No abstract

Mycothiol (MSH) functions as major low molecular weight (LMW) thiol in the industrially important Corynebacterium glutamicum. In this study, we genomically integrated an Mrx1-roGFP2 biosensor in C. glutamicum to measure dynamic changes of the MSH redox potential (EMSH) during the growth and under oxidative stress. C. glutamicum maintains a highly reducing intrabacterial EMSH throughout the growth curve with basal EMSH levels of ~− 296 mV. Consistent with its H2O2 resistant phenotype, C. glutamicum responds only weakly to 40 mM H2O2, but is rapidly oxidized by low doses of NaOCl. We further monitored basal EMSH changes and the H2O2 response in various mutants which are compromised in redox-signaling of ROS (OxyR, SigH) and in the antioxidant defense (MSH, Mtr, KatA, Mpx, Tpx). While the probe was constitutively oxidized in the mshC and mtr mutants, a smaller oxidative shift in basal EMSH was observed in the sigH mutant. The catalase KatA was confirmed as major H2O2 detoxification enzyme required for fast biosensor re-equilibration upon return to non-stress conditions. In contrast, the peroxiredoxins Mpx and Tpx had only little impact on EMSH and H2O2 detoxification. Further live imaging experiments using confocal laser scanning microscopy revealed the stable biosensor expression and fluorescence at the single cell level. In conclusion, the stably expressed Mrx1-roGFP2 biosensor was successfully applied to monitor dynamic EMSH changes in C. glutamicum during the growth, under oxidative stress and in different mutants revealing the impact of Mtr and SigH for the basal level EMSH and the role of OxyR and KatA for efficient H2O2 detoxification under oxidative stress.

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 Δ ypdA 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 Δ ypdA mutant was impaired in H 2 O 2 detoxification as measured with the novel H 2 O 2 -specific Tpx-roGFP2 biosensor. Phenotype analyses further showed that BrxA and YpdA are required for survival under NaOCl and H 2 O 2 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.

In aerobic environments, bacteria are exposed to reactive oxygen species (ROS). To avoid an excess of ROS, microorganisms are equipped with powerful enzymatic and non-enzymatic antioxidants. Corynebacterium glutamicum, a widely used industrial platform organism, uses mycothiol (MSH) as major low molecular weight (LMW) thiol and non-enzymatic antioxidant. In aerobic bioreactor cultivations, C. glutamicum becomes exposed to oxygen concentrations surpassing the air saturation, which are supposed to constitute a challenge for the intracellular MSH redox balance. In this study, the role of MSH was investigated at different oxygen levels (pO2) in bioreactor cultivations in C. glutamicum. Despite the presence of other highly efficient antioxidant systems, such as catalase, the MSH deficient ΔmshC mutant was impaired in growth in bioreactor experiments performed at pO2 values of 30%. At a pO2 level of 20%, this growth defect was abolished, indicating a high susceptibility of the MSH-deficient mutant towards elevated oxygen concentrations. Bioreactor experiments with C. glutamicum expressing the Mrx1-roGFP2 redox biosensor revealed a strong oxidative shift in the MSH redox potential (EMSH) at pO2 values above 20%. This indicates that the LMW thiol MSH is an essential antioxidant to maintain the robustness and industrial performance of C. glutamicum during aerobic fermentation processes.

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)ppGpp 0 mutant was highly susceptible to HOCl stress during the stationary phase. Transcriptome analysis of the (p)ppGpp 0 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)ppGpp 0 mutant showed a slight oxidative shift in the bacillithiol (BSH) redox potential ( E BSH ) and an impaired H 2 O 2 detoxification due to higher endogenous ROS levels. The increased ROS levels in the (p)ppGpp 0 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)ppGpp 0 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)ppGpp 0 mutant, while Fur-regulated uptake systems for iron, heme or siderophores ( efeOBU , isdABCDEFG , sirABC and sstADBCD ) were repressed. Finally, the susceptibility of the (p)ppGpp 0 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 .

No abstract

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.

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 proposed to function as BSSB reductase. Here, we investigated the role of the 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 ypdA deletion mutant under control and NaOCl stress. Monitoring the BSH redox potential (E BSH) using the Brx-roGFP2 biosensor revealed that YpdA is required for regeneration of the reduced EBSH upon recovery from oxidative stress. In addition, the ypdA 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-bacilithiolated 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. * *