Interplay between manganese and iron in pneumococcal pathogenesis: role of the orphan response regulator RitR

Streptococcus pneumoniae (the pneumococcus) is a major human pathogen that is carried asymptomatically in the nasopharynx by up to 70% of the human population. Translocation of the bacteria into internal sites can cause a range of diseases, such as pneumonia, otitis media, meningitis, and bacteremia. This transition from nasopharynx to growth at systemic sites means that the pneumococcus needs to adjust to a variety of environmental conditions, including transition metal ion availability. Although it is an important nutrient, iron potentiates oxidative stress, and it is established that in S. pneumoniae, expression of iron transport systems and proteins that protect against oxidative stress are regulated by an orphan response regulator, RitR. In this study, we investigated the effect of iron and manganese ion availability on the growth of a ritR mutant. Deletion of ritR led to impaired growth of bacteria in high-iron medium, but this phenotype could be suppressed with the addition of manganese. Measurement of metal ion accumulation indicated that manganese prevents iron accumulation. Furthermore, the addition of manganese also led to a reduction in the amount of hydrogen peroxide produced by bacterial cells. Studies of virulence in a murine model of infection indicated that RitR was not essential for pneumococcal survival and suggested that derepression of iron uptake systems may enhance the survival of pneumococci in some niches

A Novel Strategy Involved Anti-Oxidative Defense: The Conversion of NADH into NADPH by a Metabolic Network

The reduced nicotinamide adenine dinucleotide phosphate (NADPH) is pivotal to the cellular anti-oxidative defence strategies in most organisms. Although its production mediated by different enzyme systems has been relatively well-studied, metabolic networks dedicated to the biogenesis of NADPH have not been fully characterized. In this report, a metabolic pathway that promotes the conversion of reduced nicotinamide adenine dinucleotide (NADH), a pro-oxidant into NADPH has been uncovered in Pseudomonas fluorescens exposed to oxidative stress. Enzymes such as pyruvate carboxylase (PC), malic enzyme (ME), malate dehydrogenase (MDH), malate synthase (MS), and isocitrate lyase (ICL) that are involved in disparate metabolic modules, converged to create a metabolic network aimed at the transformation of NADH into NADPH. The downregulation of phosphoenol carboxykinase (PEPCK) and the upregulation of pyruvate kinase (PK) ensured that this metabolic cycle fixed NADH into NADPH to combat the oxidative stress triggered by the menadione insult. This is the first demonstration of a metabolic network invoked to generate NADPH from NADH, a process that may be very effective in combating oxidative stress as the increase of an anti-oxidant is coupled to the decrease of a pro-oxidant

Glucose-6-Phosphate Dehydrogenase Protects Escherichia coli from Tellurite-Mediated Oxidative Stress

The tellurium oxyanion tellurite induces oxidative stress in most microorganisms. In Escherichia coli, tellurite exposure results in high levels of oxidized proteins and membrane lipid peroxides, inactivation of oxidation-sensitive enzymes and reduced glutathione content. In this work, we show that tellurite-exposed E. coli exhibits transcriptional activation of the zwf gene, encoding glucose 6-phosphate dehydrogenase (G6PDH), which in turn results in augmented synthesis of reduced nicotinamide adenine dinucleotide phosphate (NADPH). Increased zwf transcription under tellurite stress results mainly from reactive oxygen species (ROS) generation and not from a depletion of cellular glutathione. In addition, the observed increase of G6PDH activity was paralleled by accumulation of glucose-6-phosphate (G6P), suggesting a metabolic flux shift toward the pentose phosphate shunt. Upon zwf overexpression, bacterial cells also show increased levels of antioxidant molecules (NADPH, GSH), better-protected oxidation-sensitive enzymes and decreased amounts of oxidized proteins and membrane lipids. These results suggest that by increasing NADPH content, G6PDH plays an important role in E. coli survival under tellurite stress

Cytosolic NADPH balancing in Penicillium chrysogenum cultivated on mixtures of glucose and ethanol

The in vivo flux through the oxidative branch of the pentose phosphate pathway (oxPPP) in Penicillium chrysogenum was determined during growth in glucose/ethanol carbon-limited chemostat cultures, at the same growth rate. Non-stationary 13C flux analysis was used to measure the oxPPP flux. A nearly constant oxPPP flux was found for all glucose/ethanol ratios studied. This indicates that the cytosolic NADPH supply is independent of the amount of assimilated ethanol. The cofactor assignment in the model of van Gulik et al. (Biotechnol Bioeng 68(6):602–618, 2000) was supported using the published genome annotation of P. chrysogenum. Metabolic flux analysis showed that NADPH requirements in the cytosol remain nearly the same in these experiments due to constant biomass growth. Based on the cytosolic NADPH balance, it is known that the cytosolic aldehyde dehydrogenase in P. chrysogenum is NAD +  dependent. Metabolic modeling shows that changing the NAD + -aldehyde dehydrogenase to NADP + -aldehyde dehydrogenase can increase the penicillin yield on substrate

