Functional Characterization of Bacterial Oligosaccharyltransferases Involved in O-Linked Protein Glycosylation▿

Protein glycosylation is an important posttranslational modification that occurs in all domains of life. Pilins, the structural components of type IV pili, are O glycosylated in Neisseria meningitidis, Neisseria gonorrhoeae, and some strains of Pseudomonas aeruginosa. In this work, we characterized the P. aeruginosa 1244 and N. meningitidis MC58 O glycosylation systems in Escherichia coli. In both cases, sugars are transferred en bloc by an oligosaccharyltransferase (OTase) named PglL in N. meningitidis and PilO in P. aeruginosa. We show that, like PilO, PglL has relaxed glycan specificity. Both OTases are sufficient for glycosylation, but they require translocation of the undecaprenol-pyrophosphate-linked oligosaccharide substrates into the periplasm for activity. Whereas PilO activity is restricted to short oligosaccharides, PglL is able to transfer diverse oligo- and polysaccharides. This functional characterization supports the concept that despite their low sequence similarity, PilO and PglL belong to a new family of “O-OTases” that transfer oligosaccharides from lipid carriers to hydroxylated amino acids in proteins. To date, such activity has not been identified for eukaryotes. To our knowledge, this is the first report describing recombinant O glycoproteins synthesized in E. coli

Exploiting the <it>Campylobacter jejuni </it>protein glycosylation system for glycoengineering vaccines and diagnostic tools directed against brucellosis

<p>Abstract</p> <p>Background</p> <p>Immune responses directed towards surface polysaccharides conjugated to proteins are effective in preventing colonization and infection of bacterial pathogens. Presently, the production of these conjugate vaccines requires intricate synthetic chemistry for obtaining, activating, and attaching the polysaccharides to protein carriers. Glycoproteins generated by engineering bacterial glycosylation machineries have been proposed to be a viable alternative to traditional conjugation methods.</p> <p>Results</p> <p>In this work we expressed the <it>C. jejuni </it>oligosaccharyltansferase (OTase) PglB, responsible for <it>N</it>-linked protein glycosylation together with a suitable acceptor protein (AcrA) in <it>Yersinia enterocolitica </it>O9 cells. MS analysis of the acceptor protein demonstrated the transfer of a polymer of N-formylperosamine to AcrA <it>in vivo</it>. Because <it>Y. enterocolitica </it>O9 and <it>Brucella abortus </it>share an identical O polysaccharide structure, we explored the application of the resulting glycoprotein in vaccinology and diagnostics of brucellosis, one of the most common zoonotic diseases with over half a million new cases annually. Injection of the glycoprotein into mice generated an IgG response that recognized the O antigen of <it>Brucella</it>, although this response was not protective against a challenge with a virulent <it>B. abortus </it>strain. The recombinant glycoprotein coated onto magnetic beads was efficient in differentiating between naïve and infected bovine sera.</p> <p>Conclusion</p> <p>Bacterial engineered glycoproteins show promising applications for the development on an array of diagnostics and immunoprotective opportunities in the future.</p