Uncovering new insights into bacterial pseudaminylation with a pan-specific antibody tool

ABSTRACT: Pseudaminic acids (Pse) are a family of nine-carbon carbohydrates produced by many bacteria, including important human pathogens. These non-mammalian carbohydrates are found within the structure of lipopolysaccharides, capsular polysaccharides, and as O-linked post-translational modifications (PTMs) of serine and threonine residues within flagellin and pili proteins. This protein O-pseudaminylation is important for the host colonisation, virulence and motility of pathogens, although only a handful of cell surface proteins bearing this PTM have been identified to date, and the functional role of this PTM remains largely unknown. Herein, we describe the synthesis of O-pseudaminylated serine and threonine amino acids with both α- and β-anomeric configuration and their installation into tryptic glycopeptide fragments of Helicobacter pylori flagellin A and B. These synthetic glycopeptides enabled assignment of the stereochemistry of O-linked Pse in H. pylori and facilitated the generation of monoclonal antibodies (mAbs) that can recognise both α- and β-configured Pse (and its C8 epimer) linked to protein or glycan. The sequences, affinities and structural epitopes of these pan-specific Pse mAbs were determined, informing the development of an immunoenrichment strategy for glycoproteomic analyses that expanded the number of known pseudaminylation sites and substrates across the proteomes of pathogenic Helicobacter pylori and Campylobacter jejuni strains. These mAbs are also capable of enriching the more complex O-glycans that contain terminal Pse in pathogenic Acinetobacter baumannii strains, enabling the discovery of hitherto unknown glycoproteins. Finally, we demonstrate that these mAbs recognise a variety of different multidrug resistant Acinetobacter baumannii capsule types and are capable of clearing infections in mice.

INSTRUMENT(S):

ORGANISM(S): Helicobacter Pylori P12

SUBMITTER: Mark Larance  

LAB HEAD: Mark Larance

PROVIDER: PXD056875 | Pride | 2025-10-25

REPOSITORIES: Pride