Identification of the ergC gene involved in polyene drug sensitivity in the Mucorales species Phycomyces blakesleeanus.

BACKGROUND: A strain of Phycomyces blakesleeanus (Mucorales, Mucoromycota) that was previously isolated after ultraviolet mutagenesis has altered responses to polyene antifungal drugs, sterol profiles, and phototropism of its sporangia. In this study, the genetic basis for these changes was sought. METHODS AND RESULTS: Two base pair substitutions were identified in the mutant within a P. blakelesleeanus gene that is homologous to others characterized from fungi, such as the Saccharomyces cerevisiae ERG3 gene, encoding sterol Δ5,6-desaturase. The polyene resistance and growth reduction phenotypes co-segregated with mutations in the gene in genetic crosses. The P. blakelesleeanus wild type ergC gene complemented a S. cerevisiae deletion strain of ERG3. CONCLUSIONS: This gene discovery may contribute towards better antifungal use in treating mucormycoses diseases caused by related species in the order Mucorales.

Inhibitory effect of berberine hydrochloride against Candida albicans and the role of the HOG-MAPK pathway.

Berberine hydrochloride (BH), an active component of Coptis chinensis and other plant taxa, has broad antimicrobial activity and may be useful for the treatment of Candida infections. In this study, the mechanisms underlying the inhibitory effect of BH against Candida albicans were evaluated, with a focus on the high-osmolarity glycerol mitogen-activated protein kinase (HOG-MAPK) pathway, which regulates multiple physiological functions. BH (256 and 64 µg ml-1) significantly increased intracellular glycerol and ROS levels in C. albicans, inhibited germ tube and hyphal formation, and increased chitin and ß-1,3-glucan exposure on the cell wall. The inhibitory effect of BH was positively correlated with its concentration, and the inhibitory effect of 256 µg ml-1 BH was greater than that of 4 µg ml-1 fluconazole (FLC). Furthermore, RT-PCR analysis showed that 256 and 64 µg ml-1 BH altered the HOG-MAPK pathway in C. albicans. In particular, the upregulation of the core genes, SLN1, SSK2, HOG1, and PBS2 may affect the expression of key downstream factors related to glycerol synthesis and osmotic pressure (GPD1), ROS accumulation (ATP11 and SOD2), germ tube and hyphal formation (HWP1), and cell wall integrity (CHS3 and GSC1). BH affects multiple biological processes in C. albicans; thus, it can be an effective alternative to conventional azole antifungal agents.

Antifungal Activity of Quinofumelin against Fusarium graminearum and Its Inhibitory Effect on DON Biosynthesis.

Fusarium graminearum, causal agent of Fusarium head blight (FHB), causes a huge economic loss. No information is available on the activity of quinofumelin, a novel quinoline fungicide, against F. graminearum or other phytopathogens. In this study, we used mycelial growth and spore germination inhibition methods to determine the inhibitory effect of quinofumelin against F. graminearum in vitro. The results indicated that quinofumelin excellently inhibited mycelial growth and spore germination of F. graminearum, with the average EC50 values of 0.019 ± 0.007 µg/mL and 0.087 ± 0.024 µg/mL, respectively. In addition, we found that quinofumelin could significantly decrease deoxynivalenol (DON) production and inhibit the expression of DON-related gene TRI5 in F. graminearum. Furthermore, we found that quinofumelin could disrupt the formation of Fusarium toxisome, a structure for producing DON. Western blot analysis demonstrated that the translation level of TRI1, a marker gene for Fusarium toxisome, was suppressed by quinofumelin. The protective and curative assays indicated that quinofumelin had an excellent control efficiency against F. graminearum on wheat coleoptiles. Taken together, quinofumelin exhibits not only an excellent antifungal activity on mycelial growth and spore germination, but also could inhibit DON biosynthesis in F. graminearum. The findings provide a novel candidate for controlling FHB caused by F. graminearum.

SIGS vs HIGS: a study on the efficacy of two dsRNA delivery strategies to silence Fusarium FgCYP51 genes in infected host and non-host plants.

