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Pathogenic fungi exhibit a heavy burden on medical care and new therapies are needed. Here, we develop the fungal specific enzyme sterylglucosidase 1 (Sgl1) as a therapeutic target. Sgl1 converts the immunomodulatory glycolipid ergosterol 3β-D-glucoside to ergosterol and glucose. Previously, we found that genetic deletion of Sgl1 in the pathogenic fungus Cryptococcus neoformans (Cn) results in ergosterol 3β-D-glucoside accumulation, renders Cn non-pathogenic, and immunizes mice against secondary infections by wild-type Cn, even in condition of CD4+ T cell deficiency. Here, we disclose two distinct chemical classes that inhibit Sgl1 function in vitro and in Cn cells. Pharmacological inhibition of Sgl1 phenocopies a growth defect of the Cn Δsgl1 mutant and prevents dissemination of wild-type Cn to the brain in a mouse model of infection. Crystal structures of Sgl1 alone and with inhibitors explain Sgl1's substrate specificity and enable the rational design of antifungal agents targeting Sgl1., (© 2021. The Author(s).)

Competing Interests: The authors have declared that no competing interests exist.

Due to the evolution and development of antifungal drug resistance, limited efficacy of existing drugs has led to high mortality in patients with serious fungal infections. To develop novel antifungal therapeutic strategies, herein a series of carboline fungal histone deacetylase (HDAC) inhibitors were designed and synthesized, which had potent synergistic effects with fluconazole against resistant Candida albicans infection. In particular, compound D12 showed excellent in vitro and in vivo synergistic antifungal efficacy with fluconazole to treat azole-resistant candidiasis. It cooperated with fluconazole in reducing the virulence of C. albicans by blocking morphological mutual transformation and inhibiting biofilm formation. Mechanism studies revealed that the reversion of drug resistance was due to downregulation of the expression of the azole target gene ERG11 and efflux gene CDR1 . Taken together, fungal HDAC inhibitor D12 offered a promising lead compound for combinational treatment of azole-resistant candidiasis.

Biocatalysis in mixtures of water and co-solvents represents an opportunity to expand the application of enzymes. However, in the presence of organic solvents, enzymes can undergo reversible inhibition, inactivation, or aggregation. In this work, we studied the effects of three co-solvents (methanol, acetone, and dimethyl sulfoxide - DMSO) on the function and structure of the recombinant Candida antarctica lipase B (rCALB), a widely used enzyme in biotechnological applications. The effects of co-solvents on rCALB were investigated by steady-state kinetics experiments, biophysical assays and by molecular dynamics simulations in the presence and upon incubation with the three co-solvents. Methanol and acetone were found to act as competitive inhibitors of rCALB and to promote its aggregation, whereas DMSO is a non-essential activator of rCALB., Competing Interests: Declaration of competing interest There are no conflicts of interest to declare., (Copyright © 2020. Published by Elsevier B.V.)

Background: Cigarette smoke is associated with higher oral Candida carriage and possible predisposition and increased susceptibility to oral candidal infection. Candida dubliniensis is associated with oral candidosis. Candidal adherence to buccal epithelial cells (BEC) and denture acrylic surfaces (DAS), germ tube (GT) formation, cell surface hydrophobicity (CSH) and hemolysin production are pathogenic traits of Candida., Objectives: The impact of exposure to cigarette smoke on the aforementioned pathogenic attributes of oral C. dubliniensis has not been studied. Hence, the impact of cigarette smoke condensate (CSC) on adhesion to BEC and DAS, GT formation, CSH and hemolysin production of 20 oral C. dubliniensis isolates after exposure to CSC for 24, 48 and 72 h was ascertained., Methods: After preparation of the CSC, using an in-house smoking device, the Candida isolates were exposed to the CSC for 24, 48 and 72 h, by a previously described in vitro method. Thereafter, the adhesion to BEC and DAS, GT formation, CSH and hemolysin production of C. dubliniensis isolates was investigated by hitherto described in vitro assays., Results: Exposure to CSC significantly increased the ability of C. dubliniensis oral isolates to adhere to BEC, DAS, GT formation, CSH and produce hemolysin following 24-h, 48-h and 72-h exposure periods to CSC (P < 0.001 for all attributes tested)., Conclusions: Exposure of oral C. dubliniensis isolates to CSC may significantly promote in vitro adhesion traits and hemolysin production of these isolates, thereby augmenting its pathogenicity in vitro in the presence of cigarette smoke.

Aim: Among the most common mechanisms of multidrug resistance (MDR) in prevalent human fungal pathogen, Candida albicans, overexpression of drug efflux pumps remains the predominant mechanism. Hence to inhibit efflux pumps and chemosensitize C. albicans against traditional antifungal drugs still represents an attractive approach. The present study aimed to analyze the effect of Vanillin (Van), a natural food flavoring agent, on drug efflux pump activity of Candida albicans., Methods and Results: We observed that Van specifically inhibits Candida drug resistance protein 2 (CaCdr2p) activity belonging to ATP Binding Cassette (ABC) superfamily as revealed by abrogated rhodamine 6G efflux and nile red accumulation assay with CaCdr2p over expressing strain. Insight studies into the mechanisms suggested that abrogated efflux by CaCdr2p is due to competitive mode of inhibition by Van as depicted by Lineweaver-Burk plot. RT-PCR, western blot and confocal microscopy further unraveled that Van leads to reduced expression of CDR2 and CaCdr2p mislocalization respectively. Furthermore, Van sensitizes the azole sensitive and resistant clinical matched pair of isolates Gu4/Gu5 and led to abrogated rhodamine 6G efflux and depleted ergosterol. Furthermore, Van synergizes with membrane targeting drugs fluconazole and amphotericin B as their fractional inhibitory coefficient index was less than 0.5., Conclusion: Van being a potent inhibitor of CaCdr2p and chemosensitizing of drug resistant C. albicans warrants further studies to be exploited as effective antifungal agent., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

The development of a tetO/TetR system in the fungus Neurospora crassa is described. The system includes (i) a synthetic gene encoding a TetR variant fused to GFP, and (ii) a standard tetO array integrated homologously, as a proof of principle, near the his-3 gene. The localization of TetR-GFP at the tetO array (observed by fluorescence microscopy) can be disrupted by the application of tetracycline. The full-length array is stable during vegetative growth, but it triggers strong repeat-induced point mutation (RIP) by the RID-dependent as well as the DIM-2-dependent pathways during the sexual phase. Thus, both RIP pathways must be inactivated to allow the faithful inheritance of the unmodified construct. In summary, this study introduces a new molecular tool into Neurospora research, and suggests that the standard tetO array can self-engage in recombination-independent homologous pairing., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Hyphal morphogenesis of Candida albicans is important for its pathogenesis. Here, we showed that the filamentous growth of C. albicans requires vacuolar H + -ATPase function. Results showed that levels of Vma4 and Vma10 increased in cells undergoing hyphal growth compared to those undergoing yeast growth. Deleting VMA4 or VMA10 abolished vacuolar functions and hyphal morphogenesis. These deletion mutants were also characterized as avirulent in a mouse model of systemic infection. Furthermore, VMA4 and VMA10 deletion strains showed hypersensitivity to fluconazole, terbinafine, and amphotericin B. Based on these findings, Vma4 and Vma10 are not only involved in vacuole biogenesis and hyphal formation, but also are good targets for antifungal drug development in C. albicans., (Copyright © 2019 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Bloodstream infections caused by non-albicans Candida species are increasing and echinocandins have been extensively used especially in patients with hemodynamic instability, previous antifungal treatment and hospital risk factors for intrinsic or acquired resistance to azoles. Candida glabrata resistance to echinocandins is reported and is generally associated with previous use of echinocandins; FKS gene mutations have been associated with a worse outcome. We report the case of a 65-year-old woman who developed candidemia and endocarditis by C. glabrata with a newly acquired FKS mutation 24 months after successful treatment of infective endocarditis by C. glabrata with a double dosage of anidulafungin (200 mg daily) followed by oral voriconazole. Driven by high echinocandin MICs the strain taken by intraoperative cultures was further analyzed in a referral microbiology laboratory, confirming the new onset of point mutation S633P of the FKS2 gene.

