Proteome Analysis of Membrane and Cell Wall Associated Proteins

An initial reference map of membrane and cell wall associated proteins of S. aureus was constructed by mass spectrometry and 72% of the identified pro...
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Proteome Analysis of Membrane and Cell Wall Associated Proteins from Staphylococcus aureus Renu Nandakumar, M. P. Nandakumar, Mark R. Marten, and Julia M. Ross* Department of Chemical and Biochemical Engineering, University of Maryland Baltimore County, 1000 Hilltop circle, Baltimore, Maryland 21250 Received August 3, 2004

Pathogenesis of Staphylococcus aureus, an opportunistic human pathogen, is complex and involves many virulence factors including an array of surface proteins (adhesins) that promote bacterial interactions with extracellular matrix components. A better understanding of these interactions can be achieved by studying the expression of membrane and cell wall associated proteins using a proteome analysis approach. To accomplish this, our goal here was to construct a reference map of membrane and cell wall associated proteins for S. aureus. Various lytic and solubilization methods have been tested to identify a suitable methodology for detection of these proteins in two-dimensional electrophoresis (2DE). Results demonstrate that cell lysis with lysostaphin, which lyses staphylococcal peptidoglycan, followed by solubilization with urea, thiourea, amidosulfobetaine 14 (ASB 14) and dithiothreitol (DTT) is an effective method, yielding a sample comprising proteins of wide molecular ranges and isoelectric points with minimum contamination from cytosolic proteins. Mass spectrometric analysis was employed to identify the membrane and cell surface proteins present in the sample and consequently an initial proteomic map of membrane and cell wall associated proteins for S. aureus is presented. Keywords: Staphylococcus aureus • cell surface proteins • sample preparation • lysostaphin • two-dimensional electrophoresis

Introduction Staphylococcus aureus, a gram positive opportunistic human pathogen is capable of causing a wide variety of diseases ranging from skin and soft tissue surface lesions to more serious conditions such as endocarditis, septic arthritis, osteomyelitis and toxic shock syndrome.1 The extensive pathogenic capability of S. aureus relies in part upon the highly regulated secretion of a vast array of virulence factors that include surface adhesins, toxins, extracellular enzymes, and polysaccharides.2 To initiate invasive infection, S. aureus must adhere to extracellular matrix substrates and eukaryotic cells via surface proteins or adhesins. These microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) are covalently attached to cell wall peptidoglycan by the membrane-associated enzyme sortase.3 Adhesins of S. aureus that interact with collagen,4 fibrinogen,5 fibronectin,6 and von Willebrand factor (vWf)7 have been well characterized and others have been identified,8,9 stressing the need for in-depth investigations on cell wall/ membrane proteins of this pathogen. In recent years, a greater understanding of the biological and pharmacological importance of membrane proteins, especially those associated with the cell wall, has prompted significant improvements in the area of membrane proteomics. Mapping of membrane-associated proteins provides a benchmark for biological experimentation such as strain comparisons of * To whom correspondence should be addressed. Fax: (410) 455-1049. E-mail: [email protected].

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pathogens with differing modes of pathogenicity and antibiotic resistance mechanisms, and can aid in the definition of vaccine and therapeutic targets.10 However, proteome analysis of membrane and cell surface proteins is complex due to their intrinsic hydrophobic nature, alkaline pI and the number of transmembrane spanning regions. These properties lead to difficulties in solubilizing proteins for 2DE. Protein solubilization has been the subject of intensive research and many solubilizing agents for membrane proteins have been used, including chaotropes, (e.g., thiourea11), novel detergents, (e.g., amidosulfobetaines; (ASB-n)12) and reducing agents (e.g., tributylphosphine13). Recent improvements in this area are summarized in detail by Santoni et al.14 and Molloy.15 In general, most proteins identified in 2DE gels to date are hydrophilic and those that are hydrophobic have relatively low grand average of hydropathicity (GRAVY) values (usually below 0.3).16 A majority of the hydrophobic proteins have remained undetected either because they precipitated during isoelectric focusing or due to their low abundance.17 Despite these difficulties, reference maps of membrane proteins have been published for Escherichia coli,18 Acinetobacter radioresistens,19 Bacillus subtilis,20 Caulobacter crescentus,21 Pseudomonas aeruginosa,12 Helicobactor pylori,22 Corynebacterium glutamicum,23 and Borrelia burgdorferi.24 A reference map for S. aureus membrane proteins has not been previously reported. Recently, 2DE was used with tryptic peptide mass mapping via matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to study the regulatory 10.1021/pr049866k CCC: $30.25

