Increasing NADPH impairs fungal H2O2 resistance by perturbing transcriptional regulation of peroxiredoxin

NADPH provides the reducing power for decomposition of reactive oxygen species (ROS), making it an indispensable part during ROS defense. It remains uncertain, however, if living cells respond to the ROS challenge with an elevated intracellular NADPH level or a more complex NADPH-mediated manner. Herein, we employed a model fungus Aspergillus nidulans to probe this issue. A conditional expression of glucose-6-phosphate dehydrogenase (G6PD)-strain was constructed to manipulate intracellular NADPH levels. As expected, turning down the cellular NADPH concentration drastically lowered the ROS response of the strain; it was interesting to note that increasing NADPH levels also impaired fungal H2O2 resistance. Further analysis showed that excess NADPH promoted the assembly of the CCAAT-binding factor AnCF, which in turn suppressed NapA, a transcriptional activator of PrxA (the key NADPH-dependent ROS scavenger), leading to low antioxidant ability. In natural cell response to oxidative stress, we noticed that the intracellular NADPH level fluctuated “down then up” in the presence of H2O2. This might be the result of a co-action of the PrxA-dependent NADPH consumption and NADPH-dependent feedback of G6PD. The fluctuation of NADPH is well correlated to the formation of AnCF assembly and expression of NapA, thus modulating the ROS defense. Our research elucidated how A. nidulans precisely controls NADPH levels for ROS defense. Graphical Abstract

Peroxiredoxin promotes longevity and H2O2-resistance in yeast through redox-modulation of protein kinase A

Peroxiredoxins are H2O2 scavenging enzymes that also carry out H2O2 signaling and chaperone functions. In yeast, the major cytosolic peroxiredoxin, Tsa1 is required for both promoting resistance to H2O2 and extending lifespan upon caloric restriction. We show here that Tsa1 effects both these functions not by scavenging H2O2, but by repressing the nutrient signaling Ras-cAMP-PKA pathway at the level of the protein kinase A (PKA) enzyme. Tsa1 stimulates sulfenylation of cysteines in the PKA catalytic subunit by H2O2 and a significant proportion of the catalytic subunits are glutathionylated on two cysteine residues. Redox modification of the conserved Cys243 inhibits the phosphorylation of a conserved Thr241 in the kinase activation loop and enzyme activity, and preventing Thr241 phosphorylation can overcome the H2O2 sensitivity of Tsa1-deficient cells. Results support a model of aging where nutrient signaling pathways constitute hubs integrating information from multiple aging-related conduits, including a peroxiredoxin-dependent response to H2O2.

Age distribution, extractability, and stability of mineral-bound organic carbon in central European soils

The largest share of total soil organic carbon (OC) is associated with minerals. The portions and turnover of stable and faster cycling mineral-associated carbon (MOC) as well as the determining factors across different soils and soil depths are still unknown. Bioavailability of MOC is supposedly regulated by desorption but instead, its stability was so far mostly tested by exposure to chemical oxidation. Therefore, we determined the extractability of MOC into a mixture of 0.1 M NaOH and 0.4 M NaF as a measure for maximal potential desorbability, and compared it with maximal potential oxidation in heated H2O2. We selected samples of three soil depth increments (0–5 cm, 10–20 cm, 30–40 cm) of five typical soils of the mid-latitudes, differing contents of clay and pedogenic oxides, and being under different land use. Extracts and residues were analyzed for OC and 14C contents, and further chemically characterized by CPMAS-13C-NMR. We hypothesized NaF-NaOH extraction to remove less and younger MOC than H2O2 oxidation, and extractable MOC to be less and relatively older in subsoils and soils with high contents of pedogenic oxides. A surprisingly constant portion of 58 ± 11 % (standard deviation) of MOC was extractable across soils, independent of depths, mineral assemblage, or land use. NMR spectra revealed strong similarities of the extracted organic matter, with more than 80 % of OC in the O/N alkyl and alkyl C region. Total MOC amounts were linked to the content of pedogenic oxides across sites, independent of variations in total clay. The uniform MOC desorption could therefore be the result of pedogenic oxides dominating the overall response of MOC to extraction. While bulk MO14C values suggested differences in OC turnover between sites, these were not linked to differences in MOC extractability. As expected, OC contents of residues had smaller 14C contents than extracts, suggesting that non-extractable OC is older. However, 14C contents of extracts and residues were strongly correlated and proportional to bulk MO14C, but not dependent on mineralogy. Also along soil profiles, where increasing MOC ages indicate slower turnover with depth, neither MOC extractability nor differences in 14C between extracts and residues changed. Increasing bonding strength with soil depths did therefore not cause the 14C depth gradients in the studied soils. Although H2O2 removed 90 ± 8 % of the MOC, the 14C content of the OC removed was similar to that of the NaF-NaOH-extracted OC, while oxidation residues were much more 14C-depleted. Different chemical treatments apparently remove OC of the same continuum, leaving increasingly older residues behind the more OC being removed. Different from the extractions, higher contents of pedogenic oxides seemingly slightly increased the oxidation-resistance of MOC, but this higher H2O2-resistance did not coincide with older MOC or oxidation residues. Our results indicate that total MOC was dominated by OC interactions with pedogenic oxides rather than clay minerals, so that no difference in MOC extraction in NaF/NaOH, and thus, bond type or strength between clay-rich and poor sites was detectable. This suggests that site-specific differences in MO14C and their depth declines are driven by the accumulation and exchange rates of OC at mineral surfaces. Accordingly, future research on M14OC should focus on soil and ecosystem properties driving dissolved organic matter formation, composition and transport along soil profiles.

The metabolome as a link in the genotype-phenotype map for peroxide resistance in the fruit fly, Drosophila melanogaster

Background Genetic association studies that seek to explain the inheritance of complex traits typically fail to explain more than a small fraction of the heritability of the trait under study. Thus we are left with a gap in the map from genotype to phenotype. Several approaches have been used to fill this gap, including those that attempt to map endophenotype such as the transcriptome, proteome or metabolome, that underlie complex traits. Here we used metabolomics to explore the nature of genetic variation for hydrogen peroxide (H­2O2) resistance in the sequenced inbred Drosophila Genetic Reference Panel (DGRP). Results We first studied genetic variation for H2O2 resistance in 180 DGRP lines and identify the insulin signaling modulator u-shaped and several regulators of feeding behavior. We then profiled a portion of the aqueous metabolome in subsets of eight ‘high resistance’ lines and eight ‘low resistance’ lines. We used these lines to represent collections of genotypes that were either resistant or sensitive to the stressor, effectively modeling a discrete trait. Across the range of genotypes in both populations, flies exhibited surprising consistency in their metabolomic signature of resistance. Metabolomic profiles were also able to distinguish stress-resistant from stress-sensitive flies with greater accuracy than the genotype of these same lines. Furthermore, we found a metabolic response to H2O2 that was shared among sensitive, but not resistant genotypes. Metabolomic data further implicated at least two pathways, glycogen and folate metabolism, as determinants of sensitivity to H2O2. We also discovered a confounding effect of feeding behavior on assays involving supplemented food. Conclusions This work suggests that the metabolome can be a point of convergence for genetic variation influencing complex traits, and efficiently elucidate the mechanisms underlying this trait variation.

Peroxiredoxin promotes longevity and H2O2-resistance in yeast through redox-modulation of protein kinase A

Peroxiredoxins are H2O2scavenging enzymes that also carry H2O2signaling and chaperone functions. In yeast, the major cytosolic peroxiredoxin, Tsa1 is required for both promoting resistance to H2O2and extending lifespan upon caloric restriction. We show here that Tsa1 effects both these functions not by scavenging H2O2, but by repressing the nutrient signaling Ras-cAMP-PKA pathway at the level of the protein kinase A (PKA) enzyme. Tsa1 stimulates sulfenylation of cysteines in the PKA catalytic subunit by H2O2and a significant proportion of the catalytic subunits are glutathionylated on two cysteine residues. Redox modification of the conserved Cys243 inhibits the phosphorylation of a conserved Thr241 in the kinase activation loop and enzyme activity, and preventing Thr241 phosphorylation can overcome the H2O2sensitivity of Tsa1-deficient cells. Results support a model of aging where nutrient signaling pathways constitute hubs integrating information from multiple aging-related conduits, including a peroxiredoxin-dependent response to H2O2.

Comparison of genes required for H2O2 resistance in Streptococcus gordonii and Streptococcus sanguinis

Hydrogen peroxide (H2O2) is produced by several members of the genus Streptococcus mainly through the pyruvate oxidase SpxB under aerobic growth conditions. The acute toxic nature of H2O2 raises the interesting question of how streptococci cope with intrinsically produced H2O2, which subsequently accumulates in the microenvironment and threatens the closely surrounding population. Here, we investigate the H2O2 susceptibility of oral Streptococcus gordonii and Streptococcus sanguinis and elucidate potential mechanisms of how they protect themselves from the deleterious effect of H2O2. Both organisms are considered primary colonizers and occupy the same intraoral niche making them potential targets for H2O2 produced by other species. We demonstrate that S. gordonii produces relatively more H2O2 and has a greater ability for resistance to H2O2 stress. Functional studies show that, unlike in Streptococcus pneumoniae, H2O2 resistance is not dependent on a functional SpxB and confirms the important role of the ferritin-like DNA-binding protein Dps. However, the observed increased H2O2 resistance of S. gordonii over S. sanguinis is likely to be caused by an oxidative stress protection machinery present even under anaerobic conditions, while S. sanguinis requires a longer period of time for adaptation. The ability to produce more H2O2 and be more resistant to H2O2 might aid S. gordonii in the competitive oral biofilm environment, since it is lower in abundance yet manages to survive quite efficiently in the oral biofilm.