Jörg Mostertz

S-Cysteinylation Is a General Mechanism for Thiol Protection of Bacillus subtilis Proteins after Oxidative Stress

S-Thiolation is crucial for protection and regulation of thiol-containing proteins during oxidative stress and is frequently achieved by the formation of mixed disulfides with glutathione. However, many Gram-positive bacteria including Bacillus subtilis lack the low molecular weight (LMW) thiol glutathione. Here we provide evidence that S-thiolation by the LMW thiol cysteine represents a general mechanism in B. subtilis. In vivo labeling of proteins with [35S]cysteine and nonreducing two-dimensional PAGE analyses revealed that a large subset of proteins previously identified as having redox-sensitive thiols are modified by cysteine in response to treatment with the thiol-specific oxidant diamide. By means of multidimensional shotgun proteomics, the sites of S-cysteinylation for six proteins could be identified, three of which are known to be S-glutathionylated in other organisms.

Fluorescence thiol modification assay: oxidatively modified proteins in Bacillus subtilis

Oxidatively modified thiol groups of cysteine residues are known to modulate the activity of a growing number of proteins. In this study, we developed a fluorescence‐based thiol modification assay and combined it with two‐dimensional gel electrophoresis and mass spectrometry to monitor the in vivo thiol state of cytoplasmic proteins. For the Gram‐positive model organism Bacillus subtilis our results show that protein thiols of growing cells are mainly present in the reduced state. Only a few proteins were found to be thiol‐modified, e.g. enzymes that include oxidized thiols in their catalytic cycle. To detect proteins that are particularly sensitive to oxidative stress we exposed growing B. subtilis cells to diamide, hydrogen peroxide or to the superoxide generating agent paraquat. Diamide mediated a significant increase of oxidized thiols in a variety of metabolic enzymes, whereas treatment with paraquat affected only a few proteins. Exposure to hydrogen peroxide forced the oxidation especially of proteins with active site cysteines, e.g. of cysteine‐based peroxidases and glutamine amidotransferase‐like proteins. Moreover, high levels of hydrogen peroxide were observed to influence the isoelectric point of proteins of this group indicating the generation of irreversibly oxidated thiols. From the overlapping set of oxidatively modified proteins, also enzymes necessary for methionine biosynthesis were identified, e.g. cobalamin‐independent methionine synthase MetE. Growth experiments revealed a methionine limitation after diamide and hydrogen peroxide stress, which suggests a thiol‐oxidation‐dependent inactivation of MetE. Finally, evidence is presented that the antibiotic nitrofurantoin mediates the formation of oxidized thiols in B. subtilis.

Primary cultures of glomerular parietal epithelial cells or podocytes with proven origin.

Parietal epithelial cells (PECs) are crucially involved in the pathogenesis of rapidly progressive glomerulonephritis (RPGN) as well as in focal and segmental glomerulosclerosis (FSGS). In this study, transgenic mouse lines were used to isolate pure, genetically tagged primary cultures of PECs or podocytes using FACsorting. By this approach, the morphology of primary glomerular epithelial cells in culture could be resolved: Primary podocytes formed either large cells with intracytoplasmatic extensions or smaller spindle shaped cells, depending on specific culture conditions. Primary PECs were small and exhibited a spindle-shaped or polygonal morphology. In the very early phases of primary culture, rapid changes in gene expression (e.g. of WT-1 and Pax-2) were observed. However, after prolonged culture primary PECs and podocytes still segregated clearly in a transcriptome analysis - demonstrating that the origin of primary cell cultures is important. Of the classical markers, synaptopodin and podoplanin expression were differentially regulated the most in primary PEC and podocyte cultures. However, no expression of any endogenous gene allowed to differentiate between the two cell types in culture. Finally, we show that the transcription factor WT1 is also expressed by PECs. In summary, genetic tagging of PECs and podocytes is a novel and necessary tool to derive pure primary cultures with proven origin. These cultures will be a powerful tool for the emerging field of parietal epithelial cell biology.

The peroxide stress response of Bacillus licheniformis

The oxidative stress response of Bacillus licheniformis after treatment with hydrogen peroxide was investigated at the transcriptome, proteome and metabolome levels. In this comprehensive study, 84 proteins and 467 transcripts were found to be up or downregulated in response to the stressor. Among the upregulated genes were many that are known to have important functions in the oxidative stress response of other organisms, such as catalase, alkylhydroperoxide reductase or the thioredoxin system. Many of these genes could be grouped into putative regulons by genomic mining. The occurrence of oxidative damage to proteins was analyzed by a 2‐DE‐based approach. In addition, we report the induction of genes with hitherto unknown functions, which may be important for the specific oxidative stress response of B. licheniformis. The genes BLi04114 and BLi04115, that are located adjacent to the catalase gene, were massively induced during peroxide stress. Furthermore, the genes BLi04207 and BLi04208, which encode proteins homologous to glyoxylate cycle enzymes, were also induced by peroxide. Metabolomic analyses support the induction of the glyoxylate cycle during oxidative stress in B. licheniformis.

