Introduction/Objectives:
Pseudaminic acid is a nonulosonic acid carbohydrate used by diverse bacterial phyla to modify proteins, capsular polysaccharides, and lipopolysaccharides. The incorporation of this carbohydrate into multiple bacterial glycoconjugates makes the development of Pseudaminic acid affinity tools an attractive approach for bacterial glycoproteomics and as a target for passive immune therapies.
Methods:
Using synthetically generated glycopeptides we have generated and characterized monoclonal antibodies specific for Pseudaminic acid suitable for glycoproteomic analysis, imaging and therapeutic intervention studies. These antibodies demonstrate high selectivity and specificity while allowing the recognition of both Pseudaminic acids directly attached to proteins and terminally exposed Pseudaminic acids within glycans.
Results and Conclusions
Using these tools, we demonstrate that the O-linked glycosylation systems of Campylobacter jejuni and Helicobacter pylori extend beyond the previously reported Flagellin substrates, confirming multiple novel O-linked glycoproteins previously suspected but not yet identified. Extending the application of these antibodies to O-linked glycosylation within Acinetobacter baumannii, we demonstrate that Pseudaminic acid incorporated into glycan chains is detectable in a stereochemistry-specific manner, providing a sensitive means to assess capsule types. For A. baumannii these antibodies allow both opsonisation and visualisation of A. baumannii infections while also allowing the expansion of the known A. baumannii glycoproteome using glycopeptide affinity enrichments. Finally, we show that Pseudaminic acid monoclonal antibodies are protective against bacterial challenges with Pseudaminic acid expressing A. baumannii using a therapeutic intervention model. Thus, Pseudaminic acid monoclonal antibodies provide a new avenue to explore the glycoproteome of multiple bacterial species as well as new tools to study the role of Pseudaminic acid-containing glycoconjugates in bacterial pathogenesis.