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Cell wall-active antibiotic induced proteins of Staphylococcus aureus identified using a proteomic approach1
FEMS Microbiology Letters 199 (2001) 79^84
www.fems-microbiology.org
Cell wall-active antibiotic induced proteins of Staphylococcus aureus identi¢ed using a proteomic approach1 Vineet K. Singh, R.K. Jayaswal, Brian J. Wilkinson * Microbiology Group, Department of Biological Sciences, Illinois State University, Normal, IL 61790-4120, USA Received 5 March 2001; received in revised form 22 March 2001; accepted 23 March 2001 First published online 17 April 2001
Abstract Proteins produced in elevated amounts in response to oxacillin challenge of Staphylococcus aureus strain RN450, were studied by comparing Coomassie blue stained two-dimensional gels of cellular proteins. At least nine proteins were produced in elevated amounts following exposure to growth inhibitory concentrations of oxacillin. N-terminal sequences were obtained for five of the proteins and the databases were searched to tentatively identify them. The proteins were identified as homologs of (i) methionine sulfoxide reductase (MsrA) ; (ii) a signal transduction protein (TRAP) involved in regulating RNAIII production encoded by the agr locus; (iii) transcription elongation factor GreA; (iv) the heat shock protein GroES; and (v) the enzyme IIA component of the phosphoenolpyruvate:sugar phosphotransferase system. A similar induction response was observed with the other cell wall-active antibiotics, but not with antibiotics that affect other cellular targets. Increased transcription of the msrA and groEL genes in response to cell wall-active antibiotics was also demonstrated. Although net protein synthesis is inhibited subsequent to inhibition of peptidoglycan biosynthesis by cell wall-active antibiotics, some proteins are induced in S. aureus, presumably in an attempt by the cell to counter the inhibitory effects of these agents. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Cell wall-active antibiotics; Stress protein; Proteomics; Staphylococcus aureus
1. Introduction It is imperative that serious Staphylococcus aureus diseases such as bacteremia and endocarditis are treated aggressively with e¡ective antimicrobial agents. However, antibiotic resistance continues to increase in S. aureus and methicillin-resistant S. aureus (MRSA) that are uniformly resistant to L-lactam antibiotics are widespread worldwide. A critical determinant of methicillin resistance is the mecA gene that encodes low a¤nity penicillin-binding protein 2a [1]. Transposon mutagenesis has identi¢ed
* Corresponding author. Tel. : +1 (309) 438-7244; Fax: +1 (309) 438-3722; E-mail: [email protected] 1
This paper is dedicated to the memory of Gerald D. Shockman, a prominent member of an earlier generation of cell wall biochemists.
various chromosomal genes, fems or aux genes, that are required for the optimum expression of methicillin resistance [2,3], several of which are involved in peptidoglycan biosynthesis. However, a connection was forced between stress biology and methicillin resistance expression by the report of Wu et al. [4] which provided evidence that an alternative sigma factor, SigB, is involved in the expression of methicillin resistance in S. aureus. Transposon inactivation of the sigB operon in homogeneous MRSA COL drastically reduced resistance to methicillin. These ¢ndings led to the hypothesis that stress gene induction was a necessary part of the response to challenge by cell wall-active antibiotics in S. aureus. Accordingly, a proteomic approach was used to identify proteins produced in response to cell wall-active antibiotic treatment of mid-exponential phase cultures of a methicillinsusceptible strain. At least nine proteins were found to be produced in elevated amounts, and ¢ve of these proteins were identi¢ed by N-terminal sequencing. The produced proteins may constitute a proteomic signature for cell wall-active antibiotics, representing a stimulon or regulon triggered by these agents [5].
0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 1 6 3 - X
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2. Materials and methods 2.1. Growth conditions S. aureus and Escherichia coli cells were grown in tryptic soy broth (Difco Laboratories, Detroit, MI, USA) and Luria^Bertani broth, respectively. When needed, ampicillin (50 Wg ml31 ) and kanamycin (30 Wg ml31 in the case of E. coli, and 100 Wg ml31 in the case of S. aureus) were added to the growth medium. 2.2. DNA manipulations Plasmid and chromosomal DNA isolation, DNA manipulations, digestion of DNA with restriction enzymes, DNA ligations and Southern blotting were carried out as described [6]. PCR was performed with the GeneAmp PCR system (Perkin-Elmer, Foster City, CA, USA). 2.3. Antibiotic stress and two-dimensional gel electrophoresis An overnight culture of S. aureus RN450 was used to inoculate 50 ml of tryptic soy broth in a 300 ml Erlenmeyer £ask, which was incubated with shaking (250 rpm) at 37³C. At turbidity (A600 ) of 0.3, 10 ml of culture was distributed to two sterile test tubes and to one of the cultures oxacillin was added to a concentration of 1.2 Wg ml31 and incubated for an additional 2.5 h. The control culture was incubated for the same time period without oxacillin. Approximately equal amounts of the biomass from the control and oxacillin treated culture adjusted via A600 determinations, were collected by centrifugation (10 000Ug, 10 min, 4³C) and washed once with 50 mM Tris^HCl bu¡er, pH 7.5 containing 0.145 M NaCl. Cells were lysed by using lysostaphin and cellular proteins were prepared and examined by two-dimensional gel electrophoresis essentially as described [7]. Following electrophoresis the gels were stained with Coomassie brilliant blue R250. Experiments were repeated at least three times to con¢rm the reproducibility of the 2-D gels. The proteins induced were designated as cell wall-active antibiotic induced proteins. Additional antibiotics with varying cell targets were also included in these studies. They were used at concentrations that inhibited the growth of S. aureus RN450 to a degree comparable to 1.2 Wg ml31 of oxacillin. To identify the cell wall-active antibiotic induced proteins, the second dimension gels were directly transferred to Sequi-Blot PVDF membranes (Bio-Rad) using Tris^ CAPS (12 mM Tris and 8 mM CAPS) bu¡er, pH 9.6 and N-terminus sequences for ¢ve of these were obtained at the Protein Science Facility of the University of Illinois, Urbana, IL, USA. Sequences were not obtained for the other four proteins as either the N-terminus amino acid was blocked (spot 8), the amount was not enough to yield
