvate-protein phosphatase (PtsI)B1,D1 1070386 63179 4.7 12 22 490
 6-Phosphofructokinase (PfkA)B2,D1 49244967 34818 5.6 11 34 327
 6-Phosphogluconate dehydrogenase, decarboxylating (Gnd)B1,D2 49483761 51770 5.0 12 30 564
 Phosphoglycerate kinase (Pgk)A1,B2,C3,D2 49483031 42575 5.2 14 47 615
 1-Pyrroline-5-carboxylate dehydrogenase (RocA)B1,D1 21205647 56833 5.0 16 39 687
 Pyruvate carboxylase, putative (PycA)B1,D1 49483277 128451 5.2 14 14 353
 Pyruvate dehydrogenase E1 component, alpha subunit, putative (PdhA)A1,C2 49244374 41357 4.9 7 32 239
 Pyruvate dehydrogenase E1 component, beta subunit, putative (PdhB)A2,B4,C1,D3 57285889 35194 4.7 10 43 525
 Pyruvate kinase (Pyk)A1,B2,C3,D1 49242068 63103 5.2 18 38 610
 Short chain dehydrogenase MW2249 (MW2249)D3 21205420 31769 4.6 4 12 179
 Succinyl-CoA synthetase (SucD)B1 49244528 31506 5.5 5 25 154
 Succinyl-CoA synthetase (SucB)D3 49483408 42060 4.9 7 18 246
 Tagatose-bisphosphate aldolase, putative (LacD1)A1,B6 49245361 30817 5.0 8 40 371
 Transaldolase, putative (MW1721)B4,D2 14247553 25742 4.8 7 37 273
 Transketolase (Tkt)B1,D1 57284532 72206 5.0 19 35 703
 Triosephosphate isomerase (Tpi)A1,B3,C2,D3 49483032 27245 4.8 8 39 477
Amino acid transport and metabolism (12)
 Alanine dehydrogenase 2 (Ald2)D1 49244722 40209 5.2 6 18 173
 Amidophosphoribosyltransferase precursor, putative (PurF)B1 49244352 54363 6.1 12 23 426
 Cysteine synthase (CysK)C2 82750220 32969 5.4 8 52 273
 3-Deoxy-7-phosphoheptulonate synthase (MW1680)B1,D1 49483977 40609 5.8 7 24 229
 Glucosamine-fructose 6-phosphate aminotransferase (GlmS)D1 14247927 65795 4.9 5 11 110
 Glutamine synthetase (GlnA)B1 1134886 50808 5.1 11 34 400
 Imidazolonepropionase (HutI)D1 87162411 45011 5.2 5 15 101
 Phosphoribosylformylglycinamidine synthase I (PurQ)B1 14246838 24541 5.0 3 21 108
 Phosphotransferase system enzyme IIA-like protein (SH1484)D1 88195154 17949 4.5 4 33 144
 SNO glutamine amidotransferase family protein (MW0475)D1 49482749 20617 5.7 6 43 211
 Thiamine pyrophosphate enzyme, putative (MW0162)B1,D1 49240559 60503 5.0 6 13 181
 Urocanate hydratase (HutU)C1 14248105 60626 5.2 4 12 55
Virulence/defence mechanisms (31)
 Aminopeptidase PepS (PepS)D1,S 87161826 46805 4.8 8 19 226
 Aureolysin (Aur)C1,D1,S 6119705 56281 5.1 3 8 102
 Chitinase (MW0945)D1,S 49483226 11338 6.6 2 32 75
 Coagulase (Coa)A4,C7,D1,S 46540 71675 8.4 20 37 920
 Collagen adhesin precursor (Cna)A2,B1,L,M,S 21205785 132921 5.9 7 7 199
 Ear (Ear)B1,D1,M,S 21203924 20322 8.6 4 19 115
 Esterase\lipase (MW2501)B1,D2,S 14248355 30986 4.7 10 52 415
 Fibrinogen-binding protein-related (MW1037)A1,C1,S 49244435 12171 10.4 3 27 115
 FmtB protein (FmtB)A1,D1 14247939 263611 4.6 9 4 229
 Fibronectin-binding protein A (FnbA)C3,L,M,S 87161146 111642 4.6 9 13 254
 Fibronectin-binding protein B (FnbB)A1,B2,C2,L,M,S 87162339 103492 4.7 9 12 265
 IgG-binding protein (Sbi)A2,S 49245643 50099 9.4 11 26 330
 IgG-binding protein A precursor (Spa)A9,C7,D4,L,M,S 83682325 49338 5.7 16 45 666
 Lipase (Lip)C3,D8,S 1095875 76845 7.1 19 30 947
 Lysophospholipase, putative (MW1732)D1,S 87162009 31019 5.1 9 53 306
 Metallo-beta-lactamase superfamily protein (YycJ)B1,S 49483948 25306 5.0 4 24 101
 Mrp protein (Mrp)C1 5834649 262876 4.6 9 4 250
 1-Phosphatidylinositol phosphodiesterase (Plc)B1,D3,S 1172527 35213 6.5 13 52 452
 Putative sulfatase (MW0681)B1,C1,D2,M,S 49244034 74353 9.0 8 17 349
 SspA, V8 protease (SspA)B2,D4,S 12025238 36304 5.0 6 27 337
 SspB, cysteine protease precursor (SspB)A1,B3,C2,D9,S 12025239 44491 5.7 15 44 738
 Serine protease SplC (SplC)B1,D2,S 88195634 26082 6.3 7 27 212
 Staphylokinase precursor (Sak)B4,C1,D6,S 21205055 18483 6.8 7 67 411
 Succinyl-diaminopimelate desuccinylase (MW1943)B1 13701801 45109 4.6 6 16 158
 Tetracycline resistance protein (TetP)B1,M,S 6094458 72677 5.3 1 2 60
 ThiJ/PfpI family protein, protease 1 (MW1815)B3,D2 49240910 32171 5.0 6 67 323
 Trigger factor (prolyl isomerase) (Tig)A2,B3,C2,D2 49483918 48565 4.3 8 24 439
 Tripeptidase, similar to (MW1465)B1 14247283 40172 5.0 4 12 88
 Truncated MHC class II analogue protein (SAOUHSC 02466)D4 88196118 15438 8.7 7 55 300
 Xaa-His dipeptidase (MW1694)B2,C1,D1,S 49245019 52775 4.6 13 34 535
 Xaa-Pro dipeptidase (MW1482)B1,D3,S 49483779 39357 5.2 9 32 314
Toxins and haemolysins (7)
 Alpha-haemolysin, chain G (Hla)C3,D4,S 2914575 33227 7.9 14 51 666
 Enterotoxin C3 (Sec3)B4,S 295149 27634 7.2 17 58 799
 Enterotoxin H (Seh)B4,S 9955226 25128 5.2 7 38 282
 Enterotoxin K (Sek)C1,D2,S 87161791 27733 8.3 6 25 252
 Enterotoxin L, extracellular (Sel2)D2,S 14247781 27479 9.0 2 4 83
 Enterotoxin Q (Seq)B2,C2,D2,S 87161054 28129 7.7 8 43 358
 Exotoxin (SAUSA300_0407)C1,S 88194194 25350 8.5 2 15 96
Stress response proteins (20)
 Alkaline shock protein 23 (Asp23)A1,B7,C5,D11 49484402 19180 5.1 8 60 301
 Alkyl hydroperoxide reductase subunit C (AhpC)A1,B2,C2,D4 49482631 20963 4.9 7 53 514
 Alkyl hydroperoxide reductase subunit F (AhpF)B1,D1 14246148 54674 4.7 5 11 164
