Hargett, A. A. | Search bioRxiv

bioRxiv2023

The structure of a C. neoformans polysaccharide motif recognized by protective antibodies: a combined NMR and MD study

Cryptococcus neoformans is a fungal pathogen responsible for cryptococcosis and cryptococcal meningitis. The C. neoformans capsular polysaccharide and shed exopolysaccharide functions both as a key virulence factor and to protect the fungal cell from phagocytosis. Currently, a glycoconjugate of these polysaccharides is being explored as a vaccine to protect against C. neoformans infection. In this combined NMR and MD study, experimentally determined NOEs and J-couplings support a structure of the synthetic decasaccharide, GXM10-Ac3, obtained by MD. 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 characterized by a 6-residue -mannan backbone repeating unit, consisting of a triad of -(1[->]3)-mannoses, modified by {beta}-(1[->]2)-xyloses on the first two mannoses and a {beta}-(1[->]2)-glucuronic acid on the third mannose. The combined NMR and MD analyses reveal that GXM10-Ac3 adopts an extended structure, with xylose/glucuronic acid branches alternating sides along the -mannan backbone. O-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 experimentally consistent GXM model displays high flexibility while maintaining a structural identity, yielding new insights to further explore intermolecular interactions between polysaccharides, interactions with anti-GXM mAbs, and the cryptococcal polysaccharide architecture. Significance StatementThis study utilized a combined NMR and MD approach to elucidate the structure of a Cryptococcus neoformans GXM synthetic decasaccharide (GXM10-Ac3), recognized by protective anti-GXM mAbs. The data revealed an extended structure in which the xylose/glucuronic acid branches and pairs of 6-O-acetyl esters predominantly alternate sides along the -mannan backbone. MD analysis of a GXM polysaccharide predicts that the decasaccharide structure is uniformly represented in the polysaccharide. Additionally, the GXM exhibits high flexibility while maintaining structural identity. These findings lay the foundation for future studies aimed at understanding anti-GXM antibody-polysaccharide interactions.

biochemistry↗

bioRxiv2022

Lyophilization induces alterations in cryptococcal exopolysaccharide resulting in reduced antibody binding

The structural, antigenic, and immunological characterization of microbial polysaccharides requires purification that often involves detergent precipitation and lyophilization. Here we examine physicochemical changes induced by lyophilization on exopolysaccharide (EPS) of the pathogenic fungus Cryptococcus neoformans. Solution 1H NMR reveals significant anomeric signal attenuation following lyophilization of native EPS while 1H ssNMR shows few changes, suggesting diminished molecular motion and consequent broadening of 1H NMR polysaccharide resonances. 13C ssNMR, dynamic light scattering, and transmission electron microscopy show that, while native EPS has rigid molecular characteristics and contains small, loosely packed polysaccharide assemblies, lyophilized and resuspended EPS is disordered and contains larger dense rosette-like aggregates, suggesting that structural water molecules in the interior of the polysaccharide assemblies are removed during extensive lyophilization. Importantly, mAbs to C. neoformans polysaccharide binds the native EPS more strongly than lyophilized EPS. Together, these observations argue for caution when interpreting the biological and immunological attributes of polysaccharides that have been lyophilized to dryness.

microbiology↗