Role In Oxidative Stress Resistance Research Articles

Decoding the role of oxidative stress resistance and alternative carbon substrate assimilation in the mature biofilm growth mode of Candida glabrata

BackgroundBiofilm formation is viewed as a vital mechanism in C. glabrata pathogenesis. Although, it plays a significant role in virulence but transcriptomic architecture and metabolic pathways governing the biofilm growth mode of C. glabrata remain elusive. The present study intended to investigate the genes implicated in biofilm growth phase of C. glabrata through global transcriptomic approach.ResultsFunctional analysis of Differentially expressed genes (DEGs) using gene ontology and pathways analysis revealed that upregulated genes are involved in the glyoxylate cycle, carbon-carbon lyase activity, pre-autophagosomal structure membrane and vacuolar parts whereas, down- regulated genes appear to be associated with glycolysis, ribonucleoside biosynthetic process, ribosomal and translation process in the biofilm growth condition. The RNA-Seq expression of eight selected DEGs (CgICL1, CgMLS1, CgPEP1, and CgNTH1, CgERG9, CgERG11, CgTEF3, and CgCOF1) was performed with quantitative real-time PCR (RT-qPCR). The gene expression profile of selected DEGs with RT-qPCR displayed a similar pattern of expression as observed in RNA-Seq. Phenotype screening of mutant strains generated for genes CgPCK1 and CgPEP1, showed that Cgpck1∆ failed to grow on alternative carbon substrate (Glycerol, Ethanol, Oleic acid) and similarly, Cgpep1∆ unable to grow on YPD medium supplemented with hydrogen peroxide. Our results suggest that in the absence of glucose, C. glabrata assimilate glycerol, oleic acid and generate acetyl coenzyme-A (acetyl-CoA) which is a central and connecting metabolite between catabolic and anabolic pathways (glyoxylate and gluconeogenesis) to produce glucose and fulfil energy requirements.ConclusionsThe study was executed using various approaches (transcriptomics, functional genomics and gene deletion) and it revealed that metabolic plasticity of C. glabrata (NCCPF-100,037) in biofilm stage modulates its virulence and survival ability to counter the stress and may promote its transition from commensal to opportunistic pathogen. The observations deduced from the present study along with future work on characterization of the proteins involved in this intricate process may prove to be beneficial for designing novel antifungal strategies.

A mitochondrial NAD/NADH kinase governs fungal virulence through an oxidative stress response and arginine biosynthesis in Fusarium graminearum

NADP/NADPH plays an indispensable role in cellular metabolism, serving as a pivotal cofactor in numerous enzymatic processes involved in anabolic pathways, antioxidant defense, and the biosynthesis of essential cellular components. NAD/NADH kinases (NADKs) phosphorylate NAD/NADH, constituting the sole de novo synthetic pathway for NADP/NADPH generation. Despite the pivotal role of NADP/NADPH in cellular functions, the physiological role of NADK remains largely unexplored in filamentous fungi. In this study, we identified three putative NADKs in Fusarium graminearum—FgNadk1, FgNadk2, and FgNadk3—responsible for NAD/NADH phosphorylation. NADK-mediated formation of intracellular NADPH proved crucial for vegetative growth, sexual reproduction, and virulence. Specifically, FgNadk2, the mitochondrial NADK, played a role in oxidative stress resistance and the maintenance of mitochondrial reactive oxygen species levels. Moreover, the deletion of FgNADK2 resulted in arginine auxotrophy, contributing to the reduced fungal virulence. These findings underscore the necessity of mitochondrial NADK in fungal virulence in F. graminearum, revealing its involvement in mitochondrial redox homeostasis and the arginine biosynthetic pathway. This study provides critical insights into the interconnectedness of metabolic pathways essential for fungal growth, stress response, and pathogenicity.

OxyR-regulated T6SS functions in coordination with siderophore to resist oxidative stress.

