Detecting Glucose Fluctuations in the Campylobacter jejuni N‑Glycan Structure

• Published in: ACS chemical biology 2021-11, Vol.16 (11), p.2690-2701
• Main Authors: Nothaft, Harald, Bian, Xiaoming, Shajahan, Asif, Miller, William G, Bolick, David T, Guerrant, Richard L, Azadi, Parastoo, Ng, Kenneth K. S, Szymanski, Christine M
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Aspartic Acid - chemistry; Campylobacter jejuni - metabolism; Carbohydrate Conformation; Carbohydrate Sequence; Enzyme-Linked Immunosorbent Assay; Escherichia coli - metabolism; Glucose - metabolism; Glycosylation; Immune Sera; Mice; Phagocytosis; Polysaccharides - chemistry; Polysaccharides - metabolism; Sequence Alignment
• ISSN: 1554-8929; 1554-8937
• DOI: 10.1021/acschembio.1c00498

Campylobacter jejuni is a significant cause of human gastroenteritis worldwide, and all strains express an N-glycan that is added to at least 80 different proteins. We characterized 98 C. jejuni isolates from infants from 7 low- and middle-income countries and identified 4 isolates unreactive with our N-glycan-specific antiserum that was raised against the C. jejuni heptasaccharide composed of GalNAc-GalNAc-GalNAc­(Glc)-GalNAc-GalNAc-diNAcBac. Mass spectrometric analyses indicated these isolates express a hexasaccharide lacking the glucose branch. Although all 4 strains encode the PglI glucosyltransferase (GlcTF), one aspartate in the DXDD motif was missing, an alteration also present in ∼4% of all available PglI sequences. Deleting this residue from an active PglI resulted in a nonfunctional GlcTF when the protein glycosylation system was reconstituted in E. coli, while replacement with Glu/Ala was not deleterious. Molecular modeling proposed a mechanism for how the DXDD residues and the structure/length beyond the motif influence activity. Mouse vaccination with an E. coli strain expressing the full-length heptasaccharide produced N-glycan-specific antibodies and a corresponding reduction in Campylobacter colonization and weight loss following challenge. However, the antibodies did not recognize the hexasaccharide and were unable to opsonize C. jejuni isolates lacking glucose, suggesting this should be considered when designing N-glycan-based vaccines to prevent campylobacteriosis.