Staphylococcus aureus PerR Is a Hypersensitive Hydrogen Peroxide Sensor using Iron-mediated Histidine Oxidation

Background: PerR is a metal-dependent H₂O₂ sensor in many Gram-positive bacteria.

Results: Staphylococcus aureus PerR_SA, previously known as a Mn²⁺-specific repressor, uses Fe²⁺ to sense very low levels of H₂O₂.

Conclusion: The apparent lack of Fe²⁺-dependent repressor activity of PerR_SA is due to the hypersensitivity of PerR_SA under aerobic conditions.

Significance: Cells expressing hypersensitive PerR_SA are less virulent than those expressing PerR_BS.

Abstract

In many Gram-positive bacteria PerR is a major peroxide sensor whose repressor activity is dependent on a bound metal cofactor. The prototype for PerR sensors, the Bacillus subtilis PerR_BS protein, represses target genes when bound to either Mn²⁺ or Fe²⁺ as corepressor, but only the Fe²⁺-bound form responds to H₂O₂. The orthologous protein in the human pathogen Staphylococcus aureus, PerR_SA, plays important roles in H₂O₂ resistance and virulence. However, PerR_SA is reported to only respond to Mn²⁺ as corepressor, which suggests that it might rely on a distinct, iron-independent mechanism for H₂O₂ sensing. Here we demonstrate that PerR_SA uses either Fe²⁺ or Mn²⁺ as corepressor, and that, like PerR_BS, the Fe²⁺-bound form of PerR_SA senses physiological levels of H₂O₂ by iron-mediated histidine oxidation. Moreover, we show that PerR_SA is poised to sense very low levels of endogenous H₂O₂, which normally cannot be sensed by B. subtilis PerR_BS. This hypersensitivity of PerR_SA accounts for the apparent lack of Fe²⁺-dependent repressor activity and consequent Mn²⁺-specific repressor activity under aerobic conditions. We also provide evidence that the activity of PerR_SA is directly correlated with virulence, whereas it is inversely correlated with H₂O₂ resistance, suggesting that PerR_SA may be an attractive target for the control of S. aureus pathogenesis.

Introduction

Reactive oxygen species, which are produced endogenously as a by-product of aerobic metabolism or exogenously by microbial competitors and eukaryotic hosts, can cause oxidative stress to bacteria by damaging cellular constituents (1, 2). To cope with reactive oxygen species, bacteria have evolved sophisticated oxidative stress response systems including transcription factors that efficiently sense specific reactive oxygen species and induce appropriate defense systems (2–4). For example, OxyR in the Gram-negative model bacterium Escherichia coli senses H₂O₂ using cysteine oxidation, and activates transcription of ∼20 genes, including genes involved in H₂O₂ detoxification (1). Whereas many Gram-negative bacteria use OxyR as the major H₂O₂ sensor, many Gram-positive bacteria use PerR as a functional equivalent of OxyR (5, 6).

Bacillus subtilis PerR (PerR_BS)⁴ is a member of Fur family of metal-dependent regulators and is the prototype for a group of metal-dependent peroxide sensing repressors (5). PerR_BS contains a structural Zn²⁺ coordinated by four cysteine residues (Cys₄:Zn site, Site 1) and a second regulatory metal binding site (Site 2) composed of three N-donor ligands (His-37, His-91, and His-93) and two O-donor ligands (Asp-85 and Asp-104). Although the binding of either Fe²⁺ (PerR_BS:Zn,Fe) or Mn²⁺ (PerR_BS:Zn,Mn) at Site 2 activates PerR_BS to bind DNA, only PerR_BS:Zn,Fe can sense low levels of H₂O₂. Unlike cysteine thiol-based peroxide sensors such as OxyR and OhrR, PerR_BS senses H₂O₂ by metal-catalyzed histidine oxidation. Reaction of Fe²⁺, bound to Site 2, with H₂O₂ leads to the rapid oxidation of either His-37 or, to a lesser degree, His-91 (two of the Site 2 ligands) with concomitant loss of iron binding (7). Structurally, this results in an opening of the DNA-binding competent caliper-like conformation, leading to a loss of DNA binding and thus allowing the induction of genes that are normally repressed by active PerR_BS:Zn,Fe (8).

Staphylococcus aureus is a major human pathogen commonly causing nosocomial and community-acquired infectious diseases worldwide. S. aureus, which can be found as part of the normal skin flora and in anterior nares of the nasal passages, can cause a spectrum of illnesses from minor skin and soft tissue infections to more invasive and serious diseases such as pneumonia, meningitis, osteomyelitis, endocarditis, toxic shock syndrome, bacteremia, and sepsis (9). As a facultative anaerobic Gram-positive bacterium, S. aureus also uses PerR for the control of oxidative stress response (10, 11). The S. aureus PerR (PerR_SA) regulon is similar to that described for PerR_BS and includes genes encoding KatA, AhpCF, MrgA, Fur, and PerR, as well as others encoding thioredoxin reductase (TrxB), bacterioferritin comigratory protein (Bcp), and an iron storage protein ferritin (Ftn). Despite the similarity of the H₂O₂-dependent regulation of the PerR_SA regulon, Fe²⁺ was reported to be completely ineffective as a corepressor for the PerR_SA-regulated genes, which were only repressed by Mn²⁺. Indeed, expression of the PerR_SA-regulated genes is induced rather than repressed by added Fe²⁺ (11–14). These observations led to the conclusion that PerR_SA is a Mn²⁺-specific repressor and further suggest that PerR_SA may use a fundamentally different and iron-independent mechanism to sense H₂O₂. Despite the importance of PerR_SA in the regulation of virulence factors of S. aureus, the mechanism by which PerR_SA senses H₂O₂ has not been elucidated.

Here we have analyzed the metal-dependent H₂O₂ sensing mechanisms of PerR_SA in vitro and in vivo in comparison with those of PerR_BS. PerR_SA, like many other Fur family proteins, contains a structural Zn²⁺ site coordinated by four cysteine residues, which is resistant to oxidation by physiologically relevant H₂O₂ concentration. Contrary to the suggestion that PerR_SA is a Mn²⁺-specific repressor, the regulatory metal binding site (composed of His-43, Asp-91, His-97, His-99, and Asp-110) can bind Fe²⁺ even with higher affinity than Mn²⁺ when measured under anaerobic conditions. Moreover, the Fe²⁺-bound PerR_SA, but not the Mn²⁺-bound form, can sense H₂O₂ by Fe²⁺-dependent oxidation of His-43 and His-97. In cells grown under aerobic conditions most of PerR_SA is detected in the fully oxidized state, whereas cells grown under oxygen-limited conditions exhibit Fe²⁺-dependent repression of the PerR_SA regulon. The exquisite sensitivity of PerR_SA to inactivation likely explains the previous observation of an apparent lack of Fe²⁺-dependent repressor activity. Finally, we provide evidence that the high H₂O₂ sensitivity of PerR_SA (in comparison to PerR_BS) is important for H₂O₂ resistance under aerobic conditions and that the low sensitivity of PerR_BS (in comparison to PerR_SA) increases virulence of S. aureus in host.

