Cellular and molecular responses of Cryptococcus to brilacidin

Cryptococcosis is a systemic mycosis caused by Cryptococcus neoformans and Cryptococcus deuterogattii. Available therapeutic options are limited and face several challenges, including toxicity, the emergence of resistant strains, high costs, and restricted distribution. In this study, we explored the cellular and molecular responses of Cryptococcus spp. to brilacidin, a recently characterized fungicidal agent with promising potential for the treatment of cryptococcosis. Brilacidin exhibited inhibitory activity with a minimum inhibitory concentration of 2.5 µM and demonstrated a high selectivity index and low cytotoxicity in murine macrophages. Synergy assays revealed that brilacidin potentiated amphotericin B activity, reducing its effective dose up to eightfold without increasing cytotoxicity. Morphological analyses by scanning, confocal, and transmission electron microscopy expanded previously described effects of brilacidin and revealed profound alterations in capsule structure, plasma membrane integrity, and intracellular organization. Brilacidin also inhibited C. neoformans biofilms in a concentration-dependent manner, accompanied by major structural disruption. We investigated the molecular response of C. neoformans and C. deuterogattii to brilacidin by quantitative proteomics, which revealed species-specific responses. Brilacidin primarily affected proteins involved in sterol metabolism, oxidative stress, and cell wall biosynthesis. These results deepen our understanding of the anti-Cryptococcus effects of brilacidin and reinforce the potential of this drug as a highly promising antifungal agent for the treatment of cryptococcosis.IMPORTANCECryptococcosis is a life-threatening fungal disease that mainly affects immunocompromised individuals and remains difficult to treat due to toxic drugs, limited therapeutic options, and the emergence of resistance. This study deepens the understanding of brilacidin as a promising antifungal candidate against the causative agents of cryptococcosis. Brilacidin exhibits potent fungicidal activity at low concentrations with minimal toxicity to host cells and enhances the efficacy of amphotericin B, allowing for reduced doses that may mitigate adverse effects. Furthermore, brilacidin disrupts key fungal structures, including the capsule and biofilms, and induces distinct cellular stress responses. By providing mechanistic and translational insights, this work supports the development of safer and more effective therapeutic strategies for this neglected and often fatal fungal infection.