CYP3RNA, a double-stranded (ds)RNA designed to concomitantly target the two sterol 14α-demethylase genes FgCYP51A and FgCYP51B and the fungal virulence factor FgCYP51C, inhibits the growth of the ascomycete fungus Fusarium graminearum (Fg) in vitro and in planta. Here we compare two different methods (setups) of dsRNA delivery, viz. transgene expression (host-induced gene silencing, HIGS) and spray application (spray-induced gene silencing, SIGS), to assess the activity of CYP3RNA and novel dsRNA species designed to target one or two FgCYP51 genes. Using Arabidopsis and barley, we found that dsRNA designed to target two FgCYP51 genes inhibited fungal growth more efficiently than dsRNA targeting a single gene, although both dsRNA species reduced fungal infection. Either dsRNA delivery method reduced fungal growth stronger than anticipated from previous mutational knock-out (KO) strategies, where single gene KO had no significant effect on fungal viability. Consistent with the strong inhibitory effects of the dsRNAs on fungal development in both setups, we detected to a large extent dsRNA-mediated co-silencing of respective non-target FgCYP51 genes. Together, our data further support the valuation that dsRNA applications have an interesting potential for pesticide target validation and gene function studies, apart from their potential for crop protection.

Integrated transcriptomic and proteomic analysis of the ethanol stress response in Saccharomyces cerevisiae Sc131.

The molecular mechanism of Saccharomyces cerevisiae tolerant to ethanol stress remains to be further elucidated. In this study, a comprehensive analysis based on RNA-seq and iTRAQ LC-MS/MS was used to investigate the global mechanism of S. cerevisiae strain Sc131 in response to ethanol stress at transcriptomic and proteomic levels. Totally, 937 differentially expressed genes (DEGs) and 457 differentially expressed proteins (DEPs) were identified in Sc131 under ethanol stress. Results revealed that 4-h ethanol stress (10% v/v) can induce filamentous growth, sexual reproduction. Mitochondria and endoplasmic reticulum (ER) were proved to be two important organelles in resisting ethanol stress. Signal transduction such as G-protein coupled receptor signaling and metal ion regulation were remarkably activated at the presence of ethanol. Moreover, silent information regulator (Sir) proteins and aromatic amino acids especially tryptophan were involved in response to ethanol and might be helpful for cell survival. This study provides a global perspective on the mechanism of S. cerevisiae tolerant to ethanol stress and sheds light on the potential application of Sc131 in Chinese bayberry wine brewing. BIOLOGICAL SIGNIFICANCE: It is of great importance for S. cerevisiae to tolerate high levels of ethanol during wine fermentation. However, the molecular mechanism of S. cerevisiae tolerant to ethanol stress remains to be further elucidated at transcriptomic and proteomic levels. In present study, we employed a comprehensive analysis based on RNA-seq and iTRAQ and found several potential pathways involving in the response of Sc131 to ethanol stress. To our knowledge, this is the first integrated analysis combining transcriptomic and proteomic technologies to study the mechanism of Sc131 under ethanol stress.

Blue light exposure and nutrient conditions influence the expression of genes involved in simultaneous hyphal knot formation in Coprinopsis cinerea.

Light and nutrients are crucial environmental factors influencing fungal sexual reproduction. Blue light induces simultaneous hyphal knot formation in Coprinopsis cinerea mycelia grown on low-glucose media but not in mycelia grown on high-glucose media. Many hyphal knots are visible in the arc near the edge of the colony one day after 15 min of blue light stimulation. These findings collectively suggest that blue light accelerates hyphal knot induction in nutrient-limited conditions. Transcriptome analysis revealed that gene expression after light exposure is divided into at least two major stages. In the first stage, genes coding for fasciclin (fas1), cyclopropane-fatty-acyl-phospholipid synthases (cfs1 and cfs2), and putative lipid exporter (nod1) are highly expressed after 1 h of light exposure in the mycelial region where the hyphal knot will be developed. These genes are upregulated by blue light and not influenced by glucose condition and mating. These results suggest that although some of the genes are critical for induction of the hyphal knots, they are not sufficient for hyphal knot development. In the second gene expression stage, genes encoding galectins (cgl1-3), farnesyl cysteine-carboxyl methyltransferases, mating pheromone-containing protein, nucleus protein (ich1), and laccase (lcc1) are specifically upregulated at 10-16 h after blue light exposure when the mycelia are cultivated on low-glucose media. These genes might be involved in the architecture of hyphal knots or signal transduction for further fruiting body development. These results contribute to the understanding of the effect of environmental factors on sexual reproduction in basidiomycetous fungi.

