Resumo

We have recently described that Golgi reassembly and stacking protein (GRASP) is required for polysaccharide secretion and virulence in the encapsulated fungal pathogen Cryptococcus neoformans, the etiologic agent of human cryptococcosis. We have also observed in previous studies that secretion of capsular polysaccharides in C. neoformans involves extracellular vesicle (EV) formation. Since GRASP is potentially involved in vesicular secretion in other eukaryotes, we hypothesized that EV-mediated polysaccharide export and GRASP are functionally connected in C. neoformans. Wild type C. neoformans cells and a mutant lacking GRASP expression (grasp) had comparable levels of EV formation. However, polysaccharide concentration in EVs produced by the grasp mutant was decreased, in comparison with WT cells. Vesicular polysaccharide fibers produced by the grasp mutant also showed reduced dimensions, as determined by dynamic light scattering. EVs obtained from cultures of the grasp mutant also differed from those produced by WT cells in diameter distribution. Analysis of the C. neoformans surface architecture by scanning electron microscopy revealed that, in comparison to WT cells, the mutant had capsular polysaccharide fibers with reduced dimensions. These results were suggestive of defects in polysaccharide traffic. We also evaluated whether lack of GRASP would interfere with polysaccharide synthesis in C. neoformans. Analysis of crude cellular extracts revealed that the grasp mutant was in fact less efficient than WT cells in synthesizing capsular polysaccharides. These results demonstrated that GRASP is required for both polysaccharide synthesis and EV-mediated traffic in C. neoformans. Since polysaccharides are determinant for virulence in this fungus, we conclude that EV formation, GRASP and pathogenesis are directly connected in the C. neoformans model.