Staphylococcus aureus svrA: a gene required for virulence and expression of the agr locusc

Steve Garvisa,1, Ji-Min Meia,1, Javier Ruiz-Albert1 and David W. Holden1

Centre for Molecular Microbiology and Infection, Imperial College of Science Technology and Medicine, The Flowers Building, Armstrong Road, London SW7 2AZ, UK1

Author for correspondence: David W. Holden. Tel: +44 207 594 3073. Fax: +44 207 594 3076. e-mail: d.holden{at}ic.ac.uk


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
A Staphylococcus aureus gene originally identified by signature-tagged mutagenesis as being required for virulence was cloned, sequenced and named svrA. Hydropathy profiles revealed that SvrA is likely to be membrane associated, having two regions with six membrane-spanning domains, the regions separated by an extended hydrophilic loop. When compared with the wild-type strain, an svrA mutant expressed greatly reduced amounts of {alpha}-, ß- and {delta}-toxins and an increased amount of protein A. Toxin production by the mutant strain was restored to wild-type levels when complemented with a plasmid expressing the svrA gene. Northern hybridization with probes specific for hla (encoding {alpha}-toxin) and spa (encoding protein A) showed that the svrA mutant strain was affected in the transcription of these genes. svrA mRNA was present in wild-type and agr strains, but agr mRNA and RNAIII were absent in the svrA mutant strain. Virulence studies suggested that the attenuation of the svrA mutant was probably due to its direct or indirect effect on the agr regulon. These results indicate that svrA is required for the expression of agr and RNAIII transcripts and is therefore a new component of the agr regulatory network controlling virulence gene expression in S. aureus.

Keywords: Gram-positive, pathogenesis, gene regulation

Abbreviations: Amp, ampicillin; Cm, chloramphenicol; Erm, erythromycin; GFP, green fluorescent protein; STM, signature-tagged mutagenesis

c The GenBank accession numbers for the svrA gene are SAV0334 (S. aureus subsp. aureus Mu50) and SA0323 (S. aureus subsp. aureus N315).

a These authors contributed equally to this work.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Staphylococcus aureus is a major human pathogen which causes a wide spectrum of infectious diseases, ranging from localized cutaneous lesions to systemic infections, such as osteomyelitis, endocarditis, pneumonia and other life-threatening complications (Sherris & Plorde, 1990 ). S. aureus produces a large number of virulence factors. These include extracellular toxins and cell-wall-associated proteins, important for colonization, immune evasion and tissue destruction. A number of regulators are involved in the production of these virulence determinants in S. aureus. These include agr (Peng et al., 1988 ), sar (Cheung et al., 1992 ), several sar homologues such as rot (McNamara et al., 2000 ), and the sae (Fournier & Hooper, 2000 ) and arl (Giraudo et al., 1997 ) two-component regulatory systems. The best characterized of all these regulators are the accessory gene regulator (agr) and the staphylococcal accessory regulator (sar).

The agr regulatory locus has been shown to be required for virulence in several infection models (Bunce et al., 1992 ; Abdelnour et al., 1993 ). At this locus, two promoters with opposing orientation, P2 and P3, produce two transcripts, RNAII and RNAIII, respectively (Peng et al., 1988 ; Kornblum et al., 1990 ). The effector RNAIII molecule is the main regulator of the agr system, and is responsible for the increased synthesis of extracellular proteins and the decreased production of cell-wall-associated proteins during the post-exponential growth phase (Novick et al., 1993 , 1995 ). The mechanism by which RNAIII regulates the expression of these genes is yet unclear, and probably involves proteins other than that encoded by the agr locus. The RNAII transcript encodes four proteins, AgrA–D. AgrA and AgrC constitute a two-component regulatory system, where AgrC acts as the signal receptor and AgrA is likely to act as the sensor regulator (Kornblum et al., 1990 ; Novick et al., 1995 ; Morfeldt et al., 1996 ). AgrD constitutes the autoinducer propeptide, which is processed in a manner that requires AgrB, to generate the small peptide that binds to and activates AgrC (Ji et al., 1995 , 1997 ). AgrC is phosphorylated in response to the autoinducer in vitro (Lina et al., 1998 ) and activates AgrA in a second phosphorylation step. Phosphorylated AgrA is then proposed to up-regulate both P2 and P3 promoters at the agr locus (Novick et al., 1995 ). This regulation is done in concurrence with SarA, a transcriptional regulator encoded by the sar locus (Cheung et al., 1992 ; Morfeldt et al., 1996 ).

