Comparative proteomics of Staphylococcus aureus and the response of methicillin-resistant and methicillin-sensitive strains to Triton X-100a

Stuart J. Cordwell1, Martin R. Larsen1, Rebecca T. Cole1 and Bradley J. Walsh1

Australian Proteome Analysis Facility, Level 4, Building F7B, Macquarie University, Sydney, Australia21091

Author for correspondence: Stuart J. Cordwell. Tel: +61 2 9850 6204. Fax: +61 2 9850 6200. e-mail: scordwell{at}proteome.org.au


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Proteomics is a powerful tool for analysing differences in gene expression between bacterial strains with alternate phenotypes. Staphylococcus aureus strains are grouped on the basis of their sensitivity to methicillin. Two-dimensional gel electrophoresis was combined with MS to compare the protein profiles of S. aureus strains COL (methicillin-resistant) and 8325 (methicillin-sensitive). Reference mapping via this approach identified 377 proteins that corresponded to 266 distinct ORFs. Amongst these identified proteins were 14 potential virulence factors. The production of 41 ‘hypothetical’ proteins was confirmed, and eight of these appeared to be unique to S. aureus. Strain COL displayed 12 protein spots, which included alkaline-shock protein 23 (Asp23) and cold-shock proteins CspABC, which either were not present in strain 8325 or were present at a significantly lower intensity in this strain. Comparative maps were used to characterize the S. aureus response to treatment with Triton X-100 (TX-100), a detergent that has been shown to reduce methicillin resistance independently of an interaction with the mecA-encoded penicillin-binding protein 2a. In response to growth of the bacteria in the presence of TX-100, 44 protein spots showed altered levels of abundance, and 11 of these spots were found only in COL. The products of genes regulated by {sigma}B (the alternative sigma factor), including Asp23 and three proteins of unknown function, and SarA (a regulator of virulence genes) were shown to be present at significantly altered levels. SarA production was induced in TX-100-treated cultures. A protein of the {sigma}B operon, RsbV, was only detected in COL and its production was down-regulated in COL when the strain was treated with TX-100, whereas RsbW was present at reduced levels in both strains. Upon growth of both strains in the presence of TX-100, no effects on the production of the essential methicillin-resistance factor FemA were detected, whereas phosphoglucosamine mutase (GlmM) production was reduced in COL alone. This study suggests that proteins of the {sigma}B and sarA regulons, as well as other factors, are involved in methicillin resistance in S. aureus.



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Fig. 1. 2-DGE of cellular proteins from S. aureus 8325. (a) Proteins separated using IPG pH 4–7 and (b) IPG pH 6–11 two-dimensional gels. Numbers refer to identifications shown in supplementary data (http://mic.sgmjournals.org). Boxes show the positions of spots only found in strain COL. Broken boxes show the positions of spots found only in cells following their growth in the presence of TX-100. NI, not identified; D suffix, potential dimer; F suffix, protein spot corresponds to a fragment of a larger translated ORF.

 
Keywords: microbial proteome, two-dimensional gel electrophoresis, mass spectrometry, SarA, {sigma}B

Abbreviations: 2-DGE, two-dimensional gel electrophoresis; IPG, immobilized pH gradient; LTA, lipoteichoic acid; MALDI/TOF, matrix-assisted laser desorption/ionization time-of-flight; TX-100, Triton X-100

a The identifications for the spots shown in Fig. 1 can be found as supplementary data in Microbiology Online (http://mic.sgmjournals.org).


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Strains of Staphylococcus aureus are responsible for a high percentage of nosocomially acquired secondary infections. S. aureus has been implicated in diseases of the retina, as well as in skin diseases such as psoriasis, wound infections leading to septicaemia and meningitis, toxic shock syndrome and secondary diseases in cystic fibrosis patients (Waldvogel, 1995 ; Projan & Novick, 1997 ). Although S. aureus is part of the normal flora of the human skin and nasopharynx, coagulase-positive strains of this organism are potential pathogens when introduced into wounds and/or the blood of susceptible patients. The emergence of methicillin-resistant strains of S. aureus (reviewed by Hiramatsu et al., 2001 ) has placed even further importance on research aimed towards understanding the pathogenicity of this organism and on the production of novel therapeutic agents against it. In recent times, strains of S. aureus have been treated with the glycopeptide antibacterial drugs vancomycin and teicoplanin; however, the use of vancomycin as a therapeutic agent is rapidly becoming ineffective due to the proliferation of newly resistant strains of S. aureus (Tenover et al., 2001 ).

The worldwide medical impact of S. aureus as an infectious agent has led to the initiation of several genome-sequencing initiatives, most of which are aiming to decipher the genetic differences between strains of S. aureus that are sensitive or resistant to the ß-lactam antibiotic methicillin. However, initial reports suggest that there are few differences between these phenotypes at the genetic level, with methicillin-sensitive S. aureus containing mobile genetic elements that encode the majority of the antibiotic-resistance genes (Kuroda et al., 2001 ). Complementary to the role of comparative genomics is the advent of similar comparisons using the technologies encompassed under the term ‘proteomics’ (Cordwell et al., 2001 ). The visual power of two-dimensional gel electrophoresis (2-DGE), combined with the sensitivity and throughput attributed to MS, has allowed significant progress to be made in characterizing proteins that allow bacteria to achieve a given phenotype. Such comparative analyses have already been undertaken for some organisms whose complete genome sequences are available, including strains of Mycobacterium tuberculosis (Jungblut et al., 1999 ) and Helicobacter pylori (Jungblut et al., 2000 ).