a sequence (spots 2 and 3), or the spot did not yield a pure protein (spot 9). The N-terminus sequence data were used to identify the open reading frames encoding the proteins from the S. aureus genome database of the University of Oklahoma's Advanced Center for Genome Technology (www.genome.ou.edu) using the BLAST program [8]. The identi¢ed ORFs were translated and the primary amino acid sequences of the putative proteins were subsequently searched for similarities in the protein databases. 2.4. Construction of an msrA promoter: :lacZ reporter strain A reporter strain was constructed using the promoter region of the strongly inducible gene msrA. Primers P1 (5P-AAACCACTACCGAATCGTCG-3P) and P2 (5P-CACCAGAAACATCCTCCTGC-3P), and S. aureus RN450 genomic DNA were used to PCR amplify an approximately 1.3 kb fragment starting 44 nt downstream of the msrA start codon and going upstream. The PCR product was cloned in the correct orientation upstream of a promoterless lacZ gene of the shuttle vector pAZ106 [9] and was introduced into the chromosome of S. aureus strain RN4220 by electroporation with selection on erythromycin. Phage 80K lysate of the resulting transformants was used to transduce the msrA promoter: :lacZ into strain S. aureus RN450. A single copy insertion of the fusion in the chromosome was con¢rmed by Southern blot analysis using the msrA promoter region as the probe (result not shown). The activity of L-galactosidase was assayed after exposing the reporter strain to di¡erent antibiotics as described above using o-nitrophenyl-L-D-galactopyranoside (ONPG) as the substrate [10]. 3. Results and discussion 3.1. Cell wall-active antibiotic induced proteins During the ¢rst 30 min of incubation the growth was indistinguishable in antibiotic treated and untreated cultures (A600 0.3^0.46). Subsequently, the growth was virtually arrested in the antibiotic treated culture, but the control culture grew exponentially (A600 to 1.4) during 150 min of incubation. The Coomassie blue stained gels of proteins from control and oxacillin treated cultures of S. aureus RN450 are shown in Fig. 1. At least nine proteins were produced in elevated amounts after 2.5 h of exposure to oxacillin stress. 3.2. N-terminal sequencing and identity of cell wall-active antibiotic induced proteins The nine cell wall-active antibiotic induced proteins were submitted for N-terminal sequencing and sequences were obtained for ¢ve of the proteins. The identities of
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Fig. 1. 2-D gels of proteins of S. aureus RN450 cultures grown (A) without and (B) with oxacillin. The proteins visibly induced are encircled in the gel of proteins from the oxacillin-stressed cells. The circles in the gels of the cells grown without oxacillin show the proteins that are reduced in amount on addition of oxacillin. The values on the right indicate the molecular mass of the reference proteins.
antibiotics with other targets had no appreciable e¡ect on msrA transcription (Table 2). MsrA is an enzyme that catalyzes the reduction of methionine sulfoxides in proteins to methionine [11]. An E. coli msrA mutant was more susceptible to the oxidative stress provided by H2 O2 compared to the parent strain [12]. However, little is known about the regulation of msrA. The ¢nding of the induced synthesis of the S. aureus MsrA homologue by cell wall-active antibiotics is novel and the mechanism is not understood. Interestingly, msrA was identi¢ed as an S. aureus viru-
these proteins based on homology searches are shown in Table 1. 1. Spot 1. The amino acid sequence of the deduced protein of an open reading frame identi¢ed based on the N-terminus sequence of this spot showed strong homology to a putative Bacillus subtilis polypeptide, methionine sulfoxide reductase, MsrA (60% identity; 74% similarity). In addition, the L-galactosidase activity of the PmsrA : :lacZ reporter strain was signi¢cantly increased upon exposure to cell wall-active antibiotics whereas
Table 1 Sequence similarities of the staphylococcal proteins induced in response to oxacillin treatment Position on the gel
N-terminus sequence
Amino acids (aa)a
Identity (%)b
No. of aa that overlap
Similar protein (no of aa)c
Spot 1
MTKEYATLAG
177
60
171
Spot 4
MKKLYTSYGT
167
Spot 5
TGEFVKIEDI
166
46
165
Spot 6
MENQKQYPMT
158
67
157
Spot 7
MLKPIGNRVI
B. subtilis putative methionine sulfoxide reductase (177) (G7446682) A signal transduction protein (TRAP) of S. aureus (G11192009) Enzyme IIA of the phosphotransferase system of E. coli (169) (G12516793) GREA (B. subtilis putative transcription cleavage factor) (157) (G7449152) S. aureus GroES (94) (G421389)
94
a
Indicates the total number of amino acids in the protein encoded by the S. aureus open reading frame identi¢ed based on the N-terminus sequence. Identity of the predicted primary sequence of the product of the S. aureus ORF to known proteins. c Numbers in the ¢rst parentheses indicate the number of amino acids in the protein to which S. aureus putative protein showed signi¢cant sequence homology and the number in the second parentheses is the GenBank PID numbers for these proteins. b
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Table 2 L-Galactosidase activity of the PmsrA: :lacZ fusion strain in response to antibiotics Sample
L-Galactosidase activity (Miller units)
Control Rifamycin Gentamicin D-cycloserine Cephalothin Oxacillin
1h
2h
9.2 þ 1.4 12.4 þ 2.2 12.1 þ 2.3 42.4 þ 6.8 90.0 þ 8.8 92.4 þ 10.6
18.0 þ 2.6 5.2 þ 1.2 8.2 þ 1.2 156.2 þ 12.4 144.4 þ 11.6 178.2 þ 15.6
Rifamycin (0.45 Wg ml31 ), gentamicin (60 Wg ml31 ), D-cycloserine (150 Wg ml31 ), cephalothin (2 Wg ml31 ), and oxacillin (1.2 Wg ml31 ) were added at A600 of 0.3. In the control samples no antibiotic was added. The experiments were carried out in triplicate. Values shown are the averages of triplicate determinations þ standard errors.
