Hyphal induction in Candida albicans: Optimizing medium parameters to accelerate hyphal growth enhanced by GlcNAc

Hyphal formation is a critical virulence trait in Candida species, contributing significantly to host tissue invasion, immune evasion, and disease progression. The morphological transition from yeast to hyphal form is therefore a key target for antifungal intervention. However, conventional hyphal induction media are often complex, expensive, and poorly standardized. In this study, we developed a simplified and cost-effective medium (MF8) containing peptone (0.16%), dextrose (0.4%), and bovine serum albumin (BSA; 0.25%), which supported moderate hyphal growth under nutrient-limited conditions. The addition of N-acetylglucosamine (GlcNAc) markedly enhanced filamentation. Using a Design of Experiments (DOE) approach via JMP software, we evaluated the effects of GlcNAc and magnesium sulfate (MgSO) on hyphal induction. GlcNAc was identified as a significant enhancer (p 0.05). Under optimized conditions (30 mM GlcNAc, 1 mM MgSO), RT-qPCR analysis revealed strong upregulation of HWP1 (100-fold at 4 h; 85-fold at 6 h) and HGC1 (>6-fold; p < 0.05). A concurrent pH shift from alkaline to acidic during 1-6 hours correlated with hyphal induction and gene activation, suggesting that acidification may serve as an additional cue regulating morphogenesis. IMPORTANCECandida species are highly adaptable opportunistic fungi that persist in diverse host niches due to numerous virulence factors. While typically commensal, they can transition to pathogenic forms, with hyphal formation being a key virulence trait. This yeast-to-hypha switch facilitates tissue invasion, immune evasion, and disease progression, making it a major target in antifungal research. Conventional hyphal induction media are often complex and costly. Developing a simplified, cost-effective, and reproducible medium would enhance studies on morphogenesis and pathogenesis, support drug screening, and potentially reveal novel virulence mechanisms under alternative environmental cues.