Carbon starvation induces coincident capsule and cell wall remodeling in Cryptococcus neoformans

The pathogenesis of Cryptococcus neoformans is largely attributed to the impact of the polysaccharide capsule on the survival of the fungus in the harsh conditions of the host. These conditions include a robust immune response and nutrient limitation in different tissues. Little is known about the survival mechanisms of C. neoformans in nutrient-deprived conditions, and a key unanswered question is whether the fungus can use capsule material for nutrition during conditions of carbon limitation. We addressed this question by measuring alterations in capsule and cell wall in response to carbon limitation and found an influence on capsule porosity and density without a change in diameter. RNA-seq analysis of the response to carbon limitation identified transcripts for enzymes with potential relevance to polysaccharide changes, including carbohydrate-active enzymes. Subsequently, the impact of a selected set of enzymes was evaluated with capsule and cell wall-relevant mutants lacking Cas1 and Cas3 (O-acetylation of capsule polysaccharide), Chs1-8 (chitin synthases), Cps1 (hyaluronic acid synthase), and Kre64 (β-glucan specific glycosidase). Overall, our findings show that C. neoformans responds to carbon starvation by increasing capsular and cell wall permeability through interactions between cell wall components (α- and β-glucans and chitin) and capsules that alter their density and porosity. The results also suggest that C. neoformans does not substantially degrade the capsule polysaccharide under the conditions of carbon limitation employed in this study.IMPORTANCEThe World Health Organization recently placed Cryptococcus neoformans in the critical priority group of fungal pathogens that threaten human health. The elaboration of a polysaccharide capsule is a major contributor to the ability of C. neoformans to cause disease. However, the mechanisms of capsule formation are not well understood, and it is unknown whether the fungus can degrade the polysaccharide upon nutrient limitation. Here, we examined capsule degradation by starving the cells for glucose and monitoring changes in capsule permeability and binding of the dye calcofluor white to the cell wall. We found that permeability and dye binding increased with starvation. A parallel transcriptome analysis revealed candidate functions involved in the response to glucose availability, and subsequent tests with the corresponding mutants indicated an intricate connection between the cell wall and the capsule.