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.

New strategies are needed to counter the escalating threat posed by drug-resistant fungi. The molecular chaperone Hsp90 affords a promising target because it supports survival, virulence and drug-resistance across diverse pathogens. Inhibitors of human Hsp90 under development as anticancer therapeutics, however, exert host toxicities that preclude their use as antifungals. Seeking a route to species-selectivity, we investigate the nucleotide-binding domain (NBD) of Hsp90 from the most common human fungal pathogen, Candida albicans. Here we report structures for this NBD alone, in complex with ADP or in complex with known Hsp90 inhibitors. Encouraged by the conformational flexibility revealed by these structures, we synthesize an inhibitor with >25-fold binding-selectivity for fungal Hsp90 NBD. Comparing co-crystals occupied by this probe vs. anticancer Hsp90 inhibitors revealed major, previously unreported conformational rearrangements. These insights and our probe's species-selectivity in culture support the feasibility of targeting Hsp90 as a promising antifungal strategy.

In recent years, incidences of invasive fungal infections have greatly increased, especially in immunosuppressed patients, but most today's antifungal drugs are not completely effective due to the development of drug resistance, as well as potential toxicity and adverse effects. Consequently, it is imperative to search for novel antifungal agents to combat fungal infections. This review will discuss the advances in the traditional antifungal therapy, and present an overview of novel strategies for the treatment of fungal infections. The papers presented here highlight new targets that could be exploited for development of new antifungal agents.

Biosynthesis of benzylpenicillin in filamentous fungi (e.g. Penicillium chrysogenum - renamed as Penicillium rubens- and Aspergillus nidulans) depends on the addition of CoA-activated forms of phenylacetic acid to isopenicillin N. Phenylacetic acid is also detoxified by means of the homogentisate pathway, which begins with the hydroxylation of phenylacetic acid to 2-hydroxyphenylacetate in a reaction catalysed by the pahA-encoded phenylacetate hydroxylase. This catabolic step has been tested in three different penicillin-producing strains of P. rubens (P. notatum, P. chrysogenum NRRL 1951 and P. chrysogenum Wisconsin 54-1255) in the presence of sucrose and lactose as non-repressing carbon sources. P. chrysogenum Wisconsin 54-1255 was able to accumulate 2-hydroxyphenylacetate at late culture times. Analysis of the P. rubens genome showed the presence of several PahA homologs, but only Pc16g01770 was transcribed under penicillin production conditions. Gene knock-down experiments indicated that the protein encoded by Pc16g01770 seems to have residual activity in phenylacetic acid degradation, this catabolic activity having no effect on benzylpenicillin biosynthesis. Proteome-wide analysis of the Wisconsin 54-1255 strain in response to phenylacetic acid revealed that this molecule has a positive effect on some proteins directly related to the benzylpenicillin biosynthetic pathway, the synthesis of amino acid precursors and other important metabolic processes., Significance: The adaptive response of Penicillium rubens to benzylpenicillin production conditions remains to be fully elucidated. This article provides important information about the molecular mechanisms interconnected with phenylacetate (benzylpenicillin side chain precursor) utilization and penicillin biosynthesis, and will contribute to the understanding of the complex physiology and adaptation mechanisms triggered by P. rubens (P. chrysogenum Wisconsin 54-1255) under benzylpenicillin production conditions., (Copyright © 2018. Published by Elsevier B.V.)

Backgroud: Though it is toxic to humans, dichlorvos is a widely used chemical pesticide and plays an important role in the control of plant pests. The application of a combination of the biocontrol agent Trichoderma with dichlorvos may reduce the need for chemical pesticides. Therefore, revealing the specific molecular mechanism of Trichoderma tolerance to dichlorvos has become particularly important., Results: In this study, using transcriptome and metabolome analyses, changes in primary and secondary metabolisms in Trichoderma asperellum TJ01 were comprehensively studied in the presence of dichlorvos. A novel C 2 H 2 zinc finger protein gene, zinc finger chimera 1 (zfc1), was discovered to be upregulated, along with a large number of oxidoreductase genes and ABC transporter genes under dichlorvos stress. In addition, gas chromatography-mass spectrometry (GC-TOF-MS), and liquid chromatography-mass spectrometry (LC-QQQ-MS) data revealed the global primary and secondary metabolic changes that occur in T. asperellum TJ01 under dichlorvos stress., Conclusions: The tolerance mechanism of T. asperellum TJ01 to dichlorvos was proposed. In addition, the absorption and residue of dichlorvos were analyzed, laying the foundation for elucidation of the mechanism by which T. asperellum TJ01 degrades pesticide residues.

A β-xylosidase from Colletotrichum graminicola (Bxcg) was purified. The enzyme showed high halotolerance, retaining about 63% of the control activity in the presence of 2.5molL -1 NaCl. The presence of NaCl has not affected the optimum reaction temperature (65°C), but the optimum pH was slightly altered (from 4.5 to 5.0) at high salt concentrations. Bxcg was fully stable at 50°C for 24h and over a wide pH range even in the presence of NaCl. In the absence of salt Bxcg hydrolyzed p-nitrophenyl-β-d-xylopyranoside with maximum velocity of 348.8±11.5Umg -1 and high catalytic efficiency (1432.7±47.3Lmmol -1 s -1 ). Bxcg revealed to be a bifunctional enzyme with both β-xylosidase and α-l-arabinofuranosidase activities, and hydrolyzed xylooligosaccharides containing up to six pentose residues. The enzyme showed high synergistic effect (3.1-fold) with an endo-xylanase for the hydrolysis of beechwood xylan, either in the absence or presence of 0.5molL -1 NaCl, and was tolerant to different organic solvents and surfactants. This is the first report of a halotolerant bifunctional β-xylosidase/α-l-arabinofuranosidase from C. graminicola, and the enzyme showed attractive properties for application in lignocellulose hydrolysis, particularly under high salinity and/or in the presence of residues of pretreatment steps., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Azole-resistant Aspergillus fumigatus (ARAF) has been reported in patients with chronic obstructive pulmonary disease (COPD) but has not been specifically assessed so far. Here, we evaluated ARAF prevalence in azole-naïve COPD patients and their homes, and assessed whether CYP51A mutations were similar in clinical and environmental reservoirs. Sixty respiratory samples from 41 COPD patients with acute exacerbation and environmental samples from 36 of these patient's homes were prospectively collected. A. fumigatus was detected in respiratory samples from 11 of 41 patients (27%) and in 15 of 36 domiciles (42%). Cyp51A sequencing and selection on itraconazole medium of clinical (n = 68) and environmental (n = 48) isolates yielded ARAF detection in 1 of 11 A. fumigatus colonized patients with COPD (9%) and 2 of 15 A. fumigatus-positive patient's homes (13%). The clinical isolate had no CYP51A mutation. Two environmental isolates from two patients harbored TR 34 /L98H mutation, and one had an H285Y mutation. Coexistence of different cyp51A genotypes and/or azole resistance profiles was detected in 3 of 8 respiratory and 2 of 10 environmental samples with more than one isolate, confirming the need for a systematic screening of all clinically relevant isolates. The high prevalence of ARAF in patients with COPD and their homes supports the need for further studies to assess the prevalence of azole resistance in patients with Aspergillus diseases in Northern France., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

We present an open-source, interactive program named Proteoform Suite that uses proteoform mass and intensity measurements from complex biological samples to identify and quantify proteoforms. It constructs families of proteoforms derived from the same gene, assesses proteoform function using gene ontology (GO) analysis, and enables visualization of quantified proteoform families and their changes. It is applied here to reveal systemic proteoform variations in the yeast response to salt stress.

