Oxidative stress alleviating potential of galactan exopolysaccharide from Weissella confusa KR780676 in yeast model system.

In the present study, galactan exopolysaccharide (EPS) from Weissella confusa KR780676 was evaluated for its potential to alleviate oxidative stress using in vitro assays and in vivo studies in Saccharomyces cerevisiae (wild type) and its antioxidant (sod1∆, sod2∆, tsa1∆, cta2∆ and ctt1∆), anti-apoptotic (pep4∆ and fis1∆) and anti-aging (sod2∆, tsa1∆ and ctt1∆)) isogenic gene deletion mutants. Galactan exhibited strong DPPH and nitric oxide scavenging activity with an IC50 value of 450 and 138 µg/mL respectively. In the yeast mutant model, oxidative stress generated by H2O2 was extensively scavenged by galactan in the medium as confirmed using spot assays followed by fluorescencent DCF-DA staining and microscopic studies. Galactan treatment resulted in reduction in the ROS generated in the yeast mutant cells as demonstrated by decreased fluorescence intensity. Furthermore, galactan exhibited protection against oxidative damage through H2O2 -induced apoptosis inhibition in the yeast mutant strains (pep4∆ and fis1∆) leading to increased survival rate by neutralizing the oxidative stress. In the chronological life span assay, WT cells treated with galactan EPS showed 8% increase in viability whereas sod2∆ mutant showed 10-15% increase indicating pronounced anti-aging effects. Galactan from W. confusa KR780676 has immense potential to be used as a natural antioxidant for nutraceutical, pharmaceutical and food technological applications. As per our knowledge, this is the first report on in-depth assessment of in vivo antioxidant properties of a bacterial EPS in a yeast deletion model system.

Nuclear envelope budding is a response to cellular stress.

Nuclear envelope budding (NEB) is a recently discovered alternative pathway for nucleocytoplasmic communication distinct from the movement of material through the nuclear pore complex. Through quantitative electron microscopy and tomography, we demonstrate how NEB is evolutionarily conserved from early protists to human cells. In the yeast Saccharomyces cerevisiae, NEB events occur with higher frequency during heat shock, upon exposure to arsenite or hydrogen peroxide, and when the proteasome is inhibited. Yeast cells treated with azetidine-2-carboxylic acid, a proline analog that induces protein misfolding, display the most dramatic increase in NEB, suggesting a causal link to protein quality control. This link was further supported by both localization of ubiquitin and Hsp104 to protein aggregates and NEB events, and the evolution of these structures during heat shock. We hypothesize that NEB is part of normal cellular physiology in a vast range of species and that in S. cerevisiae NEB comprises a stress response aiding the transport of protein aggregates across the nuclear envelope.

Aspirin Causes Lipid Accumulation and Damage to Cell Membrane by Regulating DCI1/OLE1 in Saccharomyces cerevisiae.

Aspirin is one of the most commonly used nonsteroidal anti-inflammatory drugs. Various potential pharmacological effects of aspirin, such as anticancer, antibacterial activity, and prolonging life expectancy have been discovered. However, the mechanism of aspirin is not fully elucidated. Herein, the effects of aspirin on fatty acid metabolism in yeast cell model Saccharomyces cerevisiae were studied. The results showed that aspirin can induce lipid accumulation and reduce the unsaturated fat index in cells. The assessment of cell membrane integrity demonstrated that aspirin caused damage to the cell membrane. These effects of aspirin were attributed to the alterations of the expression of DCI1 and OLE1. Similarly, aspirin was able to cause lipid accumulation and damage to the cell membrane by interfering with the expression of OLE1 in Candida albicans. These findings are expected to improve current understanding of the mode of action of aspirin and provide a novel strategy for antifungal drug design. Graphical abstract [Figure: see text].

Deletion of the Golgi Ca2+-ATPase PMR1 gene potentiates antifungal effects of dodecanol that depend on intracellular Ca2+ accumulation in budding yeast.

