Biochemical and cellular characterization of the CISD3 protein: Molecular bases of cluster release and destabilizing effects of nitric oxide.

The NEET proteins, an important family of iron-sulfur (Fe-S) proteins, have generated a strong interest due to their involvement in diverse diseases such as cancer, diabetes, and neurodegenerative disorders. Among the human NEET proteins, CISD3 has been the least studied, and its functional role is still largely unknown. We have investigated the biochemical features of CISD3 at the atomic and in cellulo levels upon challenge with different stress conditions i.e., iron deficiency, exposure to hydrogen peroxide, and nitric oxide. The redox and cellular stability properties of the protein agree on a predominance of reduced form of CISD3 in the cells. Upon the addition of iron chelators, CISD3 loses its Fe-S clusters and becomes unstructured, and its cellular level drastically decreases. Chemical shift perturbation measurements suggest that, upon cluster oxidation, the protein undergoes a conformational change at the C-terminal CDGSH domain, which determines the instability of the oxidized state. This redox-associated conformational change may be the source of cooperative electron transfer via the two [Fe2S2] clusters in CISD3, which displays a single sharp voltammetric signal at -31 mV versus SHE. Oxidized CISD3 is particularly sensitive to the presence of hydrogen peroxide in vitro, whereas only the reduced form is able to bind nitric oxide. Paramagnetic NMR provides clear evidence that, upon NO binding, the cluster is disassembled but iron ions are still bound to the protein. Accordingly, in cellulo CISD3 is unaffected by oxidative stress induced by hydrogen peroxide but it becomes highly unstable in response to nitric oxide treatment.

Targeting the redox vulnerability of acute myeloid leukaemia cells with a combination of auranofin and vitamin C.

Acute myeloid leukaemia (AML) is a heterogeneous disease characterized by complex molecular and cytogenetic abnormalities. Pro-oxidant cellular redox status is a common hallmark of AML cells, providing a rationale for redox-based anticancer strategy. We previously discovered that auranofin (AUF), initially used for the treatment of rheumatoid arthritis and repositioned for its anticancer activity, can synergize with a pharmacological concentration of vitamin C (VC) against breast cancer cell line models. In this study, we observed that this drug combination synergistically and efficiently killed cells of leukaemic cell lines established from different myeloid subtypes. In addition to an induced elevation of reactive oxygen species and ATP depletion, a rapid dephosphorylation of 4E-BP1 and p70S6K, together with a strong inhibition of protein synthesis were early events in response to AUF/VC treatment, suggesting their implication in AUF/VC-induced cytotoxicity. Importantly, a study on 22 primary AML specimens from various AML subtypes showed that AUF/VC combinations at pharmacologically achievable concentrations were effective to eradicate primary leukaemic CD34+ cells from the majority of these samples, while being less toxic to normal cord blood CD34+ cells. Our findings indicate that targeting the redox vulnerability of AML with AUF/VC combinations could present a potential anti-AML therapeutic approach.

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 1280 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 colonisation. 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. KEY POINTS: • Search with an approved fungicide to find new fungicidal synergies in drug libraries. • New combinations inhibit growth of key environmental fungi on different matrices. • The approach enables a more rapid response to demand for new biocides.

Mutational analysis of the Qi-site proton pathway in yeast cytochrome bc1 complex.

The respiratory cytochrome bc1 complex functions as a protonmotive ubiquinol:cytochrome c oxidoreductase. Lysine 228 (K228) located within the quinol reduction (Qi) site of the bc1 complex, has been reported as a key residue for proton transfer during the redox chemistry cycle to substrate quinone at Qi. In yeast, while single mutations had no effect, the combination of K228L and F225L resulted in a severe respiratory growth defect and inhibition of O2 consumption in intact cells. The inhibition was overcome by uncoupling the mitochondrial membrane or by suppressor mutations in the region of K228L-F225L. We propose that the K228L mutation introduces energetic (and kinetic) barriers into normal electron- and proton transfer chemistry at Qi, which are relieved by dissipation of the opposing protonmotive force or through the restoration of favourable intraprotein proton transfer networks via suppressor mutation.

The Candidate Antimalarial Drug MMV665909 Causes Oxygen-Dependent mRNA Mistranslation and Synergizes with Quinoline-Derived Antimalarials.

