Assessing the Anti-Cryptococcus Antifungal Potential of Artemisinin

<i>Cryptococcus neoformans</i> (<i>C. neoformans</i>) has emerged as a global pathogen of concern. While much is known about its pathobiology, its management is complicated by strains displaying non-fluconazole susceptibility. This contribution assessed the repurposing of artemisinin (ART) as an anti-<i>Cryptococcus</i> antifungal. An in vitro susceptibility assay was performed to assess the drug response of cells. To establish the ART mode of action, assays examining mitochondrial health were set up. The phagocytosis efficiency of a murine macrophage cell line towards ART-treated and non-treated cells was determined. To complement this, the immunomodulatory effects of ART were further characterised in <i>Galleria mellonella</i> (<i>G. mellonella</i>) by assessing haemocytes' phagocytosis and expression of immune genes, <i>i.e</i>., insect metalloproteinase inhibitor (IMPI) and hemolin, essential for the insect antimicrobial response. In the end, the survival rate of infected larvae was calculated. We established that ART was antifungal, with cell death triggered by the uncoupling of the cytochrome <i>c</i> (cyt <i>c</i>) from the mitochondria, leading to activation of caspase-3-dependent-like apoptosis. Moreover, treatment induced ultrastructural changes with treated cells appearing more deformed than non-treated cells (<i>p</i> < 0.05). Treatment also increased the susceptibility of cells towards both macrophage and haemocyte phagocytosis compared to non-treated cells (<i>p</i> < 0.05). Importantly, treatment seemed to weaken the cells, decreasing their virulence potential based on analysis of the expression of the immune gene markers, which translated into treatment rescuing 75% of the larvae infected with 0.1 ART-treated cells. These preliminary findings support the repurposing of ART as an anti-<i>Cryptococcus</i> antifungal.