jats:titleAbstract</jats:title>jats:sec jats:titleBackground</jats:title> jats:pWorldwide, an estimated 70.7 billion broilers were produced in 2020. With the reduction in use of prophylactic antibiotics as a result of consumer pressure and regulatory oversight alternative approaches, such as vaccination, are required to control bacterial infections. A potential way to produce a multivalent vaccine is via the generation of a glycoconjugate vaccine which consists of an antigenic protein covalently linked to an immunogenic carbohydrate. Protein-glycan coupling technology (PGCT) is an approach to generate glycoconjugates using enzymes that can couple proteins and glycan when produced in bacterial cells. Previous studies have used PGCT to generate a live-attenuated avian pathogenic jats:italicEscherichia coli</jats:italic> (APEC) strain capable of jats:italicN</jats:italic>-glycosylation of target proteins using a chromosomally integrated jats:italicCampylobacter jejuni pgl</jats:italic> locus. However, this proved ineffective against jats:italicC. jejuni</jats:italic> challenge.</jats:p> </jats:sec>jats:sec jats:titleResults</jats:title> jats:pIn this study we demonstrate the lack of surface exposure of glycosylated protein in APEC strain χ7122 carrying the jats:italicpgl</jats:italic> locusjats:italic.</jats:italic> Furthermore, we hypothesise that this may be due to the complex cell-surface architecture of jats:italicE. coli.</jats:italic> To this end, we removed the lipopolysaccharide O-antigen of APEC χ7122 jats:italicpgl</jats:italic>jats:sup+</jats:sup> via deletion of the jats:italicwecA</jats:italic> gene and demonstrate increased surface exposure of glycosylated antigens (NetB and FlpA) in this strain. We hypothesise that increasing the surface expression of the glycosylated protein would increase the chance of host immune cells being exposed to the glycoconjugate, and therefore the generation of an efficacious immune response would be more likely.</jats:p> </jats:sec>jats:sec jats:titleConclusions</jats:title> jats:pOur results demonstrate an increase in cell surface exposure and therefore accessibility of glycosylated antigens upon removal of lipopolysaccharide antigen from the APEC cell surface.</jats:p> </jats:sec>