The MerR-family regulator NmlR is involved in the defense against oxidative stress in Streptococcus pneumoniae.

Streptococcus pneumoniae has to cope with the strong oxidant hypochlorous acid (HOCl), during host-pathogen interactions. Thus, we analyzed the global gene expression profile of S. pneumoniae D39 towards HOCl stress. In the RNA-seq transcriptome, the NmlR, SifR, CtsR, HrcA, SczA and CopY regulons and the etrx1-ccdA1-msrAB2 operon were most strongly induced under HOCl stress, which participate in the oxidative, electrophile and metal stress response in S. pneumoniae. The MerR-family regulator NmlR harbors a conserved Cys52 and controls the alcohol dehydrogenase-encoding adhC gene under carbonyl and NO stress. We demonstrated that NmlR senses also HOCl stress to activate transcription of the nmlR-adhC operon. HOCl-induced transcription of adhC required Cys52 of NmlR in vivo. Using mass spectrometry, NmlR was shown to be oxidized to intersubunit disulfides or S-glutathionylated under oxidative stress in vitro. A broccoli-FLAP-based assay further showed that both NmlR disulfides significantly increased transcription initiation at the nmlR promoter by RNAP in vitro, which depends on Cys52. Phenotype analyses revealed that NmlR functions in the defense against oxidative stress and promotes survival of S. pneumoniae during macrophage infections. In conclusion, NmlR was characterized as HOCl-sensing transcriptional regulator, which activates transcription of adhC under oxidative stress by thiol switches in S. pneumoniae.

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® 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 MarR/DUF24-Family QsrR Repressor Senses Quinones and Oxidants by Thiol Switch Mechanisms in Staphylococcus aureus.

Aims: The MarR/DUF24-family QsrR and YodB repressors control quinone detoxification pathways in Staphylococcus aureus and Bacillus subtilis. In S. aureus, the QsrR regulon also confers resistance to antimicrobial compounds with quinone-like elements, such as rifampicin, ciprofloxacin, and pyocyanin. Although QsrR was shown to be inhibited by thiol-S-alkylation of its conserved Cys4 residue by 1,4-benzoquinone, YodB senses quinones and diamide by the formation of reversible intermolecular disulfides. In this study, we aimed at further investigating the redox-regulation of QsrR and the role of its Cys4, Cys29, and Cys32 residues under quinone and oxidative stress in S. aureus. Results: The QsrR regulon was strongly induced by quinones and oxidants, such as diamide, allicin, hypochlorous acid (HOCl), and AGXX® in S. aureus. Transcriptional induction of catE2 by quinones and oxidants required Cys4 and either Cys29' or Cys32' of QsrR for redox sensing in vivo. DNA-binding assays revealed that QsrR is reversibly inactivated by quinones and oxidants, depending on Cys4. Using mass spectrometry, QsrR was shown to sense diamide by an intermolecular thiol-disulfide switch, involving Cys4 and Cys29' of opposing subunits in vitro. In contrast, allicin caused S-thioallylation of all three Cys residues in QsrR, leading to its dissociation from the operator sequence. Further, the QsrR regulon confers resistance against quinones and oxidants, depending on Cys4 and either Cys29' or Cys32'. Conclusion and Innovation: QsrR was characterized as a two-Cys-type redox-sensing regulator, which senses the oxidative mode of quinones and strong oxidants, such as diamide, HOCl, and the antimicrobial compound allicin via different thiol switch mechanisms.

The two-Cys-type TetR repressor GbaA confers resistance under disulfide and electrophile stress in Staphylococcus aureus.