 2005 American Chemical Society

Staphylococcus aureus Membrane/Cell Wall Proteome

networks in pathogenicity and extracellular protein expression in S. aureus.25-27 The reference map of cytoplasmic proteins from two S. aureus strains COL and 8325 has also been published by Cordwell et al.28 and defined approximately 12% of the S. aureus proteome. Proteins of the staphylococcal cell wall envelope were poorly represented in this study reflecting the poor solubility of gram-positive peptidoglycan and cell walls. Excluding publications in which staphylococcal surface proteins have been studied to identify vaccine candidate antigens29 and cell wall active antibiotic induced proteins,30 a systematic proteome study of cell wall associated proteins is still lacking as pointed out by Hecker et al. in 2003.31 In this context, we generated an initial proteome map of membrane and cell wall associated proteins of S. aureus. To accomplish this, we compared a number of available cell lysis and protein solubilization protocols and identified a suitable methodology for the isolation of membrane/cell wall proteins. Consequently, this study details the first proteomic analysis specifically targeted toward membrane and cell wall associated proteins of S. aureus.

Experimental Section Strain and Growth Conditions. The bacterial strain used in this study, Staphylococcus aureus Phillips, was isolated from a patient diagnosed with osteomyelitis.32 Glycerol stocks were prepared from exponentially growing cultures in tryptic soy broth (TSB) (Difco, Detroit, MI) and stored at -70 °C. Experimental cultures were grown in TSB at 37 °C with constant agitation (140 rpm) until late exponential growth phase. Cell concentrations were determined using a Coulter Multisizer. Apparatus and Chemicals. The isoelectric focusing (IEF) apparatus (Multiphor II), Immobiline dry strips (pH 3-10 NL, 7 cm), Immobiline dry strip re-swelling tray and related reagents were purchased from Amersham Pharmacia Biotech (San Francisco, CA). Mini-vertical electrophoresis system (Mini protean-III) was purchased from Bio-Rad, CA. 1 mm thick precast mini gels (12% Tris- HCl) for SDS-PAGE was procured from Bio-Rad. ProteoPrep membrane extraction kit (PROTMEM), lysostaphin, raffinose, LiCl, protease inhibitor cocktail, CHAPS, DTT (dithiothreitol), and iodoacetamide were purchased from Sigma (Sigma Chemical Co, St. Louis, MO). Tributylphosphine (TBP) and Amidosulfobetaine-14 (ASB14) were purchased from Bio-Rad and CalBiochem, USA, respectively. All other chemicals were of analytical grade. Cell Lysis Procedures. Four different cell lysis procedures were compared in triplicate: (i) Enzymatic treatment with lysostaphin,29 (ii) Chemical lysis via boiling in the presence of SDS,33 (iii) extraction with LiCl,20 and (iv) extraction using a commercially available ProteoPrep membrane extraction Kit (PROT-MEM, Sigma). Lysis by Enzymatic Treatment with Lysostaphin. After harvesting bacteria, cells were washed twice with PBS and once with digestion buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 5 mM MgCl2) at 4 °C. Approximately 3 × 109 bacteria were resuspended in 3 mL of digestion buffer containing 35% raffinose, protease inhibitor cocktail and 5U of lysostaphin. After incubation at 37 °C for 30 min, the supernatant was removed by centrifugation at 8000 × g for 15 min at 4 °C. The supernatant was then treated with DNase (0.75 mg/mL) and RNase (0.5 mg/mL) by incubating for 15 min on ice to remove nucleic acid contaminants. The supernatant was further treated with 20% v/v TCA in ice for 30 min and the precipitate was collected by centrifugation at 16 000 × g for 20 min at 4 °C.