lence gene in a murine model of bacteremia using signature tagged mutagenesis [13]. 2. Spot 4. The database search identi¢ed this protein as a 21 kDa RNAIII-activating protein (TRAP) [14]. The production of staphylococcal toxins is autoinduced by RNAIII-activating protein (RAP) and by the autoinducing peptide (AIP) but is inhibited by RNAIII-inhibiting peptide (RIP). RAP phosphorylates TRAP, whereas RIP inhibits the phosphorylation of this 21 kDa protein which is unique to S. aureus [14]. A knockout mutant of the trap gene was constructed in the present study by inserting a kanamycin resistance cassette in the EcoRI site of the gene. The mutation was subsequently transferred to oxacillin-resistant S. aureus
strain COL, but the mutation did not a¡ect the expression of resistance to cell wall-active antibiotics (data not shown). Interestingly, the synthesis of the virulence regulatory molecule RNAIII was not activated by RAP in a trap mutant strain providing evidence that the RAP activates RNAIII synthesis via TRAP [14]. The authors have concluded that the trap and the agr signal transduction systems interact with one another to coordinate the production of virulence factors. Although, a TRAP deletion mutant did not show an altered response to cell wall-active antibiotics, its elevated synthesis in response to these antibiotics might be an indication of a more extensive role for this protein in signal transduction. 3. Spot 5. The 10 amino acids sequenced from the N-terminus of this protein matched with the amino acids 23^ 32 in the predicted primary sequence of the product of a potential S. aureus open reading frame. This protein showed homology to the enzyme IIA component of the phosphoenolpyruvate-dependent sugar phosphotransferase system [15]. Possibly the ¢rst 22 amino acids of this protein were cleaved o¡ by cellular proteolytic activity. 4. Spot 6. The deduced protein of the open reading frame identi¢ed by N-terminus sequencing of this spot showed high homology to a transcription elongation factor GreA (transcript cleavage factor) of E. coli [16]. The transcript cleavage factors GreA and GreB allow RNA polymerases to overcome obstacles encountered during elongation in vivo, such as pausing and
Fig. 2. 2-D gels of proteins of S. aureus RN450 cultures grown with 1.2 Wg ml31 vancomycin (A) and 150 Wg ml31
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arresting sites in the DNA sequences, nucleosomes, or DNA-binding proteins and drugs [17]. 5. Spot 7. This protein was identi¢ed as Hsp-10 (GroES) which is known to be induced by heat shock and to be present in an operon in S. aureus with GroEL [18,19]. There was uncertainty about the location of GroEL on the 2-D gel and whether it was being induced in response to cell wall-active antibiotics. Therefore, total RNA was isolated from control and oxacillin treated cultures, separated by agarose gel electrophoresis and probed with the PCR ampli¢ed groEL fragment based on the published DNA sequence (accession no. D14711) [18]. The transcription of groEL was enhanced (data not shown) in response to oxacillin, suggesting that induction of these heat shock genes is an integral part of the cell wall-active antibiotic stress response.
may represent a proteomic signature. Proteomic signatures are sets of proteins that respond speci¢cally and uniquely to particular states of the cell such as energy su¤ciency, transcriptional capacity, or envelope integrity [5]. In S. aureus presumably the cell is sensing that it is damaged and certain proteins are being produced to help rescue the cell from the potentially lethal e¡ects of cell wall-active antibiotics. However, it is possible that overexpression of certain proteins could ultimately prove to be damaging to the cell [22]. Further studies on gene expression in response to cell wall-active antibiotics, including the utilization of gene array hybridization technology, are clearly warranted. Preliminary S. aureus GeneChip studies have con¢rmed increased transcription of genes encoding MsrA, TRAP, GroES, and Enzyme IIA of the phosphotransferase system (unpublished observations).