Resistance to methicillin in S. aureus strains is dependent upon the product of the mecA gene, penicillin-binding protein 2a (Fontana, 1985 ; Reynolds & Fuller, 1986 ), as well as on the products of several other genes (fem or auxiliary genes; De Lencastre et al., 1999 ) that are not contained within mobile genetic elements and which are conserved across both methicillin-resistant and methicillin-sensitive strains. These genes include glmM (which encodes a phosphoglucosamine-mutase-like protein that is involved in peptidoglycan-precursor biosynthesis; Wu et al., 1996a ; Jolly et al., 1997 ; Glanzmann et al., 1999 ), femABCD (Berger-Bächi et al., 1989 ; Henze et al., 1993 ), fmtAB (Komatsuzawa et al., 1997 , 2000 ; Wu & De Lencastre, 1999 ) and llm (Maki et al., 1994 ). The functions of the proteins encoded by these genes are mostly unknown; however, their disruption may affect the composition of the cell wall and thus affect cell stability, while reducing, but not eliminating, resistance to antibiotics (de Jonge et al., 1992 ; Maidhof et al., 1991 ; Ornelas-Soares et al., 1993 ). Furthermore, this reduction in resistance to antibiotics occurs independently of any effects on mecA. At the cellular level, ß-lactam antibiotics are thought to act by removing acylated lipoteichoic acids (LTAs) from bacterial cell walls (Suginaka et al., 1979 ; Ohta et al., 2000 ). LTA may, in turn, regulate the production of peptidoglycan hydrolases (enzymes that break down the cell wall prior to cell division), thus its removal by ß-lactams may also induce irregular autolysis. The virulence regulators agr and sar also regulate these hydrolases (Fujimoto & Bayles, 1998 ). Furthermore, overexpression of the heat-shock regulon may inhibit cell autolysis and enhance antibiotic resistance (Qoronfleh et al., 1998 ).

The alternative sigma factor {sigma}B (sigB) regulates the production of general stress proteins in Bacillus subtilis and in other Gram-positive bacteria, including S. aureus (Hecker & Völker, 2001 ; Gertz et al., 2000 ; Hecker & Engelmann, 2000 ). General stress proteins allow bacteria to survive under extreme environmental conditions, including heat shock and oxidative damage induced by H2O2 (Hecker et al., 1996 ). In S. aureus, few {sigma}B-dependent proteins have been described, although alkaline-shock protein 23 (Asp23), coagulase and several hypothetical proteins have recently been elucidated (Gertz et al., 1999 , 2000 ). {sigma}B has also been suggested as a regulator of staphylococcal virulence factors (Kullik et al., 1998 ). Recent reports have shown that {sigma}B negatively regulates several extracellular pathogenicity factors, including staphopain, serine proteases, ß-haemolysin and leukotoxin D (Ziebandt et al., 2001 ). sigB mutants are dramatically more susceptible to methicillin than their parent wild-type strains, suggesting that {sigma}B-regulated stress-response genes and their proteins may be involved in antibiotic resistance (Wu et al., 1996b ). Conflicting studies have reported that {Delta}sigB mutants show a decrease (Gertz et al., 2000 ; Bischoff et al., 2001 ) or an increase (Cheung et al., 1999 ) in the expression of sarA (Chan & Foster, 1998 ). SarA regulates several virulence factors, either in association with agr (Heinrichs et al., 1996 ; Novick et al., 1993 ) or individually, including the repression of cell-surface proteins such as fibronectin-binding protein and protein A (Cheung et al., 1992 , 1997 ), and the positive control of extracellular factors, including ß-haemolysin, lipase and autolysin (Cheung et al., 1992 ; Ziebandt et al., 2001 ). The relationship between {sigma}B, SarA, pathogenicity and antibiotic resistance has yet to be fully elucidated.

The non-ionic detergent Triton X-100 (TX-100) reduces methicillin resistance in a range of S. aureus strains (Raychaudhuri & Chatterjee, 1985 ; Komatsuzawa et al., 1994 , 1995 ; Suzuki et al., 1997 ), with resistant strains showing the greatest increase in antibiotic sensitivity. These effects do not correlate with changes in mecA gene expression or in the ability of penicillin-binding protein 2a to bind antibiotic. Transposon mutagenesis of another gene, labelled fmt, which encodes a product with protein sequence similarity to penicillin-binding proteins, has been shown to further increase S. aureus methicillin sensitivity in the presence of TX-100 (Komatsuzawa et al., 1997 ). TX-100 stimulates autolysis and the release of acylated LTA and strains that show the greatest reduction in methicillin resistance also release significantly more LTA. These effects appear to be independent of known autolysins (Komatsuzawa et al., 1994 ; Ohta et al., 2000 ), since mutants deficient in these enzymes also show increased susceptibility to methicillin in the presence of TX-100.

In this study, we present comparative proteome mapping of a methicillin-resistant S. aureus strain (COL) and a methicillin-sensitive S. aureus strain (8325). This mapping was achieved by using a 2-DGE/MS approach. Such analysis allows further characterization of the effects of genetic and environmental challenges on S. aureus. As an example of this method of analysis, we have utilized the maps from 2-DGE/MS to compare the protein profiles generated by S. aureus strains COL and 8325 when they were grown in the presence and absence of 0·02% TX-100.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and culture conditions.
S. aureus strains 8325 and COL were kindly supplied by Professor John J. Iandolo, The University of Oklahoma Health Sciences Center, OK, USA. Strains were grown to mid-exponential phase in tryptic soy broth at 37 °C in an orbital shaker. For the TX-100 analysis, cells were grown as described but the medium was supplemented with a subinhibitory concentration of TX-100 (0·02%; Komatsuzawa et al., 1994 ). After incubation, the cultures were centrifuged at 6000 g to pellet the cells. The pellets were washed twice with PBS (0·01 M, pH 7·5), freeze-dried overnight and then stored at -80 °C until required.

