1 Microbiology Group, Department of Biological Sciences, Illinois State University, Normal, IL 61791-4120, USA
2 Infectious Disease, Wyeth Research, Pearl River, NY 10965, USA
3 Genomics, Wyeth Research, Pearl River, NY 10965, USA
Correspondence
R. K. Jayaswal
drjay{at}ilstu.edu
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ABSTRACT |
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Details of the genes upregulated by treatment of S. aureus cultures with oxacillin (Table I), bacitracin (Table II) and D-cycloserine (Table III) are available in Microbiology Online.
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INTRODUCTION |
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It is axiomatic that cell-wall-active antibiotics inhibit bacterial growth by inhibiting peptidoglycan biosynthesis (Gale et al., 1982; Walsh, 2003
). However, following inhibition of peptidoglycan biosynthesis, various secondary cellular 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) (Mychajlonka et al., 1980
). The molecular events occurring after treatment of bacteria with a cell-wall-active antibiotic have not been studied extensively. Mychajlonka et al. (1980)
showed that the gross synthesis of RNA and protein is inhibited subsequent to inhibition of peptidoglycan biosynthesis by a cell-wall-active antibiotic. However, subsequent work by Jablonski & Mychajlonka (1988)
showed increased production of certain proteins in response to oxacillin treatment of a tolerant strain of S. aureus. Recently, Singh et al. (2001a)
used a proteomic approach to study proteins produced by S. aureus in response to cell-wall-active antibiotics. Two-dimensional gel electrophoresis of oxacillin-treated S. aureus revealed at least nine proteins that were produced in elevated amounts compared to control cultures. Five of the proteins produced in response to oxacillin were identified by N-terminal sequencing. This set of proteins appeared to be produced in response to other cell-wall-active antibiotics, but not antibiotics affecting other cellular targets (Singh et al., 2001a
), suggesting there may be a proteomic signature (VanBogelen et al., 1999
) characteristic of the cellular response to cell-wall-active antibiotics.
The development of DNA microarray technology provides an opportunity to capture a genome-wide picture of changes within the bacterial transcriptome in response to environmental perturbations (Conway & Schoolnik, 2003). Recently, Dunman et al. (2001)
have described transcriptional-profiling studies utilizing a first-generation Affymetrix S. aureus GeneChipTM representing 86 % of the S. aureus strain COL genome sequence. In this report, we describe our transcriptional-profiling studies of the methicillin-susceptible strain RN450 (8325-4) genes that are upregulated in response to the cell-wall-active antibiotics oxacillin, bacitracin and D-cycloserine, which inhibit different steps in peptidoglycan biosynthesis. The studies indicate the existence of a cell-wall-stress stimulon.
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METHODS |
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RNA extraction.
Cell pellets were resuspended in 2·5 ml lysis buffer (20 mM Tris/HCl, pH 7·5, 145 mM NaCl) containing 0·5 mg lysostaphin ml-1 (Sigma) and incubated at 25 °C for 30 min for lysis to occur. Total RNA was subsequently isolated using a Qiagen RNeasy Maxi kit according to the manufacturer's recommendations for prokaryotic RNA isolation. RNA concentrations were determined by spectrophotometry (OD260 value of 1·0=40 µg RNA ml-1). Five micrograms of each RNA sample were electrophoresed in a 1·2 % agarose/0·66 M formaldehyde gel to assess RNA integrity, according to Qiagen RNA electrophoresis recommendations.
RNA processing
GeneChipTM hybridization and washing.
RNA samples were processed then hybridized and washed on S. aureus GeneChipsTM as described previously (Dunman et al., 2001).
GeneChipTM analysis.