3.3. Proteins induced in response to other cell wall-active antibiotics and antibiotics inhibiting other cellular processes
Acknowledgements
The protein response of strain RN450 to growth inhibitory concentrations of other antibiotics was determined. The cell wall-active antibiotics vancomycin (Fig. 2), D-cycloserine (Fig. 2), cephalothin (data not shown) and bacitracin (data not shown) induced the production of a similar set of proteins to that induced by oxacillin. These proteins were not induced by the RNA polymerase inhibitor rifamycin, or the protein synthesis inhibitors gentamicin and erythromycin (data not shown). 3.4. Events occurring subsequent to inhibition of growth by cell wall-active antibiotics This work provides new insights into cellular events occurring subsequent to treatment with cell wall-active antibiotics. Peptidoglycan synthesis is inhibited which is presumably responsible for the inhibition of cell growth. Subsequently, various secondary responses are possible: cells may lyse, cells may die and lyse, cells may die without lysis, or growth may be inhibited but the cells survive (tolerance) [20]. Mychajlonka et al. [20] proposed that inhibition of peptidoglycan assembly activates a global regulatory circuit that `talks back' to cytoplasmic processes. In antibiotic-tolerant Streptococcus mutans this is manifested as a dose-dependent inhibition of peptidoglycan, RNA and protein synthesis by penicillin. Thus, although the gross synthesis of RNA and protein is inhibited subsequent to inhibition of peptidoglycan synthesis, certain proteins are synthesized in elevated amounts, i.e. some genes are being selectively expressed. In an earlier study Jablonski and Mychajlonka [21] showed using one-dimensional gel electrophoresis, alterations in S. aureus protein synthesis in response to oxacillin. The set of proteins that was observed to be produced in response to the various cell wall-active antibiotics studied
We are grateful to Sarah M. Scybert for the L-galactosidase assays in the lacZ reporter strain. This work has been supported by grant AI43027-01 from the National Institutes of Health, a grant-in-aid from the American Heart Association ^ Midwest A¤liate and a postdoctoral fellowship from American Heart Association ^ Midwest A¤liate to V.K.S. References [1] Matsuhashi, M., Song, M.D., Ishino, F., Wachi, M., Doi, M., Inoue, M., Ubukata, K., Yamashita, N. and Konno, M. (1986) Molecular cloning of the gene of a penicillin-binding protein supposed to cause high resistance to beta-lactam antibiotics in Staphylococcus aureus. J. Bacteriol. 167, 975^980. [2] Berger-Ba«chi, B. (1983) Insertional inactivation of staphylococcal methicillin resistance by Tn551. J. Bacteriol. 154, 479^487. [3] De Lencastre, H., Wu, S.W., Pinho, M.G., Ludovice, A.M., Filipe, S., Gardete, S., Sobral, R., Gill, S., Chung, M. and Tomasz, A. (1999) Antibiotic resistance as a stress response : complete sequencing of a large number of chromosomal loci in Staphylococcus aureus strain COL that impact on the expression of resistance to methicillin. Microb. Drug Resist. 5, 163^175. [4] Wu, S., Lencastre, H.D. and Tomasz, A. (1996) Sigma-B, a putative operon encoding alternative sigma factor of Staphylococcus aureus RNA polymerase : Molecular cloning and cDNA sequencing. J. Bacteriol. 178, 6036^6042. [5] VanBogelen, R.A., Schiller, E.E., Thomas, J.D. and Neidhardt, F.C. (1999) Diagnosis of cellular states of microbial organisms using proteomics. Electrophoresis 20, 2149^2159. [6] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [7] Vijaranakul, U., Nadakavukaren, M.J., Bayles, D.O., Wilkinson, B.J. and Jayaswal, R.K. (1997) Characterization of an NaCl-sensitive Staphylococcus aureus mutant and rescue of the NaCl-sensitive phenotype by glycine betaine but not other compatible solutes. Appl. Environ. Microbiol. 63, 1889^1897. [8] Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J. (1990) Basic local alignment search tool. J. Mol. Biol. 215, 403^410.
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[9] Chan, P.F., Foster, S.J., Ingham, E. and Clements, M.O. (1998) The Staphylococcus aureus alternative sigma factor sigma B controls the environmental stress response but not starvation survival or pathogenicity in a mouse abscess model. J. Bacteriol. 180, 6082^6089. [10] Miller, J.M. (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [11] Brot, N., Weissbach, L., Werth, J. and Weissbach, H. (1981) Enzymatic reduction of protein-bound methionine sulfoxide. Proc. Natl. Acad. Sci. USA 78, 2155^2158. [12] Moskovitz, J., Rahman, M.A., Strassman, J., Yancey, S.O., Kushner, S.R., Brot, N. and Weissbach, H. (1995) Escherichia coli peptide methionine sulfoxide reductase gene: regulation of expression and role in protecting against oxidative damage. J. Bacteriol. 177, 502^ 507. [13] Mei, J.M., Nourbakhsh, F., Ford, C.W. and Holden, D.W.F. (1997) Identi¢cation of Staphylococcus aureus virulence genes in a murine model of bacteremia using signature-tagged mutagenesis. Mol. Microbiol. 26, 399^407. [14] Balaban, N., Goldkorn, T., Gov, Y., Hirshberg, M., Koyfman, N., Matthews, H.R., Nhan, R.T., Singh, B. and Uziel, O. (2001) Regulation of Staphylococcus aureus pathogenesis via target of RNAIIIactivating protein (TRAP). J. Biol. Chem. 276, 2658^2667. [15] Christiansen, I. and Hengstenberg, W. (1996) Cloning and sequencing of two genes from Staphylococcus carnosus coding for glucose-speci¢c
[16]
[17]
[18]
[19]
[20]
[21]
[22]
PTS and their expression in Escherichia coli K-12. Mol. Gen. Genet. 250, 375^379. Borukhov, S., Polyakov, A., Nikiforov, V. and Goldfarb, A. (1992) GreA protein: a transcription elongation factor from Escherichia coli. Proc. Natl. Acad. Sci. USA 89, 8899^8902. Uptain, S.M., Kane, C.M. and Chamberlin, M.J. (1997) Basic mechanisms of transcript elongation and its regulation. Annu. Rev. Biochem. 66, 117^172. Ohta, T., Honda, K., Kuroda, M., Saito, K. and Hayashi, H. (1993) Molecular characterization of the gene operon of heat shock proteins HSP60 and HSP10 in methicillin-resistant Staphylococcus aureus. Biochem. Biophys. Res. Commun. 193, 730^737. Qoron£eh, M.W., Streips, U. and Wilkinson, B.J. (1990) Basic features of the staphylococcal heat shock response. Antonie Van Leeuwenhoek 58, 79^86. Mychajlonka, M., McDowell, T.D. and Shockman, G.D. (1980) Inhibition of peptidoglycan, ribonucleic acid, and protein synthesis in tolerant strains of Streptococcus mutans. Antimicrob. Agents Chemother. 17, 572^582. Jablonski, P.E. and Mychajlonka, M. (1988) Oxacillin-induced inhibition of protein and RNA synthesis in a tolerant Staphylococcus aureus isolate. J. Bacteriol. 170, 1831^1836. Lewis, K. (2000) Programmed death in bacteria. Microbiol. Mol. Biol. Rev. 64, 503^514.