Staphylococcus aureus has to cope with oxidative and electrophile stress during host-pathogen interactions. The TetR-family repressor GbaA was shown to sense electrophiles, such as N-ethylmaleimide (NEM) via monothiol mechanisms of the two conserved Cys55 or Cys104 residues in vitro. In this study, we further investigated the regulation and function of the GbaA repressor and its Cys residues in S. aureus COL. The GbaA-controlled gbaAB-SACOL2595-97 and SACOL2592-nmrA-2590 operons were shown to respond only weakly 3-10-fold to oxidants, electrophiles or antibiotics in S. aureus COL, but are 57-734-fold derepressed in the gbaA deletion mutant, indicating that the physiological inducer is still unknown. Moreover, the gbaA mutant remained responsive to disulfide and electrophile stress, pointing to additional redox control mechanisms of both operons. Thiol-stress induction of the GbaA regulon was strongly diminished in both single Cys mutants, supporting that both Cys residues are required for redox-sensing in vivo. While GbaA and the single Cys mutants are reversible oxidized under diamide and allicin stress, these thiol switches did not affect the DNA binding activity. The repressor activity of GbaA could be only partially inhibited with NEM in vitro. Survival assays revealed that the gbaA mutant confers resistance under diamide, allicin, NEM and methylglyoxal stress, which was mediated by the SACOL2592-90 operon encoding for a putative glyoxalase and oxidoreductase. Altogether, our results support that the GbaA repressor functions in the defense against oxidative and electrophile stress in S. aureus. GbaA represents a 2-Cys-type redox sensor, which requires another redox-sensing regulator and an unknown thiol-reactive ligand for full derepression of the GbaA regulon genes.

The Effect of Allicin on the Proteome of SARS-CoV-2 Infected Calu-3 Cells.

Allicin (diallyl thiosulfinate) is the major thiol-reactive organosulfur compound produced by garlic plants (Allium sativum) upon tissue damage. Allicin exerts its strong antimicrobial activity against bacteria and fungi via S-thioallylation of protein thiols and low molecular weight thiols. Here, we investigated the effect of allicin on SARS-CoV-2 infected Vero E6 and Calu-3 cells. Toxicity tests revealed that Calu-3 cells showed greater allicin tolerance, probably due to >4-fold higher GSH levels compared to the very sensitive Vero E6 cells. Exposure of infected Vero E6 and Calu-3 cells to biocompatible allicin doses led to a ∼60-70% decrease of viral RNA and infectious viral particles. Label-free quantitative proteomics was used to investigate the changes in the Calu-3 proteome after SARS-CoV-2 infection and the effect of allicin on the host-virus proteome. SARS-CoV-2 infection of Calu-3 cells caused a strong induction of the antiviral interferon-stimulated gene (ISG) signature, including several antiviral effectors, such as cGAS, Mx1, IFIT, IFIH, IFI16, IFI44, OAS, and ISG15, pathways of vesicular transport, tight junctions (KIF5A/B/C, OSBPL2, CLTCL1, and ARHGAP17) and ubiquitin modification (UBE2L3/5), as well as reprogramming of host metabolism, transcription and translation. Allicin treatment of infected Calu-3 cells reduced the expression of IFN signaling pathways and ISG effectors and reverted several host pathways to levels of uninfected cells. Allicin further reduced the abundance of the structural viral proteins N, M, S and ORF3 in the host-virus proteome. In conclusion, our data demonstrate the antiviral and immunomodulatory activity of biocompatible doses of allicin in SARS-CoV-2-infected cell cultures. Future drug research should be directed to exploit the thiol-reactivity of allicin derivatives with increased stability and lower human cell toxicity as antiviral lead compounds.

The plant-derived naphthoquinone lapachol causes an oxidative stress response in Staphylococcus aureus.

