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Various species of fungi in the genus Aspergillus are the most common causative agents of invasive aspergillosis and/or producers of hepato-carcinogenic mycotoxins. Salicylaldehyde (SA), a volatile natural compound, exhibited potent antifungal and anti-mycotoxigenic activities to A. flavus and A. parasiticus. By exposure to the volatilized SA, the growth of A. parasiticus was inhibited up to 10-75% at 9.5 mM ≤ SA ≤ 16.0 mM, while complete growth inhibition was achieved at 19.0 mM ≤ SA. Similar trends were also observed with A. flavus. The aflatoxin production, i.e., aflatoxin B(1) and B(2) (AFB(1), AFB(2)) for A. flavus and AFB(1), AFB(2), AFG(1), and AFG(2) for A. parasiticus, in the SA-treated (9.5 mM) fungi was reduced by ~13-45% compared with the untreated control. Using gene deletion mutants of the model yeast Saccharomyces cerevisiae, we identified the fungal antioxidation system as the molecular target of SA, where sod1Δ [cytosolic superoxide dismutase (SOD)], sod2Δ (mitochondrial SOD), and glr1Δ (glutathione reductase) mutants showed increased sensitivity to this compound. Also sensitive was the gene deletion mutant, vph2Δ, for the vacuolar ATPase assembly protein, suggesting vacuolar detoxification plays an important role for fungal tolerance to SA. In chemosensitization experiments, co-application of SA with either antimycin A or strobilurin (inhibitors of mitochondrial respiration) resulted in complete growth inhibition of Aspergillus at much lower dose treatment of either agent, alone. Therefore, SA can enhance antifungal activity of commercial antifungal agents required to achieve effective control. SA is a potent antifungal and anti-aflatoxigenic volatile that may have some practical application as a fumigant.

A number of benzoic acid analogues showed antifungal activity against strains of Aspergillus flavus, Aspergillus fumigatus and Aspergillus terreus, causative agents of human aspergillosis, in in vitro bioassays. Structure-activity analysis revealed that antifungal activities of benzoic and gallic acids were increased by addition of a methyl, methoxyl or chloro group at position 4 of the aromatic ring, or by esterification of the carboxylic acid with an alkyl group, respectively. Thymol, a natural phenolic compound, was a potent chemosensitizing agent when co-applied with the antifungal azole drugs fluconazole and ketoconazole. The thymol-azole drug combination demonstrated complete inhibition of fungal growth at dosages far lower than the drugs alone. Co-application of thymol with amphotericin B had an additive effect on all strains of aspergilli tested with the exception of two of three strains of A. terreus, where there was an antagonistic effect. Use of two mitogen-activated protein kinase (MAPK) mutants of A. fumigatus, sakAΔ and mpkCΔ, having gene deletions in the oxidative stress response pathway, indicated antifungal and/or chemosensitization activity of the benzo analogues was by disruption of the oxidative stress response system. Results showed that both these genes play overlapping roles in the MAPK system in this fungus. The potential of safe, natural compounds or analogues to serve as chemosensitizing agents to enhance efficacy of commercial antifungal agents is discussed., (Published by Elsevier Ltd.)

The natural product dihydroferulic acid (DFA, 1) and the synthesized DFA methyl (4a), ethyl (4b), propyl (4c), hexyl (4d), octyl (4e), and decyl (4f) esters were examined for antifungal activity. Test fungi included Saccharomyces cerevisiae (wild type, and deletion mutants slt2 Delta and bck1 Delta), Aspergillus fumigatus, and A. flavus. Growth inhibition of S. cerevisiae treated with 5 mM DFA or the corresponding esters was 4a, 4b, and 4c >98%; 4d 18.8%; 1 6.4%; 4e 6.2%; and 4f 2.8%, relative to the control. The 50% minimum inhibitory concentrations for the more active propyl, methyl, and ethyl esters were 1.5, 2.1, and 4.0 mM, respectively. Compound 4c inhibited 100% growth of both aspergilli at 6.4 mM.

