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Exploiting the Campylobacter jejuni protein glycosylation system for glycoengineering vaccines and diagnostic tools directed against brucellosis.

Iwashkiw JA, Fentabil MA, Faridmoayer A, Mills DC, Peppler M, Czibener C, Ciocchini AE, Comerci DJ, Ugalde JE, Feldman MF - Microb. Cell Fact. (2012)

Bottom Line: Because Y. enterocolitica O9 and Brucella abortus 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.The recombinant glycoprotein coated onto magnetic beads was efficient in differentiating between naïve and infected bovine sera.Bacterial engineered glycoproteins show promising applications for the development on an array of diagnostics and immunoprotective opportunities in the future.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, University of Alberta, Edmonton, AB, Canada.

ABSTRACT

Background: 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.

Results: In this work we expressed the C. jejuni oligosaccharyltansferase (OTase) PglB, responsible for N-linked protein glycosylation together with a suitable acceptor protein (AcrA) in Yersinia enterocolitica O9 cells. MS analysis of the acceptor protein demonstrated the transfer of a polymer of N-formylperosamine to AcrA in vivo. Because Y. enterocolitica O9 and Brucella abortus 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 Brucella, although this response was not protective against a challenge with a virulent B. abortus strain. The recombinant glycoprotein coated onto magnetic beads was efficient in differentiating between naïve and infected bovine sera.

Conclusion: Bacterial engineered glycoproteins show promising applications for the development on an array of diagnostics and immunoprotective opportunities in the future.

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Related in: MedlinePlus

ESI-Q-TOF MS and MS/MS analysis of of Y. enterocolitica O:9 glycobioconjugates. A) MS of high molecular weight glycosylated AcrA purified from Ye O:9 WT revealed the peak 1954.7 M/Z. MS/MS of this peak showed a disaccharide of HexNAc-Hex linking a characteristic 173 M/Z pattern corresponding to the N-formylperosamine subunit to the known glycopeptide DFNR. B) MS of high molecular weight glycosylated AcrA purified from Ye O:9 O antigen mutant revealed the peak 1284.63+ M/Z. MS/MS of this peak shows the second known glycosylated site of AcrA (AVFDNNNSTLLPGAFATITSEGFIQK) modified with the hexasaccharide HexNAc-HexNAc-Hex-Hex-HexNAc-Hex.
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Figure 3: ESI-Q-TOF MS and MS/MS analysis of of Y. enterocolitica O:9 glycobioconjugates. A) MS of high molecular weight glycosylated AcrA purified from Ye O:9 WT revealed the peak 1954.7 M/Z. MS/MS of this peak showed a disaccharide of HexNAc-Hex linking a characteristic 173 M/Z pattern corresponding to the N-formylperosamine subunit to the known glycopeptide DFNR. B) MS of high molecular weight glycosylated AcrA purified from Ye O:9 O antigen mutant revealed the peak 1284.63+ M/Z. MS/MS of this peak shows the second known glycosylated site of AcrA (AVFDNNNSTLLPGAFATITSEGFIQK) modified with the hexasaccharide HexNAc-HexNAc-Hex-Hex-HexNAc-Hex.

Mentions: In order to fully characterize the carbohydrates attached to AcrA, mass spectrometry techniques were employed. AcrA was purified from Ye O9 strains, separated by SDS-PAGE, the bands of interest were excised and in-gel digested with trypsin, and the resulting peptides analyzed by ESI-Q-TOF MS and MS/MS. Examination of the higher molecular weight smear from the Ye O:9 WT glycosylated AcrA by MS revealed a peak at 1954.71+ M/Z, and subsequent analysis of this peak by MS/MS identified the known glycopeptide DFNR modified with a carbohydrate moiety (Figure 3A). We identified a HexNAc-Hex disaccharide followed by a 173 M/Z repeat, which corresponds to the N-formylperosamine homopolymer, attached to the tetrapeptide DFNR, which represents one of the known glycosylation sites of AcrA (Figure 3A). MS analysis of the glycoprotein produced by O antigen deficient strain revealed a peak of 1284.63+ M/Z. MS/MS analysis of this peak identified the second known glycosylated site of AcrA (AVFDNNNSTLLPGAFATITSEGFIQK) modified with the hexasaccharide HexNAc-HexNAc-Hex-Hex-HexNAc-Hex (Figure 3B). This hexasaccharide is known as the outer core and is also present in Y. enterocolitica O:3. Contrary to previously published work, the outer core of Ye O:9 is therefore not structurally homologous to that of Y. enterocolitica O:3, despite genetic homology [28,29].