Staphylococcus aureus is a major human pathogen, which causes life-threatening systemic and chronic infections and rapidly acquires resistance to multiple antibiotics. Thus, new antimicrobial compounds are required to combat infections with drug resistant S. aureus isolates. The 2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone lapachol was previously shown to exert antimicrobial effects. In this study, we investigated the antimicrobial mode of action of lapachol in S. aureus using RNAseq transcriptomics, redox biosensor measurements, S-bacillithiolation assays and phenotype analyses of mutants. In the RNA-seq transcriptome, lapachol caused an oxidative and quinone stress response as well as protein damage as revealed by induction of the PerR, HypR, QsrR, MhqR, CtsR and HrcA regulons. Lapachol treatment further resulted in up-regulation of the SigB and GraRS regulons, which is indicative for cell wall and general stress responses. The redox-cycling mode of action of lapachol was supported by an elevated bacillithiol (BSH) redox potential (EBSH), higher endogenous ROS levels, a faster H2O2 detoxification capacity and increased thiol-oxidation of GapDH and the HypR repressor in vivo. The ROS scavenger N-acetyl cysteine and microaerophilic growth conditions improved the survival of lapachol-treated S. aureus cells. Phenotype analyses revealed an involvement of the catalase KatA and the Brx/BSH/YpdA pathway in protection against lapachol-induced ROS-formation in S. aureus. However, no evidence for irreversible protein alkylation and aggregation was found in lapachol-treated S. aureus cells. Thus, the antimicrobial mode of action of lapachol in S. aureus is mainly caused by ROS formation resulting in an oxidative stress response, an oxidative shift of the EBSH and increased protein thiol-oxidation. As ROS-generating compound, lapachol is an attractive alternative antimicrobial to combat multi-resistant S. aureus isolates.

The alarmone (p)ppGpp confers tolerance to oxidative stress during the stationary phase by maintenance of redox and iron homeostasis in Staphylococcus aureus.

Slow growing stationary phase bacteria are often tolerant to multiple stressors and antimicrobials. Here, we show that the pathogen Staphylococcus aureus develops a non-specific tolerance towards oxidative stress during the stationary phase, which is mediated by the nucleotide second messenger (p)ppGpp. The (p)ppGpp0 mutant was highly susceptible to HOCl stress during the stationary phase. Transcriptome analysis of the (p)ppGpp0 mutant revealed an increased expression of the PerR, SigB, QsrR, CtsR and HrcA regulons during the stationary phase, indicating an oxidative stress response. The (p)ppGpp0 mutant showed a slight oxidative shift in the bacillithiol (BSH) redox potential (EBSH) and an impaired H2O2 detoxification due to higher endogenous ROS levels. The increased ROS levels in the (p)ppGpp0 mutant were shown to be caused by higher respiratory chain activity and elevated total and free iron levels. Consistent with these results, N-acetyl cysteine and the iron-chelator dipyridyl improved the growth and survival of the (p)ppGpp0 mutant under oxidative stress. Elevated free iron levels caused 8 to 31-fold increased transcription of Fe-storage proteins ferritin (ftnA) and miniferritin (dps) in the (p)ppGpp0 mutant, while Fur-regulated uptake systems for iron, heme or siderophores (efeOBU, isdABCDEFG, sirABC and sstADBCD) were repressed. Finally, the susceptibility of the (p)ppGpp0 mutant towards the bactericidal action of the antibiotics ciprofloxacin and tetracycline was abrogated with N-acetyl cysteine and dipyridyl. Taken together, (p)ppGpp confers tolerance to ROS and antibiotics by down-regulation of respiratory chain activity and free iron levels, lowering ROS formation to ensure redox homeostasis in S. aureus.

Staphylococcus aureus Uses the Bacilliredoxin (BrxAB)/Bacillithiol Disulfide Reductase (YpdA) Redox Pathway to Defend Against Oxidative Stress Under Infections.

Staphylococcus aureus is a major human pathogen and has to cope with reactive oxygen and chlorine species (ROS, RCS) during infections. The low molecular weight thiol bacillithiol (BSH) is an important defense mechanism of S. aureus for detoxification of ROS and HOCl stress to maintain the reduced state of the cytoplasm. Under HOCl stress, BSH forms mixed disulfides with proteins, termed as S-bacillithiolations, which are reduced by bacilliredoxins (BrxA and BrxB). The NADPH-dependent flavin disulfide reductase YpdA is phylogenetically associated with the BSH synthesis and BrxA/B enzymes and was recently suggested to function as BSSB reductase (Mikheyeva et al., 2019). Here, we investigated the role of the complete bacilliredoxin BrxAB/BSH/YpdA pathway in S. aureus COL under oxidative stress and macrophage infection conditions in vivo and in biochemical assays in vitro. Using HPLC thiol metabolomics, a strongly enhanced BSSB level and a decreased BSH/BSSB ratio were measured in the S. aureus COL ΔypdA deletion mutant under control and NaOCl stress. Monitoring the oxidation degree (OxD) of the Brx-roGFP2 biosensor revealed that YpdA is required for regeneration of the reduced BSH redox potential (E BSH) upon recovery from oxidative stress. In addition, the ΔypdA mutant was impaired in H2O2 detoxification as measured with the novel H2O2-specific Tpx-roGFP2 biosensor. Phenotype analyses further showed that BrxA and YpdA are required for survival under NaOCl and H2O2 stress in vitro and inside murine J-774A.1 macrophages in infection assays in vivo. Finally, NADPH-coupled electron transfer assays provide evidence for the function of YpdA in BSSB reduction, which depends on the conserved Cys14 residue. YpdA acts together with BrxA and BSH in de-bacillithiolation of S-bacillithiolated GapDH. In conclusion, our results point to a major role of the BrxA/BSH/YpdA pathway in BSH redox homeostasis in S. aureus during recovery from oxidative stress and under infections.

