Molecular architecture of the Cryptococcus neoformans capsule

Abstract

SummaryMany microbes are surrounded by phagocytosisinhibiting capsules. We took advantage of the large size of the polysaccharide capsule of the pathogenic yeast Cryptococcus neoformans to examine capsular architecture and the relationship between molecular architecture and the interaction of the capsule with potentially opsonic serum proteins. Our experimental design used complementary approaches in which (i) assessment of permeability to macromolecules of different Stokes radii; (ii) determination of the binding of Fab fragments of anticapsular antibodies as a measure of matrix density; (iii) capsular deconstruction by treatment with dimethyl sulphoxide; and (iv) evaluation of capsule plasticity, were used to probe the molecular structure of the capsule. The results showed that the capsule is a matrix with a variable porosity that increases with distance from the cell wall. A high density of the matrix at the capsule interior prevents penetration of large macromolecules to sites near the cell wall. In contrast, the capsular edge that is the interface with phagocytes presents capsular polysaccharide in a very low density that exhibits considerable plasticity and permeability to macromolecules. Notably, the capsule of yeast cells harvested from infected tissue showed a greater matrix density than yeast cells grown in vitro under capsule induction conditions.

The Physical Properties of the Capsular Polysaccharides from Cryptococcus neoformans Suggest Features for Capsule Construction

“…Indeed, monoclonal antibodies to GXM recognize cellassociated GXM and exopolysaccharide (31,63), and the ultrastructure visible in freeze-fracture images of the capsule (53) and STEM images of exopolysaccharide are similar in appearance, suggesting structural relatedness. Furthermore, the capsule contracts in response to the presence of salt (57,67), as expected given the viscoelastic properties demonstrated in this work of a polyelectrolyte molecule. The overlapping of individual polysaccharide molecules to expand the thickness of the capsule may involve either an increase in GXM production or the synthesis of GXM polymers with a higher attraction to existing fibers.…”

“…More recently, antibody penetration studies have suggested that capsular regions more distal from the cell wall also have an increased density in enlarged capsules, and capsule phenotype studies have suggested the formation of a new GXM structure at the periphery of the capsule (24,57). Although the data presented cannot distinguish between different modes of capsule growth, a capsule construction model dependent on the self-association of GXM does unify several observations.…”

“…The two different GXM structures identified by STEM were intriguing in light of recent evidence that different layers within the capsule matrix are chemically distinguishable (45,57). Structure I was most similar to the capsule architecture evident in electron microscopy of intact cells.…”

“…Assuming that capsular material is delivered to the capsule in vesicles (62,66), changes in the capsule can occur by newly synthesized GXM fibers being released and incorporated throughout the capsule by becoming entangled in existing capsular material. The density gradient of capsular material provides for more points of contact at the inner layer than at the outer layer with other existing GXM fibers (57). Capsule assembly and growth would, therefore, be a consequence of a concentration-dependent self-associative process and infers that the primary GXM structure encodes the necessary information for capsule construction.…”

Capsule Structure Changes Associated with Cryptococcus neoformans Crossing of the Blood-Brain Barrier

“…Several models could explain this apparent discrepancy. A modification in epitope accessibility could be due to changes in capsule density under different growth conditions 32 and experimental designs. In addition, the localization of a new structure outside the old one, as seen here, could also be related to the degradation of the initial structure by local factors or by aging.…”