Chitosan-induced permeabilization in yeast and its use for in situ enzymatic measurements

Abstract Chitosan is an oligosaccharide derived from chitin that is protonated at acidic pH to form a polycation. Its positive charge promotes interaction with negatively charged components of the yeast cell envelope, which has been associated with increased cell permeability and growth inhibition. In this study, we investigated chitosan interaction with cell surfaces and its permeabilizing capacity in three yeast species: Saccharomyces cerevisiae , Candida albicans, and Debaryomyces hansenii . We evaluated the correlation between differential susceptibility and chitosan association at the cell periphery, as well as cell permeabilization, by integrating growth analyses with cell-binding assays, including fluorescein isothiocyanate (FITC)-conjugated chitosan to monitor surface association and cellular integration over time, and ultrastructural examination by transmission electron microscopy (TEM). Our results showed that chitosan exhibited varying effects on the growth and permeability of each yeast strain, with D. hansenii being the most susceptible. Furthermore, we observed the incorporation of chitosan onto the cell periphery and confirmed its role as a permeabilizing agent. Finally, we used chitosan-induced permeabilization to measure the activity of selected enzymes in situ, thereby demonstrating its potential for studying metabolic functions in permeabilized yeast cells. Overall, our findings establish chitosan as a strain-dependent antifungal agent and a useful tool for functional biochemical analyses in yeast. Key points • Chitosan permeabilizes yeast cells via electrostatic interactions at the cell surface. • Permeabilization efficiency and membrane damage are species-dependent. • Functional permeabilization enabled in situ measurements of enzymatic activity.