An oxylipin signal confers protection against antifungal echinocandins in pathogenic aspergilli.

Aspergillus fumigatus is the leading causative agent of life-threatening invasive aspergillosis in immunocompromised individuals. One antifungal class used to treat Aspergillus infections is the fungistatic echinocandins, semisynthetic drugs derived from naturally occurring fungal lipopeptides. By inhibiting beta-1,3-glucan synthesis, echinocandins cause both fungistatic stunting of hyphal growth and repeated fungicidal lysis of apical tip compartments. Here, we uncover an endogenous mechanism of echinocandin tolerance in A. fumigatus whereby the inducible oxylipin signal 5,8-diHODE confers protection against tip lysis via the transcription factor ZfpA. Treatment of A. fumigatus with echinocandins induces 5,8-diHODE synthesis by the fungal oxygenase PpoA in a ZfpA dependent manner resulting in a positive feedback loop. This protective 5,8-diHODE/ZfpA signaling relay is conserved among diverse isolates of A. fumigatus and in two other Aspergillus pathogens. Our findings reveal an oxylipin-directed growth program-possibly arisen through natural encounters with native echinocandin producing fungi-that enables echinocandin tolerance in pathogenic aspergilli.

Comparative transcriptome analysis provides insights into the resistance regulation mechanism and inhibitory effect of fungicide phenamacril in Fusarium asiaticum.

Fusarium asiaticum is a destructive phytopathogenic fungus that causes Fusarium head blight of wheat (FHB), leading to serious yield and economic losses to cereal crops worldwide. Our previous studies indicated that target-site mutations (K216R/E, S217P/L, or E420K/G/D) of Type I myosin FaMyo5 conferred high resistance to phenamacril. Here, we first constructed one sensitive strain H1S and three point mutation resistant strains HA, HC and H1R. Then we conducted comparative transcriptome analysis of these F. asiaticum strains after 1 and 10 µg·mL-1 phenamacril treatment. Results indicated that 2135 genes were differentially expressed (DEGs) among the sensitive and resistant strains. The DEGs encoding ammonium transporter MEP1/MEP2, nitrate reductase, copper amine oxidase 1, 4-aminobutyrate aminotransferase, amino-acid permease inda1, succinate-semialdehyde dehydrogenase, 2, 3-dihydroxybenzoic acid decarboxylase, etc., were significantly up-regulated in all the phenamacril-resistant strains. Compared to the control group, a total of 1778 and 2097 DEGs were identified in these strains after 1 and 10 µg·mL-1 phenamacril treatment, respectively. These DEGs involved in 4-aminobutyrate aminotransferase, chitin synthase 1, multiprotein-bridging factor 1, transcriptional regulatory protein pro-1, amino-acid permease inda1, ATP-dependent RNA helicase DED1, acetyl-coenzyme A synthetase, sarcoplasmic/endoplasmic reticulum calcium ATPase 2, etc., showed significantly down-regulated expression in phenamacril-sensitive strain but not in resistant strains after phenamacril treatment. In addition, cyanide hydratase, mating-type protein MAT-1, putative purine nucleoside permease, plasma membrane protein yro2, etc., showed significantly co-down-regulated expression in all the strains after phenamacril treatment. Taken together, This study provides deep insights into the resistance regulation mechanism and the inhibitory effect of fungicide phenamacril and these new annotated proteins or enzymes are worth for the discovery of new fungicide targets.

A secondary mechanism of action for triazole antifungals in Aspergillus fumigatus mediated by hmg1.

Triazole antifungals function as ergosterol biosynthesis inhibitors and are frontline therapy for invasive fungal infections, such as invasive aspergillosis. The primary mechanism of action of triazoles is through the specific inhibition of a cytochrome P450 14-α-sterol demethylase enzyme, Cyp51A/B, resulting in depletion of cellular ergosterol. Here, we uncover a clinically relevant secondary mechanism of action for triazoles within the ergosterol biosynthesis pathway. We provide evidence that triazole-mediated inhibition of Cyp51A/B activity generates sterol intermediate perturbations that are likely decoded by the sterol sensing functions of HMG-CoA reductase and Insulin-Induced Gene orthologs as increased pathway activity. This, in turn, results in negative feedback regulation of HMG-CoA reductase, the rate-limiting step of sterol biosynthesis. We also provide evidence that HMG-CoA reductase sterol sensing domain mutations previously identified as generating resistance in clinical isolates of Aspergillus fumigatus partially disrupt this triazole-induced feedback. Therefore, our data point to a secondary mechanism of action for the triazoles: induction of HMG-CoA reductase negative feedback for downregulation of ergosterol biosynthesis pathway activity. Abrogation of this feedback through acquired mutations in the HMG-CoA reductase sterol sensing domain diminishes triazole antifungal activity against fungal pathogens and underpins HMG-CoA reductase-mediated resistance.