To increase reproducibility, each experiment was performed in duplicate (RNA extraction from control and antibiotic-treated cells). Each labelled RNA sample was then hybridized to at least one GeneChipTM. Following hybridization, washing and staining, GeneChipsTM were scanned at 570 nm, 3 µm resolution in an Affymetrix GeneChipTM scanner. Affymetrix algorithms (MICROSUITE 4.0) calculated signal intensities (mean difference) and Present or Absent determinations for each gene, as described previously (Dunman et al., 2001). To normalize for global systematic variations caused by inconsistencies in labelling and/or loading, each mean difference value was divided by the median mean difference for a given GeneChipTM. Normalized intensity values were then averaged for each gene at each assay condition. GENESPRING version 3.2.11 software (Silicon Genetics) was used to plot normalized intensity values across samples. Genes demonstrating at least a twofold increase in transcripts titres in response to each antibiotic treatment as compared to control samples were first identified. Genes that were subsequently determined to be Present by Affymetrix algorithms in antibiotic-treated samples and that were also found to be significantly different (t-test; P cut-off value of 0·05) from control samples were considered to be upregulated in response to challenge by a particular cell-wall-active antibiotic. Based on these lists of genes that were putatively upregulated by an individual antibiotic, Venn diagrams were then used to identify genes that demonstrated increased transcript titres in response to all, an individual or combinations of antibiotics tested. A similar process was used to identify genes that were downregulated in response to cell-wall-active antibiotic challenge. However, in this case, genes had to be considered Present by Affymetrix criteria in control samples as opposed to treated conditions.
Northern blot analysis.
Equal amounts of RNA (10 µg) from control and antibiotic-treated cultures were separated on a 1·2 % agarose/0·66 M formaldehyde gel and transferred to a nylon membrane by standard procedures (Sambrook et al., 1989). High-stringency hybridization was performed in a heat-sealable bag containing 10 ml of hybridization buffer [5xSSC, 5xDenhardt's, 50 % formamide, 1 % (w/v) SDS, 100 µg denatured herring sperm DNA ml-1] and radiolabelled randomly primed DNA synthesized in the presence of [
-32P]dCTP [specific activity>3000 Ci mmol-1 (>111 TBq); ICN Pharmaceuticals] products as a probe at 42 °C (Sambrook et al., 1989
). Membranes were subjected to low- and moderate-stringency washes prior to autoradiography. The following PCR primers were used to generate PCR templates. 16S rRNA (478 nt of GenBank accession no. Y15856), 5'-AAATCTTGACATCCTTTGACAACTC-3' and 5'-CTAGCTCCTAAAAGGTTACTCCACC-3'; vraS (1044 nt of GenBank GI: 9501766), 5'-TGAGCGTTCAATGGAAGG-3' and 5'-GCACAACTTTCATTGGCAC-3'; tcaA (1383 nt of GenBank GI: 15925705), 5'-GAAATCTTGCCCGAAGG-3' and 5'-TTGGTTGCTTGGTAGGTG-3'. All PCR products were gel-purified prior to being radiolabelled using the Prime-a-Gene labelling system (Promega) in the presence of [
-32P]dCTP and used to probe the membrane. The msrA gene fragment was generated from the construct pRSETa-msrA (Singh et al., 2001b
). These fragments were then radiolabelled as above before being used for probing the membrane.
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RESULTS AND DISCUSSION |
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Genes upregulated by oxacillin, bacitracin and D-cycloserine
One hundred and five genes were upregulated by all three antibiotics and are shown in Table 1. Genes belong to various functional categories, which include amino-acid transport and metabolism, carbohydrate transport and metabolism, cell-envelope biogenesis, DNA replication, recombination and repair, post-translational modification, protein turnover and chaperones, signal-transduction mechanisms, and transcription. Forty-six genes encoding hypothetical proteins were found to be putatively induced by each antibiotic.
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Several cell-wall-related genes were upregulated by each of the antibiotics. These included pbpB, the gene encoding penicillin-binding protein (PBP) 2, sgtB, encoding a hypothetical protein similar to PBP1A/1B, murZ, encoding UDP-N-acetylglucosamine-1 carboxylvinyl transferase 2, and murI, encoding glutamate racemase. Possibly also related to cell-wall metabolism was gltD, encoding the small subunit of glutamate synthase. In addition, the gene encoding Fmt, an autolysis- and methicillin-resistance-related protein, was upregulated. Furthermore, a hypothetical protein similar to the lyt divergon expression attenuator LytR was strongly upregulated. vraS, which encodes a vancomycin-resistance-associated two-component histidine kinase sensor, was strongly expressed in response to cell-wall-active antibiotics.