Members of Candida species cause significant health problems, inducing various types of superficial and deep-seated mycoses in humans. In order to prevent from Candida sp. development, essential oils are more and more frequently applied, due to their antifungal activity, low toxicity if used appropriately, and biodegrability. The aim of the study was to characterize the early alterations in Candida albicans metabolic properties in relation to proteins and chromosomal DNA profiles, after treatment with peppermint and clove oils at sub-inhibitory concentrations. The yeasts were affected by the oils even at a concentration of 0.0075% v / v , which resulted in changes in colony morphotypes and metabolic activities. Peppermint and clove oils at concentrations ranging from 0.015× MIC (minimal inhibitory concentration) to 0.5× MIC values substantially affected the enzymatic abilities of C. albicans , and these changes were primarily associated with the loss or decrease of activity of all 9 enzymes detected in the untreated yeast. Moreover, 29% isolates showed additional activity of N -acetyl-β-glucosaminidase and 14% isolates-α-fucosidase in comparison to the yeast grown without essential oils addition. In response to essential oils at 0.25-0.5× MIC, extensive changes in C. albicans whole-cell protein profiles were noted. However, the yeast biochemical profiles were intact with the sole exception of the isolate treated with clove oil at 0.5× MIC. The alterations were not attributed to gross chromosomal rearrangements in C. albicans karyotype. The predominantly observed decrease in protein fractions and the yeast enzymatic activity after treatment with the oils should be considered as a phenotypic response of C. albicans to the essential oils at their sub-inhibitory concentrations and may lead to the reduction of this yeast pathogenicity., Competing Interests: The authors declare no conflict of interest.

Background: Hospital acquired fungal infections are defined as "never events"-medical errors that should never have happened. Systemic Candida albicans infections results in 30-50% mortality rates. Typically, adhesion to abiotic medical devices and implants initiates such infections. Efficient adhesion initiates formation of aggressive biofilms that are difficult to treat. Therefore, inhibitors of adhesion are important for drug development and likely to have a broad spectrum efficacy against many fungal pathogens. In this study we further the development of a small molecule, Filastatin, capable of preventing C. albicans adhesion. We explored the potential of Filastatin as a pre-therapeutic coating of a diverse range of biomaterials., Methods: Filastatin was applied on various biomaterials, specifically bioactive glass (cochlear implants, subcutaneous drug delivery devices and prosthetics); silicone (catheters and other implanted devices) and dental resin (dentures and dental implants). Adhesion to biomaterials was evaluated by direct visualization of wild type C. albicans or a non-adherent mutant edt1 -/- that were stained or fluorescently tagged. Strains grown overnight at 30 °C were harvested, allowed to attach to surfaces for 4 h and washed prior to visualization. The adhesion force of C. albicans cells attached to surfaces treated with Filastatin was measured using Atomic Force Microscopy. Effectiveness of Filastatin was also demonstrated under dynamic conditions using a flow cell bioreactor. The effect of Filastatin under microfluidic flow conditions was quantified using electrochemical impedance spectroscopy. Experiments were typically performed in triplicate., Results: Treatment with Filastatin significantly inhibited the ability of C. albicans to adhere to bioactive glass (by 99.06%), silicone (by 77.27%), and dental resin (by 60.43%). Atomic force microcopy indicated that treatment with Filastatin decreased the adhesion force of C. albicans from 0.23 to 0.017 nN. Electrochemical Impedance Spectroscopy in a microfluidic device that mimic physiological flow conditions in vivo showed lower impedance for C. albicans when treated with Filastatin as compared to untreated control cells, suggesting decreased attachment. The anti-adhesive properties were maintained when Filastatin was included in the preparation of silicone materials., Conclusion: We demonstrate that Filastatin treated medical devices prevented adhesion of Candida, thereby reducing nosocomial infections.

Azole antifungals are potent inhibitors of fungal lanosterol 14α demethylase (CYP51) and have been used for eradication of systemic candidiasis clinically. Herein we report the design, synthesis, and biological evaluation of a series of 1-phenyl/1-(4-chlorophenyl)-2-(1H-imidazol-1-yl)ethanol esters. Many of these derivatives showed fungal growth inhibition at very low concentrations. Minimal inhibition concentration (MIC) value of 15 was 0.125 μg/mL against Candida albicans. Additionally, some of our compounds, such as 19 (MIC: 0.25 μg/mL), were potent against resistant C. glabrata, a fungal strain less susceptible to some first-line antifungal drugs. We confirmed their antifungal efficacy by antibiofilm test and their safety against human monocytes by cytotoxicity assay. To rationalize their mechanism of action, we performed computational analysis utilizing molecular docking and dynamics simulations on the C. albicans and C. glabrata CYP51 (CACYP51 and CGCYP51) homology models we built. Leu130 and T131 emerged as possible key residues for inhibition of CGCYP51 by 19., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Transport of penicillin intermediates and penicillin secretion are still poorly characterized in Penicillium chrysogenum (re-identified as Penicillium rubens). Calcium (Ca 2+ ) plays an important role in the metabolism of filamentous fungi, and casein phosphopeptides (CPP) are involved in Ca 2+ internalization. In this study we observe that the effect of CaCl 2 and CPP is additive and promotes an increase in penicillin production of up to 10-12 fold. Combination of CaCl 2 and CPP greatly promotes expression of the three penicillin biosynthetic genes. Comparative proteomic analysis by 2D-DIGE, identified 39 proteins differentially represented in P. chrysogenum Wisconsin 54-1255 after CPP/CaCl 2 addition. The most interesting group of overrepresented proteins were a peroxisomal catalase, three proteins of the methylcitrate cycle, two aminotransferases and cystationine β-synthase, which are directly or indirectly related to the formation of penicillin amino acid precursors. Importantly, two of the enzymes of the penicillin pathway (isopenicillin N synthase and isopenicillin N acyltransferase) are clearly induced after CPP/CaCl 2 addition. Most of these overrepresented proteins are either authentic peroxisomal proteins or microbody-associated proteins. This evidence suggests that addition of CPP/CaCl 2 promotes the formation of penicillin precursors and the penicillin biosynthetic enzymes in peroxisomes and vesicles, which may be involved in transport and secretion of penicillin., Significance: Penicillin biosynthesis in Penicillium chrysogenum is one of the best characterized secondary metabolism processes. However, the mechanism by which penicillin is secreted still remains to be elucidated. Taking into account the role played by Ca 2+ and CPP in the secretory pathway and considering the positive effect that Ca 2+ exerts on penicillin production, the analysis of global protein changes produced after CPP/CaCl 2 addition is very helpful to decipher the processes related to the biosynthesis and secretion of penicillin., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Azole antifungals are used not only clinically for fungal infections but also used as agricultural fungicides. Recently, azole-resistant Aspergillus fumigatus containing a tandem repeat in the promoter region of cyp51A combined with amino acid substitution(s) appears in the environment in Eurasia, especially in several European countries. Although azole fungicides have been used in Japan, especially in Hokkaido, surveillance and characterization of A. fumigatus in Hokkaido have not been reported. In this study, we collected soil samples from farms that used an azole fungicide in the Tokachi area of eastern Hokkaido, isolated 91 A. fumigatus strains, and determined the minimal inhibitory concentrations of medical azoles required for these strains. Moreover, because causative agent A. fumigatus is ubiquitous in the air and acquired from the environment, we collected 22 clinical isolates of A. fumigatus to measure their susceptibility to medical azoles in a hospital in the Tokachi area. Our data show that almost all A. fumigatus isolates retained susceptibility to medical azoles. Clinical isolates OKH34 and OKH6 showed 8 and 2 μg/mL of voriconazole, respectively, as the minimal inhibitory concentration. Both isolates did not contain tandem repeat in cyp51A promoter region. An isolate contained G448S mutation in cyp51A conferring voriconazole resistance, which is the first report from Japan. Our data shows the existence of azole-resistant and low azole-susceptible clinical isolates and highlight the necessity for continuous surveillance in Japan because resistant A. fumigatus strains can arise through clinical or environmental selection or could be introduced from overseas., (Copyright © 2016 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Tubulins are the proposed target of anticancer drugs, anthelminthics, and fungicides. In Fusarium graminearum, β2 tubulin has been reported to be the binding target of methyl benzimidazole carbamate (MBC) fungicides. However, the function of F. graminearum β1 tubulin, which shares 76% amino acid sequence identity with β2 tubulin, in MBC sensitivity has been unclear. In this study, MBC sensitivity relative to that of a parental strain (2021) was significantly reduced in a β1 tubulin deletion strain but increased in a β2 tubulin deletion strain, suggesting that β1 tubulin was involved in the MBC sensitivity of F. graminearum. When strain 2021 was grown in a medium with a low or high concentration of the MBC fungicide carbendazim (0.5 or 1.4 μg/ml), the protein accumulation levels were reduced by 47 and 87%, respectively, for β1 tubulin but only by 6 and 24%, respectively, for β2 tubulin. This result was consistent with observations that MBC fungicides are more likely to disrupt β1 tubulin microtubules rather than β2 tubulin microtubules in GFP-β tubulin fusion mutants in vivo. Furthermore, sequence analysis indicated that a difference in tubulin amino acid 240 (240L in β1 versus 240F in β2) may explain the difference in MBC binding affinity; this result was consistent with the result that an F240L mutation in β2 tubulin greatly increased sensitivity to carbendazim in F. graminearum. We suggest that β1 tubulin rather than β2 tubulin is the preferred binding target for MBC fungicides in F. graminearum.

