The structure of a <i>C. neoformans</i> polysaccharide motif recognized by protective antibodies: A combined NMR and MD study.

Abstract

<i>Cryptococcus neoformans</i> is a fungal pathogen responsible for cryptococcosis and cryptococcal meningitis. The <i>C. neoformans</i>' capsular polysaccharide and its shed exopolysaccharide function both as key virulence factors and to protect the fungal cell from phagocytosis. Currently, a glycoconjugate of these polysaccharides is being explored as a vaccine to protect against <i>C. neoformans</i> infection. In this study, NOE and <i>J</i>-coupling values from NMR experiments were consistent with a converged structure of the synthetic decasaccharide, GXM10-Ac₃, calculated from MD simulations. GXM10-Ac₃ was designed as an extension of glucuronoxylomannan (GXM) polysaccharide motif (M2) which is common in the clinically predominant serotype A strains and is recognized by protective forms of GXM-specific monoclonal antibodies. The M2 motif is a hexasaccharide with a three-residue α-mannan backbone, modified by β-(1→2)-xyloses (Xyl) on the first two mannoses (Man) and a β-(1→2)-glucuronic acid (GlcA) on the third Man. Combined NMR and MD analyses reveal that GXM10-Ac₃ adopts an extended structure, with Xyl/GlcA branches alternating sides along the α-mannan backbone. <i>O</i>-acetyl esters also alternate sides and are grouped in pairs. MD analysis of a twelve M2-repeating unit polymer supports the notion that the GXM10-Ac₃ structure is uniformly represented throughout the polysaccharide. This derived GXM model displays high flexibility while maintaining a structural identity, yielding insights to further explore intermolecular interactions between polysaccharides, interactions with anti-GXM mAbs, and the cryptococcal polysaccharide architecture.