Glycoconjugate Journal 19, 165–173, 2003 C  2003 Kluwer Academic Publishers. Manufactured in The Netherlands. Sialylglycoconjugates and sialyltransferase activity in the fungus Cryptococcus neoformans Marcio L. Rodrigues 1 , Andrey S. S. Dobroff 2 , Jos ´ e Nelson dos S. S. Couceiro 1 , Celuta S. Alviano 1∗ , Roland Schauer 3 and Luiz R. Travassos 2 1 Instituto de Microbiologia Prof. Paulo de G´ oes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, 2 Disciplina de Biologia Celular, Universidade Federal de S˜ ao Paulo, S˜ ao Paulo, Brazil, 3 Biochemisches Institut, Christian-Albrechts-Universit ¨ at, Kiel, Germany Cryptococcus neoformans is a fungal pathogen associated with systemic mycoses in up to 10% of AIDS patients. C. neoformans yeasts express sialic acids on the cell wall, where they play an anti-phagocytic role, and may represent a virulence factor at the initial phase of infection. Since the nature of the sialic acid-carrying components is undefined in C. neoformans, our aim in the present work was to identify sialylated molecules in this fungus and study the sialylation process. C. neoformans yeast forms were cultivated in a chemically defined medium free of sialic acids, to search for autologous sialylglycoconjugates. Sialylated glycolipids were not detected. Two glycoproteins with molecular masses of 38 and 67 kDa were recognized by Sambucus nigra agglutinin, an α2,6-sialic acid-specific lectin. The 67 kDa glycoprotein also interacted with Influenza C virus, but not with Limax flavus agglutinin, suggesting the presence of the 9- O-acetylated sialic acid derivative as a constituent of the oligosaccharide chains. A partially purified protein fraction from cryptococ- cal yeast forms was able to transfer sialic acid from CMP-Neu5Ac to both N-(acetyl-1- 14 C)-lactosamine and asialofetuin. Additional evidence for a sialyltransferase in C. neoformans was obtained through the reactivity of fungal proteins with rabbit anti-rat α2,6 sialyltransferase polyclonal antibody. Our results indicate that sialic acids in C. neoformans are linked to glycoproteins, which are sialylated by the action of a fungal sialyltransferase. This is the first demonstration of this biosynthetic step in pathogenic fungi. Published in 2003. Keywords: Cryptococcus neoformans, sialylglycoconjugates, O-acetylated sialic acid, sialyltransferase, lectins Introduction Cryptococcus neoformans is a yeast-like fungus commonly in- fecting immunosuppressed hosts causing cryptococcal menin- gitis, a serious and often fatal clinical condition [1]. Infection by C. neoformans begins by inhalation of fungal basidiospores or poorly encapsulated yeasts [2]. The capsular polysaccharide of C. neoformans, its major virulence factor, protects yeasts against phagocytosis by alveolar macrophages [3]. The cryp- tococcal capsule, however, reaches its complete expression at least 5 hours after the initial contact of the fungal propagules with the host [4]. The mechanisms by which C. neoformans ∗ To whom correspondence should be addressed: Dr Celuta S. Alviano, Instituto de Microbiologia Prof. Paulo de G´ oes, Universidade Federal do Rio de Janeiro. Cidade Universit´ aria, CCS, Bloco I, sala 050. 21941 590, Rio de Janeiro—RJ, Brazil. Tel.: 55 21 2562 6711; Fax: 55 21 2560 8344; E-mail: alviano@micro.ufrj.br resists phagocytosis and destruction by host cells during this period are not defined. Sialic acids are members of a family of around 50 derivatives of neuraminic acid that have many functions in a wide range of biological systems [5,6]. During the last two decades, sialic acids have been characterized in several fungal species [7–17], but little progress has been made in the identification of sialic acid-containing molecules and their biosynthesis. Their role in fungal pathogenicity is still unclear. In a previous work, we reported on the occurrence of sialic acids at the cell surface of C. neoformans [8]. Both N - acetylneuraminic acid (Neu5Ac) and its 9- O -acetylated deriva- tive (Neu5,9Ac 2 ) were detected in C. neoformans yeast cells. Neu5Ac is the most common sialic acid derivative found in nature [5], and its presence in other fungal species has been recently reviewed [7]. The occurrence, however, of Neu5,9Ac 2 in C. neoformans represents the only report on the expression of this sialic acid derivative in pathogenic fungi. Evidence for