Andrographolide Combined With Voriconazole Enhances Antifungal Activity Against Aspergillus Species

Azole resistance in Aspergillus fumigatus is increasing globally, necessitating novel combination strategies to restore the efficacy of first-line antifungals such as voriconazole (VCZ). Andrographolide (ADR), a natural diterpene lactone, has shown limited direct antifungal activity but exhibits synergistic effects with amphotericin B and immunomodulatory properties. However, its potential as an adjunct to VCZ against Aspergillus species remains unexplored. The in vitro antifungal activity of ADR combined with VCZ was evaluated against A. fumigatus, Aspergillus flavus, and Aspergillus niger using the broth microdilution checkerboard method. Biofilm formation was assessed by Congo red agar, XTT reduction, and crystal violet staining. Cytotoxicity was measured via CCK-8 assay on A549 lung epithelial cells. Transcriptomic analysis (RNA-seq) and qPCR were performed to uncover the synergistic mechanism. The ADR-VCZ combination exhibited synergy against A. fumigatus (FICI = 0.5) and additive effects against A. flavus (FICI = 0.75) and A. niger (FICI = 1.0). ADR alone showed minimal antifungal activity (MIC = 400 µg/mL). However, when combined with VCZ, it reduced the MIC of VCZ from 0.5 to 0.125 µg/mL. The combination significantly inhibited A. fumigatus biofilm formation without detectable cytotoxicity to A549 cells. Transcriptomic analysis revealed that the VCZ versus Control comparison and the ADR + VCZ versus VCZ comparison shared one common downregulated gene (AFUA_4G03760) and were co-enriched in three GO terms related to serine family amino acid catabolic processes. Four serine family metabolism-related genes (AFUA_4G03760, AFUA_1G10780, AFUA_2G07810, and AFUA_3G09320) were significantly downregulated, as confirmed by qPCR. ADR may act as an effective booster of VCZ against A. fumigatus, with transcriptomic data pointing to downregulation of genes involved in serine family metabolism and biofilm inhibition. This study provides a preliminary metabolic clue for combating aspergillosis.