The sar regulatory locus expresses three overlapping transcripts designated sarA, sarC and sarB. All three transcripts have common 3' ends, but originate from different promoters. sarA encodes a transcriptional regulator that binds to the P2 promoter and to a lesser degree the P3 promoter of the agr locus, enhancing RNAII and RNAIII transcription (Cheung et al., 1992 ; Bayer et al., 1996 ; Cheung et al., 1997 ). SarA may also act independently of the agr locus by directly regulating the expression of a number of virulence factors (Cheung & Ying, 1994 ; Cheung et al., 1994 ; Chan & Foster, 1998 ).

We previously reported the use of signature-tagged mutagenesis (STM) to identify genes important for S. aureus pathogenesis (Mei et al., 1997 ). Partial DNA sequencing of several of these genes revealed no significant similarities to characterized sequences in the DNA or protein databases. In this work we show that one of these STM mutant strains, initially designated P6C63, has a phenotype similar to that of an agr mutant strain. Furthermore, this gene appears to encode a novel staphylococcal virulence regulator because it is required for the expression of agr RNAII and RNAIII transcripts.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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Bacterial strains and plasmids.
Bacterial strains and plasmids used in this study are listed in Table 1. S. aureus strains were grown in Brain Heart Infusion (BHI) medium (Difco) with or without agar (1·5%) and antibiotics [erythromycin (Erm) at 20 µg ml-1 and/or chloramphenicol (Cm) at 20 µg ml-1). Escherichia coli strain DH5{alpha} was grown in Luria–Bertani (LB) medium (Difco) with or without ampicillin (Amp) at 50 µg ml-1.


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Table 1. Bacterial strains and plasmids used in this study

 
DNA manipulations.
Chromosomal DNA from S. aureus RN6390 was isolated as described by Pospiech & Neumann (1995) . DNA restriction and modifications were performed as described by Sambrook et al. (1989) . Plasmid DNA from S. aureus strain RN4220, a restriction-minus strain, was isolated using a Qiagen Plasmid Miniprep Kit according to the manufacturer’s protocol, except that the bacterial cells were lysed with lysostaphin (Sigma; 200 µg ml-1) at 37 °C for 30 min prior to plasmid purification.

Transduction.
Phage transductions were performed as described by Kayser et al. (1973) using {phi}80{alpha}. Recipient cells were cultured on blood agar at 37 °C overnight. Bacteria were suspended to a concentration of 0·5x1010 to 1·0x1010 c.f.u. ml-1 in 1 ml BHI broth. A total of 0·5 ml of the cell suspension was added to 0·5 ml LB broth with 5 mM CaCl2 and mixed with 0·5 ml phage lysate. The mixture was incubated for 20 min at 37 °C with shaking. Bacterial cells were collected by centrifugation and resuspended in 1 ml 20 mM sodium citrate. A 0·1 ml aliquot of this bacterial suspension was plated on BHI agar containing 20 mM sodium citrate and 10 µg Erm ml-1 and incubated for 36 h at 37 °C.

Construction of plasmids.
Plasmid pID413 is a derivative of pVA380-1 (Macrina et al., 1980 ). A DNA fragment carrying the pVA380-1 replicon was amplified by PCR from pVA380-1. Using primers F (5'-TGGAGATCTAAGCTTTGCATAACTTTCTCGTCC-3') and R (5'-TCCTGGCGATTCTGAGAC-3'), restriction sites for BglII and HindIII were introduced to the 5' end of the 2·5 kb fragment. The amplified fragment was ligated with a HindIII-digested 2·3 kb fragment carrying the tetracycline-resistance gene from pCW59 after filling in both vector and insert with DNA polymerase Klenow fragment, resulting in plasmid pID413. The DNA polylinker of plasmid pSP72 was obtained by digestion with BglII and HindIII and ligated into the BglII and HindIII sites of pID413 to generate pID413PL.

