Next Article in Journal
The NIN-Like Protein OsNLP2 Negatively Regulates Ferroptotic Cell Death and Immune Responses to Magnaporthe oryzae in Rice
Next Article in Special Issue
Transposon-Directed Insertion-Site Sequencing Reveals Glycolysis Gene gpmA as Part of the H2O2 Defense Mechanisms in Escherichia coli
Previous Article in Journal
Exogenous Betaine Enhances the Protrusion Vigor of Rice Seeds under Heat Stress by Regulating Plant Hormone Signal Transduction and Its Interaction Network
Previous Article in Special Issue
Endophytic Fungal Consortia Enhance Basal Drought-Tolerance in Moringa oleifera by Upregulating the Antioxidant Enzyme (APX) through Heat Shock Factors
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Antioxidants
Volume 11
Issue 9
10.3390/antiox11091793
antioxidants-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

The Catalase KatA Contributes to Microaerophilic H2O2 Priming to Acquire an Improved Oxidative Stress Resistance in Staphylococcus aureus

by
Nico Linzner
,
Vu Van Loi
and
Haike Antelmann
*
Institute of Biology-Microbiology, Freie Universität Berlin, 14195 Berlin, Germany
*
Author to whom correspondence should be addressed.
Antioxidants 2022, 11(9), 1793; https://doi.org/10.3390/antiox11091793
Submission received: 8 August 2022 / Revised: 29 August 2022 / Accepted: 7 September 2022 / Published: 12 September 2022
(This article belongs to the Special Issue A Lesson from Microorganisms: How to Counteract Oxidative Stress)
Browse Figures
Versions Notes

Abstract

:
Staphylococcus aureus has to cope with oxidative stress during infections. In this study, S. aureus was found to be resistant to 100 mM H2O2 during aerobic growth. While KatA was essential for this high aerobic H2O2 resistance, the peroxiredoxin AhpC contributed to detoxification of 0.4 mM H2O2 in the absence of KatA. In addition, the peroxiredoxins AhpC, Tpx and Bcp were found to be required for detoxification of cumene hydroperoxide (CHP). The high H2O2 tolerance of aerobic S. aureus cells was associated with priming by endogenous H2O2 levels, which was supported by an oxidative shift of the bacillithiol redox potential to −291 mV compared to −310 mV in microaerophilic cells. In contrast, S. aureus could be primed by sub-lethal doses of 100 µM H2O2 during microaerophilic growth to acquire an improved resistance towards the otherwise lethal triggering stimulus of 10 mM H2O2. This microaerophilic priming was dependent on increased KatA activity, whereas aerobic cells showed constitutive high KatA activity. Thus, KatA contributes to the high H2O2 resistance of aerobic cells and to microaerophilic H2O2 priming in order to survive the subsequent lethal triggering doses of H2O2, allowing the adaptation of S. aureus under infections to different oxygen environments.

1. Introduction

Staphylococcus aureus is a major human pathogen that can cause local skin and soft tissue infections, as well as life-threatening diseases, such as septicaemia, endocarditis, necrotizing pneumonia and osteomyelitis [1,2,3]. During infections, S. aureus has to cope with reactive oxygen species (ROS), such as superoxide anion (O2•−) and hydrogen peroxide (H2O2) [4], which are produced during the oxidative burst of activated macrophages and neutrophils to kill the invading pathogen [5,6,7,8]. The NADPH oxidase (NOX2) in the phagosomal membrane catalyses the one-electron transfer to molecular oxygen (O2) to generate O2•−, which is dismutated to H2O2 either spontaneously or by superoxide dismutases (SODs), including the extracellular SOD3 [6,8,9,10,11]. Both SOD3 and NOX2 are contained in secretory vesicles in macrophages and neutrophils and the fusion of these vesicles with phagosomes during phagocytosis might provide a mechanism for catalysed H2O2 production [11]. However, in neutrophils, the myeloperoxidase MPO is released from azurophilic granula into the phagosomal lumen, catalysing the dismutation of O2•− to H2O2 upon infection [6]. MPO further converts H2O2 with chloride to the highly reactive hypochlorous acid (HOCl), which is the most potent oxidant and microbicidal agent released by activated neutrophils [6,9,10]. In addition, S. aureus encounters endogenous ROS during aerobic respiration due to the stepwise one-electron transfer reactions to O2, leading to production of O2•− and H2O2 [12]. In the Fenton reaction, H2O2 reacts with free Fe2+ to generate the highly toxic hydroxyl radical (OH•), which can damage all cellular macromolecules, resulting in oxidation of proteins, lipids and carbohydrates [12,13,14]. However, H2O2 and HOCl can also function in redox signalling to activate or inactivate specific redox-sensitive regulators, which control defence mechanisms and confer resistance against the oxidants in bacterial pathogens [14,15].
S. aureus uses various enzymatic and non-enzymatic ROS and HOCl detoxification systems, such as a unique catalase (KatA), several peroxiredoxins (AhpC, Tpx, Bcp) and the low-molecular-weight (LMW) thiol bacillithiol (BSH) [14,16]. BSH associates with the bacilliredoxin/BSH/YpdA redox pathway to regenerate oxidized protein thiols and bacillithiol disulfide (BSSB) [16]. We previously constructed a Brx-roGFP2 fused biosensor to monitor the changes in the BSH redox potential (EBSH) under oxidative stress in S. aureus [17]. This study already revealed that S. aureus is highly resistant to 100 mM H2O2, since the Brx-roGFP2 biosensor responded only weakly to high H2O2 levels, leading to small EBSH changes [17]. The catalase KatA was identified as the major H2O2 detoxification enzyme, which conferred the constitutive H2O2-resistant phenotype to aerobically grown S. aureus cells [18,19]. KatA is also important for nasal colonization and mediates protection under macrophage and neutrophil infections [18,20,21,22]. The peroxiredoxin AhpCF showed compensatory roles in resistance to H2O2 and organic hydroperoxides (OHPs) and contributed to nasal colonization [20]. OHPs (ROOH) are generated during oxidation of polyunsaturated fatty acids of eukaryotic membrane lipids and are reduced by peroxiredoxins to their corresponding organic alcohols [23].
In general, AhpC, Tpx and Bcp can be classified into typical (AhpC) or atypical (Tpx, Bcp) 2-Cys peroxiredoxins based on their thiol-oxidation mechanism between the peroxidatic (CP) and resolving Cys (CR), involving inter- or intramolecular disulfides, respectively [24]. The functions and substrates of AhpC, Tpx and Bcp have been previously studied in Escherichia coli. AhpC detoxification of H2O2 leads to formation of an oxidized AhpC dimer, which aggregates to an oligomer with chaperone functions [24,25]. Regeneration of AhpC requires the NADPH-dependent flavin disulfide reductase AhpF as a redox partner [24,25]. The thiol-peroxidase Bcp of E. coli is induced by H2O2, OHPs and during aerobic growth and confers resistance against H2O2 and OHP stress [26]. The thiol-peroxidase Tpx of E. coli has been shown to catalyse detoxification of H2O2 and OHPs in vitro and is recycled by the Trx/TrxR system [27,28]. In S. aureus, Tpx responds strongly to H2O2 and other thiol-reactive compounds and was oxidized in the redox proteome under HOCl stress [14,29]. However, the detailed functions of the peroxiredoxins AhpC, Tpx and Bcp in peroxide resistance, detoxification and survival have not been studied thus far in S. aureus.
In S. aureus, transcription of katA, ahpCF and bcp is strongly induced only by high levels of 10 mM H2O2 and controlled by the peroxide-responsive PerR repressor [18,20,30]. In Bacillus subtilis, KatA is also a member of the PerR regulon but already inducible by sub-lethal doses of 100 µM H2O2 [31,32]. Pretreatment of B. subtilis cells with sub-lethal H2O2 as “priming stimulus” confers improved resistance towards subsequent lethal H2O2 doses, termed as “triggering stimulus”, which are encountered as future stress [33,34]. These terms and abbreviations—priming (P), priming plus triggering (PT) and triggering (T)—were previously introduced within our project SFB973, which was directed to priming and memory of stress responses in different organisms, including bacteria, fungi and plants [35]. In B. subtilis, the H2O2 priming effect was shown to be mediated by KatA, which is induced by a mild stress to prepare the cells for better survival when faced with future lethal oxidative stress [33]. Similarly, H2O2 priming for improved resistance towards the triggering stimulus was dependent on the OxyR-dependent enzymes KatG and AhpCF in E. coli and Salmonella Typhimurium [32,36,37]. Although S. aureus exhibits constitutive H2O2 resistance during aerobic growth, it is unknown whether priming for improved H2O2 resistance is possible under aerobic or microaerophilic conditions.
In this study, we used growth and survival phenotype analyses, Brx-roGFP2 biosensor measurements and transcriptional studies to investigate the functions of KatA and the peroxiredoxins AhpC, Tpx and Bcp in peroxide resistance, detoxification and priming during aerobic and microaerophilic growth. Our results showed that S. aureus is H2O2 primable for improved resistance only under microaerophilic conditions, which are dependent on KatA. In contrast, aerobic growth already leads to increased levels of ROS, which causes KatA-dependent aerobic priming for constitutive H2O2 resistance. While KatA confers H2O2 resistance in S. aureus, the peroxiredoxins AhpC, Tpx and Bcp were shown to contribute to survival and resistance under CHP stress and regeneration of reduced EBSH upon recovery from CHP stress.

2. Materials and Methods

2.1. Bacterial Strains, Growth and Survival Assays

Bacterial strains, plasmids and primers are described in Tables S1–S3. For genetic manipulation, E. coli was cultivated in Luria Broth (LB) medium. S. aureus COL strains were grown in RPMI medium to an optical density at 500 nm (OD500) of 0.5 and exposed to H2O2, cumene hydroperoxide (CHP) or hypochlorous acid (HOCl), followed by determination of colony-forming units (CFUs) in survival assays as previously described [38]. Each experiment was performed in at least three independent biological replicates and the results are presented as mean values with standard deviations (SD) from all biological replicates, as indicated in each figure legend. Statistical analysis was performed using Student’s unpaired two-tailed t-test with the software Graph Prism. The biochemical compounds were purchased from Sigma Aldrich. The HOCl concentration was determined as previously described [39].