To cope with growing resistance to current antimalarials, new drugs with novel modes of action are urgently needed. Molecules targeting protein synthesis appear to be promising candidates. We identified a compound (MMV665909) from the Medicines for Malaria Venture (MMV) Malaria Box of candidate antimalarials that could produce synergistic growth inhibition with the aminoglycoside antibiotic paromomycin, suggesting a possible action of the compound in mRNA mistranslation. This mechanism of action was substantiated with a Saccharomyces cerevisiae model using available reporters of mistranslation and other genetic tools. Mistranslation induced by MMV665909 was oxygen dependent, suggesting a role for reactive oxygen species (ROS). Overexpression of Rli1 (a ROS-sensitive, conserved FeS protein essential in mRNA translation) rescued inhibition by MMV665909, consistent with the drug's action on translation fidelity being mediated through Rli1. The MMV drug also synergized with major quinoline-derived antimalarials which can perturb amino acid availability or promote ROS stress: chloroquine, amodiaquine, and primaquine. The data collectively suggest translation fidelity as a novel target of antimalarial action and support MMV665909 as a promising drug candidate.

Human Mitochondrial Cytochrome b Variants Studied in Yeast: Not All Are Silent Polymorphisms.

Variations in mitochondrial DNA (mtDNA) cytochrome b (mt-cyb) are frequently found within the healthy population, but also occur within a spectrum of mitochondrial and common diseases. mt-cyb encodes the core subunit (MT-CYB) of complex III, a central component of the oxidative phosphorylation system that drives cellular energy production and homeostasis. Despite significant efforts, most mt-cyb variations identified are not matched with corresponding biochemical data, so their functional and pathogenic consequences in humans remain elusive. While human mtDNA is recalcitrant to genetic manipulation, it is possible to introduce human-associated point mutations into yeast mtDNA. Using this system, we reveal direct links between human mt-cyb variations in key catalytic domains of MT-CYB and significant changes to complex III activity or drug sensitivity. Strikingly, m.15257G>A (p.Asp171Asn) increased the sensitivity of yeast to the antimalarial drug atovaquone, and m.14798T>C (p.Phe18Leu) enhanced the sensitivity of yeast to the antidepressant drug clomipramine. We demonstrate that while a small number of mt-cyb variations had no functional effect, others have the capacity to alter complex III properties, suggesting they could play a wider role in human health and disease than previously thought. This compendium of new mt-cyb-biochemical relationships in yeast provides a resource for future investigations in humans.

Interplay between the hinge region of iron sulphur protein and the Qo site in the bc1 complex - Analysis of Plasmodium-like mutations in the yeast enzyme.

The respiratory chain bc1 complex is central to mitochondrial bioenergetics and the target of antiprotozoals. We characterized a modified yeast bc1 complex that more closely resemble Plasmodium falciparum enzyme. The mutant version was generated by replacing ten cytochrome b Qo site residues by P. falciparum equivalents. The Plasmodium-like changes caused a major dysfunction of the catalytic mechanism of the bc1 complex resulting in superoxide overproduction and respiratory growth defect. The defect was corrected by substitution of the conserved residue Y279 by a phenylalanine, or by mutations in or in the vicinity of the hinge domain of the iron-sulphur protein. It thus appears that side-reactions can be prevented by the substitution Y279F or the modification of the iron-sulphur protein hinge region. Interestingly, P. falciparum - and all the apicomplexan - contains an unusual hinge region. We replaced the yeast hinge region by the Plasmodium version and combined it with the Plasmodium-like version of the Qo site. This combination restored the respiratory growth competence. It could be suggested that, in the apicomplexan, the hinge region and the cytochrome b Qo site have co-evolved to maintain catalytic efficiency of the bc1 complex Qo site.

Iron-sulfur protein odyssey: exploring their cluster functional versatility and challenging identification.