Construction of a genomic library of S. aureus.
A partial chromosomal DNA library from S. aureus strain RN6390 was constructed in pBR322 as follows. Chromosomal DNA was partially digested with BamHI and EcoRI to a mean size of 5 kb and purified by phenol/chloroform extraction. The purified DNA fragments were ligated into pBR322 partially digested with BamHI and EcoRI. The ligation product was transformed into E. coli DH5{alpha} by electroporation and plated onto LB agar containing 50 µg Amp ml-1.

Complementation of svrA mutant strain P6C63.
A DNA fragment containing the complete coding sequence of svrA was ligated into pID413PL to complement mutant strain P6C63 (Fig. 1a). This fragment was amplified from S. aureus RN6390 genomic DNA by PCR using primers 5'-TGGGGATCCGATAAGTGTGACTGGTAG-3' and 5'-TGGAAGCTTACATTACTTCAAATAAATTA-3' based on the DNA sequence flanking the svrA gene. Restriction sites for BamHI and HindIII were introduced into the fragment at the 5' and 3' ends, respectively. The amplified fragment was digested with BamHI and HindIII and inserted into BamHI- and HindIII-digested pID413PL to generate pID437. The plasmid was transformed into strain P6C63 by electroporation. Transformants were selected by resistance to tetracycline and tested for restoration of wild-type phenotype.



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Fig. 1. (a) Map of svrA region and plasmid for complementation. Sites for restriction enzymes are indicated (A, AluI; E, EcoRI; P, PstI); the arrow represents the Tn917 insertion site. (b) Sequence alignment of SvrA and the Methanococcus jannaschii protein MJ0709, a representative member of the family of membrane proteins described by PFAM UPF0013. Identical residues (22%) are in bold type, similar residues (46%) are boxed. The arrow indicates the Tn917 insertion site. (c) Hydrophobicity plot of SvrA obtained using the program TMPred (Hofmann & Stoffel, 1993 ) using default parameters. Positive scores represent hydrophobic regions; each peak corresponds to a putative membrane-spanning domain.

 
Southern and Northern blot analysis.
Southern hybridization analysis was performed as described by Sambrook et al. (1989) using digoxigenin-labelled DNA fragments as probes. For Northern hybridization, total RNA from S. aureus RN6390 was isolated using the Qiagen RNAeasy kit according to the manufacturer’s protocol except that the bacterial cells were lysed with 200 µg lysostaphin ml-1 (Sigma) at 37 °C for 3–5 min. Equal amounts of total RNA, as estimated by gel electrophoresis and staining with ethidium bromide, were separated on 1·2% agarose gels containing 0·66 M formaldehyde and transferred onto nitrocellulose membrane. Northern hybridizations were carried out at 42 °C. All probes were radiolabelled with [{alpha}-32P]dATP by PCR amplification. Oligonucleotide primers used for PCR were as follows. For amplification of an hla fragment, primers H1 (5'-ATTTGATATGTCTCAACTGC-3') and H2 (5'-GCTCTAATTTTTAAGTGAGG-3') were used. For amplification of spa, primers S1 (5'-TATCTGGTGGCGTAACACCTG-3') and S2 (5'-GATGAAGCCGTTACGTTGTTC-3') were used. For agrA, primers A1 (5'-GCCATAAGGATGTGAATGTATG-3') and A2 (5'-GCATTTGCTAGTTATCTTG-3') were used. Primers R1 (5'-AGATCTATCAAGGATGTGATGGTT-3') and R2 (5'-GTCATTATACGATTTAGTACAATC-3') were used for the amplification of RNAIII. Differences in the expression of spa were analysed using the public domain NIH Image program (developed at the US National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/).

Colony hybridization.
DH5{alpha} harbouring pBR322 with S. aureus chromosomal fragments was plated onto LB with ampicillin. After overnight incubation at 37 °C, colonies were transferred to nylon membranes by replica plating, and hybridized with a 0·5 kb probe for the svrA gene, following procedures described by Sambrook et al. (1989) .

