ectophosphatase activity in Candida parapsilosis influences the interaction of fungi with epithelial cells

• Published in: FEMS yeast research 2007-06, Vol.7 (4), p.621-628
• Main Authors: Kiffer-Moreira, Tina, Pinheiro, Ana Acácia de Sá, Alviano, Wagner S, Barbosa, Fabiane M, Souto-Padrón, Thais, Nimrichter, Leonardo, Rodrigues, Marcio L, Alviano, Celuta S, Meyer-Fernandes, José Roberto
• Format: Article
• Language: English
• Subjects: adhesion to host cells; Animals; Candida - enzymology; Candida - pathogenicity; Candida parapsilosis; Candidemia; Cell density; Cell surface; Cell Wall - enzymology; Cell walls; CHO Cells; Cricetinae; Cricetulus; ectophosphatase; Enzymatic activity; Enzymes; Epithelial cells; Epithelial Cells - microbiology; Fungal Proteins - metabolism; Fungi; Hydrolysis; Molybdate; Nitrophenols - metabolism; Organophosphorus Compounds - metabolism; p-Nitrophenol; p-Nitrophenylphosphate; Phosphoric Monoester Hydrolases - metabolism; Phosphotyrosine; Sodium; Sodium fluoride; Sodium molybdate; Sodium orthovanadate; Transmission electron microscopy
• ISSN: 1567-1356; 1567-1364
• DOI: 10.1111/j.1567-1364.2007.00223.x

This study describes the biochemical characterization of a phosphatase activity present on the cell surface of Candida parapsilosis, a common cause of candidemia. Intact yeasts hydrolyzed p-nitrophenylphosphate to p-nitrophenol at a rate of 24.30±2.63 nmol p-nitrophenol h⁻¹ 10⁻⁷ cells. The cell wall distribution of the Ca. parapsilosis enzyme was demonstrated by transmission electron microscopy. The duration of incubation of the yeast cells with the substrate and cell density influenced enzyme activity linearly. Values of Vmax and apparent Km for p-nitrophenylphosphate hydrolysis were 26.80±1.13 nmol p-nitrophenol h⁻¹ 10⁻⁷ cells and 0.47±0.05 mM p-nitrophenylphosphate, respectively. The ectophosphatase activity was strongly inhibited at high pH as well as by classical inhibitors of acid phosphatases, such as sodium orthovanadate, sodium molybdate, sodium fluoride, and inorganic phosphate, the final product of the reaction. Only the inhibition caused by sodium orthovanadate was irreversible. Different phophorylated amino acids were used as substrates for the Ca. parapsilosis ectoenzyme, and the highest rate of phosphate hydrolysis was achieved using phosphotyrosine. A direct relationship between ectophosphatase activity and adhesion to host cells was established. In these assays, irreversible inhibition of enzyme activity resulted in decreased levels of yeast adhesion to epithelial cells.