Current antifungal interventions have often limited efficiency in treating fungal pathogens, particularly those resistant to commercial drugs or fungicides. Antifungal drug repurposing is an alternative intervention strategy, whereby new utility of various marketed, non-antifungal drugs could be repositioned as novel antifungal agents. In this study, we investigated "chemosensitization" as a method to improve the efficiency of antifungal drug repurposing, wherein combined application of a second compound (viz., chemosensitizer) with a conventional, non-antifungal drug could greatly enhance the antifungal activity of the co-applied drug. Redox-active natural compounds or structural derivatives, such as thymol (2-isopropyl-5-methylphenol), 4-isopropyl-3-methylphenol, or 3,5-dimethoxybenzaldehyde, could serve as potent chemosensitizers to enhance antifungal activity of the repurposed drug bithionol. Of note, inclusion of fungal mutants, such as antioxidant mutants, could also facilitate drug repurposing efficiency, which is reflected in the enhancement of antifungal efficacy of bithionol. Bithionol overcame antifungal (viz., fludioxonil) tolerance of the antioxidant mutants of the human/animal pathogen Aspergillus fumigatus. Altogether, our strategy can lead to the development of a high efficiency drug repurposing design, which enhances the susceptibility of pathogens to drugs, reduces time and costs for new antifungal development, and abates drug or fungicide resistance.

Populations of ochratoxin-producing Aspergillus section Circumdati species and aflatoxin-producing Aspergillus section Flavi species frequently coexist in soil and are the main sources of mycotoxin contamination of tree nuts. Identification of mycotoxigenic Aspergillus species in these sections is difficult using traditional isolation and culture methods. We developed a multiplex digital PCR (dPCR) assay to detect and quantify Aspergillus ochraceus, Aspergillus westerdijkiae , and Aspergillus steynii (section Circumdati), as well as Aspergillus flavus and Aspergillus parasiticus (section Flavi), in environmental samples based on species-specific calmodulin gene sequences. Relative quantification of each species by dPCR of mixed-species templates correlated with corresponding DNA input ratios. Target species could be detected in soil inoculated with conidia from each species. Non-target species of sections Circumdati, Flavi , and Nigri were generally not detectable using this dPCR method. Detected non-target species (Aspergillus fresenii, Aspergillus melleus, Aspergillus sclerotiorum , and Aspergillus subramanianii) were discernible from A. ochraceus in dual-template dPCR reactions based on differential fluorescence intensity. [ABSTRACT FROM AUTHOR]

Co-application of certain types of compounds to conventional antimicrobial drugs can enhance the efficacy of the drugs through a process termed chemosensitization. We show that kojic acid (KA), a natural pyrone, is a potent chemosensitizing agent of complex III inhibitors disrupting the mitochondrial respiratory chain in fungi. Addition of KA greatly lowered the minimum inhibitory concentrations of complex III inhibitors tested against certain filamentous fungi. Efficacy of KA synergism in decreasing order was pyraclostrobin > kresoxim-methyl > antimycin A. KA was also found to be a chemosensitizer of cells to hydrogen peroxide (H2O2), tested as a mimic of reactive oxygen species involved in host defense during infection, against several human fungal pathogens and Penicillium strains infecting crops. In comparison, KA-mediated chemosensitization to complex III inhibitors/H2O2 was undetectable in other types of fungi, including Aspergillus flavus, A. parasiticus, and P. griseofulvum, among others. Of note, KA was found to function as an antioxidant, but not as an antifungal chemosensitizer in yeasts. In summary, KA could serve as an antifungal chemosensitizer to complex III inhibitors or H2O2 against selected human pathogens or Penicillium species. KA-mediated chemosensitization to H2O2 seemed specific for filamentous fungi. Thus, results indicate strain- and/or drug-specificity exist during KA chemosensitization. [ABSTRACT FROM AUTHOR]

Activities of conventional antifungal agents, fludioxonil, strobilurin and antimycin A, which target the oxidative and osmotic stress response systems, were elevated by coapplication of certain benzo analogs (aldehydes and acids). Fungal tolerance to 2,3-dihydroxybenzaldehyde or 2,3-dihydroxybenzoic acid was found to rely upon mitochondrial superoxide dismutase ( SOD2) or glutathione reductase ( GLR1), genes regulated by the HOG1 signaling pathway, respectively. Thus, certain benzo analogs can be effective at targeting cellular oxidative stress response systems. The ability of these compounds to chemosensitize fungi for improved control with conventional antifungal agents is discussed. [ABSTRACT FROM AUTHOR]