Experimental Procedures

Bacterial Strains, Media, and Growth Conditions

The bacterial strains used in this study are listed in Table 1. E. coli, B. subtilis, and S. aureus were grown in Luria-Bertani (LB) media at 37 °C with appropriate antibiotics unless otherwise indicated. As metal-limited minimal media (MLMM), MOPS-buffered minimal medium was used for B. subtilis (15), and phosphate-buffered minimal medium was used for S. aureus (10). Oxygen-limited cultures were grown in 15-ml rubber screw-top tubes with the addition of 0.2% potassium nitrate. To facilitate oxygen-limited growth of S. aureus in MLMM, 1% Chelex-treated tryptone was added.

TABLE 1.

Strains used in this study

Strains	Relevant genotype and feature	Source
S. aureus
Newman	Wild type, human clinical isolate	NCTC
RN451	RN450 lysogenic for Φ11	NARSA
RN4220	Restriction-deficient transformation recipient	NARSA
LS0085	Newman perR::cat	This study
LS0088	LS0085 pLL29::perRSA-FLAG	This study
LS0093	LS0085 pLL29	This study
LS0106	Newman pCN33::PmrgA-lacZ	This study
LS0107	Newman pCN33::PkatA-lacZ	This study
LS0111	LS0085 pLL29::perRSA-FLAG pCN33::PmrgA-lacZ	This study
LS0114	LS0085 pLL29::perRBS-FLAG pCN33::PmrgA-lacZ	This study
LS0124	LS0085 pLL29 pCN33::PmrgA-lacZ	This study
LS0134	LS0085 pLL29:: perRBS-FLAG	This study
LS0166	LS0085 pCN48::perRSA-FLAG	This study
LS0241	LS0085 pLL29::perRSA-FLAG pCN33::PkatA-lacZ	This study
LS0242	LS0085 pLL29::perRSA (H43A)-FLAG pCN33::PkatA-lacZ	This study
LS0243	LS0085 pLL29::perRSA (H97A)-FLAG pCN33::PkatA-lacZ	This study
LS0244	LS0085 pLL29::perRSA (C102S)-FLAG pCN33::PkatA-lacZ	This study
LS0245	LS0085 pLL29 pCN33::PkatA-lacZ	This study
LS0304	LS0085 pLL29::perRSA (D91A)-FLAG pCN33::PkatA-lacZ	This study
LS0305	LS0085 pLL29::perRSA (H99A)-FLAG pCN33::PkatA-lacZ	This study
LS0306	LS0085 pLL29::perRSA (C105S)-FLAG pCN33::PkatA-lacZ	This study
LS0307	LS0085 pLL29::perRSA (D110A)-FLAG pCN33::PkatA-lacZ	This study
LS0308	LS0085 pLL29::perRSA (C142S)-FLAG pCN33::PkatA-lacZ	This study
LS0309	LS0085 pLL29::perRSA (C145S)-FLAG pCN33::PkatA-lacZ	This study
B. subtilis
HB9703	perR::tet	Ref. 15
HB9738	HB9703 amyE::perRBS-FLAG SPβC2Δ2::Tn917::Φ (PmrgA-cat-lacZ)	Ref. 15
LB1530	HB9703 amyE::perRSA-FLAG SPβC2Δ2::Tn917::Φ (PmrgA-cat-lacZ)	This study
LB1532	HB9703 amyE::spc SPβC2Δ2::Tn917::Φ (PmrgA-cat-lacZ)	This study
E. coli
HE9501	BL21 (DE3) pLysS pET-16b::perRBS	Ref. 15
LE0003	BL21 (DE3) pLysS pET-16b::perRSA	This study
LE0031	BL21 (DE3) pLysS pET-16b::perRSA (H43A)	This study
LE0032	BL21 (DE3) pLysS pET-16b::perRSA (D91A)	This study
LE0033	BL21 (DE3) pLysS pET-16b::perRSA (H97A)	This study
LE0034	BL21 (DE3) pLysS pET-16b::perRSA (H99A)	This study
LE0035	BL21 (DE3) pLysS pET-16b::perRSA (C102S)	This study
LE0036	BL21 (DE3) pLysS pET-16b::perRSA (C105S)	This study
LE0037	BL21 (DE3) pLysS pET-16b::perRSA (D110A)	This study
LE0038	BL21 (DE3) pLysS pET-16b::perRSA (C142S)	This study
LE0039	BL21 (DE3) pLysS pET-16b::perRSA (C145S)	This study
LE2302	BL21 (DE3) pLysS pET-11a::oxyREC	This study

Construction of a perR Deletion Mutant Strain of S. aureus Newman

The perR_SA::cat cassette was constructed by joining PCR using two 1-kb DNA fragments (upstream and downstream of perR_SA ORF) and a fragment with a chloramphenicol resistance marker (cat) from pDG1661. This cassette was cloned into the BamHI and EcoRI sites of pMAD (16) having a temperature-sensitive replication origin and a erythromycin-resistant marker (em) resulting in pJL954. Then, pJL954 was introduced into S. aureus Newman (NCTC8178) after passage through a restriction-deficient host S. aureus RN4220. S. aureus Newman strain having pJL954 integrated into the chromosome by a Campbell-type event was selected based on light blue colony color on LB agar plates containing chloramphenicol, erythromycin, and X-Gal after growing cells at 43 °C. After an overnight subculture two times in LB broth containing chloramphenicol at 30 °C, erythromycin-sensitive but chloramphenicol-resistant colonies were selected on LB plate at 43 °C. After confirmation of perR_SA deletion by PCR, the resultant strain was named LS0085.

Construction of perR-FLAG Fusion and Reporter Fusion in B. subtilis and S. aureus

For the expression of perR_SA-FLAG fusion in B. subtilis, the PCR fragment containing perR_SA ORF and about 200 bp upstream region was cloned into BamHI and EagI sites of pJL070 (15) generating pJL361. The ScaI digest of pJL361 was introduced to the perR null mutant B. subtilis strain HB9703 (15) to generate a transformant containing perR_SA-FLAG in the amyE locus. Then the P_mrgA-cat-lacZ reporter fusion stain (LB1530) was constructed by transduction with SPβ phage from HB1122 (17). For the expression of perR_SA-FLAG or perR_BS-FLAG fusions in S. aureus, the DNA fragment of pJL070 containing perR_BS-FLAG and that of pJL361 containing perR_SA-FLAG, were each cloned into BamHI and EcoRI sites of pLL29 (18) producing pJL1434 and pJL1430, respectively. Mutant alleles of perR_SA-FLAG were generated by the QuikChange method (Stratagene) using pJL1430 as templates. Each of these plasmids was integrated into the chromosome of S. aureus RN4220 with the help of int gene encoded in pLL2787, and then transferred into the perR null mutant S. aureus Newman strain (LS0085) by phage transduction using Φ11 (19). For the construction of reporter fusion plasmids, the lacZ gene from pDG1661 was PCR-amplified with the introduction of an NcoI site just after the KpnI site, and cloned into the KpnI and EcoRI sites of pCN33 resulting in pJL901. Then, DNA fragment containing the mrgA or katA promoter regions was introduced into the BamHI and NcoI sites of pJL901. The resulting reporter fusion plasmids were introduced into S. aureus Newman by electroporation after passage through S. aureus RN4220. To increase the copy number of perR_SA-FLAG, perR_SA-FLAG was cloned into the BamHI and EcoRI sites of the high copy number plasmid pCN48 (20) resulting in pJL643. This plasmid was introduced into the perR null mutant S. aureus Newman (LS0085) by electroporation after passage through S. aureus RN4220, generating a strain named LS0166. PerR_BS-FLAG and PerR_SA-FLAG proteins are fully functional as judged by reporter fusion assays, and were used for complementation of the perR null mutant strains and pulldown assays (15, 21).

