Kojic Acid Gene Clusters and the Transcriptional Activation Mechanism of Aspergillus flavus KojR on Expression of Clustered Genes.

Kojic acid (KA) is a fungal metabolite and has a variety of applications in the cosmetics and food industries. Aspergillus oryzae is a well-known producer of KA, and its KA biosynthesis gene cluster has been identified. In this study, we showed that nearly all section Flavi aspergilli except for A. avenaceus had complete KA gene clusters, and only one Penicillium species, P. nordicum, contained a partial KA gene cluster. Phylogenetic inference based on KA gene cluster sequences consistently grouped section Flavi aspergilli into clades as prior studies. The Zn(II)2Cys6 zinc cluster regulator KojR transcriptionally activated clustered genes of kojA and kojT in Aspergillus flavus. This was evidenced by the time-course expression of both genes in kojR-overexpressing strains whose kojR expression was driven by a heterologous Aspergillus nidulans gpdA promoter or a homologous A. flavus gpiA promoter. Using sequences from the kojA and kojT promoter regions of section Flavi aspergilli for motif analyses, we identified a consensus KojR-binding motif to be an 11-bp palindromic sequence of 5'-CGRCTWAGYCG-3' (R = A/G, W = A/T, Y = C/T). A CRISPR/Cas9-mediated gene-targeting technique showed that the motif sequence, 5'-CGACTTTGCCG-3', in the kojA promoter was critical for KA biosynthesis in A. flavus. Our findings may facilitate strain improvement and benefit future kojic acid production.

Fate of Aflatoxins during Almond Oil Processing.

ABSTRACT: Almonds rejected as inedible are often used for production of almond oil. However, low-quality almonds are frequently contaminated with aflatoxins, and little is known regarding transfer of aflatoxins to almond oil during processing. In this study, oil was produced from reject almonds by hexane extraction. Of 19 almond samples that were naturally contaminated with aflatoxins, 17 oil samples contained measurable amounts of aflatoxins, and aflatoxin content of contaminated oil was correlated with aflatoxin content of the nuts. However, oil aflatoxin levels were not correlated with the oxidation level of the oil as measured by percent free fatty acids and peroxide value. Adsorbents used in oil refining were tested for their ability to remove aflatoxins from contaminated oil. Fuller's earth and bentonite were the most effective, removing 96 and 86% of total aflatoxins from contaminated oil samples, respectively. Treatment with diatomaceous earth, in contrast, had no effect on aflatoxin levels in oil. These results show that oil refining steps using mineral clay adsorbents may also function to remove aflatoxins from contaminated oil.

Antifungal Drug Repurposing.

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.

Effect of Blanching on Aflatoxin Contamination and Cross-Contamination of Almonds.

ABSTRACT: Blanching of almonds was examined for reducing the aflatoxin content of contaminated nuts. Almonds with intact pellicles were spiked with aflatoxin B1 (AFB1) and blanched at 85°C. Following blanching, almond kernels and pellicles contained 20 and 19% of the spiked AFB1, respectively. The blanching water contained an additional 41% of the spiked AFB1. In a separate study, postblanching water was spiked with AFB1 and used for subsequent blanching of uncontaminated almonds. The resulting blanched kernels acquired 3.3% of the AFB1 from the spiked water, demonstrating a low level of cross-contamination from reused contaminated blanching water. The effect of the blanching temperature on partitioning of AFB1 from almonds to blanching water was significant at a 20-ppb spiking level, but not at 100 ppb. AFB1 levels that were unaccounted for in the mass balance of blanching components were presumed to be lost due to binding to water-solubilized almond components and were independent of pH and blanching time. Blanching reduced total aflatoxins in naturally contaminated almonds by 13 to 76%, depending on almond quality, as well as blanching time and temperature. These results indicate that the association between almond components and aflatoxin generated through mold contamination is more complex than in spiking experiments.

X-Ray-Based Irradiation of Larvae and Pupae of the Navel Orangeworm (Lepidoptera: Pyralidae).