2.2. Construction of the S. aureus COL ΔkatA, ΔahpC, ΔahpCΔkatA, Δtpx, Δbcp and ΔperR Mutants and Complemented Strains

The S. aureus COL ΔkatA mutant and katA complemented strains were previously constructed [40]. The S. aureus ΔahpC, Δtpx, Δbcp and ΔperR deletion mutants were constructed using the temperature-sensitive E. coli-S. aureus shuttle vector pMAD as previously described [41]. In brief, 500 bp of the up- and downstream flanking regions of the specific genes were fused by PCR, digested with BglII and SalI and ligated into pMAD. The constructs were electroporated into the restriction-negative S. aureus RN4220, followed by phage transduction using phage 81 into S. aureus COL [42]. For construction of the ΔahpCΔkatA double mutant, the plasmid pMAD-∆katA of S. aureus RN4220-pMAD-∆katA was transduced by the phage 81 into the S. aureus COL ΔahpC mutant. Selection of the ΔahpC, Δbcp, Δtpx, ΔperR and ΔahpCΔkatA deletion mutants was performed as previously described [38].
Construction of the His-tagged S. aureus ahpC, bcp and tpx complemented strains was performed using the plasmid pRB473 as previously described [17]. The genes were cloned into pRB473 after digestion with BamHI and KpnI/SacI, resulting in plasmids pRB473-ahpC-His, pRB473-bcp-His and pRB473-tpx-His, which were transduced in the ΔahpC, Δbcp and Δtpx deletion mutants. In addition, the plasmid pRB473-brx-roGFP2 [17] was introduced into the S. aureus COL ΔkatA, ΔahpC, ΔahpCΔkatA, Δtpx and Δbcp deletion mutants to construct the Brx-roGFP2 biosensor expressing catalase- and peroxiredoxin-deficient mutant strains.

2.3. Priming and Triggering Experiments

For priming and triggering, the S. aureus strains were grown aerobically under vigorous agitation in shake flasks in a shaking water bath at 150 rpm or microaerophilically in 50 mL Falcon tubes including 40 mL cultures with closed lids without shaking, as in previous publications [43,44]. At an OD500 of 0.3, naïve S. aureus cells were primed by adding sub-lethal doses of 0.1 or 1 mM H2O2, respectively, to the bacterial culture for ~30 min. Subsequently, the lethal triggering doses of 10 or 40 mM H2O2, respectively, were added to the primed bacterial cultures, followed by counting of CFUs after 2 and 4 h of growth. For triggering only, naïve cells were treated with 10 or 40 mM H2O2 at an OD500 of 0.4, followed by counting of CFUs after 2 and 4 h of growth.

2.4. Brx-roGFP2 Biosensor Measurements

To monitor the EBSH changes after H2O2 and CHP stress, we used the Brx-roGFP2 biosensor expressing WT, ΔkatA, ΔahpC, ΔahpCΔkatA, Δtpx and Δbcp mutant strains and performed injection assays with the oxidants. For measurements of Brx-roGFP2 oxidation during microaerophilic and aerobic H2O2 priming and triggering experiments, the S. aureus COL strain expressing Brx-roGFP2 was cultivated in LB medium to an OD540 of 0.3 and challenged with the priming dose of 0.1 mM H2O2 for 30 min, followed by the triggering dose of 10 mM H2O2, as described above. Samples were harvested from S. aureus cells in the naïve (C), primed (P), primed and triggered (PT) and triggered-only (T) states, alkylated with 10 mM N-ethylmaleimide (NEM), washed and resuspended in PBS with 10 mM NEM. The Brx-roGFP2 oxidation degree (OxD) and EBSH changes were determined in the S. aureus strains during oxidant injection or in samples harvested at C, P, PT and T as previously described [17,45]. For fully reduced and oxidized controls, biosensor strains were treated with 10 mM DTT and 5 mM diamide, respectively. The Brx-roGFP2 fluorescence emission was measured at 510 nm after excitation at 405 and 488 nm using the CLARIOstar microplate reader (BMG Labtech). The OxD of the Brx-roGFP2 biosensor was determined for each sample and normalized to fully reduced and oxidized controls as previously described [17,45].

2.5. Northern Blot Analyses

To analyze transcription of katA, dps and ahpCF in the S. aureus COL WT, ΔkatA, ΔahpC and ΔperR mutants using Northern blots, the S. aureus strains were grown in RPMI medium and harvested during the log phase at an OD500 of 0.4. To investigate katA and ahpC induction in the priming and triggering experiments, S. aureus WT cells were harvested in the naïve (C), primed (P), primed and triggered (PT) and triggered-only (T) states, as explained in the figure legends. RNA isolation was performed using the acid phenol extraction protocol as described previously [46]. Northern blot hybridizations were conducted using digoxigenin-labelled antisense RNA probes for katA, ahpC and dps that were synthesized in vitro using T7 RNA polymerase and the corresponding primers katA-NB-for/rev and ahpC-NB-for/rev, as previously described [46]. The dps antisense RNA probe was constructed previously [44].

2.6. Determination of the Catalase Activity Using Native PAGE and Diaminobenzidine Staining

To analyze catalase activities in S. aureus COL strains, protein extracts were prepared under native conditions and 50 µg of each sample was separated by non-denaturing 10% polyacrylamide gel electrophoresis. The gel was stained for catalase activity using 50 µg/mL horseradish peroxidase coupled with 5 mM H2O2 and 0.5 mg/mL diaminobenzidine, as described previously [47,48].

3. Results

3.1. S. aureus Exhibits KatA-Dependent H2O2 Resistance during Aerobic Growth

To investigate the roles of the catalases and peroxiredoxins in the constitutive H2O2 resistance of S. aureus COL, phenotype analyses of the ΔkatA, ΔahpC, ΔahpCΔkatA, Δtpx and Δbcp mutants and the complemented strains were performed during the aerobic growth under H2O2 stress (Figure 1; Figures S1 and S2). In agreement with previous findings [20], the ΔkatA mutant was strongly impaired in growth after exposure to 10 mM H2O2 and did not survive doses of 40 mM H2O2 (Figure 1A,E). The S. aureus COL wild type (WT) was able to grow with low doses of 1 mM H2O2 and survived to 330 and 725% after 2 and 4 h, respectively (Figure 1F; Figure S1B). However, the ΔkatA mutant was hypersensitive to peroxide stress, since the growth was inhibited by 0.4 and 1 mM H2O2 and only 32% and 4% of cells survived the 1 mM H2O2 treatment after 2 and 4 h, respectively (Figure 1F; Figure S1B). Complementation of the ΔkatA mutant with pRB473-encoded katA could only partially restore the H2O2 resistance to WT level after treatment with 0.4–1 mM H2O2 (Figure 1F; Figure S1A–C). This incomplete recovery of the WT resistance was due to lower catalase activity in the katA complemented strain, as confirmed using the diaminobenzidine gel staining method (Figure S1D).
In addition, the H2O2 resistance of WT cells was 1.5–4-fold further enhanced during the stationary phase, whereas the ΔkatA mutant did not survive 40 mM H2O2 during the log and stationary phases (Figure 1G). These data indicate that KatA also contributes to the stationary-phase H2O2 resistance in S. aureus. In contrast, the peroxiredoxin-deficient S. aureus Δtpx and Δbcp mutants were not impaired in growth or survival after exposure to 10 and 40 mM H2O2 (Figure 1C–E). However, the ΔahpC mutant displayed an increased H2O2 resistance (Figure 1B,E), which was mediated by the PerR-dependent up-regulation of KatA in the ΔahpC mutant, as shown previously [20]. To validate the derepression of PerR regulon genes in the ΔahpC mutant, we analysed the transcription of katA and the miniferritin-encoding dps gene, since both were strongly up-regulated under different thiol-stress conditions in the transcriptome of S. aureus WT cells [43,49,50]. The Northern blot results revealed the twofold up-regulation of katA, while dps transcription was induced at an 11-fold higher rate in the ΔahpC mutant, supporting the derepression of both PerR regulon genes in the ΔahpC mutant under non-stress conditions (Figure 1H).
However, the H2O2-resistant phenotype of the ΔahpC mutant could not be reversed to WT levels in growth and survival assays upon exposure to 10 and 40 mM H2O2 in the ahpC complemented strain (Figure 1E and Figure S2A). This lack of complementation might be caused by the lower plasmid-borne AhpC expression compared to the highly abundant AhpC in WT cells, as previously observed in the proteome [51]. The catalase activity was higher in the ΔahpC mutant and ahpC complemented strain than in the WT, explaining the high H2O2 resistance upon complementation (Figure S2B). To confirm whether KatA and AhpC play additive roles in H2O2 resistance, the growth and survival of the ΔahpCΔkatA double mutant was analysed. In agreement with previous data, the ΔahpCΔkatA mutant showed a slower aerobic growth (Figure S3A) [20] and displayed 3-fold and 13-fold reduced survival rates after exposure to 1 mM H2O2 for 2 h and 4 h, respectively, as compared to the ΔkatA mutant (Figure 1F).
Taken together, these results indicate that KatA plays the major role of conferring strong H2O2 resistance to aerobic S. aureus cells during the log and the stationary phases, whereas AhpC makes a minor contribution to the H2O2 resistance during the aerobic growth. Thus, KatA was identified as major determinant of the H2O2 resistance in growing and non-growing S. aureus cells.

3.2. The ΔkatA Mutant Shows a Strong Oxidative Shift in the EBSH after H2O2 Stress and Is Impaired in Its Regeneration of the Reduced State, as Revealed by the Brx-roGFP2 Biosensor

To monitor the changes in the EBSH in the catalase- and peroxiredoxin-deficient mutants, we measured the Brx-roGFP2 biosensor responses after H2O2 stress in the WT and mutant strains. Due to the strong aerobic H2O2 resistance of S. aureus WT cells, the Brx-roGFP2 biosensor responded only weakly to 10 mM H2O2 in our previous studies [17]. Thus, we first used 100 H2O2 for WT cells, leading to fast biosensor oxidation and regeneration of the reduced state of EBSH within two hours, as in our previous studies (Figure 2A) [17].
No increased biosensor oxidation was measured in WT cells after exposure to 1 mM H2O2 (Figure 2B). In contrast to WT cells, the biosensor was fully and constitutively oxidized by 1 and 100 mM H2O2 in the ΔkatA mutant, indicated by an impaired regeneration of reduced EBSH (Figure 2A,B). The katA mutant was only able to recover the reduced state of EBSH after treatment with 0.4 mM H2O2 (Figure 2C), suggesting that this low H2O2 level might be detoxified by AhpC. In support of this hypothesis, the Brx-roGFP2 biosensor was quickly oxidized and strongly delayed in the recovery of reduced EBSH after exposure to 0.4 mM H2O2 in the ΔahpCΔkatA double mutant (Figure S3B). In contrast, the ΔahpC, Δtpx and Δbcp mutants showed similar H2O2 responses and regeneration of reduced EBSH compared to the WT (Figure 2D–F). The biosensor results confirmed the hypersensitivities of the ΔkatA and ΔahpCΔkatA mutants towards H2O2 stress, supporting that KatA was responsible for the rapid detoxification of 100 mM of H2O2 and regeneration of EBSH in S. aureus WT cells, while AhpC could only detoxify low levels of 0.4 mM H2O2 in the absence of KatA. However, the peroxiredoxins Tpx and Bcp were not essential for H2O2 detoxification in S. aureus WT cells.