Effect of Cinnamaldehyde on Morphological Alterations of Aspergillus ochraceus and Expression of Key Genes Involved in Ochratoxin A Biosynthesis.

Ochratoxin A (OTA) is a potent nephrotoxic, hepatotoxic, and teratogenic compound which is a significant mycotoxin contaminates cereals during storage. Aspergillus ochraceus is the most common producer of OTA in cereals and cereal-derived products. Cinnamaldehyde is a natural substance derived from plant cinnamon playing an important role in the reduction of OTA contamination. In this study, the antifungal and antitoxigenic effect of cinnamaldehyde was investigated with its mechanisms of inhibition of fungal growth at the morphological and ultrastructural levels, and inhibition of OTA biosynthesis at the transcriptional level. Significant A. ochraceus growth was inhibited at 0.4⁻1.6 mmol/L with fumigation. A. ochraceus exposed to 0.4 mmol/L of cinnamaldehyde indicated irreversible harmful morphological and ultrastructural modifications such as the folding of the cell, the loss of integrity of the cell wall, the disruption of plasma membrane, the destruction of the mitochondria, and the absence of intracellular organelles. These alterations may be attributed to its inhibition of enzymatic reactions that regulate cell wall synthesis, thus disturbing the morphogenesis and growth of A. ochraceus. In the presence of cinnamaldehyde, the tested biosynthetic and regulatory genes like pks, nrps, veA, laeA and velB were highly downregulated. Moreover, the downregulation effect of cinnamaldehyde increased proportionally with the concentrations. These results suggest that the decrease of OTA production by cinnamaldehyde is attributed to the downregulation of the transcriptional levels of OTA biosynthetic and regulatory genes besides the inhibition of fungal growth. The study reveals the mechanisms of the antifungal and antitoxigenic activities of cinnamaldehyde against A. ochraceus, and further emphasizes that cinnamaldehyde could be a safe and effective natural agents against OTA contamination during cereals storage.

Selection of reliable reference genes for RT-qPCR during methyl jasmonate, salicylic acid and hydrogen peroxide treatments in Ganoderma lucidum.

This study aimed to identify suitable reference genes under three chemical inducers, methyl jasmonate (MeJA), salicylic acid (SA) and hydrogen peroxide (H2O2) in Ganoderma lucidum. In this study, expression stabilities of 14 candidate reference genes had been validated. Four algorithms were used: geNorm, NormFinder, BestKeeper, and RefFinder. Our results showed that, in short time, UCE2 (ubiquitin conjugating enzyme) was the most stable gene both in MeJA and H2O2 treatments, ACTIN (beta-actin) was the most suitable reference gene for SA treatment. ACTIN/UCE2 were considered the most suitable genes to normalize in MeJA, SA and H2O2 conditions. In long time, PP2A (protein phosphatase 2A regulatory subunit) was the most stable gene in MeJA and SA treatments, UCE2 was the most suitable reference gene for H2O2 treatment. PP2A/UBQ1 (polyubiquitin 1) were considered the most suitable genes to normalize in MeJA, SA and H2O2 conditions. Furthermore, target gene, oxidosqualene cyclase (osc), was selected to validate the most and least stable reference genes under different treatments. Our work provided a better support to study the regulatory mechanism of MeJA, SA and H2O2 on biological functions.

Comparative analysis of transcriptomic responses to sub-lethal levels of six environmentally relevant pesticides in Saccharomyces cerevisiae.

