Exploitation of bacterial<i>N</i>-linked glycosylation to develop a novel recombinant glycoconjugate vaccine against<i>Francisella tularensis</i>

Cuccui, Jon; Thomas, Rebecca; Moule, Madeleine G.; D’Elia, Riccardo V.; Laws, Thomas R.; Mills, Dominic C.; Williamson, Diane; Atkins, Timothy P.; Prior, Joann L.; Wren, Brendan W.

doi:10.1098/rsob.130002

Cited by 82 publications

(106 citation statements)

References 38 publications

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“…This strategy was only demonstrated in a few cases (Iwashkiw et al, 2012; Cuccui et al, 2013; Wetter et al, 2013). Since B. pseudomallei is a biosafety class III agent we were unable to directly exploit the native OPS II by expressing the N -glycosylation system in the host as previously shown (Iwashkiw et al, 2012).…”

Section: Discussionmentioning

confidence: 99%

Production of a recombinant vaccine candidate against Burkholderia pseudomallei exploiting the bacterial N-glycosylation machinery

et al. 2014

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Vaccines developing immune responses toward surface carbohydrates conjugated to proteins are effective in preventing infection and death by bacterial pathogens. Traditional production of these vaccines utilizes complex synthetic chemistry to acquire and conjugate the glycan to a protein. However, glycoproteins produced by bacterial protein glycosylation systems are significantly easier to produce, and could possible be used as vaccine candidates. In this work, we functionally expressed the Burkholderia pseudomallei O polysaccharide (OPS II), the Campylobacter jejuni oligosaccharyltransferase (OTase), and a suitable glycoprotein (AcrA) in a designer E. coli strain with a higher efficiency for production of glycoconjugates. We were able to produce and purify the OPS II-AcrA glycoconjugate, and MS analysis confirmed correct glycan was produced and attached. We observed the attachment of the O-acetylated deoxyhexose directly to the acceptor protein, which expands the range of substrates utilized by the OTase PglB. Injection of the glycoprotein into mice generated an IgG immune response against B. pseudomallei, and this response was partially protective against an intranasal challenge. Our experiments show that bacterial engineered glycoconjugates can be utilized as vaccine candidates against B. pseudomallei. Additionally, our new E. coli strain SDB1 is more efficient in glycoprotein production, and could have additional applications in the future.

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Section: Discussionmentioning

confidence: 99%

Production of a recombinant vaccine candidate against Burkholderia pseudomallei exploiting the bacterial N-glycosylation machinery

et al. 2014

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“…OTase-mediated N-glycosylation from Campylobacter jejuni has been successfully engineered in E. coli (33). Since then, numerous efforts have made to develop bioconjugate vaccines in E. coli against pathogens such as Francisella tularensis (34), Burkholderia pseudomallei (35), Shigella flexneri (36), and S. aureus (37) using this system. An OTase-mediated O-glycosylation system from Neisseria meningitides was established in Shigella spp.…”

Section: Discussionmentioning

confidence: 99%

Engineering and Dissecting the Glycosylation Pathway of a Streptococcal Serine-rich Repeat Adhesin

Zhu

Zhang²,

Yang

et al. 2016

J. Biol. Chem.

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Edited by Gerald HartSerine-rich repeat glycoproteins (SRRPs) are conserved in Gram-positive bacteria. They are crucial for modulating biofilm formation and bacterial-host interactions. Glycosylation of SRRPs plays a pivotal role in the process; thus understanding the glycosyltransferases involved is key to identifying new therapeutic drug targets. The glycosylation of Fap1, an SRRP of Streptococcus parasanguinis, is mediated by a gene cluster consisting of six genes: gtf1, gtf2, gly, gtf3, dGT1, and galT2. Mature Fap1 glycan possesses the sequence of Rha1-3Glc1-(Glc1-3GlcNAc1)-2,6-Glc1-6GlcNAc. Gtf12, Gtf3, and dGT1 are responsible for the first four steps of the Fap1 glycosylation, catalyzing the transfer of GlcNAc, Glc, Glc, and GlcNAc residues to the protein backbone sequentially. The role of GalT2 and Gly in the Fap1 glycosylation is unknown. In the present study, we synthesized the fully modified Fap1 glycan in Escherichia coli by incorporating all six genes from the cluster. This study represents the first reconstitution of an exogenous stepwise O-glycosylation synthetic pathway in E. coli. In addition, we have determined that GalT2 mediates the fifth step of the Fap1 glycosylation by adding a rhamnose residue, and Gly mediates the final glycosylation step by transferring glucosyl residues. Furthermore, inactivation of each glycosyltransferase gene resulted in differentially impaired biofilms of S. parasanguinis, demonstrating the importance of Fap1 glycosylation in the biofilm formation. The Fap1 glycosylation system offers an excellent model to engineer glycans using different permutations of glycosyltransferases and to investigate biosynthetic pathways of SRRPs because SRRP genetic loci are highly conserved.

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“…This approach leverages laboratory strains of Escherichia coli for the expression of recombinant bacterial polysaccharides (e.g., O-polysaccharide antigens), which are conjugated in vivo to a co-expressed carrier protein by the Campylobacter jejuni oligosaccharyltransferase PglB. However, while PGCT has been used to make several novel protein/glycan combinations, it is limited by variable glycan conjugation efficiency as observed for certain heterologous polysaccharide substrates (Cuccui et al, 2013; Ihssen et al, 2015; Ihssen et al, 2010) and a challenging purification of the product antigen. This is particularly pertinent in the context of producing glycoconjugates carrying mammalian-like glycans (Cuccui and Wren, 2014).…”

Section: Introductionmentioning

confidence: 99%

Immunization with Outer Membrane Vesicles Displaying Designer Glycotopes Yields Class-Switched, Glycan-Specific Antibodies

Valentine

Chen

Perregaux

et al. 2016

Cell Chemical Biology

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Summary The development of antibodies against specific glycan epitopes poses a significant challenge due to difficulties obtaining desired glycans at sufficient quantity and purity, and the fact that glycans are usually weakly immunogenic. To address this challenge, we leveraged the potent immunostimulatory activity of bacterial outer membrane vesicles (OMVs) to deliver designer glycan epitopes to the immune system. This approach involved heterologous expression of two clinically important glycans, namely polysialic acid (PSA) and Thomsen-Friedenreich antigen (T antigen) in hypervesiculating strains of non-pathogenic Escherichia coli. The resulting glycOMVs displayed structural mimics of PSA or T antigen on their surfaces, and induced high titers of glycan-specific IgG antibodies following immunization in mice. In the case of PSA glycOMVs, serum antibodies potently killed Neisseria meningitidis serogroup B (MenB), whose outer capsule is PSA, in a serum bactericidal assay. These findings demonstrate the potential of glycOMVs for inducing class-switched, humoral immune responses against glycan antigens.

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Exploitation of bacterialN-linked glycosylation to develop a novel recombinant glycoconjugate vaccine againstFrancisella tularensis

Cited by 82 publications

References 38 publications

Production of a recombinant vaccine candidate against Burkholderia pseudomallei exploiting the bacterial N-glycosylation machinery

Production of a recombinant vaccine candidate against Burkholderia pseudomallei exploiting the bacterial N-glycosylation machinery

Engineering and Dissecting the Glycosylation Pathway of a Streptococcal Serine-rich Repeat Adhesin

Immunization with Outer Membrane Vesicles Displaying Designer Glycotopes Yields Class-Switched, Glycan-Specific Antibodies

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