 ATP-dependent Clp proteinase chain (ClpP)B2,D3,M 14248322 77810 4.8 25 49 1416
 Catalase (KatA)B1,C1,D2 7161887 58287 5.3 15 31 680
 Chaperone protein DnaK, HSP70 (DnaK)A2,B1,C2,D,M 1169381 66307 4.6 15 34 875
 Chaperone protein HchA, Hsp31 (HchA)B1 49240910 32171 5.0 4 14 147
 Cold shock protein (CspA)B2,C2,D2 49483592 7317 4.5 3 78 188
 General stress protein 26 (MW2302)B1,D1 49245607 15807 5.1 4 40 199
 GroES (GroES)D1 18028156 10453 5.1 2 26 68
 NAD(P)H-flavin oxidoreductase, similar to (Frp)D1 14248297 25359 5.5 4 17 56
 Nitric oxide dioxygenase (MW0216)D1 14246007 42932 5.2 4 12 104
 OsmC-like protein (MW0781)B3,D1 49244117 15320 4.8 3 25 113
 Peroxiredoxin reductase (AhpF)D1 49243746 11325 6.4 3 42 67
 SrrA (SrrA)D1 37781574 28143 5.2 6 30 288
 Superoxide dismutase (SodA)A1,B2,C1,D2 49483802 22697 5.1 6 45 303
 Thioredoxin (TrxA)B1,D2 49483308 11433 4.4 5 56 223
 Thioredoxin (MW1870)B1,D1 49484170 21902 5.2 7 37 259
 Thioredoxin reductase (TrxB)B1,C1,D2 32468851 33595 5.2 9 30 456
 Universal stress protein, putative (MW1653)B1,D5 49483951 18463 5.6 8 63 497
Cell division and maintenance (33)
 Aminoglycoside phosphotransferase (AphA)C1 11991167 30624 4.5 3 16 64
 Autoinducer-2 production protein (LuxS)D1 49484358 17502 5.4 4 23 140
 Autolysin (Atl)A2,B1,C3,D4,S 14248419 69186 6.0 20 43 867
 Cyclophilin type peptidyl-prolyl cis-trans isomerase, putative (MW0836)D1 49483114 21605 4.6 4 18 108
 Cell division initiation protein DivIVA (MW1335)B1,D1 49483601 29963 4.4 3 11 114
 Cell division protein FtsZ (FtsZ)A1,B1,D2 38604824 41413 5.0 5 15 167
 Cytosol aminopeptidase family protein (PepA)D1 49483102 54140 5.7 9 23 215
 GAF domain protein (MW1661)D1 88195528 17100 4.9 3 22 93
 HMG-CoA synthase (MvaS)B3,D1 9937361 43191 5.0 10 31 414
 Histidine-containing phosphocarrier protein (PtsH)D2 46908 9505 4.4 2 27 109
 Ferritin (Ftn)B3,D7 49242263 19590 4.7 6 31 450
 Putative non-haem iron-containing ferritin (MW2063)D2 49484363 16681 4.6 2 19 120
 Formylmethionine deformylase homologue (Def)D1 14246861 20546 5.8 6 38 111
 Fumarylacetoacetate (FAA) hydrolase family protein (Faa)B1 49244187 33093 4.8 3 13 72
 Methionine aminopeptidase (Map)D1 49484129 27485 5.2 3 14 105
 Malonyl CoA-acyl carrier protein transacylase (FabD)D1 14247000 33628 4.9 5 23 109
 Manganese-dependent inorganic pyrophosphatase (PpaC)A1,C1,D1 492245182 34279 4.7 6 23 199
 Monofunctional biosynthetic peptidoglycan transglycosylate (MW1814)D1 49483860 11941 3.9 2 10 84
 Naphthoate synthase (MenB)D1 14246815 30392 5.4 6 18 162
 Peptidoglycan hydrolase (LytM)C2,S 2239274 35147 6.1 4 20 207
 Putative 3-methyl-2-oxobutanoate hydroxymethyltransferase (PanB)D1 49245819 29237 5.6 8 30 348
 Secretory antigen precursor SsaA (SsaA)D1 87159889 17388 5.8 1 9 73
 Stage V sporulation protein (SpoVG)B1 13700388 12312 4.9 3 33 62
 tRNA, Arginyl-tRNA synthetase (ArgS)B1,D1 49240966 62312 5.1 7 16 220
 tRNA, Aspartyl-tRNA synthetase (AspS)D1 49483875 66527 5.0 10 17 163
 tRNA, Cysteinyl-tRNA synthetase (CysS)D1 46395518 53651 5.3 9 23 280
 tRNA, Glutamyl-tRNA amidotransferase subunit B (GatB)C1,D3 82751553 53607 5.1 9 24 229
 tRNA, Glycyl-tRNA synthetase (GlyS)D1 49483813 53586 5.0 4 8 107
 tRNA, Isoleucyl-tRNA synthetase (IleS)D1 49244476 104825 5.3 8 10 179
 tRNA, Leucyl-tRNA synthetase (LeuS)B1,D1 21204871 91926 5.0 9 17 343
 tRNA, Phenylalanyl-tRNA synthetase beta subunit (PheT)C1,D1 14246909 88885 4.7 9 13 317
 tRNA, Seryl-trna synthetase (SerS)B1,D1 14245776 48609 5.0 10 27 327
 tRNA, Threonyl-tRNA synthetase 1 (ThrS)C1,D1 14247455 74341 5.3 11 20 281
Protein synthesis (18)
 Acetyltransferase (GNAT) family protein (MW2324)D2 49483339 16991 4.9 5 32 174
 Aminotransferase, putative (RocD)B1,C1 492241363 54376 6.1 7 19 229
 Branched-chain amino acid aminotransferase (IlvE)D1 82750262 40061 5.0 5 20 220
 Deblocking aminopeptidase, similar to (MW1253)D1 49483560 37848 5.3 1 3 58
 Formiminoglutamase (HutG)D1 87160628 34491 5.4 6 29 223
 Glutamine ammonia ligase (GlnA)A1,C1,D1 14247080 50822 5.1 9 34 314
 30S Ribosomal protein S1 (RpsA)A1,B1,C1,D2 14247247 43283 4.6 15 51 740
 30S Ribosomal protein S2 (RspB)B2,C1,D1 57286011 29377 5.3 7 26 306
 30S Ribosomal protein S6 (RpsF)A1,B2,C2,D3 49482595 11588 5.1 6 58 268
 S30EA Family ribosomal protein, putative (MW0714)B1,D2 49244067 22199 5.2 5 31 211
 50S Ribosomal protein L7/L12 (RplL)A2,B2,C2,D4 88194302 12704 4.6 7 72 352
 50S Ribosomal protein L10 (RplJ)B1,C1,D1 49243847 17672 4.8 5 51 242
 50S Ribosomal protein L25 (RplY)C1 49243808 23773 4.4 4 23 74
 O-acetylserine (thiol)-lyase, putative, cysteine synthase (CysK)B2,C1,D2 49243820 32955 5.4 11 58 500
 Ornithine aminotransferase (RocD1)B1,D3 49483117 43444 5.4 11 31 543
 Secretory antigen precursor (SsaA)C3 49242648 16997 5.8 1 9 86
 Secretory antigen precursor SsaA (MW0627)C1 49243980 28155 6.1 3 14 69
 Serine hydroxymethyltransferase (GlyA)B1,D1 49245349 45144 5.8 10 29 422
Nucleotide biosynthesis (16)
 Adenylosuccinate lyase (PurB)D1 49484149 49572 5.6 7 18 154
