Sensitive bioluminescence imaging of cryptococcosis in Galleria mellonella improves antifungal screening under in vivo conditions

ABSTRACT

Cryptococcus neoformans is an environmental yeast that primarily affects immunocompromised individuals, causing respiratory infections and life-threatening meningoencephalitis. Treatment is complicated by limited antifungal options, with concerns such as adverse effects, dose-limiting toxicity, blood–brain barrier permeability, and resistance development, emphasizing the critical need to optimize and expand current treatment options against invasive cryptococcosis. Galleria mellonella larvae have been introduced as an ethical intermediate for in vivo testing, bridging the gap between in vitro antifungal screening and mouse studies. However, current infection readouts in G. mellonella are indirect, insensitive, or invasive, which hampers the full potential of the model. To address the absence of a reliable non-invasive method for tracking infection, we longitudinally quantified the cryptococcal burden in G. mellonella using bioluminescence imaging (BLI). After infection with firefly luciferase-expressing C. neoformans, the resulting bioluminescence signal was quantitatively validated using colony-forming unit analysis. Longitudinal comparison of BLI to health and survival analysis revealed increased sensitivity of BLI in discriminating cryptococcal burden during early infection. Furthermore, BLI improved the detection of treatment efficacy using first-line antifungals, thereby benchmarking this model for antifungal testing. In conclusion, we introduced BLI as a real-time, quantitative readout of cryptococcal burden in G. mellonella over time, enabling more sensitive and reliable antifungal screening.

KEYWORDS:

• Galleria mellonella
• cryptococcus neoformans
• antifungal screening
• bioluminescence imaging
• cryptococcosis
• fungal infection

Acknowledgements

We thank Prof. Rob Lavigne and his team for teaching us how to work with G. mellonella larvae and Prof. Charles Van der Henst, Julien Brillard, Gaetan Clabots, and François Uytterhoeven for their help with starting up our G. mellonella rearing. Bioluminescence imaging was performed at the Molecular Small Animal Imaging Center (MoSAIC, KU Leuven).

Disclosure statement

K.L. received consultancy fees from MRM Health and MSD, speaker fees from Pfizer and Gilead, and a service fee from Thermo Fisher Scientific and TECOmedical, not related to this work. The other authors have no conflicts of interest to declare.

Data Availability statement

Data are available upon request.

Author contributions

Conceptualization: E.V., G.V.V.; data curation: E.V., L.V., L.M.; formal analysis: E.V., L.V., L.M.; funding acquisition, G.V.V.; methodology, E.V., A.R.S., G.V.V.; supervision: G.V.V., A.R.S., K.L.; writing – original draft: E.V.; writing – review and editing: E.V., L.V., L.M., A.R.S., K.L., G.V.V. All authors have read and approved the final version of the manuscript.

Additional information

Funding

This study was supported by the Flemish Research Foundation [FWO, grant numbers 1506114N and G057721N]. E.V. received a FWO aspirant mandate [1SF2222N/1SF2224N].