Unlabelled: The mycotoxin zearalenone, also known as F-2 mycotoxin or RAL is a potent estrogenic metabolite produced by some Gibberella and Fusarium species. It is a common contaminant of cereal crops, livestock and poultry products. However, detoxification of zearalenone (ZEN) remains a challenge. Recently, biological approach for ZEN detoxification is being explored. In this study, we investigated the biodegradation of ZEN by using Saccharomyces cerevisiae and the possible mechanisms involved. The findings revealed that, after 48h of incubation of S. cerevisiae in combination with ZEN, the ZEN was completely degraded by S. cerevisiae. On the contrary, heat-killed cells and cell-free culture filtrates of S. cerevisiae could not degrade ZEN. Furthermore, addition of cycloheximide to S. cerevisiae combined with ZEN at time 0h prevented ZEN degradation, while addition of cycloheximide at 12h significantly slowed down degradation. The results also indicated cellular proteomics of S. cerevisiae. Several differential proteins were identified, most of which were related to basic metabolism., Biological Significance: The findings revealed that, after 48h of incubating ZEN together with S. cerevisiae, ZEN was completely degraded by S. cerevisiae. The mechanisms involved in the degradation of ZEN by S. cerevisiae may be the production of associated intracellular and extracellular enzymes, which have the ability to degrade ZEN. In addition, there were some functional proteins produced by S. cerevisiae, indicating that the basic metabolism of S. cerevisiae was improved when ZEN was added. This novel discovery by the authors, will greatly contribute to the field of biodegradation of mycotoxin by antagonists. The authors also believed this innovation will open the grounds for further research and improvement of S. cerevisiae in the field of biodegradation., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Unlabelled: Glycoproteomics is greatly developed in recent years and big data of N-glycoproteome in mammalian tissues and cells were already established. However, the glycoproteomic studies on plant, fungus and bacteria are far left behind. In this study, we comprehensively mapped and quantified the N-glycosylation of Fusarium graminearum by combining stable isotope dimethyl labeling, hydrophilic interaction chromatography (HILIC) and high-resolution mass spectrometry. The N-glycosylation changes in Fusarium graminearum after fungicide treatment were extensively studied. Altogether we identified 927 N-glycopeptides, corresponding to 406 proteins and 774 sites and the glycosylation level was found to be largely down-regulated upon fungicide treatment. With the help of advanced bioinformatics, it was found that the N-glycoproteome changes were highly enriched in cell wall, membrane and extracellular regions. Moreover, the fungal metabolism, protein and glycosylation synthesis, and protease and glycosyl-transferase activity were all closely related with the down-regulated proteins, indicates that fungicide may affect fungal development in these aspects. These results will be useful for future studies on fungal biology. The established system for N-glycoproteome quantification has comparative or better performance compared with previous strategies and will be helpful in N-glycoproteomics of fungus and other species., Significance: We developed a robust HILIC-based system for N-glycoproteome quantification in fungus and established the largest quantitative N-glycosylation dataset in fungus, showing the high performance of the new system. The identified N-glycoproteins were proved to be high confident due to the high percentage of proteins in extracellular region and plasma. The quantification results were also accuracy and reproducible in two replicates. By the help of advanced bioinformatic tools, the obtained data was systematically analyzed. It was found that the N-glycosylation level was largely changed in cell wall, membrane and extracellular regions. Moreover, the cell metabolism, protein synthesis, and protease activity were also greatly deceased after fungicide treatment., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Unlabelled: A metal-resistant Rhodotorula mucilaginosa strain was isolated from an industrial wastewater. Effects on reduced/oxidized glutathione (GSSG/GSH), antioxidant enzymes and proteome were assessed on metal challenge (100mg/L). Increased GSH (mM/g) was found with CdCl2 (18.43±3.34), NaAsO2 (14.76±2.14), CuSO4 (14.73±2.49), and Pb(NO3)2 (15.74±5.3) versus control (7.67±0.95). GSH:GSSG ratio decreased with CdCl2, NaAsO2, and Pb(NO3)2 but not with CuSO4 and cysteine-containing protein levels increased with CdCl2 and NaAsO2. NaAsO2 exposure enhanced glutathione transferase activity but this decreased with CdCl2. Both metals significantly increased glutathione reductase and catalase activities. Metabolism-dependent uptake of Cd and As (12-day exposure) of approximately 65mg/g was observed in live cells with greater cell surface interaction for As compared to Cd. A particular role for arsenic oxidase in As resistance was identified. One dimensional electrophoresis revealed higher oxidation of protein thiols in response to NaAsO2 than to CdCl2. Two dimensional electrophoresis showed altered abundance of some proteins on metal treatment. Selected spots were excised for mass spectrometry and seven proteins identified. Under oxidative stress conditions, xylose reductase, putative chitin deacetylase, 20S proteasome subunit, eukaryotic translation elongation factor 2, valine-tRNA ligase and a metabolic enzyme F0F1 ATP synthase alpha subunit were all expressed as well as a unique hypothetical protein. These may comprise a protein expression signature for metal-induced oxidation in this yeast., Significance: Fungi are of widespread importance in agriculture, biodegradation and often show extensive tolerance to heavy metals. This makes them of interest from the perspective of bioremediation. In this study an environmental isolate of R. mucilaginosa showing extensive tolerance of a panel of heavy metals, in particular cadmium and arsenic, was studied. Several biochemical parameters such as activity of antioxidant enzymes, status of reduced and oxidized glutathione and thiols associated with proteins were all found to be affected by metal exposure. A detailed analysis with arsenic and cadmium pointed to a particular role for arsenic oxidase in arsenic bioaccumulation and tolerance. This is the first time this has been reported in R. mucilaginosa, and suggests that this isolate may have potential in biosorption of these metals in the environment. Proteomic analysis revealed that seven proteins with a variety of roles - ATP synthesis, protein degradation/synthesis, and metabolism of xylose and chitin - were differentially affected by metal exposure in a manner consistent with oxidative stress. These may therefore represent a protein expression signature for exposure to cadmium and arsenic., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Volatile organic compounds (VOCs) released by Saccharomyces cerevisiae inhibit plant pathogens, including the filamentous fungus Phyllosticta citricarpa, causal agent of citrus black spot. VOCs mediate relevant interactions between organisms in nature, and antimicrobial VOCs are promising, environmentally safer fumigants to control phytopathogens. As the mechanisms by which VOCs inhibit microorganisms are not well characterized, we evaluated the proteomic response in P. citricarpa after exposure for 12h to a reconstituted mixture of VOCs (alcohols and esters) originally identified in S. cerevisiae. Total protein was extracted and separated by 2D-PAGE, and differentially expressed proteins were identified by LC-MS/MS. About 600 proteins were detected, of which 29 were downregulated and 11 were upregulated. These proteins are involved in metabolism, genetic information processing, cellular processes, and transport. Enzymes related to energy-generating pathways, particularly glycolysis and the tricarboxylic acid cycle, were the most strongly affected. Thus, the data indicate that antimicrobial VOCs interfere with essential metabolic pathways in P. citricarpa to prevent fungal growth., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Purpose of Review: We review and offer our clinical perspectives on the emergence of echinocandin-resistant Candida., Recent Findings: Candida FKS gene mutations attenuate echinocandin activity, but overall mutation rates among clinical isolates remain low (Candida glabrata, ∼4%; other species, <1%). Rates are higher with prior echinocandin exposure, exceeding 50% among C. glabrata or Candida albicans isolates causing breakthrough invasive candidiasis. The median duration of prior echinocandin exposure among FKS mutant isolates is ∼100 days. The clinical usefulness of echinocandin susceptibility testing is limited by the low overall prevalence of resistance, and uncertainties surrounding testing methods and interpretation of minimum inhibitory concentrations (MICs). In single-center studies, caspofungin resistance (defined using institution-specific MIC breakpoints) was 32-53% sensitive and 75-95% specific for predicting treatment outcomes of C. glabrata invasive candidiasis; corresponding values for the presence of an FKS mutation were 35-41% and 90-98%. Results were similar using anidulafungin and micafungin MICs. Clinical data are scarce for non-C. glabrata species., Summary: Echinocandins remain preferred agents against invasive Candida infections. Susceptibility testing and FKS genotypic testing do not have roles in routine clinical practice, but may be useful in newly-diagnosed patients who are echinocandin-experienced or those who have not responded to echinocandin treatment.