 Adenylosuccinate synthase (PurA)B1,D2 49482270 47522 5.1 5 14 217
 Amidophosphoribosyltransferase precursor (PurF)B1,D2 49244352 54363 6.1 12 23 426
 Carbamoyl-phosphate synthase large chain (CarB)C1 14246973 117098 4.9 3 3 54
 GMP synthase (GuaA)D2 38372353 58149 4.9 6 13 200
 Inositol-monophosphate dehydrogenase (GuaB)A1,B1,C1,D1 21203531 52790 5.6 10 35 338
 Phosphoribosylamine-glycine ligase (PurD)D1 14246844 41946 5.0 2 6 50
 Phosphoribosylaminoimidazole-succinocarboxamide synthase (PurC)B1,D1 88194764 26676 5.3 12 62 494
 Phosphoribosylformylglycinamidine cyclo-ligase (PurM)D2 14246841 36966 4.8 7 26 252
 Phosphoribosylformylglycinamidine synthetase (PurL)B1,D1 14246839 79513 4.8 10 16 245
 Phosphoribosylformylglycinamidine synthase (PurS)D2 14246838 24541 5.0 7 43 215
 Phosphoribosylformylglycinamidine synthase, PurS component (MW0950)B2 49241360 9929 4.7 4 68 225
 Polyribonucleotide nucleotidyltransferase (PnpA)B1,D1 49244556 77342 4.9 14 23 530
 Purine nucleoside phosphorylase (DeoD1)A1,B3,D2 49484362 25892 4.9 6 35 329
 Pyridoxine biosynthesis protein (MW0474)A1,C1,D1 49482748 31972 5.1 8 31 267
 Uracil phosphoribosyltransferase (Upp)D1 49484336 23035 6.1 9 58 496
Fatty Acid Biosynthesis (1)
 Trans-2-enoyl-ACP reductase (FabI)B1,D1 56001093 24601 5.2 7 53 226
Transcription and Replication (18)
 2′−5′ RNA ligase (MW0896)B1,D1 49244233 19315 4.9 6 40 201
 Accessory gene regulator A (AgrA)D1 14247812 27903 5.9 5 18 92
 DNA-directed RNA polymerase alpha chain (RpoA)C2 49484440 34990 4.7 9 40 292
 DNA polymerase III, beta chain (DnaN)C1,D1 49482255 41888 4.7 8 25 194
 DNA-directed RNA polymerase delta subunit (RpoE)C1 49245364 20868 3.6 2 14 84
 Translation elongation factor G (Fus)A2,B2,C3,D2 49243855 76564 4.8 19 40 915
 Elongation factor TS (Tsf)A2,B3,C3,D5 14247027 32473 5.2 15 57 683
 Elongation factor P, putative (Efp)D1 49483778 20541 4.8 2 11 82
 RsbW (RsbW)B1 37781578 17896 4.7 3 20 70
 TatD related Dnase, putative (MW0446)D1 49482718 29263 5.1 4 17 126
 Transcription pleiotropic repressor (CodY)B1,D2 49483418 28737 5.9 8 35 354
 Transcription termination-anti-termination factor (NusA)C1,D1 14247036 43729 4.6 9 29 242
 Transcriptional regulator (MW0363)B2,D3 14246155 15127 5.0 7 65 301
 Transcriptional regulator, LytR family (SAUSA300_0958)D1 1723223 23880 5.7 5 33 170
 Transcriptional regulator, LytR family (MW0939)C1,B2 57284435 45657 6.0 6 20 144
 Translation initiation factor IF-1 (InfA)B1 49484444 8274 6.7 2 38 64
 Translation elongation factor Tu (Tuf)A2,B1,C2,D3 49243856 43077 4.7 16 59 871
 Transposase (MW2398)B2,D2 49484688 16446 5.6 4 33 191
Miscellaneous (13)
 IIIG9 protein, similar to- (LOC576703)B1 72179405 52711 9.4 1 2 51
 6,7-Dimethyl-8-ribityllumazine synthase (RibH)B1,D1 49242141 16412 5.7 7 73 342
 ABC transporter-associated protein, SufB (MW0799)D1 49483078 52512 5.1 7 18 139
 Amylase (MalA)D1 18145251 77435 5.9 1 1 54
 Aldo/keto reductase family protein (MW2127)D1 49482959 32339 5.2 4 12 114
 Arsenate reductase family protein (MW0785)D1 49483064 13591 6.7 3 49 133
 Lipoate synthase (LipA)*A1 27807337 16468 6.3 2 17 54
 Cell wall surface anchor family protein (MW2416)C1,D1 57285190 136262 5.7 16 16 804
 Short chain dehydrogenase (MW2249)B1 14248102 31777 4.7 2 5 72
 Immunodominant antigen A (IsaA)A9,B5,C13,D2 14248343 24189 5.9 4 23 259
 N-Acetylglucosamine 6-phosphate deacetylase (NagA)D1 87161324 43089 5.4 7 20 123
 4-Nitrophenylphosphatase-probable (MW0811)B1 82750544 27962 4.5 4 16 61
 SufD (SufD)D1 82750525 48518 5.4 5 18 69
Unknown (20)
 Conserved hypothetical protein (SAUSA300_0871)D1 88194663 33093 4.8 9 42 262
 Conserved hypothetical protein (SAUSA300_0916)D1 88194708 19314 5.0 7 47 249
 Conserved hypothetical protein (SAUSA300_1856)D2 88195657 19536 6.1 7 46 247
 Hypothetical exported protein (MW0347)B6,D7 49243694 21261 5.7 3 14 115
 Hypothetical exported protein (MW2606)B1,D1 82752265 18700 4.7 3 25 147
 Hypothetical cytosolic protein (MW0395)C1,S 14246202 55465 5.1 14 32 487
 Hypothetical protein (MW0542)B1 14246355 29371 5.1 3 14 97
 Hypothetical protein (MW0819)B1,D1,M 49244219 69762 5.1 14 30 604
 Hypothetical protein (MW2068)D1,S 4126674 22954 5.2 10 62 369
 Hypothetical protein (SAUSA300_0408)A1,C5,S 57285506 56443 4.8 19 40 899
 Hypothetical protein (SAUSA300_0279)**D1,M,S 77383233 24390 6.9 1 5 51
 Hypothetical protein (MW1884)A1,M,S 30043928 13044 9.3 2 26 99
 Hypothetical protein (MW0577)B1,S 49482843 18554 9.2 4 25 124
 Hypothetical cytosolic protein (MW1786)B1,C1,D1 49484087 13302 4.4 9 85 406
 Hypothetical protein (MW1795)A2,B1,D2 49484096 22344 5.3 10 58 394
 Hypothetical protein (MW2099)B1,D1 49484393 10000 6.1 3 45 95
 Hypothetical protein (SAUSA300 2327)D1 87161861 15876 4.9 7 58 336
 Hypothetical protein (SAUSA300 pUSA010004)C1,M,S 87159841 21257 9.3 2 11 55
 Putative exported protein (MW0355)B8,M 49243745 56170 5.0 17 45 620
 Putative exported protein (MW1757)D2,S 49245076 20371 6.8 3 21 100
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a

