Method for measurement of bacillithiol redox potential changes using the Brx-roGFP2 redox biosensor in Staphylococcus aureus

Recent advances in the design of genetically encoded redox biosensors, such as redox-sensitive GFP (roGFP) have facilitated the real-time imaging of the intracellular redox potential in eukaryotic cells at high sensitivity and at spatiotemporal resolution. To increase the specificity of roGFP2 for the interaction with the glutathione (GSH)/ glutathione disulfide (GSSG) redox couple, roGFP2 has been fused to glutaredoxin (Grx) to construct the Grx-roGFP2 biosensor. We have previously designed the related Brx-roGFP2 redox biosensor for dynamic measurement of the bacillithiol redox potential (E-BSH) in the human pathogen Staphylococcus aureus. Here, we describe the detailed method for measurements of the oxidation degree (OxD) of the Brx-roGFP2 biosensor in S. aureus using the microplate reader. In particularly, we provide details for determination of the E-BSH changes during the growth and after oxidative stress. For future biosensor applications at the single cell level, we recommend the design of genome-encoded roGFP2 biosensors enabling stable expression and fluorescence in bacteria. Brx-roGFP2 is specific for measurements of the bacillithiol redox potential in Staphylococcus aureus cells Control samples for fully reduced and oxidized states of Brx-roGFP2 are required for calibration during OxD measurements Easy to measure fluorescence excitation intensities at the 405 and 488 nm excitation maxima using microplate reader