Iron-sulfur (Fe-S) clusters are an essential and ubiquitous class of protein-bound prosthetic centers that are involved in a broad range of biological processes (e.g. respiration, photosynthesis, DNA replication and repair and gene regulation) performing a wide range of functions including electron transfer, enzyme catalysis, and sensing. In a general manner, Fe-S clusters can gain or lose electrons through redox reactions, and are highly sensitive to oxidation, notably by small molecules such as oxygen and nitric oxide. The [2Fe-2S] and [4Fe-4S] clusters, the most common Fe-S cofactors, are typically coordinated by four amino acid side chains from the protein, usually cysteine thiolates, but other residues (e.g. histidine, aspartic acid) can also be found. While diversity in cluster coordination ensures the functional variety of the Fe-S clusters, the lack of conserved motifs makes new Fe-S protein identification challenging especially when the Fe-S cluster is also shared between two proteins as observed in several dimeric transcriptional regulators and in the mitoribosome. Thanks to the recent development of in cellulo, in vitro, and in silico approaches, new Fe-S proteins are still regularly identified, highlighting the functional diversity of this class of proteins. In this review, we will present three main functions of the Fe-S clusters and explain the difficulties encountered to identify Fe-S proteins and methods that have been employed to overcome these issues.

Regulations of mitoNEET by the key redox homeostasis molecule glutathione.

Human mitoNEET (mNT) and CISD2 are two NEET proteins characterized by an atypical [2Fe-2S] cluster coordination involving three cysteines and one histidine. They act as redox switches with an active state linked to the oxidation of their cluster. In the present study, we show that reduced glutathione but also free thiol-containing molecules such as ß-mercaptoethanol can induce a loss of the mNT cluster under aerobic conditions, while CISD2 cluster appears more resistant. This disassembly occurs through a radical-based mechanism as previously observed with the bacterial SoxR. Interestingly, adding cysteine prevents glutathione-induced cluster loss. At low pH, glutathione can bind mNT in the vicinity of the cluster. These results suggest a potential new regulation mechanism of mNT activity by glutathione, an essential actor of the intracellular redox state.

Redox-Based Strategies against Infections by Eukaryotic Pathogens.

Redox homeostasis is an equilibrium between reducing and oxidizing reactions within cells. It is an essential, dynamic process, which allows proper cellular reactions and regulates biological responses. Unbalanced redox homeostasis is the hallmark of many diseases, including cancer or inflammatory responses, and can eventually lead to cell death. Specifically, disrupting redox balance, essentially by increasing pro-oxidative molecules and favouring hyperoxidation, is a smart strategy to eliminate cells and has been used for cancer treatment, for example. Selectivity between cancer and normal cells thus appears crucial to avoid toxicity as much as possible. Redox-based approaches are also employed in the case of infectious diseases to tackle the pathogens specifically, with limited impacts on host cells. In this review, we focus on recent advances in redox-based strategies to fight eukaryotic pathogens, especially fungi and eukaryotic parasites. We report molecules recently described for causing or being associated with compromising redox homeostasis in pathogens and discuss therapeutic possibilities.

Cellular Responses and Targets in Food Spoilage Yeasts Exposed to Antifungal Prenylated Isoflavonoids.

Prenylated isoflavonoids are phytochemicals with promising antifungal properties. Recently, it was shown that glabridin and wighteone disrupted the plasma membrane (PM) of the food spoilage yeast Zygosaccharomyces parabailii in distinct ways, which led us to investigate further their modes of action (MoA). Transcriptomic profiling with Z. parabailii showed that genes encoding transmembrane ATPase transporters, including Yor1, and genes homologous to the pleiotropic drug resistance (PDR) subfamily in Saccharomyces cerevisiae were upregulated in response to both compounds. Gene functions involved in fatty acid and lipid metabolism, proteostasis, and DNA replication processes were overrepresented among genes upregulated by glabridin and/or wighteone. Chemogenomic analysis using the genome-wide deletant collection for S. cerevisiae further suggested an important role for PM lipids and PM proteins. Deletants of gene functions involved in biosynthesis of very-long-chain fatty acids (constituents of PM sphingolipids) and ergosterol were hypersensitive to both compounds. Using lipid biosynthesis inhibitors, we corroborated roles for sphingolipids and ergosterol in prenylated isoflavonoid action. The PM ABC transporter Yor1 and Lem3-dependent flippases conferred sensitivity and resistance, respectively, to the compounds, suggesting an important role for PM phospholipid asymmetry in their MoAs. Impaired tryptophan availability, likely linked to perturbation of the PM tryptophan permease Tat2, was evident in response to glabridin. Finally, substantial evidence highlighted a role of the endoplasmic reticulum (ER) in cellular responses to wighteone, including gene functions associated with ER membrane stress or with phospholipid biosynthesis, the primary lipid of the ER membrane. IMPORTANCE Preservatives, such as sorbic acid and benzoic acid, inhibit the growth of undesirable yeast and molds in foods. Unfortunately, preservative tolerance and resistance in food spoilage yeast, such as Zygosaccharomyces parabailii, is a growing challenge in the food industry, which can compromise food safety and increase food waste. Prenylated isoflavonoids are the main defense phytochemicals in the Fabaceae family. Glabridin and wighteone belong to this group of compounds and have shown potent antifungal activity against food spoilage yeasts. The present study demonstrated the mode of action of these compounds against food spoilage yeasts by using advanced molecular tools. Overall, the cellular actions of these two prenylated isoflavonoids share similarities (at the level of the plasma membrane) but also differences. Tryptophan import was specifically affected by glabridin, whereas endoplasmic reticulum membrane stress was specifically induced by wighteone. Understanding the mode of action of these novel antifungal agents is essential for their application in food preservation.

