PeAP1-mediated oxidative stress response plays an important role in the growth and pathogenicity of Penicillium expansum.

ABSTRACTFruit blue mold disease and patulin contamination caused by Penicillium expansum lead to huge economic losses and food safety concerns worldwide. Many genes have been proven to be involved in the regulation of pathogenic and toxigenic processes of P. expansum. Histone H3 lysine 4 (H3K4) methylation is well recognized for its association with chromatin regulation and gene transcription. However, it is not clear whether H3K4 methylation is related to infection and patulin biosynthesis in Penicillium. Here, we characterized PeSet1, which is responsible for H3K4me1/me2/me3 in P. expansum. The deletion of PeSet1 caused severe defects in hyphal growth, conidiation, colonization, patulin biosynthesis, and stress responses. Moreover, we demonstrated that PeSet1 is involved in the regulation of patulin biosynthesis by mediating the expression of patulin cluster genes and crucial global regulatory factors. Likewise, PeSet1 positively regulated key genes in β-1,3-glucan biosynthesis and the reactive oxygen species scavenging process to modulate cell wall integrity and oxidative stress responses, respectively. Collectively, we have proven for the first time the function of Set1 in patulin biosynthesis and the crucial role of Set1 in colonization and stress responses in P. expansum.IMPORTANCEPenicillium expansum is one of the most important plant fungal pathogens, which not only causes blue mold rot in various fruits, leading to huge decay losses, but also produces mycotoxin patulin, posing a threat to human health. Both pathogenesis and patulin biosynthesis in P. expansum are regulated by complex and sophisticated networks. We focused on the epigenetic modification and identified a conserved histone H3K4 methyltransferase PeSet1 in P. expansum. Our work revealed the important role of PeSet1 in growth, development, colonization, patulin production, and stress responses of P. expansum. In particular, we originally described the regulation of Set1 on patulin biosynthetic pathway. These findings will provide new targets for the prevention and control of blue mold disease and patulin contamination.

SPINDLY (SPY) gene encodes a putative O-linked N-acetyl-glucosamine transferase, and yeast two-hybrid assay identified GIGANTEA (GI) as a SPY-interacting partner in Arabidopsis. GIGANTEA gene was previously shown to be involved in the regulation of oxidative stress response; however, it is unclear whether SPY gene is also involved in oxidative stress response. Here we showed that SPY plays a role in the regulation of the oxidative stress response. The spy-1 mutant was more tolerant to paraquat (PQ)-or hydrogen peroxide (H2O2)-mediated oxidative stress than wild-type plants. Analyses of endogenous H2O2 and superoxide anion radicals as well as lipid peroxidation revealed that enhanced tolerance of the spy-1 mutant to PQ-stress was not due to defects in the PQ uptake or the PQ sequestration from its site of action but rather the spy-1 mutation alleviated oxidative damage of plant cells upon PQ stress. Higher constitutive activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX) in spy-1 are more likely to be due to activation of both CSD2 gene encoding chloroplast Cu/Zn SOD and APX1 gene. Taken together, these results suggest that enhanced tolerance of the spy-1 mutant to oxidative stress is associated, at least in part, with constitutive activation of CSD2 and APX1.

bZIP transcription factor CgAP1 is essential for oxidative stress tolerance and full virulence of the poplar anthracnose fungus Colletotrichum gloeosporioides

Met-thodology

Introduction: TEAD1, the Hippo pathway-regulated transcription factor, has a critical non-redundant role in cardiomyocyte (CM) homeostasis, and its loss-of-function results in acute lethal dilated cardiomyopathy. However, the functional pathways regulated by TEAD1 in CMs remain unclear. Hypothesis: TEAD1 plays an essential role in CM oxidative stress response (OSR) via the regulation of NRF2, the master regulator of antioxidant response. Methods and Results: Conditional CM-specific TEAD1 deletion in adult mice results in acute heart failure (HF) and altered expression of OSR genes. In silico analysis of publicly available RNA-seq data revealed significant downregulation of TEAD1 in dilated and ischemic cardiomyopathy patient hearts and a positive correlation between TEAD1 and NRF2 expression. ChIP-seq with TEAD1 antibody and ATAC-seq revealed that TEAD1 occupied open promoter/enhancer regions of multiple OSR genes, including NRF2 and its targets in adult mouse hearts. Mosaic deletion of TEAD1 in ~50% CMs, which allowed assessment of TEAD1 LOF in CMs without confounding HF effect, reduced NRF2 expression, and increased myocardial oxidative stress indicated by 8OHdG staining (DNA oxidative damage) and cellROX only in TEAD1-deficient CM, but not in CM with normal TEAD1 expression. Ex vivo and in vitro TEAD1 knockout in primary murine adult and neonatal CMs and H9C2 cells resulted in significantly elevated total cellular (Boronate assay) and mitochondrial (mitoSOX) ROS due to reduced NRF2 expression and promoter activity (ARE reporter assay). TEAD1-deficient CM had a lower tolerance to chemically (Angiotensin II, DMNQ, or AntimycinA)-induced oxidative stress. Furthermore, hTEAD1 overexpression was sufficient to significantly increase NRF2-ARE activity and rescue the elevated ROS production in CMs. Conclusions: TEAD1 is a novel cell-autonomous direct transcriptional regulator of NRF2 and CM oxidative stress response.

