MALDI glycotyping of O-antigens from a single colony of gram-negative bacteria.

Polypeptide-targeted MALDI-TOF MS for microbial species identification has revolutionized microbiology. However, no practical MALDI-TOF MS identification method for O-antigen polysaccharides, a major indicator for epidemiological classification within a species of gram-negative bacteria, is available. We describe a simple MALDI glycotyping method for O-antigens that simultaneously identifies the molecular mass of the repeating units and the monosaccharide composition of the O-antigen. We analyzed the Escherichia coli O1, O6, and O157-type strains. Conventional species identification based on polypeptide patterns and O-antigen polysaccharide typing can be performed in parallel from a single colony using our MALDI-TOF MS workflow. Moreover, subtyping within the same O-antigen and parallel colony-specific O-antigen determination from mixed strains, including the simultaneous identification of multiple strains-derived O-antigens within selected colony, were performed. In MALDI glycotyping of two Enterobacteriaceae strains, a Citrobacter freundii strain serologically cross-reactive with E. coli O157 gave a MALDI spectral pattern identical to E. coli O157. On the other hand, an Edwardsiella tarda strain with no reported O-antigen cross-reactivity gave a MALDI spectral pattern of unknown O-antigen repeating units. The method described in this study allows the parallel and rapid identification of microbial genera, species, and serotypes of surface polysaccharides using a single MALDI-TOF MS instrument.

Glycoblotting-Based Ovo-Sulphoglycomics Reveals Phosphorylated N-Glycans as a Possible Host Factor of AIV Prevalence in Waterfowls.

Sulfated N-glycans play a crucial role in the interaction between influenza A virus (IAV) and its host. These glycans have been found to enhance viral replication, highlighting their significance in IAV propagation. This study investigated the expression of acidic N-glycans, specifically sulfated and phosphorylated glycans, in the egg whites of 72 avian species belonging to the Order Anseriformes (waterfowls). We used the glycoblotting-based sulphoglycomics approach to elucidate the diversity of acidic N-glycans and infer their potential role in protecting embryos from infections. Family-specific variations in sulfated and phosphorylated N-glycan profiles were identified in waterfowl egg whites. Different waterfowl species exhibited distinct expressions of sulfated trans-Gal(+) and trans-Gal(-) N-glycan structures. Additionally, species-specific expression of phosphorylated N-glycans was observed. Furthermore, it was found that waterfowl species with high avian influenza virus (AIV) prevalence displayed a higher abundance of phosphorylated hybrid and high-mannose N-glycans on their egg whites. These findings shed light on the importance of phosphorylated and sulfated N-glycans in understanding the role of acidic glycans in IAV propagation.