2-DGE.
2-DGE sample buffer [5 M urea, 2 M thiourea, 0·1% carrier ampholytes, 2% (w/v) CHAPS, 2% (w/v) sulfobetaine 3–10, 2 mM tributylphosphine (TBP); Bio-Rad] was added to 10 mg dry weight S. aureus cells and the mixture was sonicated in a tip-probe sonicator (Branson) on ice for 1 min. The procedure was repeated five times with 2 min intervals, during which time the lysate was kept on ice. The lysate was then centrifuged at 6000 g to remove unbroken cells and 150 U of Serratia marcescens endonuclease (Sigma) was added to it. A sample of the lysate (50 µl, equalling approximately 250 µg of the original dry weight of cells) was diluted in sample buffer up to a final volume of 460 µl and used to re-swell pre-cast pH 4–7 immobilized pH gradient (IPG) strip gels (Bio-Rad). Isoelectric focusing was performed using a Multiphor II (Amersham Pharmacia Biotech) or IEFCell (Bio-Rad) apparatus for a total of 80 kVh. IPG strips were detergent-exchanged, reduced and alkylated in buffer containing 6 M urea, 2% SDS, 20% (v/v) glycerol, 5 mM TBP, 2·5% (v/v) acrylamide monomer, a trace amount of bromophenol blue dye and 375 mM Tris/HCl (pH 8·8) for 20 min, prior to loading the IPG strips onto the top of an 8–18% T, 2·5% C (piperazine diacrylamide) polyacrylamide gel (20 cmx20 cm). Strips were embedded in 0·5% agarose in cathode buffer (192 mM glycine, 25 mM Tris, 0·1% SDS). Second-dimension electrophoresis was carried out at 4 °C and 3 mA gel-1 for 2 h; the current was then increased to 20 mA gel-1 and the run was continued until the bromophenol blue dye had run off the end of the gel. After the second-dimension electrophoresis, the gels were fixed in a mixture of 40% methanol and 10% acetic acid for 1 h and then stained overnight in Sypro Ruby (Molecular Probes). Gels were destained in a mixture of 10% methanol and 7% acetic acid for 1 h and imaged using a Molecular Imager Fx (Bio-Rad). To facilitate spot excision, the gels were ‘double-stained’ for a minimum of 24 h in Colloidal Coomassie blue G-250 (0·1% G-250 in 17% ammonium sulphate, 34% methanol and 3% ortho-phosphoric acid). Gels were finally destained in 1% acetic acid for a minimum of 1 h.

Image analysis.
Protein samples from each strain, and each strain grown in the presence or absence of TX-100, were acquired from separate duplicate cultures. 2-DGE was performed in triplicate for each of these sample preparations. Therefore, for each of the four growth conditions (i.e. each strain and each strain following challenge with 0·02% TX-100), six gels were generated. A single gel run consisted of four gels corresponding to each of the four samples (namely, 8325 alone, COL alone, 8325+0·02% TX-100 and COL+0·02% TX-100). Image analysis was performed to detect differences in protein production in each strain and between strains when grown in the presence of TX-100. An increase or a decrease in the visible spot intensity of greater than 1·5-fold, averaged over six gels and normalized using the intensities of 10 spots from each pH range with no apparent change in protein abundance, defined the selection of differentially expressed proteins. For comparative analyses, statistical data were acquired using the Z3 algorithm (Compugen). Each spot was assigned a value in p.p.m. that corresponded to the single spot volume amongst the total spot volume of all spots in the gel, following background subtraction and removal of fluorescence and other artefacts. The assigned values were used to determine n-fold changes in protein abundance between both of the growth conditions used. Gel spots showing reproducible n-fold changes in abundance from two of each set of three gels were selected for further analysis. Statistical analyses were performed on triplicate gels, including at least one gel from each of the two original cultures.

MS.
Protein spots were excised from the gels using a sterile scalpel and placed into a 96-well microtitre plate. Gel pieces were washed with a solution of 50 mM ammonium bicarbonate (pH 7·8) and acetonitrile (60:40) for 1 h at room temperature. The solution was removed from the wells and the gel pieces were vacuum-dried for 25 min in a SpeedVac (Savant Instruments). The gel spots were then rehydrated in 12 µl of trypsin digest solution (12 ng µl-1) [sequencing-grade modified trypsin (Promega) in 50 mM ammonium bicarbonate] at 4 °C for 1 h. Excess trypsin solution was removed from the wells and the gel pieces were suspended in 20–30 µl of 50 mM ammonium bicarbonate and incubated overnight at 37 °C. Peptides eluted from the gel pieces were concentrated and de-salted using C18 Zip-Tips (Millipore) or pre-fabricated narrow-diameter GelLoader (Eppendorf) tips packed with reversed-phase chromatography resin (Jensen et al., 1998 ; Gobom et al., 1999 ). The tips were activated and washed with 20 µl of acetonitrile and then acidified with 20 µl of 10% formic acid. Peptide solution was then slowly passed through the column using gentle air pressure. For peptide-mass mapping, bound peptides were washed with 10% formic acid and then eluted from the Zip-Tip column onto the matrix-assisted laser desorption/ionization (MALDI) target plate with 1 µl matrix solution (10 mg {alpha}-cyano-4-hydroxycinnamic acid ml-1; Sigma). For electrospray-ionization tandem MS/MS peptide sequencing, peptides were eluted in 1–2 µl of a mixture of 50% methanol and 1% formic acid directly into borosilicate nano-electrospray needles (Micromass).