The formation of reactive oxygen species is harmful and can destroy intracellular macromolecules such as lipids, proteins, and DNA, even leading to bacterial death. To cope with this situation, microbes have evolved a variety of sophisticated mechanisms, including antioxidant enzymes, siderophores, and the type VI secretion system (T6SS). However, the mechanism of oxidative stress resistance in Cupriavidus pinatubonensis is unclear. In this study, we identified Reut_A2805 as an OxyR ortholog in C. pinatubonensis, which positively regulated the expression of T6SS1 by directly binding to its operon promoter region. The study revealed that OxyR-regulated T6SS1 combats oxidative stress by importing iron into bacterial cells. Moreover, the T6SS1-mediated outer membrane vesicles-dependent iron acquisition pathway played a crucial role in the oxidative stress resistance process. Finally, our study demonstrated that the T6SS1 and siderophore systems in C. pinatubonensis exhibit different responses in combating oxidative stress under low-iron conditions, providing a comprehensive understanding of how bacterial iron acquisition systems function in diverse conditions.IMPORTANCEThe ability to eliminate reactive oxygen species is crucial for bacterial survival. Continuous formation of hydroperoxides can damage metalloenzymes, disrupt DNA integrity, and even result in cell death. While various mechanisms have been identified in other bacterial species to combat oxidative stress, the specific mechanism of oxidative stress resistance in C. pinatubonensis remains unclear. The importance of this study is that we elucidate the mechanism that OxyR-regulated T6SS1 combats oxidative stress by importing iron with the help of bacterial outer membrane vesicle. Moreover, the study highlights the contrasting responses of T6SS1- and siderophore-mediated iron acquisition systems to oxidative stress. This study provides a comprehensive understanding of bacterial iron acquisition and its role in oxidative stress resistance in C. pinatubonensis under low-iron conditions.

Structure of a superoxide dismutase from a tardigrade: Ramazzottius varieornatus strain YOKOZUNA-1.

Superoxide dismutase (SOD) is an essential and ubiquitous antioxidant protein that is widely present in biological systems. The anhydrobiotic tardigrades are some of the toughest micro-animals. They have an expanded set of genes for antioxidant proteins such as SODs. These proteins are thought to play an essential role in oxidative stress resistance in critical situations such as desiccation, although their functions at the molecular level have yet to be explored. Here, crystal structures of a copper/zinc-containing SOD (RvSOD15) from an anhydrobiotic tardigrade, Ramazzottius varieornatus strain YOKOZUNA-1, are reported. In RvSOD15, one of the histidine ligands of the catalytic copper center is replaced by a valine (Val87). The crystal structures of the wild type and the V87H mutant show that even though a histidine is placed at position 87, a nearby flexible loop can destabilize the coordination of His87 to the Cu atom. Model structures of other RvSODs were investigated and it was found that some of them are also unusual SODs, with features such as deletion of the electrostatic loop or β3 sheet and unusual metal-binding residues. These studies show that RvSOD15 and some other RvSODs may have evolved to lose the SOD function, suggesting that gene duplications of antioxidant proteins do not solely explain the high stress tolerance of anhydrobiotic tardigrades.

Characterization of FA1654: A putative DPS protein in Filifactor alocis.

The survival/adaptation of Filifactor alocis, a fastidious Gram-positive asaccharolytic anaerobe, to the inflammatory environment of the periodontal pocket requires an ability to overcome oxidative stress. Moreover, its pathogenic characteristics are highlighted by its capacity to survive in the oxidative-stress microenvironment of the periodontal pocket and a likely ability to modulate the microbial community dynamics. There is still a significant gap in our understanding of its mechanism of oxidative stress resistance and its impact on the virulence and pathogenicity of the microbial biofilm. Coinfection of epithelial cells with F. alocis and Porphyromonas gingivalis resulted in the upregulation of several genes, including HMPREF0389_01654 (FA1654). Bioinformatics analysis indicates that FA1654 has a "di-iron binding domain" and could function as a DNA starvation and stationary phase protection (DPS) protein. We have further characterized the FA1654 protein to determine its role in oxidative stress resistance in F. alocis. In the presence of hydrogen peroxide-induced oxidative stress, there was an ∼1.3 fold upregulation of the FA1654 gene in F. alocis. Incubation of the purified FA1654 protein with DNA in the presence of hydrogen peroxide and iron resulted in the protection of the DNA from Fenton-mediated degradation. Circular dichroism and differential scanning fluorimetry studies have documented the intrinsic ability of rFA1654 protein to bind iron; however, the rFA1654 protein is missing the intrinsic ability to reduce hydrogen peroxide. Collectively, the data may suggest that FA1654 in F. alocis is involved in oxidative stress resistance via an ability to protect against Fenton-mediated oxidative stress-induced damage.