Redox-Sensing-Mechanismen unter Hypochlorit-Stress in Staphylococcus aureus

Glutathione (GSH) is the major low molecular weight (LMW) thiol of eukaryotic organisms and Gram-negative bacteria to maintain the redox balance (chapters 1-2). However, Gram-positive bacteria do not produce GSH. Bacillithiol (BSH) is utilized as alternative LMW thiol in Firmicutes, such as Bacillus subtilis and Staphyloccoccus aureus. Mycothiol functions instead as major LMW thiol in all Actinomycetes, such as Mycobacteria and Corynebacteria. Under oxidative stress, LMW thiols form mixed disulfides with proteins thiols, termed as S-thiolations which function as thiol-protection and redox-control mechanism. The main goal of this work was the identification of novel thiol-switches and S-thiolated proteins in the thiol-redox proteome of the human pathogens S. aureus and Corynebacterium diphtheriae under hypochlorous acid (HOCl) stress. HOCl is a highly reactive oxidant that is produced during neutrophil infections and is the major cause of bacterial killing. Using the thiol-redox proteomics approach OxICAT, 58 NaOCl-sensitive protein thiols with >10% increased oxidations could be identified in S. aureus USA300 (chapters 3 4). Among these are five S-bacillithiolated proteins, including the two aldehyde dehydrogenases GapDH and AldA which showed the highest oxidation increase of ~29 % in the OxICAT analysis. GapDH and AldA were S-bacillithiolated at their active site Cys residues, Cys151 in GapDH and Cys279 in AldA. GapDH represents the most abundant S-bacillithiolated protein contributing with 4% to the total Cys proteome of S. aureus. The catalytic active sites of GapDH and AldA are very sensitive to overoxidation and irreversible inactivation by ROS in vitro. In the presence of BSH, S-bacillithiolation protects the active sites against irreversible oxidation and functions in reversible inactivation. Using molecular docking it was further shown that BSH can undergo disulfide formation with the GapDH and AldA active site Cys residues without major conformational changes. In C. diphtheriae, the glycolytic GapDH was identified as main target for S-mycothiolation under HOCl stress (chapter 5). In addition, GapDH is also the most abundant protein in the Cys proteome of C. diphtheriae. Exposure of purified GapDH to H2O2 and NaOCl resulted in irreversible inactivation due to overoxidation of the active site in vitro. Treatment of GapDH with H2O2 or NaOCl in the presence of MSH resulted in S-mycothiolation and reversible GapDH inactivation in vitro, which was faster compared to the overoxidation pathway. Reactivation of S mycothiolated GapDH was catalyzed by the Trx and the Mrx1 pathways in vitro. De-mycothiolation by Mrx1 was faster compared to Trx. Thus, it is interesting to note that the glycolytic GapDH is a major target for S-thiolation by BSH and MSH across Gram-positive bacteria. To identify novel redox-sensing regulators in S. aureus USA300 that could provide protection under HOCl stress, we used an RNA- seq transcriptomic approach. We identified the novel Rrf2-family redox-sensing regulator HypR as most strongly induced under NaOCl stress in the transcriptome under NaOCl stress (chapter 6). HypR was characterized as redox- sensing repressor that negatively controls expression of the hypR-merA operon and directly senses and responds to NaOCl and diamide stress by a thiol-based redox switch. Mutational analysis identified Cys33 and the conserved Cys99 as essential for NaOCl-sensing while Cys99 is also important for repressor activity of HypR in vitro and in vivo. The redox-sensing mechanism of HypR involves Cys33-Cys99' intersubunit disulfide formation by NaOCl stress both in vitro and in vivo. Moreover, the HypR-controlled flavin disulfide reductase MerA was shown to protect S. aureus against NaOCl stress and increased survival in J774A.1 macrophage infection assays. We were further interested to investigate the changes in the BSH redox potential under NaOCl stress in S. aureus. Thus, we constructed a genetically encoded bacilliredoxin-fused Brx- roGFP2 redox biosensor for dynamic live-imaging of BSH redox potential changes in S. aureus during the growth, oxidative