HDQ, a potent inhibitor of Plasmodium falciparum proliferation, binds to the quinone reduction site of the cytochrome bc1 complex.

The mitochondrial bc(1) complex is a multisubunit enzyme that catalyzes the transfer of electrons from ubiquinol to cytochrome c coupled to the vectorial translocation of protons across the inner mitochondrial membrane. The complex contains two distinct quinone-binding sites, the quinol oxidation site of the bc(1) complex (Q(o)) and the quinone reduction site (Q(i)), located on opposite sides of the membrane within cytochrome b. Inhibitors of the Q(o) site such as atovaquone, active against the bc(1) complex of Plasmodium falciparum, have been developed and formulated as antimalarial drugs. Unfortunately, single point mutations in the Q(o) site can rapidly render atovaquone ineffective. The development of drugs that could circumvent cross-resistance with atovaquone is needed. Here, we report on the mode of action of a potent inhibitor of P. falciparum proliferation, 1-hydroxy-2-dodecyl-4(1H)quinolone (HDQ). We show that the parasite bc(1) complex--from both control and atovaquone-resistant strains--is inhibited by submicromolar concentrations of HDQ, indicating that the two drugs have different targets within the complex. The binding site of HDQ was then determined by using a yeast model. Introduction of point mutations into the Q(i) site, namely, G33A, H204Y, M221Q, and K228M, markedly decreased HDQ inhibition. In contrast, known inhibitor resistance mutations at the Q(o) site did not cause HDQ resistance. This study, using HDQ as a proof-of-principle inhibitor, indicates that the Q(i) site of the bc(1) complex is a viable target for antimalarial drug development.

Novel Insights into Redox-Based Mechanisms for Auranofin-Induced Rapid Cancer Cell Death.

Auranofin (Ridaura®, AUF) is a gold complex originally approved as an antirheumatic agent that has emerged as a potential candidate for multiple repurposed therapies. The best-studied anticancer mechanism of AUF is the inhibition of thioredoxin reductase (TrxR). However, a number of reports indicate a more complex and multifaceted mode of action for AUF that could be cancer cell type- and dose-dependent. In this study, we observed that AUF displayed variable cytotoxicity in five triple-negative breast cancer cell lines. Using representative MDA-MB-231 cells treated with moderate and cytotoxic doses of AUF, we evidenced that an AUF-mediated TrxR inhibition alone may not be sufficient to induce cell death. Cytotoxic doses of AUF elicited rapid and drastic intracellular oxidative stress affecting the mitochondria, cytoplasm and nucleus. A "redoxome" proteomics investigation revealed that a short treatment with a cytotoxic dose AUF altered the redox state of a number of cysteines-containing proteins, pointing out that the cell proliferation/cell division/cell cycle and cell-cell adhesion/cytoskeleton structure were the mostly affected pathways. Experimentally, AUF treatment triggered a dose-dependent S-phase arrest and a rapid disintegration of the actin cytoskeleton structure. Our study shows a new spectrum of AUF-induced early effects and should provide novel insights into the complex redox-based mechanisms of this promising anticancer molecule.