Reverse transcription-PCR (RT-PCR).
Total RNA (1 µg) from post-exponential-phase cultures of S. aureus was reverse transcribed using the First-Strand cDNA synthesis kit (Amersham Pharmacia Biotech UK) according to the manufacturer’s instructions. PCRs were performed in a volume of 100 µl with 10 µl cDNA sample, 200 pmol each primer, 200 nM dNTPs and 2·5 U Taq DNA polymerase (Sigma). A control RT-PCR was performed under the same conditions as above after inactivating the reverse transcriptase by incubating at 95 °C for 5 min. PCR products were analysed by agarose gel electrophoresis, using procedures described above.

Infection studies.
For virulence studies, 12 20 g CD-1 female mice were injected intraperitoneally with 0·2 ml of a suspension containing 1·5x105 c.f.u. of each bacterial strain in BHI broth with 2% (w/v) Brewer’s yeast. At 96 h post-inoculation, the mice were killed and the spleens recovered for analysis. For quantification, dilution series of spleen homogenates were spread over BHI agar plates and incubated at 37 °C overnight. The Mann–Whitney non-parametric test was used to determine the significance of the distribution of the wild-type strain and each mutant strain independently.

Toxin analysis.
To analyse the production of {alpha}-, ß- and {delta}-toxins in wild-type and mutant strains, aliquots of S. aureus cultures were spotted onto either rabbit blood agar plates (for {alpha}-toxin), sheep blood agar plates (for ß-toxin) or horse blood agar plates (for {delta}-toxin) and incubated overnight at 37 °C. Cleared zones surrounding bacterial colonies were considered indicative of toxin activity.

Western blot analysis.
Western blot analysis was performed to determine expression of protein A. Whole-cell proteins were extracted from each strain. Since the strains assayed showed no noticeable difference in their respective growth rates in the conditions used, approximately 5x109 late-exponential-phase bacteria were centrifuged for each sample, resuspended in 50 µl H2O and lysed by lysostaphin treatment (200 µg ml-1), ensuring that equivalent amounts of protein were loaded for each sample. Coomassie blue staining of the protein gels showed no detectable differences in the amounts of protein loaded. Fifty microlitres of loading buffer was added to the lysates and the mixture was boiled for 10 min. Samples were separated by SDS-PAGE (12% resolving gel), electroblotted to Trans-Blot transfer media (Bio-Rad), probed with mouse anti-protein A monoclonal antibody (Sigma) and detected with the ECL detection system kit (Amersham Pharmacia Biotech). The relative quantities of protein A between the agr and the svrA mutant strains were estimated using the public domain NIH Image program.

Microscopy.
To evaluate the fluorescence of wild-type, svrA and agr strains carrying the plasmid pSB2031, containing a transcriptional fusion of the agr P3 promoter to a gene encoding the green fluorescent protein (GFP), overnight cultures grown in BHI with Erm were analysed using an Olympus BX50 fluorescence microscope. Images were captured with a high-resolution CCD camera, using analySIS 3.0 imaging software (Soft Imaging Systems).


   RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Cloning and sequencing of the svrA region
Strain P6C63 was originally identified as attenuated in virulence from an STM screen of S. aureus (Mei et al., 1997 ). It was selected for further analysis on the basis of reduced toxin production (see below). To clone the region surrounding the transposon insertion site, a partial genomic library of S. aureus P6C63 chromosomal DNA was generated in the plasmid pBR322. By hybridizing colonies from this genomic library with a probe consisting of a 0·5 kb DNA fragment flanking the transposon insertion, one positive clone was identified. Restriction analysis of the positive clone revealed a 2·5 kb AluI fragment which had been disrupted by Tn917 insertion in strain P6C63. DNA sequencing showed that the transposon insertion had disrupted the middle of three open reading frames (ORFs) in this region. This ORF is 1353 bp in length and encodes a protein of 451 amino acid residues with a predicted molecular mass of 48·8 kDa. In view of the phenotype of the mutant strain, it was designated svrA (for staphylococcal virulence regulator) (Fig. 1a, b). Sequence similarity searches of the protein domain databases revealed that SvrA shares low similarity with two families of proteins: UPF0013, an uncharacterized membrane protein family, and MVIN, a family of putative integral membrane proteins proposed to be virulence factors, including Salmonella typhimurium MviB (EMBL accession number Z26133). SvrA is predicted to have 12 membrane-spanning domains, separated by short hydrophilic loops, as determined using the program TMPred (Hofmann & Stoffel, 1993 ). The 12 domains are arranged into two groups of six, separated by an elongated hydrophilic loop (Fig. 1c). Sequence analysis of the region upstream of svrA revealed a putative promoter sequence including -35 and -10 regions similar to those found in other S. aureus genes. The intragenic region between svrA and the downstream ORF contains a predicted rho-independent transcriptional terminator ({Delta}G0=-19·2 kcal mol-1), as determined by analysis of RNA secondary structure using the program Mfold 3.1 (Lyngso et al., 1999 ), as well as a putative promoter sequence. These observations suggest that svrA and the downstream ORF are transcribed independently. Southern analysis (Fig. 2a) of the wild-type strain revealed a single band of 8·2 kb, whilst analysis of the svrA mutant strain revealed the appearance of two fragments, of 7·5 kb and 5·0 kb, and the loss of the 8·2 kb DNA fragment carrying svrA.



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Fig. 2. (a) Southern analysis of chromosomal DNA from the wild-type strain RN6390 and from the svrA mutant strain P6C63. Chromosomal DNA from strains was digested with HindIII, fractionated by agarose gel electrophoresis, transferred onto a nylon membrane and probed with digoxigenin-labelled svrA gene. Insertion of transposon Tn917 in the svrA gene introduces two HindIII sites. Digestion with this enzyme results in the loss of the 8·2 kb DNA fragment carrying svrA and the appearance of two new fragments, of 7·5 kb and 5·0 kb, in strain P6C63. (b) Southern analysis of chromosomal DNA from S. aureus strains. DNA was extracted from S. aureus strains RN6390, Newman, Wood 46, ID401 and ID402 and digested with HindIII. The digested DNA was separated by agarose gel electrophoresis, transferred onto nylon membrane and probed with digoxigenin-labelled svrA fragment. The sizes of the hybridization fragments are shown on the right in kb.

 
A Southern hybridization analysis was performed on several S. aureus strains, each with a different laboratory or clinical origin in the USA or UK, to determine if they contained svrA. Chromosomal DNAs were digested with HindIII and hybridized using the svrA gene as a probe. A single 8·2 kb fragment was observed in strain RN6390, Newman, Wood 46 and ID401, whereas a 10 kb fragment was observed in strain ID402 (Fig. 2b). Subsequent similarity searches (www.ncbi.nlm.nih.gov) of available S. aureus genome sequences (corresponding to the subspecies Mu50, N315, NCTC 8325, COL, 476 and 252) also revealed the presence of a single copy of the svrA gene, indicating that it is widely conserved in S. aureus. svrA has been designated SA0323 or SAV0334 in the annotation of the whole genome of methicillin-resistant S. aureus subspecies N315 and Mu50 (Kuroda et al., 2001 ). Interestingly, svrA is not present in the currently available Staphylococcus epidermidis genome sequence.

Phenotypic characterization
Strain P6C63 was tested for the expression of toxins, as it has been shown that loss of toxin production results in reduced virulence (Bramley et al., 1989 ). As a negative control for toxin expression, an agr mutant strain, RN6911, was used (Peng et al., 1988 ). The expression levels of {alpha}-, ß- and {delta}-toxins were examined by analysing the clearing zone around bacterial cultures spotted onto blood agar plates, a method which allows a semi-quantitative analysis of toxin production. The levels were greatly reduced in P6C63 and in the agr mutant strain RN6911 compared with their parental strain RN6390. To confirm that the toxin-deficient phenotype of strain P6C63 was due to interruption of svrA and not effects on other genes, a plasmid expressing svrA was constructed and transformed into P6C63. The complementing plasmid pID437 was able to restore {alpha}-, ß- and {delta}-toxin production (Fig. 3a), indicating that the toxin-deficient phenotype is due to mutation of svrA.



