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Aspergillus niger is an opportunistic pathogen commonly found in a variety of indoor and outdoor environments. An environmental isolate of A. niger from a pig farm were resistant to itraconazole and in-depth investigations were conducted to better understand cellular responses during growth when exposed to an antifungal. Using a combination of cultivation techniques, antibiotic stress-testing, and label-free proteomics, this study has investigated the physiological and metabolic responses of A. niger to sublethal levels of antifungal stress. Challenging A. niger with itraconazole inhibited the growth, and the minimum inhibitory concentration (MIC) was estimated to be >16 mg·L−1. Through the proteome analysis, 1305 unique proteins were identified. Differential expressed proteins during growth at 2 and 8 mg·L−1 itraconazole revealed that a total of 91 and 50 proteins were significantly differentially expressed, respectively. Challenged with itraconazole, A. niger decreased the expression of peroxidative enzymes, increased the expression of an ATP-binding cassette (ABC) transporter most likely involved as an azole efflux pump, and inhibited ergosterol synthesis, however, several ergosterol biosynthesis proteins increased in abundance. Furthermore, a reduced expression of proteins involved in the production of ATP and reducing power from both the TCA and glyoxylate cycle was observed. The mode of action of triazoles in A. niger therefore appears more complex than previously anticipated and showcase future targets for antifungal treatment.

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