Abstract：
The polysaccharide capsule of fungal pathogen Cryptococcus neoformans is a critical virulence factor that has historically evaded complete characterization. Cryptococcal polysaccharides are known to either remain attached to the cell as capsular polysaccharides (CPSs) or to be shed into the extracellular space as exopolysaccharides (EPSs). While many studies have examined the properties of EPS, far less is known about CPS. In this work, we detail the development of new physical and enzymatic methods for the isolation of CPS which can be used to explore the architecture of the capsule and isolated capsular material. We show that sonication or Glucanex enzyme cocktail digestion yields soluble CPS preparations, while use of a French pressure cell press or Glucanex digestion followed by cell disruption removed the capsule and produced cell wall-associated polysaccharide aggregates that we call \"capsule ghosts\", implying an inherent organization that allows the CPS to exist independent of the cell wall surface. Since sonication and Glucanex digestion were noncytotoxic, it was also possible to observe the cryptococcal cells rebuilding their capsule, revealing the presence of reducing end glycans throughout the capsule. Finally, analysis of dimethyl sulfoxide-extracted and sonicated CPS preparations revealed the conservation of previously identified glucuronoxylomannan motifs only in the sonicated CPS. Together, these observations provide new insights into capsule architecture and synthesis, consistent with a model in which the capsule is assembled from the cell wall outward using smaller polymers, which are then compiled into larger ones.
摘要：
真菌病原体新生隐球菌的多糖囊是历史上逃避完整表征的关键毒力因子。已知隐球菌多糖作为荚膜多糖(CPS)保持附着在细胞上,或者作为胞外多糖(EPS)释放到细胞外空间中。虽然许多研究已经检查了EPS的性质，人们对CPS知之甚少。在这项工作中,我们详细介绍了用于分离CPS的新物理和酶方法的开发，该方法可用于探索胶囊和分离的荚膜材料的结构。我们显示超声处理或葡聚糖酶混合物消化产生可溶性CPS制剂，同时使用法国压力细胞压榨或葡聚糖消化，然后进行细胞破碎，去除胶囊并产生细胞壁相关的多糖聚集体，我们称之为“胶囊鬼魂”，暗示一种固有的组织，允许CPS独立于细胞壁表面而存在。由于超声处理和葡聚糖消化是非细胞毒性的，也有可能观察到隐球菌细胞重建它们的囊，揭示整个胶囊中存在减少末端聚糖。最后,对二甲基亚砜提取和超声处理的CPS制剂的分析显示，仅在超声处理的CPS中保留了先前鉴定的葡糖醛酸木甘露聚糖基序。一起,这些观察为胶囊结构和合成提供了新的见解，与使用较小的聚合物从细胞壁向外组装胶囊的模型一致，然后被编译为更大的。
