Antifungal-induced DNA dynamics and chitin remodelling across Cryptococcus spp. and the novel broad-spectrum anti-cryptococcal candidate CPTH2.

Antifungal drug resistance is a growing global health concern, yet treatment options remain limited to just four classes: polyenes, azoles, allylamines, and echinocandins. Cryptococcus species are responsible for life-threatening meningoencephalitis and pulmonary infections, which cause over 140,000 deaths annually. The high mortality of Cryptococcus is driven by limited drug access, toxicity, persistence, and emerging resistance. To better understand antifungal resistance and identify novel therapeutics, we profiled a genetically diverse panel of Cryptococcus isolates spanning all major lineages (VNI, VNIII, VNIV, VGI-VGVI). Phenotypic assays were used to assess susceptibility to fluconazole (FLZ), 5-fluorocytosine (5-FC), and amphotericin B (AmpB), as well as the epigenetic inhibitors CPTH2 (a histone acetyltransferase inhibitor) and SAHA (a histone deacetylase inhibitor). We found lineage- and strain-specific variation in antifungal susceptibility, with disc-diffusion and microdilution assays yielding consistent patterns. Pharmacodynamic modelling revealed potent fungicidal activity of CPTH2 across all tested lineages. DNA content and chitin levels varied significantly by lineage and treatment: FLZ and CPTH2 induced DNA content increases in susceptible strains, suggesting a stress response, while 5-FC-induced chitin remodelling was most pronounced in resistant strains. Multivariate analysis identified chitin induction as a principal driver of phenotypic variance under antifungal stress. Our findings reveal lineage-specific antifungal responses in Cryptococcus, highlight the antifungal potential of epigenetic inhibitors such as CPTH2, and underscore the utility of phenotypic profiling to inform drug development and resistance surveillance.