Esculetin exhibits antifungal effects against Cryptococcus neoformans by inhibiting titan cell formation

Cryptococcus neoformans is responsible for pulmonary cryptococcosis and life-threatening cryptococcal meningitis, particularly in immunocompromised individuals. Despite its high mortality rate and the diagnostic challenges, current antifungal therapies are hampered by limitations such as side effects and the emergence of drug resistance. A critical virulence factor of C. neoformans is its ability to form titan cells, enlarged polyploid cells that promote immune evasion and dissemination. However, therapeutic interventions specifically targeting titan cell formation remain unexplored. In this study, we investigated the antifungal activity of esculetin (6,7-dihydroxycoumarin), a natural coumarin derivative with a broad range of biological activities, against C. neoformans. While esculetin exhibited modest in vitro activity (MIC = 30 μg/mL) and demonstrated synergistic effects with amphotericin B against C. neoformans in vitro, it significantly inhibited intracellular proliferation within macrophages and reduced fungal burden in murine lungs. Esculetin treatment reduced the proportion of titan cells in the infected lungs. Subsequent investigations revealed that esculetin directly inhibits titan cell formation in vitro. Limited proteolysis-mass spectrometry (Lip-MS) and molecular docking analyses identified CNAG_03983, an oxidoreductase involved in carbohydrate metabolism, as a potential molecular target of esculetin. Quantitative PCR analysis showed that esculetin treatment altered the expression of key components of the cAMP/PKA signaling pathway, including PKA1, PKR1, RIM101, GAT201, LIV3, and USV101, suggesting that esculetin may inhibit titan cell formation by perturbing cAMP/PKA signaling. These findings highlight the potential of esculetin as a novel antifungal strategy against C. neoformans, primarily through its capacity to disrupt titan cell formation and modulate associated signaling pathways.