Distribution and infection-related functions of bacillithiol in Staphylococcus aureus

Bacillithiol (Cys-GlcN-malate, BSH) serves as a major low molecular weight thiol in low GC Gram-positive bacteria including Bacillus species and a variety of Staphylococcus aureus strains. These bacteria do not produce glutathione (GSH). In this study, HPLC analyses were used to determine BSH levels in different S. aureus strains. Furthermore, the role of BSH in the resistance against oxidants and antibiotics and its function in virulence was investigated. We and others (Newton, G.L., Fahey, R.C., Rawat, M., 2012. Microbiology 158, 1117-1126) found that BSH is not produced by members of the S. aureus NCTC8325 lineage, such as strains 8325-4 and SH1000. Using bioinformatics we show that the BSH-biosynthetic gene bshC is disrupted by an 8-bp duplication in S. aureus NCTC8325. The functional bshC-gene from BSH-producing S. aureus Newman (NWMN_1087) was expressed in S. aureus 8325-4 to reconstitute BSH-synthesis. Comparison of the BSH-producing and BSH-minus strains revealed higher resistance of the BSH-producing strain against the antibiotic fosfomycin and the oxidant hypochlorite but not against hydrogen peroxide or diamide. In addition, a higher bacterial load of the BSH-producing strain was detected in human upper-airway epithelial cells and murine macrophages. This indicates a potential role of BSH in protection of S. aureus during infection.

Patterns of protein carbonylation following oxidative stress in wild-type and sigB Bacillus subtilis cells

Oxidative stress causes damage to nucleic acids, membrane lipids and proteins. One striking effect is the metal-catalyzed, site-specific carbonylation of proteins. In the gram-positive soil bacterium Bacillus subtilis, the PerR-dependent specific stress response and the σB-dependent general stress response act together to make cells more resistant to oxidative stress. In this study, we analyzed the carbonylation of cytoplasmic proteins in response to hydrogen peroxide stress in B. subtilis. Furthermore, we asked whether the σB-dependent response to oxidative stress also confers protection against protein carbonylation. To monitor the amount and specificity of protein damage, carbonyls were derivatized with 2,4-dinitrophenylhydrazine, and the resulting stable hydrazones were detected by immunoanalysis of proteins separated by one- or two-dimensional gel electrophoresis. The overall level of protein carbonylation increased strongly in cells treated with hydrogen peroxide. Several proteins, including the elongation factors EF-G, TufA and EF-Ts, were found to be highly carbonylated. Induction of the peroxide specific stress response by treatment with sub-lethal peroxide concentrations, prior to exposure to otherwise lethal levels of peroxide, markedly reduced the degree of protein carbonylation. Cells starved for glucose also showed only minor amounts of peroxide-mediated protein carbonylation compared to exponentially growing cells. We could not detect any differences between wild-type and ΔsigB cells starved for glucose or preadapted by heat treatment with respect to the amount or specificity of protein damage incurred upon subsequent exposure to peroxide stress. However, artificial preloading with proteins that are normally induced by σB-dependent mechanisms resulted in a lower level of protein carbonylation when cells were later subjected to oxidative stress.

The role of thioredoxin TrxA in Bacillus subtilis: a proteomics and transcriptomics approach.

Thiol‐disulfide oxidoreductases of the thioredoxin superfamily are crucial for maintaining the thiol redox state in living organisms. For the bacterium Bacillus subtilis thioredoxin A (TrxA) was described as the product of an essential gene indicating a key role during growth. By means of mRNA profiling Smits et al. (J. Bacteriol. 2005, 187, 3921–3930) suggested a critical function for TrxA in sulfur utilization during stationary phase. We extended the analysis of TrxA to exponential growth and characterized a trxA conditional mutant by proteome analysis complemented by transcriptomics. After TrxA‐depletion, the growth rate was dramatically decreased. The cells responded at mRNA and protein level by the increased expression of genes involved in the utilization of sulfur, which represents the most obvious response as visualized by gel‐based proteomics. Furthermore, several genes of the antioxidant response were found at higher expression levels after TrxA‐depletion. When sulfate was replaced by thiosulfate or methionine as sulfur source, the growth inhibition was abolished. In the presence of thiosulfate but in the absence of TrxA, the induction of the sulfur limitation response and the oxidative stress response was not observed. Our results show that the global change of gene expression is primarily caused by the interruption of the sulfate utilization after TrxA depletion. Thus, its function in sulfate assimilation renders TrxA an essential protein in growing B. subtilis cells.

Staphylococcus aureus alpha-toxin mediates general and cell type-specific changes in metabolite concentrations of immortalized human airway epithelial cells.