One strategy for overcoming infectious diseases caused by drug-resistant fungi involves combining drugs rendered inactive by resistance with agents targeting the drug resistance mechanism. The antifungal activity of n-dodecanol disappears as incubation time passes. In Saccharomyces cerevisiae, anethole, a principal component of anise oil, prolongs the transient antifungal effect of dodecanol by downregulating genes of multidrug efflux pumps, mainly PDR5. However, the detailed mechanisms of dodecanol's antifungal action and the anethole-induced prolonged antifungal action of dodecanol are unknown. Screening of S. cerevisiae strains lacking genes related to Ca2+ homeostasis and signaling identified a pmr1Δ strain lacking Golgi Ca2+-ATPase as more sensitive to dodecanol than the parental strain. Dodecanol and the dodecanol + anethole combination significantly increased intracellular Ca2+ levels in both strains, but the mutant failed to clear intracellular Ca2+ accumulation. Further, dodecanol and the drug combination reduced PMR1 expression and did not lead to specific localization of Pmr1p in the parental strain after 4-h treatment. By contrast with the parental strain, dodecanol did not stimulate PDR5 expression in pmr1Δ. Based on these observations, we propose that the antifungal activity of dodecanol is related to intracellular Ca2+ accumulation, possibly dependent on PMR1 function, with anethole enabling Ca2+ accumulation by restricting dodecanol efflux.

Effect of cadmium on essential metals and their impact on lipid metabolism in Saccharomyces cerevisiae.

Cadmium (Cd) is a toxic heavy metal that induces irregularity in numerous lipid metabolic pathways. Saccharomyces cerevisiae, a model to study lipid metabolism, has been used to establish the molecular basis of cellular responses to Cd toxicity in relation to essential minerals and lipid homeostasis. Multiple pathways sense these environmental stresses and trigger the mineral imbalances specifically calcium (Ca) and zinc (Zn). This review is aimed to elucidate the role of Cd toxicity in yeast, in three different perspectives: (1) elucidate stress response and its adaptation to Cd, (2) understand the physiological role of a macromolecule such as lipids, and (3) study the stress rescue mechanism. Here, we explored the impact of Cd interference on the essential minerals such as Zn and Ca and their influence on endoplasmic reticulum stress and lipid metabolism. Cd toxicity contributes to lipid droplet synthesis by activating OLE1 that is essential to alleviate lipotoxicity. In this review, we expanded our current findings about the effect of Cd on lipid metabolism of budding yeast.

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.

Molecular targets of biofabricated silver nanoparticles in Candida albicans.

We have analyzed the expressions of genes which regulate Ras-cAMP-EFG1 and CEK1-MAPK pathways involved in yeast to hyphal form morphogenesis in Candida albicans. The expression profile of genes associated with serum-induced morphogenesis showed reduced expressions of genes involved in these pathways by the treatment with biofabricated silver nanoparticles. Cell elongation gene, ECE1, was downregulated by 5.1 fold by the treatment of silver nanoparticles. Expression of hyphal inducer gene, TEC1 was downregulated by 6.28 fold. Negative regulators of yeast to hyphal transition, TUP1 and RFG1 were downregulated by 2.45 and 5.43 fold, respectively. Current study suggests that silver nanoparticles affect gene expression and may subsequently reduce virulence in C. albicans. Targeting genes involved in virulence may be an acceptable novel treatment strategy for pathogenic fungal infections.

Effect of selenium on growth and antioxidative system of yeast cells.

Selenium exhibits health-promoting properties in humans and animals. Therefore, the development of selenium-enriched dietary supplements has been growing worldwide. However, it may also exhibit toxicity at higher concentrations, causing increased oxidative stress. Different species of yeasts may exhibit different tolerances toward selenium. Therefore, in this study, we aimed to determine the effect of selenium on growth and on the antioxidative system in Candida utilis ATCC 9950 and Saccharomyces cerevisiae ATCC MYA-2200 yeast cells. The results of this study have demonstrated that high doses of selenium causes oxidative stress in yeasts, thereby increasing the process of lipid peroxidation. In addition, we obtained an increased level of GSSG from aqueous solutions of yeast biomass grown with selenium supplementation (40-60 mg/L). Increased levels of selenium in aqueous solutions resulted in an increase in the activity of antioxidant enzymes, including glutathione peroxidase and glutathione reductase. These results should encourage future research on the possibility of a thorough understanding of antioxidant system functioning in yeast cells.

