Calcium triggers Cryptococcus neoformans aggregation by forming coordination bonds with capsular glucuronoxylomannan

The cryptococcal polysaccharide capsule is a unique eukaryotic virulence factor that is a target for the immune system and the development of therapeutic antibodies. Our understanding of capsular architecture is limited to a few studies suggesting that metal dications play a role. In this work we explore a mechanism of cryptococcal aggregation that depends on calcium phosphate precipitation. We describe the chemical and biophysical properties of calcium interaction with the predominant cryptococcal polysaccharide, glucuronoxylomannan (GXM). We show that cell aggregation is a pH-dependent and occurs in a calcium dose-dependent manner. Furthermore, this cellular aggregation phenomenon as well as interpolymer capsular polysaccharide interactions are unique to calcium dications and do not occur with other mono- or dications as shown by size exclusion chromatography and circular dichroism. Diffusion ordered spectroscopy nuclear magnetic resonance and ab-initio calculations support complexation of calcium with glucuronic acid (GlcA). The ab-initio calculations also suggest that calcium ions can complex up to four GlcA monomers. Not only does calcium act as a scaffold for the cryptococcal capsule, interacting with up to four glucuronic acid residues of GXM, but calcium phosphate treatment of cells reduces the anti-phagocytic properties of the capsule, promoting ingestion by macrophages and altering antibody interactions with the capsule. This work advances our understanding of the cryptococcal capsule, its biophysical properties, by providing a model for the critical role of calcium interactions with capsular polymers of Cryptococcus neoformans including important impacts at the host-cell interface.