Detecting glucose fluctuations in the Campylobacter jejuni N-glycan structure

Authors: NOTHAFT, HARALD - University Of Alberta; BIAN, XIAOMING - University Of Georgia; SHAJAHAN, ASIF - University Of Georgia; Miller, William - Bill; BOLICK, DAVID - University Of Virginia School Of Medicine; GUERRANT, RICHARD - University Of Virginia School Of Medicine; AZADI, PARASTOO - University Of Georgia; NG, KENNETH - University Of Windsor; SZYMANSKI, CHRISTINE - University Of Georgia

Submitted to: ACS Chemical Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/18/2021
Publication Date: 11/2/2021
Citation: Nothaft, H., Bian, X., Shajahan, A., Miller, W.G., Bolick, D.T., Guerrant, R.L., Azadi, P., Ng, K.K., Szymanski, C.M. 2021. Detecting glucose fluctuations in the Campylobacter jejuni N-glycan structure. ACS Chemical Biology. 16(11):2690-2701. https://doi.org/10.1021/acschembio.1c00498.
DOI: https://doi.org/10.1021/acschembio.1c00498

Interpretive Summary: Campylobacter jejuni is an important human foodborne pathogen responsible for diarrheal disease worldwide. Proteins on the C. jejuni outer surface are modified by a conserved chain of sugars. These sugars can be used to evade the host immune response, but may be useful in the development of sugar-based vaccines against these bacterial pathogens. In a survey of C. jejuni isolated from infants in low to middle income countries, four strains did not react against antisera specific for these sugar chains. Chemical analysis of these four strains indicated that, while the surface proteins were still modified by sugars, these sugars no longer contained a glucose branch. Addition of glucose to these proteins is due to the actions of a specific enzyme, and DNA sequence analysis indicated that the gene encoding this enzyme in the four strains possessed a critical mutation in the active site of the enzyme. Mutating this site in functional enzymes resulted in non-functionality, while reversion of the mutation in one of the four isolates restored functionality.

Technical Abstract: Campylobacter jejuni is the leading bacterial cause of gastroenteritis in humans of all ages. C. jejuni species produce a conserved N-glycan heptasaccharide that is added to at least 80 different proteins. We have surveyed over 120 C. jejuni isolates from infants <1 year of age from 8 different low- and middle-income countries and identified 4 isolates that did not react with the C. jejuni N-glycan-specific antiserum serum. Mass spectrometric analyses indicated that these isolates produce an N-glycan hexasaccharide lacking the glucose branch. Although the gene encoding the glucosyltransferase, PglI was present, sequence comparisons revealed that one common feature of all four strains was a deletion of one aspartate residue in the DxDD motif, an alternation that can also be found in close to 4% of all available PglI sequences. Deletion of the corresponding D in an active PglI protein resulted in a non-functional glycosyltransferase (GTase) when the protein glycosylation system was reconstituted in E. coli, while replacing the D with an E or an A had no effect. Restoration of DxDD in an inactive PglI resulted in GTase activity in one case, in another case an additional change of I88 to a T was required. Molecular modelling suggests that the DxDD residues, as well as the length of the loop where the motif resides, is important for activity. Moreover, characterization of antibodies raised against the full length N-glycan showed that the N-glycan without the glucose was not recognized in ELISA and OPA assays. The loss of glucose in certain C. jejuni strains, either occurring through a random event or as a mechanism of immune evasion has to be taken into consideration when designing N-glycan based vaccines for broad coverage against all C. jejuni isolates.