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Fig. 3. (a) Production of {alpha}-, ß- and {delta}-toxins. Aliquots of S. aureus cultures were spotted onto rabbit blood agar plates (for {alpha}-toxin), sheep blood agar plates (for ß-toxin) and horse blood agar plates (for {delta}-toxin) and incubated overnight at 37 °C. Cleared zones surrounding bacterial colonies are indicative of toxin activity. (b) Western immunoblot analysis of protein A. Whole-cell proteins were extracted from each strain and equal amounts were separated by SDS-PAGE, electroblotted to Trans-Blot transfer media and probed with mouse anti-protein A monoclonal antibody.

 
A characteristic of agr mutant strains is the overproduction of cell-wall protein A (Kornblum et al., 1990 ). In view of the similar toxin-deficient phenotypes displayed by the agr and svrA mutant strains, we also examined expression of protein A in the svrA mutant strain by Western blot, using an anti-protein A monoclonal antibody. As shown in Fig. 3(b), after overnight culture, protein A was not detectable in the wild-type strain but was present in both agr and svrA mutant strains. Interestingly, the expression level of protein A in the svrA mutant strain was approximately 2·4 times higher than that displayed by the agr mutant strain. Protein A was barely detectable in the svrA mutant strain carrying the complementing plasmid pID437.

Effect of the svrA mutation on transcription of hla and spa
As the svrA mutant strain showed reduced production of secreted toxins and increased levels of protein A, Northern hybridizations were performed to determine whether this was due to an effect at the mRNA level. Total RNA extracted from post-exponential-phase cultures of wild-type, agr, svrA or the complemented svrA strain was subjected to Northern analysis using probes specific for hla and spa (encoding {alpha}-toxin and protein A, respectively). Hybridization with hla produced a single strong band in RNA samples from the wild-type strain and the complemented svrA mutant strain, which was absent from the agr and svrA mutant strains (Fig. 4a). When membranes were stripped and reprobed with the spa probe, a hybridizing band was detected in RNA from both agr and svrA mutant strains, but not in RNA from the wild-type or the complemented strain (Fig. 4b). The increase in the expression level of spa in the svrA mutant strain compared to the agr mutant strain was comparable to the results obtained with the Western blot. These results are consistent with the phenotypic effects of the mutation and indicate that it affects the expression of {alpha}-toxin and protein A at the mRNA level.



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Fig. 4. Northern blot analysis of hla, spa, agr and RNAIII transcription. Total RNA was isolated from 109 c.f.u. post-exponential growth phase S. aureus strains RN6390, RN6911, P6C63 and complemented strain P6C63(pID437). RNA was separated by 1·5% agarose/0·66 M formaldehyde gel electrophoresis, transferred onto nylon membrane and probed with 32P-labelled hla (a), spa (b), agrA (c) or RNAIII (d).

 
svrA is required for expression of the agr locus
The results described above suggested a link between agr and svrA. As agr is well known as a key virulence regulatory locus in S. aureus, we analysed the interaction between SvrA and the agr system to determine if svrA is regulated by agr. As we were unable to detect svrA mRNA by Northern analysis, RT-PCR was employed to examine the transcription of svrA in different strains. Total RNA extracted from post-exponential-phase culture of wild-type, agr or svrA strains was used as template for RT-PCR using primers corresponding to the svrA DNA sequence. svrA transcripts were detected in agr and wild-type strains but not in the svrA mutant strain (results not shown). A control RT-PCR was performed under the same conditions as above after heat-inactivating the reverse transcriptase. No PCR products were obtained for any of the three strains in these control experiments, indicating that the products obtained were not due to DNA contamination (results not shown). Therefore, svrA gene expression is not dependent on agr. The lack of detectable svrA mRNA by Northern hybridization may reflect a low abundance of the transcript, due to either low expression or rapid turnover.

We next determined if svrA is required for expression of the agr locus. Total RNA isolated from post-exponential-phase cultures of wild-type, agr, svrA and the complemented svrA mutant strain was subjected to Northern hybridization using probes specific for agrA and RNAIII. As shown in Fig. 4(c, d), both the agrA and RNAIII probes detected a single major band in RNA isolated from the wild-type strain and the complemented svrA mutant strain, but did not hybridize to RNA from the svrA or the agr mutant strains. This result indicates that svrA is required for the transcription of agr and RNAIII.

