Physical and structural studies of Cryptococcus neoformans polysaccharide capsule.

摘要

The polysaccharide (PS) capsule of Cryptococcus neoformans is the hallmark of this global fungal pathogen. It contributes to infection by protecting the cell against a variety of host immune defenses, interfering with phagocytosis, and suppressing both cellular and humoral immunity. Because of this, the capsule is considered a major virulence determinant and remains a major target for the development of therapeutic strategies against cryptococcal disease. The capsule's critical role in pathogenesis is well known. However, many fundamental aspects about its structure, dynamics, regulation, and mechanical properties are poorly understood due to its complex composition and conformation.;By taking a physicochemical approach and combining multiple techniques, we determined that cryptococcal capsular PS exhibits polymer solution characteristics consistent with a branched and highly entangled conformation. This structural property appears to be an important determinant of capsular PS biological activity, and presents implications for capsule synthesis, assembly, and capsular-based vaccine development. PS structure was found to be influenced by Allergen 1 (ALL1), a gene that is down-regulated in a hypervirulent phenotypic switch variant of C. neoformans. ALL1 null mutants mimic this hypervirulent phenotype and were found to secrete shorter PS with less branching and higher viscosity. These results suggest that variations in virulence, associated with ALL1 differential expression, could result from altered PS structure through phenotypic switching.;In addition, the PS capsule undergoes drastic structural modifications during chronological aging. Old C. neoformans cells exhibited capsules with increased elastic properties, lower porosity, decreased PS dimensions, decreased capsule size, decreased electrostatic potential, and altered sugar composition. PS material from old capsules contained high levels of glucose, related to the exposure of alpha(1,3)-glucans, demonstrating the complex composition of C. neoformans PS surface. Age-related capsule modifications were associated with increased resistance to phagocytosis and altered antigenic density. These results suggest that the capacity of C. neoformans to generate phenotypically and antigenically different capsules could play an important role in host immune evasion, survival, and persistence in chronic cryptococcal infections.;Several monoclonal antibodies (mAbs) generated against the capsule have shown to be protective and non-protective against in vivo models of cryptococcal disease. We determined that protective mAbs were able to affect budding of encapsulated yeasts by trapping daughter cells inside the mother's capsule. This effect is determined by the ability of the mAb to increase the elastic properties of the capsule via cross-linking of PS molecules. Results obtained from these studies are important for understanding the direct microbial-effects of capsular mAbs and the mechanisms of mAb-mediated protection.;In general, the structural and physical properties of the PS capsule described in this dissertation, further underscores its complex design and virulence capacity. These findings provide insight into fundamental capsular features important for comprehension of its biology and function. More importantly, we provide fundamental information critical for the design and development of therapeutic strategies against C. neoformans and for the study of other encapsulated pathogens.