Overexpression and Purification of Proteins

The PCR-amplified DNA fragments containing the perR_SA ORF were digested with BspHI and BamHI, and cloned into the NcoI and BamHI sites of pET-16b (Novagen) producing a plasmid named pJL203. Mutant alleles of perR_SA were generated by the QuikChange method (Stratagene) using pJL203 as template. E. coli oxyR was cloned into the NdeI and BamHI sites of pET-11a (Novagen) producing a plasmid named pJL1282. The encoded proteins were overexpressed using E. coli BL21(DE3) pLysS cells. Wild type (WT) PerR_SA proteins were purified after overexpression in E. coli BL21(DE3) pLysS cells harboring pJL203 as previously described for PerR_BS proteins (15). Briefly, the cell lysates were clarified by centrifugation and then PerR_SA was purified by heparin-Sepharose and Mono-Q chromatography using buffer A (20 mm Tris-HCl, pH 8.0, 0.1 m NaCl, and 5% glycerol (v/v)) containing 10 mm EDTA with the application of a linear gradient of 0.1–1 m NaCl. Further purification was performed using a Superdex-200 (HiLoad 16/60) column equilibrated with Chelex-treated buffer A. The concentration of PerR_SA was determined using a molar extinction coefficient of 10,430 m⁻¹ cm⁻¹ at 280 nm.

Enzyme Assays

On-gel catalase activity was assayed using 1:1 mixture of 5% ferric chloride and 5% potassium ferricyanide after gel-soaking in 2 mm H₂O₂. β-Galactosidase assays were performed with or without 100 μm H₂O₂ treatment for 30 min as described previously (15), except that lysostaphin (10 μg/ml) was used for the lysis of S. aureus cells. Measurement of Zn²⁺ release by H₂O₂ was performed as described previously (15) using 2.5 μm dimeric PerR_SA and 100 μm 4-(2-pyridylazo)resorcinol (PAR). The Zn²⁺ content of purified PerR_SA by PAR assay was measured using a molar extinction coefficient of 85,000 m⁻¹ cm⁻¹ at 494 nm for Zn²⁺-PAR complex (15).

Western Blot Analysis

At A₆₀₀ = 0.6, 10-ml cultures were harvested by centrifugation after the addition of 1.1 ml of trichloroacetic acid. Then, cells were resuspended in 500 μl of 10% trichloroacetic acid and sonicated. After recovering sonicated samples by centrifugation, the pellets were resuspended with 60 μl of alkylating buffer (100 mm iodoacetamide, 0.5 m Tris, pH 8.0, 5% glycerol, 100 mm NaCl, 2% SDS) and incubated for 1 h in the dark to alkylate-free thiols. Alkylated samples of 10 μl (75 μg of protein) were separated on 13.3% non-reducing SDS-PAGE gel using a Tris-Tricine buffer system and blotted to a polyvinylidene difluoride membrane. FLAG-tagged proteins were probed with mouse monoclonal anti-FLAG antibody and anti-mouse antibody conjugated with alkaline phosphatase (Sigma).

Fluorescence Anisotropy (FA) Experiments

FA experiments were performed using an LS55 luminescence spectrometer (PerkinElmer Life Sciences) installed in an anaerobic chamber (Coy). A 6-carboxyfluorescein (6-FAM)-labeled katA-PerR box DNA fragment was generated by annealing 5′-6-FAM-TTAAATTATAATTATTATAAATTGT-3′ (Integrated DNA Technology) and its unlabeled complement. FA measurements (λ_ex = 492 nm, slit width = 15 nm; λ_em = 520 nm, slit width = 20 nm, integration time = 1 s) were performed in 3 ml of Chelex-treated anaerobic buffer A. The percentage activity and K_d for DNA of purified PerR_SA were determined to be ∼20% and ∼1 nm, respectively, by titration of PerR_SA into 3 ml of buffer A containing 10 nm DNA and 1 mm manganese as reported previously (7). For the metal binding and H₂O₂ sensitivity assays, buffer A containing 100 nm DNA and 100 nm active dimeric PerR_SA were used, and FA was measured after each addition.

MALDI-TOF and LC-ESI MS/MS Mass Analyses

To analyze in vivo oxidation of PerR_SA (Fig. 4), LS0166 cells expressing PerR_SA-FLAG were grown in MLMM containing 50 μm FeSO₄ or 50 μm MnCl₂. At an A₆₀₀ of ∼1, cells were treated with or without 100 μm H₂O₂ for 2 min and lysed with 50 μg of lysostaphin for 1 h in 0.5 ml of buffer A. PerR_SA-FLAG protein recovered using anti-FLAG M2-agarose beads (Sigma) was incubated with 100 mm iodoacetamide in the presence of 50 mm EDTA and 1% SDS for 1 h in the dark, and separated on SDS-PAGE gel. The protein bands corresponding to PerR_SA-FLAG were analyzed by MALDI-TOF MS using a 4700 Proteomics Analyzer instrument (Applied Biosystems) after in-gel tryptic digestion as described previously (15, 22). The MALDI-TOF MS analysis of in vitro oxidation of purified PerR_SA (Fig. 3D) was performed as described previously (7, 15) using a Voyager-DE STR instrument (Applied Biosystems). The analysis of protein oxidation after overexpression in E. coli (Fig. 5) was performed as previously described (22), except that sample preparations for Fig. 5B were carried out in an anaerobic chamber. The sites of oxidation were identified by LC-MS/MS analyses using an Agilent nanoflow-1200 series HPLC system connected to a linear ion trap mass spectrometer (Thermo Scientific).

FIGURE 4.

In vivo oxidation of PerR_SA. Oxidation of PerR_SA from S. aureus cells (LS0166) grown aerobically in MLMM supplemented with no metal ion (A), 50 μm FeSO₄ (B), 50 μm MnCl₂ (C), or both 50 μm FeSO₄ and 50 μm MnCl₂ (D). PerR_SA-FLAG proteins were recovered by immunoprecipitation from S. aureus cells treated with no H₂O₂ or 100 μm H₂O₂ for ∼2 min, and analyzed by MALDI-TOF MS after SDS-PAGE separation and in-gel tryptic digestion.