research articles The precipitated protein was washed with acetone to remove traces of TCA and finally acetone was removed by speed vacuum treatment. Precipitated protein was re-suspended in sample solubilization buffer (SSB: 8 M urea, 2 M thiourea, 1% ASB 14, 1% w/v DTT and 2% v/v carrier ampholytes 3-10 NL) unless otherwise mentioned and stored at -80 °C for later use. Lysis by Boiling in SDS. Harvested staphylococcal cells were suspended in extraction buffer (125 mM Tris-HCl, pH 7.0 containing 2% SDS and protease inhibitor cocktail) at 10µL/ mg (wet weight) of cells and heated at 95 °C for 3 min. Supernatant was removed by centrifugation at 6000 × g for 15 min at 4 °C and treated with DNase/RNase as described above. Finally, protein was precipitated with 20%v/v TCA, centrifuged, washed with acetone, solubilized in SSB (as above) and stored at -80 °C for later use. Lysis by Treatment with LiCl. Cells were washed twice with 10 mM Tris-HCl, pH 7.6 and resuspended in 2 mL of extraction buffer (25 mM Tris-HCl, pH 7.6, containing 1.5 M LiCl and protease inhibitor cocktail). After incubation for 10 min on ice, the cell suspension was centrifuged again (6000 × g for 15 min, 4 °C) and the supernatant was treated with DNase/RNase as described above. Finally, protein was precipitated with 20%v/v TCA, centrifuged, washed with acetone, solubilized in SSB (as above) and stored at -80 °C for later use. Lysis by ProteoPrep Membrane Extraction Kit. Membrane protein fraction from S. aureus cells were prepared by following the instructions accompanied the PROT-MEM kit. Cells were suspended in soluble cytoplasmic and loosely bound membrane protein extraction reagent and disrupted by sonication on ice (550 Sonic Dismembrator, Fisher Scientific, Pa) for 10 min with 2 min intervals after every 1 min. After stirring the suspension slowly on ice for 1 h with DNase (0.75 mg/mL) and RNase (0.5 mg/mL), the membrane fraction was recovered by ultracentrifugation at 115 000 × g for 1 h at 4 °C, washed the precipitate twice with milliQ water and solubilized in cellular and organelle membrane solubilizing reagent aided by sonication. The suspension was centrifuged at 14 000 × g for 45 min at 15 °C to pellet cell debris. The supernatant was reduced by adding 5 mM TBP followed by alkylation with 15 mM iodoacetamide and stored at -80 °C for further analysis. 2-D Electrophoresis. Rehydration of Immobiline dry strips (IPG strip; Amersham Pharmacia Biotech) was carried out employing an Immobiline dry strip re-swelling Tray (Amersham Pharmacia Biotech) according to manufacture’s instructions. IPG strips (pH 3-10 NL), 7 cm long, were used for the present study. The Immobiline dry strips were allowed to rehydrate with the samples in 8 M urea, 2 M thiourea, 1% ASB 14, 2% v/v carrier ampholytes (pH 3-10 NL) (Amersham Pharmacia Biotech), traces of bromophenol blue and 7 mg DTT/2.5 mL of rehydration solution at room temperature for 16 h. Final sample load per strip was approximately 30 µg. The protein concentrations were measured by Plus One 2-D quant kit (Amersham Biosciences). The rehydrated strips were then subjected to isoelectric focusing (IEF) performed using Multiphor II electrophoresis unit at 20 °C. Briefly, 7 cm strips were focused for a total of 15 KVh. After focusing, the strips were stored at -80 °C for later use. Prior to the second dimension SDS-PAGE, IPG strips were equilibrated for 15 min in 5 mL of equilibration solution containing 50 mM Tris-HCl pH 8.8, 6 M urea, 30% v/v glycerol, 2% w/v SDS and traces of bromophenol blue with 100 mg/10 mL (w/v) of DTT. A second equilibration was carried out for 15 min by adding iodoacetamide (250 mg/ 10 mL) instead of DTT in equilibration solution. Second Journal of Proteome Research • Vol. 4, No. 2, 2005 251