Leptographium qinlingensis is a fungal associate of the Chinese white pine beetle (Dendroctonus armandi) and a pathogen of the Chinese white pine (Pinus armandi) that must overcome the terpenoid oleoresin defenses of host trees. L. qinlingensis responds to monoterpene flow with abundant mechanisms that include export and the use of these compounds as a carbon source. As one of the fungal cytochrome P450 proteins (CYPs), which play important roles in general metabolism, CYP51 (lanosterol 14-α demethylase) can catalyze the biosynthesis of ergosterol and is a target for antifungal drug. We have identified an L. qinlingensis CYP51F1 gene, and the phylogenetic analysis shows the highest homology with the 14-α-demethylase sequence from Grosmannia clavigera (a fungal associate of Dendroctonus ponderosae). The transcription level of CYP51F1 following treatment with terpenes and pine phloem extracts was upregulated, while using monoterpenes as the only carbon source led to the downregulation of CYP5F1 expression. The homology modeling structure of CYP51F1 is similar to the structure of the lanosterol 14-α demethylase protein of Saccharomyces cerevisiae YJM789, which has an N-terminal membrane helix 1 (MH1) and transmembrane helix 1 (TMH1). The minimal inhibitory concentrations (MIC) of terpenoid and azole fungicides (itraconazole (ITC)) and the docking of terpenoid molecules, lanosterol and ITC in the protein structure suggested that CYP51F1 may be inhibited by terpenoid molecules by competitive binding with azole fungicides.

Extracellular cellobiase activity of Termitomyces clypeatus increased from 2.9 U ml(-1) to 4.4 and 4.1 in presence of dithiothreitol (DTT) and β-mercaptoethanol (ME), respectively, with a decrease in Km from 0.4 to 0.3 mM (DTT) and 0.35 mM (ME). Catalysis was further enhanced if the reduced enzyme was alkylated and activity increased from 11.4 U ml(-1) (control) to 15.2 (DTT+N-ethylmaleimide) and 15.3 (DTT+iodoacetamide) using p-nitrophenyl-β-D-glucopyranoside and from 14.6 U ml(-1)(control) to 21.9 (DTT+N-ethylmaleimide) and 18.7 (DTT+iodoacetamide) using cellobiose. The reduced enzyme showed 17 % lesser glucose inhibition. CD and tryptophan fluorescence showed no change in secondary structure was caused by DTT up to 50 mM. Cysteine content of the enzyme was 24 %. It is postulated that reduction of disulphide bonds allows better substrate affinity for cellobiase. The studies describe a novel and simple method to increase cellobiase activity for industrial applications.

Purpose of Review: Echinocandin resistance in Candida is a great concern, as the echinocandin drugs are recommended as first-line therapy for patients with invasive candidiasis. Here, we review recent advances in our understanding of the epidemiology, underlying mechanisms, methods for detection and clinical implications., Recent Findings: Echinocandin resistance has emerged over the recent years. It has been found in most clinically relevant Candida spp., but is most common in C. glabrata with rates exceeding 10% at selected institutions. It is most commonly detected after 3-4 weeks of treatment and is associated with a dismal outcome. An extensive list of mutations in hot spot regions of the genes encoding the target has been characterized and associated with species and drug-specific loss of susceptibility. The updated antifungal susceptibility testing reference methods identify echinocandin-resistant isolates reliably, although the performance of commercial tests is somewhat more variable. Alternative technologies are being developed, including molecular detection and matrix-assisted laser desorption ionization-time of flight., Summary: Echinocandin resistance is increasingly encountered and its occurrence makes susceptibility testing essential, particularly in patients with prior exposure. The further development of rapid and user-friendly commercially available susceptibility platforms is warranted. Antifungal stewardship is important in order to minimize unnecessary selection pressure.

Pneumocystis pneumonia (PcP) is a significant cause of morbidity and mortality in immunocompromised patients. In humans, PcP is caused by the opportunistic fungal species Pneumocystis jirovecii. Progress in Pneumocystis research has been hampered by a lack of viable in vitro culture methods, which limits laboratory access to human-derived organisms for drug testing. Consequently, most basic drug discovery research for P. jirovecii is performed using related surrogate organisms such as Pneumocystis carinii, which is derived from immunosuppressed rodents. While these studies provide useful insights, important questions arise about interspecies variations and the relative utility of identified anti-Pneumocystis agents against human P. jirovecii. Our recent work has identified the histone acetyltransferase (HAT) Rtt109 in P. carinii (i.e., PcRtt109) as a potential therapeutic target for PcP, since Rtt109 HATs are widely conserved in fungi but are absent in humans. To further address the potential utility of this target in human disease, we now demonstrate the presence of a functional Rtt109 orthologue in the clinically relevant fungal pathogen P. jirovecii (i.e., PjRtt109). In a fashion similar to that of Pcrtt109, Pjrtt109 restores H3K56 acetylation and genotoxic resistance in rtt109-null yeast. Recombinant PjRtt109 is an active HAT in vitro, with activity comparable to that of PcRtt109 and yeast Rtt109. PjRtt109 HAT activity is also enhanced by the histone chaperone Asf1 in vitro. PjRtt109 and PcRtt109 showed similar low micromolar sensitivities to two reported small-molecule HAT inhibitors in vitro. Together, these results demonstrate that PjRtt109 is a functional Rtt109 HAT, and they support the development of anti-Pneumocystis agents directed at Rtt109-catalyzed histone acetylation as a novel therapeutic target for human PcP., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Nowadays, abnormal hyperpigmentation in human skin such as melasma, freckles, and chloasma has become a serious esthetic problem. Cutaneous depigmenting agents could be used to treat these hyperpigmentation-associated dieseases. Dodoviscin A is a natural product isolated from the aerial parts of Dodonaea viscosa. In the present study, we evaluated the effect of dodoviscin A on melanin production in B16-F10 melanoma cells for the first time. We found that dodoviscin A inhibited melanin biosynthesis induced by 3-isobutyl-1-methylxanthine and PD98059 significantly, and there was no obvious effect on the viability of dodoviscin A-treated B16-F10 cells. Meanwhile, dodoviscin A could suppress the activity of mushroom tyrosinase in the cell-free assay system and also decrease 3-isobutyl-1-methylxanthine-induced tyrosinase activity and expression of mature tyrosinase protein in B16-F10 cells. Western blotting analysis showed that dodoviscin A inhibited 3-isobutyl-1-methylxanthine and forskolin-induced phosphorylation of the cAMP response element binding protein in B16-F10 cells. These results indicate that dodoviscin A may be a new promising pigmentation-altering agent for cosmetic and therapeutic applications., (Georg Thieme Verlag KG Stuttgart · New York.)

