Beyond plasma membrane disruption: Novel antifungal mechanism of Neosartorya (Aspergillus) fischeri antifungal protein 2 in Candida albicans

Candida albicans is the most common human fungal pathogen and one of the major causes of drug-resistant, life-threatening fungal infections. The Neosartorya (Aspergillus) fischeri antifungal protein 2 (NFAP2), exhibits potent antifungal activity in both planktonic and biofilm-forming Candida cells in vitro and in vivo. However, the exact antifungal mechanism of NFAP2 remains unelucidated, limiting its therapeutic application. Based on our observations, we posit that NFAP2 is internalized in Candida cells and slows the growth over time, in addition to its previously characterized rapid plasma membrane disruption effect. This study aimed to understand the molecular mechanism of the long-term growth-slowing effect of NFAP2 on C. albicans. Transcriptomic analyses indicated downregulation of genes involved in metabolism and upregulation of genes associated with the cell cycle and filamentous growth. Moreover, in vivo protein-protein interaction analysis identified glutamate decarboxylase (Gad1p), ATP synthase subunit alpha (Atp1p), and enolase 1 (Eno1p) as intracellular protein targets of NFAP2. Our results support a dual mechanism of action of NFAP2. Specifically, when NFAP2 is applied below the minimum inhibitory concentration (<MIC), it enters the cells and reduces the metabolic activity by interacting with Gad1p, Atp1p, and Eno1p, resulting in slow growth. Furthermore, NFAP2 induces the yeast-hypha transition. However, when NFAP2 is applied at the MIC, it rapidly disrupts the plasma membrane.