The suitability of adult male the navel orangeworm, Amyelois transitella (Walker) for Sterile Insect Technique (SIT) has been reported for both high energy gamma (>1 MeV) and low energy x-ray (90 keV) sterilization. However, research regarding sterilization of NOW larvae and pupae by gamma irradiation indicated nonsuitability due to high mortality. Here, NOW larvae and pupae were irradiated to doses up to 50 Gy with 90 keV x-rays, then paired with nonirradiated colony mates. Sterility of surviving insects was determined by the presence or absence of hatched neonates. While presence of offspring does not guarantee viability, the absence does guarantee sterility (as is appropriate for SIT) and was thus the measure used here. Early stage larvae experienced 77% mortality at a dose of 30 Gy, versus 20% for nonirradiated control. At 40 Gy, mortality reached 98%. Of surviving early stage larvae at 30 Gy, 29% of moth pairs produced offspring. For late stage larvae, no offspring were produced at 40 Gy, but mortality was 73%. For pupae, mortality reached 53% at 30 Gy with 13% still producing neonates, while mortality reached 98% at 40 Gy. These results are consistent with reported results for gamma irradiation of NOW larvae where sterility was observed somewhere between the 30 Gy and 60 Gy data points, but mortality was high. This further confirms the lack of suitability of NOW irradiated in the larval stage, whether by gamma or x-ray, and supports the hypothesis that x-ray and gamma treatments are biologically equivalent at equal doses.

Rapid elimination of foodborne and environmental fungal contaminants by benzo analogs.

BACKGROUND: Contamination of food or the environment by fungi, especially those resistant to conventional fungicides or drugs, represents a hazard to human health. The objective of this study is to identify safe, natural antifungal agents that can remove fungal pathogens or contaminants rapidly from food and / or environmental sources. RESULTS: Fifteen antifungal compounds (nine benzo derivatives as candidates; six conventional fungicides as references) were investigated. Three benzo analogs, namely octyl gallate (OG), trans-cinnamaldehyde (CA), and 2-hydroxy-5-methoxybenzaldehyde (2H5M), at 1 g L-1 (3.54 mmol), 1 mL L-1 (7.21 mmol), 1 mL L-1 (5.39 mmol), respectively, achieved ≥99.9% fungal death after 0.5, 2.5 or 24 h of treatments, respectively, in in vitro phosphate-buffered saline (PBS) bioassay. However, when OG, CA, and 2H5M were examined in commercial food matrices, organic apple, or grape juices, only CA maintained a similar level of antifungal activity, compared with a PBS bioassay. trans-Cinnamaldehyde showed higher antifungal activity at pH 3.5, equivalent to that of commercial fruit juices, than at pH 5.6. In soil sample tests, the application of 1 mL L-1 (7.21 mmol) CA to conventional maize / tomato soil samples (pH 6.8) for 2.5 h resulted in ≥99.9% fungal death, indicating CA could also eliminate fungal contaminants in soil. While the conventional fungicide thiabendazole exerted antifungal activity comparable to CA, thiabendazole enhanced the production of carcinogenic aflatoxins by Aspergillus flavus, an undesirable side effect. CONCLUSION: trans-Cinnamaldehyde could be developed as a potent antifungal agent in food processing or soil sanitation by reducing the time / cost necessary for fungal removal. Published 2020. This article is a U.S. Government work and is in the public domain in the USA.

Curcumin and Quercetin as Potential Radioprotectors and/or Radiosensitizers for X-ray-based Sterilization of Male Navel Orangeworm Larvae.

Two natural compounds (quercetin and curcumin) were tested as sensitizing or protecting agents for Navel Orangeworm (NOW) larvae under x-ray sterilization, with the aim to reduce required doses and thus facilitate the substitution of x-ray for radioisotopes. The compounds were added to NOW diet at concentrations between 0 and 1.0 mmol kg-1 and subsequent reared male larvae were subjected to x-ray irradiation (90 keV, 9 mA) to doses up to 15 Gy. Upon emergence as adults, surviving male NOW were paired with colony virgin females and placed in isolation for observation of deformity, mortality, and fertility. Treatments included rearing larvae on infused diet before irradiation, after irradiation, and both. Results were tabulated as percentage of insects that were dead/deformed, infertile, or fertile and subjected to chi-squared analysis. While insect populations subjected to quercetin treatments were not found to be significantly different from control at any x-ray dose, all curcumin treatments yielded significant differences at an absorbed dose of 10 Gy, both in terms of decreased mortality and fertility. While none of the treatments resulted in acceptable mortality/deformity rates, the observed effects strongly support the need for continued testing of natural compounds for their efficacy to reduce required dose levels for sterilization.

