ABSTRACT: Under treatment of 4×MIC, the NT-a9 group has more differentially expressed genes (DEGs) than the FLC group, indicating NT-a9 caused more significant transcriptional changes than FLC (Figure S4B). The KEGG pathways enrichment analysis of DEGs showed that NT-a9 imposed a greater interference on sterol biosynthesis (Figure S4C). Notably, key genes involved in ergosterol biosynthesis, such as ERG1, ERG2, ERG3, ERG5, ERG6, ERG7, ERG9, ERG10, ERG11, ERG12, ERG13, ERG20, ERG24, ERG25, ERG26, ERG27, ERG28, ERG251, HMG1, IDI1 and NCP1, demonstrated markedly upregulated expression levels in NT-a9-treated group, compared to both control and FLC-treated groups (Figure S4D). These findings establish ergosterol biosynthesis inhibition as the primary mechanism underlying NT-a9's antifungal activity. In addition, KEGG pathway enrichment analysis revealed that NT-a9 profoundly impacts multiple essential metabolic pathways in C. albicans: Amino acid metabolism, including biosynthesis and catabolism of lysine, branched-chain amino acids (valine, leucine, isoleucine), histidine, and aromatic amino acids (phenylalanine, tyrosine, tryptophan); Energy metabolism, particularly pyruvate metabolic pathways; Cell proliferation processes, including DNA replication and cell cycle regulation (Figure S4C). Notably, NT-a9 significantly upregulated the terpenoid backbone biosynthesis pathway, the critical upstream precursor of sterol biosynthesis (Figure S4C). These findings suggest that NT-a9's antifungal mechanism extends beyond conventional sterol synthesis inhibition, potentially exerting multi-target effects that disrupt fundamental fungal cellular processes, ultimately leading to extensive secondary cellular damage.