Wood Utilization Is Dependent on Catalase Activities in the Filamentous Fungus Podospora anserina

Catalases are enzymes that play critical roles in protecting cells against the toxic effects of hydrogen peroxide. They are implicated in various physiological and pathological conditions but some of their functions remain unclear. In order to decipher the role(s) of catalases during the life cycle of Podospora anserina, we analyzed the role of the four monofunctional catalases and one bifunctional catalase-peroxidase genes present in its genome. The five genes were deleted and the phenotypes of each single and all multiple mutants were investigated. Intriguingly, although the genes are differently expressed during the life cycle, catalase activity is dispensable during both vegetative growth and sexual reproduction in laboratory conditions. Catalases are also not essential for cellulose or fatty acid assimilation. In contrast, they are strictly required for efficient utilization of more complex biomass like wood shavings by allowing growth in the presence of lignin. The secreted CATB and cytosolic CAT2 are the major catalases implicated in peroxide resistance, while CAT2 is the major player during complex biomass assimilation. Our results suggest that P. anserina produces external H2O2 to assimilate complex biomass and that catalases are necessary to protect the cells during this process. In addition, the phenotypes of strains lacking only one catalase gene suggest that a decrease of catalase activity improves the capacity of the fungus to degrade complex biomass

Oxygen dependence of metabolic fluxes and energy generation of Saccharomyces cerevisiae CEN.PK113-1A

<p>Abstract</p> <p>Background</p> <p>The yeast <it>Saccharomyces cerevisiae </it>is able to adjust to external oxygen availability by utilizing both respirative and fermentative metabolic modes. Adjusting the metabolic mode involves alteration of the intracellular metabolic fluxes that are determined by the cell's multilevel regulatory network. Oxygen is a major determinant of the physiology of <it>S. cerevisiae </it>but understanding of the oxygen dependence of intracellular flux distributions is still scarce.</p> <p>Results</p> <p>Metabolic flux distributions of <it>S. cerevisiae </it>CEN.PK113-1A growing in glucose-limited chemostat cultures at a dilution rate of 0.1 h^-1with 20.9%, 2.8%, 1.0%, 0.5% or 0.0% O₂in the inlet gas were quantified by ¹³C-MFA. Metabolic flux ratios from fractional [U-¹³C]glucose labelling experiments were used to solve the underdetermined MFA system of central carbon metabolism of <it>S. cerevisiae</it>.</p> <p>While ethanol production was observed already in 2.8% oxygen, only minor differences in the flux distribution were observed, compared to fully aerobic conditions. However, in 1.0% and 0.5% oxygen the respiratory rate was severely restricted, resulting in progressively reduced fluxes through the TCA cycle and the direction of major fluxes to the fermentative pathway. A redistribution of fluxes was observed in all branching points of central carbon metabolism. Yet only when oxygen provision was reduced to 0.5%, was the biomass yield exceeded by the yields of ethanol and CO₂. Respirative ATP generation provided 59% of the ATP demand in fully aerobic conditions and still a substantial 25% in 0.5% oxygenation. An extensive redistribution of fluxes was observed in anaerobic conditions compared to all the aerobic conditions. Positive correlation between the transcriptional levels of metabolic enzymes and the corresponding fluxes in the different oxygenation conditions was found only in the respirative pathway.</p> <p>Conclusion</p> <p>¹³C-constrained MFA enabled quantitative determination of intracellular fluxes in conditions of different redox challenges without including redox cofactors in metabolite mass balances. A redistribution of fluxes was observed not only for respirative, respiro-fermentative and fermentative metabolisms, but also for cells grown with 2.8%, 1.0% and 0.5% oxygen. Although the cellular metabolism was respiro-fermentative in each of these low oxygen conditions, the actual amount of oxygen available resulted in different contributions through respirative and fermentative pathways.</p

Factor H (FH) Is Released Slowly and Continuously from Human Endothelial Cells (ECs): FH Is Not Packaged in Weibel-Palade Bodies or Secreted in Response to EC Stimulation

