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.
Highlights
Paracoccidioidomycosis (PCM) is a systemic granulomatous mycosis that is endemic in rural areas of Latin America
Possible oligosaccharides, and surface terminal oligosaccharide residues in extracellular vesicles (EVs) released by the pathogenic yeast phase of Paracoccidioides representative species
Glycan microarrays revealed EV surface galactose (Gal). Residues of N-acetylglucosamine (GlcNAc)-binding lectin(s), while plant lectin microarray profiling revealed terminal Man and GlcNAc residues exposed at the EVs surface
Summary
Paracoccidioidomycosis (PCM) is a systemic granulomatous mycosis that is endemic in rural areas of Latin America It is caused by the temperature-dependent dimorphic species Paracoccidioides brasiliensis and P. lutzii[1]. Environmental fungal mycelia produce conidia that, once inhaled by the host, are transformed into the yeast pathogenic phase in the lungs[2]. This morphogenesis is followed by structural changes in cell wall polysaccharides, which are essential for establishment of the disease. Fungal extracellular vesicles (EVs) are important double-membrane structures that mediate non-conventional molecular transport across the cell wall in pathogenic fungi[10]. EV formation results in sequestration of neighboring cytoplasmic molecules to the EVs, offering a plausible explanation for the finding of proteins that lack conventional signal peptide outside the plasma membrane barrier. Non-conventional pathways are Golgi-ER-independent and group vesicular transport, and non-vesicular pathways of plasma membrane translocation across plasma membranes and transporter-based secretion[12]
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