Tetramethylpyrazine nitrone TBN extends the lifespan of C. elegans by activating the Nrf2/SKN-1 signaling pathway

SKN-1, the ortholog of mammalian Nrf2 proteins, is a transcription factor that plays an important role in oxidative stress resistance and longevity. Similar to other defense systems, the Nrf2-mediated stress response is compromised in aging and neurodegenerative diseases. Our previous studies demonstrated that tetramethylpyrazine nitrone (TBN), a derivative of tetramethylpyrazine armed with a potent free radical-scavenging nitrone moiety, exerted multifunctional neuroprotection in neurological and other diseases. However, the ability of TBN to extend a healthy lifespan and its underlying mechanisms of action are not yet clear. C. elegans have become a popular animal model in aging research. Herein, we demonstrate that TBN can extend the lifespan, promote age-associated health indicators, and restore mitochondrial function in C. elegans. TBN also significantly reduced ROS levels and superoxide accumulation in C. elegans. We show that TBN-mediated lifespan extension is SKN-1dependent. The present study provides valuable insights into the mechanisms by which TBN inhibits aging via the Nrf2/SKN-1 pathway in C. elegans.

PG1659 functions as anti-sigma factor to extracytoplasmic function sigma factor RpoE in Porphyromonas gingivalis W83.

Anti-sigma factors play a critical role in regulating the expression of sigma factors in response to environmental stress signals. PG1659 is cotranscribed with an upstream gene PG1660 (rpoE), which encodes for a sigma factor that plays an important role in oxidative stress resistance and the virulence regulatory network of P. gingivalis. PG1659, which is annotated as a hypothetical gene, is evaluated in this study. PG1659, composed of 130 amino acids, is predicted to be transmembrane protein with a single calcium (Ca2+ ) binding site. In P. gingivalis FLL358 (ΔPG1659::ermF), the rpoE gene was highly upregulated compared to the wild-type W83 strain. RpoE-induced genes were also upregulated in the PG1659-defective isogenic mutant. Both protein-protein pull-down and bacterial two-hybrid assays revealed that the PG1659 protein could interact with/bind RpoE. The N-terminal domain of PG1659, representing the cytoplasmic fragment of the protein, is critical for interaction with RpoE. In the presence of PG1659, the initiation of transcription by the RpoE sigma factor was inhibited. Taken together, our data suggest that PG1659 is an anti-sigma factor which plays an important regulatory role in the modulation of the sigma factor RpoE in P. gingivalis.

DNA-binding protein from starvation cells traps intracellular free-divalent iron and plays an important role in oxidative stress resistance in Acetobacter pasteurianus NBRC 3283

Acetobacter pasteurianus accumulates reactive oxygen species (ROS). ROS are produced by electron and oxygen coupling in the electron transport chain in the intracellular environment during the stationary and in the acetic acid over-oxidation phases in the presence of ethanol, thereby exposing cell to oxidative stress. In this study, to reveal the resistance mechanism to oxidative stress in A.pasteurianus, we focused on DNA-binding protein from starvation cells (Dps) and analyzed the function of Dps against oxidative stress. When Dps under the copresence of plasmid DNA was exposed to H2O2 and divalent iron, plasmid DNA fragmentation was suppressed under the presence of Dps; however, DNA binding was not observed, revealing a defensive activity for oxidative damage. In addition, this finding revealed that Dps incorporates a divalent iron intracellularly, forming a ferroxidase center. Moreover, levels of hydroxyl radicals produced by Fenton reaction under the presence of H2O2 and divalent iron were decreased by the addition of Dps, resulting in the suppression of the Fenton reaction. Through fluorescence microscopy using a divalent-iron-specific fluorescent probe, we found that, in dps gene disruptants, the accumulation of the divalent iron increased, and the dps gene disruptants showed higher sensitivity to H2O2 than the wild-type. These result strongly suggested that Dps traps intracellular free-divalent iron and plays an important role in the oxidative stress resistance of A.pasteurianus NBRC 3283 after the acetic acid fermentation phase.