Degradation kinetics of aflatoxin B1 and B2 in solid medium by using pulsed light irradiation.

BACKGROUND: Pulsed light (PL) is a new potential technology to degrade aflatoxin. The objective of this study was to investigate the degradation characters of aflatoxin B1 (AFB1 ) and B2 (AFB2 ) treated under PL irradiation. A kinetic degradation study of AFB1 and AFB2 in solid medium was performed under PL irradiation at different initial concentrations of AFB1 (229.9, 30.7 and 17.8 µg kg-1 ) and AFB2 (248.2, 32.2 and 19.5 µg kg-1 ) and irradiation intensities (2.86, 1.60 and 0.93 W cm-2 ) of PL. A second-order reaction model was applied to describe degradation of AFB1 and AFB2 . RESULTS: The results showed that the degradation of AFB1 and AFB2 followed the second-order reaction kinetic model well (R2  > 0.97). The degradation rate was proportional to the intensities of PL irradiation and the initial concentrations of aflatoxins. CONCLUSION: It is concluded that the degradation of AFB1 and AFB2 with the use of PL could be accurately described using the second-order reaction kinetic model. © 2018 Society of Chemical Industry.

Silo-Stored Pistachios at Varying Humidity Levels Produce Distinct Volatile Biomarkers.

Fungal-contaminated tissues are known to produce volatile profiles that are different from uncontaminated tissues. Fungi require certain water activity levels before growth can occur. For nonxerophilic fungi, a water activity of 0.85 is typical for growth, and for extreme xerophilic fungi, the water activity can be as low as 0.64. Recent investigations with stored pistachios (kernels in shell, no hull tissue) at varying relative humidities showed differences among the collected volatile profiles at the tested humidities (ambient, 63, 75, and 84%). Water activities of the kernel and shell were also measured. Results showed significant changes in volatile profiles as a function of water activity of the corresponding pistachio tissue with measured water activity levels at or below that of what is considered extreme xerophilic activities. Because fungal growth, including mycotoxigenic fungi, is dependent upon water activity, the detected volatile profiles could be used for early detection of fungal presence. Multivariate analysis of the volatile data demonstrated significant differences among the volatile profiles at the tested relative humidity levels, and several volatiles were identified as biomarkers of increased humidity and likely fungal development.

Differentiation of Volatile Profiles from Stockpiled Almonds at Varying Relative Humidity Levels Using Benchtop and Portable GC-MS.

Contamination by aflatoxin, a toxic metabolite produced by Aspergillus fungi ubiquitous in California almond and pistachio orchards, results in millions of dollars of lost product annually. Current detection of aflatoxin relies on destructive, expensive, and time-intensive laboratory-based methods. To explore an alternative method for the detection of general fungal growth, volatile emission profiles of almonds at varying humidities were sampled using both static SPME and dynamic needle-trap SPE followed by benchtop and portable GC-MS analysis. Despite the portable SPE/GC-MS system detecting fewer volatiles than the benchtop system, both systems resolved humidity treatments and identified potential fungal biomarkers at extremely low water activity levels. This ability to resolve humidity levels suggests that volatile profiles from germinating fungal spores could be used to create an early warning, nondestructive, portable detection system of fungal growth.

Simultaneous decontamination and drying of rough rice using combined pulsed light and holding treatment.

BACKGROUND: Pulsed light (PL) technology has been proven effective in food disinfection. However, increasing the light intensity or treatment time could swiftly increase the temperature of the food product. Using the thermal effect in an appropriate way may achieve a simultaneous disinfection and drying effect. The objective of this study was to investigate the feasibility of simultaneous disinfection and drying of rough rice using PL and holding treatment. RESULTS: Freshly harvested rice samples were inoculated by Aspergillus flavus (A. flavus) and treated using PL under different intensities and durations followed by holding treatment. The PL treatment under intensity of 1.08 W cm(-2) for 21 s led to a reduction of 0.29 log cfu g(-1) on the population size of A. flavus spores. After holding treatment, a 5.2 log cfu g(-1) reduction was achieved. The corresponding total moisture removal reached 3.3% points. No adverse effect on milling quality was detected after the treatment. CONCLUSION: The obtained results revealed that the combined PL and holding treatment had good potential for successful application in the rice industry to simultaneously achieve disinfection and drying. © 2015 Society of Chemical Industry.

