Mitochondrial ferredoxin determines vulnerability of cells to copper excess

The essential micronutrient copper is tightly regulated in organisms, as environmental exposure or homeostasis defects can cause toxicity and neurodegenerative disease. The principal target(s) of copper toxicity have not been pinpointed, but one key effect is impaired supply of iron-sulphur (FeS) clusters to the essential protein Rli1 (ABCE1). Here, to find upstream FeS-biosynthesis/delivery protein(s) responsible for this, we compared copper sensitivity of yeast overexpressing candidate targets. Overexpression of the mitochondrial ferredoxin Yah1 produced copper hyper-resistance. 55Fe turnover assays revealed that FeS1-integrity of Yah1 was particularly vulnerable to copper among the test proteins. Furthermore, destabilization of the FeS domain of Yah1 produced copper hypersensitivity, and YAH1 overexpression rescued Rli1 dysfunction. This copper-resistance function was conserved in the human ferredoxin, Fdx2. The data indicate that the essential mitochondrial ferredoxin is an important copper target, determining a tipping point where plentiful copper supply becomes excess. This knowledge could help in tackling copper-related diseases

Discovery of Natural Products With Antifungal Potential Through Combinatorial Synergy

The growing prevalence of antifungal drug resistance coupled with the slow development of new, acceptable drugs and fungicides has raised interest in natural products (NPs) for their therapeutic potential and level of acceptability. However, a number of well-studied NPs are considered promiscuous molecules. In this study, the advantages of drug–drug synergy were exploited for the discovery of pairwise NP combinations with potentiated antifungal activity and, potentially, increased target specificity. A rational approach informed by previously known mechanisms of action of selected NPs did not yield novel antifungal synergies. In contrast, a high-throughput screening approach with yeast revealed 34 potential synergies from 800 combinations of a diverse NP library with four selected NPs of interest (eugenol, EUG; β-escin, ESC; curcumin, CUR; berberine hydrochloride, BER). Dedicated assays validated the most promising synergies, namely, EUG + BER, CUR + sclareol, and BER + pterostilbene (PTE) [fractional inhibitory concentrations (FIC) indices ≤ 0.5 in all cases], reduced to as low as 35 (BER) and 7.9 mg L–1 (PTE). These three combinations synergistically inhibited a range of fungi, including human or crop pathogens Candida albicans, Aspergillus fumigatus, Zymoseptoria tritici, and Botrytis cinerea, with synergy also against azole-resistant isolates and biofilms. Further investigation indicated roles for mitochondrial membrane depolarization and reactive oxygen species (ROS) formation in the synergistic mechanism of EUG + BER action. This study establishes proof-of-principle for utilizing high-throughput screening of pairwise NP interactions as a tool to find novel antifungal synergies. Such NP synergies, with the potential also for improved specificity, may help in the management of fungal pathogens

Method for RNA extraction and transcriptomic analysis of single fungal spores

Transcriptomic analysis of single cells has been increasingly in demand in recent years, thanks to technological and methodological advances as well as growing recognition of the importance of individuals in biological systems. However, the majority of these studies have been performed in mammalian cells, due to their ease of lysis and high RNA content. No single cell transcriptomic analysis has yet been applied to microbial spores, even though it is known that heterogeneity at the phenotype level exists among individual spores. Transcriptomic analysis of single spores is challenging, in part due to the physically robust nature of the spore wall. This precludes the use of methods commonly used for mammalian cells. Here, we describe a simple method for extraction and amplification of transcripts from single fungal conidia (asexual spores), and its application in single-cell transcriptomics studies. The method can also be used for studies of small numbers of fungal conidia, which may be necessary in the case of limited sample availability, low-abundance transcripts or interest in small subpopulations of conidia.• The method allows detection of transcripts from single conidia of Aspergillus niger• The method allows detection of genomic DNA from single conidia of Aspergillus nige

Potentiated inhibition of Trichoderma virens and other environmental fungi by new biocide combinations

