The WalKR system controls major staphylococcal virulence genes and is involved in triggering the host inflammatory response

Abstract

The WalKR two-component system is essential for the viability of Staphylococcus aureus, playing a central role in controlling cell wall metabolism. We produced a constitutively active form of WalR in S. aureus through a phosphomimetic amino acid replacement (WalR(c), D55E). The strain displayed significantly increased biofilm formation and alpha-hemolytic activity. Transcriptome analysis was used to determine the full extent of the WalKR regulon, revealing positive regulation of major virulence genes involved in host matrix interactions (efb, emp, fnbA, and fnbB), cytolysis (hlgACB, hla, and hlb), and innate immune defense evasion (scn, chp, and sbi), through activation of the SaeSR two-component system. The impact on pathogenesis of varying cell envelope dynamics was studied using a murine infection model, showing that strains producing constitutively active WalR(c) are strongly diminished in their virulence due to early triggering of the host inflammatory response associated with higher levels of released peptidoglycan fragments. Indeed, neutrophil recruitment and proinflammatory cytokine production were significantly increased when the constitutively active walR(c) allele was expressed, leading to enhanced bacterial clearance. Taken together, our results indicate that WalKR play an important role in virulence and eliciting the host inflammatory response by controlling autolytic activity.

Fig 1

Constitutive WalR activity strongly increases expression of WalKR-regulated genes. The relative levels of walR, walR/walR^c, atlA, and lytM transcripts were measured by qRT-PCR during growth of the HG001 wild-type strain carrying either the pCN51 empty vector (strain ST1033), the pSD3-14 or pSD3-12 plasmids, respectively, expressing the walR or walR^c alleles from the Pcad promoter (strains ST1035 and ST1034). The expression levels were normalized using 16S rRNA as an internal standard and are indicated as the n-fold change with respect to the control strain (HG001/pCN51), expressed as means and standard deviations.

Fig 2

Expression of the constitutive walR^c allele leads to increased autolysis and enhanced biofilm formation. (A) Growth curves of S. aureus HG001 derivatives carrying the pCN51 empty vector (strain ST1033, ○), the pSD3-14 plasmid with the wild-type walR gene (strain ST1035, □), or the pSD3-12 plasmid with the walR^c allele (strain ST1034, ■). Bacteria were grown in TSB plus 0.25 μM CdCl₂. (B) Strains carrying the pCN51 empty vector (ST1033; ○) or the pSD3-12 walR^c expression plasmid (ST1034; ■) were grown until reaching an OD₆₀₀ of 1. Cells were resuspended in PBS and incubated at 37°C under aeration. Autolysis was measured as the decline of OD₆₀₀ over time, indicated as a percentage of the initial OD. (C) Biofilm assays were performed in microtiter plates after growth at 37°C for 24 h (strain ST1033 [A] and strain ST1034 [B]). Adherent biomass was quantified, normalized to the OD₆₀₀ of each cell culture, and is represented as the n-fold variation compared to the control strain. Experiments were carried out in quadruplicate, and standard deviations are indicated.

Fig 3

The WalR^c (D55E) regulator negatively regulates icaR and spa transcription. Relative levels of icaR and spa transcripts were measured by qRT-PCR in S. aureus strains carrying either the pCN51 empty vector (ST1033) or the pSD3-12 plasmid with the walR^c allele (ST1034). Expression levels were normalized using 16S rRNA as an internal standard and are indicated as the n-fold change with respect to the control strain (HG001/pCN51), expressed as means and standard deviations.

Fig 4

The sle1 and SAOUHSC_02855 cell wall degradation genes are new members of the WalKR regulon. (A) Relative levels of sle1 and SAOUHSC_02855 transcripts were measured by qRT-PCR and expressed as the n-fold change with respect to the control strain (HG001/pCN51), indicated as means and standard deviations. (B) DNase I footprinting analysis of S. aureus WalR binding to the sle1 promoter region. Radiolabeled DNA fragments were incubated with increasing amounts of WalR as follows: lane 1, no protein; lane 2, 3.5 pmol; lane 3, 7 pmol; lane 4, 14 pmol; and lane 5, 21 pmol. The corresponding Sanger dideoxy chain termination sequencing reactions (GATC) are shown. Brackets indicate the extent of regions protected by WalR from DNase I cleavage. (C) Nucleotide sequence of the sle1 and SAOUHSC_02855 promoter regions. Regions protected by WalR are indicated by gray bars, and the conserved direct repeats are indicated by arrows. The WalR consensus operator sequence is aligned with the direct repeats in the sle1 and SAOUHSC_02855 upstream regions, with mismatches underlined.