Matrix-assisted laser desorption/ionization time-of-flight (MALDI/TOF) MS peptide-mass mapping was performed using a PerSeptive Biosystems Voyager DE-STR apparatus and a Micromass TofSpec 2E apparatus. Both instruments were equipped with 337 nm nitrogen lasers. Mass spectra were obtained in reflectron/delayed extraction mode, averaging 256 laser shots per sample. Two-point internal calibration of spectra was performed using trypsin autolysis peaks of 842·5 and 2211·1 Da. Lists of mono-isotopic peaks corresponding to the masses of generated tryptic peptides for each gel-purified protein spot were used to search the translated S. aureus N315 genome (Kuroda et al., 2001 ; http://www.bio.nite.go.jp/cgi-bin/dogan/) using the program PROTEINLYNX (Micromass). Parameters for the database search were as follows, 0·08 Da mass accuracy, one missed cleavage, and cysteine-acrylamide and methionine sulfoxide modifications allowed. The quality of matches was defined by the number of matching peptide masses and the percentage of protein sequence covered by those masses in comparison to other potential matches. No molecular mass or isoelectric point ‘window’ was used, enabling the identification of cleavage products. Generally, a sequence ‘coverage’ of approximately 25% was required for match confidence; however, protein fragments and high-mass proteins may be identified with significantly lower coverage yet a high number of matching peptides, whereas low-mass proteins may not be identified with significantly higher coverage and few matching peptides. Proteins not identified using this approach were subjected to a second round of MS, as described below.

Tandem electrospray-ionization MS was performed using a Q-Tof hybrid quadrupole/orthogonal-acceleration TOF mass spectrometer (Micromass). Nano-electrospray needles containing the sample were mounted in the source and stable flow was obtained using capillary voltages of 900–1200 V. Precursor ion scans were performed to detect mass to charge (m/z) values for peptides within the mixture. The m/z value of each individual precursor ion was selected for fragmentation and collided with argon gas using collision energies of 18–30 eV. Fragment ions (corresponding to the loss of amino acids from the precursor peptide) were recorded and processed using MASSLYNX version 3.4 (Micromass). Amino-acid sequences were deduced by the mass differences between y-ion or b-ion ‘ladder’ series using the program MASSESEQ (Micromass) and confirmed by manual interpretation. Peptide sequences were then used to search the S. aureus N315 and Mu50 genomes as well as the unfinished 8325 and COL genomes using the program BLASTP (Altschul et al., 1990 ; http://www.ncbi.nlm.gov/Microb_blast/unfinishedgenome).


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Protein identification and strain comparison
Proteins isolated from exponential phase cultures of S. aureus COL and 8325 were solubilized for 2-DGE and arrayed using pH 4–7 and pH 6–11 IPG two-dimensional gels (Fig. 1). The majority of S. aureus proteins clustered between pH 4·5 and 6·0 and between 20 and 45 kDa. A total of 377 two-dimensional gel-purified proteins were isolated and analysed using MALDI/TOF MS, following tryptic digest. These ‘spots’ represented 266 expressed ORFs, corresponding to approximately 12% of the S. aureus proteome (the identifications for the spots shown in Fig. 1 can be found as supplementary data at http://mic.sgmjournals.org). On average, each identified gene was present as 1·42 spots on the two-dimensional gels. The most extensively modified S. aureus proteins included elongation factor Tu (four pI variants and one C-terminal fragment) and fructose-bisphosphate aldolase (SA1927, four pI variants). In total, 36 proteins were identified in more than one two-dimensional gel spot, mostly as a result of pI variation. Each of the identified proteins was placed in a functional category as defined by Riley (1993) and assigned the ORF number defined in the S. aureus N315 genome sequencing project (Kuroda et al., 2001 ). The complete set of glycolytic enzymes (12 proteins) was detected as well as an entire complement of chaperones (DnaK, GroEL, GroES, GrpE, Tig and DnaJ). Twenty-two ribosomal proteins were identified; the remainder were most likely absent due to their basic isoelectric points. The major class of characterized protein spots was the so-called ‘hypotheticals’, or proteins of unknown function. Forty-one previously hypothetical proteins (15·5% of all of the identified ORFs) were characterized for the first time; eight of these appeared to be unique to S. aureus. A further 53 proteins were designated ‘hypothetical’ but showed some sequence similarity to known functional classes. Proteins of the cell envelope were underrepresented in this study, reflecting the poor solubility of Gram-positive peptidoglycan and cell walls. However, 20 potential virulence and resistance factors were detected (Table 1). Five pairs of paralogous proteins were also identified – fructose-bisphosphate aldolase (SA1927 and SA2399), L-lactate dehydrogenase (SA0232 and SA2395), superoxide dismutase (SodA) (SA0128 and SA1382), SarA (SA0573 and SA2089) and Asp23 (SA1356 and SA1984).


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Table 1. Potential S. aureus virulence factors and proteins associated with antibiotic and other resistance phenotypes identified by 2-DGE/MS

 
The protein-spot patterns of the two strains were compared using image analysis. Similarly abundant proteins showed a high degree of spot positional correlation between the two strains, indicating that the genomes of COL and 8325 are highly conserved within the majority of encoded amino-acid sequences. Only a handful of proteins showed altered position between the strains, typically as a result of minor amino-acid-sequence variations. For example, ATP synthase {alpha}-chain (SA1907) shifts to the acidic side in COL due to a single amino-acid substitution (lysine to asparagine) at position 373 (data not shown). 3-Methyl-2-oxobutanoate hydroxymethyltransferase (SA2392) also shifts to the acidic side in COL (pI 5·50 versus pI 5·63 in 8325). Less than 10 proteins were detected that showed visible alteration in their spot-migration pattern between the strains. The pattern of strain COL contained 12 visible spots between pH 4–11 that either could not be detected in 8325 or were present at a significantly reduced intensity in this strain, whereas 11 such protein spots were identified for strain 8325 (Table 2). The COL-associated spots included pI variants of all three cold-shock proteins (CspABC), two thiamin-biosynthesis proteins (ThiL and ThiF) and a single potential virulence factor (fibronectin–fibrinogen binding protein). Three proteins linked to the alternative sigma factor {sigma}B (sigB) – Asp23 (two pI variants), anti-anti-{sigma} factor RsbV and conserved hypothetical protein SA0772 (described as Csb8 in Gertz et al., 2000 ; Figs 2 and 3) – were also present at significantly higher intensities in the pattern of COL. However, only RsbV was entirely absent from two-dimensional gels of cellular proteins from strain 8325. Comparisons of the RsbV sequences from the unfinished COL and 8325 genomes, made using the BLAST algorithm, revealed 100% sequence identity, suggesting that transcriptional or post-translational modification of this protein may account for the lack of visible RsbV in strain 8325.