Fungi cause diverse, serious socio-economic problems, including biodeterioration of valuable products and materials that spawns a biocides industry worth ~$11 billion globally. To help combat environmental fungi that commonly colonise material products, this study tested the hypothesis that combination of an approved fungicide with diverse agents approved by the FDA (Food and Drug Administration) could reveal potent combinatorial activities with promise for fungicidal applications. The strategy to use approved compounds lowers potential development risks for any effective combinations. A high-throughput assay of 1,280 FDA-approved compounds was conducted to find those that potentiate the effect of iodopropynyl-butyl-carbamate (IPBC) on the growth of Trichoderma virens; IPBC is one of the two most widely used Biocidal Products Regulations-approved fungicides. From this library, 34 compounds in combination with IPBC strongly inhibited fungal growth. Low-cost compounds that gave the most effective growth inhibition were tested against other environmental fungi that are standard biomarkers for resistance of synthetic materials to fungal colonization. Trifluoperazine (TFZ) in combination with IPBC enhanced growth-inhibition of three of the five test fungi. The antifungal hexetidine (HEX) potentiated IPBC action against two of the test organisms. Testable hypotheses on the mechanisms of these combinatorial actions are discussed. Neither IPBC + TFZ nor IPBC + HEX exhibited a combinatorial effect against mammalian cells. These combinations retained strong fungal growth-inhibition properties after incorporation to a polymer matrix (alginate) with potential for fungicide delivery. The study reveals the potential of such approved compounds for novel combinatorial applications in the control of fungal environmental-opportunists

Repurposing Nonantifungal Approved Drugs for Synergistic Targeting of Fungal Pathogens

With the spread of drug resistance, new antimicrobials are urgently needed. Here, we set out to tackle this problem by high-throughput exploration for novel antifungal synergies among combinations of approved, nonantifungal drugs; a novel strategy exploiting the potential of alternative targets, low chemicals usage and low development risk. We screened the fungal pathogen Candida albicans by combining a small panel of nonantifungal drugs (all in current use for other clinical applications) with 1280 compounds from an approved drug library. Screens at sublethal concentrations of the antibiotic paromomycin (PM), the antimalarial primaquine (PQ), or the anti-inflammatory drug ibuprofen (IF) revealed a total of 17 potential strong, synergistic interactions with the library compounds. Susceptibility testing with the most promising combinations corroborated marked synergies [fractional inhibitory concentration (FIC) indices ≤0.5] between PM + β-escin, PQ + celecoxib, and IF + pentamidine, reducing the MICs of PM, PQ, and IF in C.albicans by >64-, 16-, and 8-fold, respectively. Paromomycin + β-escin and PQ + celecoxib were effective also against C.albicans biofilms, azole-resistant clinical isolates, and other fungal pathogens. Actions were specific, as no synergistic effect was observed in mammalian cells. Mode of action was investigated for one of the combinations, revealing that PM + β-escin synergistically increase the error-rate of mRNA translation and suggesting a different molecular target to current antifungals. The study unveils the potential of the described combinatorial strategy in enabling acceleration of drug-repurposing discovery for combatting fungal pathogens

Novel combinations of agents targeting translation that synergistically inhibit fungal pathogens

A range of fungicides or antifungals are currently deployed to control fungi in agriculture or medicine, but resistance to current agents is growing so new approaches and molecular targets are urgently needed. Recently, different aminoglycoside antibiotics combined with particular transport inhibitors were found to produce strong, synergistic growth-inhibition of fungi, by synergistically increasing the error rate of mRNA translation. Here, focusing on translation fidelity as a novel target for combinatorial antifungal treatment, we tested the hypothesis that alternative combinations of agents known to affect the availability of functional amino acids would synergistically inhibit growth of major fungal pathogens. We screened 172 novel combinations against three phytopathogens (Rhizoctonia solani, Zymoseptoria tritici, Botrytis cinerea) and three human pathogens (Cryptococcus neoformans, Candida albicans, Aspergillus fumigatus), showing that 48 combinations synergistically inhibited growth. Of these, 23 combinations were effective against more than one pathogen, including combinations comprising food-and-drug approved compounds, e.g., quinine with bicarbonate, and quinine with hygromycin. These combinations (fractional inhibitory combination [FIC] index [less than] 0.5) gave up to 100% reduction of fungal growth yield at concentrations of agents which, individually, had negligible effect. No synergy was evident against bacterial, plant or mammalian cells, indicating specificity for fungi. Mode-of-action analyses for quinine + hygromycin indicated that synergistic mistranslation was the antifungal mechanism. That mechanism was not universal as bicarbonate exacerbated quinine action by increasing drug uptake. The study unveils chemical combinations and a target process with potential for control of diverse fungal pathogens, and suggests repurposing possibilities for several current therapeutics