research articles dimension vertical SDS-PAGE was performed using precast mini-gels (12% Tris-HCl) of 1 mm in thickness (Bio-Rad, CA). Mini-gel 2DE was carried out employing Mini-vertical electrophoresis system (Mini protean-III) according to the manufacture’s instructions. Briefly, electrophoresis was performed at constant current of 6 mA/gel for 30 min followed by 12 mA/ gel for one and half hours until the bromophenol band had exited the gel. Gels were stained with neutral silver stain as described.34 Electropherogram images were obtained with GS800 imaging densitometer (Bio-Rad) in gray scale mode. Molecular weight and pI values were estimated using SDSPAGE markers and IEF mixes obtained from Sigma Chemical Co, St. Louis, USA. The gel images were compared and the spots were counted using Z3 image matching software (Compugen, Israel) at the maximum confidence level after removing the spike spots. Spot confirmation was accomplished both by visual observation and the reproducibility between gel sets and cultivations. For analyzing the results of reproducibility experiments, images of three replicate gels were used to construct a composite or raw master gel, which serves to eliminate noise and minor discrepancies between gels. The spots on the gel were then identified and each was assigned a spot quantity (q). The q value characterizes spot size and intensity is defined as the sum of the gray level values of all the pixels in a spot and is expressed in terms of PPM of the total spot quantity on the gel (Compugen Z3 manual 2002). Thus q is an approximate fractional representation of the amount of protein in a particular spot and the ratio of q values for the same spot on two different gels gives an approximation of differential expression. Mass Spectrometry for Protein Identification. For mass spectrometric identification, gel spots were arbitrarily selected, excised, destained, and digested with sequencing grade trypsin (Promega, Madison, WI) as described by Gharahdaghi et al.35 Peptides were extracted by adding 70% acetonitrile in water containing 0.1% trifluoroacetic acid (TFA). The extracts from multiple extractions were pooled together and desalted using Zip-Tip C18 reverse phase peptide separation matrix (Millipore, USA). Samples were either used directly or concentrated under speed vacuum and subjected to matrix assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometry (Bruker Daltonics Autoflex, Bruker, USA). To prepare samples, aliquots of 0.6 µL of peptide solution was mixed with an equal volume of matrix (50 mM R-cyano-4-hydroxycinnamic acid in 60% acetonitrile/0.1% TFA solution), deposited on the instrument target plate and air-dried at room temperature. Mass spectra of peptide digests were acquired as the average of the ion signals generated by the irradiation of the target with 50-100 laser pulses, in positive linear mode with an acceleration voltage of 20 kV. Mass calibration was performed using standard peptides bradkinin and insulin as external standards. The resulting peptide mass fingerprints were used to search the translated S. aureus Mu50, MW2, and N315 genomes36,37 using Mascot (Matrix Science, London, UK). The database search was conducted using a mass accuracy of 50 ppm and carbamidomethyl modification of cysteine residues were allowed. The quality of the matches was defined by the number of matching peptide masses and the percentage of protein sequence covered by those masses. Generally, sequence coverage of at least 20% and a minimum of 4 matching peptide masses was required for a match confidence. Subcellular localization of the identified proteins was determined using PSORT program (http://psort.org). The presence of predicted signal peptides was determined using the program SignalP V2 252

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(http://www.cbs.dtu.dk/services/SignalP-2.0/). The theoretical molecular weights and pI before or after removal of signal peptides as well as grand average hydropathy (GRAVY) values were calculated using the ProtParam tool at ExPASy (http://expasy.cbr.nrc.ca/tools/protparam.html). Mapping of transmembrane regions for proteins was conducted using the program TMPred (http://www.ch.embnet.org/ software/TMPRED_form.html) as well as SOSUI (http:// sosui.proteome.bio.tuat.ac.jp/sosuiframe0.html).

Results and Discussion Cell Lysis. Figure 1 shows results obtained from 2DE of S. aureus membrane preparations by lysostaphin treatment (Figure 1A), boiling in the presence of SDS (Figure 1B), incubation with LiCl (Figure 1C) and the commercially available ProteoPrep membrane extraction kit (Figure 1D). Multiple 2DE (>20) were carried out using the samples prepared by the above-mentioned methods. Samples prepared via treatment with lysostaphin consistently gave comparably better 2DE images both in terms of the number of spots and the overall image quality judged by resolution and absence of streaking. Quantitative image comparison with Z3 software (Compugen, Israel) showed that lysostaphin treatment (Figure 1A) reproducibly displayed 121 spots compared to 101 spots obtained by boiling with SDS (Figure 1B) and 85 spots obtained for incubation with LiCl (Figure 1C). 85 spots were common in all the gels prepared by the three different isolation methods. For all methods, more proteins were generally found in the acidic range (pH 4-7) and many proteins in the 50-150 kDa range were represented by large trains of spots that vary by pI. This pattern is indicative of multiple forms of the same protein and may be caused by posttranslational processing or experimental artifacts such as deamidation.21 Figure 1D shows the 2DE image of the sample prepared by ProteoPrep membrane extraction kit, which is significantly different in spot pattern and number of spots. This is likely attributed to the different extraction and solubilization methods and reagents. The ProteoPrep membrane extraction kit (Figure 1D) did not yield reproducible gels and was not considered further. Results from the first three treatments were similar with respect to the overall spot pattern and the location of major proteins in the gel. We note however that gels of SDS (Figure 1B) and LiCl treatment (Figure 1C) lack a number of prominent proteins present with lysostaphin treatment. Treatment with SDS resulted in protein spots in the low molecular weight, acidic region of the gel that were absent in the lysostaphin/ LiCl treatment. However, the overall reduced number of spots in SDS treatment can be attributed to its deleterious effects on IEF due to the aggregation of some proteins after SDS is replaced by nonionic detergents.38 In previous studies, preparation of membrane proteins for 2DE has often involved rather vigorous cell disruption methods such as sonication12 or French pressure cell treatment.21 These methods can cause heating of the sample or significant contamination by cytoplasmic proteins, and require timeconsuming ultracentrifugation steps that may lead to the loss of low abundant proteins. In contrast, lysostaphin (a glycylglycine endopeptidase from Staphylococcus stapholyticus) enzymatically cleaves the pentaglycine cross bridges of the staphylococcal peptidoglycan, predominantly at their central glycine residue.39 Lysostaphin treatment has been used in a number of studies for the isolation of cell wall components such as penicillin binding proteins, transferrin-binding proteins40-42

