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
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ABSTRACT |
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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.
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INTRODUCTION |
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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, AgrAD. 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.
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METHODS |
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Transduction.
Phage transductions were performed as described by Kayser et al. (1973) using
80
. 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 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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Colony hybridization.
DH5 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 manufacturers 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) Brewers 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 MannWhitney 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 -, ß- and
-toxins in wild-type and mutant strains, aliquots of S. aureus cultures were spotted onto either rabbit blood agar plates (for
-toxin), sheep blood agar plates (for ß-toxin) or horse blood agar plates (for
-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).
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RESULTS AND DISCUSSION |
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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
-, ß- and
-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
-, ß- and
-toxin production (Fig. 3a
), indicating that the toxin-deficient phenotype is due to mutation of svrA.
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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 -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
-toxin and protein A at the mRNA level.
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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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ACKNOWLEDGEMENTS |
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Received 18 March 2002;
revised 30 May 2002;
accepted 7 June 2002.