Characterization of the structurally diverse N-linked glycans of Campylobacter species

Abstract

The Gram-negative bacterium Campylobacter jejuni encodes an extensively characterized N-linked protein glycosylation system that modifies many surface proteins with a heptasaccharide glycan. In C. jejuni, the genes that encode the enzymes required for glycan biosynthesis and transfer to protein are located at a single pgl gene locus. Similar loci are also present in the genome sequences of all other Campylobacter species, although variations in gene content and organization are evident. In this study, we have demonstrated that only Campylobacter species closely related to C. jejuni produce glycoproteins that interact with both a C. jejuni N-linked-glycan-specific antiserum and a lectin known to bind to the C. jejuni N-linked glycan. In order to further investigate the structure of Campylobacter N-linked glycans, we employed an in vitro peptide glycosylation assay combined with mass spectrometry to demonstrate that Campylobacter species produce a range of structurally distinct N-linked glycans with variations in the number of sugar residues (penta-, hexa-, and heptasaccharides), the presence of branching sugars, and monosaccharide content. These data considerably expand our knowledge of bacterial N-linked glycan structure and provide a framework for investigating the role of glycosyltransferases and sugar biosynthesis enzymes in glycoprotein biosynthesis with practical implications for synthetic biology and glycoengineering.

Fig 1

Inferred phylogeny (A) and putative pgl loci (B) of Campylobacter species. (A) A phylogenetic tree was generated using approximately 1,300 bases of 16S rRNA gene sequence from Campylobacter species and E. coli K-12 strain W3110 as an outlier. Sequences were aligned using ClustalW2 (27) and a neighbor-joining phylogenetic tree produced using MEGA5 software (24). Species in bold type were analyzed further in this study, and the genome sequences of those underlined are available. (B) The C. jejuni NCTC 11168 PglB amino acid sequence was used as the query in a BLAST search against the nonredundant protein database to identify further Campylobacter pglB genes. For species whose genomes have been sequenced, predicted amino acid sequences from ORFs surrounding the pglB genes were used as queries in BLAST searches to identify potential functions. Where BLAST searches resulted in hits with C. jejuni Pgl proteins, a Needleman-Wunsch pairwise global alignment (NCBI) of the query and the corresponding C. jejuni NCTC 11168 orthologue was performed. Where the amino acid identity was >30% (gaps, <10%), ORFs were labeled according to the C. jejuni pgl nomenclature (data not shown). Those with predicted functions involved in polysaccharide biosynthesis are identified as follows: putative N-linked OTases or PglB proteins, black shading; sugar biosynthesis enzymes, no shading; glycosyltransferases, light shading; transporters, dark shading; unrelated genes or genes of unknown function, grey outline with no shading. Excluded are Campylobacter rectus, which has sequencing errors in the pglB gene (37), and C. fetus subsp. venerealis, which has the same gene arrangement in this locus as C. fetus subsp. fetus.

Fig 2

Reactivity of Campylobacter proteins with C. jejuni N-linked-glycan-specific reagents. Western blot assays of whole-cell lysates from C. jejuni (C. jej.) and the corresponding isogenic pglB knockout mutant (C. jej. pglB::kan) along with C. coli (C. col.), C. lari (C. lar.), C. hyointestinalis (C. hyo.), C. helveticus (C. hel.), C. upsaliensis (C. ups.), C. fetus (C. fet.), C. lanienae (C. lan.), C. sputorum (C. spu.), and C. concisus (C. con.) were probed with C. jejuni N-linked-glycan-specific hR6 antiserum (A) or SBA lectin (B).

Fig 3

OTase activities of Campylobacter species. Campylobacter membrane preparations were solubilized in detergent and assayed for N-linked OTase activity using a fluorescently labeled peptide (ADQNATA). Reaction products were separated by Tris-Tricine SDS-PAGE. The unmodified peptide (arrow) runs toward the bottom of the gel, while derived glycosylated peptides display reduced mobility. For reference, the lowest-mobility product produced by C. jejuni membranes corresponds to the peptide modified with a heptasaccharide (15). The OTase activity displayed by membrane preparations from a range of Campylobacter species (A) was, in a number of cases, significantly increased when membranes prepared from E. coli expressing C. jejuni pglB were added to reaction mixtures (B). The species tested were C. jejuni (C. jej), C. coli (C. col.), C. lari (C. lar.), C. hyointestinalis (C. hyo.), C. helveticus (C. hel.), C. upsaliensis (C. ups.), C. fetus (C. fet.), C. lanienae (C. lan.), C. sputorum (C. spu.), and C. concisus (C. con.).

Fig 4

MS analysis of Campylobacter in vitro-generated N-linked glycans. In vitro glycosylation of a biotin-labeled fluorescent peptide (FITC-ADQNATAK-biotin) was analyzed by MALDI-TOF MS. Panels A through to I are MALDI-LIFT-TOF/TOF MS-generated spectra derived from glycopeptides produced by the indicated Campylobacter species. Below the spectra are the corresponding inferred N-linked glycan structures. Fragments corresponding to peptides with no glycan (arrows) generated peaks with an m/z value of either 1,556 or 1,199, depending on the presence or absence of the labile FITC moiety. The m/z values of peaks corresponding to fragment ions resulting from the sequential loss of sugar residues are indicated in the spectra. a.u., arbitrary units.

Fig 5

Schematic overview of epsilonproteobacterial N-linked glycan structures. The C. jejuni N-linked glycan structure is from reference , the C. lari structure is from reference and this study, the H. pullorum structure is from reference , and the H. winghamensis and W. succinogenes structures are unpublished data from our laboratory. All other structures are from this study. The abbreviated species names C. jej., C. col., C. ups., C. hel., C. fet., C. hyo., C. lar., C. lan., C. spu., C. con., H. pul., H. win., and W. suc. correspond to C. jejuni, C. coli, C. upsaliensis, C. helveticus, C. fetus, C. hyointestinalis, C. lari, C. lanienae, C. sputorum, C. concisus, H. pullorum, H. winghamensis, and W. succinogenes, respectively. Unusual sugar residues are indicated by their respective masses in daltons.