Studies on the biological role of the antifungal protein PeAfpA from Penicillium expansum by functional gene characterization and transcriptomic profiling.

Antifungal proteins (AFPs) from filamentous fungi have enormous potential as novel biomolecules for the control of fungal diseases. However, little is known about the biological roles of AFPs beyond their antifungal action. Penicillium expansum encodes three phylogenetically different AFPs (PeAfpA, PeAfpB and PeAfpC) with diverse profiles of antifungal activity. PeAfpA stands out as a highly active AFP that is naturally produced at high yields. Here, we provide new data about the function of PeAfpA in P. expansum through phenotypical characterization and transcriptomic studies of null mutants of the corresponding afpA gene. Mutation of afpA did not affect axenic growth, conidiation, virulence, stress responses or sensitivity towards P. expansum AFPs. However, RNA sequencing evidenced a massive transcriptomic change linked to the onset of PeAfpA production. We identified two large gene expression clusters putatively involved in PeAfpA function, which correspond to genes induced or repressed with the production of PeAfpA. Functional enrichment analysis unveiled significant changes in genes related to fungal cell wall remodeling, mobilization of carbohydrates and plasma membrane transporters. This study also shows a putative co-regulation between the three afp genes. Overall, our transcriptomic analyses provide valuable insights for further understanding the biological functions of AFPs.

The Candida albicans Cdk8-dependent phosphoproteome reveals repression of hyphal growth through a Flo8-dependent pathway.

Ssn3, also known as Cdk8, is a member of the four protein Cdk8 submodule within the multi-subunit Mediator complex involved in the co-regulation of transcription. In Candida albicans, the loss of Ssn3 kinase activity affects multiple phenotypes including cellular morphology, metabolism, nutrient acquisition, immune cell interactions, and drug resistance. In these studies, we generated a strain in which Ssn3 was replaced with a functional variant of Ssn3 that can be rapidly and selectively inhibited by the ATP analog 3-MB-PP1. Consistent with ssn3 null mutant and kinase dead phenotypes, inhibition of Ssn3 kinase activity promoted hypha formation. Furthermore, the increased expression of hypha-specific genes was the strongest transcriptional signal upon inhibition of Ssn3 in transcriptomics analyses. Rapid inactivation of Ssn3 was used for phosphoproteomic studies performed to identify Ssn3 kinase substrates associated with filamentation potential. Both previously validated and novel Ssn3 targets were identified. Protein phosphorylation sites that were reduced specifically upon Ssn3 inhibition included two sites in Flo8 which is a transcription factor known to positively regulate C. albicans morphology. Mutation of the two Flo8 phosphosites (threonine 589 and serine 620) was sufficient to increase Flo8-HA levels and Flo8 dependent transcriptional and morphological changes, suggesting that Ssn3 kinase activity negatively regulates Flo8.Under embedded conditions, when ssn3Δ/Δ and efg1Δ/Δ mutants were hyperfilamentous, FLO8 was essential for hypha formation. Previous work has also shown that loss of Ssn3 activity leads to increased alkalinization of medium with amino acids. Here, we show that the ssn3Δ/Δ medium alkalinization phenotype, which is dependent on STP2, a transcription factor involved in amino acid utilization, also requires FLO8 and EFG1. Together, these data show that Ssn3 activity can modulate Flo8 and its direct and indirect interactions in different ways, and underscores the potential importance of considering Ssn3 function in the control of transcription factor activities.

Mitochondrial fusion and fission are required for proper mitochondrial function and cell proliferation in fission yeast.

