Modification of the Campylobacter jejuni N-linked glycan by EptC protein-mediated addition of phosphoethanolamine

Abstract

Campylobacter jejuni is the major worldwide cause of bacterial gastroenteritis. C. jejuni possesses an extensive repertoire of carbohydrate structures that decorate both protein and non-protein surface-exposed structures. An N-linked glycosylation system encoded by the pgl gene cluster mediates the synthesis of a rigidly conserved heptasaccharide that is attached to protein substrates or released as free oligosaccharide in the periplasm. Removal of N-glycosylation results in reduced virulence and impeded host cell attachment. Since the N-glycan is conserved, the N-glycosylation system is also an attractive option for glycoengineering recombinant vaccines in Escherichia coli. To determine whether non-canonical N-glycans are present in C. jejuni, we utilized high throughput glycoproteomics to characterize C. jejuni JHH1 and identified 93 glycosylation sites, including 34 not previously reported. Interrogation of these data allowed the identification of a phosphoethanolamine (pEtN)-modified variant of the N-glycan that was attached to multiple proteins. The pEtN moiety was attached to the terminal GalNAc of the canonical N-glycan. Deletion of the pEtN transferase eptC removed all evidence of the pEtN-glycan but did not globally influence protein reactivity to patient sera, whereas deletion of the pglB oligosaccharyltransferase significantly reduced reactivity. Transfer of eptC and the pgl gene cluster to E. coli confirmed the addition of the pEtN-glycan to a target C. jejuni protein. Significantly reduced, yet above background levels of pEtN-glycan were also observed in E. coli not expressing eptC, suggesting that endogenous E. coli pEtN transferases can mediate the addition of pEtN to N-glycans. The addition of pEtN must be considered in the context of glycoengineering and may alter C. jejuni glycan-mediated structure-function interactions.

FIGURE 1.

Identification of a pEtN-modified N-linked glycan attached to antigenic protein HisJ (Cj0734c). A, shown is peptide fragmentation (lowercase y and b) by HCD MS/MS supporting the assignment of the peptide sequence ²⁷ESNASVELK³⁵. B, fragmentation of pEtN leads to the loss of ethanolamine (43.04 Da) followed by phosphate (79.97 Da). C, shown is pEtN and N-glycan fragmentation (uppercase Y and B) supporting the terminal location of pEtN attached to GalNAc. Oxonium ions of N-glycan sugars and pEtN are also annotated.

FIGURE 2.

CID MS/MS of pEtN-glycan-modified glycopeptide ²⁷ESNASVELK³⁵. A, a fragmentation map shows complete coverage of the pEtN-glycan. B, shown is fragmentation of the doubly-charged ion of ²⁷ESNASVELK³⁵ (m/z = 1253.03597). Highlighted regions of both 1120–1240 m/z (I) and 450–1100 m/z (II) are provided to enable complete glycan annotation.

FIGURE 3.

A pEtN-glycan-modified glycopeptide (⁵²⁹QDLNSTLPVVNTNHAK⁵⁴⁴) from NCTC 11168 O. A, HCD MS/MS denoting peptide fragment ions confirming the identity (MASCOT ion score, 36) of XIC of pEtN-glycan-modified (B) and canonical N-glycan-modified (C) ⁵²⁹QDLNSTLPVVNTNHAK⁵⁴⁴.

FIGURE 4.

Comparison of glycoproteomes from C. jejuni JHH1 wild type and ΔpglB and ΔeptC deletion mutants. Western blotting with anti-N-linked glycan antibodies (A) and anti-JlpA (0G, 1G, and 2G refer to the number of occupied N-glycosites (47) (B) is shown. C, shown is XIC of pEtN-glycan and canonical N-glycan attached to peptide ²⁷ESNASVELK³⁵. CID MS/MS spectra of pEtN- glycan-modified (D) and canonical N-glycan-modified (E) forms of peptide ²⁷ESNASVELK³⁵, taken from wild-type and ΔeptC C. jejuni JHH1, respectively.

FIGURE 5.

Expression of the C. jejuni N-linked glycosylation system, EptC-HA, and AcrA-His in E. coli. A, upper, induction (I) of EptC with arabinose from pNS3 compared with non-induced controls (NI) and empty vector control pMLBAD is shown. Lower, comparison of AcrA glycosylation (0G, 1G, and 2G refer to the number of occupied N-glycosites). B, XIC of pEtN- glycan-modified (i and iii), E. coli heptasaccharide-modified (i and ii) and canonical C. jejuni N- glycan-modified (iii and iv) glycopeptides from AcrA ¹¹³ATFENASKDFNR¹²⁴ are shown. C, confirmation of glycan structures by CID MS/MS (i–iv).

FIGURE 6.

The effect of ΔpglB and ΔeptC on C. jejuni phenotypes. A, shown is a Western blot using patient serum. B, shown are motility assays of C. jejuni JHH1 wild type, ΔeptC, and ΔpglB. C, polymyxin B sensitivity of C. jejuni JHH1 wild-type, ΔpglB, and ΔeptC. D, shown is restoration of polymyxin B resistance in C. jejuni NCTC 11168 ΔpglB::pglB.