Augmenting the Activity of Monoterpenoid Phenols against Fungal Pathogens Using 2-Hydroxy-4-methoxybenzaldehyde that Target Cell Wall Integrity.

Disruption of cell wall integrity system should be an effective strategy for control of fungal pathogens. To augment the cell wall disruption efficacy of monoterpenoid phenols (carvacrol, thymol), antimycotic potency of benzaldehyde derivatives that can serve as chemosensitizing agents were evaluated against strains of Saccharomyces cerevisiae wild type (WT), slt2Δ and bck1Δ (mutants of the mitogen-activated protein kinase (MAPK) and MAPK kinase kinase, respectively, in the cell wall integrity pathway). Among fourteen compounds investigated, slt2Δ and bck1Δ showed higher susceptibility to nine benzaldehydes, compared to WT. Differential antimycotic activity of screened compounds indicated "structure-activity relationship" for targeting the cell wall integrity, where 2-hydroxy-4-methoxybenzaldehyde (2H4M) exhibited the highest antimycotic potency. The efficacy of 2H4M as an effective chemosensitizer to monoterpenoid phenols (viz., 2H4M + carvacrol or thymol) was assessed in yeasts or filamentous fungi (Aspergillus, Penicillium) according to European Committee on Antimicrobial Susceptibility Testing or Clinical Laboratory Standards Institute M38-A protocols, respectively. Synergistic chemosensitization greatly lowers minimum inhibitory or fungicidal concentrations of the co-administered compounds. 2H4M also overcame the tolerance of two MAPK mutants (sakAΔ, mpkCΔ) of Aspergillus fumigatus to fludioxonil (phenylpyrrole fungicide). Collectively, 2H4M possesses chemosensitizing capability to magnify the efficacy of monoterpenoid phenols, which improves target-based (viz., cell wall disruption) antifungal intervention.

Semiochemicals from ex situ abiotically stressed cactus tissue: a contributing role of fungal spores?

Semiochemicals play a central role in communication between plants and insects, such as signaling the location of a suitable host. Fungi on host plants can also play an influential role in communicating certain plant vulnerabilities to an insect. The spiroketal conophthorin is an important semiochemical produced by developing fungal spores. Spiroketals are also used as signals for scolytid communication. Plants and fungi are known to emit varying volatile profiles under biotic and abiotic stress. This paper reports distinctive temporal-volatile profiles from three abiotic treatments, room temperature (control), -15 °C (cold), and -15 °C to room temperature (shock), of cactus tissue plugs. Volatiles from the three treatments included monoterpenes from control plugs, compounds of varying classes and origin at later stages for cold plugs, and known semiochemicals, including spiroketals, at later stages for shock plugs. The results highlight several important findings: a unique tissue source of the spiroketals; abiotic cold-shock stress is indicated as the cause of spiroketal production; and, given previous findings of spirogenesis, fungal spore involvement is a probable biosynthetic origin of the spiroketals. These findings suggest an important role of fungal volatiles as signaling plant vulnerability to insects.

Use of benzo analogs to enhance antimycotic activity of kresoxim methyl for control of aflatoxigenic fungal pathogens.