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.

Calcineurin Silencing in Dictyostelium discoideum Leads to Cellular Alterations Affecting Mitochondria, Gene Expression, and Oxidative Stress Response.

Calcineurin is involved in development and cell differentiation of the social amoeba Dictyostelium discoideum. However, since knockouts of the calcineurin-encoding genes are not possible in D. discoideum it is assumed that the phosphatase also plays a crucial role during vegetative growth of the amoebae. Therefore, we investigated the role of calcineurin during vegetative growth in D. discoideum. RNAi-silenced calcineurin mutants showed cellular alterations with an abnormal morphology of mitochondria and had increased content of mitochondrial DNA (mtDNA). In contrast, mitochondria showed no substantial functional impairment. Calcineurin-silencing led to altered expression of calcium-regulated genes as well as mitochondrially-encoded genes. Furthermore, genes related to oxidative stress were higher expressed in the mutants, which correlated to an increased resistance towards reactive oxygen species (ROS). Most of the changes observed during vegetative growth were not seen after starvation of the calcineurin mutants. We show that impairment of calcineurin led to many subtle, but in the sum crucial cellular alterations in vegetative D. discoideum cells. As these alterations were not observed after starvation we propose a dual role for calcineurin during growth and development. Our results imply that calcineurin is one player in the mutual interplay between mitochondria and ROS during vegetative growth.

The aldehyde dehydrogenase AldA contributes to the hypochlorite defense and is redox-controlled by protein S-bacillithiolation in Staphylococcus aureus.

Staphylococcus aureus produces bacillithiol (BSH) as major low molecular weight (LMW) thiol which functions in thiol-protection and redox-regulation by protein S-bacillithiolation under hypochlorite stress. The aldehyde dehydrogenase AldA was identified as S-bacillithiolated at its active site Cys279 under NaOCl stress in S. aureus. Here, we have studied the expression, function, redox regulation and structural changes of AldA of S. aureus. Transcription of aldA was previously shown to be regulated by the alternative sigma factor SigmaB. Northern blot analysis revealed SigmaB-independent induction of aldA transcription under formaldehyde, methylglyoxal, diamide and NaOCl stress. Deletion of aldA resulted in a NaOCl-sensitive phenotype in survival assays, suggesting an important role of AldA in the NaOCl stress defense. Purified AldA showed broad substrate specificity for oxidation of several aldehydes, including formaldehyde, methylglyoxal, acetaldehyde and glycol aldehyde. Thus, AldA could be involved in detoxification of aldehyde substrates that are elevated under NaOCl stress. Kinetic activity assays revealed that AldA is irreversibly inhibited under H2O2 treatment in vitro due to overoxidation of Cys279 in the absence of BSH. Pre-treatment of AldA with BSH prior to H2O2 exposure resulted in reversible AldA inactivation due to S-bacillithiolation as revealed by activity assays and BSH-specific Western blot analysis. Using molecular docking and molecular dynamic simulation, we further show that BSH occupies two different positions in the AldA active site depending on the AldA activation state. In conclusion, we show here that AldA is an important target for S-bacillithiolation in S. aureus that is up-regulated under NaOCl stress and functions in protection under hypochlorite stress.