The Mascot search results displayed above (Entrez Protein number, MW, pI, MP, SC%, and MS/MS MOWSE score) are from the best protein match to published S. aureus genomes. In many instances, database searches were performed before the genome sequence of USA300 was published and the best match is to a protein from another published S. aureus strain. However, the Entrez Protein name indicates the name of the likely MW2 or USA300 protein. SC, sequence coverage. MP, matched peptides.

AMW2 supernatants from mid-exponential phase of growth.

BMW2 supernatants from stationary phase of growth.

CLAC supernatants from mid-exponential phase of growth.

DLAC supernatants from stationary phase of growth.

ETheoretical or predicted.

LContains an LPXTG cell wall anchoring signal sequence.

MContains probable transmembrane regions.

SContains a probable N-terminal signal peptide sequence.

1–8Number after the letter A,B,C or D indicates number of times a protein was identified.

*

Best Mascot search homology was to Cathelicidin 4 [indolicidin] [Bos taurus].

**

Best Mascot search homology was to Pfl_3008 [Pseudomonas fluorescens PfO-1] (ABA74746).

Identification of CA-MRSA exoproteins associated with virulence

Twenty exoproteins (20%) identified at mid-exponential growth and 33 (15%) of those at early stationary phase of growth are known to be associated with virulence (Fig. 1 and Table 1). There were essentially three subcategories of virulence determinants found in culture media. Proteases or enzymes, including aminopeptidase (PepS), aureolysin (Aur), staphylokinase (Sak), V8 protease (SspA), cysteine protease (SspB), serine protease (SplC), lipase (Lip) and Xaa-Pro dipeptidase, were produced by each strain at either phase of growth (Fig. 1 and Table 1). These enzymes degrade and/or modify proteins and lipids present in the growth environment (McGavin et al., 1997; Karlsson et al., 2001; Massimi et al., 2002; Imamura et al., 2005). For example, cysteine protease/staphopain B (SspB), which directly cleaves kininogen, also works in concert with staphopain A (ScpA) to promote vascular leakage and lower blood pressure, thereby facilitating septic shock (Massimi et al., 2002; Imamura et al., 2005). SspA causes release of cell surface fibronectin-binding protein (FnbB) and immunoglobulin G (IgG)-binding protein A (protein A, Spa), modifying capacity for host interaction and increasing free FnbB and Spa (McGavin et al., 1997; Karlsson et al., 2001).

A second group of molecules identified in CA-MRSA culture media were those involved in bacteria–host interaction or adhesion, such as coagulase (Coa), collagen adhesin (Cna), enolase (Eno), fibrinogen-binding protein, FnbA, FnbB, 1-phosphatidylinositol phosphodiesterase (Plc), Spa and IgG-binding protein (Sbi) (Figs 1 and 2, and Table 1). These molecules can activate the clotting cascade (Coa) (Panizzi et al., 2006), mediate binding to host tissues (Cna, Eno, FnbA and FnbB) (Patti et al., 1992; Greene et al., 1995; Carneiro et al., 2004), and sequester host antibody (Spa and Sbi) (Forsgren and Sjoquist, 1966; Zhang et al., 1998). Although the function of S. aureus Plc is uncharacterized, that of Listeria monocytogenes promotes adhesion to epithelial cells and mediates escape of the pathogen from phagosomes (Krawczyk-Balska and Bielecki, 2005; Wei et al., 2005).

The toxins and haemolysins, namely alpha-haemolysin (Hla), enterotoxin C3 (Sec3), enterotoxin H (Seh), enterotoxin K (Sek), enterotoxin L (Sel2) and enterotoxin Q (Seq), comprised at most 4% of the exoproteins produced by MW2 and/or LAC in mid-exponential or early stationary phases of growth (4/98 at mid-exponential phase and 6/229 at early stationary phase of growth respectively) (Fig. 1 and Table 1). Unexpectedly, we failed to detect gamma-haemolysin subunits (HlgA, HlgB and HlgC), LukD/E, LukM, or either component of PVL (LukS-PV and LukF-PV) in culture supernatants under the two growth conditions tested.

Proteins involved in metabolism, biosynthesis, transcription and replication are present in MW2 and LAC culture supernatants

CA-MRSA culture supernatants contained 73 proteins known to be involved in the transport and utilization of carbohydrates or amino acids for energy (Table 1). Thirty-five proteins known to participate in biosynthesis of nucleotides, proteins and fatty acids, and 51 proteins involved in cell division, transcription and replication, were also identified in culture media (Fig. 1 and Table 1). The observation that cytoplasmic proteins were found in culture supernatant is not unexpected, as numerous cycles of autolysis would have occurred thereby releasing proteins into culture medium (Lei et al., 2000; Chaussee et al., 2001; Trost et al., 2005).

Stress response proteins are present in culture supernatants

Twenty proteins associated with the response to environmental stress were identified in MW2 and LAC culture supernatants (Fig. 1 and Table 1). For example, alkyl hydroperoxide reductase (AhpC and AhpF), catalase (KatA), superoxide dismutase (SodA), thioredoxin (TrxA) and thioredoxin reductase (TrxB), proteins that function to inactivate reactive oxygen species, and heat shock proteins, GroES and DnaK, were identified in culture supernatants at both phases of growth (Fig. 1 and Table 1). Consistent with this observation, genes encoding these proteins are induced in MW2 and LAC during phagocytosis by human neutrophils (Voyich et al., 2005). Further, heat shock proteins such as DnaK and GroEL have been shown to be immunogenic in patients with S. aureus endocarditis (Qoronfleh et al., 1993; 1998), suggesting they are exoproteins in vivo.