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Table 2. Proteins with significant differences in abundance in S. aureus strains 8325 and COL

 


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Fig. 2. Changes in the production of CspABC, RsbVW, Rot, PtsH, stage V sporulation protein G (SA0456) and hypothetical proteins SA0919, SA0271 and SA0772 under the different growth conditions. Proteins were extracted from strains grown under the following conditions: (a) strain 8325 grown in medium with no TX-100; (b) strain COL grown in medium with no TX-100; (c) strain 8325 grown in medium supplemented with 0·02% TX-100; (d) strain COL grown in medium supplemented with 0·02% TX-100. The images shown represent magnified areas of representative 2-DGE gels for the S. aureus strains.

 


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Fig. 3. Changes in the production of Asp23, UreB, pyrimidine operon repressor (SA1041) and hypothetical protein SA0755 under the different growth conditions. Panels (a–d) are as described in the legend to Fig. 2.

 
Image analysis was also used to determine the most abundant proteins in each strain based on summed spot volumes via the program Z3. Where more than one spot represented a particular identified ORF, the intensities of each spot were summed to provide an ‘expression’ value for that ORF. The 30 most abundant proteins for each strain are shown in Table 3 and account for approximately 45% of the entire visible protein separated on S. aureus two-dimensional gels. The most abundant proteins included metabolic enzymes (ATP synthase ß-chain, glyceraldehyde-3-phosphate dehydrogenase, fructose-bisphosphate aldolase, pyruvate dehydrogenase E1 ß-subunit, inositol monophosphate dehydrogenase and alcohol dehydrogenase) and several ribosomal proteins. Three hypothetical proteins (SA0941, SA1528 and SA1663) were also present amongst the most highly produced proteins under the chosen conditions. Abundant proteins were remarkably conserved between the two strains, with only seven differences noted. Despite their absence from either list, the majority of these proteins were not present at significantly different levels (1·5-fold change in abundance) between the two strains. In strain COL, CspC and Asp23 were amongst the most abundant cellular proteins and were present at significantly greater levels than in strain 8325; CspA was also present at significantly greater levels in COL, yet it still ranked as the second most abundant protein in strain 8325.


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Table 3. Comparison of the 30 most abundant proteins from S. aureus strains 8325 and COL

 
TX-100-associated protein production
TX-100 added to cultures at subinhibitory concentrations has been shown to reduce levels of methicillin resistance in a number of S. aureus strains (Raychaudhuri & Chatterjee, 1985 ; Komatsuzawa et al., 1994 , 1995 ; Suzuki et al., 1997 ). We studied the effects of 0·02% TX-100 on protein production in a methicillin-sensitive strain (8325) and a methicillin-resistant strain (COL) of S. aureus. Growth curves for both strains were essentially as described previously (Komatsuzawa et al., 1994 ; data not shown). Proteins isolated from harvested exponential-phase cells were used to perform comparative 2-DGE on both pH 4–7 and pH 6–11 two-dimensional gels. A total of 44 differentially produced proteins were detected (Table 4). Of these, 25 were detected in both strains, 11 were detected only in COL and a further eight were detected only in 8325.


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Table 4. Proteins whose production was up- or down-regulated in S. aureus strains 8325 and/or COL grown in the presence of 0·02% TX-100

 
Of the proteins detected as being TX-100-associated in both strains, 16 were present at reduced levels or were absent altogether from S. aureus grown in the presence of TX-100. These included regulatory proteins, such as Rot (repressor of toxins; Fig. 2) and pyrimidine operon repressor, isoforms of fructose-bisphosphate aldolase (SA1927; Fig. 3) and glyceraldehyde-3-phosphate dehydrogenase, phosphotransacetylase, {sigma}B-associated proteins RsbW and Asp23, and several hypothetical proteins (including SA1423, 0271, 0372 and 1671). Two of these hypothetical proteins (SA0372 and SA1671) have previously been shown to be {sigma}B-regulated (Gertz et al., 2000 ). A further nine proteins were present at increased levels of production and included urease ß-chain (Fig. 3), a fragment of cell-division protein FtsZ (Fig. 4), heat-shock protein HslU and secretory antigen SsaA. An increase in the production of two variants of SarA was also observed (Fig. 5).



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Fig. 4. Changes in the production of GlmM and a fragment of FtsZ under the different growth conditions. Panels (a–d) are as described in the legend to Fig. 2.

 


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Fig. 5. Changes in the production of SarA under the different growth conditions. Panels (a–d) are as described in the legend to Fig. 2.

