Sclareolide as Antifungal Strategy Against <i>Cryptococcus neoformans</i>: Unveiling Its Mechanisms of Action

Cryptococcal infection commonly begins as an opportunistic infection in humans, however, this can escalate to a systemic or life-threatening form in immunocompromised individuals. Here, we aim to identify novel antifungal molecules from plants resources. Sclareolide, a phytochemical classified as a sesquiterpene lactone, was assessed against <i>Cryptococcus neoformans</i> H99. Sclareolide exhibited promising antifungal properties with a minimum inhibitory concentration (MIC) of 16 µg/mL. Additionally, the <i>C. neoformans</i> growth rate was significantly affected by sclareolide treatment in a concentration-dependent manner, as observed through a time killing assay, with a significant reduction at MIC × 8 compared to the control by 48 h. To elucidate the underlying mechanisms of sclareolide antifungal activity, fluorescence-based methods were employed. Propidium iodide (PI) accumulation assay indicated a reduction in C. <i>neoformans</i> membrane integrity, with values as low as 6.62 ± 0.18% after treatment. Moreover, sclareolide at MIC × 4 and MIC × 8 significantly increased the production of reactive oxygen species (ROS) and reduced the mitochondrial membrane potential (MMP), suggesting oxidative stress and mitochondrial dysfunction in <i>C. neoformans</i>. Sclareolide did not induce caspase-dependent apoptosis, suggesting a non-apoptotic mechanism. Further, a checkerboard experiment was performed to assess potential synergistic interaction with Amphotericin B, however, no synergism was observed. Moving on, sclareolide at 128 µg/mL did not exhibit toxicity in <i>Galleria mellonella,</i> further supporting its potential as a safe antifungal agent. These findings suggest that the antifungal activity of sclareolide against <i>C. neoformans</i> is mediated by oxidative stress. Further in vivo and pharmacokinetic studies are recommended to explore the potential of sclareolide as a prototype for the development of novel anti-cryptococcal therapies.