Campylobacter is a medically and industrially important genus of bacteria that is responsible for food spoilage and various diseases. Campylobacter jejuni was the first bacterial species studied containing an N-linked glycosylation pathway for the modification of proteins, where a heptasaccharide is added onto asparagine residues of more than 60 proteins.1,2 A huge diversity of modifications can take place, often involving long and variable chains of differing glycan groups that form elaborate structures on the surface of the protein. These modifications affect DNA uptake, pathogenesis in chickens and mouse models, and recognition by various cells of the immune system.1
The question remains, how widespread are these modifications and mechanisms within the genus? Campylobacter species can be sorted into groups based on serotype, a reaction with a specific antibody. It is curious that antibodies raised against the glycosylated surface proteins in C. jejuni fail to elicit a response to a large variety of Campylobacter species. This result taken alone may indicate either that a large portion of this genus does not glycosylate its surface proteins or that the modifications are present yet unique enough to avoid detection by the antibody. In Nothaft et al. (2012),3 analysis of 29 different complete genomes of Campylobacter species reveals that enzymes responsible for glycosylation are widespread in the genus. Further analysis of the phylogeny and enzymology of these systems reveals that N-linked glycosylation can be classified into two large groups. In group I, the more thermotolerant organisms resemble the jejuni-like modifications of a heptasaccharide chain attached to an asparagine residue. In group II, the range of different modifications is widespread, not necessarily adhering to the previously established seven-carbohydrate chain seen in the group I organisms, the jejuni-like group.3
The overall conservation of the pgl gene through the Campylobacter genus indicates that all the species can perform the N-linked glycosylation. The failure of the anti-sera specific to the C. jejuni linkages to detect any modifications in the non-thermotolerant group II indicates that the sequence of carbohydrates or the linkages may be unique to that group. Precursor ion scanning using an LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific) for the presence of different carbohydrate chains and moieties revealed that organisms in group I, the thermotolerant group, did add the jejuni-like heptasaccharide structure.3 This was achieved through the use of the free oligosaccharides (fOS) method of detection for soluble chains in cell lysate after treatment with pronase E digestion and permethylation.4 There were small modifications to the overall structure, hydration and phosphorylation in one species in this group, but the heptasaccharide backbone was present. Mass spectrometry (MS) and fOS analysis of members from group II indicated that at least two different glycan chains were being used as modifications. Campylobacter fetus, as well as a few other species in this subgroup, contained both chains at the same time in the cell, sometimes attached to the same proteins. The chains were not entirely dissimilar to the jejuni-like chains but were different enough to not be recognized by the antibody. Tandem MS of the chains revealed that three of the glycan residues at the reducing end of the chain were identical to the jejuni-like chains, while the subsequent residues were unique. Further analysis of group II glycoproteins revealed the presence of 65 different modified peptides.3 Each of these peptides was modified with one of the two previously identified chains.
Interestingly, the modifications in group II could be further divided into eight separate classes, based on the nature of the modifications and the antigenicity of the modified proteins to a variety of antibodies raised against the cell surface proteins. The 29 species from the Campylobacter genus were screened with new antibodies raised against C. fetus (group II) specific glycosylation chains. Members from group I failed to elicit responses, as expected, while proteins from C. fetus and C. hyointestinalis strongly reacted with the group II antibodies.3 This is perhaps not surprising, but additional organisms from group II had variable reactions to the antibodies. Again, the lack of or reduced immunogenicity of the anti-sera toward these modified surface proteins was thought to possibly indicate additional glycosylation motifs not recognized by the group II antibodies and, in fact, this was the case. Precursor ion scanning of fOS-treated lysates from species weakly or non-reactive to the group II anti-sera revealed a wide variety of additional residues present in chains, including various modified forms such as acetylated and branched chains, or shorter chains of only three carbohydrate residues ([Hex]2-HexNAc) in the case of the C. sputorum subgroup.3
This systematic study of protein modification by glycosylation in 29 different Campylobacter species ultimately reveals a conserved glycosylation pathway with a wide diversity of modifications possible at the glycan level. This suggests that even though the overall pathway is present in this group of organisms, it has evolved apart via the specific types of modification, through the use of various glycosyltransferases. The presence of different chains on the proteins of the Campylobacter species is important to understand due to the nature of the display of these modified proteins and their role in the immunogenic responses of organisms they colonize.1,3 An increased understanding of these structures could potentially lead to additional diagnostic tools or vaccines against these different groups and species.
1. Szymanski, C.M., et al. (2003) “Campylobacter–A tale of two protein glycosylation systems,” Trends in Microbiology, 11(5) (pp. 233–8).
2. Linton, D., et al. (2005) “Functional analysis of the Campylobacter jejuni N-linked protein glycosylation pathway,” Molecular Microbiology, 55(6) (pp. 1695–1703).
3. Nothaft, H., et al. (2012) “Diversity in the protein N-glycosylation pathways within the campylobacter genus,” Molecular and Cellular Proteomics, 11(11) (pp. 1203–19).
4. Nothaft, H., et al. (2010) “N-linked protein glycosylation in a bacterial system,” Methods in Molecular Biology, 600(1) (pp. 227–43).
Post Author: Adam Humbard.