Mitochondria form a branched tubular network in many types of cells, depending on a balance between mitochondrial fusion and fission. How mitochondrial fusion and fission are involved in regulating mitochondrial function and cell proliferation is not well understood. Here, we dissected the roles of mitochondrial fusion and fission in mitochondrial function and cell proliferation in fission yeast. We examined mitochondrial membrane potential by staining cells with DiOC6 and assessed mitochondrial respiration by directly measuring oxygen consumption of cells with a dissolved oxygen respirometer. We found that defects in mitochondrial fission or fusion reduce mitochondrial membrane potential and compromise mitochondrial respiration while the absence of both mitochondrial fusion and fission restores wild type-like respiration, normal membrane potential, and tubular networks of mitochondria. Moreover, we found that the absence of either mitochondrial fission or fusion prolongs the cell cycle and that the absence of both mitochondrial fusion and fission significantly delays cell cycle progression after nitrogen replenishment. The prolonged/delayed cell cycle is likely due to the deregulation of Cdc2 activation. Hence, our work not only establishes an intimate link between mitochondrial morphology and function but also underscores the importance of mitochondrial dynamics in regulating the cell cycle.

Assessment of genotoxic chemicals using chemogenomic profiling based on gene-knockout library in Saccharomyces cerevisiae.

Understanding the adverse effects of genotoxic chemicals and identifying them effectively from non-genotoxic chemicals are of great worldwide concerns. Here, Saccharomyces cerevisiae (yeast) genome-wide single-gene knockout screening approach was conducted to assess two genotoxic chemicals (4-nitroquinoline-1-oxide (4-NQO) and formaldehyde (FA)) and environmental pollutant dichloroacetic acid (DCA, genotoxicity is controversial). DNA repair was significant enriched in the gene ontology (GO) biology process (BP) terms and KEGG pathways when exposed to low concentrations of 4-NQO and FA. Higher concentrations of 4-NQO and FA influenced some RNA metabolic and biosynthesis pathways. Moreover, replication and repair associated pathways were top ranked KEGG pathways with high fold-change for low concentrations of 4-NQO and FA. The similar gene profiles perturbed by DCA with three test concentrations identified, the common GO BP terms associated with aromatic amino acid family biosynthetic process and ubiquitin-dependent protein catabolic process via the multivesicular body sorting pathway. DCA has no obvious genotoxicity as there was no enriched DNA damage and repair pathways and fold-change of replication and repair KEGG pathways were very low. Five genes (RAD18, RAD59, MUS81, MMS4, and BEM4) could serve as candidate genes for genotoxic chemicals. Overall, the yeast functional genomic profiling showed great performance for assessing the signatures and potential molecular mechanisms of genotoxic chemicals.

Inhibition of LPMOs by Fermented Persimmon Juice.

Fermented persimmon juice, Kakishibu, has traditionally been used for wood and paper protection. This protective effect stems at least partially from inhibition of microbial cellulose degrading enzymes. The inhibitory effect of Kakishibu on lytic polysaccharide monooxygenases (LPMOs) and on a cocktail of cellulose hydrolases was studied, using three different cellulosic substrates. Dose dependent inhibition of LPMO activity by a commercial Kakishibu product was assessed for the well-characterized LPMO from Thermoascus aurantiacus TaAA9A, and the inhibitory effect was confirmed on five additional microbial LPMOs. The model tannin compound, tannic acid exhibited a similar inhibitory effect on TaAA9A as Kakishibu. It was further shown that both polyethylene glycol and tannase can alleviate the inhibitory effect of Kakishibu and tannic acid, indicating a likely mechanism of inhibition caused by unspecific tannin-protein interactions.

Phosphoproteomic responses of TORC1 target kinases reveal discrete and convergent mechanisms that orchestrate the quiescence program in yeast.

The eukaryotic TORC1 kinase assimilates diverse environmental cues, including growth factors and nutrients, to control growth by tuning anabolic and catabolic processes. In yeast, TORC1 stimulates protein synthesis in response to abundant nutrients primarily through its proximal effector kinase Sch9. Conversely, TORC1 inhibition following nutrient limitation unlocks various distally controlled kinases (e.g., Atg1, Gcn2, Npr1, Rim15, Slt2/Mpk1, and Yak1), which cooperate through poorly defined circuits to orchestrate the quiescence program. To better define the signaling landscape of the latter kinases, we use in vivo quantitative phosphoproteomics. Through pinpointing known and uncharted Npr1, Rim15, Slt2/Mpk1, and Yak1 effectors, our study examines the architecture of the distally controlled TORC1 kinase network. Accordingly, this is built on a combination of discrete, convergent, and multilayered feedback regulatory mechanisms, which likely ensure homeostatic control of and/or robust responses by TORC1 and its effector kinases under fluctuating nutritional conditions.

