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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

Cross reactivity between Y. enterocolitica O:9 and Brucella spp. LPS samples. (0.2 OD/sample loaded) on 15% SDS-PAGE: A) LPS silver stain analysis of samples of Y. enterocolitica 1) OC-/HP-, 2)HP-, 3) OC-, 4) WT; Brucella 5) B. abortus, 6) B. melintensis, and 7) B. suis. B) Immunoblot against the same samples with monoclonal α-Yersinia (Yst 9-2). C) Immunoblot of the same samples using monoclonal α-Brucella (M84). Cross reactivity between the two different genus' LPS is observed by both monoclonal antibodies reacting against the higher molecular weight homopolymeric N-formylperosamine polysaccharide.
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Figure 1: Cross reactivity between Y. enterocolitica O:9 and Brucella spp. LPS samples. (0.2 OD/sample loaded) on 15% SDS-PAGE: A) LPS silver stain analysis of samples of Y. enterocolitica 1) OC-/HP-, 2)HP-, 3) OC-, 4) WT; Brucella 5) B. abortus, 6) B. melintensis, and 7) B. suis. B) Immunoblot against the same samples with monoclonal α-Yersinia (Yst 9-2). C) Immunoblot of the same samples using monoclonal α-Brucella (M84). Cross reactivity between the two different genus' LPS is observed by both monoclonal antibodies reacting against the higher molecular weight homopolymeric N-formylperosamine polysaccharide.

Mentions: The B. abortus and B. suis O antigens were previously characterized by genomic analysis, NMR, and serological assays and appear to be identical to that of Yersinia enterocolitica O:9 (Ye O:9) [15,24,25]. Ye O:9 is a Class II biosafety hazard organism and is easily manipulated and cultured, making it a suitable host for the production of the glycoconjugate protein with the N-formylperosamine homopolymer, which we hypothesize could cross-protect against brucellosis [26]. In some Y. enterocolitica strains, an additional "outer core" (OC) consisting of a shorter glycan chain is assembled onto the Und-PP carrier and subsequently ligated to lipid A. To confirm cross reactivity of the Ye O:9 (Table 1) and the B. abortus O antigens, LPS of both species were analyzed by SDS-PAGE and immunoblot (Figure 1). Our analysis included the wild-type Ye O:9 strain, plus three derivatives lacking the OC, the O antigen, or both glycan structures. LPS samples from of the Ye O:9 strains exhibited a different electrophoretic pattern according to the mutation carried by each strain. The double mutant strain only displayed a band corresponding to lipid A core (lane 1). The O antigen deficient strain exhibited a unique band that migrated slower than the lipid A core, as expected for the presence of the low molecular weight OC structure attached to the lipid A (lane 2). The OC minus strain only produced the high molecular weight homopolymer (lane 3), and the WT strain produced both glycan structures (lane 4; Figure 1A). Analysis of the LPS extracts using monoclonal antibodies (Figure 1B, C) demonstrated that only the high molecular weight carbohydrate of Ye O:9 was reactive towards the Yst9-2 (anti-Ye O:9 antigen) monoclonal antibody (mAb; Figure 1B, lanes 3 and 4). The Yst9-2 mAb antibody also recognized the O antigen of B abortus, B. melitensis and B. suis, confirming the cross reactivity of the LPS between the species (Figure 1B, Lanes 5-7). The M84 mAb directed against B. abortus O antigen also reacted with the Ye O:9 polysaccharide (Figure 1C, lanes 3 and 4). Interestingly, B. melitensis LPS did not react towards the M84 antibody (Figure 1C, lane 6). This result was not totally unexpected because although the B. abortus and B. melitensis polysaccharides have a similar composition, there are structural differences between their O antigens [27]. These results confirmed that the O antigens of Ye 0:9 and B. abortus have a similar structure, and suggested that a conjugate carrying the Ye O:9 antigen could mount an immune response that may be cross-protective against B. abortus.