stress and under antibiotics treatment (chapter 7-8). The Brx-roGFP2 biosensor showed a specific and rapid response to low levels BSSB in vitro which required the active-site Cys of Brx. However, the biosensor was unresponsive to other LMW thiol disulfides in vitro. Dynamic live-imaging in two MRSA isolates USA300 and COL revealed fast and dynamic responses of the Brx-roGFP2 biosensor under NaOCl and H2O2 stress and constitutive oxidation of the probe in different BSH-deficient mutants. Using confocal laser scanning microscopy, the changes in the BSH redox potential in S. aureus were confirmed at the single cell level. In phagocytosis assays with THP-1 macrophages, the biosensor was 87 % oxidized in S. aureus COL. However, no changes in the BSH redox potential were measured after treatment with different antibiotics classes indicating that antibiotics do not cause oxidative stress in S. aureus. Our studies demonstrate that this novel Brx-roGFP2 biosensor catalyzes specific equilibration between the BSH and roGFP2 redox couples and can be used for dynamic live imaging of the BSH redox potential inside S. aureus. Future studies are directed to apply this Brx-roGFP2 biosensor for screening of the BSH redox potential across S. aureus isolates of different clonal complexes to reveal the differences in pathogen fitness and in their ROS detoxification capacities as defense mechanisms against the host immune system. In addition, this biosensor can be applied in drug research to screen for new ROS-generating antibiotics that affect the BSH redox potential in S. aureus.Glutathion (GSH) ist die wichtigste niedermolekulare Thiolverbindung in eukaryontischen Organismen und Gram-negativen Bakterien, um die Redoxbalance aufrechtzuerhalten (Kapitel 1-2). Gram-positive Bakterien produzieren kein GSH, sondern dafür alternative Thiolverbindungen. Bacillithiol (BSH) fungiert als alternative Thiolverbindung in Firmicutes, wie z.B. in Bacillus subtilis und Staphyloccoccus aureus. Mycothiol kommt dagegen als wichtigste Thiolverbindung in allen Actinomycetes vor, wie z.B. in Mycobakterien und Corynebakterien. Niedermolekulare Thiolverbindungen spielen eine wichtige Rolle bei post-translationalen Modifikationen nach oxidativem Stress, wobei Cysteine zu S-Thiolierungen oxidiert werden können. S-Thiolierungen schützen die Thiolgruppe vor irreversibler Oxidation zur Cystein-Sulfonsäure und fungieren als Redox-Schalter. Das Hauptziel dieser Arbeit war es, neue Thiolschalter und S-Thiolierungen im Thiolredoxproteom in den pathogenen Bakterien S. aureus and Corynebacterium diphtheriae nach HOCl stress zu identifizieren. HOCl ist ein sehr reaktives Oxidant und wird von Neutrophilen während der Infektion produziert. HOCl ist deshalb für die Abwehr des angeborenen Immunsystems gegen Bakterien von großer Bedeutung. Im Thiolredoxproteom von S. aureus USA300 konnten mittels der OxICAT-Methode 58 NaOCl-sensitive Cysteine identifiziert werden, die >10% erhöhte Oxidation nach NaOCl-Stress aufwiesen (Kapitel 3 4). Dazu zählten fünf S-bacillithiolierte Proteine, wie z.B. die Aldehyd-Dehydrogenasen GapDH und AldA, die ca. 29 % stärker oxidiert waren in der OxICAT-Analyse. GapDH und AldA sind in ihrem katalytischen Zentrum S-bacillithioliert, am Cys151 von GapDH und am Cys279 von AldA. GapDH ist das am häufigsten vorkommende S-bacillithiolierte Protein, welches mit 4% zum Gesamt-Cystein-Proteom in S. aureus beiträgt. Die katalytischen aktiven Zentren von GapDH und AldA sind sehr sensitiv gegenüber Überoxidationen und irreversiblen Inaktivierungen durch ROS in vitro. In Gegenwart von BSH und ROS kommt es zur S-Bacillithiolierung der aktiven Zentren von GapDH und AldA. Die S-Bacillithiolierung dient als Schutz der Thiolgruppe vor Überoxidation und führt ebenfalls zur reversiblen Inaktivierung der Enzyme. Durch molekulares Docking konnte weiterhin gezeigt werden, dass die S-Bacillithiolierung der Cysteine in den aktiven Zentren von GapDH und AldA keine Konformationsänderungen erfordert. In C. diphtheriae wurde die glykolytische GapDH als S-mycothioliert nach HOCl-Stress identifiziert (Kapitel 5). GapDH ist ebenfalls das am häufigsten vorkommende Protein im Cystein-Proteom von