To confirm this, a reporter gene consisting of a transcriptional fusion between the agr P3 promoter and a gene encoding GFP was introduced into wild-type, svrA and agr mutant strains, on plasmid pSB2031 (Qazi et al., 2001 ). After growth in BHI with Erm overnight, virtually all wild-type bacterial cells carrying this plasmid expressed GFP but neither the svrA nor the agr bacteria expressed detectable levels of the protein (Fig. 5).



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Fig. 5. Fluorescent microscopic analysis of GFP expression. RN6390 carrying plasmid pSB2031 (GFP under control of the RNAIII P3 promoter) is able to express GFP protein, while svrA and agr bacteria carrying the same plasmid are not.

 
The svrA mutant was originally isolated by STM, and LD50 analysis indicated that it is highly attenuated in virulence (Mei et al., 1997 ). To determine if the attenuation could be explained by its effect on the agr system, mice were inoculated intraperitoneally with approximately 1·5x105 c.f.u. of either wild-type, svrA, agr or an agr svrA double-mutant strain, constructed by transduction of the svrA mutation from P6C63 into an agr mutant, RN6911. Bacteria were recovered from spleens 96 h after inoculation and numbers were determined by plating a dilution series on LB agar (Table 2). Both single mutants and the double mutant were recovered in significantly lower numbers than the wild-type strain. The numbers of the double-mutant strain recovered were not significantly different from those of the svrA single-mutant strain, indicating that the effect of an additional mutation in agr does not contribute to further attenuate the virulence of an svrA mutant. The results obtained with the single infections were confirmed by analysing the relative virulence of these two strains by Competitive Index (CI) (Beuzón & Holden, 2001 ). The CI obtained was 0·96, indicating that the attenuation of the svrA single mutant is not greatly different from that of the svrA agr double mutant. This result, along with the differences observed in the expression of spa between the svrA and the agr single mutants, suggests that the phenotypes displayed by the svrA mutant can be explained mostly by its effect on agr, but that svrA may have additional functions unconnected with the agr regulon.


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Table 2. In vivo survival of S. aureus wild-type and mutant strains

 
SvrA is predicted to have several membrane-spanning domains and is therefore likely to be localized in the bacterial plasma membrane. How it influences transcription of agr is unclear, but it is possible that it could act in the RAP/TRAP signalling pathway. RAP is an RNAIII autoinducing protein secreted by S. aureus (Balaban & Novick, 1995 ). It induces the phosphorylation of a protein called TRAP, which is important for activation of RNAIII synthesis, possibly by up-regulation of agr and RNAII (Balaban et al., 1998 ). However, since TRAP is likely to be a globular cytosolic protein, it has been proposed that it might interact with extracellular RAP indirectly, via a membrane-associated intermediate (Balaban et al., 2001 ). It is conceivable that SvrA could provide this function. If so, phosphorylation of TRAP should be prevented in an svrA mutant. Further work is necessary to determine if SvrA functions in this or some other signalling pathway, to influence the complex regulation of virulence determinants of this important pathogen.


   ACKNOWLEDGEMENTS
 
We are grateful to Dr Jerry Slightholm of Pharmacia and Upjohn Inc. for providing the DNA sequence of svrA and flanking regions. The agr::gfp plasmid pSB2031 was generously provided by Dr P. Hill, University of Nottingham. We thank C. R. Beuzón for critical reading of the manuscript. We gratefully acknowledge the use of the network service at HGMP Resource Centre, Hinxton, UK. This work was supported by grants from the Wellcome Trust and from Pharmacia and Upjohn Inc.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Abdelnour, A., Arvidson, S., Bremell, T., Ryden, C. & Tarkowski, A. (1993). The accessory gene regulator (agr) controls Staphylococcus aureus virulence in a murine arthritis model. Infect Immun 61, 3879-3885.[Abstract]

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Received 18 March 2002; revised 30 May 2002; accepted 7 June 2002.