Francisella tularensis causes a lethal human disease known as tularemia. As an intracellular pathogen, Francisella survives and replicates in phagocytic cells, such as macrophages. However, to establish an intracellular niche, Francisella must overcome the oxidative stress posed by the reactive oxygen species (ROS) produced by the infected macrophages. OxyR and SoxR/S are two well-characterized transcriptional regulators of oxidative stress responses in several bacterial pathogens. Only the OxyR homolog is present in F. tularensis, while the SoxR homologs are absent. The functional role of OxyR has not been established in F. tularensis. We demonstrate that OxyR regulates oxidative stress responses and provides resistance against ROS, thereby contributing to the survival of the F. tularensis subsp. holarctica live vaccine strain (LVS) in macrophages and epithelial cells and contributing to virulence in mice. Proteomic analysis reveals the differential production of 128 proteins in the oxyR gene deletion mutant, indicating its global regulatory role in the oxidative stress response of F. tularensis. Moreover, OxyR regulates the transcription of the primary antioxidant enzyme genes by binding directly to their putative promoter regions. This study demonstrates that OxyR is an important virulence factor and transcriptional regulator of the oxidative stress response of the F. tularensis LVS.

SETD6 is a negative regulator of oxidative stress response

Control of activity and functionality of microbial starter and probiotic cultures under industrial fermentation conditions is essential in order to provide a tasty, appealing, healthy, and safe product. Oxidative stress is one of the harsh conditions that fermentative microbes have managed to endure during their use in industrial fermentation processes. A widely-used lactic acid bacterium in food fermentations is Lactobacillus plantarum. Hence, understanding oxidative stress response in this micro-organism can be used for engineering robustness towards oxidative stress. There are two systems known to be involved in oxidative stress response and redox homeostasis in bacteria: the thioredoxin and glutaredoxin systems. In this study, we constructed a set of L. plantarum WCFS1 strains with alterations in these systems. Using the constructed strains under different oxidative stress conditions (hydrogen peroxide, diamide (thiol-stress), aerobic growth, and respiratory growth), global transcriptome analysis was performed. Subsequently, the functional role of the thioredoxin- and glutaredoxin system was analyzed and validated using a number of different techniques including comparative genomics, enzyme assays, and q-PCR. The main results obtained in this study include the role of the thioredoxin system in oxidative stress response in L. plantarum as well as in adaptation to aerobic cultivation; characterization and overlap of the thioredoxin and glutaredoxin system in this bacterium; insight into transcriptome regulation of oxidative stress response, and unravelling of stress-response networks present in L. plantarum WCFS1. The comparative genomics and global transcriptome analysis of the oxidative stress response of L. plantarum WCFS1 presented in this thesis can be used for optimization of the performance of lactic acid bacteria in industrial fermentations.

NADPH-oxidase mediated production of Reactive Oxygen Species (ROS) by alveolar macrophages and neutrophils is a critical mechanism for immune defence against Aspergillus fumigatus. Fungal oxidative stress response includes enzymatic response by superoxide dismutases (SOD), catalases, and enzymes from the thioredoxin and glutathione systems, which are regulated by the transcription factor Yap1. Secondary metabolites are also involved in defense against ROS. Some of the secondary metabolite clusters are controlled by the transcriptional regulator LaeA. The redundancy of antioxidant systems, and the variable impact of SOD or catalase gene deletions on in vitro oxidative stress sensitivity and in vivo virulence suggest a complex regulation of oxidative stress response in A. fumigatus, making high-throughput approaches, such as microarray or next generation sequencing (NGS), highly relevant to study their respective role. These approaches have been widely applied to A fumigatus, in order to characterize its metabolic response to different stresses mimicking in vivo conditions (such as antifungals, or neutrophils), or to transcription factor deletion (including LaeA). In some studies, oxidative stress response process and antioxidant enzymes have been identified as key metabolic pathways. However, oxidative stress response has not been analyzed systematically and a further data analysis could be helpful to clarify the role of A. fumigatus antioxidant systems and, potentially, to identify new drug targets. In this review, we synthesized available A. fumigatus microarrays and NGS data, focusing on the role of antioxidant systems. We analyzed the different methodologies that were used for transcriptomic analysis, and we compared biological processes and antioxidant system modulations in A. fumigatus exposed to stress.