In addition, two genes involved in DNA replication, recombination and repair were strongly expressed. These were recU, encoding a recombination protein U homologue, and one encoding a conserved hypothetical protein.
Two genes involved in the regulation of transcription were strongly induced. These were the above-mentioned lytR homologue and hrcA, encoding a heat-inducible transcriptional repressor.
The three cell-wall-active antibiotics studied are each active at different steps in peptidoglycan biosynthesis (Gale et al., 1982; Walsh, 2003
). Oxacillin binds to PBPs and inhibits the final cross-linking step of peptidoglycan biosynthesis. D-cycloserine inhibits alanine racemase and D-alanine : D-alanine synthetase in a true competitive manner. Bacitracin binds to the carrier molecule in peptidoglycan biosynthesis C55-isoprenyl pyrophosphate and prevents its dephosphorylation. Even though the agents affect different stages of peptidoglycan biosynthesis, 105 genes were upregulated when S. aureus cells were challenged with each of the three antibiotics. This suggests that the cell responds to inhibition of peptidoglycan biosynthesis in general, and the genes comprise a cell-wall-stress stimulon. A stimulon refers to the entire set of genes responding together to an environmental stimulus (Smith & Neidhardt, 1983
), the environmental stimulus being a cell-wall-active antibiotic in this case. Cao et al. (2002)
have referred to a vancomycin stimulon in Bacillus subtilis. Two prominent functional categories to which genes belonged were cell-wall-related genes, and post-translational modification, protein turnover and chaperones.
Each antibiotic stimulated transcription of the pbpB gene encoding PBP2 (Table 1). Murakami et al. (1999)
and Boyle-Vavra et al. (2003)
have demonstrated by Northern blot analysis that oxacillin and vancomycin increase the level of pbpB transcripts. Vancomycin also upregulated pbpB in transcriptional-profiling studies (data not shown). PBP2 plays a critical role in the peptidoglycan metabolism of S. aureus. Cooperation of the transglycosylase domain of PBP2 is required for the PBP2a of methicillin-resistant S. aureus to carry out cell-wall synthesis in the presence of methicillin (Pinho et al., 2001
). PBP2 is believed to play a role in borderline resistance to methicillin in the absence of mecA (Chambers, 1997
). Increased PBP production has been noted in laboratory and clinical glycopeptide-intermediate S. aureus (Hanaki et al., 1998
; Moreira et al., 1997
). We can propose that the cell responds to inhibition of peptidoglycan synthesis by increasing the transcription of the pbpB gene in order to boost PBP2 production, and presumably then the rate of peptidoglycan synthesis, to restore the damaged and missing wall. Perhaps glucose-specific-enzyme-IIA induction serves to increase the rate of glucose transport into the cell to provide necessary energy for increased peptidoglycan biosynthesis. Peptidoglycan synthesis is believed to be activated in a clinical glycopeptide-intermediate S. aureus (Hanaki et al., 1998
).
murZ is upregulated by the cell-wall-active antibiotics and is the first step committed to biosynthesis of UDP-N-acetylmuramyl pentapeptide. The enzyme UDP-N-acetylglucosamine 1-carboxylvinyl transferase 2 catalyses the condensation of phosphoenolpyruvate with UDP-N-acetylglucosamine (Marquardt et al., 1992). This may also be a response by the cell to increase the rate of peptidoglycan biosynthesis. Two genes related to D-glutamate production that were upregulated were murI, encoding glutamate racemase, and gltD, encoding the small subunit of glutamate synthase.
tca has been described by Brandenberger et al. (2000) as being a three-cistronic operon that increases teicoplanin resistance in S. aureus. It is speculated that tcaRAB may be involved in cell-wall biosynthesis.
Two genes possibly related to the suppression of autolysis, which would serve to protect the cell from damage caused by the triggering of these enzymes by cell-wall-active antibiotics, were fmt and lytR. Fmt is a protein whose inactivation leads to a greater rate of autolysis of S. aureus in the presence of 0·02 % Triton X-100 (Komatsuzawa et al., 1997). Fmt is also believed to be involved in the expression of oxacillin resistance in the presence of Triton X-100. LytR is a potential response regulator of a two-component regulatory system that regulates a dicistronic operon encoding two proteins, LrgA and LrgB. They are hypothesized to control peptidoglycan hydrolase activity (Brunskill & Bayles, 1996
; Groicher et al., 2000
).
vraS is a vancomycin-resistance-associated gene with homology to the histidine kinase family (Kuroda et al., 2000), located immediately upstream of vraR, a response-regulator homologue. The transcription of vra was upregulated in a clinical vancomycin-resistant isolate. Overexpression of vraR in a susceptible strain increased vancomycin resistance.