Infección por Saccharomyces cerevisiae en un individuo inmunocompetente / Saccharomyces cerevisiae infection in an immunocompetent host

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Regulation of the inositol transporter Itr1p by hydrogen peroxide in Saccharomyces cerevisiae.

Myo-inositol is a precursor of several membrane phospholipids and sphingolipids and plays a key role in gene regulation in Saccharomyces cerevisiae (S. cerevisiae). Here, we tested whether H2O2 was affecting the levels of the inositol transporters and thus inositol uptake. In S. cerevisiae cells adapted to H2O2 Itr1-GFPp accumulated in the plasma membrane until 20 min, concomitantly with an inhibition of its internalization. Exposure to H2O2 did not alter Itr2-GFPp cellular levels and induced only an 8% decrease at 10 min in the plasma membrane. Therefore, decreased inositol intracellular levels are not caused by decreased levels of inositol transporters in the plasma membrane. However, results show that H2O2 adaptation affects Itr1p turnover and, consequently, H2O2-adapted yeast cells display an inositol transporter phenotype comparable to cells grown in the absence of inositol in growth medium, i.e. accumulation in the plasma membrane and decreased degradation.

Impact of the timing and the nature of nitrogen additions on the production kinetics of fermentative aromas by Saccharomyces cerevisiae during winemaking fermentation in synthetic media.

During alcoholic fermentation, many parameters, including the nitrogen composition of the must, can affect aroma production. The aim of this study was to examine the impact of several types of nitrogen sources added at different times during fermentation. Nitrogen was added as ammonium or a mixture of amino acids at the beginning of fermentation or at the start of the stationary phase. These conditions were tested with two Saccharomyces cerevisiae strains that have different nitrogen requirements. The additions systematically reduced the fermentation duration. The aroma production was impacted by both the timing of the addition and the composition of the nitrogen source. Propanol appeared to be a metabolic marker of the presence of assimilable nitrogen in the must. The production of ethyl esters was slightly higher after the addition of any type of nitrogen; the production of higher alcohols other than propanol was unchanged, and acetate esters were overproduced due to the overexpression of the genes ATF1 and ATF2. Finally the parameter affecting the most the synthesis of beneficial aromas was the addition timing: The supply of organic nitrogen at the beginning of the stationary phase was more favorable for the synthesis of beneficial aromas.

Synergistic Effect of Endogenous and Exogenous Aldehydes on Doxorubicin Toxicity in Yeast.

Anthracyclines are frequently used to treat many cancers including triple negative breast cancer, which is commonly observed in African-American women (AA), and tend to be more aggressive, carry worse prognoses, and are harder to manage because they lack molecular targets. Although effective, anthracyclines use can be limited by serious side effects and eventually the development of drug resistance. In S. cerevisiae, mutants of HOM6 display hypersensitivity to doxorubicin. HOM6 is required for synthesis of threonine and interruption of the pathway leads to accumulation of the threonine intermediate L-aspartate-semialdehyde. This intermediate may synergize with doxorubicin to kill the cell. In fact, deleting HOM3 in the first step, preventing the pathway to reach the HOM6 step, rescues the sensitivity of the hom6 strain to doxorubicin. Using several S. cerevisiae strains (wild type, hom6, hom3, hom3hom6, ydj1, siz1, and msh2), we determined their sensitivity to aldehydes and to their combination with doxorubicin, cisplatin, and etoposide. Combination of formaldehyde and doxorubicin was most effective at reducing cell survival by 31-fold-39-fold (in wild type cells) relative to doxorubicin and formaldehyde alone. This effect was dose dependent on doxorubicin. Cotreatment with formaldehyde and doxorubicin also showed increased toxicity in anthracycline-resistant strains siz1 and msh2. The hom6 mutant also showed sensitivity to menadione with a 2.5-fold reduction in cell survival. The potential use of a combination of aldehydes and cytotoxic drugs could potentially lead to applications intended to enhance anthracycline-based therapy.