FIGURE 3.

Metal-dependent DNA binding and H₂O₂-mediated oxidation of PerR_SAin vitro. A, metal-dependent DNA binding activity of PerR_SA. Various concentrations of metal ions (open circle for Fe²⁺ and filled circle for Mn²⁺) are added to samples containing 100 nm DNA and 100 nm active PerR_SA dimer, and metal-dependent DNA-binding of PerR_SA was monitored by fluorescence anisotropy change. B, sensitivity of PerR_SA to H₂O₂ in the presence of Fe²⁺. Protein (100 nm active PerR_SA:Zn dimer), FeSO₄, H₂O₂, and MnCl₂ were sequentially added to buffer A containing 100 nm DNA with an interval of 2 min between each addition, and FA was measured after each addition. 10 μm H₂O₂ rapidly (<20 s) inactivated PerR_SA in the presence of 2 μm Fe²⁺ and the loss of PerR_SA activity was not recovered by the addition of Mn²⁺. C, sensitivity of PerR_SA to H₂O₂ in the presence of Mn²⁺. Protein (100 nm active PerR_SA:Zn dimer), MnCl₂, H₂O₂, and EDTA were sequentially added to buffer A containing 100 nm DNA with an interval of 2 min between each addition except for 10 min between H₂O₂ and EDTA, and FA was measured after each addition. PerR_SA was insensitive to 1 mm H₂O₂ for 10 min but lost DNA-binding activity rapidly by addition of 1 mm EDTA. D, metal-dependent oxidation of PerR_SA. Oxidation of PerR_SA by H₂O₂ in the presence of 10 μm Fe²⁺ or 100 μm Mn²⁺ was monitored by MALDI-TOF MS after tryptic digestion. Note that no cysteine oxidation was observed as judged by fully alkylated T9 peptide (T9*) containing Cys-102 and Cys-105.

FIGURE 5.

Comparison of oxidation sensitivity among PerR_BS, E. coli OxyR and PerR_SA. Oxidation of PerR_SA (A), PerR_BS (C), or E. coli OxyR (D) in E. coli cells (LE0003, PerR_SA; HE9501, PerR_BS; LE2302, E. coli OxyR) grown under aerobic conditions, or PerR_SA (B) in E. coli cells (LE0003) grown under oxygen-limited conditions. E. coli cells were grown in LB media under aerobic conditions or oxygen-limited conditions, and treated with or without 100 μm H₂O₂ for 1 min. Oxidation status of proteins was analyzed by MALDI-TOF MS after SDS-PAGE fractionation and in-gel tryptic digestion as reported previously (22). Asterisks represent peptides containing carboxyamidomethylated cysteine residue(s).

Caenorhabditis elegans Killing Assay

NGM agar plates (5.5 cm diameter) spread with 30 μl of A₆₀₀ = 1 culture of E. coli OP50 (laboratory nematode food), S. aureus expressing no PerR (LS0093), S. aureus expressing PerR_SA (LS0088), or S. aureus expressing PerR_BS (LS0134) were used. For each assay 90 L4 stage C. elegans were used in triplicate of 30 worms/plate. The plates were incubated at 25 °C, and scored for live and dead worms at least every 24 h as described previously (23). For each assay, the survival of worms was calculated by the Kaplan-Meier method, and survival differences were tested by using OASIS (24).

Results

Structural Zn²⁺ and Regulatory Metal Binding Sites of PerR_SA

PerR_SA is highly similar (67% sequence identity) to PerR_BS, but previous results have highlighted some striking differences in their response to added metal ions (11–14). To provide a structural context for our investigation of PerR_SA reactivity we generated a homology model of PerR_SA based on the known structure of PerR_BS (25). As shown in Fig. 1, A and B, PerR_SA retains four highly conserved cysteine residues (Cys-102, Cys-105, Cys-142, and Cys-145) corresponding to those involved in high affinity structural Zn²⁺ binding (Site 1) in most Fur family proteins as well as in PerR_BS (5, 15, 26). PerR_SA also has five other residues (His-43, Asp-91, His-97, His-99, and Asp-110), which correspond to the N/O donor ligands for the regulatory metal binding (Site 2) in PerR_BS (7, 8). To investigate the role of these predicted metal-binding residues in protein function, we generated PerR_SA mutants and examined the in vivo repressor activities of WT and mutants using a PerR-regulated katA promoter-lacZ fusion (P_katA-lacZ) (Fig. 1C). As expected, the P_katA-lacZ reporter fusion was repressed in cells expressing WT PerR_SA-FLAG but derepressed in the perR null mutant cells. Note that the repression levels of P_katA-lacZ reporter fusion by WT PerR_SA-FLAG were similar to those observed with the WT S. aureus strain, indicating that the perR null mutant strain complemented with WT PerR_SA-FLAG behaves like WT strain. All nine mutants exhibited no repressor activity for the P_katA-lacZ reporter fusion, and furthermore, these mutant proteins were present at levels greater than WT protein (Fig. 1D) indicative of a loss of repression of the autoregulated perR promoter. These results indicate that these amino acid residues proposed to be metal ligands are essential for in vivo repressor function.

FIGURE 1.

Metal binding sites of PerR_SA. A, sequence alignment of PerR_SA with PerR_BS. The candidate ligands for Zn²⁺ (yellow) and Fe²⁺/Mn²⁺ (red) are conserved in PerR_SA. The two tryptic peptides of PerR_SA, T5 (blue) containing His-43 and T9 (green) containing His-97, are the sites of oxidation. The tryptic peptides of PerR_BS, T5 containing His-37 and T11 containing His-91, are also shown. B, predicted structure of PerR_SA monomer based on PerR_BS structure (Protein Data Bank code 3F8N) (25) using Swiss Model (40). C, mutational analyses of PerR_SA. Wild type S. aureus cells (WT PerR, LS0107), S. aureus cells expressing no PerR_SA (ΔperR, LS0245), or S. aureus cells expressing PerR_SA-FLAG variants as indicated, were grown in LB medium and treated with 100 μm H₂O₂ (+H₂O₂) for 30 min or not (−H₂O₂). Repressor activities of PerR_SA variants were measured using P_katA-lacZ reporter fusion and were expressed as Miller units of β-galactosidase activity (values represent the mean ± S.D. from three separate experiments). Statistical analysis was performed with a Student's t test (*, p < 0.05). D, analysis of expression levels for PerR_SA variants. S. aureus cells, expressing no PerR_SA (ΔperR) or expressing PerR_SA-FLAG variants as indicated, were grown in LB medium. The expression levels of FLAG-tagged PerR variants were analyzed by Western blot using anti-FLAG antibody. E, structural Zn²⁺-binding assay of PerR_SA variants on SDS-PAGE gel. The crude extracts of E. coli cells, overexpressing no PerR_SA (Blank) or overexpressing PerR_SA variants as indicated, were preincubated with 10 mm DTT and separated by SDS-PAGE using Tris glycine buffer system. Protein bands were detected by Coomassie Brilliant Blue R staining. F, oxidation of Cys₄:Zn site by H₂O₂. Release of Zn²⁺ from PerR_SA:Zn (5 μm purified PerR_SA) was measured by monitoring Zn²⁺-PAR complex at 494 nm after treatment of 0, 1, 10, and 100 mm H₂O₂ as described previously (15). The Zn²⁺ content was calculated using a molar extinction coefficient of 85,000 m⁻¹ cm⁻¹ at 494 nm for Zn²⁺-PAR complex (15).