Staphylococcus aureus Membrane/Cell Wall Proteome

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Figure 1. Comparison of protein isolation methods for the 2DE of membrane proteins from Staphylococcus aureus. Samples were prepared by (A) lysostaphin (B) boiling in the presence of SDS (C) incubation with LiCl (D) ProteoPrep Membrane Extraction Kit (Sigma). See Materials and Methods for details. Spot identification numbers in Figure 1A,B correspond to listings in Table 2.

and also for identifying vaccine candidate antigens using proteome analysis.29 In the present study, we found lysis of staphylococcal cells by lysostaphin provided the most detailed 2DE gels making it an effective, reproducible and relatively easy extraction method for the preparation of membrane and cell wall associated proteins. We also studied the effect of incubation time on the degree of cell lysis by lysostaphin (images not shown). Incubation with 5U of lysostaphin for 5 and 15 min yielded 60 and 71 protein spots respectively while incubation for 30 and 45 min yielded many more spots (121-130). We also evaluated how lysostaphin concentration affects the degree of lysis (images not shown). As lysostaphin concentration increases there is a corresponding increase in the degree of cell lysis at a constant incubation time of 30 min. Concentrations between 3U and 10U of lysostaphin yield a significant increase in number of spots, from 72 to 139. This could be due to an enrichment in the concentration of membrane proteins as well as the release of proteins closely associated with the inner surface of cytoplasmic membrane or those that are cytoplasmic. For further studies, we selected a lysostaphin concentration of 5U and an incubation time of 30 min as lysis parameters for the bacterial concentration used here (3 × 109 cells). We find these conditions maximize the number of protein spots while minimizing contamination by cyosolic proteins. These parameters are in

agreement with those used by Vytvytska et al.29 for the preparation of S. aureus surface proteins. Reproducibility of the lysostaphin protocol was assessed by running multiple gels on protein samples from three different bacterial cultivations. The gels (images not shown) appear nearly identical in qualitative aspects such as overall image and resolution and are quantitatively similar in the number of protein spots present (120 ( 5). Image analysis software showed a high degree of similarity in spot position and abundance between gels and gel sets. To analyze the reproducibility of the protocol in a quantitative manner we used spot quantity to measure protein expression ratios between cultivations. As illustrated in Figure 2, the log of protein expression ratios were very close to zero showing the similarities in the protein expression between cultivations and between the gels. Comparison of Solubilization Conditions. In the present study, we evaluated various detergents, chaotropes and reducing agent combinations to determine an effective solubilization procedure for the membrane and cell wall associated proteins from Staphylococcus. Figure 3 shows images obtained with four of these solubilization solutions. Among the various other combinations tested (Table 1), there were significant variations in the number of spots, degree of streaking, overall quality, and resolution of the images. We note the use of the chaotrope thiourea in conjunction with urea significantly increased the Journal of Proteome Research • Vol. 4, No. 2, 2005 253

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Nandakumar et al. Table 1. Analysis of Solubilization Conditions for the 2-D Gel Electrophoresis of Membrane Proteins from Staphylococcus aureusa no. of subjective method urea thiourea chaps ASB14 TBP DTT tris total image no. (M) (M) (%) (1%) (mM) (%) (mM) spots qualityb

1 2 3 4 5 6 7 8 9 10 11 Figure 2. Comparison of gel images obtained from different cultivations. For image analysis, gel image from cultivation 1 was taken as reference. Protein expression ratio is the logarithm of spot volume in gel 1/spot volume in gel 2 (logq1/qi; where ‘i’ is gel 2 or 3). Protein samples were prepared by enzymatic digestion with lysostaphin and solubilized with urea 8M, thiourea 2M, ASB 14 1% and DTT 1%.

extracting power of the solubilizing solutions. In addition, the use of phosphine reducing agent TBP instead of DTT resulted in an improved resolution of proteins especially in the low