Molecular characterization and overexpression of the difenoconazole resistance gene CYP51 in Lasiodiplodia theobromae field isolates.

Stem-end rot (SER) caused by Lasiodiplodia theobromae is an important disease of mango in China. Demethylation inhibitor (DMI) fungicides are widely used for disease control in mango orchards. The baseline sensitivity to difenoconazole of 138 L. theobromae isolates collected from mango in the field in 2019 was established by the mycelial growth rate method. The cross-resistance to six site-specific fungicides with different modes of action were investigated using 20 isolates randomly selected. The possible mechanism for L. theobromae resistance to difenoconazole was preliminarily determined through gene sequence alignment and quantitative real-time PCR analysis. The results showed that the EC50 values of 138 L. theobromae isolates to difenoconazole ranged from 0.01 to 13.72 µg/mL. The frequency of difenoconazole sensitivity formed a normal distribution curve when the outliers were excluded. Difenoconazole showed positive cross-resistance only with the DMI tebuconazole but not with non-DMI fungicides carbendazim, pyraclostrobin, fludioxonil, bromothalonil, or iprodione. Some multifungicide-resistant isolates of L. theobromae were found. Two amino acid substitutions (E209k and G207A) were found in the CYP51 protein, but they were unlikely to be related to the resistance phenotype. There was no alteration in the promoter region of the CYP51 gene. However, difenoconazole significantly increased the expression of the CYP51 gene in the resistant isolates compared to the susceptible isolates. These results are vital to develop effective mango disease management strategies to avoid the development of further resistance.

Polyamines Upregulate Cephalosporin C Production and Expression of ß-Lactam Biosynthetic Genes in High-Yielding Acremonium chrysogenum Strain.

The high-yielding production of pharmaceutically significant secondary metabolites in filamentous fungi is obtained by random mutagenesis; such changes may be associated with shifts in the metabolism of polyamines. We have previously shown that, in the Acremonium chrysogenum cephalosporin C high-yielding strain (HY), the content of endogenous polyamines increased by four- to five-fold. Other studies have shown that the addition of exogenous polyamines can increase the production of target secondary metabolites in highly active fungal producers, in particular, increase the biosynthesis of ß-lactams in the Penicillium chrysogenum Wis 54-1255 strain, an improved producer of penicillin G. In the current study, we demonstrate that the introduction of exogenous polyamines, such as spermidine or 1,3-diaminopropane, to A. chrysogenum wild-type (WT) and HY strains, leads to an increase in colony germination and morphological changes in a complete agar medium. The addition of 5 mM polyamines during fermentation increases the production of cephalosporin C in the A. chrysogenum HY strain by 15-20% and upregulates genes belonging to the beta-lactam biosynthetic cluster. The data obtained indicate the intersection of the metabolisms of polyamines and beta-lactams in A. chrysogenum and are important for the construction of improved producers of secondary metabolites in filamentous fungi.

RPD3 and UME6 are involved in the activation of PDR5 transcription and pleiotropic drug resistance in ρ0 cells of Saccharomyces cerevisiae.

BACKGROUND: In Saccharomyces cerevisiae, the retrograde signalling pathway is activated in ρ0/- cells, which lack mitochondrial DNA. Within this pathway, the activation of the transcription factor Pdr3 induces transcription of the ATP-binding cassette (ABC) transporter gene, PDR5, and causes pleiotropic drug resistance (PDR). Although a histone deacetylase, Rpd3, is also required for cycloheximide resistance in ρ0/- cells, it is currently unknown whether Rpd3 and its DNA binding partners, Ume6 and Ash1, are involved in the activation of PDR5 transcription and PDR in ρ0/- cells. This study investigated the roles of RPD3, UME6, and ASH1 in the activation of PDR5 transcription and PDR by retrograde signalling in ρ0 cells. RESULTS: ρ0 cells in the rpd3∆ and ume6∆ strains, with the exception of the ash1∆ strain, were sensitive to fluconazole and cycloheximide. The PDR5 mRNA levels in ρ0 cells of the rpd3∆ and ume6∆ strains were significantly reduced compared to the wild-type and ash1∆ strain. Transcriptional expression of PDR5 was reduced in cycloheximide-exposed and unexposed ρ0 cells of the ume6∆ strain; the transcriptional positive response of PDR5 to cycloheximide exposure was also impaired in this strain. CONCLUSIONS: RPD3 and UME6 are responsible for enhanced PDR5 mRNA levels and PDR by retrograde signalling in ρ0 cells of S. cerevisiae.

