Extracellular vesicles from Paracoccidioides pathogenic species transport polysaccharide and expose ligands for DC-SIGN receptors

Abstract

Extracellular vesicles (EVs) mediate non-conventional transport of molecules across the fungal cell wall. We aimed at describing the carbohydrate composition and surface carbohydrate epitopes of EVs isolated from the pathogenic fungi Paracoccidioides brasiliensis and P. lutzii using standard procedures. Total EV carbohydrates were ethanol-precipitated from preparations depleted of lipids and proteins, then analyzed by chemical degradation, gas chromatography-mass spectrometry, nuclear magnetic resonance and size-exclusion chromatography. EV glycosyl residues of Glc, Man, and Gal comprised most probably two major components: a high molecular mass 4,6-α-glucan and a galactofuranosylmannan, possibly an oligomer, bearing a 2-α-Manp main chain linked to β-Galf (1,3) and α-Manp (1,6) end units. The results also suggested the presence of small amounts of a (1→6)-Manp polymer, (1→3)-glucan and (1→6)-glucan. Glycan microarrays allowed identification of EV surface lectin(s), while plant lectin microarray profiling revealed terminal Man and GlcNAc residues exposed at the EVs surface. Mammalian lectin microarray profiling showed that DC-SIGN receptors recognized surface carbohydrate in Paracoccidioides EVs. Our results suggest that oligosaccharides, cytoplasmic storage, and cell wall polysaccharides can be exported in fungal EVs, which also expose surface PAMPs and lectins. The role of these newly identified components in the interaction with the host remains to be unraveled.

Fungal Extracellular Vesicles with a Focus on Proteomic Analysis

“…EVs have only been isolated from yeast‐phase Paracoccidioides and they contain carbohydrates, proteins, lipids, and RNA. The EV carbohydrate is composed of glucose, mannose, and galactose residues predicted to form long α‐4,6‐glucan chains together with galactofuranosylmannan polymer or oligomers with an 2‐α‐Manp backbone linked to β‐Galf (1,3) and α‐Manp (1,6) residues, leading to speculation that P. brasiliensis EVs function in cell wall biosynthesis . Immunogenic α‐linked galactopyranosyl epitopes are present both on the surface of and within P. brasiliensis EVs .…”

“…Immunogenic α‐linked galactopyranosyl epitopes are present both on the surface of and within P. brasiliensis EVs . In addition, lectin microarrays have revealed the presence of mannose and N‐acetylgluosamine residues on the surface of P. brasiliensis and P. lutzii EVs that were recognized by the innate immune receptor DC‐SIGN (Dendritic Cell‐Specific Intercellular adhesion molecule‐3‐Grabbing Non‐integrin) . EVs from P. brasiliensis also induce murine macrophages to produce pro‐inflammatory molecules .…”

A novel protocol for the isolation of fungal extracellular vesicles reveals the participation of a putative scramblase in polysaccharide export and capsule construction in Cryptococcus gattii.

“…In fungi, these structures were first isolated in the human pathogen Cryptococcus neoformans (2). EVs have been further described in at least eleven additional species and their functions in fungi include the molecular transport across the cell wall (2), induction of drug resistance (3), prion transmission (4, 5), delivery of virulence factors (6, 7), immunological stimulation (8–12), RNA export (13), transfer of virulence traits (14), and trans-kingdom communication followed by regulation of expression of virulence-related genes (15). Although it is now well recognized that EVs play multiple and essential roles in fungal physiology, many questions remain unanswered (16).…”

Antifungal Innate Immunity: A Perspective from the Last 10 Years

“…In addition to its role in the recognition of mannoproteins from C. albicans , it has also been suggested to interact with polysaccharides from Paracoccidioides extracellular vesicles and to mediate internalization of Aspergillus conidia [24, 39]. SIGNR3, the closest murine homolog of DC-SIGN, has been implicated in sensing fungi present in the microbiota [40].…”