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. 2012 Jan 25:11:13.
doi: 10.1186/1475-2859-11-13.

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

Affiliations

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

Jeremy A Iwashkiw et al. Microb Cell Fact. .

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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Figures

Figure 1
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.
Figure 2
Figure 2
Proteins carrying Y. enterocolitica O:9 O-antigens are immunoreactive against α-Yersinia and α-Brucella monoclonal antibodies. Each strain was transformed with pMAF10 (pglBCj) and pMH5 (acrA), and glycosylated AcrA was purified from 1 L of culture using Ni2+ affinity chromatography. After purification, samples were loaded onto a 10% SDS-PAGE gel and analyzed by: A) Coomassie brilliant blue (5 μg/sample), immunoblot (2 μg/sample) using B) α-AcrA, C) α-Ye O:9 (Yst9-2) mAb, or D) α-Brucella O antigen M84 mAb. Samples were purified from the following strains: 1) OC-/HP-, 2) HP-, 3) OC-, 4) WT, and 3*) 10 × loaded volume of 3 (OC-).
Figure 3
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.
Figure 4
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.
Figure 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.
Figure 6
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.

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