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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

Y. enterocolitica O:9 bioconjugate as a promising antigen for the diagnosis of bovine brucellosis. A) Magnetic bead-based immunoassay for detection of antibodies against Brucella abortus O-antigen. Magnetic beads coated with the AcrA-OAg glycoconjugate were incubated with the indicated bovine serum samples (dilution 1/200). Bound antibodies were detected using a Cy5-conjugated goat anti-bovine IgG. The bar graph data represents the means and standard deviation for two separate determinations. Control: magnetic beads incubated with PBS-Tween 0.1%. B) Immunoblot of the same bovine serum samples.
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Figure 6: Y. enterocolitica O:9 bioconjugate as a promising antigen for the diagnosis of bovine brucellosis. A) Magnetic bead-based immunoassay for detection of antibodies against Brucella abortus O-antigen. Magnetic beads coated with the AcrA-OAg glycoconjugate were incubated with the indicated bovine serum samples (dilution 1/200). Bound antibodies were detected using a Cy5-conjugated goat anti-bovine IgG. The bar graph data represents the means and standard deviation for two separate determinations. Control: magnetic beads incubated with PBS-Tween 0.1%. B) Immunoblot of the same bovine serum samples.

Mentions: Because vaccination with the glycosylated AcrA induced the production of a specific IgG immune response against the O-antigen, we asked if this glycoconjugate could be used as an antigen for the diagnosis of the infection in cows. To test this, we immobilized AcrA (control) or AcrA-O:9 on paramagnetic microbeads (see Materials and Methods) and tested the reactivity towards sera from non-infected animals, as well as from cows vaccinated with the B. abortus Δpgm or infected with B. abortus 2308 strain. These animals are part of an efficacy trial to test the protective capacity of the Δpgm strain [10,30] (manuscript in preparation). As mentioned earlier, Δpgm is a rough strain that does not induce the production of anti-O-antigen specific immunoglobulin titers in mice. As can be observed in Figure 6A, the assay clearly differentiates non-infected from infected animals and does not react with sera from animals vaccinated with a strain that lacks a complete LPS. Additionally, it is shown that none of these sera reacted against the non-glycosylated form of AcrA in a immunoblot indicating that the IgG response detected is directed specifically towards the carbohydrate moiety of the antigen (Figure 6B). Taken together, these results strongly suggest that this novel antigen could be used for the development of new diagnostic tools for brucellosis.


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)

Y. enterocolitica O:9 bioconjugate as a promising antigen for the diagnosis of bovine brucellosis. A) Magnetic bead-based immunoassay for detection of antibodies against Brucella abortus O-antigen. Magnetic beads coated with the AcrA-OAg glycoconjugate were incubated with the indicated bovine serum samples (dilution 1/200). Bound antibodies were detected using a Cy5-conjugated goat anti-bovine IgG. The bar graph data represents the means and standard deviation for two separate determinations. Control: magnetic beads incubated with PBS-Tween 0.1%. B) Immunoblot of the same bovine serum samples.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Y. enterocolitica O:9 bioconjugate as a promising antigen for the diagnosis of bovine brucellosis. A) Magnetic bead-based immunoassay for detection of antibodies against Brucella abortus O-antigen. Magnetic beads coated with the AcrA-OAg glycoconjugate were incubated with the indicated bovine serum samples (dilution 1/200). Bound antibodies were detected using a Cy5-conjugated goat anti-bovine IgG. The bar graph data represents the means and standard deviation for two separate determinations. Control: magnetic beads incubated with PBS-Tween 0.1%. B) Immunoblot of the same bovine serum samples.
Mentions: Because vaccination with the glycosylated AcrA induced the production of a specific IgG immune response against the O-antigen, we asked if this glycoconjugate could be used as an antigen for the diagnosis of the infection in cows. To test this, we immobilized AcrA (control) or AcrA-O:9 on paramagnetic microbeads (see Materials and Methods) and tested the reactivity towards sera from non-infected animals, as well as from cows vaccinated with the B. abortus Δpgm or infected with B. abortus 2308 strain. These animals are part of an efficacy trial to test the protective capacity of the Δpgm strain [10,30] (manuscript in preparation). As mentioned earlier, Δpgm is a rough strain that does not induce the production of anti-O-antigen specific immunoglobulin titers in mice. As can be observed in Figure 6A, the assay clearly differentiates non-infected from infected animals and does not react with sera from animals vaccinated with a strain that lacks a complete LPS. Additionally, it is shown that none of these sera reacted against the non-glycosylated form of AcrA in a immunoblot indicating that the IgG response detected is directed specifically towards the carbohydrate moiety of the antigen (Figure 6B). Taken together, these results strongly suggest that this novel antigen could be used for the development of new diagnostic tools for brucellosis.

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