Methods: 3 echinocandin-susceptible bloodstream isolates of Cg were grown in liquid RPMI with or w/out inhibitory concentrations of micafungin (MFG; 0.004 µg/mL) or caspofungin (CAS; 0.008 µg/mL). Cells were stained with fluorescent markers specific for cell wall chitin, mannan, and viability, then imaged utilizing high-content single-cell techniques. Phenotypic characteristics of Cg cells that survive echinocandin exposure were determined by comparing the morphology and abundance cell wall components among the viable and non-viable cell subpopulations. To identify cellular characteristics associated with reduced macrophage phagocytosis, CAS or MFG treated cells were co-incubated RAW 264.7 macrophage and imaged as above. Phenotypic characteristics of the non-phagocytized yeast cells before and after co-incubation with macrophage was compared.
Results: Compared to untreated controls, growth in MFG and CAS significantly increased the proportion of cells with multiple-buds (50% ± 10% and 40% ± 18% vs. 12% ± 6%; p<0.001) and induced cellular enlargement (biovolume; 35 ± 9 µm3 and 80 ± 58 µm3 vs. 26±5 µm3; p<0.001). Cell enlargement, reduced cell wall mannan, and increased chitin were highly correlated with survival to MFG and CAS exposure (p<0.001). Comparison of the drug-exposed yeast cell population before and after co-incubation with macrophage found an increased proportion of viable cells and cells with a large diameter (≥ 7µM) remained un-phagocytized, indicating strong phagocytic preference for small, non-viable yeast cells.
Conclusion: Cg cells that survive echinocandins have distinct cell wall changes and are large in size. These cells tend to evade phagocytosis by macrophages, suggesting a potential mechanism by which Cg may persist despite echinocandin treatment.
N. D. Beyda, Astellas: Grant Investigator and Scientific Advisor , Consulting fee and Research grant .