Abstract: Caffeic acid (3,4-dihydroxycinnamic acid, 12 mM) added to a fat-based growth medium reduces >95% of aflatoxin production by Aspergillus flavus NRRL 3357, without affecting fungal growth. Microarray analysis of caffeic acid-treated A. flavus indicated expression of almost all genes in the aflatoxin biosynthetic cluster were down-regulated, ranging from a log2 ratio of caffeic acid treated and untreated of −1.12 (medium) to −3.13 (high). The only exceptions were genes norB and the aflatoxin pathway regulator-gene, aflJ, which showed low expression levels in both treated and control fungi. The secondary metabolism regulator-gene, laeA, also showed little change in expression levels between the fungal cohorts. Alternatively, expression of genes in metabolic pathways (i.e., amino acid biosynthesis, metabolism of aromatic compounds, etc.) increased (log2 ratio >1.5). The most notable up-regulation of A. flavus expression occurred in four genes that are orthologs of the Saccharomyces cerevisiae AHP1 family of genes. These genes encode alkyl hydroperoxide reductases that detoxify organic peroxides. These increases ranged from a log2 ratio of 1.08 to 2.65 (moderate to high), according to real-time quantitative reverse transcription-PCR (qRT-PCR) assays. Based on responses of S. cerevisiae gene deletion mutants involved in oxidative stress response, caffeic, chlorogenic, gallic and ascorbic acids were potent antioxidants under oxidative stress induced by organic peroxides, tert-butyl and cumene hydroperoxides. Differential hypersensitivity to these peroxides and hydrogen peroxide occurred among different mutants in addition to their ability to recover with different antioxidants. These findings suggest antioxidants may trigger induction of genes encoding alkyl hydroperoxide reductases in A. flavus. The possibilities that induction of these genes protects the fungus from oxidizing agents (e.g., lipoperoxides, reactive oxygen species, etc.) produced during host-plant infection and this detoxification attenuates upstream signals triggering aflatoxigenesis are discussed. [Copyright &y& Elsevier]

The activity of fludioxonil, a phenylpyrrole fungicide, is elevated by coapplication of the aspirin/salicylic acid metabolite, 2,5-dihydroxybenzoic acid (2,5-DHBA). Fludioxonil activity is potentiated through a mitogen-activated protein kinase (MAPK) pathway that regulates osmotic/oxidative stress-responses. 2,5-DHBA disrupts cellular GSH (reduced glutathione)/GSSG (oxidized glutathione) homeostasis, further stressing the oxidative stress-response system. This stress enhances fludioxonil activity. 2,5-DHBA treatment also prevents tolerance of MAPK mutants resistant to fludioxonil. [ABSTRACT FROM AUTHOR]

Control of fungal pathogens is increasingly problematic due to the limited number of effective drugs available for antifungal therapy. Conventional antifungal drugs could also trigger human cytotoxicity associated with the kidneys and liver, including the generation of reactive oxygen species. Moreover, increased incidences of fungal resistance to the classes of azoles, such as fluconazole, itraconazole, voriconazole, or posaconazole, or echinocandins, including caspofungin, anidulafungin, or micafungin, have been documented. Of note, certain azole fungicides such as propiconazole or tebuconazole that are applied to agricultural fields have the same mechanism of antifungal action as clinical azole drugs. Such long-term application of azole fungicides to crop fields provides environmental selection pressure for the emergence of pan-azole-resistant fungal strains such as Aspergillus fumigatus having TR34/L98H mutations, specifically, a 34 bp insertion into the cytochrome P450 51A (CYP51A) gene promoter region and a leucine-to-histidine substitution at codon 98 of CYP51A. Altogether, the emerging resistance of pathogens to currently available antifungal drugs and insufficiency in the discovery of new therapeutics engender the urgent need for the development of new antifungals and/or alternative therapies for effective control of fungal pathogens. We discuss the current needs for the discovery of new clinical antifungal drugs and the recent drug repurposing endeavors as alternative methods for fungal pathogen control. [ABSTRACT FROM AUTHOR]