MW2 and LAC produced numerous exoproteins of unknown function

We identified 20 culture supernatant proteins with no characterized function, including four putative exported proteins (Fig. 1, Table 1 and Table S1) (Baba et al., 2002; Diep et al., 2006). Fourteen of these proteins were conserved across 10 sequenced strains of S. aureus (Table S1). Genes encoding MW0395 and MW1757 reside within Type II genomic islands of MW2 known as νSaα and νSaβ respectively, and each is located near or among putative virulence determinants (Baba et al., 2002). MW1884 is encoded by MW2 prophage ΦSa3 and is juxtaposed to the gene encoding staphylokinase (sak), a known virulence factor in S. aureus (Baba et al., 2002). Several other exoproteins with no characterized function, such as MW0542, MW1795, MW2068, SAUSA300_0871, SAUSA300_0916 and SAUSA300_2327, have homology to enzymes that participate in metabolism or replication, or respond to stress (Table S1). It will be important to determine whether some of these proteins have a role in virulence and/or if they are potential vaccine targets.

Selected MW2 and LAC exoproteins differ in abundance

Although MW2 and LAC produced many common exoproteins, 11 exoproteins detected at either phase of growth differed in abundance between the strains (Figs 3 and 4). Hla and a putative surface protein (SAUSA300_0408) were in greater abundance in LAC culture supernatants at mid-exponential growth phase, and multiple repeat polypeptide (Mrp), Sak, and Coa were found exclusively in the same supernatants (Fig. 3A). By comparison, Cna, Sec3 and a putative exported protein (MW0355) were identified as exoproteins only in MW2 culture supernatants at mid-exponential growth (Fig. 3B).

Fig. 3.

Fig. 3

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Quantitative analysis of CA-MRSA culture supernatant proteins produced during growth in vitro. Differential analysis of MW2 and LAC culture supernatant proteins was performed as described under Experimental procedures. Proteins more abundant in/found only in LAC (A and D) or MW2 (B and E) supernatants. Panels C and F represent proteins equally abundant in MW2 and LAC. The phase of growth at which the analysis was performed is indicted to the left of the panels. Results are the mean ± SD of three separate experiments at each phase of growth.

Fig. 4.

Fig. 4

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Exoproteins produced by MW2 and LAC during infection. Culture supernatant proteins made by MW2 or LAC at the indicated phase of growth were separated by 2-DGE, transferred to nitrocellulose membranes, and probed with convalescent sera from mice infected with each strain or non-immune sera (not shown). Proteins immunoreactive only with sera from infected mice are indicated (boxed). Unidentified immunoreactive proteins are annotated with Arabic numbers. Immunoblots are representative of three separate experiments.

At stationary phase of growth, Hla, Sak, Lip, Seq, SplC, SspA, SspB and Sek were either increased ≥ 2-fold in LAC supernatants or were found only in those supernatants (Fig. 3D). There was far more Cna (39.5-fold) in MW2 supernatants at this phase of growth, and Sec3 and MW0577, a protein of unknown function, were detected only in MW2 culture media (Fig. 3E). Differences in exoproteins between these strains may underlie in part the noted variances in disease phenotypes (Baba et al., 2002; Kazakova et al., 2005; Miller et al., 2005; Voyich et al., 2005; Diep et al., 2006).

Exoproteins made during infection in vivo

To reconcile exoproteins produced by MW2 and LAC in vitro and those made during infection in vivo, we used a mouse abscess model to generate immune/convalescent sera from mice infected with MW2 and LAC (Voyich et al., 2006). Following 2-DGE and transfer to nitrocellulose, MW2 and LAC exoproteins were probed with convalescent serum from mice infected with either MW2 or LAC. Several proteins from these CA-MRSA strains were commonly immunogenic in mice (Fig. 4). For example, AhpC, Atl, formate tetrahydrofolate ligase (Fhs), glyceraldehyde 3-phosphate dehydrogenase (Gap), Lip and SspB were immunogenic in mice infected with either strain (Fig. 4 and Table 2). Cna, Sec3 and Sak are known virulence factors that were immunogenic in mice infected with either MW2 (Cna and Sec3) or LAC (Sak) (Fig. 4 and Table 2), findings consistent with the differential analysis of exoproteins produced in vitro (compare Figs 3 and 4). Although many other immunogenic proteins were detected by our analysis, many were cross-reactive with non-immune sera (unboxed, unmarked protein spots, Fig. 4) or could not be identified with absolute certainty (indicated by numbers, Fig. 4). Taken together, these data provide strong support to the idea that proven or putative virulence factors, such as Atl, Cna, Lip, Sak, Sec3 and SspB, are made during CA-MRSA infection in vivo.

Table 2.

Immunogenic (in vivo expressed) exoproteins of MW2 and LAC.

Proteina MW2 LAC Immunoreactivity
AhpC, alkyl hydroperoxide reductase ME, S ME, S NI, I
AroA, chorismate mutase – S I
Asp23, alkaline shock protein 23 – S NI, I
Atl, autolysin ME, S ME, S I
ClpP, ATP-dependent Clp protease S S NI, I
Cna, collagen adhesin precursor ME, S – I
Coa, coagulase ME – NI, I
DeoD, purine nucleoside phosphorylase ME ME NI, I
Ear S S I
Eno, enolase S S NI, I
Fba, fructose bisphosphate aldolase S – NI, I
Fhs, formate tetrahydrofolate ligase ME, S ME, S I
Ftn, ferritin – S I
Gap, glyceraldehyde 3-phosphate dehydrogenase ME, S ME, S I
GlyA, serine hydroxymethyltransferase – S I
Gpi, glucose 6-phosphate isomerase ME ME NI, I
GuaB, inositol-monophosphate dehydrogenase ME – NI, I
Idh1, isocitrate dehydrogenase – S NI, I
Lip, lipase S S I
MW0525, hexulose 6-phosphate synthase – S I
MW0896, 2′−5′ RNA ligase – S NI, I
MW1795, hypothetical protein – S NI, I
MW1870, thioredoxin – S NI, I
PdhA, pyruvate dehydrogenase subunit A ME ME NI, I
PdhB, pyruvate dehydrogenase subunit B S S NI, I
Pgd, phosphogluconate dehydrogenase ME ME NI, I
Pgi, phosphoglucose isomerase S S NI, I
Plc, 1-phosphatidylinositol phosphodiesterase S S I
Pta, phosphate acetyltransferase – S I
RocD, ornithine aminotransferase S – NI, I
Sak, staphylokinase ME ME, S I
Sec3, staphylococcal enterotoxin C3 S – I
Seq, staphylococcal enterotoxin Q ME ME I
SodA, superoxide dismutase S S I
Spa, immunoglobulin-binding protein A ME, S ME, S NI, I
SspB, cysteine protease precursor ME, S ME, S I
Sulfatase S – NI, I
Tkt, transketolase S – I
TpiA, triosephosphate isomerase – ME I
Tsf, translation elongation factor Ts ME S I
Tuf, translation elongation factor Tu ME, S ME, S NI, I
Unknown 1 ME – I
Unknown 2 ME – I
Unknown 3 – ME I
Unknown 4 – ME I
Unknown 5 – ME I
Unknown 6 – ME I
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Protein identities were obtained by overlay analysis as described under Experimental procedures. Results are representative of three experiments using serum pooled from 10 to 15 mice.

