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Replicative aging is an underexplored field of research in medical mycology. Cryptococcus neoformans ( Cn ) and Candida glabrata ( Cg ) are dreaded fungal pathogens that cause fatal invasive infections. The fungal cell wall is essential for yeast viability and pathogenesis. In this study, we provide data characterizing age-associated modifications to the cell wall of Cn and Cg . Here, we report that old yeast cells upregulate genes of cell wall biosynthesis, leading to cell wall reorganization and increased levels of all major components, including glucan, chitin, and its derivatives, as well as mannan. This results in a significant thickening of the cell wall in aged cells. Old-generation yeast cells exhibited drastic ultrastructural changes, including the presence of abundant vesicle-like particles in the cytoplasm, and enlarged vacuoles with altered pH homeostasis. Our findings suggest that the cell wall modifications could be enabled by augmented intracellular trafficking. This work furthers our understanding of the cell phenotype that emerges during aging. It highlights differences in these two fungal pathogens and elucidates mechanisms that explain the enhanced resistance of old cells to antifungals and phagocytic attacks. IMPORTANCE Cryptococcus neoformans and Candida glabrata are two opportunistic human fungal pathogens that cause life-threatening diseases. During infection, both microorganisms have the ability to persist for long periods, and treatment failure can occur even if standard testing identifies the yeasts to be sensitive to antifungals. Replicative life span is a trait that is measured by the number of divisions a cell undergoes before death. Aging in fungi is associated with enhanced tolerance to antifungals and resistance to phagocytosis, and characterization of old cells may help identify novel antifungal targets. The cell wall remains an attractive target for new therapies because it is essential for fungi and is not present in humans. This study shows that the organization of the fungal cell wall changes remarkably during aging and becomes thicker and is associated with increased intracellular trafficking as well as the alteration of vacuole morphology and pH homeostasis.

Chronic meningoencephalitis is caused by Cryptococcus neoformans and is treated in many parts of the world with fluconazole (FLC) monotherapy, which is associated with treatment failure and poor outcome. In the host, C. neoformans propagates predominantly under low glucose growth conditions. We investigated whether low glucose, mimicked by growing in synthetic media (SM) with 0.05% glucose (SMlowglu), affects FLC-resistance. A > 4-fold increase in FLC tolerance was observed in seven C. neoformans strains when minimum inhibitory concentration (MIC) was determined in SMlowglu compared to MIC in SM with normal (2%) glucose (SMnlglu). In SMlowglu, C. neoformans cells exhibited upregulation of efflux pump genes AFR1 (8.7-fold) and AFR2 (2.5-fold), as well as decreased accumulation (2.6-fold) of Nile Red, an efflux pump substrate. Elevated intracellular ATP levels (3.2-fold and 3.4-fold), as well as decreased mitochondrial reactive oxygen species levels (12.8-fold and 17-fold), were found in the presence and absence of FLC, indicating that low glucose altered mitochondrial function. Fluorescence microscopy revealed that mitochondria of C. neoformans grown in SMlowglu were fragmented, whereas normal glucose promoted a reticular network of mitochondria. Although mitochondrial membrane potential (MMP) was not markedly affected in SMlowglu, it significantly decreased in the presence of FLC (12.5-fold) in SMnlglu, but remained stable in SMlowglu-growing C. neoformans cells. Our data demonstrate that increased FLC tolerance in low glucose-growing C. neoformans is the result of increased efflux pump activities and altered mitochondrial function, which is more preserved in SMlowglu. This mechanism of resistance is different from FLC heteroresistance, which is associated with aneuploidy of chromosome 1 (Chr1). [ABSTRACT FROM AUTHOR]