Staphylococcus aureus alpha-toxin (Hla) is a potent pore-forming cytotoxin that plays an important role in the pathogenesis of S. aureus infections, including pneumonia. The impact of Hla on the dynamics of the metabolome in eukaryotic host cells has not been investigated comprehensively. Using 1H-NMR, GC-MS and HPLC-MS, we quantified the concentrations of 51 intracellular metabolites and assessed alterations in the amount of 25 extracellular metabolites in the two human bronchial epithelial cell lines S9 and 16HBE14o− under standard culture conditions and after treatment with sub-lethal amounts (2 µg/ml) of recombinant Hla (rHla) in a time-dependent manner. Treatment of cells with rHla caused substantial decreases in the concentrations of intracellular metabolites from different metabolic pathways in both cell lines, including ATP and amino acids. Concomitant increases in the extracellular concentrations were detected for various intracellular compounds, including nucleotides, glutathione disulfide and NAD+. Our results indicate that rHla has a major impact on the metabolome of eukaryotic cells as a consequence of direct rHla-mediated alterations in plasma membrane permeability or indirect effects mediated by cellular signalling. However, cell-specific changes also were observed. Glucose consumption and lactate production rates suggest that the glycolytic activity of S9 cells, but not of 16HBE14o− cells, is increased in response to rHla. This could contribute to the observed higher level of resistance of S9 cells against rHla-induced membrane damage.

Effects of irbesartan on gene expression revealed by transcriptome analysis of left atrial tissue in a porcine model of acute rapid pacing in vivo

BACKGROUND Atrial fibrillation (AF) is characterized by electrical and structural remodeling of the atria with atrial fibrosis being one hallmark. Angiotensin II (AngII) is a major contributing factor and blockage of its type I receptor (AT1R) prevents remodeling to some extent. Here we explored the effects of the AT1R antagonist irbesartan on global gene expression and profibrotic signaling pathways after induction of rapid atrial pacing (RAP) in vivo in pigs. METHODS AND RESULTS Microarray-based RNA profiling was used to screen left atrial (LA) tissue specimens for differences in atrial gene expression in a model of acute RAP. RAP caused an overall expression profile that reflected AngII-induced ROS production, tissue remodeling, and energy depletion. Of special note, the mRNA levels of EDN1, SGK1, and CTGF encoding pro-endothelin, stress- and glucocorticoid activated kinase-1, and of connective tissue growth factor were identified to be significantly increased after 7h of rapid pacing. These specific expression changes were additionally validated by RT-qPCR or immunoblot analyses in LA, RA, and partly in LV samples. All RAP-induced differential gene expression patterns were partially attenuated in the presence of irbesartan. Similar results were obtained after RAP of HL-1 cardiomyocytes in vitro. Furthermore, exogenously added endothelin-1 (ET1) induced CTGF expression concomitant to the transcriptional activation of SGK1 in HL-1 cells. CONCLUSIONS RAP provokes substantial changes in atrial and ventricular myocardial gene expression that could be partly reversed by irbesartan. ET1 contributes to AF-dependent atrial fibrosis by synergistic activity with AngII to stimulate SGK1 expression and enhance phosphorylation of the SGK1 protein which, in turn, induces CTGF. The latter has been consistently associated with tissue fibrosis. These findings suggest ETR antagonists as being beneficial in AF treatment.

Redox regulation of mitochondrial proteins and proteomes by cysteine thiol switches.

Mitochondria are hotspots of cellular redox biochemistry. Respiration as a defining mitochondrial function is made up of a series of electron transfers that are ultimately coupled to maintaining the proton motive force, ATP production and cellular energy supply. The individual reaction steps involved require tight control and flexible regulation to maintain energy and redox balance in the cell under fluctuating demands. Redox regulation by thiol switching has been a long-standing candidate mechanism to support rapid adjustment of mitochondrial protein function at the posttranslational level. Here we review recent advances in our understanding of cysteine thiol switches in the mitochondrial proteome with a focus on their operation in vivo. We assess the conceptual basis for thiol switching in mitochondria and discuss to what extent insights gained from in vitro studies may be valid in vivo, considering thermodynamic, kinetic and structural constraints. We compare functional proteomic approaches that have been used to assess mitochondrial protein thiol switches, including thioredoxin trapping, redox difference gel electrophoresis (redoxDIGE), isotope-coded affinity tag (OxICAT) and iodoacetyl tandem mass tag (iodoTMT) labelling strategies. We discuss conditions that may favour active thiol switching in mitochondrial proteomes in vivo, and appraise recent advances in dissecting their impact using combinations of in vivo redox sensing and quantitative redox proteomics. Finally we focus on four central facets of mitochondrial biology, aging, carbon metabolism, energy coupling and electron transport, exemplifying the current emergence of a mechanistic understanding of mitochondrial regulation by thiol switching in living plants and animals.