Hedera rhombea inhibits the biofilm formation of Candida, thereby increases the susceptibility to antifungal agent, and reduces infection.

Candida is an opportunistic pathogen and a common cause of fungal infections worldwide. Anti-fungal use against Candida infections has resulted in the appearance of resistant strains. The limited choice of anti-fungal therapy means alternative strategies are needed to control fungal infectious diseases. The aim of this study was to evaluate the inhibition of Candida biofilm formation by Hedera rhombea (Korean name: songak) extract. Biofilm formation was assessed using the crystal violet assay which showed a dose dependent reduction in the presence of extract with the biofilm formation inhibitory concentration of C. albicans (IC50 = 12.5µg/ml), C. tropicalis var. tropicalis (IC50 = 25µg/ml), C. parapsilosis var. parapsilosis (IC50 = 6.25µg/ml), C. glabrata (IC50 = 6.25µg/ml), C. tropicalis (IC50 = 12.5µg/ml), and C. parapsilosis (IC50 = 12.5µg/ml) without directly reducing Candida growth. Treatment with 6.25µg/mL of extract increased the antifungal susceptibility to miconazole from 32% decreasing of fungal growth to 98.8% of that based on the fungal growth assay. Treatment of extract dose-dependently reduced the dimorphic transition of Candida based on the dimorphic transition assay and treatment of 3.125µg/mL of extract completely blocked the adherence of Candida to the HaCaT cells. To know the molecular mechanisms of biofilm formation inhibition by extract, qRT-PCR analysis was done, and the extract was found to dose dependently reduce the expression of hyphal-associated genes (ALS3, ECE1, HWP1, PGA50, and PBR1), extracellular matrix genes (GSC1, ZAP1, ADH5, and CSH1), Ras1-cAMP-PKA pathway genes (CYR1, EFG1, and RAS1), Cph2-Tec1 pathway gene (TEC1) and MAP kinases pathway gene (HST7). In this study, Hedera rhombea extract showed inhibition of fungal biofilm formation, activation of antifungal susceptibility, and reduction of infection. These results suggest that fungal biofilm formation is good screen for developing the antifungal adjuvant and Hedera rhombea extract should be a good candidate against biofilm-related fungal infection.

Are Point Mutations in HMG-CoA Reductases (Hmg1 and Hmg2) a Step towards Azole Resistance in Aspergillus fumigatus?

Invasive aspergillosis, mainly caused by Aspergillus fumigatus, can lead to severe clinical outcomes in immunocompromised individuals. Antifungal treatment, based on the use of azoles, is crucial to increase survival rates. However, the recent emergence of azole-resistant A. fumigatus isolates is affecting the efficacy of the clinical therapy and lowering the success rate of azole strategies against aspergillosis. Azole resistance mechanisms described to date are mainly associated with mutations in the azole target gene cyp51A that entail structural changes in Cyp51A or overexpression of the gene. However, strains lacking cyp51A modifications but resistant to clinical azoles have recently been detected. Some genes have been proposed as new players in azole resistance. In this study, the gene hmg1, recently related to azole resistance, and its paralogue hmg2 were studied in a collection of fifteen azole-resistant strains without cyp51A modifications. Both genes encode HMG-CoA reductases and are involved in the ergosterol biosynthesis. Several mutations located in the sterol sensing domain (SSD) of Hmg1 (D242Y, G307D/S, P309L, K319Q, Y368H, F390L and I412T) and Hmg2 (I235S, V303A, I312S, I360F and V397C) were detected. The role of these mutations in conferring azole resistance is discussed in this work.

Transcriptional Profiling of the Candida auris Response to Exogenous Farnesol Exposure.