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Cell wall-active antibiotic induced proteins of Staphylococcus aureus identi¢ed using a proteomic approach1 Vineet K. Singh, R.K. Jayaswal, Brian J. Wilkinson * Microbiology Group, Department of Biological Sciences, Illinois State University, Normal, IL 61790-4120, USA Received 5 March 2001; received in revised form 22 March 2001; accepted 23 March 2001 First published online 17 April 2001
Abstract Proteins produced in elevated amounts in response to oxacillin challenge of Staphylococcus aureus strain RN450, were studied by comparing Coomassie blue stained two-dimensional gels of cellular proteins. At least nine proteins were produced in elevated amounts following exposure to growth inhibitory concentrations of oxacillin. N-terminal sequences were obtained for five of the proteins and the databases were searched to tentatively identify them. The proteins were identified as homologs of (i) methionine sulfoxide reductase (MsrA) ; (ii) a signal transduction protein (TRAP) involved in regulating RNAIII production encoded by the agr locus; (iii) transcription elongation factor GreA; (iv) the heat shock protein GroES; and (v) the enzyme IIA component of the phosphoenolpyruvate:sugar phosphotransferase system. A similar induction response was observed with the other cell wall-active antibiotics, but not with antibiotics that affect other cellular targets. Increased transcription of the msrA and groEL genes in response to cell wall-active antibiotics was also demonstrated. Although net protein synthesis is inhibited subsequent to inhibition of peptidoglycan biosynthesis by cell wall-active antibiotics, some proteins are induced in S. aureus, presumably in an attempt by the cell to counter the inhibitory effects of these agents. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Cell wall-active antibiotics; Stress protein; Proteomics; Staphylococcus aureus
1. Introduction It is imperative that serious Staphylococcus aureus diseases such as bacteremia and endocarditis are treated aggressively with e¡ective antimicrobial agents. However, antibiotic resistance continues to increase in S. aureus and methicillin-resistant S. aureus (MRSA) that are uniformly resistant to L-lactam antibiotics are widespread worldwide. A critical determinant of methicillin resistance is the mecA gene that encodes low a¤nity penicillin-binding protein 2a [1]. Transposon mutagenesis has identi¢ed
* Corresponding author. Tel. : +1 (309) 438-7244; Fax: +1 (309) 438-3722; E-mail: [email protected] 1
This paper is dedicated to the memory of Gerald D. Shockman, a prominent member of an earlier generation of cell wall biochemists.
various chromosomal genes, fems or aux genes, that are required for the optimum expression of methicillin resistance [2,3], several of which are involved in peptidoglycan biosynthesis. However, a connection was forced between stress biology and methicillin resistance expression by the report of Wu et al. [4] which provided evidence that an alternative sigma factor, SigB, is involved in the expression of methicillin resistance in S. aureus. Transposon inactivation of the sigB operon in homogeneous MRSA COL drastically reduced resistance to methicillin. These ¢ndings led to the hypothesis that stress gene induction was a necessary part of the response to challenge by cell wall-active antibiotics in S. aureus. Accordingly, a proteomic approach was used to identify proteins produced in response to cell wall-active antibiotic treatment of mid-exponential phase cultures of a methicillinsusceptible strain. At least nine proteins were found to be produced in elevated amounts, and ¢ve of these proteins were identi¢ed by N-terminal sequencing. The produced proteins may constitute a proteomic signature for cell wall-active antibiotics, representing a stimulon or regulon triggered by these agents [5].
0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 1 6 3 - X
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2. Materials and methods 2.1. Growth conditions S. aureus and Escherichia coli cells were grown in tryptic soy broth (Difco Laboratories, Detroit, MI, USA) and Luria^Bertani broth, respectively. When needed, ampicillin (50 Wg ml31 ) and kanamycin (30 Wg ml31 in the case of E. coli, and 100 Wg ml31 in the case of S. aureus) were added to the growth medium. 2.2. DNA manipulations Plasmid and chromosomal DNA isolation, DNA manipulations, digestion of DNA with restriction enzymes, DNA ligations and Southern blotting were carried out as described [6]. PCR was performed with the GeneAmp PCR system (Perkin-Elmer, Foster City, CA, USA). 2.3. Antibiotic stress and two-dimensional gel electrophoresis An overnight culture of S. aureus RN450 was used to inoculate 50 ml of tryptic soy broth in a 300 ml Erlenmeyer £ask, which was incubated with shaking (250 rpm) at 37³C. At turbidity (A600 ) of 0.3, 10 ml of culture was distributed to two sterile test tubes and to one of the cultures oxacillin was added to a concentration of 1.2 Wg ml31 and incubated for an additional 2.5 h. The control culture was incubated for the same time period without oxacillin. Approximately equal amounts of the biomass from the control and oxacillin treated culture adjusted via A600 determinations, were collected by centrifugation (10 000Ug, 10 min, 4³C) and washed once with 50 mM Tris^HCl bu¡er, pH 7.5 containing 0.145 M NaCl. Cells were lysed by using lysostaphin and cellular proteins were prepared and examined by two-dimensional gel electrophoresis essentially as described [7]. Following electrophoresis the gels were stained with Coomassie brilliant blue R250. Experiments were repeated at least three times to con¢rm the reproducibility of the 2-D gels. The proteins induced were designated as cell wall-active antibiotic induced proteins. Additional antibiotics with varying cell targets were also included in these studies. They were used at concentrations that inhibited the growth of S. aureus RN450 to a degree comparable to 1.2 Wg ml31 of oxacillin. To identify the cell wall-active antibiotic induced proteins, the second dimension gels were directly transferred to Sequi-Blot PVDF membranes (Bio-Rad) using Tris^ CAPS (12 mM Tris and 8 mM CAPS) bu¡er, pH 9.6 and N-terminus sequences for ¢ve of these were obtained at the Protein Science Facility of the University of Illinois, Urbana, IL, USA. Sequences were not obtained for the other four proteins as either the N-terminus amino acid was blocked (spot 8), the amount was not enough to yield