Accidental spills and misuse of pesticides may lead to current and/or legacy environmental contamination and may pose concerns regarding possible risks towards non-target microbes and higher eukaryotes in ecosystems. The present study was aimed at comparing transcriptomic responses to effects of sub-lethal levels of six environmentally relevant pesticide active substances in the Saccharomyces cerevisiae eukaryotic model. The insecticide carbofuran, the fungicide pyrimethanil and the herbicides alachlor, S-metolachlor, diuron and methyl(4-chloro-2-methylphenoxy)acetate were studied. Some are currently used agricultural pesticides, while others are under restricted utilization or banned in Europe and/or North America albeit being used in other geographical locations. In the present work transcriptional profiles representing genome-wide responses in a standardized yeast population upon 2 h of exposure to concentrations of each compound exerting equivalent toxic effects, i.e., inhibition of growth by 20% relative to the untreated control cells, were examined. Hierarchical clustering and Venn analyses of the datasets of differentially expressed genes pointed out transcriptional patterns distinguishable between the six active substances. Functional enrichment analyses allowed predicting mechanisms of pesticide toxicity and response to pesticide stress in the yeast model. In general, variations in transcript numbers of selected genes assessed by Real-Time quantitative reverse transcription polymerase chain reaction confirmed microarray data and correlated well with growth inhibitory effects. A possible biological relevance of mechanistic predictions arising from these comparative transcriptomic analyses is discussed in the context of better understanding potential modes of action and adverse side-effects of pesticides.

Development of Candida albicans Biofilms Is Diminished by Paeonia lactiflora via Obstruction of Cell Adhesion and Cell Lysis.

Candida albicans infections are often problematic to treat owing to antifungal resistance, as such infections are mostly associated with biofilms. The ability of C. albicans to switch from a budding yeast to filamentous hyphae and to adhere to host cells or various surfaces supports biofilm formation. Previously, the ethanol extract from Paeonia lactiflora was reported to inhibit cell wall synthesis and cause depolarization and permeabilization of the cell membrane in C. albicans. In this study, the P. lactiflora extract was found to significantly reduce the initial stage of C. albicans biofilms from 12 clinical isolates by 38.4%. Thus, to assess the action mechanism, the effect of the P. lactiflora extract on the adhesion of C. albicans cells to polystyrene and germ tube formation was investigated using a microscopic analysis. The density of the adherent cells was diminished following incubation with the P. lactiflora extract in an acidic medium. Additionally, the P. lactiflora-treated C. albicans cells were mostly composed of less virulent pseudohyphae, and ruptured debris was found in the serum-containing medium. A quantitative real-time PCR analysis indicated that P. lactiflora downregulated the expression of C. albicans hypha-specific genes: ALS3 by 65% (p = 0.004), ECE1 by 34.9% (p = 0.001), HWP1 by 29.2% (p = 0.002), and SAP1 by 37.5% (p = 0.001), matching the microscopic analysis of the P. lactiflora action on biofilm formation. Therefore, the current findings demonstrate that the P. lactiflora ethanol extract is effective in inhibiting C. albicans biofilms in vitro, suggesting its therapeutic potential for the treatment of biofilm-associated infections.

Transcriptome analysis reveals novel insights into the response to Pb exposure in Phanerochaete chrysosporium.

Metals released into the environment continue to be of concern for human health. Using white-rot fungi as biosorbents for heavy metals removal is an attractive alternative owing to its good performance and low cost. However, the molecular mechanism underlying heavy metal tolerance in white-rot fungi has not yet been fully elucidated. This study identified and analyzed the lead (Pb)-induced transcriptional changes in Phanerochaete chrysosporium, a well-known heavy metal hyperaccumulating white-rot fungus. The results confirmed its outstanding ability in Pb tolerance and effective defense system. By comparative analysis of gene expression profiles obtained from cDNA-amplified fragment length polymorphism (cDNA-AFLP), we isolated 43 transcript-derived fragments (TDFs) differentially regulated by Pb exposure in P. chrysosporium, and 23 TDFs presented significant similarities to genes encoding known or putative proteins which belong to different functional categories involving ion binding, energy and carbohydrate metabolism, and signal transduction. The detailed characterization of these Pb-responsive genes was presented and the expression patterns of some interesting genes were validated by quantitative RT-PCR. This work provides the first evidence of Pb-responsive genes along with their putatively functional annotations in P. chrysosporium, which may help to understand the mechanism underlying heavy metal accumulation and tolerance in P. chrysosporium.