Elucidation of bacterial and fungal interactions in multispecies biofilms will have major impacts on understanding the pathophysiology of infections. The objectives of this study were to (i) evaluate the effect of Pseudomonas aeruginosa lipopolysaccharide (LPS) on Candida albicans hyphal development and transcriptional regulation, (ii) investigate protein expression during biofilm formation, and (iii) propose likely molecular mechanisms for these interactions. The effect of LPS on C. albicans biofilms was assessed by XTT-reduction and growth curve assays, light microscopy, scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM). Changes in candidal hypha-specific genes (HSGs) and transcription factor EFG1 expression were assessed by real-time polymerase chain reaction and two-dimensional gel electrophoresis, respectively. Proteome changes were examined by mass spectrometry. Both metabolic activities and growth rates of LPS-treated C. albicans biofilms were significantly lower (P < 0.05). There were higher proportions of budding yeasts in test biofilms compared with the controls. SEM and CLSM further confirmed these data. Significantly upregulated HSGs (at 48 h) and EFG1 (up to 48 h) were noted in the test biofilms (P < 0.05) but cAMP levels remained unaffected. Proteomic analysis showed suppression of candidal septicolysin-like protein, potential reductase-flavodoxin fragment, serine hydroxymethyltransferase, hypothetical proteins Cao19.10301(ATP7), CaO19.4716(GDH1), CaO19.11135(PGK1), CaO19.9877(HNT1) by P. aeruginosa LPS. Our data imply that bacterial LPS inhibit C. albicans biofilm formation and hyphal development. The P. aeruginosa LPS likely target glycolysis-associated mechanisms during candidal filamentation., (© 2012 John Wiley & Sons A/S.)

The biofilm of Candida albicans has been implicated as a source of bloodstream infections. Dispersal cells, as the final biofilm stage, are responsible for its spread. The aim of this study was to compare the susceptibility of biofilm and dispersal cells vs. planktonic cells (overnight liquid culture) of C. albicans to caspofungin (CAS) and fluconazole (FLU) when the drugs were added: i) at the beginning of the experiment; ii) after 1.5 h (adherence stage); iii) after 24 h (early mature biofilm). The findings were evaluated after 48 h (mature biofilm) using the XTT reduction assay. Later administration of the drug increased biofilm sessile minimal inhibitory concentration (SMIC(80)) of both FLU and CAS from 1 μg mL(-1) to over 64 μg mL(-1) and from 0.125 μg mL(-1) to over 16 μg mL(-1), respectively. Susceptibility of dispersal cells also decreased with time of administration. We also determined the expression of the Als1 and Als3 genes in 48-h sessile biofilm and dispersal cells of C. albicans SC5314 and compared it to planktonic cells. The expression was normalised to the standard Act1 gene in every condition tested. Quantitative real-time PCR revealed a strong up-regulation of the Als1 gene in the dispersal cells but not in biofilm and high expression of the Als3 gene in both biofilm and dispersal cells. High expression of both Als1 and Als3 genes supports the hypothesis that dispersal cells pose a high-risk of infection.

In an environment with simulated elevated atmospheric ozone, two genotypes of snap bean (Phaseolus vulgaris L.) that differed in O3 sensitivity (O3-sensitive: S156; O3-tolerant: R123) were selected as host plants for arbuscular mycorrhizal (AM) fungi. The objective was to investigate the effect of elevated O3 on the AM structure and glomalin production in two genotypes, and to understand the effect of elevated O3 the growth of AM fungi and formation of AM structure. The results showed that in comparison with ambient O3 (20 nL x L(-1)), elevated O3 (70 nL x L(-1)) significantly decreased the mycorrhizal colonization rate in both genotypes, particularly the S156 plant (decreased by 43.6%). Elevated O3 exposed a great negative effect on the AM structure in both genotypes. For example, the arbuscule number per unit root, and the length of external hyphae in root compartment and hyphal compartment were significantly decreased, especially in S156 plant. However, the effect of elevated atmospheric O3 was not significant on the vesicule number per unit root in both genotypes. Total glomalin production in mycorrhizosphere and hyphosphere of both genotypes was only slightly affected by elevated O3, however, the production of easily extractable glomalin was significantly increased. In addition, no evident difference in glomalin concentration was observed between two genotypes at either ambient O3 or elevated O3. This study showed that the mycorrhizal colonization rate, AM structure and production of easily extractable glomalin gave great response to elevated O3 especially in the O3-sensitive plant.

Signal transduction systems comprising histidine kinases are suggested as new molecular targets of antibiotics. The important human fungal pathogen Candida albicans possesses three histidine kinases, one of which is the type III histidine kinase CaNik1, which activates the MAP kinase Hog1. We established a screening system for inhibitors of this class of histidine kinases by functional expression of the CaNIK1 gene in S. cerevisiae. This transformant was susceptible to fungicides to which the wild type strain was resistant, such as fludioxonil and ambruticin. Growth inhibition correlated with phosphorylation of Hog1 and was dependent on an intact Hog1 pathway. At the N-terminus the histidine kinase CaNik1 has four amino acid repeats of 92 amino acids each and one truncated repeat of 72 amino acids. Within these repeats we identified 9 HAMP domains with a paired structure. We constructed mutants in which one or two pairs of these domains were deleted. S. cerevisiae transformants expressing the full-length CaNIK1 showed the highest sensitivity to the fungicides, any truncation reduced the susceptibility of the transformants to the fungicides. This indicates that the HAMP domains are decisive for the mode of action of the antifungal compounds., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Aim: Candida glabrata is a major pathogen in humans known to be intrinsically resistant to fluconazole. However, genotypic, phenotypic, and proteomic changes associated with reduced susceptibility to fluconazole are not properly understood. The aim of this study was to observe specific phenotypic, chromosomal, and proteomic alterations in a Candida glabrata strain sequentially exposed to fluconazole., Methods: Candida glabrata was exposed to increased concentrations of fluconazole in RPMI for 55 days. Phenotypic changes were evaluated using standard assays. Molecular/proteomic changes in C. glabrata were analyzed by contour-clamped homogeneous electric field electrophoresis, reverse transcription-polymerase chain reaction, and mass spectrometry., Results:   Candida glabrata demonstrated increased fluconazole resistance (>256 μg/mL), with extensive cross-resistance to ketoconazole (0.38-3.0 μg), itraconazole (8 to >32 μg), and voriconazole (0.125-1.5 μg). Morphologically dissimilar colonies on RPMI/fluconazole agar demonstrated variable chromosomal profiles compared with the control isolate. Stable chromosomal changes were associated with a significantly higher (P<0.05) mRNA level of the hemolysin gene compared with the control. Phenotypic switching on CuSO4 agar was associated with variable metallothionein mRNA transcription levels. The proteome analysis of a fluconazole-resistant offshoot demonstrated a total of 98 protein spots, 25 showing a twofold upregulation., Conclusion: Fluconazole exposure initiates the chance evolution of a new colonizing population with specific virulence traits., (© 2011 Blackwell Publishing Asia Pty Ltd.)