a sequence (spots 2 and 3), or the spot did not yield a pure protein (spot 9). The N-terminus sequence data were used to identify the open reading frames encoding the proteins from the S. aureus genome database of the University of Oklahoma's Advanced Center for Genome Technology (www.genome.ou.edu) using the BLAST program [8]. The identi¢ed ORFs were translated and the primary amino acid sequences of the putative proteins were subsequently searched for similarities in the protein databases. 2.4. Construction of an msrA promoter: :lacZ reporter strain A reporter strain was constructed using the promoter region of the strongly inducible gene msrA. Primers P1 (5P-AAACCACTACCGAATCGTCG-3P) and P2 (5P-CACCAGAAACATCCTCCTGC-3P), and S. aureus RN450 genomic DNA were used to PCR amplify an approximately 1.3 kb fragment starting 44 nt downstream of the msrA start codon and going upstream. The PCR product was cloned in the correct orientation upstream of a promoterless lacZ gene of the shuttle vector pAZ106 [9] and was introduced into the chromosome of S. aureus strain RN4220 by electroporation with selection on erythromycin. Phage 80K lysate of the resulting transformants was used to transduce the msrA promoter: :lacZ into strain S. aureus RN450. A single copy insertion of the fusion in the chromosome was con¢rmed by Southern blot analysis using the msrA promoter region as the probe (result not shown). The activity of L-galactosidase was assayed after exposing the reporter strain to di¡erent antibiotics as described above using o-nitrophenyl-L-D-galactopyranoside (ONPG) as the substrate [10]. 3. Results and discussion 3.1. Cell wall-active antibiotic induced proteins During the ¢rst 30 min of incubation the growth was indistinguishable in antibiotic treated and untreated cultures (A600 0.3^0.46). Subsequently, the growth was virtually arrested in the antibiotic treated culture, but the control culture grew exponentially (A600 to 1.4) during 150 min of incubation. The Coomassie blue stained gels of proteins from control and oxacillin treated cultures of S. aureus RN450 are shown in Fig. 1. At least nine proteins were produced in elevated amounts after 2.5 h of exposure to oxacillin stress. 3.2. N-terminal sequencing and identity of cell wall-active antibiotic induced proteins The nine cell wall-active antibiotic induced proteins were submitted for N-terminal sequencing and sequences were obtained for ¢ve of the proteins. The identities of
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Fig. 1. 2-D gels of proteins of S. aureus RN450 cultures grown (A) without and (B) with oxacillin. The proteins visibly induced are encircled in the gel of proteins from the oxacillin-stressed cells. The circles in the gels of the cells grown without oxacillin show the proteins that are reduced in amount on addition of oxacillin. The values on the right indicate the molecular mass of the reference proteins.
antibiotics with other targets had no appreciable e¡ect on msrA transcription (Table 2). MsrA is an enzyme that catalyzes the reduction of methionine sulfoxides in proteins to methionine [11]. An E. coli msrA mutant was more susceptible to the oxidative stress provided by H2 O2 compared to the parent strain [12]. However, little is known about the regulation of msrA. The ¢nding of the induced synthesis of the S. aureus MsrA homologue by cell wall-active antibiotics is novel and the mechanism is not understood. Interestingly, msrA was identi¢ed as an S. aureus viru-
these proteins based on homology searches are shown in Table 1. 1. Spot 1. The amino acid sequence of the deduced protein of an open reading frame identi¢ed based on the N-terminus sequence of this spot showed strong homology to a putative Bacillus subtilis polypeptide, methionine sulfoxide reductase, MsrA (60% identity; 74% similarity). In addition, the L-galactosidase activity of the PmsrA : :lacZ reporter strain was signi¢cantly increased upon exposure to cell wall-active antibiotics whereas
Table 1 Sequence similarities of the staphylococcal proteins induced in response to oxacillin treatment Position on the gel
N-terminus sequence
Amino acids (aa)a
Identity (%)b
No. of aa that overlap
Similar protein (no of aa)c
Spot 1
MTKEYATLAG
177
60
171
Spot 4
MKKLYTSYGT
167
Spot 5
TGEFVKIEDI
166
46
165
Spot 6
MENQKQYPMT
158
67
157
Spot 7
MLKPIGNRVI
B. subtilis putative methionine sulfoxide reductase (177) (G7446682) A signal transduction protein (TRAP) of S. aureus (G11192009) Enzyme IIA of the phosphotransferase system of E. coli (169) (G12516793) GREA (B. subtilis putative transcription cleavage factor) (157) (G7449152) S. aureus GroES (94) (G421389)
94
a
Indicates the total number of amino acids in the protein encoded by the S. aureus open reading frame identi¢ed based on the N-terminus sequence. Identity of the predicted primary sequence of the product of the S. aureus ORF to known proteins. c Numbers in the ¢rst parentheses indicate the number of amino acids in the protein to which S. aureus putative protein showed signi¢cant sequence homology and the number in the second parentheses is the GenBank PID numbers for these proteins. b
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Table 2 L-Galactosidase activity of the PmsrA: :lacZ fusion strain in response to antibiotics Sample
L-Galactosidase activity (Miller units)
Control Rifamycin Gentamicin D-cycloserine Cephalothin Oxacillin
1h
2h
9.2 þ 1.4 12.4 þ 2.2 12.1 þ 2.3 42.4 þ 6.8 90.0 þ 8.8 92.4 þ 10.6
18.0 þ 2.6 5.2 þ 1.2 8.2 þ 1.2 156.2 þ 12.4 144.4 þ 11.6 178.2 þ 15.6
Rifamycin (0.45 Wg ml31 ), gentamicin (60 Wg ml31 ), D-cycloserine (150 Wg ml31 ), cephalothin (2 Wg ml31 ), and oxacillin (1.2 Wg ml31 ) were added at A600 of 0.3. In the control samples no antibiotic was added. The experiments were carried out in triplicate. Values shown are the averages of triplicate determinations þ standard errors.