A multilayered repair system protects the mycobacterial chromosome from endogenous and antibiotic-induced oxidative damage

Oxidative damage to DNA is a threat to the genomic integrity and coding accuracy of the chromosomes of all living organisms. Guanine is particularly susceptible to oxidation, and 8-oxo-dG (OG), when produced in situ or incorporated by DNA polymerases, is highly mutagenic due to mispairing with adenine. In many bacteria, defense against OG depends on MutT enzymes, which sanitize OG in the nucleotide pool, and the MutM/Y system, which counteracts OG in chromosomal DNA. In Escherichia coli, antibiotic lethality has been linked to oxidative stress and the downstream consequences of OG processing. However, in mycobacteria, the role of these systems in genomic integrity and antibiotic lethality is not understood, in part because mycobacteria encode four MutT enzymes and two MutMs, suggesting substantial redundancy. Here, we definitively probe the role of OG handling systems in mycobacteria. We find that, although MutT4 is the only MutT enzyme required for resistance to oxidative stress, this effect is not due to OG processing. We find that the dominant system that defends against OG-mediated mutagenesis is MutY/MutM1, and this system is dedicated to in situ chromosomal oxidation rather than correcting OG incorporated by accessory polymerases (DinB1/DinB2/DinB3/DnaE2). In addition, we uncover that mycobacteria resist antibiotic lethality through nucleotide sanitization by MutTs, and in the absence of this system, accessory DNA polymerases and MutY/M contribute to antibiotic-induced lethality. These results reveal a complex, multitiered system of OG handling in mycobacteria with roles in oxidative stress resistance, mutagenesis, and antibiotic lethality.

Loss of Oxidation Resistance 1, OXR1, Is Associated with an Autosomal-Recessive Neurological Disease with Cerebellar Atrophy and Lysosomal Dysfunction

We report an early-onset autosomal-recessive neurological disease with cerebellar atrophy and lysosomal dysfunction. We identified bi-allelic loss-of-function (LoF) variants in Oxidative Resistance 1 (OXR1) in five individuals from three families; these individuals presented with a history of severe global developmental delay, current intellectual disability, language delay, cerebellar atrophy, and seizures. While OXR1 is known to play a role in oxidative stress resistance, its molecular functions are not well established. OXR1 contains three conserved domains: LysM, GRAM, and TLDc. The gene encodes at least six transcripts, including some that only consist of the C-terminal TLDc domain. We utilized Drosophila to assess the phenotypes associated with loss of mustard (mtd), the fly homolog of OXR1. Strong LoF mutants exhibit late pupal lethality or pupal eclosion defects. Interestingly, although mtd encodes 26 transcripts, severe LoF and null mutations can be rescued by a single short human OXR1 cDNA that only contains the TLDc domain. Similar rescue is observed with the TLDc domain of NCOA7, another human homolog of mtd. Loss of mtd in neurons leads to massive cell loss, early death, and an accumulation of aberrant lysosomal structures, similar to what we observe in fibroblasts of affected individuals. Our data indicate that mtd and OXR1 are required for proper lysosomal function; this is consistent with observations that NCOA7 is required for lysosomal acidification.