Many organisms have adaptive responses to oxidative stress, and the levels of several antioxidant defense enzymes have been shown to be induced by changes in the levels of hydrogen peroxide or superoxide. Reactive oxygen species have also been proposed to be second messengers and to signal cellular fates such as proliferation and apoptosis. These observations suggest that cells have mechanisms to sense reactive oxygen species and induce specific responses. The mechanisms by which hydrogen peroxide and superoxide are sensed are not well understood, but a number of transcription factors that regulate the expression of antioxidant genes and/or whose activities are modulated by oxidation and reduction are known. In this chapter, we provide an overview of these transcriptional regulators in bacteria, yeast, and mammalian cells. REGULATORS IN PROKARYOTES As for many genetic responses, the regulators of oxidative stress responses have been best characterized in Escherichia coli. Wild-type cells show distinct adaptive responses to hydrogen peroxide and superoxide anion, and the key regulators of these two responses are OxyR and SoxR together with SoxS (see Table 1). In addition to these transcription factors, several SoxS homologs and the rpoS -encoded σ s subunit of RNA polymerase have also recently been shown to regulate the expression of antioxidant defense genes. OxyR The expression of at least nine of the hydrogen-peroxide-inducible proteins is controlled by OxyR in E. coli and Salmonella typhimurium (Christman et al. 1985). Several of the genes whose expression is activated by OxyR have been identified and include katG (hydroperoxidase I), ahpCF (an...

Oxidative stress response in pathogenic mycobacteria is believed to be of significance for host-pathogen interactions at various stages of infection. It also plays a role in determining the intrinsic susceptibility to isoniazid in mycobacterial species. In this work, we characterized the oxyR-ahpC and furA-katG loci in the nontuberculous pathogen Mycobacterium marinum. In contrast to Mycobacterium smegmatis and like Mycobacterium tuberculosis and Mycobacterium leprae, M. marinum was shown to possess a closely linked and divergently oriented equivalents of the regulator of peroxide stress response oxyR and its subordinate gene ahpC, encoding a homolog of alkyl hydroperoxide reductase. Purified mycobacterial OxyR was found to bind to the oxyR-ahpC promoter region from M. marinum and additional mycobacterial species. Mobility shift DNA binding analyses using OxyR binding sites from several mycobacteria and a panel of in vitro-generated mutants validated the proposed consensus mycobacterial recognition sequence. M. marinum AhpC levels detected by immunoblotting, were increased upon treatment with H2O2, in keeping with the presence of a functional OxyR and its binding site within the promoter region of ahpC. In contrast, OxyR did not bind to the sequences upstream of the katG structural gene, and katG expression did not follow the pattern seen with ahpC. Instead, a new open reading frame encoding a homolog of the ferric uptake regulator Fur was identified immediately upstream of katG in M. marinum. The furA-katG linkage and arrangement are ubiquitous in mycobacteria, suggesting the presence of additional regulators of oxidative stress response and potentially explaining the observed differences in ahpC and katG expression. Collectively, these findings broaden our understanding of oxidative stress response in mycobacteria. They also suggest that M. marinum will be useful as a model system for studying the role of oxidative stress response in mycobacterial physiology, intracellular survival, and other host-pathogen interactions associated with mycobacterial diseases.

