Influence of N-glycosylation on the morphogenesis and growth of Paracoccidioides brasiliensis and on the biological activities of yeast proteins.
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.
Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil.
Fausto Bruno
FB
Dos Reis Almeida
Fernanda Caroline
FC
Carvalho
Vânia Sammartino
VS
Mariano
Ana Claudia Paiva
AC
Alegre
Roberto do Nascimento
Rdo N
Silva
Ebert Seixas
ES
Hanna
Maria Cristina
MC
Roque-Barreira
D000118:Acetylglucosaminidase / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism / Q000502:physiology*; D006031:Glycosylation; D037102:Lectins / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism; D009024:Morphogenesis; D010228:Paracoccidioides / Q000187:drug effects / Q000201:enzymology / Q000254:growth & development* / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis; D014415:Tunicamycin / Q000494:pharmacology
D005656:Fungal Proteins; D037102:Lectins; D014409:Tumor Necrosis Factor-alpha; C548066:paracoccin, Paracoccidioides brasiliensis; D014415:Tunicamycin; D000118:Acetylglucosaminidase