The antifungal resistance threat posed by Candida auris necessitates bold and innovative therapeutic options. Farnesol is a quorum-sensing molecule with a potential antifungal and/or adjuvant effect; it may be a promising candidate in alternative treatment regimens. To gain further insights into the farnesol-related effect on C. auris, genome-wide gene transcription analysis was performed using transcriptome sequencing (RNA-Seq). Farnesol exposure resulted in 1,766 differentially expressed genes. Of these genes, 447 and 304 genes with at least 1.5-fold increase or decrease in transcription, respectively, were selected for further investigation. Genes involved in morphogenesis, biofilm events (maturation and dispersion), gluconeogenesis, iron metabolism, and regulation of RNA biosynthesis showed downregulation, whereas those related to antioxidative defense, transmembrane transport, glyoxylate cycle, fatty acid ß-oxidation, and peroxisome processes were upregulated. In addition, farnesol treatment increased the transcription of certain efflux pump genes, including MDR1, CDR1, and CDR2. Growth, measured by the change in the number of CFU, was significantly inhibited within 2 h of the addition of farnesol (5.8 × 107 ± 1.1 × 107 and 1.1 × 107 ± 0.3 × 107 CFU/ml for untreated control and farnesol-exposed cells, respectively) (P < 0.001). In addition, farnesol treatment caused a significant reduction in intracellular iron (152.2 ± 21.1 versus 116.0 ± 10.0 mg/kg), manganese (67.9 ± 5.1 versus 18.6 ± 1.8 mg/kg), and zinc (787.8 ± 22.2 versus 245.8 ± 34.4 mg/kg) (P < 0.05 to 0.001) compared to untreated control cells, whereas the level of cooper was significantly increased (274.6 ± 15.7 versus 828.8 ± 106.4 mg/kg) (P < 0.001). Our data demonstrate that farnesol significantly influences the growth, intracellular metal ion contents, and gene transcription related to fatty acid metabolism, which could open new directions in developing alternative therapies against C. auris. IMPORTANCE Candida auris is a dangerous fungal pathogen that causes outbreaks in health care facilities, with infections associated with a high mortality rate. As conventional antifungal drugs have limited effects against the majority of clinical isolates, new and innovative therapies are urgently needed. Farnesol is a key regulator molecule of fungal morphogenesis, inducing phenotypic adaptations and influencing biofilm formation as well as virulence. Alongside these physiological modulations, it has a potent antifungal effect alone or in combination with traditional antifungals, especially at supraphysiological concentrations. However, our knowledge about the mechanisms underlying this antifungal effect against C. auris is limited. This study has demonstrated that farnesol enhances the oxidative stress and reduces the fungal survival strategies. Furthermore, it inhibits manganese, zinc transport, and iron metabolism as well as increases fungal intracellular copper content. In addition, metabolism was modulated toward ß-oxidation. These results provide definitive explanations for the observed antifungal effects.

The Candida glabrata Upc2A transcription factor is a global regulator of antifungal drug resistance pathways.

The most commonly used antifungal drugs are the azole compounds, which interfere with biosynthesis of the fungal-specific sterol: ergosterol. The pathogenic yeast Candida glabrata commonly acquires resistance to azole drugs like fluconazole via mutations in a gene encoding a transcription factor called PDR1. These PDR1 mutations lead to overproduction of drug transporter proteins like the ATP-binding cassette transporter Cdr1. In other Candida species, mutant forms of a transcription factor called Upc2 are associated with azole resistance, owing to the important role of this protein in control of expression of genes encoding enzymes involved in the ergosterol biosynthetic pathway. Recently, the C. glabrata Upc2A factor was demonstrated to be required for normal azole resistance, even in the presence of a hyperactive mutant form of PDR1. Using genome-scale approaches, we define the network of genes bound and regulated by Upc2A. By analogy to a previously described hyperactive UPC2 mutation found in Saccharomyces cerevisiae, we generated a similar form of Upc2A in C. glabrata called G898D Upc2A. Analysis of Upc2A genomic binding sites demonstrated that wild-type Upc2A binding to target genes was strongly induced by fluconazole while G898D Upc2A bound similarly, irrespective of drug treatment. Transcriptomic analyses revealed that, in addition to the well-described ERG genes, a large group of genes encoding components of the translational apparatus along with membrane proteins were responsive to Upc2A. These Upc2A-regulated membrane protein-encoding genes are often targets of the Pdr1 transcription factor, demonstrating the high degree of overlap between these two regulatory networks. Finally, we provide evidence that Upc2A impacts the Pdr1-Cdr1 system and also modulates resistance to caspofungin. These studies provide a new perspective of Upc2A as a master regulator of lipid and membrane protein biosynthesis.

Genome organization and evolution of a eukaryotic nicotinate co-inducible pathway.