Abstract Introduction Ultra-large von Willebrand factor (ULVWF) strings are synthesized in ECs, packaged in Weibel-Palade Bodies (WPBs), and secreted by stimulated ECs. Complement components studied to date [C3, factor (F) B, FD, FP, FH, FI, C5] are released slowly and continuously from human umbilical vein endothelial cell (HUVEC) cytoplasm and are not packaged in WPBs (PLoS One. 2013;8(3):e59372). In contrast, a recent report (Blood. 2014;123(1):121-5) contended that FH co-localizes with VWF in the WPBs. If this were so, it could have therapeutic importance for the treatment of atypical hemolytic uremic syndrome (aHUS) resulting from deficiency of FH because it might be possible to increase circulating FH levels transiently by administration of the WPB secretagogue, des-amino-D-arginine vasopressin (DDAVP). Hypothesis FH is not co-localized with VWF in WBPs, but rather is released slowly and continuously from EC cytoplasm regardless of cell stimulation. Methods Immunofluorescent Microscopy HUVECs were stimulated with histamine and stained with rabbit anti-VWF plus secondary donkey anti-rabbit Alexa Fluor IgG-488. The cells were then fixed and stained with goat-anti FH plus secondary chicken anti-goat Alexa Fluor IgG-647. The nuclei were detected with DAPI. In vitro VWF and FH from HUVECs HUVECs either were, or were not, stimulated with histamine. Supernatant was collected a variety of times over 7 hrs and assayed for VWF and FH antigen levels by ELISA. VWF assay antibodies (polyclonal): (1) capture, rabbit anti-human VWF (Ramco); (2) detection, goat anti-human VWF (Bethyl) and rabbit anti-goat IgG-HRP (Invitrogen). FH assay antibodies: (1) capture, polyclonal goat anti-human FH (Advanced Research Technologies); (2) detection, monoclonal mouse anti-human FH (Pierce, Thermo Scientific) and polyclonal goat anti-mouse IgG-HRP (Invitrogen). In vivo VWF and FH Plasma samples were obtained from 6 pediatric patients with von Willebrand disease (VWD) being tested for EC release of WPB VWF in response to DDAVP. For each patient, 1 sample was obtained prior to DDAVP administration, and 2 other samples were obtained 1 and 4 hours later. VWF levels were measured in each sample using standard clinical laboratory procedure at an affiliated hospital. FH antigen levels were quantified by ELISA, as above. Results Using non-overlapping spectral secondary detection antibody pairs, VWF was seen in clusters in HUVEC WPBs (Fig. 1A). In contrast, FH was distributed throughout the HUVEC cytoplasm (Fig. 1B). The VWF and FH images did not overlap, indicating that VWF and FH did not co-localize in the WPBs. Fig 1. Immunofluorescent images of HUVECs stained for VWF and FH. Fig 1. Immunofluorescent images of HUVECs stained for VWF and FH. Histamine addition to HUVECs in vitro resulted in ~ 4-fold increases in VWF secreted from HUVEC WPBs at 30 min and 1 hour, and 2-fold increases at 3 hours (Fig 2A). In contrast, FH release was slow and continuous, regardless of histamine stimulation, suggesting that FH is located in EC cytoplasm and is not stored in WPBs (Fig. 2B). Fig 2. In vitro VWF and FH release from ECs under non-stimulated and histamine-stimulated conditions. Fig 2. In vitro VWF and FH release from ECs under non-stimulated and histamine-stimulated conditions. In all 6 patient samples, VWF antigen increased significantly at 1-hour post-DDAVP administration (Fig. 3A). In contrast, FH antigen levels did not change significantly at hour 1 or hour 4, compared to hour 0, indicating that FH is not co-localized and secreted along with VWF from the WPBs of stimulated ECs in vivo (Fig. 3B). Fig 3. (A) Mean responses of VWF antigen to DDAVP, in vivo, with 95% CIs.The mean response was significantly greater 1-hour and 4-hours post-DDAVP compared with baseline (P=0.0085 and 0.0079, respectively). After 1-hour post-DDAVP, the mean response was 201% greater (95% CI: 129%, 314%) than baseline. After 4-hours, the mean response was 174% greater (95% CI: 123%, 247%) than baseline. (B) Responses of FH to DDAVP, in vivo. There was no statistically significant difference in FH response between time points (P=0.77). Fig 3. (A) Mean responses of VWF antigen to DDAVP, in vivo, with 95% CIs.The mean response was significantly greater 1-hour and 4-hours post-DDAVP compared with baseline (P=0.0085 and 0.0079, respectively). After 1-hour post-DDAVP, the mean response was 201% greater (95% CI: 129%, 314%) than baseline. After 4-hours, the mean response was 174% greater (95% CI: 123%, 247%) than baseline. (B) Responses of FH to DDAVP, in vivo. There was no statistically significant difference in FH response between time points (P=0.77). Conclusions We used immunofluorescent microscopy and ELISA assays on samples obtained in vitro and in vivo to demonstrate that FH is not packaged in, or secreted from, the WPBs of stimulated human ECs. FH is, therefore, similar to all other complement components studied to date in that it is released slowly and continuously from ECs and is not influenced by cell stimulation. DDAVP is unlikely to be a viable treatment option for patients with aHUS secondary to deficiency or inhibition of FH. Disclosures Sartain: Hemostasis and Thrombosis Research Society: Research Funding. Turner:Mary R Gibson Foundation: Research Funding; Hinkson Memorial Fund : Research Funding. Moake:Mary R Gibson Foundation: Research Funding; Hinkson Memorial Fund: Research Funding. </jats:sec