Show simple item record

dc.contributor.authorTerra, Vanessa S
dc.contributor.authorMauri, Marta
dc.contributor.authorSannasiddappa, Thippeswamy H
dc.contributor.authorSmith, Alexander A
dc.contributor.authorStevens, Mark P
dc.contributor.authorGrant, Andrew
dc.contributor.authorWren, Brendan W
dc.contributor.authorCuccui, Jon
dc.contributor.authorGlycoengineering of Veterinary Vaccines consortium (GoVV)
dc.date.accessioned2022-01-10T12:48:10Z
dc.date.available2022-01-10T12:48:10Z
dc.date.issued2022-01-05
dc.date.submitted2021-11-07
dc.identifier.issn1475-2859
dc.identifier.others12934-021-01728-7
dc.identifier.other1728
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/332523
dc.description.abstractBACKGROUND: Campylobacter is an animal and zoonotic pathogen of global importance, and a pressing need exists for effective vaccines, including those that make use of conserved polysaccharide antigens. To this end, we adapted Protein Glycan Coupling Technology (PGCT) to develop a versatile Escherichia coli strain capable of generating multiple glycoconjugate vaccine candidates against Campylobacter jejuni. RESULTS: We generated a glycoengineering E. coli strain containing the conserved C. jejuni heptasaccharide coding region integrated in its chromosome as a model glycan. This methodology confers three advantages: (i) reduction of plasmids and antibiotic markers used for PGCT, (ii) swift generation of many glycan-protein combinations and consequent rapid identification of the most antigenic proteins or peptides, and (iii) increased genetic stability of the polysaccharide coding-region. In this study, by using the model glycan expressing strain, we were able to test proteins from C. jejuni, Pseudomonas aeruginosa (both Gram-negative), and Clostridium perfringens (Gram-positive) as acceptors. Using this pgl integrant E. coli strain, four glycoconjugates were readily generated. Two glycoconjugates, where both protein and glycan are from C. jejuni (double-hit vaccines), and two glycoconjugates, where the glycan antigen is conjugated to a detoxified toxin from a different pathogen (single-hit vaccines). Because the downstream application of Live Attenuated Vaccine Strains (LAVS) against C. jejuni is to be used in poultry, which have a higher body temperature of 42 °C, we investigated the effect of temperature on protein expression and glycosylation in the E. coli pgl integrant strain. CONCLUSIONS: We determined that glycosylation is temperature dependent and that for the combination of heptasaccharide and carriers used in this study, the level of PglB available for glycosylation is a step limiting factor in the glycosylation reaction. We also demonstrated that temperature affects the ability of PglB to glycosylate its substrates in an in vitro glycosylation assay independent of its transcriptional level.
dc.languageen
dc.publisherSpringer Science and Business Media LLC
dc.subjectResearch
dc.subjectBiological conjugation
dc.subjectPglB
dc.subjectTemperature
dc.subjectPGCT
dc.subjectOne health
dc.subjectPoultry
dc.subjectVaccine
dc.titlePglB function and glycosylation efficiency is temperature dependent when the pgl locus is integrated in the Escherichia coli chromosome.
dc.typeArticle
dc.date.updated2022-01-10T12:48:09Z
prism.issueIdentifier1
prism.publicationNameMicrob Cell Fact
prism.volume21
dc.identifier.doi10.17863/CAM.79973
dcterms.dateAccepted2021-12-17
rioxxterms.versionofrecord10.1186/s12934-021-01728-7
rioxxterms.versionVoR
rioxxterms.licenseref.urihttp://creativecommons.org/licenses/by/4.0/
dc.contributor.orcidTerra, Vanessa S [0000-0002-2734-4036]
dc.contributor.orcidGrant, Andrew [0000-0001-9746-2989]
dc.identifier.eissn1475-2859
pubs.funder-project-idBiotechnology and Biological Sciences Research Council (BB/N001591/1)
cam.issuedOnline2022-01-05


Files in this item

Thumbnail
Thumbnail
Thumbnail

This item appears in the following Collection(s)

Show simple item record