The cell-wall-active antibiotics also caused strong induction of genes encoding the stress molecular chaperones/proteases HtrA and Hsp33. HtrA is a protease (Pallen & Wren, 1997). Hsp33 (encoded by hslO) is a potent molecular chaperone (Graf & Jakob, 2002
) that is under heat-shock control at the transcriptional level. On the post-translational level, Hsp33 is under oxidative stress control (Jakob et al., 1999
). The redox sensor is a zinc-coordinating cysteine centre that forms intramolecular disulfide bonds under oxidizing, activating conditions. msrA also encodes a protein that protects against oxidative damage, reducing methionine sulfoxide residues to methionine (Singh et al., 2001b
). An implication of these results is that treatment of cells with cell-wall-active antibiotics causes the accumulation of damaged, misfolded and aggregated proteins, necessitating the production of stress-chaperone and protease proteins to deal with the accumulation of aberrant proteins. Furthermore, the results imply that treatment with cell-wall-active antibiotics causes oxidative damage to proteins, as indicated by the induction of msrA and hslO (Storz & Zheng, 2000
). This appears to be a previously unappreciated aspect of the mode of action of cell-wall-active agents. prsA encodes a peptidyl-prolyl cis/trans isomerase. Such enzymes catalyse cistrans isomerization around X-Pro peptide bonds and facilitate envelope protein folding (Raivio & Silhavy, 2000
). These proteins participate in the response to envelope stress in Escherichia coli. In B. subtilis, PrsA is an extracellular lipoprotein and is considered to be dedicated to assisting the folding and stability of exported proteins at the cytoplasmic membranecell wall interface (Wahlström et al., 2003
).
Northern blot analysis of gene induction
Northern blot analysis was used to further validate the results obtained from transcriptional profiling. As expected, the msrA gene was demonstrated to show increased transcription in response to oxacillin, bacitracin and D-cycloserine as revealed by Northern blot analysis (Fig. 1). This is consistent with the transcriptional-profiling results which demonstrated that msrA is upregulated in response to cell-wall-active antibiotic stress. Northern analysis also showed that transcripts from vraS and tcaA were increased when bacteria were challenged with oxacillin (Fig. 2
). This is in agreement with the transcriptional-profiling results that these genes are highly induced in response to oxacillin stress.
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Genes upregulated by bacitracin but not by oxacillin or D-cycloserine
Forty-six genes were up regulated twofold or more by bacitracin but not by the other antibiotics (Table II, http://mic.sgmjournals.org). Strikingly, ORF 1764 was upregulated 50-fold, and this ORF is believed to represent an integral recombinase, core domain family protein. Three genes encoding proteins involved in lysine biosynthesis were upregulated, dapB, dapD and lysA. Lysine is a component of S. aureus peptidoglycan. The pbp4 gene was increased in expression, as was mvaK1, encoding mevalonate kinase.
Genes upregulated by D-cycloserine but not by oxacillin or bacitracin
an enormous number (218) of genes were upregulated by D-cycloserine (Table III, http://mic.sgmjournals.org), including genes involved in amino-acid transport and metabolism (27), carbohydrate transport and metabolism (6), cell-envelope biogenesis (6), cell motility and secretion (5), coenzyme metabolism (4), DNA replication, recombination and repair (4), energy production and conversion (4), hypothetical protein (106), inorganic-ion transport and metabolism (8), lipid metabolism (8), miscellaneous (18), nucleotide transport and metabolism (2), post-translational modification, protein turnover and chaperones (2), secondary metabolites biosynthesis, transport and catabolism (6), transcription (7), and translation, ribosomal structure and biogenesis (1).