In vitro and in vivo antifungal efficacy of plant based lawsone against Fusarium oxysporum species complex.

Fusarium oxysporum is an ascomycete facultative fungus which generally affects to plants. However, it is recently known as a serious emerging opportunistic pathogen of human and other animals. F. oxysporum shows broad resistance to commonly used antifungal agents and therefore development of alternative therapeutic agents is required. In this study, we investigated the antifungal efficacy of plant based natural lawsone against pathogenic F. oxysporum. Antifungal susceptibility test determined the concentration dependent growth inhibition of lawsone against F. oxysporum with minimum inhibitory concentration (MIC) at 100µg/mL. Ultra-structural analysis indicates the prominent damage on cell wall of the mycelium after lawsone treatment, and suggests that it could increase the membrane permeability and disintegration of cells leading to cellular death. Propidium iodide (PI) uptake assay results showed the higher level of cell death in lawsone treated F. oxysporum which further confirms the loss of plasma membrane integrity. Also, detection of reactive oxygen species (ROS) using DCFH-DA has clearly indicated that lawsone (100µg/mL) can induce the ROS level in the filaments of F. oxysporum. MTT assay results showed the loss of viability and germination capacity of F. oxysporum spores by lawsone in concentration dependent manner. Moreover, lawsone treatment induced the mRNA expression of two autophagy related genes (ATG1 and ATG8) indicating that lawsone may activate the autophagy related pathways in F. oxysporum due to the oxidative stress generated by ROS. F. oxysporum infected zebrafish has recovered after lawsone therapy as a topical treatment suggesting that lawsone is a potential natural antifusariosis agent.

Overexpression of OLE1 Enhances Cytoplasmic Membrane Stability and Confers Resistance to Cadmium in Saccharomyces cerevisiae.

The heavy metal cadmium is widely used and released into the environment, posing a severe threat to crops and humans. Saccharomyces cerevisiae is one of the most commonly used organisms in the investigation of environmental metal toxicity. We investigated cadmium stress and the adaptive mechanisms of yeast by screening a genome-wide essential gene overexpression library. A candidate gene, OLE1, encodes a delta-9 desaturase and was associated with high anti-cadmium-stress activity. The results demonstrated that the expression of OLE1 was positively correlated with cadmium stress tolerance and induction was independent of Mga2p and Spt23p (important regulatory factors for OLE1). Moreover, in response to cadmium stress, cellular levels of monounsaturated fatty acids were increased. The addition of exogenous unsaturated fatty acids simulated overexpression of OLE1, leading to cadmium resistance. Such regulation of OLE1 in the synthesis of unsaturated fatty acids may serve as a positive feedback mechanism to help cells counter the lipid peroxidation and cytoplasmic membrane damage caused by cadmium. IMPORTANCE: A S. cerevisiae gene encoding a delta-9 desaturase, OLE1, was associated with high anti-cadmium-stress activity. The data suggest that the regulation of OLE1 in the synthesis of unsaturated fatty acids may serve as a positive feedback mechanism to help yeast cells counter the lipid peroxidation and cytoplasmic membrane damage caused by cadmium. The discovery of OLE1 involvement in membrane stability may indicate a novel defense strategy against cadmium stress.

Antifungal activity, kinetics and molecular mechanism of action of garlic oil against Candida albicans.

