Detecting Glucose Fluctuations in the Campylobacter jejuni N-Glycan Structure

Abstract

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.

Bacterial glycosylation, it’s complicated

“…Even with a model microbe such as C. jejuni for which we have a comprehensive understanding of the glycan structures that the microbe is capable of synthesizing-and extensive knowledge about the enzymes involved in the biosynthesis of these structures [for C. jejuni 11168, see (MicroGlycoDB, 2022)], we still have very little understanding about which structures are actually expressed in the microbe's natural environments and how they are impacted in vivo-and we know even less about other C. jejuni strains, or Campylobacter species (Bian et al, 2020). For example, we recently discovered that even the invariant C. jejuni N-glycan structure can be altered through phase-variation to synthesize a hexasaccharide lacking the glucose branch, and yet this variation is not regularly observed (Nothaft et al, 2021a). Interestingly, rather than a homopolymeric G-tract, the pglI gene encoding the glucosyltransferase, possessed a repetitive GAT stretch encoding aspartate residues responsible for the DxDD motif, found in 3-glycosyltransferase GT2 CAZy family members and needed for divalent cation coordination and nucleotide phosphodiester interaction of the donor (Brockhausen, 2014).…”

“…Interestingly, rather than a homopolymeric G-tract, the pglI gene encoding the glucosyltransferase, possessed a repetitive GAT stretch encoding aspartate residues responsible for the DxDD motif, found in 3-glycosyltransferase GT2 CAZy family members and needed for divalent cation coordination and nucleotide phosphodiester interaction of the donor (Brockhausen, 2014). In this case, functional PglI enzymes have a DDDDE sequence in the catalytic region, but variants with one less aspartate are nonfunctional (Nothaft et al, 2021a). More studies are needed to determine the impact of this alteration and why it is so infrequent.…”