8 8 8 8 8 8 8 8 8 8 8

0 0 0 0 0 0 2 2 2 2 2

2 2 2 0 4 4 2 4 0 0 0

1 1 0 1 0 0 0 0 1 1 1

2 0 2 2 0 2 2 2 2 2 0

1 1 1 0 1 1 1 1 0 1 1

0 0 0 0 40 0 0 0 0 40 0

29 31 101 103 34 35 32 33 40 73 121

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

a Evaluated as described in Materials and Methods. b +: Poor, ++: Fair, +++: Good.

molecular weight acidic proteins. However, TBP also resulted in horizontal streaking, which made the overall gel image less appealing and concealed the protein spots in that region. Excellent resolution and recovery of protein spots, especially high molecular weight proteins, was obtained when the detergent CHAPS was replaced by ASB14. For further studies we selected the solubilization solution with the composition of urea 8 M, thiourea 2 M, ASB 14 1% and DTT 1% (line 11,

Figure 3. 2-D gel electrophoresis of membrane proteins from Staphylococcus aureus under different solubilization conditions. Solubilization/rehydration conditions in A,B,C,D correspond to methods no: 11, 3, 4, and 10 in Table 1, respectively. Spot identification numbers in Figure 3B correspond to listings in Table 2. 254

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Staphylococcus aureus Membrane/Cell Wall Proteome Table 2: Identification of Proteins by Mass Spectrometry spot ID

1 2 3 4 5 6 7 8 10 11 12 13 14 15 16 19 20 21 22 23 30 39 49 50 52 53 54 56

58 65 66 67 77 78 80 81

protein

signal peptidase IB ABC transporter ATP-binding protein homlogue ABC transporter ATP-binding protein homlogue ABC transporter ATP-binding protein homlogue spoVG protein Hypothetical protein SAV0387 SA2198 precursor (lipoprotein) Staphylokinase precursor AGRD protein Serine protease SpID Serine protease SpIA Enterotoxin YENT2 SIGB gene (Hypothetical protein SA1873 Staphylococcal accessory regulator A Methicillin resistance regulatory protein Immunoglobulin G binding protein A precursor Ser-Asp rich fibrinogen binding, bone sialoportein binding protein Xaa-Pro dipeptidase Intercellular adhesion protein B Lipoprotein signal peptidase Hypothetical protein SAV2144 SA1584 protein (Lysophospholipase homolog) Vitronectin binding protein Cell-division protein Chaperone protein dnaK (Heat shock protein 70) Zinc metalloproteinase aureolysin SA1683 protein (ABC transporter homolog) SA0769 protein (ABC transporter ATP-binding protein homologue) ATP synthase B chain Amino acid carrier protein CSPC (Cold-shock protein C) 10 kDa chaperonin (Protein Cpn10) (Heat shock protein 10 Probable thiol peroxidase GrpE protein (HSP-70 cofactor) (HSP20) Hypothetical protein SAV2693 Chromosome segregation SMC protein

seq.cov. (%)

theoretical pI/mass

65 62

9.02/21692 4.90/ 28275

50

GRAVY valued

gene

accession no.

-0.436 -0.303

SPSB SAV0842

P72365 Q99VG3

4.90/ 28275

-0.303

SAV0842

Q99VG3

67

4.90/ 28275

-0.303

SAV0842

Q99VG3

55 56 62 71 74 44 22 47 42

4.85/ 12320 4.96/15136 5.78/ 14386 6.75/18490 4.86/ 5206 9.16/ 25678 9.10/25441 8.48/16002 9.55/13442