Gene dosage effects in yeast support broader roles for the LOG1, HAM1 and DUT1 genes in detoxification of nucleotide analogues.

Purine and pyrimidine analogues have important uses in chemotherapies against cancer, and a better understanding of the mechanisms that cause resistance to these drugs is therefore of importance in cancer treatment. In the yeast Saccharomyces cerevisiae, overexpression of the HAM1 gene encoding inosine triphosphate pyrophosphatase confers resistance to both the purine analogue 6-N-hydroxylaminopurine (HAP) and the pyrimidine analogue 5-fluorouracil (5-FU) (Carlsson et al., 2013, PLoS One 8, e52094). To find out more about the mechanisms of resistance to nucleotide analogues, and possible interdependencies between purine and pyrimidine analogue resistance mechanisms, we screened a plasmid library in yeast for genes that confer HAP resistance when overexpressed. We cloned four such genes: ADE4, DUT1, APT2, and ATR1. We further looked for genetic interactions between these genes and genes previously found to confer resistance to 5-FU. We found that HMS1, LOG1 (YJL055W), HAM1, and ATR1 confer resistance to both 5-FU and HAP, whereas ADE4, DUT1 and APT2 are specific for HAP resistance, and CPA1 and CPA2 specific for 5-FU resistance. Possible mechanisms for 5-FU and HAP detoxification are discussed based on the observed genetic interactions. Based on the effect of LOG1 against both 5-FU and HAP toxicity, we propose that the original function of the LOG (LONELY GUY) family of proteins likely was to degrade non-canonical nucleotides, and that their role in cytokinin production is a later development in some organisms.

Histatin-5 induces the reversal of Pdr5p mediated fluconazole resistance in Saccharomyces cerevisae.

BACKGROUND: Candidiasis is a major opportunistic fungal infection in humans. The low number of antifungal drugs available to treat Candida infections and the increasing incidence of multidrug resistant (MDR) strains point to an urgent need of identifying new therapeutic options. The role of salivary components can provide insights for the development of new methodologies of control. OBJECTIVE: The aim of this study was to evaluate the ability of histatin-5, a constitutive immunological peptide present in saliva, in reversing fungal MDR phenotype, using a resistant Saccharomyces cerevisiae strain as model of study. RESULTS: A total of 2.5µg and 5µg of histatin-5 revealed to be able to chemosensitize (to revert antifungal resistance) a MDR strain to fluconazole impairing its intrinsic resistance. The presence of histatin-5 decreased the strain growth when associated to fluconazole, and also assisted in the retention of rhodamine 6G within cell cytoplasm. The ATPase activity of Pdr5p, an ABC efflux transporter, was significantly reduced up to 65% within physiological concentration of the peptide. CONCLUSION: Results revealed that histatin-5 is able to revert MDR phenotype and may be considered a potential alternative MDR inhibitor. Since Pdr5p is homologous to Candida albicans CaCdr1p and CaCdr2p, data obtained might be extrapolated to these transporters, inferring that associating fluconazole and histatin-5 may be a useful tool to circumvent failure treatments of infections caused by Candida MDR strains.

[Molecular bases of diseases caused by mutations in genes encoding subunits of ATP synthase]. / Molekularne podloze chorób spowodowanych mutacjami w genach kodujacych podjednostki syntazy ATP.