3.3. S. aureus Shows KatA-Dependent Microaerophilic H2O2 Priming to Acquire an Improved Resistance towards Lethal H2O2 Doses

The previous data revealed that KatA confers strong H2O2 resistance during aerobic growth. However, the role of KatA in the priming of S. aureus for improved H2O2 resistance during microaerophilic conditions was not investigated. Thus, the S. aureus WT and the ΔkatA mutant were grown under microaerophilic conditions to the log phase and primed with 0.1 mM H2O2 for 30 min, followed by triggering with 10 mM H2O2 (Figure 3A). The growth and survival were analysed in naïve (C), primed (P), primed and triggered (PT) and triggered bacteria (T) (Figure 3A).
The primed S. aureus WT and ΔkatA mutant (P) were not impaired in growth and survival under 0.1 mM H2O2 (Figure 3B–E). However, the primed and triggered WT (PT) could acquire an improved resistance towards the triggering stimulus of 10 mM H2O2 compared to the triggering-only state (T) (Figure 3B,D). Specifically, PT bacteria showed survival rates of 52–72%, whereas T bacteria were almost killed and survived only to <0.07% after 10 mM H2O2 treatment. This indicates that S. aureus is primable for improved oxidative stress resistance during microaerophilic growth (Figure 3B,D). However, in contrast to the WT, the primed ΔkatA mutant strain was unable to acquire the improved resistance towards otherwise lethal doses of 10 mM H2O2 under microaerophilic conditions (Figure 3C,E). Both PT and T bacteria of the ΔkatA mutant were strongly impaired in growth and completely killed after treatment with 10 mM H2O2 as a triggering stimulus (Figure 3C,E).
However, due to its lower catalase activity from plasmid-based KatA expression (Figure S1D), the katA complemented strain did not recover the improved H2O2 resistance upon microaerophilic priming, resulting in growth inhibition and killing of PT and T bacteria (Figure S4). Overall, these results indicate that KatA is responsible for S. aureus priming for improved resistance against upcoming lethal H2O2 stress under microaerophilic conditions. Thus, KatA confers the constitutive resistance during aerobic growth and prepares S. aureus for future oxidative stress under microaerophilic conditions.

3.4. S. aureus Is Not Primable for Improved H2O2 Resistance during Aerobic Growth

Next, priming and triggering experiments were performed in aerobically grown S. aureus cells to analyse whether the constitutive H2O2 resistance could be further enhanced in primed cells (Figure 4). First, we used the same H2O2 doses for the priming (0.1 mM) and triggering (10 mM) experiments as applied in the microaerophilic experiments (Figure 4A). As expected, there were no differences in growth and survival between PT and T bacteria after exposure to 10 mM H2O2 during the aerobic growth (Figure 4B,C). Both PT and T bacteria were similarly resistant and fully survived the 10 mM H2O2 triggering dose. Thus, the constitutive resistance of aerobic S. aureus cells towards 10 mM H2O2 could not be further enhanced by pre-exposure to the priming stimulus of 0.1 mM H2O2 (Figure 4B,C). As shown before, this constitutive H2O2 resistance of aerobically grown S. aureus cells was dependent on KatA (Figure 1A,E,F).
We further increased the H2O2 doses for priming (1 mM H2O2) and triggering (40 mM) of S. aureus during the aerobic growth (Figure 4D). However, higher priming doses also could not improve the growth and survival of PT bacteria in response to the subsequent 40 mM H2O2 stress compared to the T bacteria treated with 40 mM H2O2 only (Figure 4E,F). Both PT and T bacteria were strongly impaired in growth under 40 mM H2O2 stress and showed survival rates of <10% after 2 h.
Small survival differences of 4% were observed after 4 h in T versus PT bacteria but not at the 2 h time point (Figure 4F). In conclusion, these different priming setups support that S. aureus is not primable for enhanced H2O2 resistance under aerobic conditions. We hypothesize that ROS production during aerobic respiration acts as a priming stimulus to induce KatA, which confers the high H2O2 resistance.

3.5. Microaerophilic H2O2 Priming Causes Increased Transcription of KatA and Elevated KatA Activity, which Confers Improved Resistance towards Lethal H2O2 Doses in S. aureus

Northern blot analyses were used to study whether microaerophilic H2O2 priming induces transcription of the PerR-dependent katA gene and the ahpCF operon in S. aureus (Figure 5). The results revealed that transcription of katA was significantly up-regulated by 1.8-fold upon microaerophilic priming with 0.1 mM H2O2 (Figure 5B,D), whereas the basal level of katA transcription was already higher under aerobic conditions and could not be further induced during aerobic H2O2 priming (Figure 5C,E). Transcription of the ahpCF operon was not significantly induced during microaerophilic priming (Figure 5B,G). However, transcription of katA and ahpCF was strongly reduced after triggering by 10 mM H2O2 under microaerophilic conditions, since the triggering dose was lethal (Figure 5B,D,G). Transcription of katA decreased even in the PT bacteria compared to P cells, which highlights the high efficiency of the KatA protein for fast removal of H2O2 in PT bacteria (Figure 5B,D). Under aerobic conditions, PT and T bacteria did not show significantly enhanced transcription of katA and ahpCF, supporting that the constitutive H2O2 resistance could not be further enhanced by pre-exposure to the priming dose (Figure 5C,E,H).
The katA transcript levels could be confirmed by catalase activities during the microaerophilic and aerobic priming experiments (Figure 5F). Specifically, the basal activity of KatA was very low during the microaerophilic growth, whereas aerobic cells showed a constitutive high catalase activity. The KatA activity could be only enhanced upon microaerophilic priming but not during aerobic priming due to the high constitutive resistance (Figure 5F). Further consistent with the Northern blots, the KatA activity decreased strongly in PT and T bacteria during microaerophilic priming. Together, these results reveal that microaerophilic priming induces katA transcription and KatA activity, which confers improved resistance against otherwise lethal H2O2 stress.

3.6. Microaerophilic H2O2 Priming Leads to a Strong Oxidative Shift in the EBSH

We were interested in the EBSH differences between aerobic and microaerophilic growth conditions, supporting the enhanced ROS levels in aerobic cells as endogenous priming stimuli. Moreover, we aimed to analyse the Brx-roGFP2 biosensor response upon microaerophilic priming and triggering to investigate if the KatA induction upon microaerophilic priming is accompanied by a change in the Brx-roGFP2 biosensor oxidation. The comparison of the basal biosensor oxidation revealed a more reducing basal OxD of 0.1 and an EBSH of −310 mV during microaerophilic growth (C1) compared to the OxD of 0.3 and the EBSH of −291 mV during aerobic growth (C1) (Figure 5I,J). Thus, the higher basal oxidation in aerobic cells accounted for the increased ROS level due to aerobic respiration. Upon microaerophilic priming, the Brx-roGFP2 biosensor showed a fivefold increased OxD and an oxidized EBSH of −281 mV, which was further oxidized in PT and T bacteria. In contrast, the biosensor did not respond to aerobic priming and showed increased oxidation only in aerobic PT and T bacteria (Figure 5I,J). These results support that microaerophilic priming leads to an oxidative shift in the EBSH from −310 mV to −281 mV, resulting in increased catalase expression that primes the cells for improved H2O2 resistance. Due to the higher basal oxidation in aerobic cells, priming did not change the high EBSH of −291 mV, which is consistent with the constitutive KatA expression (Figure 5I,J). These results on the EBSH differences of ~20 mV between microaerophilic and aerobic cells strongly support that respiratory H2O2 primes aerobic cells for constitutive H2O2 resistance.

3.7. The Peroxiredoxins AhpC, Tpx and Bcp Mediate CHP Resistance in S. aureus

While the role of KatA in aerobic H2O2 resistance and microaerophilic H2O2 priming was clearly revealed, the peroxiredoxins AhpC, Tpx and Bcp could also function in the resistance to organic hydroperoxides in S. aureus. Using growth and survival assays, the phenotypes of the catalase and peroxiredoxin-deficient mutants were analysed after CHP treatment (Figure 6). While the growth of the ΔkatA and Δbcp mutants was not affected by 0.15 mM CHP stress, the CHP-treated ΔahpC and Δtpx mutants showed slightly reduced growth rates (Figure 6A–D), which could be restored to WT levels upon complementation (Figure 6E,F). In addition, the ΔahpC, Δtpx and Δbcp mutants showed significantly decreased survival rates after 4 h of CHP stress, supporting that the peroxiredoxins confer protection against CHP stress in S. aureus (Figure 6G). These CHP-sensitive survival phenotypes of the peroxiredoxin-deficient mutants could be restored to WT levels in the ahpC, tpx and bcp complemented strains (Figure 6H). However, the slightly increased CHP resistance of the ΔkatA mutant could not be reverted to WT levels in the katA complemented strain (Figure 6G), probably due to the partial complementation by plasmid-based KatA expression (Figure S1D).

3.8. Peroxiredoxin-Deficient ΔahpC, Δtpx and Δbcp Mutants Are Delayed in CHP Detoxification as Revealed by Brx-roGFP2 Measurements

To investigate the impact of the peroxiredoxins in the maintenance of the reduced state of EBSH in S. aureus, Brx-roGFP2 biosensor measurements were performed after 0.5 mM CHP stress (Figure 7). The Brx-roGFP2 biosensor was similarly quickly oxidized by 0.5 mM CHP in the WT, ΔkatA, ΔahpC, Δtpx and Δbcp mutants. However, while the WT and ΔkatA mutant could regenerate the reduced state of EBSH within 2 h, the ΔahpC mutant was unable to regenerate the basal level of EBSH during the recovery phase from CHP stress (Figure 7A,B). In addition, both the Δbcp and Δtpx mutants showed significant delays in recovery of the reduced state of EBSH upon CHP stress as compared to the WT (Figure 7C,D). These biosensor results support that the peroxiredoxins AhpC, Tpx and Bcp are important for CHP detoxification and contribute to regeneration of EBSH during the recovery phase from CHP stress in S. aureus. In contrast, KatA does not contribute to CHP detoxification and resistance in S. aureus.

3.9. KatA and Peroxiredoxins Do Not Contribute to Protection against HOCl Stress

Previous transcriptome analyses revealed an increased transcription of the PerR regulon under HOCl stress in S. aureus [38]. Thus, growth curves and survival assays were used to analyse the phenotypes of the ΔkatA, ΔahpC, Δtpx and Δbcp mutants under HOCl stress. However, none of these mutants showed significant defects in growth or survival after HOCl stress compared to the WT, indicating that the catalase and peroxiredoxins do not contribute to HOCl detoxification and resistance in S. aureus (Figure S5A–E).