Aim: To synthesize a novel polyamide SL-A92 and evaluate its bioactivity against drug resistance in Candida albicans., Methods: SL-A92 was synthesized using N-hydroxybenzotriazole (HOBT)/N,N'-dicyclohexylcarbodiimide (DCC) in solution phase. Its antifungal activities and effects on strain growth were tested using the micro-broth dilution method and growth curves, respectively. Induced drug resistance in the C. albicans collection strain SC5314 was obtained by incubation with fluconazole (12 microg/mL) for 21 passages. Meanwhile, incubations with SL-A92 plus fluconazole were also carried out in SC5314 strains, and the MIC(80)s were used to evaluate the inhibitory effects of SL-A92 on drug resistance during the induction process. Real time RT-PCR was performed to investigate the CDR1 and CDR2 mRNA levels in induced SC5314 strains., Results: SC5314 strain induced by the combination of fluconazole and SL-A92 (200 microg/mL) did not develop drug resistance. On day 24, the CDR1 and CDR2 mRNA levels in SC5314 strain co-treated with fluconazole and SL-A92 relative to fluconazole alone were 26% and 24%, respectively, and on day 30 the CDR1 and CDR2 mRNA levels were 43% and 31%, respectively., Conclusion: SL-A92 can block the development of drug resistance during the fluconazole induction process, which partially results from the down-regulation of CDR1 and CDR2.

Background: The filamentous fungus Aspergillus niger is well-known as a producer of primary metabolites and extracellular proteins. For example, glucoamylase is the most efficiently secreted protein of Aspergillus niger, thus the homologous glucoamylase (glaA) promoter as well as the glaA signal sequence are widely used for heterologous protein production. Xylose is known to strongly repress glaA expression while maltose is a potent inducer of glaA promoter controlled genes. For a more profound understanding of A. niger physiology, a comprehensive analysis of the intra- and extracellular proteome of Aspergillus niger AB1.13 growing on defined medium with xylose or maltose as carbon substrate was carried out using 2-D gel electrophoresis/Maldi-ToF and nano-HPLC MS/MS., Results: The intracellular proteome of A. niger growing either on xylose or maltose in well-aerated controlled bioreactor cultures revealed striking similarities. In both cultures the most abundant intracellular protein was the TCA cycle enzyme malate-dehydrogenase. Moreover, the glycolytic enzymes fructose-bis-phosphate aldolase and glyceraldehyde-3-phosphate-dehydrogenase and the flavohemoglobin FhbA were identified as major proteins in both cultures. On the other hand, enzymes involved in the removal of reactive oxygen species, such as superoxide dismutase and peroxiredoxin, were present at elevated levels in the culture growing on maltose but only in minor amounts in the xylose culture. The composition of the extracellular proteome differed considerably depending on the carbon substrate. In the secretome of the xylose-grown culture, a variety of plant cell wall degrading enzymes were identified, mostly under the control of the xylanolytic transcriptional activator XlnR, with xylanase B and ferulic acid esterase as the most abundant ones. The secretome of the maltose-grown culture did not contain xylanolytic enzymes, instead high levels of catalases were found and glucoamylase (multiple spots) was identified as the most abundant extracellular protein. Surprisingly, the intracellular proteome of A. niger growing on xylose in bioreactor cultures differed more from a culture growing in shake flasks using the same medium than from the bioreactor culture growing on maltose. For example, in shake flask cultures with xylose as carbon source the most abundant intracellular proteins were not the glycolytic and the TCA cycle enzymes and the flavohemoglobin, but CipC, a protein of yet unknown function, superoxide dismutase and an NADPH dependent aldehyde reductase. Moreover, vacuolar proteases accumulated to higher and ER-resident chaperones and foldases to lower levels in shake flask compared to the bioreactor cultures., Conclusions: The utilization of xylose or maltose was strongly affecting the composition of the secretome but of minor influence on the composition of the intracellular proteome. On the other hand, differences in culture conditions (pH control versus no pH control, aeration versus no aeration and stirring versus shaking) have a profound effect on the intracellular proteome. For example, lower levels of ER-resident chaperones and foldases and higher levels of vacuolar proteases render shake flask conditions less favorable for protein production compared to controlled bioreactor cultures.

The Penicillium chrysogenum antifungal protein PAF inhibits polar growth and induces apoptosis in Aspergillus nidulans. We report here that two signalling cascades are implicated in its antifungal activity. PAF activates the cAMP/protein kinase A (Pka) signalling cascade. A pkaA deletion mutant exhibited reduced sensitivity towards PAF. This was substantiated by the use of pharmacological modulators: PAF aggravated the effect of the activator 8-Br-cAMP and partially relieved the repressive activity of caffeine. Furthermore, the Pkc/mitogen-activated protein kinase (Mpk) signalling cascade mediated basal resistance to PAF, which was independent of the small GTPase RhoA. Non-functional mutations of both genes resulted in hypersensitivity towards PAF. PAF did not increase MpkA phosphorylation or induce enzymes involved in the remodelling of the cell wall, which normally occurs in response to activators of the cell wall integrity pathway. Notably, PAF exposure resulted in actin gene repression and a deregulation of the chitin deposition at hyphal tips of A. nidulans, which offers an explanation for the morphological effects evoked by PAF and which could be attributed to the interconnection of the two signalling pathways. Thus, PAF represents an excellent tool to study signalling pathways in this model organism and to define potential fungal targets to develop new antifungals.

We investigated the effects of divalent alkaline earth and first-row transition metal and zinc ions on α-glucosidase activity in vitro and in vivo. CuSO₄ and ZnSO₄ exhibited a high α-glucosidase inhibitory effect in vitro. The IC(50) values of CuSO₄ were 0.77 ± 0.01 (substrate; maltose) and 0.78 ± 0.01 (substrate; sucrose), and those of ZnSO₄ were 5.49 ± 0.14 (substrate; maltose) and 4.70 ± 0.06 (substrate; sucrose) for yeast α-glucosidase. On the basis of Lineweaver-Burk plots, both CuSO₄ and ZnSO₄ exhibited different modes of inhibition against α-glucosidase. Subsequently, oral glucose and sucrose tolerance tests (OGTT and OSTT) were performed on non-diabetic ddY mice to examine the effect of the metal ions on their blood glucose levels. As a result of single oral administration of CuSO₄ in non-diabetic ddY mice, a significant and potent lowering of the blood glycemic response toward disaccharide, sucrose, ingestion was observed at 45 min after doses of 0.08 and 0.24 mmol kg(-1) body weight. In contrast, the CuSO₄ administration showed no suppression of the elevation of blood glucose levels in mice after a monosaccharide, glucose, administration. These results indicate that CuSO₄ suppresses disaccharide digestion by inhibiting α-glucosidase activity in the epithelium of the small intestine, suggesting that antidiabetic Cu complexes with some ligands have a similar action mechanism to that of α-glucosidase inhibitor, acarbose, currently used for clinical purposes.

G protein-coupled receptors (GPCRs) are important targets for various drugs that are on the market. An important area in which we urgently need novel drug targets is the field of antifungals. Recently a new class of nutrient sensing G protein-coupled receptors have been identified in fungi. The founding member of this novel class of GPCRs is Gpr1, the sucrose/glucose sensing receptor of Saccharomyces cerevisiae. This receptor activates the cAMP-PKA pathway that is important for the yeast-to-pseudohypha transition and for stress tolerance. The capacity to change morphology is an important virulence factor for pathogenic fungi and it has been shown in these fungi that morphogenesis also depends on the cAMP-PKA pathway. Therefore, functional Gpr1 homologous receptors in these fungi may be interesting targets for antifungals. Despite the fact that these receptors are not essential for growth, modification of their activity may affect fitness of the fungus allowing the human or plant defense systems to win the battle. In addition, synthetic avirulent interactions, such as the one observed between the C. albicans Gpr1 and the trehalose-6-phosphate phosphatase enzyme, may be a tool to come up with a good combination therapy approach. It will be necessary to identify antagonists or inverse agonists for these receptors, which will require good screening systems and large compound libraries.