Deleterious effect of the Qo inhibitor compound resistance-conferring mutation G143A in the intron-containing cytochrome b gene and mechanisms for bypassing it.

The mutation G143A in the inhibitor binding site of cytochrome b confers a high level of resistance to fungicides targeting the bc(1) complex. The mutation, reported in many plant-pathogenic fungi, has not evolved in fungi that harbor an intron immediately after the codon for G143 in the cytochrome b gene, intron bi2. Using Saccharomyces cerevisiae as a model organism, we show here that a codon change from GGT to GCT, which replaces glycine 143 with alanine, hinders the splicing of bi2 by altering the exon/intron structure needed for efficient intron excision. This lowers the levels of cytochrome b and respiratory growth. We then investigated possible bypass mechanisms that would restore the respiratory fitness of a resistant mutant. Secondary mutations in the mitochondrial genome were found, including a point mutation in bi2 restoring the correct exon/intron structure and the deletion of intron bi2. We also found that overexpression of nuclear genes MRS2 and MRS3, encoding mitochondrial metal ion carriers, partially restores the respiratory growth of the G143A mutant. Interestingly, the MRS3 gene from the plant-pathogenic fungus Botrytis cinerea, overexpressed in an S. cerevisiae G143A mutant, had a similar compensatory effect. These bypass mechanisms identified in yeast could potentially arise in pathogenic fungi.

Inkjet 3D Printing of Polymers Resistant to Fungal Attachment.

Inkjet 3D printing is an additive manufacturing method that allows the user to produce a small batch of customized devices for comparative study versus commercial products. Here, we describe the use of a commercial 2D ink development system (Dimatix material printing) to manufacture small batches of 3D medical or other devices using a recently characterized fungal anti-attachment material. Such printed devices may resist problems that beset commercial medical products due to colonization by the fungal pathogen Candida albicans. By sequentially introducing the cross-section bitmaps of the product's CAD model and elevating the print head height using the auto-clicking script, we were able to create complex self-support geometries with the 2D ink development system. The use of this protocol allows researchers to produce a small batch of specimens for characterization from only a few grams of raw material. Additionally, we describe the testing of manufactured specimens for fungal anti-attachment. In comparison with most commercial AM systems, which require at least a few hundred grams of ink for printing trials, our protocol is well suited for smaller-scale production in material studies.

Evolving challenges and strategies for fungal control in the food supply chain.

Fungi that spoil foods or infect crops can have major socioeconomic impacts, posing threats to food security. The strategies needed to manage these fungi are evolving, given the growing incidence of fungicide resistance, tightening regulations of chemicals use and market trends imposing new food-preservation challenges. For example, alternative methods for crop protection such as RNA-based fungicides, biocontrol, or stimulation of natural plant defences may lessen concerns like environmental toxicity of chemical fungicides. There is renewed focus on natural product preservatives and fungicides, which can bypass regulations for 'clean label' food products. These require investment to find effective, safe activities within complex mixtures such as plant extracts. Alternatively, physical measures may be one key for fungal control, such as polymer materials which passively resist attachment and colonization by fungi. Reducing or replacing traditional chlorine treatments (e.g. of post-harvest produce) is desirable to limit formation of disinfection by-products. In addition, the current growth in lower sugar food products can alter metabolic routing of carbon utilization in spoilage yeasts, with implications for efficacy of food preservatives acting via metabolism. The use of preservative or fungicide combinations, while involving more than one chemical, can reduce total chemicals usage where these act synergistically. Such approaches might also help target different subpopulations within heteroresistant fungal populations. These approaches are discussed in the context of current challenges for food preservation, focussing on pre-harvest fungal control, fresh produce and stored food preservation. Several strategies show growing potential for mitigating or reversing the risks posed by fungi in the food supply chain.

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 + ß-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 + ß-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 + ß-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.

The Preservative Sorbic Acid Targets Respiration, Explaining the Resistance of Fermentative Spoilage Yeast Species.