ME, mid-exponential phase of growth; S, stationary phase of growth; NI, non-immune sera; I, immune sera.

Phagocytosis of CA-MRSA by human neutrophils triggers production/secretion of virulence factors

To determine if production of selected exoproteins is triggered by interaction of S. aureus with host cells and/or if the proteins are made within phagosomes, we used confocal laser-scanning microscopy to evaluate production of Aur, Hla, SspA and SspB after phagocytosis by human polymorphonuclear leucocytes (PMNs) (Figs 5 and 6). Notably, there was a time-dependent increase in Aur, Hla, SspA and SspB produced by MW2 and/or LAC within neutrophil phagocytic vacuoles (Figs 5 and 6, yellow arrowheads). There was also redistribution of each molecule over time; at 15 or 60 min proteins were typically localized only to S. aureus, whereas at 3 or 4 h after phagocytosis each molecule was diffused within larger, more mature phagosomes or distributed throughout the cell (Figs 5 and 6, yellow arrowheads). Accumulation of these molecules late during phagocytosis correlates well with the noted PMN lysis caused by MW2 and LAC (Figs 5 and 6) (Voyich et al., 2005). Production of virulence factors within phagosomes is consistent with the notion that these proteins are made during infection in vivo.

Fig. 5.

Fig. 5

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Production and distribution of selected MW2 (USA400) virulence factors during phagocytosis by human PMNs. Following phagocytosis of MW2, aureolysin (Aur), SspA and SspB were visualized by confocal laser-scanning microscopy. White arrowheads indicate bacteria. Yellow arrowheads indicate areas enriched with the S. aureus protein of interest. The image labelled ‘Merge’ illustrates distribution of neutrophil actin-related protein (ARP, green) and nuclei (blue). DIC, differential interference contrast.

Fig. 6.

Fig. 6

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Production and distribution of selected LAC (USA300) virulence factors during phagocytosis by human PMNs. Following phagocytosis of LAC, Hla, SspA and SspB were visualized by confocal laser-scanning microscopy. Labelling for this figure is otherwise identical to the legend for Fig. 5.

Discussion

The striking increase in CA-MRSA infections over the past few years has prompted an intense search for the underlying molecular determinants. To date, few virulence factors are associated specifically with CA-MRSA disease and no single determinant appears to account for the increased incidence and severity of CA-MRSA infections (Lina et al., 1999; Baba et al., 2002; de Bentzmann et al., 2004; Diep et al., 2004; 2006; Fridkin et al., 2005; Voyich et al., 2006). It is almost certain that a combination of virulence determinants, including S. aureus exoproteins, and host susceptibility promote disease in otherwise healthy subjects. Inasmuch as exoproteins produced by CA-MRSA probably facilitate evasion of innate host defence (Voyich et al., 2005) and thereby contribute to disease, we performed a comprehensive analysis of exoproteins produced by MW2 and LAC in vitro and during infection.

A limited number of proteomics-based studies have investigated exoproteins of S. aureus, typically using strain COL or laboratory-derived strains (Ziebandt et al., 2001; 2004; Nakano et al., 2002). For example, Ziebrandt et al. recently compared S. aureus strains RN6390 and RN6911 and identified 43 exoproteins produced in vitro, including many controlled by accessory gene regulator (agr) and/or alternative sigma factor óB (sigB) (Ziebandt et al., 2004). Nakano et al. identified 29 exoproteins produced by MRSA strains using 2-DGE coupled with N-terminal peptide sequencing (Nakano et al., 2002). By comparison, numerous studies have reported individual S. aureus exoproteins that promote pathogenesis, including proteases (McGavin et al., 1997; Karlsson et al., 2001; Imamura et al., 2005), enterotoxins and exotoxins (Dinges et al., 2000; McCormick et al., 2001), and leukotoxins and haemolysins (Kaneko and Kamio, 2004). Although progress has been made towards identification and characterization of many important S. aureus exoproteins, there is a noted paucity of information regarding exoproteins produced by CA-MRSA strains.

Our analysis of MW2 (USA400) and LAC (USA300) culture supernatants identified 250 exoproteins (out of 600+ resolved protein spots) between two phases of growth in vitro, at present the single most comprehensive view of S. aureus exoproteins. Differential analysis of MW2 and LAC exoproteins revealed key differences between the strains (Fig. 3). These differences were not due to differences in rate of growth between the strains, because MW2 and LAC have essentially identical growth curves in vitro (Fig. 1A). Although many of the differentially abundant exoproteins, including Atl, Coa, Hla, Lip, Mrp, Sak, Sek, Seq, Sec3, SspA, SspB and SplC, are relatively ubiquitous among S. aureus, it is possible that the observed variances in protein levels relate to distinct strain pathologies. For example, Cna is linked to necrotizing pneumonia (de Bentzmann et al., 2004) and there were higher levels of this exoprotein in MW2 culture supernatants (Fig. 3). MW2 is a strain known to cause lethal pneumonia (Centers for Disease Control and Prevention, 1999). Compared with MW2, more Hla was present in LAC culture supernatants (7.5 ± 1.8- and 9.2 ± 1.8-fold more Hla at mid-exponential and stationary phases of growth respectively). Consistent with that observation, Hla appeared to accumulate more rapidly in LAC-containing neutrophil phagosomes or was more highly diffused in and around deteriorating PMNs after phagocytosis of LAC compared with MW2 containing cells (accumulation of Hla typically occurred by 180 min in LAC-containing PMNs versus 240 min in those with MW2). We recently found dramatic differences in pathophysiology between MW2 and LAC in a mouse skin infection/abscess model (Voyich et al., 2006). LAC produced rapid and pronounced dermonecrosis in infected animals, whereas mice infected with MW2 developed intact abscesses (Voyich et al., 2006). Thus, differences in exoprotein abundance, such as that for Hla, may underlie the differences in strain pathology. Additional studies are needed to test this hypothesis.

