H2O2 Resistance Research Articles

Heme metabolism in Strigomonas culicis: Implications of H2O2 resistance induction and symbiont elimination

Monoxenous trypanosomatid Strigomonas culicis harbors an endosymbiotic bacterium, which enables the protozoa to survive without heme supplementation. The impact of H2O2 resistance and symbiont elimination on intracellular heme and Fe2+ availability was analyzed through a comparison of WT strain with both WT H2O2-resistant (WTR) and aposymbiotic (Apo) protozoa. The relative quantification of the heme biosynthetic pathway through label-free parallel reaction monitoring targeted mass spectrometry revealed that H2O2 resistance does not influence the abundance of tryptic peptides. However, the Apo strain showed increased coproporphyrinogen III oxidase and ferrochelatase levels. A putative ferrous iron transporter, homologous to LIT1 and TcIT from Leishmania major and Trypanosoma cruzi, was identified for the first time. Label-free parallel reaction monitoring targeted mass spectrometry also showed that S.culicis Iron Transporter (ScIT) increased 1.6- and 16.4-fold in WTR and Apo strains compared to WT. Accordingly, antibody-mediated blockage of ScIT decreased by 28.0% and 40.0% intracellular Fe2+concentration in both WTR and Apo strains, whereas no effect was detected in WT. In a heme-depleted medium, adding 10μM hemin decreased ScIT transcript levels in Apo, whereas 10μM PPIX, the substrate of ferrochelatase, increased intracellular Fe2+ concentration and ferric iron reduction. Overall, the data suggest mechanisms dependent on de novo heme synthesis (and its substrates) in the Apo strain to overcome reduced heme availability. Given the importance of heme and Fe2+ as cofactors in metabolic pathways, including oxidative phosphorylation and antioxidant systems, this study provides novel mechanistic insights associated with H2O2 resistance in S.culicis.

Dpr-mediated H2O2 resistance contributes to streptococcus survival in a cystic fibrosis airway model system.

Streptococcus spp. are recognized as a highly prevalent pathogen in cystic fibrosis (CF) airway infections. However, the role of this microbe in clinical outcomes for persons with CF is poorly understood. Here, we leverage a polymicrobial community system previously developed by our group to model CF airway infections as a tool to investigate a Pseudomonas-Streptococcus interaction involving reactive oxygen species (ROS). We show that protection against ROS is required for Streptococcus sanguinis survival in a clinically relevant polymicrobial system. Using this model system to study interspecies interactions contributes to our broader understanding of the complex role of Streptococcus spp. in the CF lung.

Natural variation in yeast reveals multiple paths for acquiring higher stress resistance

BackgroundOrganisms frequently experience environmental stresses that occur in predictable patterns and combinations. For wild Saccharomyces cerevisiae yeast growing in natural environments, cells may experience high osmotic stress when they first enter broken fruit, followed by high ethanol levels during fermentation, and then finally high levels of oxidative stress resulting from respiration of ethanol. Yeast have adapted to these patterns by evolving sophisticated “cross protection” mechanisms, where mild ‘primary’ doses of one stress can enhance tolerance to severe doses of a different ‘secondary’ stress. For example, in many yeast strains, mild osmotic or mild ethanol stresses cross protect against severe oxidative stress, which likely reflects an anticipatory response important for high fitness in nature.ResultsDuring the course of genetic mapping studies aimed at understanding the mechanisms underlying natural variation in ethanol-induced cross protection against H2O2, we found that a key H2O2 scavenging enzyme, cytosolic catalase T (Ctt1p), was absolutely essential for cross protection in a wild oak strain. This suggested the absence of other compensatory mechanisms for acquiring H2O2 resistance in that strain background under those conditions. In this study, we found surprising heterogeneity across diverse yeast strains in whether CTT1 function was fully necessary for acquired H2O2 resistance. Some strains exhibited partial dispensability of CTT1 when ethanol and/or salt were used as mild stressors, suggesting that compensatory peroxidases may play a role in acquired stress resistance in certain genetic backgrounds. We leveraged global transcriptional responses to ethanol and salt stresses in strains with different levels of CTT1 dispensability, allowing us to identify possible regulators of these alternative peroxidases and acquired stress resistance in general.ConclusionsUltimately, this study highlights how superficially similar traits can have different underlying molecular foundations and provides a framework for understanding the diversity and regulation of stress defense mechanisms.

Identification and Functional Analysis of Lysine 2-Hydroxyisobutyrylation in Cyanobacteria.

Cyanobacteria are the oldest prokaryotic photoautotrophic microorganisms and have evolved complicated post-translational modification (PTM) machinery to respond to environmental stress. Lysine 2-hydroxyisobutyrylation (Khib) is a newly identified PTM that is reported to play important roles in diverse biological processes, however, its distribution and function in cyanobacteria have not been reported. Here, we performed the first systematic studies of Khib in a model cyanobacterium Synechococcus sp. strain PCC 7002 (Syn7002) using peptide prefractionation, pan-Khib antibody enrichment, and high-accuracy mass spectrometry (MS) analysis. A total of 1875 high-confidence Khib sites on 618 proteins were identified, and a large proportion of Khib sites are present on proteins in the cellular metabolism, protein synthesis, and photosynthesis pathways. Using site-directed mutagenesis and functional studies, we showed that Khib of glutaredoxin (Grx) affects the efficiency of the PS II reaction center and H2O2 resistance in Syn7002. Together, this study provides novel insights into the functions of Khib in cyanobacteria and suggests that reversible Khib may influence the stress response and photosynthesis in both cyanobacteria and plants.

Enhanced oxidative stress resistance in Ustilago maydis and its implications on the virulence

The phytopathogenic fungus Ustilago maydis causes corn smut by suppressing host plant defenses, including the oxidative burst response. While many studies have investigated how U. maydis responds to oxidative stress during infection, the consequences of heightened resistance to oxidative stress on virulence remain understudied. This study aimed to identify the effects on virulence in U. maydis strains exhibiting enhanced resistance to hydrogen peroxide (H2O2).To achieve this, we exposed U. maydis SG200 to 20 escalating H2O2 shocks, resulting in an adapted strain resistant to concentrations as high as 60 mM of H2O2, a lethal dose for the initial strain. Genetic analysis of the adapted strain revealed five nucleotide substitutions, two minor copy number variants, and a large amplification event on chromosome nine (1–149 kb) encompassing the sole catalase gene. Overexpressing catalase increased resistance to H2O2; however, this resistance was lower than that observed in the adapted strain. Additionally, virulence was reduced in both strains with enhanced H2O2 resistance.In summary, enhanced H2O2 resistance, achieved through either continuous exposure to the oxidative agent or through catalase overexpression, decreased virulence. This suggests that the response to the oxidative stress burst in U. maydis is optimal and that increasing the resistance to H2O2 does not translate into increased virulence. These findings illuminate the intricate relationship between oxidative stress resistance and virulence in U. maydis, offering insights into its infection mechanisms.

The role of iron-uptake factor PiuB in pathogenicity of soybean pathogen Xanthomonas axonopodis pv. glycines

Iron is an essential element for living organisms that plays critical roles in the process of bacterial growth and metabolism. However, it remains to be elucidated whether piuB encoding iron-uptake factor is involved in iron uptake and pathogenicity of Xanthomonas axonopodis pv. glycines (Xag). To investigate the function of piuB, we firstly generated a piuB deletion mutant (ΔpiuB) by homologous recombination. Compared with the wild-type, the piuB mutant exhibited significantly reduced growth and virulence in host soybean. The mutant displayed markedly increased siderophore secretory volume, and its sensitivity to Fe3+, Cu2+, Zn2+ and Mn2+ was significantly enhanced. Additionally, the H2O2 resistance, exopolysaccharide yield, biofilm formation, and cell mobility of ΔpiuB were significantly diminished compared to that of the wild-type. The addition of exogenous Fe3+ cannot effectively restore the above characteristics of ΔpiuB. However, expressing piuB in trans rescued the properties lost by ΔpiuB to the levels in the wild-type. Taken together, our results demonstrated that PiuB is a potential factor for Xag to assimilate Fe3+, and is necessary for Xag to be pathogenic in host soybean.

The secondary metabolite hydrogen cyanide protects Pseudomonas aeruginosa against sodium hypochlorite-induced oxidative stress.

The high pathogenicity of Pseudomonas aeruginosa is attributed to the production of many virulence factors and its resistance to several antimicrobials. Among them, sodium hypochlorite (NaOCl) is a widely used disinfectant due to its strong antimicrobial effect. However, bacteria develop many mechanisms to survive the damage caused by this agent. Therefore, this study aimed to identify novel mechanisms employed by P. aeruginosa to resist oxidative stress induced by the strong oxidizing agent NaOCl. We analyzed the growth of the P. aeruginosa mutants ΔkatA, ΔkatE, ΔahpC, ΔahpF, ΔmsrA at 1 μg/mL NaOCl, and showed that these known H2O2 resistance mechanisms are also important for the survival of P. aeruginosa under NaOCl stress. We then conducted a screening of the P. aeruginosa PA14 transposon insertion mutant library and identified 48 mutants with increased susceptibility toward NaOCl. Among them were 10 mutants with a disrupted nrdJa, bvlR, hcnA, orn, sucC, cysZ, nuoJ, PA4166, opmQ, or thiC gene, which also exhibited a significant growth defect in the presence of NaOCl. We focussed our follow-up experiments (i.e., growth analyzes and kill-kinetics) on mutants with defect in the synthesis of the secondary metabolite hydrogen cyanide (HCN). We showed that HCN produced by P. aeruginosa contributes to its resistance toward NaOCl as it acts as a scavenger molecule, quenching the toxic effects of NaOCl.

Cytosol Peroxiredoxin and Cell Surface Catalase Differentially Respond to H2O2 Stress in Aspergillus nidulans.

Both catalase and peroxiredoxin show high activities of H2O2 decomposition and coexist in the same organism; however, their division of labor in defense against H2O2 is unclear. We focused on the major peroxiredoxin (PrxA) and catalase (CatB) in Aspergillus nidulans at different growth stages to discriminate their antioxidant roles. The dormant conidia lacking PrxA showed sensitivity to high concentrations of H2O2 (>100 mM), revealing that PrxA is one of the important antioxidants in dormant conidia. Once the conidia began to swell and germinate, or further develop to young hyphae (9 h to old age), PrxA-deficient cells (ΔprxA) did not survive on plates containing H2O2 concentrations higher than 1 mM, indicating that PrxA is an indispensable antioxidant in the early growth stage. During these early growth stages, absence of CatB did not affect fungal resistance to either high (>1 mM) or low (<1 mM) concentrations of H2O2. In the mature hyphae stage (24 h to old age), however, CatB fulfills the major antioxidant function, especially against high doses of H2O2. PrxA is constitutively expressed throughout the lifespan, whereas CatB levels are low in the early growth stage of the cells developing from swelling conidia to early growth hyphae, providing a molecular basis for their different contributions to H2O2 resistance in different growth stages. Further enzyme activity and cellular localization analysis indicated that CatB needs to be secreted to be functionalized, and this process is confined to the growth stage of mature hyphae. Our results revealed differences in effectiveness and timelines of two primary anti-H2O2 enzymes in fungus.

Berberine Hydrochloride-loaded Liposomes Gel: Preparation, Characterization and Antioxidant Activity

Abstract: Aim: Berberine hydrochloride is a Traditional Chinese Medicine component with antibacterial, antiviral, anti-inflammatory, antioxidant and lowering blood sugar effects. However, the application of berberine hydrochloride has been hindered for a long time due to its poor solubility and low bioavailability. This study aims to design a liposomes-gel to enhance the bioavailability and antioxidant activity of berberine hydrochloride. Materials and Methods: The thin-film dispersion hydration method was used to prepare liposomes, and liposomes-gel was prepared by the natural swelling method using sodium alginate as the hydrogel matrix. Berberine hydrochloride-loaded liposomes-gel was characterized by the encapsulation efficiency, particle size, potential, and transmission electron microscopy, and studied by in vitro release and antioxidant activity of the preparations. Results: The results revealed the encapsulation efficiency of berberine hydrochloride-loaded liposomes was 79.62±4.20% and the mean particle size was 153.7±11.2 nm. The release of berberine hydrochloride from liposomes-gel conformed to a first-order release model. The scavenging rate of DPPH free radicals and H2O2 by berberine hydrochloride-loaded liposomes-gel exceeds 70%. Conclusion: The liposomes-gel has a sustained-release effect on berberine hydrochloride. Berberine hydrochloride-loaded liposomes-gel hin vitroad good antioxidant properties by measuring the scavenging ability on DPPH free radicals, H2O2 and lipid peroxidation resistance. Keywords: Berberine hydrochloride, Liposomes, Sodium alginate, Hydrogel, Antioxidant.

H2O2-Driven Hydroxylation of Steroids Catalyzed by Cytochrome P450 CYP105D18: Exploration of the Substrate Access Channel.

CYP105D18 supports H2O2 as an oxygen surrogate for catalysis well and shows high H2O2 resistance capacity. We report the hydroxylation of different steroids using H2O2 as a cosubstrate. Testosterone was regiospecifically hydroxylated to 2β-hydroxytestosterone. Based on the experimental data and molecular docking, we predicted that hydroxylation of methyl testosterone and nandrolone would occur at position 2 in the A-ring, while hydroxylation of androstenedione and adrenosterone was predicted to occur in the B-ring. Further, structure-guided rational design of the substrate access channel was performed with the mutagenesis of residues S63, R82, and F184. Among the mutants, S63A showed a marked decrease in product formation, while F184A showed a significant increase in product formation in testosterone, nandrolone, methyl testosterone, androstenedione, and adrenosterone. The catalytic efficiency (kcat/Km) toward testosterone was increased 1.36-fold in the F184A mutant over that in the wild-type enzyme. These findings might facilitate the potential use of CYP105D18 and further engineering to establish the basis of biotechnological applications. IMPORTANCE The structural modification of steroids is a challenging chemical reaction. Modifying the core ring and the side chain improves the biological activity of steroids. In particular, bacterial cytochrome P450s are used as promiscuous enzymes for the activation of nonreactive carbons of steroids. In the present work, we reported the H2O2-mediated hydroxylation of steroids by CYP105D18, which also overcomes the use of expensive cofactors. Further, exploring the substrate access channel and modifying the bulky amino acid F184A increase substrate conversion while modifying the substrate recognizing amino acid S63 markedly decreases product formation. Exploring the substrate access channel and the rational design of CYP105D18 can improve the substrate conversion, which facilitates the engineering of P450s for industrial application.

The Catalase KatA Contributes to Microaerophilic H2O2 Priming to Acquire an Improved Oxidative Stress Resistance in Staphylococcus aureus.

Staphylococcus aureus has to cope with oxidative stress during infections. In this study, S. aureus was found to be resistant to 100 mM H2O2 during aerobic growth. While KatA was essential for this high aerobic H2O2 resistance, the peroxiredoxin AhpC contributed to detoxification of 0.4 mM H2O2 in the absence of KatA. In addition, the peroxiredoxins AhpC, Tpx and Bcp were found to be required for detoxification of cumene hydroperoxide (CHP). The high H2O2 tolerance of aerobic S. aureus cells was associated with priming by endogenous H2O2 levels, which was supported by an oxidative shift of the bacillithiol redox potential to −291 mV compared to −310 mV in microaerophilic cells. In contrast, S. aureus could be primed by sub-lethal doses of 100 µM H2O2 during microaerophilic growth to acquire an improved resistance towards the otherwise lethal triggering stimulus of 10 mM H2O2. This microaerophilic priming was dependent on increased KatA activity, whereas aerobic cells showed constitutive high KatA activity. Thus, KatA contributes to the high H2O2 resistance of aerobic cells and to microaerophilic H2O2 priming in order to survive the subsequent lethal triggering doses of H2O2, allowing the adaptation of S. aureus under infections to different oxygen environments.

Differential Sensitivity of Two Leukemia Cell Lines towards Two Major Gas Plasma Products Hydrogen Peroxide and Hypochlorous Acid

Oxidative stress has major implications for health and disease. At the same time, the term collectively describes the reactions to different types of reactive oxygen species (ROS) and oxidants, including hydrogen peroxide (H2O2) and hypochlorous acid (HOCl). However, how both compare in terms of cytotoxicity and mechanism of action is less known. Using two leukemia cell lines, Jurkat and THP-1, as model systems at similar cell concentrations, we found an 8-fold greater sensitivity of the former over the latter for H2O2 exposure. Unexpectantly, this was not the case with HOCl exposure. Jurkat cells were 2-fold more resistant to HOCl-induced cytotoxicity than THP-1 cells. In each cell type, the relatively more toxic oxidant also induced activation of caspases 3 and 7 at earlier time points, as time-lapse fluorescence microscopy revealed. The effects observed did not markedly correlate with changes in intracellular GSH and GSSG levels. In addition, siRNA-mediated knockdown of the Nrf2 target HMOX-1 encoding for HO-1 protein and the growth and survival factor IL-8 revealed Jurkat cells to become more sensitive to HOCl, while HO-1 and IL-8 siRNA-mediated knockdown in THP-1 cells produced greater sensitivity towards H2O2. siRNA-mediated knockdown of catalase increased oxidant sensitivity only negligibly. Collectively, the data suggest striking HOCl-resistance of Jurkat and H2O2 resistance of THP-1 cells, showing similar protective roles of HO-1 and IL-8, while caspase activation kinetics differ.

Improved Hydrogen Peroxide Stress Resistance of Zymomonas mobilis NADH Dehydrogenase (ndh) and Alcohol Dehydrogenase (adhB) Mutants

Unintended shifts in stress resistance of microbial strains with engineered central metabolism may impact their growth and production performance under oxidative, lignocellulosic, solvent, and other stress conditions, and as such, must be taken into account in bioprocess design. In the present work, we studied oxidative stress resistance in mutant strains of the facultatively anaerobic, ethanologenic bacterium Zymomonas mobilis with modified respiratory (inactivated NADH dehydrogenase Ndh, by disruption of ndh) and ethanologenic (inactivated iron-containing alcohol dehydrogenase isoenzyme ADH II, by disruption of adhB) catabolism, using exogenously added H2O2 in the concentration range of 2–6 mM as the oxidative stressor. Both mutations improved H2O2 resistance and enhanced catalase activity by a factor of 2–5, while the overexpression of Ndh had an opposite effect. Strains with a catalase-negative background were unable to grow already at 1 mM hydrogen peroxide, and their H2O2 resistance did not depend on AdhB or Ndh expression levels. Hence, the improved resistance of the ndh and adhB mutants to H2O2 resulted from their elevated catalase activity. The interrelation between these mutations, the catabolic redox balance, catalase activity, and oxidative stress defense in Z. mobilis is discussed.

Light Regulates Acinetobacter baumannii Chromosomal and pAB3 Plasmid Genes at 37°C.

The opportunistic pathogen A. baumannii has a remarkable capacity to persist in the hospital environment and cause devastating human infections. This capacity can be attributed partly to the sensing and regulatory systems that enable this pathogen to modify its physiology based on environmental cues. One of the signals that A. baumannii senses and responds to is light through the sensing and regulatory roles of the BlsA photoreceptor protein in cells cultured at temperatures below 30°C. This report presents evidence that a light stimulon is operational at 37°C, a condition at which the BlsA production and activity are drastically impaired. Global transcriptional analysis showed that the 37°C light stimulon includes the differential expression of chromosomal genes encoding a wide range of functions that are known to be involved in the adaptation to different metabolic conditions, as well as virulence and persistence in the host and the medical environment. Unexpectedly, the 37°C light stimulon also includes the differential expression of conjugation functions encoded by pAB3 plasmid genes. Our work further demonstrates that the TetR1 and H-NS regulators encoded by this conjugative plasmid control the expression of H2O2 resistance and surface motility, respectively. Furthermore, our data showed that pAB3 has an overall negative effect on the expression of these phenotypes and plays no significant virulence role. Although the nature of the bacterial factors and the mechanisms by which the regulation is attained at 37°C remain unknown, taken together, our work expands the current knowledge about light sensing and gene regulation in A. baumannii. IMPORTANCE As a facultative pathogen, Acinetobacter baumannii persists in various environments by sensing different environmental cues, including light. This report provides evidence of light-dependent regulation at 37°C of the expression of genes coding for a wide range of functions, including those involved in the conjugation of the pAB3 plasmid. Although this plasmid affects the expression of virulence traits when tested under laboratory conditions, it does not have a significant impact when tested using ex vivo and in vivo experimental models. These findings provide a better understanding of the interplay between light regulation and plasmid persistence in the pathobiology of A. baumannii.

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

Enhancing H2O2 Tolerance and Separation Performance through the Modification of the Polyamide Layer of a Thin-Film Composite Nanofiltration Membrane by Using Graphene Oxide

Through interfacial polymerization (IP), a polyamide (PA) layer was synthesized on the top of a commercialized polysulfone substrate to form a thin-film composite (TFC) nanofiltration membrane. Graphene oxide (GO) was dosed during the IP process to modify the NF membrane, termed TFC-GO, to enhance oxidant resistance and membrane performance. TFC-GO exhibited increased surface hydrophilicity, water permeability, salt rejection, removal efficiency of pharmaceutical and personal care products (PPCPs), and H2O2 resistance compared with TFC. When H2O2 exposure was 0–96,000 ppm-h, the surfaces of the TFC and TFC-GO membranes were damaged, and swelling was observed using scanning electron microscopy. However, the permeate flux of TFC-GO remained stable, with significantly higher NaCl, MgSO4, and PPCP rejection with increasing H2O2 exposure intensity than TFC, which exhibited a 3.5-fold flux increase with an approximate 50% decrease in salt and PPCP rejection. GO incorporated into a PA layer could react with oxidants to mitigate membrane surface damage and increase the negative charge on the membrane surface, resulting in the enhancement of the electrostatic repulsion of negatively charged PPCPs. This hypothesis was confirmed by the significant decrease in PPCP adsorption onto the surface of TFC-GO compared with TFC. Therefore, TFC-GO membranes exhibited superior water permeability, salt rejection, and PPCP rejection and satisfactory resistance to H2O2, indicating its great potential for practical applications.

Regulatory involvement of the PerR and SloR metalloregulators in the Streptococcus mutans oxidative stress response.

Streptococcus mutans is a commensal of the human oral microbiome that can promote dental caries under conditions of dysbiosis. This study investigates metalloregulators and their involvement in the S. mutans oxidative stress response. Oxidative stress in the human mouth can derive from temporal increases in reactive oxygen species (ROS) after meal consumption and from endogenous bacterial ROS-producers that colonize the dentition. We hypothesize that the S. mutans PerR (SMU.593) and SloR (SMU.186) metalloregulatory proteins contribute to the regulation of oxidative stress genes and their products. Expression assays with S. mutans UA159 wild type cultures exposed to H2O2 reveal that H2O2 upregulates perR, and that PerR represses sloR transcription upon binding directly to Fur and PerR consensus sequences within the sloR operator. In addition, the results of Western blot experiments implicate the Clp proteolytic system in SloR degradation under conditions of H2O2-stress. To reveal a potential role for SloR in the H2O2-resistant phenotype of S. mutans GMS802 (a perR-deficient strain), we generated a sloR/perR double knockout mutant, GMS1386, where we observed upregulation of the tpx and dpr antioxidant genes. These results are consistent with GMS802 H2O2 resistance and with a role for PerR as a transcriptional repressor. Cumulatively, these findings support a reciprocal relationship between PerR and SloR during the S. mutans oxidative stress response and begin to elucidate the fitness strategies that evolved to foster S. mutans persistence in the transient environments of the human oral cavity.IMPORTANCEIn 2020, untreated dental caries, especially in the permanent dentition, ranked among the most prevalent infectious diseases worldwide, disproportionately impacting individuals of low socioeconomic status. Untreated caries can lead to systemic health problems and has been associated with extended school and work absences, inappropriate use of emergency departments, and an inability for military forces to deploy. Together with public health policy, research aimed at alleviating S. mutans -induced tooth decay is important because it can improve oral health (and overall health), especially in underserved populations. This research, focused on S. mutans metalloregulatory proteins and their gene targets, is significant because it can promote virulence gene control in an important oral pathogen, and contribute to the development of an anti-caries therapeutic that can reduce tooth decay.

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

Abstract. The largest share of total soil organic carbon (OC) is associated with minerals. However, the factors that determine the amount and turnover of slower- versus faster-cycling components of mineral-associated carbon (MOC) are still poorly understood. Bioavailability of MOC is thought to be regulated by desorption, which can be facilitated by displacement and mobilization by competing ions. However, MOC stability is usually determined by exposure to chemical oxidation, which addresses the chemical stability of the organic compounds rather than the bonding strength of the OC–mineral bond. We used a solution of NaOH, a strong agent for desorption due to high pH, and NaF, adding F−, a strongly sorbing anion that can replace anionic organic molecules on mineral surfaces, to measure the maximum potentially desorbable MOC. For comparison, we measured maximal potential oxidation of MOC using heated H2O2. We selected MOC samples (&gt; 1.6 g cm3) obtained from density fractionation of samples from three soil depth increments (0–5, 10–20, and 30–40 cm) of five typical soils of central Europe, with a range of clay and pedogenic oxide contents, and under different ecosystem types (one coniferous forest, two deciduous forests, one grassland, and one cropland). Extracts and residues were analysed for OC and 14C contents, and further chemically characterized by cross-polarization magic angle spinning 13C-nuclear magnetic resonance (CPMAS-13C-NMR). We expected that NaF–NaOH extraction would remove less and younger MOC than H2O2 oxidation and that the NaF–NaOH extractability of MOC is reduced in subsoils and soils with high pedogenic oxide contents. The results showed that a surprisingly consistent proportion of 58 ± 11 % (standard deviation) of MOC was extracted with NaF–NaOH across soils, independent of depth, mineral assemblage, or land use conditions. NMR spectra revealed strong similarities in the extracted organic matter, with more than 80 % of OC in the O/N (oxygen and/or nitrogen) alkyl and alkyl C region. Total MOC amounts were correlated with the content of pedogenic oxides across sites, independent of variations in total clay, and the same was true for OC in extraction residues. Thus, the uniform extractability of MOC may be explained by dominant interactions between OC and pedogenic oxides across all study sites. While Δ14C values of bulk MOC suggested differences in OC turnover between sites, these were not linked to differences in MOC extractability. As expected, OC contents of residues had more negative Δ14C values than extracts (an average difference between extracts and residues of 78 ± 36 ‰), suggesting that non-extractable OC is older. Δ14C values of extracts and residues were strongly correlated and proportional to Δ14C values of bulk MOC but were not dependent on mineralogy. Neither MOC extractability nor differences in Δ14C values between extracts and residues changed with depth along soil profiles, where declining Δ14C values might indicate slower OC turnover in deeper soils. Thus, the 14C depth gradients in the studied soils were not explained by increasing stability of organic–mineral associations with soil depth. Although H2O2 removed 90 ± 8 % of the MOC, the Δ14C values of oxidized OC (on average −50 ± 110 ‰) were similar to those of OC extracted with NaF–NaOH (−51 ± 122 ‰), but oxidation residues (−345 ± 227 ‰) were much more depleted in 14C than residues of the NaF–NaOH extraction (−130 ± 121 ‰). Accordingly, both chemical treatments removed OC from the same continuum, and oxidation residues were older than extraction residues because more OC was removed. In contrast to the NaF–NaOH extractions, higher contents of pedogenic oxides slightly increased the oxidation resistance of MOC, but this higher H2O2 resistance did not coincide with more negative Δ14C values of MOC nor its oxidation residues. Therefore, none of the applied chemical fractionation schemes were able to explain site-specific differences in Δ14C values. Our results indicate that total MOC was dominated by OC interactions with pedogenic oxides rather than clay minerals, as we detected no difference in bond strength between clay-rich and clay-poor sites. This suggests that site-specific differences in Δ14C values of bulk MOC and depth profiles are driven by the accumulation and exchange rates of OC at mineral surfaces.

Functional Characterization of Two Thioredoxin Proteins of Toxoplasma gondii Using the CRISPR-Cas9 System.

Toxoplasmosis caused by infection with Toxoplasma gondii is an important parasitic zoonosis with a worldwide distribution. In this study, we examined the functions of two thioredoxins (namely CTrp26 and CTrx1) of T. gondii tachyzoites by generation of HA tag strains or gene deficient parasites in Type I RH strain (ToxoDB#10). Immunofluorescence analysis (IFA) was used to investigate the subcellular localization of the thioredoxins (Trxs). Results of IFA showed that both CTrp26 and CTrx1 were located in the cytoplasm of T. gondii. Functional characterizations of CTrp26 and CTrx1-deficient parasites were performed by plaque assay, intracellular replication, egress, H2O2 resistance, detection of reactive oxygen species (ROS) level and total antioxidant capacity (T-AOC) assays in vitro, as well as mouse infection in vivo. Our results showed that deletion of CTrp26 or CTrx1 did not influence the ability of T. gondii RH strain to replicate, egress, form plaque, resist H2O2 exposure, maintain the ROS level, and T-AOC, and also did not serve as virulence factors in Kunming mice. Taken together, these results provide new properties of the two Trxs. Although they are not essential for RH strain, they may have roles in other strains of this parasite due to their different expression patterns, which warrants future research.

Free Rather Than Total Iron Content Is Critically Linked to the Fur Physiology in Shewanella oneidensis.

Ferric uptake regulator (Fur) is a transcriptional regulator playing a central role in iron homeostasis of many bacteria, and Fur inactivation commonly results in pleiotropic phenotypes. In Shewanella oneidensis, a representative of dissimilatory metal-reducing γ-proteobacteria capable of respiring a variety of chemicals as electron acceptors (EAs), Fur loss substantially impairs respiration. However, to date the mechanism underlying the physiological phenomenon remains obscure. This investigation reveals that Fur loss compromises activity of iron proteins requiring biosynthetic processes for their iron cofactors, heme in particular. We then show that S. oneidensis Fur is critical for maintaining heme homeostasis by affecting both its biosynthesis and decomposition of the molecule. Intriguingly, the abundance of iron-containing proteins controlled by H2O2-responding regulator OxyR increases in the fur mutant because the Fur loss activates OxyR. By comparing suppression of membrane-impermeable, membrane-permeable, and intracellular-only iron chelators on heme deficiency and elevated H2O2 resistance, our data suggest that the elevation of the free iron content by the Fur loss is likely to be the predominant factor for the Fur physiology. Overall, these results provide circumstantial evidence that Fur inactivation disturbs bacterial iron homeostasis by altering transcription of its regulon members, through which many physiological processes, such as respiration and oxidative stress response, are transformed.