Evaluation Of The Inhibitory Effects Of Artemisinin On Cryptococcus Neoformans

Introduction: Cryptococcus (C.) neoformans has emerged as a pathogen of global importance and has been included in the critical group of the WHO fungal priority pathogens list. This inclusion is mainly due to drug resistance and ill-tolerance. This study sought to evaluate the impact of the anti-mitochondrial drug artemisinin on the growth, mitochondrial function, and virulence of cryptococcal cells. We theorise that the strictly aerobic cryptococcal cells will suffer deleterious effects when exposed to artemisinin, as they cannot switch to an alternate glycolytic fermentative pathway to generate energy.

Methods: A susceptibility assay was performed to assess the inhibitory properties of artemisinin of three C. neoformans isolates and H99, the reference laboratory strain. The ultrastructural changes were examined using SEM and TEM. Mitochondrial function was evaluated by monitoring the overproduction of ROS, cytochrome c release, and the activation of caspase 3, an early marker of apoptosis. Infection was established in Galleria mellonella, where infected larvae were monitored for cocoon formation. Expression of the IMPI gene in the larvae was also assessed.

Results: A 10-fold increase in artemisinin concentration led to growth inhibition in a dose-dependent manner. In this study, 0.1 mM was defined as the MIC_50. Treated cells showed elevated ROS levels and cytochrome c in the cytoplasm, indicating dysfunctional mitochondria. The activation of caspase 3 was notably higher in treated cells than in non-treated cells. The larvae with treated cells could form cocoons, while those infected with non-treated cells could not. Moreover, the expression levels of IMPI were significantly higher in treated cells than in non-treated cells, suggesting enhanced clearance of artemisinin-treated cells.

Conclusions: The preliminary data presented in this study present empirical evidence of artemisinin serving as a repurposed control agent that could inhibit cryptococcal growth and assist an infected animal in clearing infection.