Influence of N-Glycosylation on the Morphogenesis and Growth of Paracoccidioides brasiliensis and on the Biological Activities of Yeast Proteins

Abstract

The fungus Paracoccidioides brasiliensis is a human pathogen that causes paracoccidioidomycosis, the most prevalent systemic mycosis in Latin America. The cell wall of P. brasiliensis is a network of glycoproteins and polysaccharides, such as chitin, that perform several functions. N-linked glycans are involved in glycoprotein folding, intracellular transport, secretion, and protection from proteolytic degradation. Here, we report the effects of tunicamycin (TM)-mediated inhibition of N-linked glycosylation on P. brasiliensis yeast cells. The underglycosylated yeasts were smaller than their fully glycosylated counterparts and exhibited a drastic reduction of cell budding, reflecting impairment of growth and morphogenesis by TM treatment. The intracellular distribution in TM-treated yeasts of the P. brasiliensis glycoprotein paracoccin was investigated using highly specific antibodies. Paracoccin was observed to accumulate at intracellular locations, far from the yeast wall. Paracoccin derived from TM-treated yeasts retained the ability to bind to laminin despite their underglycosylation. As paracoccin has N-acetyl-β-d-glucosaminidase (NAGase) activity and induces the production of TNF-α and nitric oxide (NO) by macrophages, we compared these properties between glycosylated and underglycosylated yeast proteins. Paracoccin demonstrated lower NAGase activity when underglycosylated, although no difference was detected between the pH and temperature optimums of the two forms. Murine macrophages stimulated with underglycosylated yeast proteins produced significantly lower levels of TNF-α and NO. Taken together, the impaired growth and morphogenesis of tunicamycin-treated yeasts and the decreased biological activities of underglycosylated fungal components suggest that N-glycans play important roles in P. brasiliensis yeast biology.

Paracoccin overexpression in Paracoccidioides brasiliensis reveals the influence of chitin hydrolysis on fungal virulence and host immune response

“…These results show a faster ov-PCN yeast transition compared to wt-PCN or EV. Our data supports the critical effect of PCN in the morphological transition from yeast to mycelium and cell wall biogenesis of P. brasiliensis [21, 22, 32].…”

“…We described PCN from P. brasiliensis as a major protein component of a GlcNAc-binding fraction of yeast extracts [19, 20]. The preparations obtained by affinity to immobilized GlcNAc or chitin allowed us to identify several properties of PCN: ( i ) it contributes to fungal adhesion to extracellular matrix (ECM) components, such as laminin, in a carbohydrate recognition- and concentration-dependent manner [19]; (ii) it induces macrophages to produce TNF-α and nitric oxide [19]; (iii) it exerts N-acetylglucosaminidase (NAGase) activity, which accounts for yeast growth and cell wall biogenesis [21, 22].…”

Virulence-Associated Enzymes of Cryptococcus neoformans

“…As nature's "recyclers," environmental fungi secrete a number of degradative enzymes to breakdown macromolecules and obtain nutrients in the environment (7,(66)(67)(68)(69). C. neoformans is no exception and releases a number of lipases, proteases, and DNases.…”

Recombinant Paracoccin Reproduces the Biological Properties of the Native Protein and Induces Protective Th1 Immunity against Paracoccidioides brasiliensis Infection

“…This protein binds to laminin in a sugar recognition-dependent manner, induces macrophages to produce TNF-α and NO, and is related to fungal growth [16] and morphogenesis [17]. In addition, paracoccin exerts N -acetylglucosaminidase activity [18].…”