4. Discussion

Here, we investigated the roles of catalase and peroxiredoxins in the peroxide stress resistance and priming of S. aureus during aerobic and microaerophilic growth. Our results revealed that S. aureus is not primable towards improved H2O2 resistance during aerobic growth due to its constitutive H2O2 resistance, which was shown to be dependent on the catalase KatA. Moreover, we found that microaerophilic S. aureus cells can be primed to acquire an enhanced resistance towards lethal H2O2 doses, which was also mediated by KatA (Figure 3B,D). In addition, the peroxiredoxins AhpC, Tpx and Bcp were shown to contribute to the CHP detoxification and resistance in S. aureus to ensure the maintenance of the redox balance upon recovery from stress.
The roles of KatA and AhpC in the peroxide resistance of S. aureus have been previously demonstrated [20]. In this work, we additionally used Brx-roGFP2 biosensor measurements, showing the impact of KatA and AhpC on the level of H2O2 detoxification and regeneration of reduced EBSH under oxidative stress. Without KatA, S. aureus cells are highly sensitive to oxidants and only able to remove low doses of 0.4 mM H2O2, which were shown to be detoxified by AhpC. In addition, the ΔahpCΔkatA double mutant showed an increased sensitivity towards H2O2 stress in survival assays as compared to the ΔkatA mutant. These data confirm that KatA and AhpCF have compensatory roles in H2O2 resistance to ensure the survival of S. aureus under oxidative stress [20]. Expression of katA, bcp, dps and the ahpCF operon is controlled by the peroxide-sensing PerR repressor, which is inactivated by H2O2 due to Fe2+-catalysed histidine oxidation in S. aureus [18,52,53]. Due to PerR derepression in the ΔahpC mutant, katA and dps expression was elevated, as confirmed here using Northern blots and shown previously [20]. Thus, the higher KatA expression level in the ΔahpC mutant mediates the enhanced H2O2 resistance, confirming previous results in S. aureus and B. subtilis [20,54,55].
In addition, we showed that aerobically grown S. aureus acquire an improved resistance to H2O2 during the stationary phase, which also depends on KatA. The oxidative stress resistance was also enhanced during the stationary phase in other bacteria, such as B. subtilis and E. coli [56,57]. In addition, KatA activity was elevated during the stationary phase in S. aureus and B. subtilis [20,48]. Altogether, our results on aerobic S. aureus cells reveal that KatA is the major player that confers the strong constitutive H2O2 resistance during the log and stationary phases, while the peroxiredoxin AhpC plays an additional role of scavenging 0.4 mM H2O2 in the absence of KatA. Apart from its major role as an H2O2 scavenger, the catalase also provides heme and iron as cofactors for cellular metabolism, which could contribute to S. aureus survival under oxidative stress.
We further showed that S. aureus is primable with sub-lethal doses of 0.1 mM H2O2 to acquire an improved resistance towards otherwise lethal doses of 10 mM H2O2 during the microaerophilic growth. This microaerophilic H2O2 priming was found to be dependent on KatA, which was transcriptionally induced and showed a higher catalase activity upon challenge with the priming dose. These results are consistent with previous data showing increased KatA activity by exposure to 100 µM H2O2 during oxygen limitation [52]. We showed that microaerophilic priming by KatA prepares the cells to better survive the lethal triggering stress, resulting in improved growth and survival of S. aureus. In contrast, due to their constitutive H2O2 resistance, aerobic S. aureus cells were not primable to acquire higher resistance towards lethal H2O2 doses. Aerobic priming was not possible with 0.1 mM or 1 mM H2O2 since katA transcription and catalase activity were already elevated in naïve cells and could not be further increased in primed cells. The higher basal level of EBSH of −291 mV in aerobic cells compared to the more reducing basal EBSH of −310 mV in microaerophilic cells strongly indicates an increased ROS level in aerobic cells due to aerobic respiration. These biosensor data support that S. aureus is primed by endogenous ROS generated by aerobic respiration to achieve their constitutive H2O2 resistance phenotype (Figure 8). Thus, the PerR regulon is already up-regulated during aerobic growth due to ROS generated during aerobic respiration [52], resulting in the H2O2-resistant phenotype.
This up-regulation of the PerR regulon in S. aureus during the aerobic growth was caused by the hypersensitive PerR repressor, which is poised by very low endogenous levels of H2O2 generated during aerobic respiration [52]. The endogenous H2O2 concentration in aerobic E. coli cells was determined as ~50 nM [58]. While PerR of S. aureus is hypersensitive to endogenous H2O2 levels during aerobic growth, the PerR protein of B. subtilis is less sensitive and cannot sense such low H2O2 levels originating from aerobic respiration [52]. Thus, B. subtilis can be primed during aerobic growth with 0.1 mM H2O2, leading to PerR inactivation and derepression of the PerR-controlled KatA, which confers an adaptive resistance against the otherwise lethal triggering dose of 10 mM H2O2 [33,34,52,55,59].
Similarly, aerobic priming is possible in E. coli and S. Typhimurium after exposure to 100 µM H2O2, leading to activation of the OxyR regulon, including the major catalase, which confers an adaptive and improved resistance towards lethal concentrations of 10 mM H2O2 [36,57,60]. In conclusion, priming of bacteria towards improved H2O2 resistance depends on the sensitivity of the redox-sensing peroxide regulators. While many bacteria harbour less sensitive H2O2 sensors and are primable by sub-lethal H2O2 doses during aerobic growth, S. aureus PerR is hypersensitive to endogenous H2O2 levels during aerobic growth [52], resulting in constitutive aerobic H2O2 resistance and microaerophilic priming to acquire improved H2O2 resistance only in oxygen-limited conditions (Figure 8).
While KatA is responsible for detoxification of up to 100 mM H2O2 and confers the high resistance to 100 mM H2O2 in aerobic S. aureus cells, the peroxiredoxin AhpC was shown to enable the detoxification of low levels of 0.4 mM H2O2, which could originate from the aerobic respiration. Thus, our results support that catalases are scavengers of high mM levels of H2O2, whereas peroxiredoxins can only detoxify physiological µM H2O2 levels. However, the levels of H2O2 experienced by S. aureus during the oxidative burst were determined in the range of ~2 µM inside the phagosomes of neutrophils and macrophages [13,61,62]. In mammalian cells, the physiological intracellular H2O2 concentration was estimated in the range of 1–100 nM, while the extracellular H2O2 was 100-fold higher [63]. Thus, S. aureus might not experience such high doses of 10–100 mM H2O2 during interaction with immune cells. However, as a commensal bacterium, S. aureus colonizes the anterior nares and the nasopharynx together with competing microbes, such as Streptococcus pneumoniae, which generates millimolar levels of H2O2 by the lactate and pyruvate oxidases to kill competitive bacteria [64,65,66,67,68,69]. In a mouse infection model, KatA was required for nasal colonization of S. aureus [18]. Furthermore, the katA mutant was impaired in survival towards H2O2 produced by S. pneumoniae during nasal colonization [20,70]. Thus, microaerophilic H2O2 priming of S. aureus might provide an advantage in its ecological niche to resist the high H2O2 levels generated by competing microbes.
In contrast, KatA and the peroxiredoxins are not directly involved in the defence against HOCl stress in S. aureus. However, KatA might contribute to lowering external H2O2 and, subsequently, HOCl levels in the neutrophil phagosome. In support of this notion, KatA was found to be induced upon macrophage infection and to be essential for survival of S. aureus inside macrophages [21]. In addition, S. aureus strains with high catalase activity were more resistant to killing by neutrophils compared to strains with lower catalase activity [22]. These data support that KatA is an important defence mechanism in S. aureus against the respiratory burst of macrophages and neutrophils.
In addition, the peroxiredoxins AhpC, Tpx and Bcp were found to confer protection against CHP stress in S. aureus cells. Using Brx-roGFP2 biosensor measurements, the peroxiredoxin-deficient ΔahpC, Δtpx and Δbcp mutants showed delayed regeneration of the reduced state of EBSH after recovery from CHP stress. Thus, AhpC, Tpx and Bcp function in CHP detoxification and contribute to the maintenance of the cellular redox balance. The role of AhpC in CHP resistance has been previously demonstrated in S. aureus [20]. Similarly, the AhpC homologs of B. subtilis, E. coli and S. Typhimurium conferred resistance towards CHP stress [55,71]. In E. coli, the Δtpx and Δbcp mutants were more sensitive towards various OHPs, indicating that these peroxiredoxins are more specific to reduction of organic peroxide substrates [26,28]. Kinetic assays of the E. coli Tpx protein demonstrated the substrate specificity towards alkyl hydroperoxides over H2O2 [27]. Similarly, Bcp of E. coli has a fivefold higher Vmax/Km value for linoleic acid hydroperoxide as a substrate compared to H2O2 [26].

5. Conclusions

Taken together, we have shown that the catalase KatA is the major player in aerobic H2O2 resistance in S. aureus and mediates priming to endogenous ROS levels generated during aerobic respiration to confer the constitutive H2O2 resistance towards the triggering stimulus in aerobic cells. In addition, KatA mediates microaerophilic priming by low H2O2 levels to prepare S. aureus cells for improved and adaptive resistance against otherwise lethal H2O2 doses. Furthermore, the peroxiredoxins AhpC, Tpx and Bcp were shown to contribute to CHP detoxification and resistance to ensure the survival and regeneration of the reduced EBSH in S. aureus (Figure 8). In future studies, we aim to elucidate the functions of KatA and the peroxiredoxins in signal transduction, as redox-active chaperones and in cellular metabolism during aerobic growth and under oxidative stress in S. aureus.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/antiox11091793/s1: Figure S1: Complementation of KatA increases H2O2 resistance in S. aureus, but the catalase activity in the katA complemented strain is lower compared to the WT. Figure S2: Growth and catalase activity of the ahpC complemented strain. Figure S3: The ΔkatAΔahpC double mutant is hypersensitive towards H2O2 and delayed in the recovery of reduced EBSH after 0.4 mM H2O2. Figure S4: The katA complemented strain is not primable towards increased H2O2 resistance during microaerophilic growth. Figure S5: KatA and the peroxiredoxins AhpC, Tpx and Bcp are not involved in HOCl detoxification in S. aureus. Figure S6: The Northern blot images using the katA specific RNA probe show the katA transcripts (images of Figure 1H; two bioreplicates, three technical replicates). Figure S7: The Northern blot images using the ahpC specific RNA probe show the ahpCF operon transcripts (images of Figure 1H; two bioreplicates, three technical replicates). Figure S8: The Northern blot images using the dps specific RNA probe show the dps operon transcripts (images of Figure 1H; two bioreplicates, three technical replicates). Figure S9: The Northern blot images of priming experiments using the katA specific RNA probe show the katA transcripts (images of Figure 5B,C; three bioreplicates). Figure S10: The Northern blot images of priming experiments using the ahpC specific RNA probe show the ahpCF transcripts (images of Figure 5B,C; three bioreplicates). Figure S11: Native gels of catalase stains used for KatA activity assays (images of Figure 5F; two bioreplicates, two technical replicates). Figure S12: Native gels of catalase stains used for KatA activity assays (images of Figure S1D; three bioreplicates). Figure S13: Native gels of catalase stains used for KatA activity assays (images of Figure S2B; three bioreplicates). Table S1: Bacterial strains; Table S2: Plasmids; Table S3: Oligonucleotide primers. Table S4: CFU counts for the H2O2 survival assays. Table S5: CFU counts for microaerophilic H2O2 priming. Table S6: CFU counts for aerobic H2O2 priming. Table S7: CFU counts for the CHP survival assays. Table S8: CFU counts for HOCl survival assays.