Exploiting the Campylobacter jejuni protein glycosylation system for glycoengineering vaccines and diagnostic tools directed against brucellosis.

Iwashkiw JA, Fentabil MA, Faridmoayer A, Mills DC, Peppler M, Czibener C, Ciocchini AE, Comerci DJ, Ugalde JE, Feldman MF - Microb. Cell Fact. (2012)

ESI-Q-TOF MS and MS/MS analysis of of Y. enterocolitica O:9 glycobioconjugates. A) MS of high molecular weight glycosylated AcrA purified from Ye O:9 WT revealed the peak 1954.7 M/Z. MS/MS of this peak showed a disaccharide of HexNAc-Hex linking a characteristic 173 M/Z pattern corresponding to the N-formylperosamine subunit to the known glycopeptide DFNR. B) MS of high molecular weight glycosylated AcrA purified from Ye O:9 O antigen mutant revealed the peak 1284.63+ M/Z. MS/MS of this peak shows the second known glycosylated site of AcrA (AVFDNNNSTLLPGAFATITSEGFIQK) modified with the hexasaccharide HexNAc-HexNAc-Hex-Hex-HexNAc-Hex.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3298491&req=5

Figure 3: ESI-Q-TOF MS and MS/MS analysis of of Y. enterocolitica O:9 glycobioconjugates. A) MS of high molecular weight glycosylated AcrA purified from Ye O:9 WT revealed the peak 1954.7 M/Z. MS/MS of this peak showed a disaccharide of HexNAc-Hex linking a characteristic 173 M/Z pattern corresponding to the N-formylperosamine subunit to the known glycopeptide DFNR. B) MS of high molecular weight glycosylated AcrA purified from Ye O:9 O antigen mutant revealed the peak 1284.63+ M/Z. MS/MS of this peak shows the second known glycosylated site of AcrA (AVFDNNNSTLLPGAFATITSEGFIQK) modified with the hexasaccharide HexNAc-HexNAc-Hex-Hex-HexNAc-Hex.
Mentions: In order to fully characterize the carbohydrates attached to AcrA, mass spectrometry techniques were employed. AcrA was purified from Ye O9 strains, separated by SDS-PAGE, the bands of interest were excised and in-gel digested with trypsin, and the resulting peptides analyzed by ESI-Q-TOF MS and MS/MS. Examination of the higher molecular weight smear from the Ye O:9 WT glycosylated AcrA by MS revealed a peak at 1954.71+ M/Z, and subsequent analysis of this peak by MS/MS identified the known glycopeptide DFNR modified with a carbohydrate moiety (Figure 3A). We identified a HexNAc-Hex disaccharide followed by a 173 M/Z repeat, which corresponds to the N-formylperosamine homopolymer, attached to the tetrapeptide DFNR, which represents one of the known glycosylation sites of AcrA (Figure 3A). MS analysis of the glycoprotein produced by O antigen deficient strain revealed a peak of 1284.63+ M/Z. MS/MS analysis of this peak identified the second known glycosylated site of AcrA (AVFDNNNSTLLPGAFATITSEGFIQK) modified with the hexasaccharide HexNAc-HexNAc-Hex-Hex-HexNAc-Hex (Figure 3B). This hexasaccharide is known as the outer core and is also present in Y. enterocolitica O:3. Contrary to previously published work, the outer core of Ye O:9 is therefore not structurally homologous to that of Y. enterocolitica O:3, despite genetic homology [28,29].

Bottom Line: Because Y. enterocolitica O9 and Brucella abortus 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.The recombinant glycoprotein coated onto magnetic beads was efficient in differentiating between naïve and infected bovine sera.Bacterial engineered glycoproteins show promising applications for the development on an array of diagnostics and immunoprotective opportunities in the future.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, University of Alberta, Edmonton, AB, Canada.

ABSTRACT

Background: 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.

Results: In this work we expressed the C. jejuni oligosaccharyltansferase (OTase) PglB, responsible for N-linked protein glycosylation together with a suitable acceptor protein (AcrA) in Yersinia enterocolitica O9 cells. MS analysis of the acceptor protein demonstrated the transfer of a polymer of N-formylperosamine to AcrA in vivo. Because Y. enterocolitica O9 and Brucella abortus 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 Brucella, although this response was not protective against a challenge with a virulent B. abortus strain. The recombinant glycoprotein coated onto magnetic beads was efficient in differentiating between naïve and infected bovine sera.

Conclusion: Bacterial engineered glycoproteins show promising applications for the development on an array of diagnostics and immunoprotective opportunities in the future.

Show MeSH
Related in: MedlinePlus