The fungal threat to global food security

Fungi threaten the security of food supply to human populations on several fronts. They destroy up to 30 % of crop products through disease and spoilage processes, while mycotoxin-producing fungi and opportunistic pathogens endanger food safety. Control of these fungi is vital for improving food security, but current measures are inadequate and further challenges due to human-population growth and climate change are escalating. Investment and innovation in research on strategies to control fungal growth, harnessed through international, inter-disciplinary collaboration across socio-economic boundaries, provides one key to rising to this challenge

Phenotypic heterogeneity is a selected trait in natural yeast populations subject to environmental stress

Summary Populations of genetically uniform microorganisms exhibit phenotypic heterogeneity, where individual cells have varying phenotypes. Such phenotypes include fitness-determining traits. Phenotypic heterogeneity has been linked to increased population-level fitness in laboratory studies, but its adaptive significance for wild microorganisms in the natural environment is unknown. Here, we addressed this by testing heterogeneity in yeast isolates from diverse environmental sites, each polluted with a different principal contaminant, as well as from corresponding control locations. We found that cell-to-cell heterogeneity (in resistance to the appropriate principal pollutant) was prevalent in the wild yeast isolates. Moreover, isolates with the highest heterogeneity were consistently observed in the polluted environments, indicating that heterogeneity is positively related to survival in adverse conditions in the wild. This relationship with survival was stronger than for the property of mean resistance (IC 50) of an isolate. Therefore, heterogeneity could be the major determinant of microbial survival in adverse conditions. Indeed, growth assays indicated that isolates with high heterogeneities had a significant competitive advantage during stress. Analysis of yeasts after cultivation for ≥ 500 generations additionally showed that high heterogeneity evolved as a heritable trait during stress. The results showed that environmental stress selects for wild microorganisms with high levels of phenotypic heterogeneity

Soil aggregates by design: Manufactured aggregates with defined microbial composition for interrogating microbial activities in soil microhabitats

Differences in the structure of microbial communities are reported to exist between the inside and outside of soil aggregates, but the impacts of soil aggregation on microbial activity in soils, essential for soil health, have proven difficult to study in a controlled manner. We have developed a new method to manufacture soil macroaggregates in the laboratory with the ability to introduce microorganisms of choice to the endo- or exo-aggregate environments, offering new avenues for experimental research. X-ray Computed Tomography imaging confirmed the manufactured aggregates had similar overall porosity, pore size and pore connectivity to naturally-formed aggregates. We exploited this new approach to test the hypothesis that microorganisms within aggregates are protected from environmental stresses, in comparison to organisms located near aggregate surfaces. Soil isolates of yeasts introduced to the interior or exterior of manufactured soil aggregates showed no significant difference in the survival of metal- or anoxic-stresses, but organisms within aggregates were protected from heat stress in a time-dependent manner. The results indicate that microbial communities may be protected from particular environmental perturbations by the complex porous architecture that arises from the aggregated soil structure, and underscore the value of this new approach for improving our understanding of the interactions between the soil physical environment and the associated soil biology

Extreme osmotolerance and halotolerance in food-relevant yeasts and the role of glycerol-dependent cell individuality

Osmotolerance and halotolerance are used to describe resistance to sugars and salt, respectively. Here, a comprehensive screen of more than 600 different yeast isolates revealed that osmosensitive species were equally affected by NaCl and glucose. However, the relative toxicity of salt became increasingly prominent in more osmoresistant species. We confirmed that growth inhibition by glucose in a laboratory strain of Saccharomyces cerevisiae occurred at a lower water activity (Aw) than by salt (NaCl), and pre-growth in high levels of glucose or salt gave enhanced cross-resistance to either. Salt toxicity was largely due to osmotic stress but with an additive enhancement due to effects of the relevant cation. Almost all of the yeast isolates from the screen were also noted to exhibit hetero-resistance to both salt and sugar, whereby high concentrations restricted growth to a small minority of cells within the clonal populations. Rare resistant colonies required growth for up to 28 days to become visible. This cell individuality was more marked with salt than sugar, a possible further reflection of the ion toxicity effect. In both cases, heteroresistance in S. cerevisiae was strikingly dependent on the GPD1 gene product, important for glycerol synthesis. In contrast, a tps1? deletant impaired for trehalose showed altered MIC but no change in heteroresistance. Effects on heteroresistance were evident in chronic (but not acute) salt or glucose stress, particularly relevant to growth on low Aw foods. The study reports diverse osmotolerance and halotolerance phenotypes and heteroresistance across an extensive panel of yeast isolates, and indicates that Gpd1-dependent glycerol synthesis is a key determinant enabling growth of rare yeast subpopulations at low Aw, brought about by glucose and in particular salt