-0.55 -0.523 -0.922 -0.307 0.483 -0.273 -0.399 -0.893 -0.389

SPOVG SAV0387 SAV2410 SAK AGRD SPLD SPLA YENT2 SAV2068

Q99WA5 Q99WJ2 Q99RL9 P00802 O33586 Q9KH48 Q99T60 Q99T49 O05341

40

7.77/14775

-0.496

SARA

Q53600

56

8.94/14838

-0.752

MECI

Q932L5

32

5.60/48873

2

225

-1.131

SPA

Q99XA2

24

4.14/103293

2 signal anchor

476

-1.156

SDRC

Q99W48

59 65 55 35 57

5.02/11407 9.40/34105 6.82/18342 5.04/24072 5.43/31866

1 4 1 1

1-30 1-26

290 40 221 275

-0.164 -0.601 0.802 -0.275 -0.362

SAV1529 ICAB LSPA SAV2144 SAV1765

Q931R2 Q8NUI6 Q99US2 Q99SB5 Q99TA3

47 52 59

4.80/76767 5.42/77984 4.65/66418

3

1-25

232

-0.352 -0.51 -0.523

FUSA FTSH DNAK

P81683 Q99W92 Q99TR7

40

5.14/56427

1

1-27

509

-0.699

AUR

Q99R00

39

6.72/64920

5

115

0.196

SAV1866

Q99T13

60

6.82/38373

1

341

-0.142

SAV0837

Q99VG8

63 38 77 67

5.04/19539 8.98/52256 4.45/7374 4.92/10416

1 9

173 54

-0.514 0.688 -0.514 -0.439

ATPF ALST CSPC GROS

Q99SF1 Q99UC3 O33592 Q08841

70 40

4.57/18162 4.42/24022

-0.12 -1.033

TPX GRPE

Q99TF0 P45553

74 20

4.79/17541 5.35/136700

-0.137 -0.758

SAV2693 SMC

Q99QU8 Q99UN6

TMRa

SPb

1

1 1 1 2 2

1 1

res/TMc

191

1-26 1-27 1-36 1-35

1-36

120 163 47 119 117

154 1188

a TMR: No of Transmembrane helices (TmPred). b SP: Presence and location of signal peptide (SignalP). c res/TM: ratio of protein residue number to the putative number of transmembrane helices. d Gravy Value: Grand Average of Hydropathicity.

Table 1) by balancing the recovery of spots, resolution and clarity of the overall image. The incorporation of zwitterionic detergent, alkyl aminosulfobetaine ASB 14 in the 2DE sample solution described originally by Chevallet et al.43 and Rabilloud et al.44 has been shown to improve the solubility of membrane proteins when compared to sample solutions containing more conventional detergents such as CHAPS and SB 3-10. Our results support the observations of Nouwens et al.12 that a combination of urea, thiourea, TBP and ASB14 is superior for solubilizing membrane proteins from P. aeruginosa.

Protein Identification by MALDI-TOF-MS. We have employed the above-described extraction and sample preparation protocol to construct a membrane protein reference map of S. aureus. Table 2 lists the identification of 36 protein spots by database search of primarily the S. aureus Mu50 genome and incorporates the GRAVY values, length of the signal peptide and the number of transmembrane helices. The positions of the spots are shown in Figure 1A,B and Figure 3B. Among these identified proteins, 72% (26 spots) were membrane and cell wall associated proteins as per the subcellular localization predicted by the program PSORT.45 Nine protein spots were Journal of Proteome Research • Vol. 4, No. 2, 2005 255

research articles predicted to be of cytoplasmic origin and 1 protein was of extracellular nature. Three spots were identified as hypothetical proteins whose functions are unknown; with the subcellular localization of two proteins predicted to be the membrane. Despite the care taken to minimize contamination by cytosolic proteins, a number of heat and cold shock proteins such as DnaK, HSP20, Cpn10, and CSPC appear in the membrane fraction. However, it has been noted that abundant bacterial proteins such as ubiquitous heat shock proteins may often be found in the enriched fractions despite the unlikely occurrence of these proteins on the cell surface.10 There is some immunological and biochemical evidence to suggest that at least the chaperonin GroEl (HSP60) may cycle between the cytosol and membrane compartments in its role as a cellular scavenger of degradable proteins and carrier of newly synthesized proteins to their correct cellular location.46 The extraordinary adhesive and serum binding properties of staphylococci have been traced back to the expression of surface proteins.47 In the present study, four spots were identified as adhesins such as ser-rich fibrinogen binding, bone sialoprotein binding protein (spot 20), intercellular adhesion protein B (spot 22), vitronectin binding protein (spot 49), and immunoglobulin binding protein A (spot 19). Intercellular adhesion protein plays a major role in cell-cell adhesion and is required for biofilm formation.48 It is interesting to note that the database search of peptide masses obtained for spot 20 in the S. aureus MW2 genome also identified it as collagen adhesin although with very low sequence coverage (less than 10%). This is significant as the strain used in the present study, S. aureus Phillips, is well documented to produce collagen adhesin.49 One possible reason for this could be the similarities various MSCRAMMs share in sequence, structural and functional aspects. Collagen adhesin was previously reported in at least 2 forms with molecular masses of approximately 135 and 110 kDa in different strains32 and collagen adhesin of 106 kDa was found in the extracellular proteome of S. aureus.27 However, fibrinogenbinding protein (103 kDa) was accepted as the protein identity for spot 20 based on the higher sequence coverage and more than 90% matching of the peptide masses searched. The grand average hydropathy values (a measure of overall protein hydrophobicity) of the identified proteins imply that many are not very hydrophobic overall, but rather have domains of high hydrophobicity that allow them to associate with the lipid bilayer. The most hydrophobic protein detected in this study had a GRAVY value of 0.802 (spot 23, lipoprotein signal peptidase), however approximately 85% of the membrane proteins identified had negative GRAVY values. The presence of predominantly hydrophilic proteins in the membrane fraction suggests that other factors, such as their location in the membrane and the number of transmembrane spanning regions (TMRs), may be of importance to their solubility for 2DE. A transmembrane map was generated for each of the identified proteins using the program TmPred,50 which predict the likelihood that a protein traverses a membrane, and the most likely orientation of the protein in the membrane. For 20 membrane proteins identified in this study, a high score was obtained for the presence of at least one transmembrane region, suggesting them to be integral membrane proteins. The other identified nontransmembrane proteins may belong to the class of peripheral membrane proteins often found in association with the integral membrane proteins. It is likely that the overall hydrophobicity of a protein determined its ability to be solubilized for 2DE while the number of 256

