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
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ABSTRACT |
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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).
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INTRODUCTION |
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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 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
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
).
B has also been suggested as a regulator of staphylococcal virulence factors (Kullik et al., 1998
). Recent reports have shown that
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
B-regulated stress-response genes and their proteins may be involved in antibiotic resistance (Wu et al., 1996b
). Conflicting studies have reported that
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
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.
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METHODS |
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2-DGE.
2-DGE sample buffer [5 M urea, 2 M thiourea, 0·1% carrier ampholytes, 2% (w/v) CHAPS, 2% (w/v) sulfobetaine 310, 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 47 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 818% 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 2030 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
-cyano-4-hydroxycinnamic acid ml-1; Sigma). For electrospray-ionization tandem MS/MS peptide sequencing, peptides were eluted in 12 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 9001200 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 1830 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).
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RESULTS |
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DISCUSSION |
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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
B (e.g. Asp23 and conserved hypothetical protein SA0772) or part of the sigB operon (RsbV). Strain 8325 and its derivatives resemble
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
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 (411) investigated in this study. Without RsbU, strain 8325 produces only trace amounts of
B and, therefore, has impaired stress tolerance (Giachino et al., 2001
). asp23 is exclusively regulated by
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
B regulon (Csb8) by Gertz et al. (2000)
. In this study, 18
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
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
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 B regulon in Gram-positive bacteria includes proteins associated with the general stress response (Hecker et al., 1996
; Hecker & Völker, 2000
). In B. subtilis,
B is regulated by rsb genes in the following way. During exponential growth, the anti-
factor RsbW binds to the RNA-polymerase-binding site of
B and inactivates it (Benson & Haldenwang, 1993
).
B is then activated by primary signals which allow anti-anti-
factor RsbV to bind RsbW (Dufour & Haldenwang, 1994
) and release functional
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
B activation post-heat-shock, but is not required during stationary phase or nutrient limitation (Völker et al., 1995
). S. aureus
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
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
B activity. Of the six
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
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
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.
B is only one of several potential sarA regulators (e.g. sarR repressor; Manna & Cheung, 2001
) and the exact role of
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 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
B are present in equivalent amounts, at least in B. subtilis (Benson & Haldenwang, 1993
); however, we could not identify
B itself in this study. RsbX may negatively regulate expression of the sigB operon (Benson & Haldenwang, 1993
) and increased activity of
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
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
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
sigB, suggesting that
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
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
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
B-dependent promoter (Gertz et al., 2000
); however,
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
B results in increased cell-wall thickness and hyper-resistance to ß-lactams (Morikawa et al., 2001
), providing further evidence that
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, 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.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 11 February 2002;
revised 5 May 2002;
accepted 16 May 2002.