lence gene in a murine model of bacteremia using signature tagged mutagenesis [13]. 2. Spot 4. The database search identi¢ed this protein as a 21 kDa RNAIII-activating protein (TRAP) [14]. The production of staphylococcal toxins is autoinduced by RNAIII-activating protein (RAP) and by the autoinducing peptide (AIP) but is inhibited by RNAIII-inhibiting peptide (RIP). RAP phosphorylates TRAP, whereas RIP inhibits the phosphorylation of this 21 kDa protein which is unique to S. aureus [14]. A knockout mutant of the trap gene was constructed in the present study by inserting a kanamycin resistance cassette in the EcoRI site of the gene. The mutation was subsequently transferred to oxacillin-resistant S. aureus
strain COL, but the mutation did not a¡ect the expression of resistance to cell wall-active antibiotics (data not shown). Interestingly, the synthesis of the virulence regulatory molecule RNAIII was not activated by RAP in a trap mutant strain providing evidence that the RAP activates RNAIII synthesis via TRAP [14]. The authors have concluded that the trap and the agr signal transduction systems interact with one another to coordinate the production of virulence factors. Although, a TRAP deletion mutant did not show an altered response to cell wall-active antibiotics, its elevated synthesis in response to these antibiotics might be an indication of a more extensive role for this protein in signal transduction. 3. Spot 5. The 10 amino acids sequenced from the N-terminus of this protein matched with the amino acids 23^ 32 in the predicted primary sequence of the product of a potential S. aureus open reading frame. This protein showed homology to the enzyme IIA component of the phosphoenolpyruvate-dependent sugar phosphotransferase system [15]. Possibly the ¢rst 22 amino acids of this protein were cleaved o¡ by cellular proteolytic activity. 4. Spot 6. The deduced protein of the open reading frame identi¢ed by N-terminus sequencing of this spot showed high homology to a transcription elongation factor GreA (transcript cleavage factor) of E. coli [16]. The transcript cleavage factors GreA and GreB allow RNA polymerases to overcome obstacles encountered during elongation in vivo, such as pausing and
Fig. 2. 2-D gels of proteins of S. aureus RN450 cultures grown with 1.2 Wg ml31 vancomycin (A) and 150 Wg ml31
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D-cycloserine
(B).
V.K. Singh et al. / FEMS Microbiology Letters 199 (2001) 79^84
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arresting sites in the DNA sequences, nucleosomes, or DNA-binding proteins and drugs [17]. 5. Spot 7. This protein was identi¢ed as Hsp-10 (GroES) which is known to be induced by heat shock and to be present in an operon in S. aureus with GroEL [18,19]. There was uncertainty about the location of GroEL on the 2-D gel and whether it was being induced in response to cell wall-active antibiotics. Therefore, total RNA was isolated from control and oxacillin treated cultures, separated by agarose gel electrophoresis and probed with the PCR ampli¢ed groEL fragment based on the published DNA sequence (accession no. D14711) [18]. The transcription of groEL was enhanced (data not shown) in response to oxacillin, suggesting that induction of these heat shock genes is an integral part of the cell wall-active antibiotic stress response.
may represent a proteomic signature. Proteomic signatures are sets of proteins that respond speci¢cally and uniquely to particular states of the cell such as energy su¤ciency, transcriptional capacity, or envelope integrity [5]. In S. aureus presumably the cell is sensing that it is damaged and certain proteins are being produced to help rescue the cell from the potentially lethal e¡ects of cell wall-active antibiotics. However, it is possible that overexpression of certain proteins could ultimately prove to be damaging to the cell [22]. Further studies on gene expression in response to cell wall-active antibiotics, including the utilization of gene array hybridization technology, are clearly warranted. Preliminary S. aureus GeneChip studies have con¢rmed increased transcription of genes encoding MsrA, TRAP, GroES, and Enzyme IIA of the phosphotransferase system (unpublished observations).