This is one of the first reports of transcriptional profiling of the response of a human bacterial pathogen to cell-wall-active antibiotics. We believe that our studies have uncovered a cell-wall-stress stimulon, which may have characteristic transcriptome and proteome signatures. Although the focus of this body of work was to identify genes that are collectively upregulated by cell-wall-active antibiotics, Table 2 lists 87 genes that were determined to be downregulated by each antibiotic. Recently, Ng et al. (2003)
have reported on microarray analysis of the response of Streptococcus pneumoniae to challenge with sublethal concentrations of translation inhibitors. Transcript levels of ribosomal proteins and translation factors were upregulated, whereas tRNA charging and amino-acid biosynthesis enzyme transcript levels were downregulated. Global transcription patterns formed a signature that could be used to classify the mode of action of translation inhibitors. Subinhibitory concentrations of erythromycin and rifampicin were shown to alter global transcription patterns through studies of promoterlux reporter constructs in a Salmonella typhimurium library (Goh et al., 2002
).
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The B. subtilis SigW and SigM sigma factors are part of the extracytoplasmic function subfamily of sigma factors, members of which control cell-envelope-related functions (Helmann, 2002). Cao et al. (2002)
showed that vancomycin induced 19 SigW-dependent genes as revealed by DNA microarray studies. In addition, vancomycin induced genes that are not known to be controlled by extracytoplasmic function sigma factors, including 10 cell-wall-related genes. Thirty-three members of the large SigB general stress regulon were induced later during vancomycin treatment.
Extracytoplasmic function sigma factors have not been discovered in Staphylococcus aureus genome sequencing (Kuroda et al., 2001), with the only sigma factors described to date being housekeeping sigma factor SigA and SigB, which regulates the expression of various virulence factors and stress genes (Cheung et al., 1999
; Gertz et al., 2000
). The extent of the involvement of SigB in the expression of the cell-wall-stress stimulon we have described is unknown at this point.
The strain that we used in these studies, S. aureus 8325-4, is a derivative of strain 8325, which is the line of strains most frequently used in genetics-based staphylococcal studies (Novick, 1991). Giachino et al. (2001)
reported that 8325 derivatives have an 11 bp deletion in the 5' part of the rsbU gene. This generates a stop codon giving an ORF of 74 aa compared to the uninterrupted 323 aa ORF of intact RbsU. RbsU is a protein activator of SigB. Thus, 8325 derivatives are defective in SigB activation, although SigB is detected in the strains by Western blotting (Giachino et al., 2001
). Our proteomic studies of the response of S. aureus to cell-wall-active antibiotics were carried out using strain 8325-4, as were the present transcriptomic studies. Clearly, future studies should evaluate the role of SigB in the expression of the putative cell-wall-stress stimulon we have described. Horsburgh et al. (2002)
have recently described the construction of an RbsU+ derivative of strain 8325-4. The msrA gene, which is strongly induced by oxacillin in strain 8325-4 (Singh et al., 2001a
, b
), is also strongly induced in 8325-4 RbsU+ (R. Pechous, N. Ledala, B. J. Wilkinson & R. K. Jayaswal, unpublished observations).
Recently, Chan et al. (2003) have reported on their studies on the microarray identification of genes responsive to cell-wall-active antibiotics using a different strain of S. aureus, which is presumably SigB+, and different cell-wall-active antibiotics to the ones used in the present study. They found several of the same genes to be upregulated that we found to be induced, including vraS, vraR, murZ, sgtB, prsA, msrA, lytR and tcaA.
We expect that full delineation and definition of the cell-wall-stress stimulon will be an iterative process. The mechanism of sensing cell-wall stress or damage and the signal-transduction pathways remain to be worked out. Chan et al. (2003) have provided evidence that a VraS/VraR controlled regulon is part of the cell-wall-stress stimulon.
Recognition of a cell-wall-stress stimulon should prove useful in antibacterial drug development, including allowing investigators to identify a cell-wall-inhibitory mode of action of novel active compounds. Also, it will be interesting to evaluate the transcription response to cell-wall-active antibiotics in strains resistant to these agents.
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ACKNOWLEDGEMENTS |
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Received 16 April 2003;
revised 23 June 2003;
accepted 23 June 2003.