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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

BALB/c mice elicit an IgG immune response against Y. enterocolitica LPS, but is not protective against B. abortus infection. A) ELISA response of the sera obtained from the third bleed (1/200 dilution) of the different mouse groups against Y. enterocolitica O:9 LPS. Microtiter plates were coated with 12.5 μg of Y. enterocolitica O:9 LPS. Each datum point represents the average of three replicate wells. Response was read after 1 h @ 37°C at OD405 nm. The bar in each set of data corresponds to the average of each group.
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Figure 5: BALB/c mice elicit an IgG immune response against Y. enterocolitica LPS, but is not protective against B. abortus infection. A) ELISA response of the sera obtained from the third bleed (1/200 dilution) of the different mouse groups against Y. enterocolitica O:9 LPS. Microtiter plates were coated with 12.5 μg of Y. enterocolitica O:9 LPS. Each datum point represents the average of three replicate wells. Response was read after 1 h @ 37°C at OD405 nm. The bar in each set of data corresponds to the average of each group.

Mentions: The generation of antibodies against the Ye O:9 antigen was further analyzed by ELISA. Each well was coated with 12.5 μg of Ye O:9 LPS (Figure 5). Of the three groups of mice, only the sera from mice belonging to the two groups inoculated with glycosylated AcrA showed an IgG response directed towards the polysaccharide. However, a high level of variation in the absorbance values was observed, with some animals showing no significant response. Interestingly, the group of mice inoculated with the lower amount of glycoprotein (1.5 μg) exhibited a higher average OD405 nm than the group inoculated with 3 μg. Nevertheless, the immune response was insufficient or inefficient in protecting the mice against a challenge with B. abortus, as no statistical difference was observed in bacterial load in the spleen of infected mice irrespective of whether they were injected with glycosylated or unglycosylated AcrA (data not shown).


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)

BALB/c mice elicit an IgG immune response against Y. enterocolitica LPS, but is not protective against B. abortus infection. A) ELISA response of the sera obtained from the third bleed (1/200 dilution) of the different mouse groups against Y. enterocolitica O:9 LPS. Microtiter plates were coated with 12.5 μg of Y. enterocolitica O:9 LPS. Each datum point represents the average of three replicate wells. Response was read after 1 h @ 37°C at OD405 nm. The bar in each set of data corresponds to the average of each group.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: BALB/c mice elicit an IgG immune response against Y. enterocolitica LPS, but is not protective against B. abortus infection. A) ELISA response of the sera obtained from the third bleed (1/200 dilution) of the different mouse groups against Y. enterocolitica O:9 LPS. Microtiter plates were coated with 12.5 μg of Y. enterocolitica O:9 LPS. Each datum point represents the average of three replicate wells. Response was read after 1 h @ 37°C at OD405 nm. The bar in each set of data corresponds to the average of each group.
Mentions: The generation of antibodies against the Ye O:9 antigen was further analyzed by ELISA. Each well was coated with 12.5 μg of Ye O:9 LPS (Figure 5). Of the three groups of mice, only the sera from mice belonging to the two groups inoculated with glycosylated AcrA showed an IgG response directed towards the polysaccharide. However, a high level of variation in the absorbance values was observed, with some animals showing no significant response. Interestingly, the group of mice inoculated with the lower amount of glycoprotein (1.5 μg) exhibited a higher average OD405 nm than the group inoculated with 3 μg. Nevertheless, the immune response was insufficient or inefficient in protecting the mice against a challenge with B. abortus, as no statistical difference was observed in bacterial load in the spleen of infected mice irrespective of whether they were injected with glycosylated or unglycosylated AcrA (data not shown).

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