 
The production of seven proteins was significantly induced when strain COL was grown in the presence of TX-100; these proteins included three variants of immunodominant antigen A (IsaA; Fig. 6) and three variants of a C-terminal 40 kDa fragment of a conserved hypothetical protein (SA0674) with sequence similarity to anion-binding proteins from a variety of bacteria. A single spot corresponding to IsaA was detected in strain 8325 (Fig. 6), but this was not as intense as the same spot found in COL. Spots for four proteins whose production had been down-regulated were also observed; these included anti-anti-{sigma} factor RsbV and 3-hydroxy-3-methylglutaryl CoA synthase. The production of a conserved hypothetical protein (SA0755) with sequence similarity to general stress proteins and of phosphoglucosamine mutase (GlmM) was repressed in COL; however, no repression of these proteins was observed in 8325 (Figs 3 and 4). In strain 8325, spots for two hypothetical proteins (SA1528 and SA1529) were present at reduced intensity after growth of the culture in the presence of TX-100. Two other proteins detected in strain 8325 when it was grown without TX-100 were absent from 8325 when it was grown in the presence of TX-100 – these were a protein with similarity to ferritin and a second isoform of alcohol dehydrogenase. Two protein spots were induced in 8325 when it was grown in the presence of TX-100 [protein-export protein PrsA and 3-oxoacyl-(acyl carrier protein) reductase]. The second of these was the only protein spot to show a statistically significant opposite effect between the two strains.



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Fig. 6. Changes in the production of IsaA and an isoform of fructose-bisphosphate aldolase under the different growth conditions. Panels (a–d) are as described in the legend to Fig. 2.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Protein identification and strain comparison
Proteins from phenotypically different strains of S. aureus were separated over two IPG two-dimensional gels covering the range pH 4–11. An effective separation area of 35 cm in width was utilized in the first dimension with a single overlapping pH unit present between pH 6 and 7. Fluorescence-colloidal Coomassie blue double-staining allowed 377 protein spots to be excised and characterized by MS. Only 11 spots (2·9% of the total number of spots) could not be identified. These unidentified spots were either of very low mass, and thus generated few tryptic peptides within the mass range covered by MALDI/TOF MS, or were poorly abundant spots contaminated with human keratins, and thus had poor peptide signal to noise ratios. The 377 two-dimensional gel-purified spots corresponded to 266 S. aureus ORFs. Thirty-six of these ORFs were expressed as two or more spots. Previously, we have separated approximately 70% of the S. aureus proteome by using narrow-range (single pH unit) two-dimensional gels to enrich for lower abundance proteins that are usually beneath the visible dynamic range of protein abundance (Cordwell et al., 2000 ). However, a number of putative regulatory proteins (including Rot, RsbW, RsbV, pyrimidine operon repressor, SarA, CodY, LexA, Fur homologue, and conserved hypotheticals resembling response regulators, SA1080, SA1700, SA0017 and SA0641) that are often amongst the proteins present in the lowest abundance in bacteria were identified here. Proteins of the staphylococcal cell envelope were poorly represented; hence, techniques for the isolation of staphylococcal membrane fractions suitable for 2-DGE are currently being pursued. Proteins from culture supernatants were not investigated; however, at least 100–200 secreted proteins have been identified previously (Ziebandt et al., 2001 ). Taking into account the cellular proteins identified in this study, it appears that at least 20–25% of the theoretical S. aureus proteome has now been defined.

2-DGE patterns of cellular proteins from S. aureus strains 8325 and COL were highly conserved. This differs from cellular proteins of the gut pathogen Helicobacter pylori, where very few spots migrate identically across strains (Jungblut et al., 2000 ). Only 23 protein spots (12 in strain COL and 11 in strain 8325) were present at significantly altered levels in either strain. Sequence comparisons for each of these proteins revealed that they were identical, suggesting that changes in spot position caused by sequence-based charge variations did not occur. However, since neither the 8325 nor the COL genome has been finalized and published, we cannot rule out the possibility that such variations occur for those proteins that were apparently entirely absent from one strain alone. Several proteins present at greater levels in the COL strain were products of genes either regulated by {sigma}B (e.g. Asp23 and conserved hypothetical protein SA0772) or part of the sigB operon (RsbV). Strain 8325 and its derivatives resemble {Delta}sigB mutants due to an 11 bp deletion in the rsbU gene (Kullik & Giachino, 1997 ; Kullik et al., 1998 ). RsbU is a positive regulator of {sigma}B and is necessary for the activation of this alternate sigma factor following environmental stress in B. subtilis (Völker et al., 1995 ). RsbU dephosphorylates RsbV, thus allowing RsbV to bind RsbW. RsbV is an acidic protein with a pI value of 4·4; in a phosphorylated form it would have a pI value of approximately 4·0 and might not be detectable in the protein pattern of strain 8325 due to the pH range (4–11) investigated in this study. Without RsbU, strain 8325 produces only trace amounts of {sigma}B and, therefore, has impaired stress tolerance (Giachino et al., 2001 ). asp23 is exclusively regulated by {sigma}B and is absent from SDS-PAGE gels of strain 8325 and its derivatives (Gertz et al., 1999 ). Using a higher resolution 2-DGE approach we were able to detect trace levels of Asp23 in strain 8325; however, significantly greater levels of this protein were detected in strain COL. SA0772 was described as part of the {sigma}B regulon (Csb8) by Gertz et al. (2000) . In this study, 18 {sigma}B-regulated proteins were characterized by N-terminal sequencing. We used these sequences to probe the N315 database for potential homologues (data not shown), and 15 sequences corresponding to ORFs encoded by the S. aureus genome were detected. Six of these 15 proteins were identified in our study (hypothetical proteins SA0372, SA0528, SA0772, SA0774, SA1671 and SarA); however, only SA0772 was found at a significantly higher level in the COL strain. The association of three cold-shock proteins (CspABC) with COL may also suggest that the cold-shock response is {sigma}B-dependent in S. aureus, as has been suggested for Listeria monocytogenes (Becker et al., 2000 ).