A novel role for the glutamate decarboxylase system in Listeria monocytogenes; protection against oxidative stress

The GAD system is widely present in several types of organisms and is known to play an important role in bacterial acid tolerance. There is only one account of this system playing a role in oxidative stress in bacteria and one in yeasts. Here we show for first time that it affects the oxidative stress resistance of a Gram-positive bacterium, (L. monocytogenes, tested in three strains; 10403S, EGD-e, and LO28). We found a statistically significant reduction in survival after H2O2 exposure in ΔgadD3 and ΔgadD2 of EGD-e and in ΔgadD1 of LO28. Furthermore, we observed a lag phase prolongation in ΔgadD3 of 10403S and EGD-e and a larger inhibition zone in disk diffusion assay for ΔgadD1 and ΔgadD3 of EGD-e upon H2O2 exposure. All GAD genes playing a role in oxidative stress resistance are part of GADi system and this occurs partly through catalase activity, while the most potent GADe system plays no role. The latter effects could occur through the GABA shunt, but we show here that mutants in succinate semialdehyde dehydrogenase do not show a phenotype suggesting that either effects are through the GABA transaminase or, this pathway is not involved. Our study highlights for first time the role of the GAD system in oxidative stress resistance of a Gram-positive bacterium, which could be used in Food Hurdle Technology to eliminate pathogens such as L. monocytogenes, while it gives an insight on the general mechanism.

Oxidative stress resistance as a factor in aging: evidence from an extended longevity phenotype of Drosophila melanogaster.

Longevity of a species is a multifactorial quantitative trait influenced by genetic background, sex, age and environment of the organism. Extended longevity phenotypes (ELP) from experimental evolution in the laboratory can be used as model systems to investigate the mechanisms underlying aging and senescence. ELPs of Drosophila are correlated with various life history attributes such as resistance to environmental stressors (starvation, desiccation, cold and paraquat), developmental time, biochemical defenses, etc. The association between oxidative stress resistance and longevity is not clear and ELPs offer an opportunity to examine the role of oxidative stress resistance in longevity. Here, we have investigated the hypothesis that enhanced oxidative stress resistance and elevated antioxidant defense system play a positive role in longevity using an ELP of Drosophila melanogaster. An ELP of D. melanogaster isolated and characterized in our laboratory through artificial selection (inbred laboratory strain of Oregon K) is employed in this study. Our ELP, named as long lifespan (LLS) flies, shows marked extension in lifespan when compared to the progenitor population (normal lifespan, NLS) and makes a suitable model to study the role of mitochondrial genome in longevity because of its least heterogeneity. In this study, sensitivity to ethanol with age was employed as a measure of resistance to oxidative stress in NLS and LLS flies. Effect of age and oxidative stress on longevity was examined by employing NLS and LLS flies of different age groups against ethanol-induced oxidative stress. Results show that the lower mortality against ethanol was associated with enhanced oxidative stress resistance, higher antioxidant defenses, lower reactive oxygen species (ROS) levels, enhanced alcohol dehydrogenase activity and better locomotor ability attributes of LLS flies. In addition, age-related changes like locomotor impairments, decreased antioxidant defenses, higher ROS levels and sensitivity to oxidative stress were delayed in LLS flies when compared to NLS. Our study supports the hypothesis that higher oxidative stress resistance and enhanced antioxidant defenses are significant factors in extending longevity.

Oxidized OxyR Up-Regulates ahpCF Expression to Suppress Plating Defects of oxyR- and Catalase-Deficient Strains.

It is well established that in bacteria, such as Escherichia coli, OxyR is a transcriptional regulator that mediates the response to H2O2 by activating the OxyR regulon, which consists of many genes that play vital roles in oxidative stress resistance. In Shewanella, OxyR regulates, however, in both reduced and oxidized states, the production of H2O2 scavengers, including major catalase KatB and NADH peroxidase AhpCF. Here we showed that the oxyR mutant carried a plating defect manifested as division arresting, a phenotype that can be completely suppressed by an OxyR variant constitutively existing in oxidized form (OxyRL197P). This effect of OxyRL197P could not be solely attributed to the increment in KatB production, since the suppression was also observed in the absence of KatB. Although expression of peroxidase CcpA was greatly activated by OxyRL197P, the contribution of the protein in alleviating plating defect was negligible. We eventually identified AhpCF as the critical factor, when produced at substantially elevated levels by OxyRL197P, to protect the cell from H2O2 attack. Our data indicate that AhpCF is a particularly important peroxidase in oxidative stress resistance in Shewanella, not only playing a compensatory role for catalase, but also by itself providing sufficient protection from killing of H2O2 generated abiotically.