The aim of this study was to examine two benzo analogs, octylgallate (OG) and veratraldehyde (VT), as antifungal agents against strains of Aspergillus parasiticus and A.flavus (toxigenic or atoxigenic). Both toxigenic and atoxigenic strains used were capable of producing kojic acid, another cellular secondary product. A. fumigatus was used as a genetic model for this study. When applied independently, OG exhibits considerably higher antifungal activity compared to VT. The minimum inhibitory concentrations (MICs) of OG were 0.3-0.5 mM, while that of VT were 3.0-5.0 mM in agar plate-bioassays. OG or VT in concert with the fungicide kresoxim methyl (Kre-Me; strobilurin) greatly enhanced sensitivity of Aspergillus strains to Kre-Me. The combination with OG also overcame the tolerance of A. fumigatus mitogen-activated protein kinase (MAPK) mutants to Kre-Me. The degree of compound interaction resulting from chemosensitization of the fungi by OG was determined using checkerboard bioassays, where synergistic activity greatly lowered MICs or minimum fungicidal concentrations. However, the control chemosensitizer benzohydroxamic acid, an alternative oxidase inhibitor conventionally applied in concert with strobilurin, did not achieve synergism. The level of antifungal or chemosensitizing activity was also "compound-strain" specific, indicating differential susceptibility of tested strains to OG or VT, and/or heat stress. Besides targeting the antioxidant system, OG also negatively affected the cell wall-integrity pathway, as determined by the inhibition of Saccharomyces cerevisiae cell wall-integrity MAPK pathway mutants. We concluded that certain benzo analogs effectively inhibit fungal growth. They possess chemosensitizing capability to increase efficacy of Kre-Me and thus, could reduce effective dosages of strobilurins and alleviate negative side effects associated with current antifungal practices. OG also exhibits moderate antiaflatoxigenic activity.

Spread of Aspergillus flavus by Navel Orangeworm (Amyelois transitella) on Almond.

Navel orangeworm (NOW) damage to almond is correlated with increased incidence of aflatoxin contamination caused by Aspergillus flavus. However, no reports demonstrate a causative relationship between NOW feeding and A. flavus infection. To demonstrate the potential of NOW to act as a vector of A. flavus on almond, NOW eggs were dusted with A. flavus and incubated in microchambers adjacent to but not touching agar plates or almond kernels. Following egg hatch, A. flavus colonies developed on agar along trails left by NOW larvae. Almond kernels damaged with A. flavus-carrying NOW showed higher incidence of A. flavus colonization and aflatoxin contamination than control treatments. Interestingly, levels of aflatoxin in NOW-damaged, A. flavus-infected almond were significantly higher than control treatments, even in the absence of visible fungal growth. Commercial almond orchards had a relatively low level of contamination with Aspergillus section Flavi in spring and early summer and a high level during summer, corresponding with the higher level of NOW infestation of the crop. Our study demonstrates that NOW is capable of vectoring A. flavus to almond, and that monitoring and sorting of almond kernels for insect damage is warranted to limit aflatoxin contamination potential both before and after harvesting.

Synergism of antifungal activity between mitochondrial respiration inhibitors and kojic acid.

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.

Enhancement of commercial antifungal agents by Kojic Acid.

Natural compounds that pose no significant medical or environmental side effects are potential sources of antifungal agents, either in their nascent form or as structural backbones for more effective derivatives. Kojic acid (KA) is one such compound. It is a natural by-product of fungal fermentation commonly employed by food and cosmetic industries. We show that KA greatly lowers minimum inhibitory (MIC) or fungicidal (MFC) concentrations of commercial medicinal and agricultural antifungal agents, amphotericin B (AMB) and strobilurin, respectively, against pathogenic yeasts and filamentous fungi. Assays using two mitogen-activated protein kinase (MAPK) mutants, i.e., sakA∆, mpkC∆, of Aspergillus fumigatus, an agent for human invasive aspergillosis, with hydrogen peroxide (H(2)O(2)) or AMB indicate such chemosensitizing activity of KA is most conceivably through disruption of fungal antioxidation systems. KA could be developed as a chemosensitizer to enhance efficacy of certain conventional antifungal drugs or fungicides.

Generation of the volatile spiroketals conophthorin and chalcogran by fungal spores on polyunsaturated fatty acids common to almonds and pistachios.

The spiroketal (E)-conophthorin has recently been reported as a semiochemical of the navel orangeworm moth, a major insect pest of California pistachios and almonds. Conophthorin and the isomeric spiroketal chalcogran are most commonly known as semiochemicals of several scolytid beetles. Conophthorin is both an insect- and plant-produced semiochemical widely recognized as a nonhost plant volatile from the bark of several angiosperm species. Chalcogran is the principal aggregation pheromone component of the six-spined spruce bark beetle. Recent research has shown conophthorin is produced by almonds undergoing hull-split, and both spiroketals are produced by mechanically damaged almonds. To better understand the origin of these spiroketals, the volatile emissions of orchard fungal spores on fatty acids common to both pistachios and almonds were evaluated. The volatile emission for the first 13 days of spores placed on a fatty acid was monitored. The spores investigated were Aspergillus flavus (atoxigenic), A. flavus (toxigenic), Aspergillus niger, Aspergillus parasiticus, Penicillium glabrum, and Rhizopus stolonifer. The fatty acids used as growth media were palmitic, oleic, linoleic, and linolenic. Spores on linoleic acid produced both spiroketals, those on linolenic acid produced only chalcogran, and those on palmitic and oleic acid did not produce either spiroketal. This is the first report of the spiroketals conophthorin and chalcogran from a fungal source.