In Aspergillus nidulans a regulon including 11 hxn genes (hxnS, T, R, P, Y, Z, X, W, V, M and N) is inducible by a nicotinate metabolic derivative, repressible by ammonium and under stringent control of the nitrogen-state-sensitive GATA factor AreA and the specific transcription factor HxnR. This is the first report in a eukaryote of the genomic organization of a possibly complete pathway of nicotinate utilization. In A. nidulans the regulon is organized in three distinct clusters, this organization is variable in the Ascomycota. In some Pezizomycotina species all 11 genes map in a single cluster; in others they map in two clusters. This variable organization sheds light on cluster evolution. Instances of gene duplication followed by or simultaneous with integration in the cluster, partial or total cluster loss, and horizontal gene transfer of several genes (including an example of whole cluster re-acquisition in Aspergillus of section Flavi) were detected, together with the incorporation in some clusters of genes not found in the A. nidulans co-regulated regulon, which underlie both the plasticity and the reticulate character of metabolic cluster evolution. This study provides a comprehensive phylogeny of six members of the cluster across representatives of all Ascomycota classes.

The HMGB Protein KlIxr1, a DNA Binding Regulator of Kluyveromyces lactis Gene Expression Involved in Oxidative Metabolism, Growth, and dNTP Synthesis.

In the traditional fermentative model yeast Saccharomyces cerevisiae, ScIxr1 is an HMGB (High Mobility Group box B) protein that has been considered as an important regulator of gene transcription in response to external changes like oxygen, carbon source, or nutrient availability. Kluyveromyces lactis is also a useful eukaryotic model, more similar to many human cells due to its respiratory metabolism. We cloned and functionally characterized by different methodologies KlIXR1, which encodes a protein with only 34.4% amino acid sequence similarity to ScIxr1. Our data indicate that both proteins share common functions, including their involvement in the response to hypoxia or oxidative stress induced by hydrogen peroxide or metal treatments, as well as in the control of key regulators for maintenance of the dNTP (deoxyribonucleotide triphosphate) pool and ribosome synthesis. KlIxr1 is able to bind specific regulatory DNA sequences in the promoter of its target genes, which are well conserved between S. cerevisiae and K. lactis. Oppositely, we found important differences between ScIrx1 and KlIxr1 affecting cellular responses to cisplatin or cycloheximide in these yeasts, which could be dependent on specific and non-conserved domains present in these two proteins.

Mechanistic analysis of cadmium toxicity in Saccharomyces cerevisiae.

As a potentially toxic heavy metal, Cadmium (Cd) can cause endoplasmic reticulum and oxidative stress, and thus lead to cell death. To explore the mechanisms of Cd toxicity, we investigated the UPRE-lacZ expression, the intracellular reactive oxygen species (ROS) and cell death in the 151 Cd-sensitive mutants of Saccharomyces cerevisiae in response to Cd stress. We identified 101 genes regulating UPRE-lacZ expression were involved in preventing ROS production and/or cell death from increasing to high levels, while mutants for 72 genes caused both elevated ROS production and cell death, indicating the Cd-induced ROS production and cell death are mediated by UPR. Genes involved in cell wall integrity (CWI) pathway, vacuolar protein sorting (VPS) and vacuolar transport, calcium/calcineurin pathway and PHO pathways were all required for the Cd-induced UPR, intracellular ROS and cell death. To conclude, this study highlights the importance of Cd-induced UPR, intracellular ROS levels and cell death that may play crucial roles in Cd-induced toxicity.

Global view of dynamic expression and precise mapping of mitochondrial tRNAs-derived fragments during stressed conditions in S. pombe.

In this study, we provide a global view of population and processing of mitochondrial tRNAs-derived fragments (mt-tRFs) in fission yeast Schizosaccharomyces pombe. Here, mt-tRFs of 15-30 nucleotides were retrieved from S. pombe small RNA libraries obtained from unstressed, stress, and during stationary phase conditions. We demonstrate that production of these fragments increase during heat stress and stationary phase conditions in S. pombe, especially (most notably) in stationary phase. Analysis of data also reveals depending on the tRNA, either 5'-mt-tRF or 3'-mt-tRF was found and major mt-tRNA processing sites have been precisely identified. Furthermore, RNA-seq reveals that inactivation of trz2 encoding S. pombe mitochondrial tRNase ZL globally impairs mt-tRF processing. Finally, our result showed mt-tRFs were predicted to target mitochondrial genome mapping mtDNA-encoded protein gene. These observations suggest that mitochondrial tRFs may play an important regulatory role in response to stress and development.