CosR is an oxidative stress sensing a MarR-type transcriptional repressor in Corynebacterium glutamicum

The MarR family is unique to both bacteria and archaea. The members of this family, one of the most prevalent families of transcriptional regulators in bacteria, enable bacteria to adapt to changing environmental conditions, such as the presence of antibiotics, toxic chemicals, or reactive oxygen species (ROS), mainly by thiol-disulfide switches. Although the genome of Corynebacterium glutamicum encodes a large number of the putative MarR-type transcriptional regulators, their physiological and biochemical functions have so far been limited to only two proteins, regulator of oxidative stress response RosR and quinone oxidoreductase regulator QosR. Here, we report that the ncgl2617 gene (cosR) of C. glutamicum encoding an MarR-type transcriptional regulator plays an important role in oxidative stress resistance. The cosR null mutant is found to be more resistant to various oxidants and antibiotics, accompanied by a decrease in ROS production and protein carbonylation levels under various stresses. Protein biochemical function analysis shows that two Cys residues presenting at 49 and 62 sites in CosR are redox-active. They form intermolecular disulfide bonds in CosR under oxidative stress. This CosR oxidation leads to its dissociation from promoter DNA, depression of the target DNA, and increased oxidative stress resistance of C. glutamicum. Together, the results reveal that CosR is a redox-sensitive regulator that senses peroxide stress to mediate oxidative stress resistance in C. glutamicum.

Exposure to N-chlorotaurine induces oxidative stress responses in Aspergillus fumigatus

The neutrophil-derived oxidant N-chlorotaurine (NCT) displays remarkable in vivo tolerability and efficacy against a range of pathogens. The aim of this study was to characterize the response of the pulmonary pathogen Aspergillus fumigatus to NCT. The effect of NCT on the growth and viability of A. fumigatus was characterized. NCT-induced alteration of amino acids and gliotoxin from A. fumigatus mycelium was assessed. Label-free shotgun quantitative proteomic analysis was performed on A. fumigatus exposed to NCT for 24 h. Incubation of A. fumigatus with NCT at concentrations ranging from 6.8 to 55 mM decreased conidial growth and viability, and mycelium biomass relative to the controls. Exposure to NCT (13.77 mM) resulted in increased amino acids and gliotoxin levels from A. fumigatus mycelium. Exposure of A. fumigatus mycelium to NCT (6.8 mM) revealed an enrichment in proteins associated with the ribosome, transcription and translation and non-ribosomal peptide biosynthesis (e.g. Pes1, Pes3), which play an essential role in oxidative stress resistance in A. fumigatus. A decrease in the abundance of proteins associated with fumagillin and pseurotin biosynthesis highlighted the anti-virulence activity of NCT. These results indicate that NCT induces an oxidative stress response in A. fumigatus as evidenced by alterations in the proteome and inhibits conidial and mycelial growth. Clinical investigations of topical application of NCT to treat Aspergillus infections are encouraged.

Myo-inositol-1-phosphate synthase (Ino-1) functions as a protection mechanism in Corynebacterium glutamicum under oxidative stress.

Reactive oxygen species (ROS) generated in aerobic metabolism and oxidative stress lead to macromolecules damage, such as to proteins, lipids, and DNA, which can be eliminated by the redox buffer mycothiol (AcCys‐GlcN‐Ins, MSH). Myo‐inositol‐phosphate synthase (Ino‐1) catalyzes the first committed step in the synthesis of MSH, thus playing a critical role in the growth of the organism. Although Ino‐1s have been systematically studied in eukaryotes, their physiological and biochemical functions remain largely unknown in bacteria. In this study, we report that Ino‐1 plays an important role in oxidative stress resistance in the gram‐positive Actinobacteria Corynebacterium glutamicum. Deletion of the ino‐1 gene resulted in a decrease in cell viability, an increase in ROS production, and the aggravation of protein carbonylation levels under various stress conditions. The physiological roles of Ino‐1 in the resistance to oxidative stresses were corroborated by the absence of MSH in the Δino‐1 mutant. In addition, we found that the homologous expression of Ino‐1 in C. glutamicum yielded a functionally active protein, while when expressed in Escherichia coli BL21(DE3), it lacked measurable activity. An examination of the molecular mass (Mr) suggested that Ino‐1 expressed in E. coli BL21(DE3) was not folded in a catalytically competent conformation. Together, the results unequivocally showed that Ino‐1 was important for the mediation of oxidative resistance by C. glutamicum.