Aspergillus fumigatus is a major fungal pathogen that is responsible for approximately 90% of human aspergillosis. Cofilin is an actin depolymerizing factor that plays crucial roles in multiple cellular functions in many organisms. However, the functions of cofilin in A. fumigatus are still unknown. In this study, we constructed an A. fumigatus strain overexpressing cofilin (cofilin OE). The cofilin OE strain displayed a slightly different growth phenotype, significantly increased resistance against H2O2 and diamide, and increased activation of the high osmolarity glycerol pathway compared to the wild-type strain (WT). The cofilin OE strain internalized more efficiently into lung epithelial A549 cells, and induced increased transcription of inflammatory factors (MCP-1, TNF-α and IL-8) compared to WT. Cofilin overexpression also resulted in increased polysaccharides including β-1, 3-glucan and chitin, and increased transcription of genes related to oxidative stress responses and polysaccharide synthesis in A. fumigatus. However, the cofilin OE strain exhibited similar virulence to the wild-type strain in murine and Galleria mellonella infection models. These results demonstrated for the first time that cofilin, a regulator of actin cytoskeleton dynamics, might play a critical role in the regulation of oxidative stress responses and cell wall polysaccharide synthesis in A. fumigatus.

Candida albicans is a major fungal pathogen of humans, causing approximately 400,000 life-threatening systemic infections world-wide each year in severely immunocompromised patients. An important fungicidal mechanism employed by innate immune cells involves the generation of toxic reactive oxygen species (ROS), such as superoxide and hydrogen peroxide. Consequently, there is much interest in the strategies employed by C. albicans to evade the oxidative killing by macrophages and neutrophils. Our understanding of how C. albicans senses and responds to ROS has significantly increased in recent years. Key findings include the observations that hydrogen peroxide triggers the filamentation of this polymorphic fungus and that a superoxide dismutase enzyme with a novel mode of action is expressed at the cell surface of C. albicans. Furthermore, recent studies have indicated that combinations of the chemical stresses generated by phagocytes can actively prevent C. albicans oxidative stress responses through a mechanism termed the stress pathway interference. In this review, we present an up-date of our current understanding of the role and regulation of oxidative stress responses in this important human fungal pathogen.

Small ubiquitin-related modifiers (SUMOs) are post-translationally conjugated to other proteins and are thereby essential regulators of a wide range of cellular processes. Sumoylation, and enzymes of the sumoylation pathway, are conserved in the malaria causing parasite, Plasmodium falciparum. However, the specific functions of sumoylation in P. falciparum, and the degree of functional conservation between enzymes of the human and P. falciparum sumoylation pathways, have not been characterized. Here, we demonstrate that sumoylation levels peak during midstages of the intra-erythrocyte developmental cycle, concomitant with hemoglobin consumption and elevated oxidative stress. In vitro studies revealed that P. falciparum E1- and E2-conjugating enzymes interact effectively to recognize and modify RanGAP1, a model mammalian SUMO substrate. However, in heterologous reactions, P. falciparum E1 and E2 enzymes failed to interact with cognate human E2 and E1 partners, respectively, to modify RanGAP1. Structural analysis, binding studies, and functional assays revealed divergent amino acid residues within the E1-E2 binding interface that define organism-specific enzyme interactions. Our studies identify sumoylation as a potentially important regulator of oxidative stress response during the P. falciparum intra-erythrocyte developmental cycle, and define E1 and E2 interactions as a promising target for development of parasite-specific inhibitors of sumoylation and parasite replication.

Candida albicans is the most frequently encountered fungal pathogen in humans, capable of causing mucocutaneous and systemic infections in immunocompromised individuals. C. albicans virulence is influenced by multiple factors. Importantly, iron acquisition and avoidance of the immune oxidative burst are two critical barriers for survival in the host. Prior studies using whole genome microarray expression data indicated that the CCAAT-binding factor is involved in the regulation of iron uptake/utilization and the oxidative stress response. This study examines directly the role of the CCAAT-binding factor in regulating the expression of oxidative stress genes in response to iron availability. The CCAAT-binding factor is a heterooligomeric transcription factor previously shown to regulate genes involved in respiration and iron uptake/utilization in C. albicans. Since these pathways directly influence the level of free radicals, it seemed plausible the CCAAT-binding factor regulates genes necessary for the oxidative stress response. In this study, we show the CCAAT-binding factor is involved in regulating some oxidative stress genes in response to iron availability, including CAT1, SOD4, GRX5, and TRX1. We also show that CAT1 expression and catalase activity correlate with the survival of C. albicans to oxidative stress, providing a connection between iron obtainability and the oxidative stress response. We further explore the role of the various CCAAT-binding factor subunits in the formation of distinct protein complexes that modulate the transcription of CAT1 in response to iron. We find that Hap31 and Hap32 can compensate for each other in the formation of an active transcriptional complex; however, they play distinct roles in the oxidative stress response during iron limitation. Moreover, Hap43 was found to be solely responsible for the repression observed under iron deprivation.