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TMR influences the ability of a detergent based solution to efficiently extract the protein from within the highly insoluble lipid membranes.12 The fact that we were able to isolate proteins with as many as nine transmembrane helices in the present study (spot 65, amino acid carrier protein) illustrates the effectiveness of the employed protocol. This is a significant improvement over the three TMR proteins that are thought to be the limit of standard CHAPS/DTT solubilization solutions.51 Proteins such as AGRD, amino acid carrier protein and lipoprotein signal peptidase with positive GRAVY values also exhibited a relatively low Res/TM ratio (amino acid residues/ transmembrane region) of 40-54 confirming their hydrophobicity. Presence of spots representing proteins with very high GRAVY values such as 0.802 and low Res/TM ratio as 40 clearly highlights the power of 2DE protocol used in the present study. However, the presence of membrane proteins with mainly negative GRAVY values and few transmembrane regions in the sample supports the previous observations that only mildly hydrophobic proteins can be solubilized with detergents commercially available today.52 Among the proteins identified, signal peptide sequencess which target proteins for secretion, were present in 10 proteins. Many of them were also predicted to have more than one transmembrane region. It is noted that many of the TM prediction methods have the tendency to predict signal peptide as the first transmembrane segment.53 Also interesting is that Enterotoxin YENT2 (spot 13), whose location listed by SwissProt as extracellular, is predicted not to have a signal peptide. Both prokaryotes and eukaryotes are known to produce a number of proteins that lack a typical hydrophobic leader peptide, which are nevertheless actively secreted by a mechanism that does not require the standard secretory pathway to be functional.54 It has been suggested that export of at least some of these proteins is accomplished through the action of substrate specific ATP driven membrane translocators.55 In our analysis most of the identified proteins migrated according to their predicted pI and molecular weights. We also observed a few proteins that migrated on the 2D gels slightly differently than predicted. However, with signal peptides removed, their pI and molecular weights correspond more closely to the experimental pI and molecular weights (data not shown). Typically these signal sequences consist of positively charged and hydrophobic amino acids, which influence the predicted pI of the protein when included in the sequences.12

Concluding Remarks To mount a successful infection, gram-positive pathogens display surface proteins that adhere to specific receptors on host tissues or provide for microbial escape from the host’s immune responses. A comprehensive knowledge of surface and extracellular proteins produced by S. aureus will provide a basis for future studies on the development of vaccines and diagnostic tools. The major limiting factor in membrane proteome analysis is the complexity of protein solubilization and separation procedures due to their chemical character and membrane compartmentalization in addition to the secretion of cell wall proteins in a growth phase dependent manner. In the present study, we utilized a cell lysis protocol based on the enzymatic digestion by lysostaphin followed by solubilization with urea, thiourea, ASB 14 and DTT as a relatively easy and effective method for preparing a sample comprising proteins of wide molecular ranges and pI with minimal contamination from cytosolic proteins. We also constructed an initial reference map

research articles

Staphylococcus aureus Membrane/Cell Wall Proteome

of membrane and cell wall associated proteins of S. aureus by mass spectrometric identification of 36 protein spots, of which 72% appear to be of membrane origin including a number of cell surface adhesins. The fact that a number of cell surface adhesion proteins and proteins involved in quorum sensing and biofilm formation were identified in this initial membrane/ cell wall proteome map encourages the use of a proteomics approach in future studies.

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