Investigation of the effect of lipid droplets on echinocandin resistance in Cryptococcus neoformans

Cryptococcus are environmental opportunistic fungal agents that can cause infections, especially in immunosuppressed individuals. In recent years, the incidence of these infections has increased in parallel with the rising number of immunosuppressed individuals. The fact that the drugs used in treatment are not fully effective in eradicating the infection and the rise in drug resistance has led to the search for new treatment options. The most recently developed antifungals, the echinocandins, are safe and effective drugs for fungal infections. However, these drugs are ineffective against cryptococci. Although many studies have been conducted on why cryptococci are resistant to echinocandins, the exact resistance mechanisms have not yet been fully elucidated. Calcineurin is an important molecule involved in the virulence, growth, and drug resistance development of microorganisms. Calcineurin inhibitors (CNIs) have been studied for years in various fungal pathogens due to their antifungal activity. However, there are limited data in the literature examining the relationship between CNIs and intracellular lipid metabolism in Cryptococcus. The aim of this study is to investigate the effect of CNIs and lipid droplets (LDs) on echinocandin resistance in Cryptococcus neoformans strains. A total of 30 genetically identified and previously stocked C. neoformans strains from Pamukkale University Microbiology Laboratory were included in the study. The MIC values of strains grown in shaking water baths in media with and without CNIs were measured comparatively using the microdilution method. ΔMIC values were recorded. Simultaneously grown strains were evaluated using flow cytometry. The effects of CNIs on cell size, cell granularity, and lipid droplets were assessed through flow cytometry data. The MIC50 values for the 30 Cryptococcus neoformans strains were found to be 4 for caspofungin (CAS), 32 for micafungin (MCF), and 16 for anidulafungin (ANF); the MIC90 values were 8, 32, and 32, respectively. In the presence of tacrolimus (TAC), increased susceptibility was observed in 2 strains to CAS and in 1 strain to ANF. In the presence of cyclosporine (CSP), increased susceptibility was detected in 3 strains to CAS and 8 strains to ANF. In the presence of voclosporin (VOC), susceptibility increased in 2 strains to CAS, 5 strains to MCF, and 6 strains to ANF. In the presence of ascomycin (ASC), susceptibility increased in 6 strains to CAS, 4 strains to MCF, and 11 strains to ANF. The effects of CNIs on cell size were significant with ASC, CSP, and TAC, which caused notable increases (p<0.05). VOC did not show a significant effect on cell size. Loss of homogeneity in cell size matrices was observed in 71.4% of strains under ASC, 5.9% under CSP, 66.6% under TAC, and 12.5% under VOC. Under ASC, cell granularity increased significantly (p<0.05). No significant difference in granularity was observed with CSP, TAC, or VOC. All CNIs significantly reduced cell granularity in oleic acid-enriched media (p<0.001 for ASC, CSP, VOC; p<0.05 for TAC). Loss of homogeneity in cell granularity matrices was found in 76.2% of strains under ASC, 5.9% under CSP, 50% under TAC, and 12.5% under VOC. In strains showing CAS MIC changes in the presence of ASC, lipid droplets increased significantly (p<0.05). In strains showing ANF MIC changes under ASC, intracellular lipid droplets significantly decreased (p<0.05). This suggests that molecular differences between ANF and CAS may account for this observation. Correlation analysis showed that LD changes were not correlated with cell size or cell granularity. Our study found that CNIs altered echinocandin susceptibilities in C. neoformans strains. Although there are hypotheses regarding the mechanisms underlying this result, the molecular differences between echinocandins and CNIs and the distribution of our data suggest that no single mechanism fully explains the findings. Further studies involving more strains in both in vitro and in vivo settings may help clarify these mechanisms. We believe that our data may aid in developing new treatment strategies using existing options for cryptococcosis, which remains challenging to treat. Keywords: Cryptococcosis, calcineurin inhibitors, echinocandin resistance, lipid droplets