ATP synthase is the last enzyme of the OXPHOS system synthesizing ATP. Mutations in either mitochondrial or nuclear genes encoding subunits of this enzyme (17 polypeptides) cause neurodegenerative diseases. The ATP synthase subunits 8 (ATP8, alias A6L) and a (ATP6) are encoded by the MT-ATP8 and MT-ATP6 mitochondrial genes, respectively. 17 diseases associated mutations were identified in five nuclear genes coding for subunits of this enzyme. 58 mutations were described in the MT-ATP6 and MT-ATP8 genes, among them 36 were deposited in MITOMAP database. For most of them neither their pathogenic character nor the mechanisms are known. This review summarizes what is known about the molecular basis of the ATP synthase deficiencies. We review the mutations in the ATP synthase genes as well as biochemical data obtained from studies of patient's cells and cybrid or yeast models. We include yeast research about drugs selection and their mechanism of action. Moreover we position the mutations into a recently published structural model of the Fo complex and discuss their structural/functional consequences.

Opi1p translocation to the nucleus is regulated by hydrogen peroxide in Saccharomyces cerevisiae.

During exposure of yeast cells to low levels of hydrogen peroxide (H2 O2 ), the expression of several genes is regulated for cells to adapt to the surrounding oxidative environment. Such adaptation involves modification of plasma membrane lipid composition, reorganization of ergosterol-rich microdomains and altered gene expression of proteins involved in lipid and vesicle traffic, to decrease permeability to exogenous H2 O2 . Opi1p is a transcriptional repressor that is inactive when present at the nuclear membrane/endoplasmic reticulum, but represseses transcription of inositol upstream activating sequence (UASINO )-containing genes, many of which are involved in the synthesis of phospholipids and fatty acids, when it is translocated to the nucleus. We investigated whether H2 O2 in concentrations inducing adaptation regulates Opi1p function. We found that, in the presence of H2 O2 , GFP-Opi1p fusion protein translocates to the nucleus and, concomitantly, the expression of UASINO -containing genes is affected. We also investigated whether cysteine residues of Opi1p were implicated in the H2 O2 -mediated translocation of this protein to the nucleus and identified cysteine residue 159 as essential for this process. Our work shows that Opi1p is redox-regulated and establishes a new mechanism of gene regulation involving Opi1p, which is important for adaptation to H2 O2 in yeast cells. Copyright © 2017 John Wiley & Sons, Ltd.

TORC1-dependent sumoylation of Rpc82 promotes RNA polymerase III assembly and activity.

Maintaining cellular homeostasis under changing nutrient conditions is essential for the growth and development of all organisms. The mechanisms that maintain homeostasis upon loss of nutrient supply are not well understood. By mapping the SUMO proteome in Saccharomyces cerevisiae, we discovered a specific set of differentially sumoylated proteins mainly involved in transcription. RNA polymerase III (RNAPIII) components, including Rpc53, Rpc82, and Ret1, are particularly prominent nutrient-dependent SUMO targets. Nitrogen starvation, as well as direct inhibition of the master nutrient response regulator target of rapamycin complex 1 (TORC1), results in rapid desumoylation of these proteins, which is reflected by loss of SUMO at tRNA genes. TORC1-dependent sumoylation of Rpc82 in particular is required for robust tRNA transcription. Mechanistically, sumoylation of Rpc82 is important for assembly of the RNAPIII holoenzyme and recruitment of Rpc82 to tRNA genes. In conclusion, our data show that TORC1-dependent sumoylation of Rpc82 bolsters the transcriptional capacity of RNAPIII under optimal growth conditions.

Cell cycle-dependent positive and negative functions of Fun30 chromatin remodeler in DNA damage response.