We used sera from mice infected with MW2 or LAC to identify exoproteins made during infection in vivo (Fig. 4). Previous serological proteome studies using strain COL identified 15 immunogenic proteins made during human infections, although only four of these proteins (alkaline shock protein, hexose 6-phosphate synthase, PdhB and Tuf) are common with our analysis (Table 2) (Vytvytska et al., 2002). In more recent work, Clarke et al. used bacteriophage expression libraries to identify S. aureus antigens produced during human infections (Clarke et al., 2006). Several of those immunogenic proteins, i.e. chorismate mutase (AroA), autolysin (Atl), Coa, Fhs, Gap, transketolase (Tkt) and triosephosphate isomerase (Tpi), were also identified as in vivo expressed S. aureus antigens by our studies (Table 2). Our work revealed many additional exoproteins produced during infection, such as AhpC, Cna, Ear, ferritin (Ftn), Lip, Plc, phosphate acetyltransferase (Pta), Sak, Sec3, Seq and SspB (Fig. 4). Importantly, these proteins were immunoreactive only with sera from MW2- or LAC-infected animals (as opposed to sera from uninfected animals). Variances in antigenic exoproteins between MW2 and LAC are likely explained in part by differences in gene content or phase of growth used to test antigenicity (Fig. 4). The relative importance of these in vivo expressed proteins remains to be determined.

At least four of the exoproteins identified by our proteomic analysis (Aur, Hla, SspA and SspB) were produced within phagosomes of human neutrophils following uptake (phagocytosis) (Figs 5 and 6). These findings are notable because MW2 and LAC are known to cause rapid lysis of PMNs (Voyich et al., 2005) and the factors responsible for the dramatic host cell lysis remain elusive (Voyich et al., 2006). Consistent with these data, we determined previously that the gene encoding Hla was upregulated during phagocytosis (Voyich et al., 2005). Although our studies do not demonstrate that Aur, Hla, SspA and SspB are directly related to the observed PMN lysis, increased accumulation of these virulence determinants accompanied initial stages of neutrophil destruction (Figs 5 and 6).

The S. aureus genome consists of ∼2600 proteins of which more than 40% have no similarity to proteins of known function (Kuroda et al., 2001). Moreover, 33% of identified proteins are unique to S. aureus (Kuroda et al., 2001). Therefore, it is not surprising that 8.5% of the proteins identified in our study have no known function (Fig. 1). Some of these exoproteins are of significant interest based upon homology to known S. aureus proteins (e.g. SAUSA300_0407 as an exotoxin homologue) or given their location within the genome (Table 1 and Table S1). Several exoproteins identified by our analysis are putative exported or surface proteins, e.g. SAUSA300_0408 and MW0355 (rather than metabolism enzymes, etc.) and also require characterization in the context of CA-MRSA pathogenesis (Fig. 3).

We used a proteomics-based approach to generate a comprehensive view of exoproteins made by prominent CA-MRSA strains, including identification of proteins that are immunogenic and thus produced during infection in vivo. Identification of these exoproteins is an important first step towards development of vaccines, prophylactics, and enhanced therapeutics designed to control CA-MRSA infections.

Experimental procedures

Growth of S. aureus and generation of culture supernatants

Staphylococcus aureus strains MW2 (USA400) (Baba et al., 2002) and LAC (USA300-0114) (Kazakova et al., 2005; Miller et al., 2005; Voyich et al., 2005; Diep et al., 2006) were cultured in tryptic soy broth containing 0.25% dextrose (TSB, Becton, Dickenson, and Company, Franklin Lakes, NJ) filtered sequentially through 10 kDa cut-off and 0.22 μm filters. Cultures were inoculated with a 1:200 dilution of overnight culture (500 μl of culture into 100 ml of TSB in a 1 l flask) and incubated at 37°C with shaking (250 rpm). All strains were cultured to mid-exponential (OD600 = 0.75) or stationary (OD600 = 2.0) phases of growth and placed on ice until used. Bacteria were removed from culture media by two sequential rounds of centrifugation at 2851 g for 10 min at 4°C. This procedure yielded clarified culture supernatants for subsequent proteomic analyses.

Precipitation and preparation of culture supernatant proteins

Precipitation of proteins from clarified culture supernatants was performed in polypropylene flasks to reduce protein loss. One hundred millilitres of clarified supernatant was added to 300 ml of 100% ethanol (Molecular Grade, Sigma-Aldrich, St Louis, MO) and chilled at −20°C overnight. Precipitated protein was transferred to Oakridge centrifuge tubes and sedimented by centrifugation at 27 216 g for 30 min at 4°C. To optimize yield, protein in polypropylene flasks (residual) and centrifuge tubes was air-dried for approximately 1 h. Protein in the flasks were solubilized with 3 ml of 2-D solubilization solution (7 M urea, 2 M thiourea, 4% CHAPS) with gentle swirling. These samples were transferred to Oakridge tubes containing precipitated protein pellets and swirled gently to dissolve pellets. Polypropylene flasks were rinsed with an additional 600 μl of 2-D solubilization solution.

Culture supernatant proteins were clarified further with a second precipitation in 30% trifluoroacetic acid (Sigma-Aldrich) and incubated on ice for a minimum of 30 min. Precipitated proteins were collected by centrifugation at 14 100 g for 5 min at room temperature. The protein pellet was dispersed by vortexing in 25 μl of distilled H2O for 10 s followed by the addition of 1 ml of acetone (−20°C). Proteins were washed in acetone for a minimum of 30 min at −20°C and then pelleted by centrifugation at 14 100 g for 5 min at room temperature. Proteins were air dried for ∼1 h or until pellets appeared dry. Pellets were solubilized in 400 μl of Destreak Rehydration Solution (GE Healthcare, Piscataway, NJ) containing tributylphosphine (200 mM) and ampholytes (pH 3–10, 4 μl of 100× solution) (Bio-Rad, Hercules, CA) at room temperature for 1 h with constant swirling. Samples were used immediately or stored briefly at −20°C.

Isolation of human neutrophils

Human polymorphonuclear leucocytes were isolated from fresh venous blood of healthy individuals using a published method (Kobayashi et al., 2002; Burlak et al., 2006). Studies were performed in accordance with a protocol approved by the Institutional Review Board for Human Subjects, National Institute of Allergy and Infectious Diseases. PMN preparations typically contained ∼94% neutrophils, with the remaining cells being predominantly eosinophils. All reagents used contained < 25.0 pg ml−1 endotoxin (Limulus Amebocyte Lysate assay, Fisher Scientific, Suwannee, GA).

Neutrophil phagocytosis

MW2 and LAC were cultured to mid-exponential phase of growth and phagocytosis experiments were performed with serum-opsonized bacteria as described (Kobayashi et al., 2003; Voyich et al., 2005). At the indicated times, phagocytosis was terminated either by chilling PMNs on ice or adding cold paraformaldehyde (4%) to assay wells.

Generation of immune sera

Female Crl:SKH1-hrBR mice (Charles River Laboratories, Wilmington, MA) were anaesthetized with isoflurane and inoculated with 50 μl of DPBS containing 107 cfu of MW2 or LAC by subcutaneous injection in the right flank. Abscesses typically formed within 4 days and resolved 14 days after infection (Voyich et al., 2006). At 28 days post infection, mice were euthanized and blood was collected from 10 to 15 mice to prepare pooled immune serum. Blood from uninfected animals was processed in parallel (non-immune serum). All studies conformed to guidelines set forth by the National Institutes of Health and were reviewed and approved by the Animal Use Committee at Rocky Mountain Laboratories, NIAID.