Fig 5

WalR^c enhances S. aureus hemolytic activity. S. aureus strains carrying either the pCN51 empty vector or the pSD3-12 walR^c expression plasmid were grown until reaching an OD₆₀₀ of 1. Then, 20 μl of each culture was spotted onto Columbia horse blood agar plates, followed by incubation at 37°C. The clear thin halo surrounding bacteria corresponds to delta-hemolysin activity, whereas the larger cloudy halo is due to alpha-hemolysin activity.

Fig 6

Constitutive WalR activity promotes bacterial clearance and host survival in a murine sepsis model. (A) Kaplan-Meier survival curves of mice inoculated i.p. with 10⁷ CFU/g mouse body weight of either the control ST1033 strain (▲) or the ST1034 WalR^c-producing strain (■). A total of 22 mice were used in each group in three independent experiments and survival was monitored over a 7-day period. ***, P < 0.001 (compared to control strain as determined by log-rank [Mantel-Cox] test). (B) S. aureus burden was measured in peritoneal lavage fluids at 1.5 h and 3 h postinfection (i.p.) with 5 × 10⁶ CFU/g mouse body weight. The data (means ± the standard error of the mean [SEM]) are representative of three independent experiments of five mice/group (n = 15). Gray bars, ST1033 (HG001/pCN51) control strain; black bars, ST1034 WalR^c-producing strain (HG001/pSD3-14). ***, P < 0.0005 (compared to control strain as determined by Mann-Whitney U test).

Fig 7

Constitutive WalR activity leads to increased neutrophil recruitment and enhanced cytokine production during the early phases of infection. (A) Peritoneal lavage fluids were collected at 1.5 h and 3 h postinfection (i.p.) with 5 × 10⁶ CFU/g mouse body weight. Macrophage and neutrophil counts were determined by flow cytometry. The data (means ± the SEM) are representative of three independent experiments with five mice/group (n = 15). Gray bars, ST1033 (HG001/pCN51) control strain; black bars, ST1034 WalR^c-producing strain (HG001/pSD3-14). *, P < 0.05 compared to control strain as determined by Mann-Whitney U test. (B) Cytokine production was measured by ELISA in peritoneal lavage fluids collected at 1.5 h and 3 h postinfection (i.p.) with 5 × 10⁶ CFU/g mouse body weight. The data (means ± the SEM) are representative of three independent experiments with five mice/group (n = 15). Gray bars, ST1033 (HG001/pCN51) control strain; black bars, ST1034 WalR^c-producing strain (HG001/pSD3-14). *, P < 0.05; **, P < 0.01 (compared to control strain as determined by Mann-Whitney U test).

Fig 8

The S. aureus walR^c strain is strongly lowered in macrophage survival. RAW264.7 macrophages were infected with the HG001 strain carrying either the pCN51 empty vector (gray bars) or the pSD3-12 walR^c expression plasmid (black bars). Viable intracellular bacteria were quantified at the time of internalization (T0), 24 and 48 h after infection. A representative experiment (out of 3) is shown performed in triplicate (means ± the SD). *, P < 0.05; **, P < 0.00005 (as determined by using the Student t test).

Fig 9

WalR-dependent PGN release promotes inflammatory cytokine secretion in whole human blood. S. aureus culture supernatants were collected after growth until reaching an OD₆₀₀ of 1 and used to elicit cytokine production in whole human blood. (A and B) Whole human blood was incubated at 37°C with diluted S. aureus supernatants (1:32) and plasma cytokine levels were determined by ELISA after 3 h. (A) S. aureus HG001 background. Gray bars, ST1033 control strain; black bars, ST1034 WalR^c-producing strain. (B) S. aureus ST1160 ΔsaeRS background. Gray bars, ST1161 control strain; black bars, ST1162 WalR^c-producing strain. (C) PGN levels were assayed using the silkworm larvae plasma test in culture supernatants of S. aureus HG001 or ΔsaeSR strains (gray bars, pCN51 control; black bars, pSD3-14 walR^c), presented as the n-fold change with respect to the control strain.

Fig 10

Impact of the WalKR two-component system (TCS) on S. aureus virulence. The WalKR TCS is activated through phosphorylation of the WalR response regulator by the WalK histidine kinase, leading to increased expression of several genes involved in cell wall (CW) degradation and turnover. Higher release rates of CW degradation products after infection can then trigger the host innate immune response through activation of the NF-κB pathway, resulting in more efficient bacterial clearance and decreased virulence. Increased WalKR activity also leads to stimulation of the SaeSR TCS (dotted arrow) and higher expression of virulence genes involved in host-pathogen interactions and innate immune system evasion. Fine-tuning of WalKR activity must therefore play an important role in the switch between S. aureus commensal and pathogenic lifestyles.