Azoles are the mainstay of oral therapy for aspergillosis. Azole resistance in Aspergillus has been reported infrequently. The first resistant isolate was detected in 1999 in Manchester, UK. In a clinical collection of 519 A. fumigatus isolates, the frequency of itraconazole resistance was 5%, a significant increase since 2004 (p<0.001). Of the 34 itraconazole-resistant isolates we studied, 65% (22) were cross-resistant to voriconazole and 74% (25) were cross-resistant to posaconazole. Thirteen of 14 evaluable patients in our study had prior azole exposure; 8 infections failed therapy (progressed), and 5 failed to improve (remained stable). Eighteen amino acid alterations were found in the target enzyme, Cyp51A, 4 of which were novel. A population genetic analysis of microsatellites showed the existence of resistant mutants that evolved from originally susceptible strains, different cyp51A mutations in the same strain, and microalterations in microsatellite repeat number. Azole resistance in A. fumigatus is an emerging problem and may develop during azole therapy.

The chemical-genetic profile can be defined as quantitative values of deletion strains' growth defects under exposure to chemicals. In yeast, the compendium of chemical-genetic profiles of genomewide deletion strains under many different chemicals has been used for identifying direct target proteins and a common mode-of-action of those chemicals. In the previous study, valuable biological information such as protein-protein and genetic interactions has not been fully utilized. In our study, we integrated this compendium and biological interactions into the comprehensive collection of approximately 490 protein complexes of yeast for model-based prediction of a drug's target proteins and similar drugs. We assumed that those protein complexes (PCs) were functional units for yeast cell growth and regarded them as hidden factors and developed the PC-based Bayesian factor model that relates the chemical-genetic profile at the level of organism phenotypes to the hidden activities of PCs at the molecular level. The inferred PC activities provided the predictive power of a common mode-of-action of drugs as well as grouping of PCs with similar functions. In addition, our PC-based model allowed us to develop a new effective method to predict a drug's target pathway, by which we were able to highlight the target-protein, TOR1, of rapamycin. Our study is the first approach to model phenotypes of systematic deletion strains in terms of protein complexes. We believe that our PC-based approach can provide an appropriate framework for combining and modeling several types of chemical-genetic profiles including interspecies. Such efforts will contribute to predicting more precisely relevant pathways including target proteins that interact directly with bioactive compounds.

The effects of acidified-nitrite stress on the growth initiation and intracellular pH (pH(i)) of individual cells of Debaryomyces hansenii and Candida zeylanoides were investigated. Our results show that 200 microg/ml of nitrite caused pronounced growth inhibition and intracellular acidification of D. hansenii at an external pH (pH(ex)) value of 4.5 but did not at pH(ex) 5.5. These results indicate that nitrous acid as such plays an important role in the antifungal effect of acidified nitrite. Furthermore, both yeast species experienced severe growth inhibition and a pH(i) decrease at pH(ex) 4.5, suggesting that at least some of the antifungal effects of acidified nitrite may be due to intracellular acidification. For C. zeylanoides, this phenomenon could be explained in part by the uncoupling effect of energy generation from growth. Debaryomyces hansenii was more tolerant to acidified nitrite at pH(ex) 5.5 than C. zeylanoides, as determined by the rate of growth initiation. In combination with the fact that D. hansenii was able to maintain pH(i) homeostasis at pH(ex) 5.5 but C. zeylanoides was not, our results suggest that the ability to maintain pH(i) homeostasis plays a role in the acidified-nitrite tolerance of D. hansenii and C. zeylanoides. Possible mechanisms underlying the different abilities of the two yeast species to maintain their pH(i) homeostasis during acidified-nitrite stress, comprising the intracellular buffer capacity and the plasma membrane ATPase activity, were investigated, but none of these mechanisms could explain the difference.

Numerous hydrophobic and amphipathic compounds including several detergents are known to inhibit the ubiquinone reductase reaction of respiratory chain complex I (proton pumping NADH:ubiquinone oxidoreductase). Guided by the X-ray structure of the peripheral arm of complex I from Thermus thermophilus we have generated a large collection of site-directed mutants in the yeast Yarrowia lipolytica targeting the proposed ubiquinone and inhibitor binding pocket of this huge multiprotein complex at the interface of the 49-kDa and PSST subunits. We could identify a number of residues where mutations changed I(50) values for representatives from all three groups of hydrophobic inhibitors. Many mutations around the domain of the 49-kDa subunit that is homologous to the [NiFe] centre binding region of hydrogenase conferred resistance to DQA (class I/type A) and rotenone (class II/type B) indicating a wider overlap of the binding sites for these two types of inhibitors. In contrast, a region near iron-sulfur cluster N2, where the binding of the n-alkyl-polyoxyethylene-ether detergent C(12)E(8) (type C) was exclusively affected, appeared comparably well separated. Taken together, our data provide structure-based support for the presence of distinct but overlapping binding sites for hydrophobic inhibitors possibly extending into the ubiquinone reduction site of mitochondrial complex I.

Object: To determine if tetracycline, previously reported to increase the probability of developing symptomatic vaginal yeast infections, has a direct effect on Candida albicans growth or induction of virulent phenotypes., Method: In vitro, clinical isolates of yeast were cultivated with sublethal concentrations of tetracycline and yeast cell counts, hyphal formation, drug efflux pump activity, biofilm production, and hemolysin production were determined by previously reported methods., Results: Tetracycline concentrations above 150 microg/mL inhibited Candida albicans, but at submicrogram/mL, a modest growth increase during the early hours of the growth curve was observed. Tetracycline did not inhibit hyphal formation at sublethal concentrations. Hypha formation appeared augmented by exposure to tetracycline in the presence of chemically defined medium and especially in the presence of human serum. Efflux pump CDR1 was upregulated and a nonsignificant trend toward increased biofilm formation was noted., Conclusion: Tetracycline appears to have a small growth enhancing effect and may influence virulence through augmentation of hypha formation, and a modest effect on drug efflux and biofilm formation, although tetracycline did not affect hemolysin. It is not clear if the magnitude of the effect is sufficient to attribute vaginitis following tetracycline treatment to direct action of tetracycline on yeast.

Candida albicans hypha formation which has been stimulated via the Ras1-cAMP-Efg1 signalling cascade is inhibited by farnesol, a C. albicans autoregulatory factor, and small molecules such as dodecanol. In cultures containing farnesol or dodecanol, hypha formation was restored upon addition of dibutyryl-cAMP. The CAI4-Ras1(G13V) strain, which carries a dominant-active variant of Ras1 and forms hyphae in the absence of inducing stimuli, grew as yeast in medium with farnesol or dodecanol; the heat shock sensitivity of the CAI4-Ras1(G13V) strain was also suppressed by these compounds. Neither Pde1 nor Pde2 was necessary for the repression of hyphal growth by farnesol or dodecanol. Two transcripts, CTA1 and HSP12, which are at higher levels upon mutation of Ras1 or Cdc35, were increased in abundance in cells grown with farnesol or dodecanol. Microscopic analysis of strains carrying CTA1 and HWP1 promoter fusions grown with intermediate concentrations of farnesol or dodecanol indicated a link between cells with the increased expression of cAMP-repressed genes and cells repressed for hypha formation. Because several cAMP-controlled outputs are affected by farnesol and dodecanol, our findings suggest that these compounds impact activity of the Ras1-Cdc35 pathway, thus leading to an alteration of C. albicans morphology.

The mitochondrial outer membrane contains protein import machineries, the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). It has been speculated that TOM or SAM are required for Bax-induced release of intermembrane space (IMS) proteins; however, experimental evidence has been scarce. We used isolated yeast mitochondria as a model system and report that Bax promoted an efficient release of soluble IMS proteins while preproteins were still imported, excluding an unspecific damage of mitochondria. Removal of import receptors by protease treatment did not inhibit the release of IMS proteins by Bax. Yeast mutants of each Tom receptor and the Tom40 channel were not impaired in Bax-induced protein release. We analyzed a large collection of mutants of mitochondrial outer membrane proteins, including SAM, fusion and fission components, but none of these components was required for Bax-induced protein release. The released proteins included complexes up to a size of 230 kDa. We conclude that Bax promotes efficient release of IMS proteins through the outer membrane of yeast mitochondria while the inner membrane remains intact. Inactivation of the known protein import and sorting machineries of the outer membrane does not impair the function of Bax at the mitochondria.