TX-100-associated protein production
The non-ionic detergent TX-100 reduces levels of methicillin resistance in all strains of S. aureus when added to cultures at subinhibitory concentrations, with the greatest reductions in resistance seen in previously highly resistant strains (Komatsuzawa et al., 1994 , 1995 ; Suzuki et al., 1997 ). We investigated the effects of this detergent on the proteome of two S. aureus strains, COL (methicillin-resistant) and 8325 (methicillin-sensitive). The essential methicillin-resistance factor FemA appeared as a series of three pI variants under all conditions; however, no change in femA expression was detected in either strain when grown in the presence of TX-100, in accordance with previous results (Komatsuzawa et al., 1994 ; Suzuki et al., 1997 ). The products of the mecA, fmtAB and llm genes were not detected. The production of 44 proteins (as detected by the intensity of their respective spots) was up- or down-regulated when the strains were grown in the presence of TX-100. These 44 proteins included proteins involved in regulatory functions (repressor of toxins, pyrimidine operon repressor, RsbVW, SarA and SA2089), cell division (FtsZ and stage V sporulation protein G) and cell-wall biosynthesis (alanine dehydrogenase and GlmM). Several proteins of the {sigma}B regulon, including the product of sarA, were altered in their spot patterns when the strains were grown in the presence of TX-100, suggesting that these global regulators may be involved in the optimal expression of methicillin resistance (Wu et al., 1996b ; De Lencastre et al., 1999 ).

The {sigma}B regulon in Gram-positive bacteria includes proteins associated with the general stress response (Hecker et al., 1996 ; Hecker & Völker, 2000 ). In B. subtilis, {sigma}B is regulated by rsb genes in the following way. During exponential growth, the anti-{sigma} factor RsbW binds to the RNA-polymerase-binding site of {sigma}B and inactivates it (Benson & Haldenwang, 1993 ). {sigma}B is then activated by primary signals which allow anti-anti-{sigma} factor RsbV to bind RsbW (Dufour & Haldenwang, 1994 ) and release functional {sigma}B that in turn regulates the production of over 100 different proteins (Petersohn et al., 2001 ; Price et al., 2001 ). The majority of this regulation occurs post-transcriptionally and involves the phosphorylation (inactive form) and dephosphorylation (active form) of RsbV. RsbU phosphatase is required for {sigma}B activation post-heat-shock, but is not required during stationary phase or nutrient limitation (Völker et al., 1995 ). S. aureus {Delta}sigB mutants show a significant decline in methicillin resistance (Wu et al., 1996b ), and recent reports suggest that overexpression of sigB leads to hyper-resistance (Morikawa et al., 2001 ). Asp23 has been proposed as a model protein for tracking {sigma}B activity (Giachino et al., 2001 ). After growth of strain COL in the presence of TX-100, we noted a significant reduction in asp23 expression in this strain, suggesting that TX-100 treatment resulted in lower {sigma}B activity. Of the six {sigma}B-dependent proteins previously characterized in S. aureus (Gertz et al., 2000 ) and identified in this study, five showed either no difference in the presence of TX-100 or were down-regulated. This was in accordance with our hypothesis that less functional {sigma}B was present. The production of the sixth protein, SarA, was significantly up-regulated following growth in the presence of TX-100. These results are in agreement with those of Cheung et al. (1999) who found that SarA levels increased in sigB mutants, and are in conflict with others who have shown that the loss of functional {sigma}B results in reduced sarA expression (Deora et al., 1997 ; Manna et al., 1998 ; Gertz et al., 2000 ; Bischoff et al., 2001 ). The increase in SarA abundance was more pronounced in strain 8325 when it was grown in the presence of TX-100, although similar levels of this protein were seen in both strains when grown without TX-100. {sigma}B is only one of several potential sarA regulators (e.g. sarR repressor; Manna & Cheung, 2001 ) and the exact role of {sigma}B in sarA expression remains to be elucidated.

Significantly lower levels of RsbW and RsbV were detected in the protein-spot patterns of the strains grown in the presence of TX-100, suggesting that TX-100 influences {sigma}B activity by reducing sigB expression. The rsbVW genes (as well as rsbX) are co-transcribed with sigB in B. subtilis. Previous reports have suggested that RsbW and {sigma}B are present in equivalent amounts, at least in B. subtilis (Benson & Haldenwang, 1993 ); however, we could not identify {sigma}B itself in this study. RsbX may negatively regulate expression of the sigB operon (Benson & Haldenwang, 1993 ) and increased activity of {sigma}B occurs in rsbX mutants (Völker et al., 1995 ); however, no rsbX homologue has been found in S. aureus nor has a putative sigB operon repressor been determined. The regulation of sigB in S. aureus is certainly different to the regulation of sigB in B. subtilis, since {sigma}B-regulated genes are constitutively expressed rather than solely induced upon environmental stress (Gertz et al., 1999 ). Recent work (Palma & Cheung, 2001 ) has also suggested that {sigma}B is regulated by RsbU-independent mechanisms in S. aureus.

The staphylococcal accessory regulator SarA influences the production of cell-surface and extracellular proteins. Both sar and agr mutants show increased susceptibility to ß-lactam antibiotics (Píriz Durán et al., 1996 ; Fujimoto & Bayles, 1998 ). Recent global analyses of the sarA regulon have been conducted using DNA micro-arrays (Dunman et al., 2001 ) and proteomics (Ziebandt et al., 2001 ) and suggest that over 100 genes are controlled by SarA. We found that growth of the S. aureus strains in the presence of TX-100 resulted in increased expression of sarA and the appearance of a further two SarA variants. The presence of a 28 kDa spot identified as SarA provides further evidence that this protein is present as a dimer in vivo (Rechtin et al., 1999 ). The increase in SarA levels, coupled with a significant reduction in the level of Rot present (repressor of toxins; McNamara et al., 2000 ), suggests that TX-100 not only reduces antibiotic resistance, but that this reduction in resistance may also be coupled to an increase in extracellular toxin production.