C. diphtheriae. Nach Exposition von gereinigtem GapDH mit H2O2 und NaOCl kam es zur Überoxidation des aktiven Zentrums zur Sulfonsäure, was zur irreversiblen Inaktivierung führte. Die Oxidation von GapDH durch H2O2 und NaOCl in Gegenwart von MSH führte zur S-mycothiolierung und reversiblen GapDH Inaktivierung in vitro. Kinetische Messungen zeigten weiterhin, dass die S-Mycothiolierung schneller ablief als die Überoxidation zur Sulfonsäure. Die Reaktivierung von S mycothiolierten GapDH konnte sowohl durch den Trx-Pathway als auch durch Mrx1 katalysiert werden in vitro. Die Reduktion der Mycothiolierungen mittels Mrx1 verlief wesentlich schneller im Vergleich zur Reduktion durch Trx. Somit wurde hiermit die glykolytische Glyceraldehyd-3-Phosphat-Dehydrogenase GapDH als ein wichtiges S-thioliertes metabolisches Enzym in verschiedenen Gram-positiven Bakterien identifiziert und charakterisiert. Wir waren weiterhin interessiert, neue HOCl-spezifische redox-sensitive Regulatoren zu identifizieren. Dazu wurde eine RNA-seq Transkriptomanalyse nach NaOCl-Stress durchgeführt. Wir konnten einen neuen Regulator der Rrf2-Familie identifizieren, der sehr stark durch HOCl-Stress im Transkriptom induziert wurde (Kapitel 6). HypR wurde als neuer redox- sensitiver Repressor charakterisiert, der die Expression des hypR-merA-Operons negativ reguliert. HypR wird direkt nach NaOCl und Diamid-Stress über eine reversible Thioloxidation reguliert. Durch Mutagenese wurde gezeigt, dass Cys33 und das konservierte Cys99 essential für das Redox-sensing nach NaOCl- Stress sind. Cys99 ist ebenfalls wichtig für die Repressor-Aktivität von HypR in vitro und in vivo. HypR wird nach NaOCl-Stress durch eine intermolekulare Disufidbrückenbildung zwischen Cys33 und Cys99' in vitro und in vivo reguliert. HypR reguliert die Flavin-Disulfid-Oxidoreduktase MerA. Es konnte gezeigt werden, dass MerA am Schutz von S. aureus gegenüber NaOCl-Stress beteiligt ist und zum Überleben in Infektionsassays mit Makrophagen beiträgt. Unsere weiteren Untersuchungen zielten darauf ab, die Veränderungen im BSH- Redoxpotential in S. aureus nach oxidativen Stress zu messen. Dafür wurde ein genetisch-kodierter Bacilliredoxin-fusionierter Brx-roGFP2-Biosensor konstruiert für die Analyse des BSH-Redoxpotentials in S. aureus während des Wachstums, nach oxidativem Stress und nach Antibiotika-Behandlung (Kapitel 7-8). Der Brx-roGFP2-Biosensor zeigte eine spezifische und schnelle Oxidation nach Inkubation mit geringen Mengen BSSB in vitro, welche auf das aktive Zentrum von Brx zurückzuführen war. Keine Oxidation des Biosensors wurde nach Inkubation mit anderen niedermolekularen Thiolverbindungen gemessen. Biosensor-Messungen in zwei MRSA-Isolaten USA300 und COL zeigten eine schnelle und dynamische Oxidation des Brx-roGFP2 Biosensors nach NaOCl und H2O2-Stress. Der Biosensor war konstitutiv oxidiert in verschiedenen BSH-negativen S. aureus Mutanten. Durch konfokale Laser-Scanning-Mikroskopie konnten die Veränderungen im BSH-Redoxpotential in S. aureus auf Einzelzell-Ebene bestätigt werden. Nach Infektionsversuchen mit THP-1 Makrophagen wurde eine 87 %-ige Oxidation des Biosensors in S. aureus COL gemessen. Jedoch wurden keinen Veränderungen des BSH-Redoxpotentials nach Behandlung mit verschiedenen Antibiotika nachgewiesen. Dies weist darauf hin, dass Antibiotika in S. aureus keinen oxidativen Stress verursachen. Unsere Untersuchungen zeigten, dass der neue Brx-roGFP2 Biosensor eine spezifische Äquilibrierung zwischen den BSH und roGFP2 Redoxpaaren katalysiert. Deshalb kann der Biosensor weiterhin in S. aureus angewandt werden für dynamische Messungen des BSH-Redoxpotentials. In zukünftigen Studien soll der Brx-roGFP2 Biosensor für das Screening des BSH- Redoxpotentials in S. aureus-Isolaten verschiedender klonaler Komplexe eingesetzt werden. Somit könnten Unterschiede in der Fitness und Entgiftung von ROS zwischen verschiedenen S. aureus-Isolaten untersucht werden als Abwehrmechanismen gegen das Immunsystem des Wirts. Der Biosensor kann ebenfalls in der Antibiotika-Forschung eingesetzt werden, um nach neuen ROS- produzierenden Antibiotika zu screenen, die einen Einfluss auf das BSH- Redoxpotential von S. aureus haben