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)

Cross reactivity between Y. enterocolitica O:9 and Brucella spp. LPS samples. (0.2 OD/sample loaded) on 15% SDS-PAGE: A) LPS silver stain analysis of samples of Y. enterocolitica 1) OC-/HP-, 2)HP-, 3) OC-, 4) WT; Brucella 5) B. abortus, 6) B. melintensis, and 7) B. suis. B) Immunoblot against the same samples with monoclonal α-Yersinia (Yst 9-2). C) Immunoblot of the same samples using monoclonal α-Brucella (M84). Cross reactivity between the two different genus' LPS is observed by both monoclonal antibodies reacting against the higher molecular weight homopolymeric N-formylperosamine polysaccharide.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Cross reactivity between Y. enterocolitica O:9 and Brucella spp. LPS samples. (0.2 OD/sample loaded) on 15% SDS-PAGE: A) LPS silver stain analysis of samples of Y. enterocolitica 1) OC-/HP-, 2)HP-, 3) OC-, 4) WT; Brucella 5) B. abortus, 6) B. melintensis, and 7) B. suis. B) Immunoblot against the same samples with monoclonal α-Yersinia (Yst 9-2). C) Immunoblot of the same samples using monoclonal α-Brucella (M84). Cross reactivity between the two different genus' LPS is observed by both monoclonal antibodies reacting against the higher molecular weight homopolymeric N-formylperosamine polysaccharide.
Mentions: The B. abortus and B. suis O antigens were previously characterized by genomic analysis, NMR, and serological assays and appear to be identical to that of Yersinia enterocolitica O:9 (Ye O:9) [15,24,25]. Ye O:9 is a Class II biosafety hazard organism and is easily manipulated and cultured, making it a suitable host for the production of the glycoconjugate protein with the N-formylperosamine homopolymer, which we hypothesize could cross-protect against brucellosis [26]. In some Y. enterocolitica strains, an additional "outer core" (OC) consisting of a shorter glycan chain is assembled onto the Und-PP carrier and subsequently ligated to lipid A. To confirm cross reactivity of the Ye O:9 (Table 1) and the B. abortus O antigens, LPS of both species were analyzed by SDS-PAGE and immunoblot (Figure 1). Our analysis included the wild-type Ye O:9 strain, plus three derivatives lacking the OC, the O antigen, or both glycan structures. LPS samples from of the Ye O:9 strains exhibited a different electrophoretic pattern according to the mutation carried by each strain. The double mutant strain only displayed a band corresponding to lipid A core (lane 1). The O antigen deficient strain exhibited a unique band that migrated slower than the lipid A core, as expected for the presence of the low molecular weight OC structure attached to the lipid A (lane 2). The OC minus strain only produced the high molecular weight homopolymer (lane 3), and the WT strain produced both glycan structures (lane 4; Figure 1A). Analysis of the LPS extracts using monoclonal antibodies (Figure 1B, C) demonstrated that only the high molecular weight carbohydrate of Ye O:9 was reactive towards the Yst9-2 (anti-Ye O:9 antigen) monoclonal antibody (mAb; Figure 1B, lanes 3 and 4). The Yst9-2 mAb antibody also recognized the O antigen of B abortus, B. melitensis and B. suis, confirming the cross reactivity of the LPS between the species (Figure 1B, Lanes 5-7). The M84 mAb directed against B. abortus O antigen also reacted with the Ye O:9 polysaccharide (Figure 1C, lanes 3 and 4). Interestingly, B. melitensis LPS did not react towards the M84 antibody (Figure 1C, lane 6). This result was not totally unexpected because although the B. abortus and B. melitensis polysaccharides have a similar composition, there are structural differences between their O antigens [27]. These results confirmed that the O antigens of Ye 0:9 and B. abortus have a similar structure, and suggested that a conjugate carrying the Ye O:9 antigen could mount an immune response that may be cross-protective against B. abortus.

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