Previously we have demonstrated that the structural Zn²⁺ binding status of PerR_BS can be monitored by mobility difference on non-reducing SDS-PAGE: monomeric PerR_BS containing bound Zn²⁺ migrates faster than PerR_BS lacking bound Zn²⁺ (15). To investigate the Zn²⁺ binding status of PerR_SA, WT and mutant proteins were separated on SDS-PAGE after overexpression in E. coli. WT and Site 2 mutants migrated with the mobility characteristic of the Zn²⁺-bound form, whereas all four Site 1 mutants migrated with the mobility characteristic of the Zn²⁺-lacking form (Fig. 1E). This result indicates that PerR_SA contains a tightly bound Zn²⁺ coordinated by four cysteine residues, and that mutations at the proposed regulatory metal binding site do not affect the Zn²⁺ binding.

PerR_SA was shown previously to be a Mn²⁺-specific repressor, which suggested that this protein might use an Fe²⁺-independent H₂O₂ sensing mechanism (11, 14). We therefore wondered whether the cysteine residues coordinating Zn²⁺ might serve a role in peroxide sensing. To test this, we measured the rate of Zn²⁺ release from purified PerR_SA upon H₂O₂ treatment (Fig. 1F) by monitoring the formation of a Zn²⁺-PAR complex as reported previously (15). The second-order rate constant of Zn²⁺ release and the Zn²⁺ content of PerR_SA were determined to be ∼0.05 m⁻¹ s⁻¹ and ∼0.8 Zn²⁺/monomer, respectively, which are comparable with those of PerR_BS (15). The slow rate of H₂O₂-mediated Zn²⁺ release, along with the retention of Zn²⁺ despite the use of 10 mm EDTA during the purification procedures, further supports the idea that the Zn²⁺ site of PerR_SA plays a structural rather than a peroxide sensing role. All these data together indicate that PerR_SA has a structural Zn²⁺ site coordinated by four cysteine residues and a regulatory metal binding site composed of His-43, Asp-91, His-97, His-99, and Asp-110.

In Vivo Repressor Activity of PerR_SA in Comparison with PerR_BS

To investigate the difference in metal- and H₂O₂-sensing ability of PerR_BS and PerR_SA, the repressor activities of PerR proteins were examined in MLMM using an mrgA promoter lacZ-fusion (P_mrgA-lacZ). As reported previously, PerR_BS repressed the P_mrgA-lacZ reporter fusion in the presence of either Fe²⁺ or Mn²⁺, and Fe²⁺-dependent repression was relieved upon H₂O₂ treatment (Fig. 2A) (7). PerR_SA repressed the P_mrgA-lacZ reporter fusion in the presence of Mn²⁺ but not in the presence of Fe²⁺, consistent with the previous finding that PerR_SA is a Mn²⁺-dependent repressor (Fig. 2B) (11). Interestingly, however, β-galactosidase expression was increased by about 2-fold in the presence of Fe²⁺ as reported previously (12) and further increased upon H₂O₂ treatment. The previous observation that this Fe²⁺-dependent induction of the mrgA gene is not observed with the perR null mutant S. aureus (12) suggests that the Fe²⁺- and H₂O₂-dependent increase of β-galactosidase expression is mediated by PerR_SA, although it is not clear whether Fe²⁺ directly interacts with PerR_SA or not.

FIGURE 2.

Comparison of activities between PerR_SA and PerR_BSin vivo. A and B, metal-dependent repressor activities of PerR_BS (A) and PerR_SA (B). B. subtilis cells expressing PerR_BS-FLAG (HB9738) and S. aureus cells expressing PerR_SA-FLAG (LS0111) were grown aerobically in MLMM supplemented with 10 μm FeSO₄ or 10 μm MnCl₂, and treated with 100 μm H₂O₂ (+H₂O₂) for 30 min or not (−H₂O₂). Repressor activities of PerR_BS and PerR_SA were measured using B. subtilis P_mrgA-lacZ and S. aureus P_mrgA-lacZ reporter fusions, respectively. Statistical analysis was performed with a Student's t test (*, p < 0.05; NS, not significant). C and D, repressor activities of PerR_BS and PerR_SA in B. subtilis (C) and in S. aureus (D). B. subtilis cells expressing no PerR (LB1532), PerR_BS-FLAG (HB9738), or PerR_SA-FLAG (LB1530), and S. aureus cells expressing no PerR (LS0124), PerR_BS-FLAG (LS0114), or PerR_SA-FLAG (LS0111) were used. Cells were grown aerobically in LB medium and treated with 100 μm H₂O₂ (+H₂O₂) for 30 min or not (−H₂O₂). Repressor activities of PerR proteins in B. subtilis were measured using B. subtilis P_mrgA-lacZ and those in S. aureus were measured using S. aureus P_mrgA-lacZ reporter fusions. A–D, repressor activities were expressed as Miller units of β-galactosidase activity (values represent the mean ± S.D. from three separate experiments). Statistical analysis was performed with a Student's t test (*, p < 0.05; NS, not significant). E and F, Western blot analysis of PerR_BS and PerR_SA in B. subtilis (E) and in S. aureus (F). B. subtilis cells expressing no PerR (LB1532), PerR_BS-FLAG (HB9738), or PerR_SA-FLAG (LB1530), and S. aureus cells expressing no PerR (LS0124), PerR_BS-FLAG (LS0114), or PerR_SA-FLAG (LS0111) were used. Cells were grown aerobically in LB medium and treated with 100 μm H₂O₂ (+H₂O₂) for 30 min or not (−H₂O₂). The FLAG-tagged PerR proteins were probed by anti-FLAG antibody.

