Opsonin-free, real-time imaging of Cryptococcus neoformans capsule during budding

Abstract

Cryptococcus neoformans is a unicellular fungal pathogen that causes meningoencephalitis, killing hundreds of thousands of patients each year. Its most distinctive characteristic is a polysaccharide capsule that envelops the whole cell. It is the major virulence attribute and the antigen for serologic diagnosis. We have developed a method for easy observation of the capsule and its growth dynamics using the cell-separation reagent Percoll and differential interference contrast (DIC) microscopy. Percoll suspension is far less disruptive of cell physiology than methods relying on antibody binding to the capsule, and measurements made with it are equivalent with India ink. Time-lapse microscopy observations using this method suggest that during budding, a dividing cell can regulate whether the capsule polysaccharide it produces is deposited on the capsule of the bud or on its own. This observation has important implications for our understanding of the C. neoformans capsule induction process during budding.List of abbreviations and acronymsCSFCerebrospinal fluidDICDifferential interference microscopyNANumerical apertureCCDCharge-coupled deviceMMMinimal mediumCIMCO2-independent mediumMOPS3-Morpholinopropane-1-sulfonic acidSDStandard deviation