A Paracoccidioides brasiliensis glycan shares serologic and functional properties with cryptococcal glucuronoxylomannan

Abstract

The cell wall of the yeast form of the dimorphic fungus Paracoccidioides brasiliensis is enriched with α1,3-glucans. In Cryptococcus neoformans, α1,3-glucans interact with glucuronoxylomannan (GXM), a hetero polysaccharide that is essential for fungal virulence. In this study, we investigated the occurrence of P. brasiliensis glycans sharing properties with cryptococcal GXM. Protein database searches in P. brasiliensis revealed the presence of sequences homologous to those coding for enzymes involved in the synthesis of GXM and capsular architecture in C. neoformans. In addition, monoclonal antibodies (mAbs) raised to cryptococcal GXM bound to P. brasiliensis cells. Using protocols that were previously established for extraction and analysis of C. neoformans GXM, we recovered a P. brasiliensis glycan fraction composed of mannose and galactose, in addition to small amounts of glucose, xylose and rhamnose. In comparison with the C. neoformans GXM, the P. brasiliensis glycan fraction components had smaller molecular dimensions. The P. brasiliensis components, nevertheless, reacted with different GXM-binding mAbs. Extracellular vesicle fractions of P. brasiliensis also reacted with a GXM-binding mAb, suggesting that the polysaccharide-like molecule is exported to the extracellular space in secretory vesicles. An acapsular mutant of C. neoformans incorporated molecules from the P. brasiliensis extract onto the cell wall, resulting in the formation of surface networks that resembled the cryptococcal capsule. Coating the C. neoformans acapsular mutant with the P. brasiliensis glycan fraction resulted in protection against phagocytosis by murine macrophages. These results suggest that P. brasiliensis and C. neoformans share metabolic pathways required for the synthesis of similar polysaccharides and that P. brasiliensis yeast cell walls have molecules that mimic certain aspects of C. neoformans GXM. These findings are important because they provide additional evidence for the sharing of antigenically similar components across phylogenetically distant fungal species. Since GXM has been shown to be important for the pathogenesis of C. neoformans and to elicit protective antibodies, the finding of similar molecules in P. brasiliensis raises the possibility that these glycans play similar functions in paracoccidiomycosis.

Role of the Apt1 Protein in Polysaccharide Secretion by Cryptococcus neoformans

“…Fungal extracellular vesicles (EVs) were isolated from culture supernatants as described in a number of studies by sequential centrifugation steps (8,9,11,13,14,35). Briefly, the cells and debris were removed from culture fluids by centrifugation at 5,000 and 15,000 ϫ g (15 min, 4°C).…”

“…Remarkably, most of the molecules trafficked by fungi to the extracellular milieu lack secretion signals (5-7). Extracellular fungal molecules include numerous proteins (8)(9)(10)(11) but also pigments (12) and polysaccharides (13,14). It is now well accepted that these molecules are at least partially exported to the outer space in EVs (15).…”

The impact of proteomics on the understanding of functions and biogenesis of fungal extracellular vesicles

“…This information and the fact that molecules that do not contain any molecular tag directing them to conventional processes of secretion (e.g. polysaccharides and pigments) are components of fungal EV [20, 24, 26, 33] strongly suggests, once more, the participation of unconventional and maybe still unknown mechanisms of secretion and vesicle biogenesis. Therefore, one can conclude that the still embryonic proteomic and biochemical studies on EV produced by fungi revealed the existence of membrane-containing compartments at least composed of lipids, polysaccharides, and proteins that, in general, did not engage the conventional pathway of secretion.…”

“…The combined use of serology, biochemistry, proteomics, and lipidomics led to the identification of polysaccharides, phospholipids, neutral lipids, and proteins as extracellular vesicle components in fungi [20, 24-26, 33-35, 37, 38]. Therefore, it is now well accepted that fungal EV are carriers of distinct molecules to the extracellular space.…”

Cryptococcus Neoformans Glucuronoxylomannan Fractions of Different Molecular Masses are Functionally Distinct

“…GXM samples had their dimensions determined by dynamic light scattering in a 90Plus/BI-MAS Multi Angle Particle Sizing analyzer (Brookhaven Instruments Corp., NY, USA), as described [13]. The serological reactivity of GXM in the different fractions obtained by ultrafiltration was analyzed by ELISA and dot blot, as previously described [14,20,21]. …”