Author Contributions

N.L. and V.V.L. performed the experiments and analysed the data. V.V.L. constructed the mutants and provided advice for biosensor measurements. N.L. wrote the initial draft of the manuscript. H.A. designed the concept of the project, supervised the experiments, provided funding and wrote the final manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by an ERC Consolidator grant (GA 615585) MYCOTHIOLOME and grants from the Deutsche Forschungsgemeinschaft (AN746/4-1 and AN746/4-2) within the project SPP1710, by the SFB973 project C08 and by the SFB/TR84 project B06 to H.A.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All reported data are available in the manuscript.

Acknowledgments

We are very grateful to Franziska Kiele for excellent technical assistance. We further would like to thank Quach Ngoc Tung and Lukas Olff for their support in mutant constructions and phenotype analyses. The publication fee of this article was funded by the Open Access Initiative of the Freie Universität Berlin.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

References

  1. Archer, G.L. Staphylococcus aureus: A well-armed pathogen. Clin. Infect Dis. 1998, 26, 1179–1181. [Google Scholar] [CrossRef] [PubMed]
  2. Lowy, F.D. Staphylococcus aureus infections. N. Engl. J. Med. 1998, 339, 520–532. [Google Scholar] [CrossRef] [PubMed]
  3. Boucher, H.W.; Corey, G.R. Epidemiology of methicillin-resistant Staphylococcus aureus. Clin. Infect Dis. 2008, 46 (Suppl. S5), S344–S349. [Google Scholar] [CrossRef] [PubMed]
  4. Sies, H.; Belousov, V.V.; Chandel, N.S.; Davies, M.J.; Jones, D.P.; Mann, G.E.; Murphy, M.P.; Yamamoto, M.; Winterbourn, C. Defining roles of specific reactive oxygen species (ROS) in cell biology and physiology. Nat. Rev. Mol. Cell Biol. 2022, 23, 499–515. [Google Scholar] [CrossRef] [PubMed]
  5. Dupre-Crochet, S.; Erard, M.; Nubetae, O. ROS production in phagocytes: Why, when, and where? J. Leukoc. Biol. 2013, 94, 657–670. [Google Scholar] [CrossRef]
  6. Winterbourn, C.C.; Kettle, A.J.; Hampton, M.B. Reactive oxygen species and neutrophil function. Annu. Rev. Biochem. 2016, 85, 765–792. [Google Scholar] [CrossRef] [PubMed]
  7. Nguyen, G.T.; Green, E.R.; Mecsas, J. Neutrophils to the ROScue: Mechanisms of NADPH Oxidase Activation and Bacterial Resistance. Front. Cell Infect Microbiol. 2017, 7, 373. [Google Scholar] [CrossRef] [PubMed]
  8. Herb, M.; Schramm, M. Functions of ROS in Macrophages and Antimicrobial Immunity. Antioxidants 2021, 10, 313. [Google Scholar] [CrossRef]
  9. Ulfig, A.; Leichert, L.I. The effects of neutrophil-generated hypochlorous acid and other hypohalous acids on host and pathogens. Cell Mol. Life Sci. 2021, 78, 385–414. [Google Scholar] [CrossRef]
  10. Winterbourn, C.C.; Kettle, A.J. Redox reactions and microbial killing in the neutrophil phagosome. Antioxid Redox Signal 2013, 18, 642–660. [Google Scholar] [CrossRef]
  11. Petersen, S.V.; Poulsen, N.B.; Linneberg Matthiesen, C.; Vilhardt, F. Novel and Converging Ways of NOX2 and SOD3 in Trafficking and Redox Signaling in Macrophages. Antioxidants 2021, 10, 172. [Google Scholar] [CrossRef]
  12. Imlay, J.A. Pathways of oxidative damage. Annu. Rev. Microbiol. 2003, 57, 395–418. [Google Scholar] [CrossRef] [PubMed]
  13. Imlay, J.A. Cellular defenses against superoxide and hydrogen peroxide. Annu. Rev. Biochem. 2008, 77, 755–776. [Google Scholar] [CrossRef] [PubMed]
  14. Linzner, N.; Loi, V.V.; Fritsch, V.N.; Antelmann, H. Thiol-based redox switches in the major pathogen Staphylococcus aureus. Biol. Chem. 2021, 402, 333–361. [Google Scholar] [CrossRef] [PubMed]
  15. Hillion, M.; Antelmann, H. Thiol-based redox switches in prokaryotes. Biol. Chem. 2015, 396, 415–444. [Google Scholar] [CrossRef]
  16. Linzner, N.; Loi, V.V.; Fritsch, V.N.; Tung, Q.N.; Stenzel, S.; Wirtz, M.; Hell, R.; Hamilton, C.J.; Tedin, K.; Fulde, M.; et al. Staphylococcus aureus uses the bacilliredoxin (BrxAB)/bacillithiol disulfide reductase (YpdA) redox pathway to defend against oxidative stress under infections. Front. Microbiol. 2019, 10, 1355. [Google Scholar] [CrossRef]
  17. Loi, V.V.; Harms, M.; Müller, M.; Huyen, N.T.T.; Hamilton, C.J.; Hochgräfe, F.; Pane-Farre, J.; Antelmann, H. Real-time imaging of the bacillithiol redox potential in the human pathogen Staphylococcus aureus using a genetically encoded bacilliredoxin-fused redox biosensor. Antioxid Redox Signal 2017, 26, 835–848. [Google Scholar] [CrossRef]
  18. Horsburgh, M.J.; Clements, M.O.; Crossley, H.; Ingham, E.; Foster, S.J. PerR controls oxidative stress resistance and iron storage proteins and is required for virulence in Staphylococcus aureus. Infect Immun. 2001, 69, 3744–3754. [Google Scholar] [CrossRef]
  19. Weber, H.; Engelmann, S.; Becher, D.; Hecker, M. Oxidative stress triggers thiol oxidation in the glyceraldehyde-3-phosphate dehydrogenase of Staphylococcus aureus. Mol. Microbiol. 2004, 52, 133–140. [Google Scholar] [CrossRef]
  20. Cosgrove, K.; Coutts, G.; Jonsson, I.M.; Tarkowski, A.; Kokai-Kun, J.F.; Mond, J.J.; Foster, S.J. Catalase (KatA) and alkyl hydroperoxide reductase (AhpC) have compensatory roles in peroxide stress resistance and are required for survival, persistence, and nasal colonization in Staphylococcus aureus. J. Bacteriol. 2007, 189, 1025–1035. [Google Scholar] [CrossRef] [Green Version]
  21. Das, D.; Saha, S.S.; Bishayi, B. Intracellular survival of Staphylococcus aureus: Correlating production of catalase and superoxide dismutase with levels of inflammatory cytokines. Inflamm. Res. 2008, 57, 340–349. [Google Scholar] [CrossRef] [PubMed]
  22. Mandell, G.L. Catalase, superoxide dismutase, and virulence of Staphylococcus aureus. In vitro and in vivo studies with emphasis on staphylococcal--leukocyte interaction. J. Clin. Invest. 1975, 55, 561–566. [Google Scholar] [CrossRef] [PubMed]
  23. Meireles, D.A.; da Silva Neto, J.F.; Domingos, R.M.; Alegria, T.G.P.; Santos, L.C.M.; Netto, L.E.S. Ohr–OhrR, a neglected and highly efficient antioxidant system: Structure, catalysis, phylogeny, regulation, and physiological roles. Free Radic Biol. Med. 2022, 185, 6–24. [Google Scholar] [CrossRef] [PubMed]
  24. Poole, L.B.; Hall, A.; Nelson, K.J. Overview of peroxiredoxins in oxidant defense and redox regulation. Curr. Protoc. Toxicol. 2011, 7, 9. [Google Scholar] [CrossRef] [PubMed]
  25. Parsonage, D.; Nelson, K.J.; Ferrer-Sueta, G.; Alley, S.; Karplus, P.A.; Furdui, C.M.; Poole, L.B. Dissecting peroxiredoxin catalysis: Separating binding, peroxidation, and resolution for a bacterial AhpC. Biochemistry 2015, 54, 1567–1575. [Google Scholar] [CrossRef] [PubMed]
  26. Jeong, W.; Cha, M.K.; Kim, I.H. Thioredoxin-dependent hydroperoxide peroxidase activity of bacterioferritin comigratory protein (BCP) as a new member of the thiol-specific antioxidant protein (TSA)/Alkyl hydroperoxide peroxidase C (AhpC) family. J. Biol. Chem. 2000, 275, 2924–2930. [Google Scholar] [CrossRef]
  27. Baker, L.M.; Poole, L.B. Catalytic mechanism of thiol peroxidase from Escherichia coli. Sulfenic acid formation and overoxidation of essential CYS61. J. Biol. Chem. 2003, 278, 9203–9211. [Google Scholar] [CrossRef]
  28. Cha, M.K.; Kim, W.C.; Lim, C.J.; Kim, K.; Kim, I.H. Escherichia coli periplasmic thiol peroxidase acts as lipid hydroperoxide peroxidase and the principal antioxidative function during anaerobic growth. J. Biol. Chem. 2004, 279, 8769–8778. [Google Scholar] [CrossRef]
  29. Imber, M.; Huyen, N.T.T.; Pietrzyk-Brzezinska, A.J.; Loi, V.V.; Hillion, M.; Bernhardt, J.; Thärichen, L.; Kolsek, K.; Saleh, M.; Hamilton, C.J.; et al. Protein S-bacillithiolation functions in thiol protection and redox regulation of the glyceraldehyde-3-phosphate dehydrogenase Gap in Staphylococcus aureus under hypochlorite stress. Antioxid Redox Signal 2018, 28, 410–430. [Google Scholar] [CrossRef]
  30. Chelikani, P.; Fita, I.; Loewen, P.C. Diversity of structures and properties among catalases. Cell Mol. Life Sci. 2004, 61, 192–208. [Google Scholar] [CrossRef]
  31. Mongkolsuk, S.; Helmann, J.D. Regulation of inducible peroxide stress responses. Mol. Microbiol. 2002, 45, 9–15. [Google Scholar] [CrossRef] [PubMed]
  32. Faulkner, M.J.; Helmann, J.D. Peroxide stress elicits adaptive changes in bacterial metal ion homeostasis. Antioxid Redox Signal 2011, 15, 175–189. [Google Scholar] [CrossRef] [PubMed]
  33. Engelmann, S.; Hecker, M. Impaired oxidative stress resistance of Bacillus subtilis sigB mutants and the role of katA and katE. FEMS Microbiol. Lett. 1996, 145, 63–69. [Google Scholar] [CrossRef] [PubMed]
  34. Murphy, P.; Dowds, B.C.; McConnell, D.J.; Devine, K.M. Oxidative stress and growth temperature in Bacillus subtilis. J. Bacteriol. 1987, 169, 5766–5770. [Google Scholar] [CrossRef]
  35. Hilker, M.; Schwachtje, J.; Baier, M.; Balazadeh, S.; Baurle, I.; Geiselhardt, S.; Hincha, D.K.; Kunze, R.; Mueller-Roeber, B.; Rillig, M.C.; et al. Priming and memory of stress responses in organisms lacking a nervous system. Biol. Rev. Camb. Philos. Soc. 2016, 91, 1118–1133. [Google Scholar] [CrossRef]
  36. Christman, M.F.; Morgan, R.W.; Jacobson, F.S.; Ames, B.N. Positive control of a regulon for defenses against oxidative stress and some heat-shock proteins in Salmonella typhimurium. Cell 1985, 41, 753–762. [Google Scholar] [CrossRef]
  37. Chiang, S.M.; Schellhorn, H.E. Regulators of oxidative stress response genes in Escherichia coli and their functional conservation in bacteria. Arch. Biochem. Biophys. 2012, 525, 161–169. [Google Scholar] [CrossRef]