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

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

Application of genetically encoded redox biosensors to measure dynamic changes in the glutathione, bacillithiol and mycothiol redox potentials in pathogenic bacteria

Gram-negative bacteria utilize glutathione (GSH) as their major LMW thiol. However, most Gram-positive bacteria do not encode enzymes for GSH biosynthesis and produce instead alternative LMW thiols, such as bacillithiol (BSH) and mycothiol (MSH). BSH is utilized by Firmicutes and MSH is the major LMW thiol of Actinomycetes. LMW thiols are required to maintain the reduced state of the cytoplasm, but are also involved in virulence mechanisms in human pathogens, such as Staphylococcus aureus, Mycobacterium tuberculosis, Streptococcus pneumoniae, Salmonella enterica subsp. Typhimurium and Listeria monocytogenes. Infection conditions often cause perturbations of the intrabacterial redox balance in pathogens, which is further affected under antibiotics treatments. During the last years, novel glutaredoxin-fused roGFP2 biosensors have been engineered in many eukaryotic organisms, including parasites, yeast, plants and human cells for dynamic live-imaging of the GSH redox potential in different compartments. Likewise bacterial roGFP2-based biosensors are now available to measure the dynamic changes in the GSH, BSH and MSH redox potentials in model and pathogenic Gram-negative and Gram-positive bacteria. In this review, we present an overview of novel functions of the bacterial LMW thiols GSH, MSH and BSH in pathogenic bacteria in virulence regulation. Moreover, recent results about the application of genetically encoded redox biosensors are summarized to study the mechanisms of host-pathogen interactions, persistence and antibiotics resistance. In particularly, we highlight recent biosensor results on the redox changes in the intracellular food-borne pathogen Salmonella Typhimurium as well as in the Gram-positive pathogens S. aureus and M. tuberculosis during infection conditions and under antibiotics treatments. These studies established a link between ROS and antibiotics resistance with the intracellular LMW thiol-redox potential. Future applications should be directed to compare the redox potentials among different clinical isolates of these pathogens in relation to their antibiotics resistance and to screen for new ROS-producing drugs as promising strategy to combat antimicrobial resistance

The Role of Bacillithiol in Gram-Positive Firmicutes

Significance: Since the discovery and structural characterization of bacillithiol (BSH), the biochemical functions of BSH-biosynthesis enzymes (BshA/B/C) and BSH-dependent detoxification enzymes (FosB, Bst, GlxA/B) have been explored in Bacillus and Staphylococcus species. It was shown that BSH plays an important role in detoxification of reactive oxygen and electrophilic species, alkylating agents, toxins, and antibiotics. Recent Advances: More recently, new functions of BSH were discovered in metal homeostasis (Zn buffering, Fe-sulfur cluster, and copper homeostasis) and virulence control in Staphylococcus aureus. Unexpectedly, strains of the S. aureus NCTC8325 lineage were identified as natural BSH-deficient mutants. Modern mass spectrometry-based approaches have revealed the global reach of protein S-bacillithiolation in Firmicutes as an important regulatory redox modification under hypochlorite stress. S-bacillithiolation of OhrR, MetE, and glyceraldehyde-3-phosphate dehydrogenase (Gap) functions, analogous to S-glutathionylation, as both a redox-regulatory device and in thiol protection under oxidative stress. Critical Issues: Although the functions of the bacilliredoxin (Brx) pathways in the reversal of S-bacillithiolations have been recently addressed, significantly more work is needed to establish the complete Brx reduction pathway, including the major enzyme(s), for reduction of oxidized BSH (BSSB) and the targets of Brx action in vivo. Future Directions: Despite the large number of identified S-bacillithiolated proteins, the physiological relevance of this redox modification was shown for only selected targets and should be a subject of future studies. In addition, many more BSH-dependent detoxification enzymes are evident from previous studies, although their roles and biochemical mechanisms require further study. This review of BSH research also pin-points these missing gaps for future research. Antioxid. Redox Signal. 28, 445–462

