381. Morphologic Changes Associated With Echinocandin Tolerance Enhance Immunoevasion of Candida glabrata

Abstract

BackgroundActivation of the cell wall integrity pathway and enhanced cell wall chitin synthesis are compensatory mechanisms associated with the incomplete killing of Candida glabrata by echinocandins. Echinocandin-induced morphologic changes in C. glabrata have also been described, yet their correlation with cell wall composition and macrophage responses to echinocandin treated C. glabrata are not well characterized. Elucidating these relationships is needed to understand how C. glabrata is capable of resisting both echinocandin killing and host immune responses.MethodsThree echinocandin-susceptible bloodstream isolates of C. glabrata were grown in liquid RPMI with or without 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 C. glabrata cells that survive echinocandin exposure were determined by comparing the morphology and abundance cell wall components among the viable and nonviable 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 nonphagocytized yeast cells before and after co-incubation with macrophage was compared.ResultsCompared with 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 (≥7 µM) remained un-phagocytized, indicating strong phagocytic preference for small, nonviable yeast cells.Conclusion C. glabrata 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 C. glabrata may persist despite echinocandin treatment.Disclosures N. D. Beyda, Astellas: Grant Investigator and Scientific Advisor, Consulting fee and Research grant.