  38. Loi, V.V.; Busche, T.; Tedin, K.; Bernhardt, J.; Wollenhaupt, J.; Huyen, N.T.T.; Weise, C.; Kalinowski, J.; Wahl, M.C.; Fulde, M.; et al. Redox-sensing under hypochlorite stress and infection conditions by the Rrf2-family repressor HypR in Staphylococcus aureus. Antioxid Redox Signal 2018, 29, 615–636. [Google Scholar] [CrossRef]
  39. Winter, J.; Ilbert, M.; Graf, P.C.; Ozcelik, D.; Jakob, U. Bleach activates a redox-regulated chaperone by oxidative protein unfolding. Cell 2008, 135, 691–701. [Google Scholar] [CrossRef]
  40. Linzner, N.; Fritsch, V.N.; Busche, T.; Tung, Q.N.; Loi, V.V.; Bernhardt, J.; Kalinowski, J.; Antelmann, H. The plant-derived naphthoquinone lapachol causes an oxidative stress response in Staphylococcus aureus. Free Radic. Biol. Med. 2020, 158, 126–136. [Google Scholar] [CrossRef]
  41. Arnaud, M.; Chastanet, A.; Debarbouille, M. New vector for efficient allelic replacement in naturally nontransformable, low-GC-content, gram-positive bacteria. Appl. Environ. Microbiol. 2004, 70, 6887–6891. [Google Scholar] [CrossRef] [PubMed]
  42. Rosenblum, E.D.; Tyrone, S. Serology, density, and morphology of staphylococcal phages. J. Bacteriol. 1964, 88, 1737–1742. [Google Scholar] [CrossRef]
  43. Fritsch, V.N.; Loi, V.V.; Busche, T.; Sommer, A.; Tedin, K.; Nürnberg, D.J.; Kalinowski, J.; Bernhardt, J.; Fulde, M.; Antelmann, H. The MarR-type repressor MhqR confers quinone and antimicrobial resistance in Staphylococcus aureus. Antioxid Redox Signal 2019, 31, 1235–1252. [Google Scholar] [CrossRef] [PubMed]
  44. Fritsch, V.N.; Loi, V.V.; Busche, T.; Tung, Q.N.; Lill, R.; Horvatek, P.; Wolz, C.; Kalinowski, J.; Antelmann, H. The alarmone (p)ppGpp confers tolerance to oxidative stress during the stationary phase by maintenance of redox and iron homeostasis in Staphylococcus aureus. Free Radic. Biol. Med. 2020, 161, 351–364. [Google Scholar] [CrossRef] [PubMed]
  45. Loi, V.V.; Antelmann, H. Method for measurement of bacillithiol redox potential changes using the Brx-roGFP2 redox biosensor in Staphylococcus aureus. MethodsX 2020, 7, 100900. [Google Scholar] [CrossRef]
  46. Wetzstein, M.; Volker, U.; Dedio, J.; Lobau, S.; Zuber, U.; Schiesswohl, M.; Herget, C.; Hecker, M.; Schumann, W. Cloning, sequencing, and molecular analysis of the dnaK locus from Bacillus subtilis. J. Bacteriol. 1992, 174, 3300–3310. [Google Scholar] [CrossRef]
  47. Clare, D.A.; Duong, M.N.; Darr, D.; Archibald, F.; Fridovich, I. Effects of molecular oxygen on detection of superoxide radical with nitroblue tetrazolium and on activity stains for catalase. Anal. Biochem. 1984, 140, 532–537. [Google Scholar] [CrossRef]
  48. Loewen, P.C.; Switala, J. Multiple catalases in Bacillus subtilis. J. Bacteriol. 1987, 169, 3601–3607. [Google Scholar] [CrossRef]
  49. Loi, V.V.; Busche, T.; Preuss, T.; Kalinowski, J.; Bernhardt, J.; Antelmann, H. The AGXX antimicrobial coating causes a thiol-specific oxidative stress response and protein S-bacillithiolation in Staphylococcus aureus. Front. Microbiol. 2018, 9, 3037. [Google Scholar] [CrossRef]
  50. Loi, V.V.; Huyen, N.T.T.; Busche, T.; Tung, Q.N.; Gruhlke, M.C.H.; Kalinowski, J.; Bernhardt, J.; Slusarenko, A.J.; Antelmann, H. Staphylococcus aureus responds to allicin by global S-thioallylation–role of the Brx/BSH/YpdA pathway and the disulfide reductase MerA to overcome allicin stress. Free Radic. Biol. Med. 2019, 139, 55–69. [Google Scholar] [CrossRef]
  51. Wolf, C.; Hochgräfe, F.; Kusch, H.; Albrecht, D.; Hecker, M.; Engelmann, S. Proteomic analysis of antioxidant strategies of Staphylococcus aureus: Diverse responses to different oxidants. Proteomics 2008, 8, 3139–3153. [Google Scholar] [CrossRef] [PubMed]
  52. Ji, C.J.; Kim, J.H.; Won, Y.B.; Lee, Y.E.; Choi, T.W.; Ju, S.Y.; Youn, H.; Helmann, J.D.; Lee, J.W. Staphylococcus aureus PerR Is a hypersensitive hydrogen peroxide sensor using iron-mediated histidine oxidation. J. Biol. Chem. 2015, 290, 20374–20386. [Google Scholar] [CrossRef] [PubMed]
  53. Lee, J.W.; Helmann, J.D. The PerR transcription factor senses H2O2 by metal-catalysed histidine oxidation. Nature 2006, 440, 363–367. [Google Scholar] [CrossRef] [PubMed]
  54. Bsat, N.; Chen, L.; Helmann, J.D. Mutation of the Bacillus subtilis alkyl hydroperoxide reductase (ahpCF) operon reveals compensatory interactions among hydrogen peroxide stress genes. J. Bacteriol. 1996, 178, 6579–6586. [Google Scholar] [CrossRef]
  55. Antelmann, H.; Engelmann, S.; Schmid, R.; Hecker, M. General and oxidative stress responses in Bacillus subtilis: Cloning, expression, and mutation of the alkyl hydroperoxide reductase operon. J. Bacteriol. 1996, 178, 6571–6578. [Google Scholar] [CrossRef] [PubMed]
  56. Dowds, B.C.; Murphy, P.; McConnell, D.J.; Devine, K.M. Relationship among oxidative stress, growth cycle, and sporulation in Bacillus subtilis. J. Bacteriol. 1987, 169, 5771–5775. [Google Scholar] [CrossRef] [PubMed]
  57. Farr, S.B.; Kogoma, T. Oxidative stress responses in Escherichia coli and Salmonella typhimurium. Microbiol. Rev. 1991, 55, 561–585. [Google Scholar] [CrossRef]
  58. Seaver, L.C.; Imlay, J.A. Hydrogen peroxide fluxes and compartmentalization inside growing Escherichia coli. J. Bacteriol. 2001, 183, 7182–7189. [Google Scholar] [CrossRef]
  59. Pinochet-Barros, A.; Helmann, J.D. Redox Sensing by Fe2+ in Bacterial Fur Family Metalloregulators. Antioxid. Redox Signal 2018, 29, 1858–1871. [Google Scholar] [CrossRef]
  60. Åslund, F.; Zheng, M.; Beckwith, J.; Storz, G. Regulation of the OxyR transcription factor by hydrogen peroxide and the cellular thiol-disulfide status. Proc. Natl. Acad. Sci. USA 1999, 96, 6161–6165. [Google Scholar] [CrossRef] [Green Version]
  61. Winterbourn, C.C.; Hampton, M.B.; Livesey, J.H.; Kettle, A.J. Modeling the reactions of superoxide and myeloperoxidase in the neutrophil phagosome: Implications for microbial killing. J. Biol. Chem. 2006, 281, 39860–39869. [Google Scholar] [CrossRef] [PubMed]
  62. Imlay, J.A. Where in the world do bacteria experience oxidative stress? Environ. Microbiol. 2019, 21, 521–530. [Google Scholar] [CrossRef] [PubMed]
  63. Sies, H.; Jones, D.P. Reactive oxygen species (ROS) as pleiotropic physiological signalling agents. Nat. Rev. Mol. Cell Biol. 2020, 21, 363–383. [Google Scholar] [CrossRef] [PubMed]
  64. Bogaert, D.; de Groot, R.; Hermans, P.W.M. Streptococcus pneumoniae colonisation: The key to pneumococcal disease. Lancet Infect. Dis. 2004, 4, 144–154. [Google Scholar] [CrossRef]
  65. Liu, C.M.; Price, L.B.; Hungate, B.A.; Abraham, A.G.; Larsen, L.A.; Christensen, K.; Stegger, M.; Skov, R.; Andersen, P.S. Staphylococcus aureus and the ecology of the nasal microbiome. Sci. Adv. 2015, 1, e1400216. [Google Scholar] [CrossRef]
  66. Udaka, S.; Koukol, J.; Vennesland, B. Lactic oxidase of Pneumococcus. J. Bacteriol. 1959, 78, 714–725. [Google Scholar] [CrossRef]
  67. Spellerberg, B.; Cundell, D.R.; Sandros, J.; Pearce, B.J.; Idanpaan-Heikkila, I.; Rosenow, C.; Masure, H.R. Pyruvate oxidase, as a determinant of virulence in Streptococcus pneumoniae. Mol. Microbiol. 1996, 19, 803–813. [Google Scholar] [CrossRef]
  68. Pericone, C.D.; Bae, D.; Shchepetov, M.; McCool, T.; Weiser, J.N. Short-sequence tandem and nontandem DNA repeats and endogenous hydrogen peroxide production contribute to genetic instability of Streptococcus pneumoniae. J. Bacteriol. 2002, 184, 4392–4399. [Google Scholar] [CrossRef]
  69. Lisher, J.P.; Tsui, H.T.; Ramos-Montanez, S.; Hentchel, K.L.; Martin, J.E.; Trinidad, J.C.; Winkler, M.E.; Giedroc, D.P. Biological and chemical adaptation to endogenous hydrogen peroxide production in Streptococcus pneumoniae D39. mSphere 2017, 2, e00291-16. [Google Scholar] [CrossRef]
  70. Park, B.; Nizet, V.; Liu, G.Y. Role of Staphylococcus aureus catalase in niche competition against Streptococcus pneumoniae. J. Bacteriol. 2008, 190, 2275–2278. [Google Scholar] [CrossRef] [Green Version]
  71. Storz, G.; Jacobson, F.S.; Tartaglia, L.A.; Morgan, R.W.; Silveira, L.A.; Ames, B.N. An alkyl hydroperoxide reductase induced by oxidative stress in Salmonella typhimurium and Escherichia coli: Genetic characterization and cloning of ahp. J. Bacteriol. 1989, 171, 2049–2055. [Google Scholar] [CrossRef] [PubMed] [Green Version]
Antioxidants 11 01793 g001 550
Figure 1. KatA confers strong H2O2 resistance during aerobic growth, while Tpx and Bcp are not required for H2O2 resistance. (A–D) Growth curves of S. aureus COL WT, ΔkatA (A), ΔahpC (B), Δtpx (C) and Δbcp mutants (D) in RPMI medium before (co) and after exposure to 10 mM H2O2 at an OD500 of 0.5. (E) Survival rates were determined as CFU counts for S. aureus COL WT, ΔkatA, ΔahpC, Δtpx and Δbcp mutants and the ahpC complemented strain at 2 and 4 h after treatment with 40 mM H2O2. (F) Survival rates were analysed for the WT, ΔkatA and ΔahpCΔkatA mutants and the katA complemented strain (katA) after 2 and 4 h of exposure to 1 mM H2O2 based on CFU counts. (G) S. aureus COL WT and ΔkatA mutant cells were exposed to 40 mM H2O2 during the log and stationary phases at OD500 of 0.5 and 2–3, respectively. In (E–G), the survival rates were calculated relative to the untreated control, which was set to 100%. Mean values and standard deviation (SD) from three to four biological replicates are shown. (H) Northern blot analyses of the katA, dps and ahpCF specific transcripts in the S. aureus WT, ΔkatA, ΔahpC and ΔperR mutants. The methylene blue stains below the Northern blot images denote the bands of the 16S and 23S rRNAs used as RNA loading controls. Quantification of the intensities of the Northern blot bands was performed from two biological and three technical replicates using Image J and is shown in the diagrams as fold changes (FCs) of induction of the katA, dps and ahpCF transcripts in the mutants relative to the WT. Error bars represent the SD. The statistics were analysed using Student’s unpaired two-tailed t-test in Graph Prism. Symbols: ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001.