A cluster of four spots of approximately 25 kDa and identified as immunodominant antigen A (IsaA; Lorenz et al., 2000 ) was induced in COL when it was grown in the presence of TX-100. Only a single spot corresponding to IsaA was detected in strain 8325. IsaA encodes a protein of unknown function; however, it contains a signal sequence and was previously identified in culture supernatants of S. aureus COL (Ziebandt et al., 2001 ). In sarA mutants, two significant spots corresponding to IsaA were absent from COL extracellular fractions, indicating that this protein is positively regulated by SarA. Intriguingly, and not discussed by Ziebandt et al. (2001) , IsaA appears as only a single spot in COL {Delta}sigB, suggesting that {sigma}B is involved in pathways that post-translationally modify IsaA. We are currently attempting to characterize potential modifications in IsaA. The increase in production of variants of IsaA is consistent with the induction of SarA production upon treatment of cultures with TX-100; however, IsaA has previously been detected as a secreted protein and we are unable to explain its intracellular dominance. One hypothesis is that TX-100 modifies COL-specific secretory mechanisms for the translocation of this protein, or that SarA-induced extracellular serine proteases (Karlsson et al., 2001 ) increase IsaA turnover. We also found enhanced intracellular levels of secretory antigen precursor SsaA, which has previously been shown to be negatively regulated by both SarA (Ziebandt et al., 2001 ) and Agr (Dunman et al., 2001 ).

Cellular effects of TX-100
TX-100 induces the release of acylated LTA from the surface of S. aureus cells (Komatsuzawa et al., 1994 ). LTA may be involved in the regulation of peptidoglycan hydrolases, enzymes (autolysins) that break down the cell wall during cell division (Höltje & Tomasz, 1975 ). When LTA is released from the cell surface by TX-100, the production of these hydrolases is no longer tightly regulated and autolysis is enhanced. Deletion of at least one gene encoding a lytic enzyme leads to hyper-methicillin resistance (Fujimura & Murakami, 1997 ); however, autolytic enzyme mutants of S. aureus also show increased susceptibility to ß-lactams in the presence of TX-100 (Komatsuzawa et al., 1994 ). sarA mutants show increased levels of autolysis (Fujimoto & Bayles, 1998 ), while recent reports have shown that the expression of atl (autolysin) is repressed under sarA (Dunman et al., 2001 ). SarA also positively regulates a cluster of genes (SA0244, SA1103 and SA0523) involved in LTA biosynthesis (Dunman et al., 2001 ). We hypothesize that TX-100 induces the release of LTA and hence the activation of autolysis, as previously shown. The cell may then respond to this signal by increasing expression of sarA, which in turn inhibits the synthesis of further autolysin and increases the production of LTA biosynthetic enzymes. Furthermore, recent data suggest that {sigma}B may control the production of a murein hydrolase post-transcriptional regulatory protein in B. subtilis (Price et al., 2001 ). This protein shares sequence similarity to the S. aureus LrgAB regulator of murein hydrolase (Groicher et al., 2000 ). Loss of LrgAB reduces ß-lactam resistance. TX-100 reduces {sigma}B activity while altering the phenotype of methicillin resistance, perhaps partly due to secondary effects on regulators such as LrgAB.

A series of genes involved in peptidoglycan and cell-wall biosynthesis have been implicated in S. aureus methicillin resistance (Berger-Bächi et al., 1992 ). One of these genes is glmM, which encodes the phosphoglucosamine mutase that catalyses the reaction glucosamine 6-phosphate to glucosamine 1-phosphate during the initial stages of peptidoglycan biosynthesis (Wu et al., 1996a ; Jolly et al., 1997 ; Glanzmann et al., 1999 ). We found that the abundance of GlmM was reduced by more than threefold in COL following its growth in the presence of TX-100, but that its level remained constant in 8325. The production of a second protein involved in peptidoglycan biosynthesis (alanine dehydrogenase, Ald) was dramatically repressed in both strains following their growth in the presence of TX-100. This study is the first to suggest that antibiotic resistance may be linked to L-alanine biosynthesis as a precursor for peptidoglycan formation. Neither ald nor glmM appear to contain a {sigma}B-dependent promoter (Gertz et al., 2000 ); however, {sigma}B has been shown to control the production of at least 14 regulatory proteins in B. subtilis (Price et al., 2001 ). A similarly large complement in S. aureus may contain regulators that control the expression of ald or the glmM operon, which also contains two hypothetical proteins and is part of a co-transcribed gene cluster containing fmtB (Wu & de Lencastre, 1999 ; Komatsuzawa et al., 2000 ). Recent reports have suggested that overexpression of {sigma}B results in increased cell-wall thickness and hyper-resistance to ß-lactams (Morikawa et al., 2001 ), providing further evidence that {sigma}B plays a significant role in cell-wall biosynthesis and antibiotic resistance in S. aureus.

TX-100 clearly reduces methicillin resistance using a complex regulatory network. It appears that this relies on a cellular signal (release of LTA), which in turn triggers a response involving two global regulatory proteins, {sigma}B and SarA, and a cascade of secondary events that leads to altered expression of genes associated with cell-wall biosynthesis, cell division and energy metabolism. We have presented the first global analysis of phenotypically different S. aureus strains and have characterized a significant percentage of their proteomes. This work has provided a unique tool for analysing the effects of genetic and environmental challenges on gene expression in S. aureus, such as the response of this organism to subinhibitory concentrations of TX-100. The results from this analysis provide further evidence of the significance of regulatory proteins in the expression of antibiotic resistance and potential mechanisms for combating this increasing medical problem.


   ACKNOWLEDGEMENTS
 
This work was facilitated by access to the Australian Proteome Analysis Facility (APAF), established in 1995 under the Australian Government Major National Research Facility (MNRF) programme. M.R.L. is supported by a Danish Natural Science Research Council Post-Doctoral Fellowship. S.J.C. and B.J.W. wish to acknowledge Bio-Rad and Micromass for their support.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 11 February 2002; revised 5 May 2002; accepted 16 May 2002.