To test whether differences in cellular milieu between B. subtilis and S. aureus affect the repressor activity of PerR proteins, PerR_BS and PerR_SA were expressed both in B. subtilis (Fig. 2, C and E) and S. aureus (Fig. 2, D and F). PerR_BS expressed in S. aureus repressed the S. aureus P_mrgA-lacZ reporter fusion with even higher repressor activity than PerR_SA, and responded normally to H₂O₂ as in B. subtilis (Fig. 2D). PerR_SA expressed in B. subtilis or in S. aureus repressed the B. subtilis P_mrgA-lacZ fusion or S. aureus P_mrgA-lacZ fusion, respectively, although not quite as efficiently as PerR_BS (Fig. 2, C and D). Interestingly, the repression levels of both the B. subtilis and S. aureus P_mrgA-lacZ reporter fusions by PerR_SA were similar to those by PerR_BS treated with H₂O₂ for 30 min. Furthermore, PerR_SA responded poorly to H₂O₂ (∼1.2-fold induction) both in B. subtilis and S. aureus when compared with the responsiveness of PerR_BS to H₂O₂ (more than 3-fold induction). Thus it is likely that the difference in responsiveness to metal and H₂O₂ between PerR_SA and PerR_BS is due to differences between the PerR proteins rather than the cellular environments. In summary the in vivo data indicate that Fe²⁺ addition appears to lead to apparent activation or derepression, rather than repression, of PerR_SA-regulated genes under our experimental conditions.

In Vitro PerR_SA Senses H₂O₂ by Iron-mediated Histidine Oxidation

To test whether PerR_SA is activated to bind DNA by both Mn²⁺ and Fe²⁺, we measured the apparent affinity of PerR_SA for Fe²⁺ and Mn²⁺ using a fluorescence anisotropy-based DNA-binding assay (Fig. 3A). Because PerR is immediately oxidized in the presence of Fe²⁺ under aerobic conditions as reported previously (7), all the FA experiments were performed under anaerobic conditions. Consistent with the observed Mn²⁺-dependent repressor activity of PerR_SA in vivo, the DNA-binding affinity of PerR_SA was increased by the addition of Mn²⁺. The apparent K_d for the Mn²⁺-dependent activation of PerR_SA was determined to be 9 μm, which is slightly weaker than that of PerR_BS (∼3 μm). Interestingly, despite the apparent lack of Fe²⁺-dependent repressor activity of PerR_SA in vivo, Fe²⁺ could also increase the DNA binding of PerR_SA in a concentration-dependent manner. The apparent K_d for the Fe²⁺-dependent activation of PerR_SA (0.1 μm) appeared to be the same as that of PerR_BS. These results therefore suggest that the apparent lack of Fe²⁺-dependent repressor activity and poor responsiveness to H₂O₂ of PerR_SA in vivo is not due to a decreased Fe²⁺ binding affinity of PerR_SA per se.

Because both Fe²⁺ and Mn²⁺ increase the DNA binding affinity of PerR_SA, we next investigated the effect of H₂O₂ on the DNA-binding activity of different metal-bound forms of PerR_SA (PerR_SA:Zn,Fe and PerR_SA:Zn,Mn) under anaerobic conditions (Fig. 3, B and C). Upon addition of 10 μm H₂O₂, PerR_SA:Zn,Fe completely lost DNA-binding activity in 20 s, whereas PerR_SA:Zn,Mn retained DNA-binding activity for 10 min even in the presence of 1 mm H₂O₂, indicating that PerR_SA utilizes Fe²⁺, but not Mn²⁺, for the sensing of low levels of H₂O₂ in vitro. Furthermore, loss of the DNA-binding ability of PerR_SA:Zn,Fe by H₂O₂ treatment could not be restored by the addition of Mn²⁺, indicating that H₂O₂ sensing by PerR_SA:Zn,Fe likely accompanies protein modification rather than simply the loss of bound Fe²⁺.

To test the hypothesis that H₂O₂ leads to a metal-dependent modification of PerR_SA, we analyzed the effect of H₂O₂ on different metallated forms of PerR_SA using MALDI-TOF MS (Fig. 3D). PerR_SA:Zn,Mn displayed no detectable changes in tryptic peptide peaks after 100 μm H₂O₂ treatment. However, H₂O₂-treated PerR_SA:Zn,Fe exhibited a significant decrease in the intensity of the T5 peptide (Tyr-36 to Arg-70) and T9* peptide (Phe-90 to Lys-107) with a concomitant increase in the intensity of two tryptic peptides corresponding to T5 + 16 and T9* + 16. The sites of oxidation responsible for this 16-Da mass increase were mapped to His-43 (corresponding to His-37 in PerR_BS) in the T5 peptide and His-97 (corresponding to His-91 in PerR_BS) in the T9* peptide using LC-ESI MS analysis (Fig. 1A and data not shown). Note that the T9* peptide also contains Cys-102 and Cys-105, which were detected in their fully alkylated form, indicative of no oxidation at cysteine residues after 100 μm H₂O₂ treatment, consistent with the structural role for these zinc-coordinating cysteine residues. These data indicate that PerR_SA, like PerR_BS, senses low levels of H₂O₂ by Fe²⁺-mediated oxidation of either of two histidine residues, His-43 and/or His-97, which are used as regulatory metal binding ligands.

Apparent Lack of Fe²⁺-dependent Repressor Activity of PerR_SA in Vivo Is Due to the Hypersensitivity of PerR_SA to Iron-mediated Oxidation under Aerobic Conditions

Since we observed the Fe²⁺-dependent DNA-binding activity and Fe²⁺-mediated histidine oxidation of PerR_SA in vitro, we wondered whether H₂O₂ sensing by histidine oxidation would also occur in vivo. To monitor the oxidation of PerR_SA in vivo, we analyzed the oxidation status of PerR_SA recovered by immunoprecipitation from S. aureus cells grown in MLMM supplemented with Fe²⁺ or Mn²⁺ using MALDI-TOF MS (Fig. 4). Interestingly, even without H₂O₂ treatment, almost all of the T5 peptide was detected as oxidized form (T5 + 16) and a significant amount of the T9* peptide was also detected as oxidized form (T9* + 16) for PerR_SA from cells grown in MLMM supplemented with Fe²⁺ or both Fe²⁺ and Mn²⁺. However, less oxidation was observed at both T5 and T9* peptides for PerR_SA from cells grown in MLMM supplemented with Mn²⁺ or no metal ion, and no significant further oxidation of these peptides was detected upon H₂O₂ treatment. These observations indicate that under aerobic growth conditions the majority of PerR_SA:Zn,Fe exists in an oxidized form even without external addition of H₂O₂.

To compare the sensitivity of PerR_SA with those of well known H₂O₂ sensors, E. coli OxyR and PerR_BS, we analyzed protein oxidation in E. coli using MALDI-TOF MS as described previously (22). As noted for PerR_SA recovered from S. aureus cells (Fig. 4), PerR_SA from aerobically grown E. coli cells exhibited a significant oxidation at the T5 peptide even in the absence of H₂O₂ treatment (Fig. 5A). However, PerR_SA from E. coli cells grown under oxygen-limited conditions exhibited no detectable oxidation at both T5 and T9* peptides, and oxidation of these peptides was observed upon external H₂O₂ treatment (Fig. 5B). In contrast, under aerobic conditions, PerR_BS exhibited less oxidation at both T5 and T11* peptides when compared with PerR_SA (Fig. 5C), and E. coli OxyR exhibited no detectable oxidation of both T19 and T20 peptides, which contain the peroxidatic cysteine (Cys-199) and resolving cysteine (Cys-208), respectively (Fig. 5D). These results indicate that PerR_SA is more sensitive than PerR_BS or E. coli OxyR to oxidation by low levels of H₂O₂, which are normally encountered during the aerobic growth of E. coli.