A small number (10 to 20) of yeast species cause major spoilage in foods. Spoilage yeasts of soft drinks are resistant to preservatives like sorbic acid, and they are highly fermentative, generating large amounts of carbon dioxide gas. Conversely, many yeast species derive energy from respiration only, and most of these are sorbic acid sensitive and so prevented from causing spoilage. This led us to hypothesize that sorbic acid may specifically inhibit respiration. Tests with respirofermentative yeasts showed that sorbic acid was more inhibitory to both Saccharomyces cerevisiae and Zygosaccharomyces bailii during respiration (of glycerol) than during fermentation (of glucose). The respiration-only species Rhodotorula glutinis was equally sensitive when growing on either carbon source, suggesting that ability to ferment glucose specifically enables sorbic acid-resistant growth. Sorbic acid inhibited the respiration process more strongly than fermentation. We present a data set supporting a correlation between the level of fermentation and sorbic acid resistance across 191 yeast species. Other weak acids, C2 to C8, inhibited respiration in accordance with their partition coefficients, suggesting that effects on mitochondrial respiration were related to membrane localization rather than cytosolic acidification. Supporting this, we present evidence that sorbic acid causes production of reactive oxygen species, the formation of petite (mitochondrion-defective) cells, and Fe-S cluster defects. This work rationalizes why yeasts that can grow in sorbic acid-preserved foods tend to be fermentative in nature. This may inform more-targeted approaches for tackling these spoilage organisms, particularly as the industry migrates to lower-sugar drinks, which could favor respiration over fermentation in many spoilage yeasts.IMPORTANCE Spoilage by yeasts and molds is a major contributor to food and drink waste, which undermines food security. Weak acid preservatives like sorbic acid help to stop spoilage, but some yeasts, commonly associated with spoilage, are resistant to sorbic acid. Different yeasts generate energy for growth by the processes of respiration and/or fermentation. Here, we show that sorbic acid targets the process of respiration, so fermenting yeasts are more resistant. Fermentative yeasts are also those usually found in spoilage incidents. This insight helps to explain the spoilage of sorbic acid-preserved foods by yeasts and can inform new strategies for effective control. This is timely as the sugar content of products like soft drinks is being lowered, which may favor respiration over fermentation in key spoilage yeasts.

Discovery of (meth)acrylate polymers that resist colonization by fungi associated with pathogenesis and biodeterioration.

Fungi have major, negative socioeconomic impacts, but control with bioactive agents is increasingly restricted, while resistance is growing. Here, we describe an alternative fungal control strategy via materials operating passively (i.e., no killing effect). We screened hundreds of (meth)acrylate polymers in high throughput, identifying several that reduce attachment of the human pathogen Candida albicans, the crop pathogen Botrytis cinerea, and other fungi. Specific polymer functional groups were associated with weak attachment. Low fungal colonization materials were not toxic, supporting their passive, anti-attachment utility. We developed a candidate monomer formulation for inkjet-based 3D printing. Printed voice prosthesis components showed up to 100% reduction in C. albicans biofilm versus commercial materials. Furthermore, spray-coated leaf surfaces resisted fungal infection, with no plant toxicity. This is the first high-throughput study of polymer chemistries resisting fungal attachment. These materials are ready for incorporation in products to counteract fungal deterioration of goods, food security, and health.

Epoxy-amine oligomers from terpenes with applications in synergistic antifungal treatments.

A bis-epoxide monomer was synthesised in two steps from (R)-carvone, a terpenoid renewable feedstock derived from spearmint oil, and used to prepare ß-aminoalcohol oligomers in polyaddition reactions with bis-amines without requiring solvent or catalyst. A sub-set of the resultant materials were readily water soluble and were investigated for antifungal activity in combination with the fungicide iodopropynyl-butylcarbamate (IPBC) or the antifungal drug amphotericin B. The oligo-(ß-aminoalcohol)s alone were inactive against Trichoderma virens and Candida albicans but in combination with IPBC and amphotericin B demonstrated synergistic growth-inhibition of both fungi. Quantitative analysis showed that the presence of the terpene-based oligomers decreased the minimum inhibitory concentration (MIC) of IPBC by up to 64-fold and of amphotericin B by 8-fold. The efficacy of the combined formulation was further demonstrated with agar disk diffusion assays, which revealed that IPBC and amphotericin B reduced the growth of the fungi, as shown by zones of inhibition, to a greater extent when in the presence of the oligo-(ß-aminoalcohol)s. These data suggest potential future use of these renewable feedstock derived oligomers in antifungal material and related biomedical applications.