3.3. Proteins induced in response to other cell wall-active antibiotics and antibiotics inhibiting other cellular processes
Acknowledgements
The protein response of strain RN450 to growth inhibitory concentrations of other antibiotics was determined. The cell wall-active antibiotics vancomycin (Fig. 2), D-cycloserine (Fig. 2), cephalothin (data not shown) and bacitracin (data not shown) induced the production of a similar set of proteins to that induced by oxacillin. These proteins were not induced by the RNA polymerase inhibitor rifamycin, or the protein synthesis inhibitors gentamicin and erythromycin (data not shown). 3.4. Events occurring subsequent to inhibition of growth by cell wall-active antibiotics This work provides new insights into cellular events occurring subsequent to treatment with cell wall-active antibiotics. Peptidoglycan synthesis is inhibited which is presumably responsible for the inhibition of cell growth. Subsequently, various secondary responses are possible: cells may lyse, cells may die and lyse, cells may die without lysis, or growth may be inhibited but the cells survive (tolerance) [20]. Mychajlonka et al. [20] proposed that inhibition of peptidoglycan assembly activates a global regulatory circuit that `talks back' to cytoplasmic processes. In antibiotic-tolerant Streptococcus mutans this is manifested as a dose-dependent inhibition of peptidoglycan, RNA and protein synthesis by penicillin. Thus, although the gross synthesis of RNA and protein is inhibited subsequent to inhibition of peptidoglycan synthesis, certain proteins are synthesized in elevated amounts, i.e. some genes are being selectively expressed. In an earlier study Jablonski and Mychajlonka [21] showed using one-dimensional gel electrophoresis, alterations in S. aureus protein synthesis in response to oxacillin. The set of proteins that was observed to be produced in response to the various cell wall-active antibiotics studied
We are grateful to Sarah M. Scybert for the L-galactosidase assays in the lacZ reporter strain. This work has been supported by grant AI43027-01 from the National Institutes of Health, a grant-in-aid from the American Heart Association ^ Midwest A¤liate and a postdoctoral fellowship from American Heart Association ^ Midwest A¤liate to V.K.S. References [1] Matsuhashi, M., Song, M.D., Ishino, F., Wachi, M., Doi, M., Inoue, M., Ubukata, K., Yamashita, N. and Konno, M. (1986) Molecular cloning of the gene of a penicillin-binding protein supposed to cause high resistance to beta-lactam antibiotics in Staphylococcus aureus. J. Bacteriol. 167, 975^980. [2] Berger-Ba«chi, B. (1983) Insertional inactivation of staphylococcal methicillin resistance by Tn551. J. Bacteriol. 154, 479^487. [3] De Lencastre, H., Wu, S.W., Pinho, M.G., Ludovice, A.M., Filipe, S., Gardete, S., Sobral, R., Gill, S., Chung, M. and Tomasz, A. (1999) Antibiotic resistance as a stress response : complete sequencing of a large number of chromosomal loci in Staphylococcus aureus strain COL that impact on the expression of resistance to methicillin. Microb. Drug Resist. 5, 163^175. [4] Wu, S., Lencastre, H.D. and Tomasz, A. (1996) Sigma-B, a putative operon encoding alternative sigma factor of Staphylococcus aureus RNA polymerase : Molecular cloning and cDNA sequencing. J. Bacteriol. 178, 6036^6042. [5] VanBogelen, R.A., Schiller, E.E., Thomas, J.D. and Neidhardt, F.C. (1999) Diagnosis of cellular states of microbial organisms using proteomics. Electrophoresis 20, 2149^2159. [6] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [7] Vijaranakul, U., Nadakavukaren, M.J., Bayles, D.O., Wilkinson, B.J. and Jayaswal, R.K. (1997) Characterization of an NaCl-sensitive Staphylococcus aureus mutant and rescue of the NaCl-sensitive phenotype by glycine betaine but not other compatible solutes. Appl. Environ. Microbiol. 63, 1889^1897. [8] Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J. (1990) Basic local alignment search tool. J. Mol. Biol. 215, 403^410.
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[9] Chan, P.F., Foster, S.J., Ingham, E. and Clements, M.O. (1998) The Staphylococcus aureus alternative sigma factor sigma B controls the environmental stress response but not starvation survival or pathogenicity in a mouse abscess model. J. Bacteriol. 180, 6082^6089. [10] Miller, J.M. (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [11] Brot, N., Weissbach, L., Werth, J. and Weissbach, H. (1981) Enzymatic reduction of protein-bound methionine sulfoxide. Proc. Natl. Acad. Sci. USA 78, 2155^2158. [12] Moskovitz, J., Rahman, M.A., Strassman, J., Yancey, S.O., Kushner, S.R., Brot, N. and Weissbach, H. (1995) Escherichia coli peptide methionine sulfoxide reductase gene: regulation of expression and role in protecting against oxidative damage. J. Bacteriol. 177, 502^ 507. [13] Mei, J.M., Nourbakhsh, F., Ford, C.W. and Holden, D.W.F. (1997) Identi¢cation of Staphylococcus aureus virulence genes in a murine model of bacteremia using signature-tagged mutagenesis. Mol. Microbiol. 26, 399^407. [14] Balaban, N., Goldkorn, T., Gov, Y., Hirshberg, M., Koyfman, N., Matthews, H.R., Nhan, R.T., Singh, B. and Uziel, O. (2001) Regulation of Staphylococcus aureus pathogenesis via target of RNAIIIactivating protein (TRAP). J. Biol. Chem. 276, 2658^2667. [15] Christiansen, I. and Hengstenberg, W. (1996) Cloning and sequencing of two genes from Staphylococcus carnosus coding for glucose-speci¢c
[16]
[17]
[18]
[19]
[20]
[21]
[22]
PTS and their expression in Escherichia coli K-12. Mol. Gen. Genet. 250, 375^379. Borukhov, S., Polyakov, A., Nikiforov, V. and Goldfarb, A. (1992) GreA protein: a transcription elongation factor from Escherichia coli. Proc. Natl. Acad. Sci. USA 89, 8899^8902. Uptain, S.M., Kane, C.M. and Chamberlin, M.J. (1997) Basic mechanisms of transcript elongation and its regulation. Annu. Rev. Biochem. 66, 117^172. Ohta, T., Honda, K., Kuroda, M., Saito, K. and Hayashi, H. (1993) Molecular characterization of the gene operon of heat shock proteins HSP60 and HSP10 in methicillin-resistant Staphylococcus aureus. Biochem. Biophys. Res. Commun. 193, 730^737. Qoron£eh, M.W., Streips, U. and Wilkinson, B.J. (1990) Basic features of the staphylococcal heat shock response. Antonie Van Leeuwenhoek 58, 79^86. Mychajlonka, M., McDowell, T.D. and Shockman, G.D. (1980) Inhibition of peptidoglycan, ribonucleic acid, and protein synthesis in tolerant strains of Streptococcus mutans. Antimicrob. Agents Chemother. 17, 572^582. Jablonski, P.E. and Mychajlonka, M. (1988) Oxacillin-induced inhibition of protein and RNA synthesis in a tolerant Staphylococcus aureus isolate. J. Bacteriol. 170, 1831^1836. Lewis, K. (2000) Programmed death in bacteria. Microbiol. Mol. Biol. Rev. 64, 503^514.
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