Figure 1. KatA confers strong H2O2 resistance during aerobic growth, while Tpx and Bcp are not required for H2O2 resistance. (A–D) Growth curves of S. aureus COL WT, ΔkatA (A), ΔahpC (B), Δtpx (C) and Δbcp mutants (D) in RPMI medium before (co) and after exposure to 10 mM H2O2 at an OD500 of 0.5. (E) Survival rates were determined as CFU counts for S. aureus COL WT, ΔkatA, ΔahpC, Δtpx and Δbcp mutants and the ahpC complemented strain at 2 and 4 h after treatment with 40 mM H2O2. (F) Survival rates were analysed for the WT, ΔkatA and ΔahpCΔkatA mutants and the katA complemented strain (katA) after 2 and 4 h of exposure to 1 mM H2O2 based on CFU counts. (G) S. aureus COL WT and ΔkatA mutant cells were exposed to 40 mM H2O2 during the log and stationary phases at OD500 of 0.5 and 2–3, respectively. In (E–G), the survival rates were calculated relative to the untreated control, which was set to 100%. Mean values and standard deviation (SD) from three to four biological replicates are shown. (H) Northern blot analyses of the katA, dps and ahpCF specific transcripts in the S. aureus WT, ΔkatA, ΔahpC and ΔperR mutants. The methylene blue stains below the Northern blot images denote the bands of the 16S and 23S rRNAs used as RNA loading controls. Quantification of the intensities of the Northern blot bands was performed from two biological and three technical replicates using Image J and is shown in the diagrams as fold changes (FCs) of induction of the katA, dps and ahpCF transcripts in the mutants relative to the WT. Error bars represent the SD. The statistics were analysed using Student’s unpaired two-tailed t-test in Graph Prism. Symbols: ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001.
Antioxidants 11 01793 g001
Antioxidants 11 01793 g002 550
Figure 2. The ΔkatA mutant can only detoxify 0.4 mM H2O2 stress, as revealed by the Brx-roGFP2 biosensor in S. aureus. (A–F) Brx-roGFP2 biosensor responses to 100, 1 and 0.4 mM H2O2 were monitored in the S. aureus COL WT, ΔkatA (A–C), ΔahpC (D), Δtpx (E) and Δbcp mutants (F) expressing Brx-roGFP2 from plasmid pRB473. H2O2 injection assays were performed in microplates using the CLARIOstar microplate reader, as described previously [45]. The oxidation degrees (OxD) of the Brx-roGFP2 responses were calculated based on the 405/488 nm excitation ratios and normalized to fully reduced (DTT-treated) and fully oxidized (diamide-treated) controls, as described previously [45]. Mean values and SD of the OxD values are presented from three independent biological replicates.
Figure 2. The ΔkatA mutant can only detoxify 0.4 mM H2O2 stress, as revealed by the Brx-roGFP2 biosensor in S. aureus. (A–F) Brx-roGFP2 biosensor responses to 100, 1 and 0.4 mM H2O2 were monitored in the S. aureus COL WT, ΔkatA (A–C), ΔahpC (D), Δtpx (E) and Δbcp mutants (F) expressing Brx-roGFP2 from plasmid pRB473. H2O2 injection assays were performed in microplates using the CLARIOstar microplate reader, as described previously [45]. The oxidation degrees (OxD) of the Brx-roGFP2 responses were calculated based on the 405/488 nm excitation ratios and normalized to fully reduced (DTT-treated) and fully oxidized (diamide-treated) controls, as described previously [45]. Mean values and SD of the OxD values are presented from three independent biological replicates.
Antioxidants 11 01793 g002
Antioxidants 11 01793 g003 550
Figure 3. Microaerophilic H2O2 priming confers improved resistance against otherwise lethal H2O2, which depends on KatA. (A) Setup for microaerophilic priming and triggering experiments. The S. aureus WT and ΔkatA mutant strains were grown microaerophilically and primed during the log phase with 0.1 mM H2O2 for ~30 min (P), followed by treatment with 10 mM H2O2 as a triggering stimulus (PT). The growth curves (B,C) and survival rates (D,E) were measured in naïve (C), primed (P), primed and triggered (PT) and triggered-only bacteria (T). The survival rates were calculated after 2 and 4 h of H2O2 stress relative to untreated control cells. The results are from three to four biological replicates. Error bars represent the SD. The statistics were calculated using Student’s unpaired two-tailed t-test in Graph Prism. Symbols: ns p > 0.05, * p < 0.05, ** p ≤ 0.01 and *** p ≤ 0.001.
Figure 3. Microaerophilic H2O2 priming confers improved resistance against otherwise lethal H2O2, which depends on KatA. (A) Setup for microaerophilic priming and triggering experiments. The S. aureus WT and ΔkatA mutant strains were grown microaerophilically and primed during the log phase with 0.1 mM H2O2 for ~30 min (P), followed by treatment with 10 mM H2O2 as a triggering stimulus (PT). The growth curves (B,C) and survival rates (D,E) were measured in naïve (C), primed (P), primed and triggered (PT) and triggered-only bacteria (T). The survival rates were calculated after 2 and 4 h of H2O2 stress relative to untreated control cells. The results are from three to four biological replicates. Error bars represent the SD. The statistics were calculated using Student’s unpaired two-tailed t-test in Graph Prism. Symbols: ns p > 0.05, * p < 0.05, ** p ≤ 0.01 and *** p ≤ 0.001.
Antioxidants 11 01793 g003
Antioxidants 11 01793 g004 550
Figure 4. S. aureus is not primable for improved H2O2 resistance during aerobic growth. (A,D) Setup for aerobic priming and triggering experiments. The aerobically grown S. aureus WT was primed during the log phase with either 0.1 mM H2O2 (A) or 1 mM H2O2 (D) for ~30 min (P) and subsequently treated with either 10 mM H2O2 (A) or 40 mM H2O2 (D), respectively, as triggering stimuli (PT). The growth curves (B,E) and survival rates (C,F) were measured for naïve (C), primed (P), primed and triggered (PT) and triggered-only bacteria (T). Survival rates of the H2O2-treated cells were calculated relative to the untreated control. The results are from three to four biological replicates. Error bars represent the SD. The statistics were calculated using Student’s unpaired two-tailed t-test in Graph Prism. Symbols: ns p > 0.05 and * p < 0.05.
Figure 4. S. aureus is not primable for improved H2O2 resistance during aerobic growth. (A,D) Setup for aerobic priming and triggering experiments. The aerobically grown S. aureus WT was primed during the log phase with either 0.1 mM H2O2 (A) or 1 mM H2O2 (D) for ~30 min (P) and subsequently treated with either 10 mM H2O2 (A) or 40 mM H2O2 (D), respectively, as triggering stimuli (PT). The growth curves (B,E) and survival rates (C,F) were measured for naïve (C), primed (P), primed and triggered (PT) and triggered-only bacteria (T). Survival rates of the H2O2-treated cells were calculated relative to the untreated control. The results are from three to four biological replicates. Error bars represent the SD. The statistics were calculated using Student’s unpaired two-tailed t-test in Graph Prism. Symbols: ns p > 0.05 and * p < 0.05.
Antioxidants 11 01793 g004
Antioxidants 11 01793 g005 550
Figure 5. Microaerophilic H2O2 priming leads to increased katA transcription and KatA activity, as well as fast Brx-roGFP2 biosensor oxidation. (A) The setup for microaerophilic and aerobic priming and triggering experiments included priming with 0.1 mM H2O2 and triggering with 10 mM H2O2. (B,C) To analyze transcription of katA and ahpCF using Northern blots, RNA was isolated from S. aureus WT cells in the naïve (C1, C2), primed (P), primed and triggered (PT) and triggered (T) states. The band intensities of the katA (D,E) and ahpCF specific transcripts (G,H) were quantified from two biological replicates using ImageJ. The transcriptional induction of katA and ahpCF was calculated as fold change relative to the control C1, which was set to 1. Error bars represent the SD. The statistics were calculated using ordinary one-way ANOVA and Dunnet’s multiple comparisons test in Graph Prism. Symbols: ns p > 0.05; * p ≤ 0.05; ** p ≤ 0.01 and *** p≤ 0.001. (F) The catalase activity was analysed in cell extracts of the S. aureus WT during microaerophilic and aerobic H2O2 priming for C2, P, PT and T states using native PAGE and diaminobenzidine staining. The catalase activity assays were performed in two biological and two technical replicates. (I,J) The response of the Brx-roGFP2 biosensor was measured in S. aureus COL grown in LB medium under microaerophilic and aerobic conditions in naïve (C1, C2), primed (P), primed and triggered (PT) and triggered (T) cells. The C1 and C2 samples were harvested at OD500 of 0.3 and 0.4, respectively, and the P, PT and T bacteria were harvested after 10 min of H2O2 exposure (maximum biosensor oxidation). Samples were blocked with 10 mM NEM and the fluorescence excitation maxima were measured at 405 and 488 nm using the microplate reader. OxD values and the EBSH of Brx-roGFP2 were calculated using the 405/408 nm excitation ratio, as described in the Section 2. Mean values of three biological replicates are shown, error bars represent the SD and p-values were calculated using Student’s unpaired two-tailed t-test in Graph Prism software. Symbols: ns p > 0.05; * p ≤0.05; ** p ≤ 0.01; *** p ≤ 0.001 and **** p ≤ 0.0001.