The evolutionally conserved Fun30 chromatin remodeler in Saccharomyces cerevisiae has been shown to contribute to cellular resistance to genotoxic stress inflicted by camptothecin (CPT), methyl methanesulfonate (MMS) and hydroxyurea (HU). Fun30 aids in extensive DNA resection of DNA double stranded break (DSB) ends, which is thought to underlie its role in CPT-resistance. How Fun30 promotes MMS- or HU-resistance has not been resolved. Interestingly, we have recently found Fun30 to also play a negative role in cellular tolerance to MMS and HU in the absence of the Rad5-dependent DNA damage tolerance pathway. In this report, we show that Fun30 acts to down regulate Rad9-dependent DNA damage checkpoint triggered by CPT or MMS, but does not affect Rad9-independent intra-S phase replication checkpoint induced by MMS or HU. These results support the notion that Fun30 contributes to cellular response to DSBs by preventing excessive DNA damage checkpoint activation in addition to its role in facilitating DNA end resection. On the other hand, we present evidence suggesting that Fun30's negative function in MMS- and HU-tolerance in the absence of Rad5 is not related to its regulation of checkpoint activity. Moreover, we find Fun30 to be cell cycle regulated with its abundance peaking in G2/M phase of the cell cycle. Importantly, we demonstrate that artificially restricting Fun30 expression to G2/M does not affect its positive or negative function in genotoxin-resistance, but confining Fun30 to S phase abolishes its functions. These results indicate that both positive and negative functions of Fun30 in DNA damage response occur mainly in G2/M phase.

Macelignan inhibits bee pathogenic fungi Ascophaera apis growth through HOG1 pathway.

Ascosphaera apis is a bee pathogen that causes bee larvae infection disease, to which treatment is not yet well investigated. The aim of this study was to investigate antifungal susceptibility in vitro against A. apis and to identify a new antifungal agent for this pathogen through minimal inhibitory concentration (MIC) assay and western blot analysis. Macelignan had 1.56 and 3.125 µg/mL MIC against A. apis after 24 and 48 h, respectively, exhibiting the strongest growth inhibition against A. apis among the tested compounds (corosolic acid, dehydrocostus lactone, loganic acid, tracheloside, fangchinoline and emodin-8-O-ß-D-glucopyranoside). Furthermore, macelignan showed a narrow-ranged spectrum against various fungal strains without any mammalian cell cytotoxicity. In spite of miconazole having powerful broad-ranged anti-fungal activity including A. apis, it demonstrated strong cytotoxicity. Therefore, even if macelignan alone was effective as an antifungal agent to treat A. apis, combined treatment with miconazole was more useful to overcome toxicity, drug resistance occurrence and cost effectiveness. Finally, HOG1 was revealed as a target molecule of macelignan in the anti-A. apis activity by inhibiting phosphorylation using S. cerevisiae as a model system. Based on our results, macelignan, a food-grade antimicrobial compound, would be an effective antifungal agent against A. apis infection in bees.

Macelignan inhibits bee pathogenic fungi Ascophaera apis growth through HOG1 pathway

Ascosphaera apis is a bee pathogen that causes bee larvae infection disease, to which treatment is not yet well investigated. The aim of this study was to investigate antifungal susceptibility in vitro against A. apis and to identify a new antifungal agent for this pathogen through minimal inhibitory concentration (MIC) assay and western blot analysis. Macelignan had 1.56 and 3.125 μg/mL MIC against A. apis after 24 and 48 h, respectively, exhibiting the strongest growth inhibition against A. apis among the tested compounds (corosolic acid, dehydrocostus lactone, loganic acid, tracheloside, fangchinoline and emodin-8-O-β-D-glucopyranoside). Furthermore, macelignan showed a narrow-ranged spectrum against various fungal strains without any mammalian cell cytotoxicity. In spite of miconazole having powerful broad-ranged anti-fungal activity including A. apis, it demonstrated strong cytotoxicity. Therefore, even if macelignan alone was effective as an antifungal agent to treat A. apis, combined treatment with miconazole was more useful to overcome toxicity, drug resistance occurrence and cost effectiveness. Finally, HOG1 was revealed as a target molecule of macelignan in the anti-A. apis activity by inhibiting phosphorylation using S. cerevisiae as a model system. Based on our results, macelignan, a food-grade antimicrobial compound, would be an effective antifungal agent against A. apis infection in bees.