Thiol-based redox switches in the major pathogen Staphylococcus aureus

Staphylococcus aureus is a major human pathogen, which encounters reactive oxygen, nitrogen, chlorine, electrophile and sulfur species (ROS, RNS, RCS, RES and RSS) by the host immune system, during cellular metabolism or antibiotics treatments. To defend against redox active species and antibiotics, S. aureus is equipped with redox sensing regulators that often use thiol switches to control the expression of specific detoxification pathways. In addition, the maintenance of the redox balance is crucial for survival of S. aureus under redox stress during infections, which is accomplished by the low molecular weight (LMW) thiol bacillithiol (BSH) and the associated bacilliredoxin (Brx)/BSH/bacillithiol disulfide reductase (YpdA)/NADPH pathway. Here, we present an overview of thiol-based redox sensors, its associated enzymatic detoxification systems and BSH-related regulatory mechanisms in S. aureus, which are important for the defense under redox stress conditions. Application of the novel Brx-roGFP2 biosensor provides new insights on the impact of these systems on the BSH redox potential. These thiol switches of S. aureus function in protection against redox active desinfectants and antimicrobials, including HOCl, the AGXX (R) antimicrobial surface coating, allicin from garlic and the naphthoquinone lapachol. Thus, thiol switches could be novel drug targets for the development of alternative redox-based therapies to combat multi-drug resistant S. aureus isolates

The neutrophil oxidant hypothiocyanous acid causes a thiol-specific stress response and an oxidative shift of the bacillithiol redox potential in Staphylococcus aureus

During infections, Staphylococcus aureus is exposed to hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN), which are produced by the neutrophil myeloperoxidase as potent antimicrobial killing agents. In this work, we applied RNAseq transcriptomics, Brx-roGFP2 biosensor measurements, and phenotype analyses to investigate the stress responses and defense mechanisms of S. aureus COL toward HOSCN stress. Based on the RNAseq transcriptome profile, HOSCN exerts strong thiol-specific oxidative, electrophile, and metal stress responses as well as protein damage in S. aureus, which is indicated by the strong induction of the HypR, TetR1, PerR, QsrR, MhqR, CstR, CsoR, CzrA, AgrA, HrcA, and CtsR regulons. Phenotype analyses of various mutants in HOSCN-responsive genes revealed that the HOSCN reductase MerA conferred the highest resistance toward HOSCN stress in S. aureus COL, whereas the QsrR and MhqR electrophile stress regulons do not contribute to protection. Brx-roGFP2 biosensor measurements and bacillithiol (BSH)-specific Western blot analyses revealed a strong oxidative shift of the bacillithiol redox potential (EBSH) and increased S-bacillithiolations in S. aureus, indicating that BSH is oxidized to bacillithiol disulfide (BSSB) under HOSCN stress. While the ΔmerA mutant was delayed in recovery of the reduced EBSH, overproduction of MerA in the ΔhypR mutant enabled faster recovery of EBSH due to efficient HOSCN detoxification. Moreover, both MerA and BSH were shown to contribute to HOSCN resistance in growth assays. In summary, HOSCN provokes a thiol-specific oxidative, electrophile, and metal stress response, an oxidative shift in EBSH and increased S-bacillithiolation in S. aureus

Analysis on the performance of reconfigurable intelligent surface-aided free-space optical link under atmospheric turbulence and pointing errors

Free-space optical (FSO) communication can provide the cost-efficient, secure, high data-rate communication links required for applications. For example, it provides broadband Internet access and backhauling for the fifth-generation (5G) and the sixth-generation (6G) communication networks. However, previous solutions to deal with signal loss caused by obstructions and atmospheric turbulence. In these solutions, reconfigurable intelligent surfaces (RISs) are considered hardware technology to improve the performance of optical wireless communication systems. This study investigates the pointing error effects for RIS-aided FSO links under atmospheric turbulence channels. We analyze the performance of RIS-aided FSO links influenced by pointing errors, atmospheric attenuation, and turbulence for the subcarrier quadrature amplitude modulation (SC-QAM) technique. Atmospheric turbulence is modeled using log-normal distribution for weak atmospheric turbulence. Several numerical outcomes obtained for different transmitter beam waist radius and pointing error displacement standard deviation are shown to quantitatively illustrate the average symbol error rate (ASER)

The MarR-Type Repressor MhqR Confers Quinone and Antimicrobial Resistance in Staphylococcus aureus