The above observations suggest that the poor H₂O₂ responsiveness and the apparent lack of Fe²⁺-dependent repressor activity of PerR_SA can be overcome under oxygen-limited growth conditions where limited amounts of H₂O₂ are generated. Consistent with this hypothesis, PerR_SA exhibited an increased repressor activity under oxygen-limited growth conditions (Fig. 6A) when compared with that under aerobic growth conditions (Fig. 2D). Furthermore, under these conditions PerR_SA responded to H₂O₂ (∼3-fold induction) much like PerR_BS, as judged by an increased β-galactosidase activity upon H₂O₂ treatment. We also investigated the metal-dependent H₂O₂-sensing ability of PerR_SA by growing cells in MLMM under oxygen-limited growth conditions. As shown in Fig. 6B, addition of either Fe²⁺ or Mn²⁺ enabled PerR_SA to repress the P_mrgA-lacZ reporter fusion, and β-galactosidase activity was increased by more than 2-fold upon H₂O₂ treatment in the presence of Fe²⁺ or in the presence of both Fe²⁺ and Mn²⁺, but only slightly (∼1.2-fold) in the presence of only Mn²⁺. These data demonstrate that PerR_SA functions as a Fe²⁺-dependent repressor and senses H₂O₂ in an Fe²⁺-dependent manner in vivo under oxygen-limited growth conditions (Fig. 6), as observed in vitro under anaerobic conditions (Fig. 3). We also note that the efficient sensing of H₂O₂ in the presence of both Fe²⁺ and Mn²⁺ is consistent with the higher affinity of PerR_SA for Fe²⁺ than Mn²⁺ as observed in vitro (Fig. 3A). In general, these results indicate that PerR_SA behaves in oxygen-limited cells much like PerR_BS does in B. subtilis, and that the apparent lack of Fe²⁺-dependent repressor activity (and thus poor H₂O₂ responsiveness) of PerR_SA under aerobic conditions is due to an efficient oxidation of PerR_SA by low levels of endogenous H₂O₂.

FIGURE 6.

In vivo repressor activities of PerR_SA under oxygen-limited conditions. A, repressor activities and H₂O₂ sensing abilities of PerR_BS and PerR_SA under oxygen-limited conditions. S. aureus cells expressing no PerR (LS0124), PerR_BS-FLAG (LS0114), or PerR_SA-FLAG (LS0111) were grown in LB medium under oxygen-limited conditions, and treated with 100 μm H₂O₂ (+H₂O₂) for 30 min or not (−H₂O₂). Repressor activities of PerR proteins were measured using S. aureus P_mrgA-lacZ reporter fusions. B, metal-dependent repressor activities of PerR_SA under oxygen-limited conditions. S. aureus cells expressing PerR_SA-FLAG (LS0111) were grown in MLMM supplemented with no metal ion, 10 μm FeSO₄, 10 μm MnCl₂, or both 10 μm FeSO₄ and 10 μm MnCl₂ under oxygen-limited conditions, and treated with 100 μm H₂O₂ (+H₂O₂) for 30 min or not (−H₂O₂). Repressor activities were measured using S. aureus P_mrgA-lacZ reporter fusion. A and B, repressor activities were expressed as Miller units of β-galactosidase activity (values represent the mean ± S.D. from three separate experiments). Statistical analysis was performed with a Student's t test (*, p < 0.05; NS, not significant).

Fe²⁺-mediated H₂O₂ Susceptibility of PerR_SA Is Important for H₂O₂ Resistance and Pathogenicity of S. aureus

It has been suggested that resistance to oxidative stress is an important factor for the survival and persistence of S. aureus (14). To investigate whether the difference in sensitivity of PerR proteins to oxidation affects the H₂O₂ resistance of S. aureus, we measured the growth of cells in the presence and absence of H₂O₂ (Fig. 7A). As expected the perR null mutant S. aureus cells exhibited an increased H₂O₂ resistance compared with those expressing PerR_SA. Also, compared with cells expressing PerR_SA, S. aureus cells expressing PerR_BS (which is less sensitive to Fe-mediated oxidation than PerR_SA) exhibited an increased H₂O₂ sensitivity. These data indicate that the ability of PerR_SA to respond to very low levels of H₂O₂ encountered during aerobic growth is important for the H₂O₂ resistance of S. aureus. Consistent with this, high levels of KatA activity were detected in the perR null mutant S. aureus cells, but low levels of KatA activity were detected from cells expressing PerR_BS, when compared with those expressing PerR_SA (Fig. 7B).

FIGURE 7.

Effects of PerR activity on H₂O₂ resistance and virulence of S. aureus. A, effects of PerR activity on the survival of S. aureus in the absence or presence of H₂O₂. S. aureus cells (3 μl of A₆₀₀ = 1 culture, with indicated dilutions) expressing no PerR (LS0093), PerR_BS-FLAG (LS0134), or PerR_SA-FLAG (LS0088) were spotted on LB-agar plate containing no H₂O₂ or 200 μm H₂O₂, and grown under aerobic conditions at 37 °C. Note that fresh LB agar plate shielded from light was used to prevent the generation of H₂O₂ by photochemical reactions (41). B, effects of PerR activity on the expression of KatA. S. aureus cells expressing no PerR (LS0093), PerR_BS-FLAG (LS0134), or PerR_SA-FLAG (LS0088) were grown in LB medium under either aerobic or oxygen-limited conditions, and treated with 100 μm H₂O₂ or not. Catalase activity staining was performed after separation of cell extract (10 μg protein) on native PAGE gel. C, effects of PerR activity on the pathogenicity of S. aureus using C. elegans as a model host. C. elegans killing was presented as Kaplan-Meier survival plots of worms fed with E. coli OP50 (negative control) (n = 86), S. aureus strain expressing no PerR (LS0093) (n = 86), PerR_BS-FLAG (LS0134) (n = 77), or PerR_SA-FLAG (LS0088) (n = 84).

Although the H₂O₂ defense enzymes, such as KatA and AhpC, which are under the control of PerR_SA play important roles in the nasal colonization and infection by S. aureus, it has been reported that they are not important for virulence (11, 14). However, interestingly, PerR is known to be required for virulence in other models of infection including murine skin abscess (11, 14), fruit fly (27), and zebrafish (28). We used a C. elegans model, which has been widely used as an invertebrate animal model for S. aureus pathogenesis (23), to investigate whether the difference in sensitivity of PerR proteins affects the virulence of S. aureus (Fig. 7C). As noted for other models of infection, the perR null mutant S. aureus was attenuated in the C. elegans model. Unexpectedly, S. aureus cells expressing PerR_BS killed C. elegans more rapidly than did those expressing PerR_SA. These results suggest that the virulence of S. aureus is somewhat directly correlated with the activity of PerR, given that heterologous PerR_BS, which is less sensitive to oxidation than PerR_SA, increases the virulence of S. aureus.