Loss of msnA, a putative stress regulatory gene, in Aspergillus parasiticus and Aspergillus flavus increased production of conidia, aflatoxins and kojic acid.

Production of the harmful carcinogenic aflatoxins by Aspergillus parasiticus and Aspergillus flavus has been postulated to be a mechanism to relieve oxidative stress. The msnA gene of A. parasiticus and A. flavus is the ortholog of Saccharomyces cerevisiae MSN2 that is associated with multi-stress response. Compared to wild type strains, the msnA deletion (∆msnA) strains of A. parasiticus and A. flavus exhibited retarded colony growth with increased conidiation. The ∆msnA strains also produced slightly higher amounts of aflatoxins and elevated amounts of kojic acid on mixed cereal medium. Microarray assays showed that expression of genes encoding oxidative stress defense enzymes, i.e., superoxide dismutase, catalase, and cytochrome c peroxidase in A. parasiticus ∆msnA, and the catalase A gene in A. flavus ∆msnA, was up-regulated. Both A. parasiticus and A. flavus ∆msnA strains produced higher levels of reactive oxygen species (ROS), and ROS production of A. flavus msnA addback strains was decreased to levels comparable to that of the wild type A. flavus. The msnA gene appears to be required for the maintenance of the normal oxidative state. The impairment of msnA resulted in the aforementioned changes, which might be used to combat the increased oxidative stress in the cells.

Antifungal activity of redox-active benzaldehydes that target cellular antioxidation.

BACKGROUND: Disruption of cellular antioxidation systems should be an effective method for control of fungal pathogens. Such disruption can be achieved with redox-active compounds. Natural phenolic compounds can serve as potent redox cyclers that inhibit microbial growth through destabilization of cellular redox homeostasis and/or antioxidation systems. The aim of this study was to identify benzaldehydes that disrupt the fungal antioxidation system. These compounds could then function as chemosensitizing agents in concert with conventional drugs or fungicides to improve antifungal efficacy. METHODS: Benzaldehydes were tested as natural antifungal agents against strains of Aspergillus fumigatus, A. flavus, A. terreus and Penicillium expansum, fungi that are causative agents of human invasive aspergillosis and/or are mycotoxigenic. The yeast Saccharomyces cerevisiae was also used as a model system for identifying gene targets of benzaldehydes. The efficacy of screened compounds as effective chemosensitizers or as antifungal agents in formulations was tested with methods outlined by the Clinical Laboratory Standards Institute (CLSI). RESULTS: Several benzaldehydes are identified having potent antifungal activity. Structure-activity analysis reveals that antifungal activity increases by the presence of an ortho-hydroxyl group in the aromatic ring. Use of deletion mutants in the oxidative stress-response pathway of S. cerevisiae (sod1Δ, sod2Δ, glr1Δ) and two mitogen-activated protein kinase (MAPK) mutants of A. fumigatus (sakAΔ, mpkCΔ), indicates antifungal activity of the benzaldehydes is through disruption of cellular antioxidation. Certain benzaldehydes, in combination with phenylpyrroles, overcome tolerance of A. fumigatus MAPK mutants to this agent and/or increase sensitivity of fungal pathogens to mitochondrial respiration inhibitory agents. Synergistic chemosensitization greatly lowers minimum inhibitory (MIC) or fungicidal (MFC) concentrations. Effective inhibition of fungal growth can also be achieved using combinations of these benzaldehydes. CONCLUSIONS: Natural benzaldehydes targeting cellular antioxidation components of fungi, such as superoxide dismutases, glutathione reductase, etc., effectively inhibit fungal growth. They possess antifungal or chemosensitizing capacity to enhance efficacy of conventional antifungal agents. Chemosensitization can reduce costs, abate resistance, and alleviate negative side effects associated with current antifungal treatments.