The antifungal activity, kinetics, and molecular mechanism of action of garlic oil against Candida albicans were investigated in this study using multiple methods. Using the poisoned food technique, we determined that the minimum inhibitory concentration of garlic oil was 0.35 µg/mL. Observation by transmission electron microscopy indicated that garlic oil could penetrate the cellular membrane of C. albicans as well as the membranes of organelles such as the mitochondria, resulting in organelle destruction and ultimately cell death. RNA sequencing analysis showed that garlic oil induced differential expression of critical genes including those involved in oxidation-reduction processes, pathogenesis, and cellular response to drugs and starvation. Moreover, the differentially expressed genes were mainly clustered in 19 KEGG pathways, representing vital cellular processes such as oxidative phosphorylation, the spliceosome, the cell cycle, and protein processing in the endoplasmic reticulum. In addition, four upregulated proteins selected after two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE) analysis were identified with high probability by mass spectrometry as putative cytoplasmic adenylate kinase, pyruvate decarboxylase, hexokinase, and heat shock proteins. This is suggestive of a C. albicans stress responses to garlic oil treatment. On the other hand, a large number of proteins were downregulated, leading to significant disruption of the normal metabolism and physical functions of C. albicans.

New-to-nature sophorose analog: a potent inducer for gene expression in Trichoderma reesei.

Controlled hydrolysis of lactonic sophorolipids from Starmerella bombicola yields a previously undescribed sophorose analog that potently induces cellulase in Trichoderma reesei Rut-C30. Acid treatment of natural sophorolipids results in a mixture of monoacetylated, deacetylated, and diacetylated sophorolipids in acidic and lactonic forms. Isolation of the active components of the mixture, followed by structure determination by MS and NMR, reveals a new chemical entity, in which the lactone ring has been opened at the C-1' rather than at the C-4″ position of the sophorose moiety. This sophorose ester is resistant to degradation by the host and is at least 28 times more powerful an inducer than sophorose in shake-flask culture. Even at low concentrations (0.05 mM), the chemically modified sophorolipid effectively induces cellulase. With further improvements, this highly enabling technology can potentially reduce the cost of enzymes produced in T. reesei and can facilitate the rapid deployment of enzyme plants to support the nascent cellulosic biofuels and biochemicals industries.

Lysine enhances the effect of amphotericin B against Candida albicans in vitro.

Amphotericin B (AmB) is a polyene antibiotic produced by Streptomyces nodosus and has been used for >50 years in the treatment of acute systemic fungal infections. In the present study, we demonstrated that lysine, an essential amino acid, could enhance the effect of AmB against Candida albicans in vitro, although lysine itself did not exert a fungicidal effect. In addition, the combination of AmB with lysine could provide an enhanced action against Candida parapsilosis and Cryptococcus neoformans compared with AmB alone. Lysine could also enhance the antifungal effect of caspofungin or nystatin. An enhanced effect of the combination of lysine with AmB was observed for the prevention of biofilm and hypha formation. Furthermore, our results demonstrated that lysine-mediated oxidative damage, such as the generation of endogenous reactive oxygen species, may be the mechanism underlying the enhancing effect of lysine on AmB. Our results also showed that CaMCA1 gene plays an important role in increasing the sensitivity of C. albicans cells upon AmB treatment. Using AmB together with lysine may be a promising strategy for the therapy of disseminated candidiasis.

Inhibitory effect of eugenol on aflatoxin B1 production in Aspergillus parasiticus by downregulating the expression of major genes in the toxin biosynthetic pathway.

Aflatoxin contamination of grains and agro-products is a serious food safety issue and a significant economic concern worldwide. In the present study, the effects of eugenol on Aspergillus parasiticus growth and aflatoxin production were studied in relation to the expression of some essential genes involved in aflatoxin biosynthetic pathway. The fungus was cultured in presence of serial two-fold concentrations of eugenol (15.62-500 µg mL(-1)) for 3 days at 28 °C. Mycelia dry weight was determined as an index of fungal growth, while aflatoxin production was assessed by high performance liquid chromatography. The expression of aflatoxin biosynthetic genes including ver-1, nor-1, pksA, omtA and aflR were evaluated by real-time PCR. Eugenol strongly inhibited A. parasiticus growth in the range of 19.16-95.83 % in a dose-dependent manner. Aflatoxin B1 production was also inhibited by the compound in the range of 15.07-98.0 %. The expressions of ver-1, nor-1, pksA, omtA and aflR genes were significantly suppressed by eugenol at concentrations of 62.5 and 125 µg mL(-1). These results indicate that eugenol may be considered as a good candidate to control toxigenic fungal growth and the subsequent contamination of food, feed and agricultural commodities by carcinogenic aflatoxins.