Intersection of phosphate transport, oxidative stress and TOR signalling in Candida albicans virulence.

Phosphate is an essential macronutrient required for cell growth and division. Pho84 is the major high-affinity cell-surface phosphate importer of Saccharomyces cerevisiae and a crucial element in the phosphate homeostatic system of this model yeast. We found that loss of Candida albicans Pho84 attenuated virulence in Drosophila and murine oropharyngeal and disseminated models of invasive infection, and conferred hypersensitivity to neutrophil killing. Susceptibility of cells lacking Pho84 to neutrophil attack depended on reactive oxygen species (ROS): pho84-/- cells were no more susceptible than wild type C. albicans to neutrophils from a patient with chronic granulomatous disease, or to those whose oxidative burst was pharmacologically inhibited or neutralized. pho84-/- mutants hyperactivated oxidative stress signalling. They accumulated intracellular ROS in the absence of extrinsic oxidative stress, in high as well as low ambient phosphate conditions. ROS accumulation correlated with diminished levels of the unique superoxide dismutase Sod3 in pho84-/- cells, while SOD3 overexpression from a conditional promoter substantially restored these cells’ oxidative stress resistance in vitro. Repression of SOD3 expression sharply increased their oxidative stress hypersensitivity. Neither of these oxidative stress management effects of manipulating SOD3 transcription was observed in PHO84 wild type cells. Sod3 levels were not the only factor driving oxidative stress effects on pho84-/- cells, though, because overexpressing SOD3 did not ameliorate these cells’ hypersensitivity to neutrophil killing ex vivo, indicating Pho84 has further roles in oxidative stress resistance and virulence. Measurement of cellular metal concentrations demonstrated that diminished Sod3 expression was not due to decreased import of its metal cofactor manganese, as predicted from the function of S. cerevisiae Pho84 as a low-affinity manganese transporter. Instead of a role of Pho84 in metal transport, we found its role in TORC1 activation to impact oxidative stress management: overexpression of the TORC1-activating GTPase Gtr1 relieved the Sod3 deficit and ROS excess in pho84-/- null mutant cells, though it did not suppress their hypersensitivity to neutrophil killing or hyphal growth defect. Pharmacologic inhibition of Pho84 by small molecules including the FDA-approved drug foscarnet also induced ROS accumulation. Inhibiting Pho84 could hence support host defenses by sensitizing C. albicans to oxidative stress.

OxyR of Haemophilus parasuis is a global transcriptional regulator important in oxidative stress resistance and growth

Haemophilus parasuis is an opportunistic pathogen and the causative agent of Glässer's disease in swine. This disease has high morbidity and mortality rates in swine populations, and is responsible for major economic losses worldwide. Survival of H. parasuis within the host requires mechanisms for coping with oxidative stress conditions. In many bacteria, OxyR is known to mediate protection against oxidative stress; however, little is known about the role of OxyR in H. parasuis. In the current study, an oxyR mutant strain was constructed in H. parasuis strain SC1401 and designated H. parasuis SC1401∆oxyR. The oxyR mutant strain had a slower growth rate and impaired biofilm formation compared to the wild type strain. Complementation restored the growth-associated phenotypes to wild type levels. Oxidative stress susceptibility testing, using a range of concentrations of H2O2, indicated that H. parasuis SC1401∆oxyR was more sensitive to oxidative stress than the wild type strain. RNA sequencing transcriptome analysis comparing H. parasuis SC1401 with H. parasuis SC1401∆oxyR identified 466 differentially expressed genes. These genes were involved in a wide range of biological processes, including: oxidative stress, transcriptional regulation, and DNA replication, recombination, and repair. These findings provide a foundation for future research to examine the role of OxyR as a global transcriptional regulator and to better define its role in oxidative stress resistance in H. parasuis.