Figure 5. Microaerophilic H2O2 priming leads to increased katA transcription and KatA activity, as well as fast Brx-roGFP2 biosensor oxidation. (A) The setup for microaerophilic and aerobic priming and triggering experiments included priming with 0.1 mM H2O2 and triggering with 10 mM H2O2. (B,C) To analyze transcription of katA and ahpCF using Northern blots, RNA was isolated from S. aureus WT cells in the naïve (C1, C2), primed (P), primed and triggered (PT) and triggered (T) states. The band intensities of the katA (D,E) and ahpCF specific transcripts (G,H) were quantified from two biological replicates using ImageJ. The transcriptional induction of katA and ahpCF was calculated as fold change relative to the control C1, which was set to 1. Error bars represent the SD. The statistics were calculated using ordinary one-way ANOVA and Dunnet’s multiple comparisons test in Graph Prism. Symbols: ns p > 0.05; * p ≤ 0.05; ** p ≤ 0.01 and *** p≤ 0.001. (F) The catalase activity was analysed in cell extracts of the S. aureus WT during microaerophilic and aerobic H2O2 priming for C2, P, PT and T states using native PAGE and diaminobenzidine staining. The catalase activity assays were performed in two biological and two technical replicates. (I,J) The response of the Brx-roGFP2 biosensor was measured in S. aureus COL grown in LB medium under microaerophilic and aerobic conditions in naïve (C1, C2), primed (P), primed and triggered (PT) and triggered (T) cells. The C1 and C2 samples were harvested at OD500 of 0.3 and 0.4, respectively, and the P, PT and T bacteria were harvested after 10 min of H2O2 exposure (maximum biosensor oxidation). Samples were blocked with 10 mM NEM and the fluorescence excitation maxima were measured at 405 and 488 nm using the microplate reader. OxD values and the EBSH of Brx-roGFP2 were calculated using the 405/408 nm excitation ratio, as described in the Section 2. Mean values of three biological replicates are shown, error bars represent the SD and p-values were calculated using Student’s unpaired two-tailed t-test in Graph Prism software. Symbols: ns p > 0.05; * p ≤0.05; ** p ≤ 0.01; *** p ≤ 0.001 and **** p ≤ 0.0001.
Antioxidants 11 01793 g005
Antioxidants 11 01793 g006 550
Figure 6. The peroxiredoxins AhpC, Tpx and Bcp contribute to CHP resistance in S. aureus. (A–F) Growth phenotypes were analysed for the S. aureus COL WT, ΔkatA (A), ΔahpC (B), Δtpx (C) and Δbcp mutants (D) and the ahpC (E) and tpx (F) complemented strains in RPMI medium before (co) and after exposure to 0.15 mM CHP stress at an OD500 of 0.5. (G,H) The survival rates of the S. aureus COL WT, ΔkatA, ΔahpC, Δtpx and Δbcp mutants (G) and the katA (G), ahpC, tpx and bcp (H) complemented strains were determined at 2 and 4 h after exposure to 0.25 mM CHP relative to the untreated control, which was set to 100%. Mean values and SD of four to six biological replicates are presented. The statistics were calculated using Student’s unpaired two-tailed t-test in Graph Prism. Symbols: ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001.
Figure 6. The peroxiredoxins AhpC, Tpx and Bcp contribute to CHP resistance in S. aureus. (A–F) Growth phenotypes were analysed for the S. aureus COL WT, ΔkatA (A), ΔahpC (B), Δtpx (C) and Δbcp mutants (D) and the ahpC (E) and tpx (F) complemented strains in RPMI medium before (co) and after exposure to 0.15 mM CHP stress at an OD500 of 0.5. (G,H) The survival rates of the S. aureus COL WT, ΔkatA, ΔahpC, Δtpx and Δbcp mutants (G) and the katA (G), ahpC, tpx and bcp (H) complemented strains were determined at 2 and 4 h after exposure to 0.25 mM CHP relative to the untreated control, which was set to 100%. Mean values and SD of four to six biological replicates are presented. The statistics were calculated using Student’s unpaired two-tailed t-test in Graph Prism. Symbols: ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001.
Antioxidants 11 01793 g006
Antioxidants 11 01793 g007 550
Figure 7. AhpC, Tpx and Bcp function in CHP detoxification and regeneration of reduced EBSH during the recovery phase, as revealed by the Brx-roGFP2 biosensor. (A–D) Brx-roGFP2 responses to 0.5 mM CHP in S. aureus COL WT, ΔkatA (A), ΔahpC (B), Δtpx (C) and Δbcp mutants expressing Brx-roGFP2 from plasmid pRB473. H2O2 injection assays were performed in microplates and the biosensor responses measured using the CLARIOstar microplate reader, as described previously [45]. (D) The oxidation degree (OxD) of the Brx-roGFP2 response was calculated based on the 405/488 nm excitation ratio and normalized to fully reduced (DTT-treated) and oxidized (diamide-treated) controls. Mean values and SD of three biological replicates are shown. The statistics were calculated using Student’s unpaired two-tailed t-test in Graph Prism. Symbols: ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001.
Figure 7. AhpC, Tpx and Bcp function in CHP detoxification and regeneration of reduced EBSH during the recovery phase, as revealed by the Brx-roGFP2 biosensor. (A–D) Brx-roGFP2 responses to 0.5 mM CHP in S. aureus COL WT, ΔkatA (A), ΔahpC (B), Δtpx (C) and Δbcp mutants expressing Brx-roGFP2 from plasmid pRB473. H2O2 injection assays were performed in microplates and the biosensor responses measured using the CLARIOstar microplate reader, as described previously [45]. (D) The oxidation degree (OxD) of the Brx-roGFP2 response was calculated based on the 405/488 nm excitation ratio and normalized to fully reduced (DTT-treated) and oxidized (diamide-treated) controls. Mean values and SD of three biological replicates are shown. The statistics were calculated using Student’s unpaired two-tailed t-test in Graph Prism. Symbols: ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001.
Antioxidants 11 01793 g007
Antioxidants 11 01793 g008 550
Figure 8. Summary schematics of microaerophilic H2O2 priming (right) and constitutive aerobic H2O2 resistance (left) in S. aureus. We found that microaerophilic priming with 100 µM H2O2 induces improved resistance towards otherwise lethal doses of 10 mM H2O2 in S. aureus (right), while aerobic cells are already primed by endogenous ROS originating from respiration, resulting in the constitutive high H2O2 resistance (left). The higher ROS levels during the aerobic growth were revealed by the ~20 mV increased basal EBSH compared to microaerophilic cells. The catalase KatA was shown to be induced upon microaerophilic H2O2 priming (right) and by endogenous H2O2 during aerobic respiration (left), mediating improved and constitutive H2O2 resistance, respectively, in S. aureus. In addition, the peroxiredoxins AhpC, Tpx and Bcp were shown to confer resistance towards organic hydroperoxides (R-OOH). Furthermore, KatA and the peroxiredoxins contribute to the regeneration of reduced EBSH upon recovery from oxidative stress. MQ-H2 and MQ indicate reduced and oxidized menaquinone. BSH and BSSB are reduced bacillithiol and oxidized bacillithiol disulfide, respectively.
Figure 8. Summary schematics of microaerophilic H2O2 priming (right) and constitutive aerobic H2O2 resistance (left) in S. aureus. We found that microaerophilic priming with 100 µM H2O2 induces improved resistance towards otherwise lethal doses of 10 mM H2O2 in S. aureus (right), while aerobic cells are already primed by endogenous ROS originating from respiration, resulting in the constitutive high H2O2 resistance (left). The higher ROS levels during the aerobic growth were revealed by the ~20 mV increased basal EBSH compared to microaerophilic cells. The catalase KatA was shown to be induced upon microaerophilic H2O2 priming (right) and by endogenous H2O2 during aerobic respiration (left), mediating improved and constitutive H2O2 resistance, respectively, in S. aureus. In addition, the peroxiredoxins AhpC, Tpx and Bcp were shown to confer resistance towards organic hydroperoxides (R-OOH). Furthermore, KatA and the peroxiredoxins contribute to the regeneration of reduced EBSH upon recovery from oxidative stress. MQ-H2 and MQ indicate reduced and oxidized menaquinone. BSH and BSSB are reduced bacillithiol and oxidized bacillithiol disulfide, respectively.
Antioxidants 11 01793 g008
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Linzner, N.; Loi, V.V.; Antelmann, H. The Catalase KatA Contributes to Microaerophilic H2O2 Priming to Acquire an Improved Oxidative Stress Resistance in Staphylococcus aureus. Antioxidants 2022, 11, 1793. https://doi.org/10.3390/antiox11091793

AMA Style

Linzner N, Loi VV, Antelmann H. The Catalase KatA Contributes to Microaerophilic H2O2 Priming to Acquire an Improved Oxidative Stress Resistance in Staphylococcus aureus. Antioxidants. 2022; 11(9):1793. https://doi.org/10.3390/antiox11091793

Chicago/Turabian Style

Linzner, Nico, Vu Van Loi, and Haike Antelmann. 2022. "The Catalase KatA Contributes to Microaerophilic H2O2 Priming to Acquire an Improved Oxidative Stress Resistance in Staphylococcus aureus" Antioxidants 11, no. 9: 1793. https://doi.org/10.3390/antiox11091793

APA Style

Linzner, N., Loi, V. V., & Antelmann, H. (2022). The Catalase KatA Contributes to Microaerophilic H2O2 Priming to Acquire an Improved Oxidative Stress Resistance in Staphylococcus aureus. Antioxidants, 11(9), 1793. https://doi.org/10.3390/antiox11091793

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop