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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

Sera of BALB/c mice immunized with bioconjugate shows a directed IgG immune response against N-formylperosamine of Y. enterocolitica O:9 and Brucella spp. A) Control sera and B) Immune sera raised by injecting purified glycoproteins containing 3 μg glycan: 1) Unglycosylated AcrA, 2) HP-, 3) OC-, 4) WT. Both sets of sera react with each glycoprotein due to the high immunostimulatory characteristic of AcrA. C) Control sera and D); immune serum (IgG response) blotted against Y. enterocolitica 0:9 LPS from different strains from Figure 1. 5) OC-/HP-, 6) HP-, 7) OC-, 8) WT. Only the test serum was reactive against the higher molecular weight portion corresponding to the homopolymer of N-formylperosamine. E) Control sera and F) immune serum blotted against Brucella spp. LPSs: 9) B. abortus, 10) B. melitensis, and 11) B. suis. Only the immune sera are reactive against the Brucella LPS. Interestingly, although each LPS is comprised of N-formylperosamine, different linkages are present which may cause the difference in reactivity of the sera.
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Figure 4: Sera of BALB/c mice immunized with bioconjugate shows a directed IgG immune response against N-formylperosamine of Y. enterocolitica O:9 and Brucella spp. A) Control sera and B) Immune sera raised by injecting purified glycoproteins containing 3 μg glycan: 1) Unglycosylated AcrA, 2) HP-, 3) OC-, 4) WT. Both sets of sera react with each glycoprotein due to the high immunostimulatory characteristic of AcrA. C) Control sera and D); immune serum (IgG response) blotted against Y. enterocolitica 0:9 LPS from different strains from Figure 1. 5) OC-/HP-, 6) HP-, 7) OC-, 8) WT. Only the test serum was reactive against the higher molecular weight portion corresponding to the homopolymer of N-formylperosamine. E) Control sera and F) immune serum blotted against Brucella spp. LPSs: 9) B. abortus, 10) B. melitensis, and 11) B. suis. Only the immune sera are reactive against the Brucella LPS. Interestingly, although each LPS is comprised of N-formylperosamine, different linkages are present which may cause the difference in reactivity of the sera.

Mentions: To evaluate the potential use of the glycoprotein as conjugate vaccine, the purified AcrA containing the Ye O:9 antigen was injected intraperitoneally into mice to measure the immune response as well as test subsequent protection against a challenge with B. abortus. The concentration of purified glycosylated AcrA was quantified as 1.77 mg/mL for protein and 0.71 mg/mL for carbohydrate, giving a protein: carbohydrate ratio of 2.48. Three separate groups of mice were injected, one with unglycosylated AcrA and two with different amounts (1.5 μg and 3 μg of carbohydrate per mouse) of glycosylated AcrA, respectively. After a second injection of glycoprotein, sera obtained from the control (Figure 4A, C, and 4E) and 3 μg group (Figure 4B, D, and 4F) were analyzed for IgG immune response by immunoblot. As expected, both sets of sera reacted strongly against AcrA (Figure 4A, b). However, when the sera were assayed against the purified Y. enterocolitica LPS, only the groups injected with glycosylated AcrA were reactive against purified LPS samples from Y. enterocolitica O:9 OC mutant and WT strains, indicating that IgG antibodies against the N-formylperosamine homopolymer were generated (Figure 4C, D). No reactivity was observed for the control sera towards the Brucella LPS, whereas the sera from the mice injected with glycosylated AcrA showed a strong immunoreactivity towards the B. abortus and B. suis LPS (Figure 4E, F). A very weak response was observed against the B. melintensis LPS.


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)

Sera of BALB/c mice immunized with bioconjugate shows a directed IgG immune response against N-formylperosamine of Y. enterocolitica O:9 and Brucella spp. A) Control sera and B) Immune sera raised by injecting purified glycoproteins containing 3 μg glycan: 1) Unglycosylated AcrA, 2) HP-, 3) OC-, 4) WT. Both sets of sera react with each glycoprotein due to the high immunostimulatory characteristic of AcrA. C) Control sera and D); immune serum (IgG response) blotted against Y. enterocolitica 0:9 LPS from different strains from Figure 1. 5) OC-/HP-, 6) HP-, 7) OC-, 8) WT. Only the test serum was reactive against the higher molecular weight portion corresponding to the homopolymer of N-formylperosamine. E) Control sera and F) immune serum blotted against Brucella spp. LPSs: 9) B. abortus, 10) B. melitensis, and 11) B. suis. Only the immune sera are reactive against the Brucella LPS. Interestingly, although each LPS is comprised of N-formylperosamine, different linkages are present which may cause the difference in reactivity of the sera.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Sera of BALB/c mice immunized with bioconjugate shows a directed IgG immune response against N-formylperosamine of Y. enterocolitica O:9 and Brucella spp. A) Control sera and B) Immune sera raised by injecting purified glycoproteins containing 3 μg glycan: 1) Unglycosylated AcrA, 2) HP-, 3) OC-, 4) WT. Both sets of sera react with each glycoprotein due to the high immunostimulatory characteristic of AcrA. C) Control sera and D); immune serum (IgG response) blotted against Y. enterocolitica 0:9 LPS from different strains from Figure 1. 5) OC-/HP-, 6) HP-, 7) OC-, 8) WT. Only the test serum was reactive against the higher molecular weight portion corresponding to the homopolymer of N-formylperosamine. E) Control sera and F) immune serum blotted against Brucella spp. LPSs: 9) B. abortus, 10) B. melitensis, and 11) B. suis. Only the immune sera are reactive against the Brucella LPS. Interestingly, although each LPS is comprised of N-formylperosamine, different linkages are present which may cause the difference in reactivity of the sera.
Mentions: To evaluate the potential use of the glycoprotein as conjugate vaccine, the purified AcrA containing the Ye O:9 antigen was injected intraperitoneally into mice to measure the immune response as well as test subsequent protection against a challenge with B. abortus. The concentration of purified glycosylated AcrA was quantified as 1.77 mg/mL for protein and 0.71 mg/mL for carbohydrate, giving a protein: carbohydrate ratio of 2.48. Three separate groups of mice were injected, one with unglycosylated AcrA and two with different amounts (1.5 μg and 3 μg of carbohydrate per mouse) of glycosylated AcrA, respectively. After a second injection of glycoprotein, sera obtained from the control (Figure 4A, C, and 4E) and 3 μg group (Figure 4B, D, and 4F) were analyzed for IgG immune response by immunoblot. As expected, both sets of sera reacted strongly against AcrA (Figure 4A, b). However, when the sera were assayed against the purified Y. enterocolitica LPS, only the groups injected with glycosylated AcrA were reactive against purified LPS samples from Y. enterocolitica O:9 OC mutant and WT strains, indicating that IgG antibodies against the N-formylperosamine homopolymer were generated (Figure 4C, D). No reactivity was observed for the control sera towards the Brucella LPS, whereas the sera from the mice injected with glycosylated AcrA showed a strong immunoreactivity towards the B. abortus and B. suis LPS (Figure 4E, F). A very weak response was observed against the B. melintensis LPS.

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