Polyphosphate kinase 1-coding gene (ppk1) is involved in the oxidative stress resistance in uropathogenic Escherichia coli by modulating the expression of katG and katE genes

Objective To investigate the role and the mechanism of ppk1 gene (coding for polyphosphate kinase 1) in oxidative stress resistance in uropathogenic Escherichia coli (UPEC). Methods Mutant strains with ppk1-deletion (△pk1) and complemented strains (△pk1-C) were constructed based on the UPEC strain CFT073. A comparative analysis was conducted to analyze survival rates of CFT073, △pk1 and △pk1-C strains at different time points while they were under oxidative stress. Differences in protein expression between CFT073 and △pk1 strains were analyzed using mass spectrometric analysis. Differences between CFT073 and △pk1 strains in expression of katG and katE genes were analyzed using real-time quantitative RT-PCR. Results The survival rate of △pk1 strains was lower than that of CFT073 strains at every time point, while the survival rate of △pk1-C strains was basically the same as that of CFT073 strains. Gel image analysis and mass spectrometric analysis revealed that six proteins were down-regulated and one was up-regulated in △pk1 strains as compared with those in CFT073 strains. Expression of the catalase-coding genes katG and katE in △pk1 strains were respectively (20.5±8.2)% and (20.9±6.9)% of those in CFT073 strains (P<0.05). Conclusion The ppk1 gene plays an important role in oxidative stress resistance in UPEC by modulating the expression of catalase-coding genes katG and katE. Key words: Urinary tract infection; Escherichia coli; Polyphosphate kinase 1; Oxidative stress

NapA Mediates a Redox Regulation of the Antioxidant Response, Carbon Utilization and Development in Aspergillus nidulans.

The redox-regulated transcription factors (TFs) of the bZIP AP1 family, such as yeast Yap1 and fission yeast Pap1, are activated by peroxiredoxin proteins (Prxs) to regulate the antioxidant response. Previously, Aspergillus nidulans mutants lacking the Yap1 ortholog NapA have been characterized as sensitive to H2O2 and menadione. Here we study NapA roles in relation to TFs SrrA and AtfA, also involved in oxidant detoxification, showing that these TFs play different roles in oxidative stress resistance, catalase gene regulation and development, during A. nidulans life cycle. We also uncover novel NapA roles in repression of sexual development, normal conidiation, conidial mRNA accumulation, and carbon utilization. The phenotypic characterization of ΔgpxA, ΔtpxA, and ΔtpxB single, double and triple peroxiredoxin mutants in wild type or ΔnapA backgrounds shows that none of these Prxs is required for NapA function in H2O2 and menadione resistance. However, these Prxs participate in a minor NapA-independent H2O2 resistance pathway and NapA and TpxA appear to regulate conidiation along the same route. Using transcriptomic analysis we show that during conidial development NapA-dependent gene expression pattern is different from canonical oxidative stress patterns. In the course of conidiation, NapA is required for regulation of at least 214 genes, including ethanol utilization genes alcR, alcA and aldA, and large sets of genes encoding proteins involved in transcriptional regulation, drug detoxification, carbohydrate utilization and secondary metabolism, comprising multiple oxidoreductases, membrane transporters and hydrolases. In agreement with this, ΔnapA mutants fail to grow or grow very poorly in ethanol, arabinose or fructose as sole carbon sources. Moreover, we show that NapA nuclear localization is induced not only by oxidative stress but also by growth in ethanol and by carbon starvation. Together with our previous work, these results show that SakA-AtfA, SrrA and NapA oxidative stress-sensing pathways regulate essential aspects of spore physiology (i.e., cell cycle arrest, dormancy, drug production and detoxification, and carbohydrate utilization).