Artemisinin mimics calorie restriction to extend yeast lifespan via a dual-phase mode: a conclusion drawn from global transcriptome profiling.

Calorie restriction (CR) promotes longevity among distinct organisms from yeast to mammals. Although CR-prolonged lifespan is believed to associate with enhanced respiratory activity, it is apparently controversial for accelerated energy consumption regardless of insufficient nutrient intake. In reconciling the contradiction of less food supply versus much metabolite dispense, we revealed a CR-based mode of dual-phase responses that encompass a phase of mitochondrial enhancement (ME) and a phase of post-mitochondrial enhancement (PME), which can be distinguished by the expression patterns and activity dynamics of mitochondrial signatures. ME is characterized by global antioxidative activation, and PME is denoted by systemic metabolic modulation. CR-mediated aging-delaying effects are replicated by artesunate, a semi-synthetic derivative of the antimalarial artemisinin that can alkylate heme-containing proteins, suggesting artesunate-heme conjugation functionally resembles nitric oxide-heme interaction. A correlation of artesunate-heme conjugation with cytochrome c oxidase activation has been established from adduct formation and activity alteration. Exogenous hydrogen peroxide also mimics CR to trigger antioxidant responses, affect signaling cascades, and alter respiratory rhythms, implying hydrogen peroxide is engaged in lifespan extension. Conclusively, artesunate mimics CR-triggered nitric oxide and hydrogen peroxide to induce antioxidative networks for scavenging reactive oxygen species and mitigating oxidative stress, thereby directing metabolic conversion from anabolism to catabolism, maintaining essential metabolic functionality, and extending life expectancy in yeast.

Iron binding modulates candidacidal properties of salivary histatin 5.

Salivary protein histatin 5 (Hst 5) is fungicidal toward Candida albicans, the causative agent of oropharyngeal candidiasis. However, its activity in saliva is compromised by salivary protease-mediated degradation and interaction with salivary salts. Hst 5 has also been shown to bind various metals in saliva-namely, Zn, Cu, and Ni. Surprisingly, interactions of Hst 5 with Fe have not been studied, although iron is one of the most abundant metals present in saliva. Using circular dichroism, we show that Hst 5 can bind up to 10 equivalents of iron as measured by loss of its alpha-helical secondary structure that is normally observed for it in trifluoroethylene. A significant decrease in the candidacidal ability of Hst 5 was observed upon iron binding, with increasing iron concentrations being inversely proportional to Hst 5 killing activity. Binding assays showed that the decrease in killing was likely a result of reduced binding (10-fold reduction) of Fe-Hst 5 to C. albicans cells. Protease stability analysis showed that Fe-Hst 5 was completely resistant to trypsin digestion. In contrast, zinc binding had limited effects on Hst 5 fungicidal activity or protease susceptibility. RNA sequencing results identified changes in iron uptake genes in Hst 5-treated C. albicans cells. Our findings thus suggest that consequences of Hst 5 binding iron not only affect candidacidal ability and proteolyic stability of Hst 5, but may also contribute to a novel killing mechanism involving interference with cellular iron metabolism.

[Molecular biological approach to screening essential genes as potential targets for antifungal targets in pathogenic yeast Candida].

  We established a system, named ETS system, for screening and identification of essential genes from the pathogenic haploid yeast Candida glabrata by using temperature-sensitive (ts) mutants. Based on the general concepts that ts mutations are generated within essential genes in the genome by virtue of point mutation, the ETS system enabled us to screen and identify a variety of essential genes from the C. glabrata genomic DNA library as the genes that complement ts mutations. The ETS system established in the present study may provide novel potential antifungal targets.