Investigation of the Staphylococcus aureus GraSR regulon reveals novel links to virulence, stress response and cell wall signal transduction pathways - PubMed (original) (raw)
Investigation of the Staphylococcus aureus GraSR regulon reveals novel links to virulence, stress response and cell wall signal transduction pathways
Mélanie Falord et al. PLoS One. 2011.
Abstract
The GraS/GraR two-component system has been shown to control cationic antimicrobial peptide (CAMP) resistance in the major human pathogen Staphylococcus aureus. We demonstrated that graX, also involved in CAMP resistance and cotranscribed with graRS, encodes a regulatory cofactor of the GraSR signaling pathway, effectively constituting a three-component system. We identified a highly conserved ten base pair palindromic sequence (5' ACAAA TTTGT 3') located upstream from GraR-regulated genes (mprF and the dlt and vraFG operons), which we show to be essential for transcriptional regulation by GraR and induction in response to CAMPs, suggesting it is the likely GraR binding site. Genome-based predictions and transcriptome analysis revealed several novel GraR target genes. We also found that the GraSR TCS is required for growth of S. aureus at high temperatures and resistance to oxidative stress. The GraSR system has previously been shown to play a role in S. aureus pathogenesis and we have uncovered previously unsuspected links with the AgrCA peptide quorum-sensing system controlling virulence gene expression. We also show that the GraSR TCS controls stress reponse and cell wall metabolism signal transduction pathways, sharing an extensive overlap with the WalKR regulon. This is the first report showing a role for the GraSR TCS in high temperature and oxidative stress survival and linking this system to stress response, cell wall and pathogenesis control pathways.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Figure 1. The graXRS operon is transcribed from a σA promoter.
(A) The graXRS/vraFG locus of S. aureus HG001. (B) Primer extension analysis of graXRS mRNA was carried out using total RNA extracted from S. aureus strain HG001 during mid-exponential growth in TSB at 37°C. Primer extension experiments were performed using the _graX_-specific oligonucleotide MF63 (lane 1). The corresponding Sanger dideoxy chain termination sequencing reactions (GATC) were carried out on a PCR-generated DNA fragment fragment corresponding to the graX upstream region (MF62/MF63). The transcriptional start site is boxed. (C) Nucleotide sequence of the graXRS operon upstream region. Potential σA-type -35 and −10 sequences are boxed and the transcriptional start site is labelled +1.
Figure 2. GraXSR do not control their own synthesis.
Expression of the graXRS operon was followed using a graX'-lacZ transcriptional fusion in S. aureus strains HG001, ST1036 (Δ_graRS_) and ST1070 (Δ_graX_). β-Galactosidase assays were performed as described in Materials and Methods and measured during mid-exponential growth at 37°C in TSB (grey bars) or after treatment with 200 µg ml−1 colistin for the HG001 strain (black bar). Means and standard deviations values are presented from at least three independent experiments.
Figure 3. Identification of potential GraR-binding sites in the promoters of known GraR-regulated genes.
(A) Primer extension analysis of mprF, dltXABCD and vraFG transcripts was carried out using total RNA extracted from S. aureus strain HG001 treated with 200 µg ml−1 colistin during mid-exponential growth at 37°C in TSB, using specific oligonucleotides for mprF, dltX and vraF (lanes 1 to 3 respectively). The corresponding Sanger dideoxy chain termination sequencing reactions (GATC) were carried out on PCR-generated DNA fragments corresponding to the respective upstream regions (see Table 5). The transcriptional start sites are boxed. (B) Alignment of the potential GraR DNA-binding sites in the mprF, dltXABCD and vraFG promoter regions. The potential GraR-binding site is shaded and conserved nucleotides are shown in white. Potential −35 and −10 sequences are underlined and the transcriptional start sites are indicated in bold.
Figure 4. GraSR-dependent gene expression requires GraX, colistin and the consensus binding site.
vraFG (A) and mprF (B) expression was followed using transcriptional lacZ fusions, with or without the upstream GraR operator sequence (vraF'-lacZ, mprF'-lacZ and ΔA_vraF_'-lacZ, ΔA_mprF_'-lacZ, respectively). The fusions were introduced in S. aureus strains HG001, ST1036 (Δ_graRS_) and ST1070 (Δ_graX_). Expression was measured during mid-exponential growth in TSB at 37°C (grey bars) or after treatment with 50 µg ml−1 colistin (black bars). β-Galactosidase assays were performed as described in Materials and Methods. Means and standard deviation values are presented from three independent experiments.
Figure 5. Point mutations in the GraR-binding site prevent vraFG expression and colistin induction.
(A) Alignment of the DNA sequences used to construct the _vraF_2'-lacZ and _vraF_2*-lacZ fusions. Potential −35 and −10 sequences are underlined, the identified transcriptional start site is indicated in bold and the GraR-binding site is shaded. Point mutations introduced in the _vraF_2*-lacZ fusion are shown in white. (B) _vraF_2'-lacZ and _vraF_2*-lacZ fusion expression was measured in S. aureus HG001 (strains ST1168 and ST1169, respectively) during mid-exponential growth at 37°C in TSB (grey bars) or after treatment with 200 µg ml−1 colistin (black bars). β-Galactosidase assays were performed as described in Materials and Methods. Means and standard deviations values are presented from three independent experiments.
Figure 6. The GraR operator consensus is a perfect inverted repeat obtained by alignment of regulon gene upstream sequences.
The consensus sequence for the GraR-binding site was generated using the WebLogo tool (
) by alignment of the upstream sequences of the 29 potential regulon genes identified by in silico analysis (Table 2).
Figure 7. Correlation between microarray and qRT-PCR experiments for expression of GraSR-dependent genes.
The expression levels of qoxA, ssaA, SAOUHSC_00669 and agrB genes were analyzed by qRT-PCR in the HG001 and ST1036 (Δ_graRS_) strains. RNA samples were prepared from cultures during mid exponential growth after treatment with 50 µg ml−1 colistin. Comparative analysis (fold-change) of transcriptome analysis (black bars) and qRT-PCR experiments (grey bars) are shown. Means and standard deviation values for the qRT-PCR data are presented from at least three independent experiments.
Figure 8. GraXSR are involved in oxidative stress resistance.
The effect of 40 mM paraquat was analyzed on HG001 (•, ○), ST1036 (▪, □; Δ_graRS_), ST1070 (▴, ▵; Δ_graX_) strains grown in TSB at 37°C, and diluted to a final OD 600 nm of 0.025. Growth was followed at 600 nm using a microtiter plate reader in the presence (closed symbols) or absence (open symbols) of 40 mM paraquat (methylviologen). A representative curve of three independent experiments is shown for each strain.
Figure 9. GraSR are required for growth of Staphylococcus aureus at high temperature.
The effect of high temperature was tested on growth of S. aureus strains ST1120 (HG001 pMK4-Pprot), ST1117 (Δ_graRS_ pMK4-Pprot) and the complemented Δ_graRS_ mutant, Δ_graRS_-c (strain ST1116; Δ_graRS_ pMK4-Pprot_-graR_). Strains were grown at 37°C in TSB and diluted to an OD 600 nm of 0.2. Serial dilutions were then carried out and 10 µl of each dilution was spotted on TSA plates, and incubated at 37°C or 44°C for 48 h.
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