Discussion

PerR and PerR-like regulators have been described in a wide variety of organisms since the first characterization in B. subtilis (4, 5, 17, 29, 30). However, to date, the H₂O₂-sensing mechanism of PerR proteins has only been extensively studied for PerR_BS (7, 15, 21). Here we demonstrate that PerR_SA, previously regarded as a Mn²⁺-specific repressor, senses H₂O₂ using the same Fe²⁺-dependent histidine oxidation mechanism previously described for PerR_BS. Moreover we show that the apparent lack of Fe²⁺-dependent repressor activity, and the consequent Mn²⁺-specific repressor activity of PerR_SA in vivo, is due to the hypersensitivity of PerR_SA to H₂O₂ under aerobic conditions, rather than due to a decreased Fe²⁺ binding affinity of PerR_SA per se.

Several lines of evidence indicate that PerR_SA is a more sensitive H₂O₂ sensor than either PerR_BS or E. coli OxyR. The majority of PerR_SA in aerobically grown S. aureus is detected in an oxidized form (Fig. 4), whereas only partial oxidation is observed with PerR_BS from aerobically grown B. subtilis (7). However, the interpretation of this result can be complicated by potential differences in the levels of endogenous H₂O₂ between these two species. Therefore, we directly compared the levels of oxidized PerR proteins when both were expressed in either S. aureus or B. subtilis. Indeed the direct measurement of KatA activity (Fig. 7B) and reporter fusion assays (Fig. 2, C and D) indicate that the expression levels of PerR_SA-regulated genes are higher in cells expressing PerR_SA than in those expressing PerR_BS. This indicates that under otherwise identical conditions, oxidation levels of PerR_SA are higher than those of PerR_BS in both B. subtilis and S. aureus, consistent with the hypothesis that PerR_SA is intrinsically more susceptible to oxidation than PerR_BS. Furthermore, we also used E. coli, where H₂O₂ detoxification systems are under the control of OxyR, as neutral host to directly compare the sensitivity of PerR proteins and OxyR protein. The rate of H₂O₂ generation in aerobically growing E. coli is ∼10 μm per s (31), however, the steady-state concentration of H₂O₂ is kept ∼50 nm by the action of scavenging enzymes such as AhpC (1). Normally, under these routine aerobic growth conditions, OxyR is inactive: OxyR is activated when the intracellular H₂O₂ concentration reaches ∼200 nm (1, 32, 33). PerR_BS has a second-order rate constant of ∼10⁵ m⁻¹ s⁻¹ for inactivation by H₂O₂, which is comparable with that of E. coli OxyR (7). Consistent with this, PerR_BS and E. coli OxyR exhibit no significant oxidation in aerobically grown E. coli (Fig. 5, C and D). In contrast, a significant oxidation of PerR_SA is observed in aerobically grown E. coli without external H₂O₂ treatment, indicating that endogenously produced H₂O₂ is sufficient to oxidize PerR_SA (Fig. 5A). Collectively these indicate that PerR_SA senses very low levels of H₂O₂ (as little as ∼50 nm) as generated during normal aerobiosis in E. coli, levels that do not significantly oxidize PerR_BS or E. coli OxyR. Corroborating with this, it has recently been reported that OxyR2 from Vibrio vulnificus is activated under normal aerobic growth conditions, whereas OxyR1, an E. coli OxyR homologue, is only activated by exogenous H₂O₂ (34).

The efficient sensing of H₂O₂ and induction of defense enzymes have been considered crucial for pathogens that have to fight against H₂O₂ assault by macrophages or neutrophils (35, 36). However, the perR null mutant S. aureus strain, which is more resistant to H₂O₂ than the wild type by constitutive expression of H₂O₂ defense enzymes, exhibits attenuated virulence in our C. elegans model of infection (Fig. 7C) as observed with other models of infection (11, 27, 28). Moreover, the H₂O₂-sensitive S. aureus strain by the expression of PerR_BS (Fig. 7, A and B) is not attenuated in C. elegans (Fig. 7C), consistent with the previous finding that neither KatA nor AhpC are required for resistance to neutrophil-dependent killing or virulence of S. aureus (14). Instead, the expression of PerR_BS, which is less sensitive to H₂O₂ compared with PerR_SA, increases the virulence suggesting that the activity of PerR positively correlates with the virulence of S. aureus. This may imply that the inactivation of PerR by H₂O₂, rather than the direct poisoning of bacteria by H₂O₂, can be exploited by phagocytic cells that wish to reduce the virulence of S. aureus. It is not clear why inactivation of PerR activity reduces the virulence of S. aureus. One possible explanation would be poor growth of S. aureus in the iron-limited host environment. Derepression of PerR-regulon is likely to lead to Fe²⁺ deficiency due to the elevated expression of KatA, which consumes Fe²⁺, and Fur, which represses Fe²⁺ uptake, as observed with B. subtilis (37). Alternatively, or in addition, active PerR may be involved in the induction of the virulence factor, either directly or indirectly. Indeed it has been shown that Streptococcus pyogenes PerR regulates an extracellular virulence factor, MF3, directly (38). All together, our findings that the activity of PerR is directly linked to the virulence of S. aureus suggests that PerR_SA can be an attractive target for a novel approach to design new drugs for S. aureus treatment (39).

In contrast to virulence, H₂O₂ resistance is inversely correlated with the activity of PerR because the H₂O₂ defense systems are derepressed by H₂O₂-mediated PerR inactivation. Our results clearly show this relationship (Fig. 7, A and B). As reported previously the perR null mutant S. aureus exhibits increased resistance to H₂O₂, whereas S. aureus expressing PerR_BS exhibits an increased sensitivity to H₂O₂ than that expressing PerR_SA, presumably due to the hyper-repression of PerR_SA-regulated genes by PerR_BS. Although KatA and AhpC are not required for virulence, they are known to play important roles for survival under aerobic conditions and especially for colonization at the anterior nares, which are the primary ecological niche for S. aureus (14). Considering that most of the PerR_SA is fully oxidized and no significant further derepression of PerR regulon is triggered by H₂O₂ treatment under aerobic conditions, it is likely that S. aureus, as a facultative anaerobic bacterium, has evolved PerR_SA to sense low levels of endogenous H₂O₂ normally encountered under aerobic environment, rather than to sense higher levels of external H₂O₂ produced by microbial competitor or by host immune system.

Author Contributions

C. J., J. K., J. H., and J. L. conceived and designed the experiments. C. J., J. K., Y. W., Y. L., T. C., and S. J. performed the experiments. C. J., J. K., H. Y., J. H., and J. L. analyzed the data and wrote the paper.