In vitro antifungal and antibiofilm activities of auranofin against itraconazole-resistant Aspergillus fumigatus

Background: Infections caused by azole-resistant Aspergillus are a rising public health threat with high mortality rates, high treatment costs and limited available antifungals, indicating an urgent need for new antifungals or strategies. Our aim was to investigate antifungal and antibiofilm activities of auranofin, an FDAapproved anti-antirheumatic drug. Methods: Fungal susceptibility testing for auranofin was carried out by the broth-based microdilution methods. Cell viability treated by auranofin was tested by resazurin dye testing. The synergistic effect of auranofin and antifungal drugs was evaluated using checkboard assay. The inhibitory of biofilms were measured by crystal violet staining. Gene expression level analysis and enzyme activity was investigated with qRT-PCR analysis and DTNB assay. The key amino acid residues in the binding of auranofin with A. fumigatus thioredoxin reductase (AfTrxR) were indicated by structural analyses, site-directed mutagenesis, and microscale thermophoresis (MST) assays. Results: Auranofin has fungicidal activity and in vitro antifungal spectrum including Aspergillus flavus, Aspergillus fumigatus, Aspergillus terreus, Aspergillus niger, even itraconazole (ITC)-resistant A. fumigatus. Additionally, it has antibiofilm activities against ITC-resistant A. fumigatus by reducing the expression level of SomA and MedA. Moreover, we discovered a synergistic effect of auranofin and ITC or amphotericin B against ITCresistant A. fumigatus. Auranofin downregulated the gene transcription of AfTrxR, and strongly inhibited the enzyme activity of AfTrxR through interacting with residues C145 and C148. Conclusions: Auranofin has fungicidal and antibiofilm activities in Aspergillus spp. and is also a potentiator of ITC or amphotericin B in vitro.