Aims: Quinone compounds are electron carriers and have antimicrobial and toxic properties due to their mode of actions as electrophiles and oxidants. However, the regulatory mechanism of quinone resistance is less well understood in the pathogen Staphylococcus aureus. Results: Methylhydroquinone (MHQ) caused a thiol-specific oxidative and electrophile stress response in the S. aureus transcriptome as revealed by the induction of the PerR, QsrR, CstR, CtsR, and HrcA regulons. The SACOL2531-29 operon was most strongly upregulated by MHQ and was renamed as mhqRED operon based on its homology to the Bacillus subtilis locus. Here, we characterized the MarR-type regulator MhqR (SACOL2531) as quinone-sensing repressor of the mhqRED operon, which confers quinone and antimicrobial resistance in S. aureus. The mhqRED operon responds specifically to MHQ and less pronounced to pyocyanin and ciprofloxacin, but not to reactive oxygen species (ROS), hypochlorous acid, or aldehydes. The MhqR repressor binds specifically to a 9–9 bp inverted repeat (MhqR operator) upstream of the mhqRED operon and is inactivated by MHQ in vitro, which does not involve a thiol-based mechanism. In phenotypic assays, the mhqR deletion mutant was resistant to MHQ and quinone-like antimicrobial compounds, including pyocyanin, ciprofloxacin, norfloxacin, and rifampicin. In addition, the mhqR mutant was sensitive to sublethal ROS and 24 h post-macrophage infections but acquired an improved survival under lethal ROS stress and after long-term infections. Innovation: Our results provide a link between quinone and antimicrobial resistance via the MhqR regulon of S. aureus. Conclusion: The MhqR regulon was identified as a novel resistance mechanism towards quinone-like antimicrobials and contributes to virulence of S. aureus under long-term infections

The AGXX (R) Antimicrobial Coating Causes a Thiol-Specific Oxidative Stress Response and Protein S-bacillithiolation in Staphylococcus aureus

Van Loi V, Busche T, Preuss T, Kalinowski J, Bernhardt J, Antelmann H. The AGXX (R) Antimicrobial Coating Causes a Thiol-Specific Oxidative Stress Response and Protein S-bacillithiolation in Staphylococcus aureus. FRONTIERS IN MICROBIOLOGY. 2018;9: 3037.Multidrug-resistant pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA) pose an increasing health burden and demand alternative antimicrobials to treat bacterial infections. The surface coating AGXX (R) is a novel broad-spectrum antimicrobial composed of two transition metals, silver and ruthenium that can be electroplated on various surfaces, such as medical devices and implants. AGXX (R) has been shown to kill nosocomial and waterborne pathogens by production of reactive oxygen species (ROS), but the effect of AGXX (R) on the bacterial redox balance has not been demonstrated. Since treatment options for MRSA infections are limited, ROS-producing agents are attractive alternatives to combat multi-resistant strains. In this work, we used RNA-seq transcriptomics, redox biosensor measurements and phenotype analyses to study the mode of action of AGXX (R) microparticles in S. aureus USA300. Using growth and survival assays, the growth-inhibitory amount of AGXX (R) microparticles was determined as 5 mu g/ml. In the RNA-seq transcriptome, AGXX (R) caused a strong thiol-specific oxidative stress response and protein damage as revealed by the induction of the PerR, HypR, QsrR, MhqR, CstR, CtsR, and HrcA regulons. The derepression of the Fur, Zur, and CsoR regulons indicates that AGXX (R) also interferes with the metal ion homeostasis inducing Fe-2(+)- and Zn-2(+)-starvation responses as well as export systems for toxic Ag+ ions. The induction of the SigB and GraRS regulons reveals also cell wall and general stress responses. AGXX (R). stress was further shown to cause protein S-bacillithiolation, protein aggregation and an oxidative shift in the bacillithiol (BSH) redox potential. In phenotype assays, BSH and the HypR-controlled disulfide reductase MerA were required for protection against ROS produced under AGXX (R) stress in S. aureus. Altogether, our study revealed a strong thiol-reactive mode of action of AGXX (R) in S. aureus USA300 resulting in an increased BSH redox potential and protein S-bacillithiolation