Isoelectric focusing and second-dimension SDS-PAGE

Culture supernatant proteins were precipitated in cold acetone and then solubilized with isoelectric focusing (IEF) buffer (7% urea, 2% thiourea, 4% CHAPS and 200 mM tributylphosphine) as described (Burlak et al., 2006). Protein concentration was measured with the 2-D Quant kit (GE Healthcare) and purified proteins were stored at −80°C.

For IEF, samples were treated with Destreak Rehydration solution (25% of total sample volume) (GE Healthcare), 200 mM tributylphosphine and 1% ampholytes. IEF was performed with 11 cm IPG Ready Strips for 40 kVh. IPG Ready Strips were rehydrated actively at 50 V overnight prior to first dimension separation. Moistened filter paper wicks (Whatman no. 1 paper, Whatman, Florham Park, NJ) were added between each electrode and strip prior to focusing (after rehydration). Wicks were changed four times in the first 4 h of IEF, after which the voltage was maintained at 8000 V (11 cm IPG Ready Strips). Following IEF, IPG Ready Strips were stored at −80°C until used for SDS-PAGE.

Second-dimension SDS-PAGE was performed essentially as described (Burlak et al., 2006), except electrophoresis was performed at 50 mA per gel until the dye front reached the bottom of each gel (∼1 h for 11 cm gels). Protein spots were excised and peptides were prepared for ADI-MS/MS analysis as described previously for liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Burlak et al., 2006). For simplicity, the combination of IEF and second-dimension SDS-PAGE is abbreviated as 2-DGE.

Mass spectrometry

Peptide samples (tryptic digests, as described/referenced above) were analysed by automated direct infusion (ADI) using Nanomate (Advion BioSciences, Ithaca, NY), an automated chip-based nano-electrospray interface source, coupled to a quadrupole-time of flight mass spectrometer, QStarXL MS/MS System (Applied Biosystems/Sciex, Framingham, MA). Computer-controlled data-dependent automated switching to MS/MS provided peptide sequence information. AnalystQS software (Applied Biosystems/Sciex) was used for data acquisition. Data processing and databank searching were performed with Mascot software version 2.1 (Matrix Science, Beachwood, OH). The National Center for Biotechnology Information non-redundant protein database (NCBInr, updated 12 May, 2006 at 18:01:48 GMT), National Library of Medicine, NIH was used for the search analysis. Search criteria were limited to double- and triple-charge ions and included monoisotopic masses, analysis of peptides for carbamidomethylation and/or propionamidylation of cysteine, oxidation of methionine, peptide and MS/MS tolerances of 0.2 Da and 0.8 Da respectively, and a maximum of one missed tryptic cleavage. Significance threshold for positive identification was determined by the Mascot Search program.

Amino acid sequence analysis

Proteins involved in virulence/defence mechanisms, stress response proteins, toxins, haemolysins and proteins of unknown function were evaluated for presence of an LPXTG motif, which predicts a cell wall anchor, and/or for sequences that predict an N-terminal signal peptide or transmembrane region(s). We used the NCBInr database to query the full sequence of each protein identified by ADI/MS/MS for the presence of LPXTG motifs. The presence of membrane spanning domains and N-terminal signal peptide sequences was deduced by searching protein sequences with PSORT software provided by the PSORT WWW server (http://psort.nibb.ac.jp/). Although none of the 20 hypothetical proteins identified in this study contain LPXTG motifs, eight proteins contain probable N-terminal signal peptide sequences and five have predicted transmembrane regions (Table 1 and Table S1).

In addition, sequences of the hypothetical proteins were compared to 902 genomes (Bacterial, Archaea, Viral, and Eukarya) using NCBI and ERGO, a curated NIAID database containing public and proprietary DNA.

SDS-PAGE and immunoblotting

Proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and membranes were blocked with 10% normal goat serum in Tris buffered saline 150 mM NaCL, 50 mM Tris base, pH 7.5, 1% Tween 20 and 0.02% sodium azide (Sigma-Aldrich) overnight at 4°C. Blots were probed with immune or non-immune mouse sera for 1–2 h at ambient temperature or overnight at 4°C. Blots were washed in buffer containing 250 mM NaCl, 10 mM Hepes and 2% Tween 20 (Sigma-Aldrich) and incubated with secondary antibodies conjugated to horseradish peroxidase for 1–2 h at ambient temperature. Immunoreactive proteins were visualized with enhanced chemiluminescence (SuperSignal West Pico, Pierce Biotechnology, Rockford, IL) using Kodak X-Omat film (Eastman Kodak, Rochester, NY).

Immunofluorescence and confocal laser-scanning microscopy

Acid washed coverslips (No. 1, 13 mm, round) were flamed and coated with 100% normal human serum in 24 well tissue culture plates for 1 h. Coverslips were washed twice with DPBS and synchronized phagocytosis was performed in 24 well plates as described above. Fixed PMNs were washed three times in DPBS and then permeabilized with 0.2% Triton X-100 for 5 min. After three more washes in DPBS, cells were incubated with blocking buffer (DPBS containing 5% BSA, 0.02% sodium azide) for 1 h. Samples were labelled with a 1:1000 dilution of rabbit antiserum containing antibodies specific for Hla (Sigma-Aldrich), SspA, SspB, Aur and 2 μg ml−1 of goat polyclonal antibodies specific for human actin-related protein 2 μg ml−1 (Santa Cruz Biotechnologies, Santa Cruz, CA) overnight at 4°C. Samples were washed and subsequently labelled with donkey anti-rabbit antibody conjugated with phycoerythrin 1:1000 (Jackson Immunoresearch, West Grove, PA) or donkey anti-goat antibody conjugated to AlexaFluor488 (1:1000) (Molecular Probes, Eugene, OR). PMNs were stained with DAPI (300 nM in DPBS, Molecular Probes) and DRAQ5 (1.25 μM in DPBS, Biostatus Limited, Leicestershire, UK) prior to mounting coverslips onto slides. Slides were analysed with a Zeiss LSM510 confocal laser-scanning microscope coupled to an Axiovert 200M inverted microscope (Carl Zeiss, Thornwood, NY). Images were acquired using a 100× Plan-Apochromat oil immersion objective (1.4 NA) at 512 × 512 pixel resolution with 2.7× digital magnification. Images were adjusted equally for brightness and contrast in Adobe Photoshop CS (Adobe Systems Incorporated, San Jose, CA).

Acknowledgments

This research was supported by the Intramural Research Program of the NIH, National Institutes of Allergy and Infectious Diseases.

Supplementary material

The following supplementary material is available for this article online:

Table S1

Inter-strain conservation of proteins of unknown function.

This material is available as part of the online article from http://www.blackwell-synergy.com

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Associated Data

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Supplementary Materials

Table S1

Inter-strain conservation of proteins of unknown function.

cmi0009-1172_TableS1.doc (46KB, doc)

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