Spatial and temporal sequence of capsule construction in Cryptococcus neoformans

The pathogenic yeast Cryptococcus neoformans is distinguished by an extensive polysaccharide cap-sule, which impedes host defences and is absolutely required for fungal virulence. Despite the biological importance of the capsule, nothing is known about how it is assembled. Substantial capsule growth occurs in two distinct situations relevant to crypto-coccal pathogenesis: formation of new buds and induction of capsule on mature cells. We developed pulse–chase protocols to examine these events in a dynamic way using a variety of microscopy tech-niques. We show that the capsule overlying buds is newly synthesized and differs physically from the corresponding parental material. New capsule formed by mature cells upon induction of synthesis is added at the inner aspect of the existing structure, displac-ing pre-existing material outwards. Surprisingly, new polysaccharide material is also deposited throughout the capsule, yielding a progressively denser struc-ture. These results yield the first model of capsule synthesis and open new lines of investigation into the underlying mechanisms