Modification of avian pathogenic Escherichia coli χ7122 lipopolysaccharide increases accessibility to glycoconjugate antigens

Abstract

Background Worldwide, 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 Escherichia coli (APEC) strain capable of N-glycosylation of target proteins using a chromosomally integrated Campylobacter jejuni pgl locus. However, this proved ineffective against C. jejuni challenge. Results In this study we demonstrate the lack of surface exposure of glycosylated protein in APEC strain χ7122 carrying the pgl locus. Furthermore, we hypothesise that this may be due to the complex cell-surface architecture of E. coli. To this end, we removed the lipopolysaccharide O-antigen of APEC χ7122 pgl+ via deletion of the wecA 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. Conclusions Our results demonstrate an increase in cell surface exposure and therefore accessibility of glycosylated antigens upon removal of lipopolysaccharide antigen from the APEC cell surface.

A systematic review and meta-analysis on the efficacy of vaccination against colibacillosis in broiler production

“…efficacy and broader coverage. Exploring innovative technology, such as irradiation [75] or glycoconjugate vaccines [76], may hold promise for improving vaccine delivery and bird protection against colibacillosis. Increased investment in research and development along with collaborative efforts between academic, industry and regulatory agencies, can expedite the translation of promising vaccine candidates into commercial products.…”

Extracellular vesicles produced by avian pathogenic Escherichia coli (APEC) activate macrophage proinflammatory response and neutrophil extracellular trap (NET) formation through TLR4 signaling

“…To determine whether the LPS on APEC EVs activates the NLRP3 inflammasome in THP-1 macrophages, we removed the LPS from APEC EVs as follows. The wecA gene encodes the O-antigen transferase that initiates the attachment of the E. coli O-antigen to polysaccharides, and the deletion of the wecA gene causes the loss of E. coli LPS [41,42]. Therefore, we truncated the LPS of APEC strain CT265 by deleting the wecA gene and thus removing the O-antigen.…”

“…APEC strain CT265 was cultured in Luria-Bertani (LB) broth at 37 °C [74]. APEC mutant CT265ΔwecA, in which the wecA gene was deleted, was constructed as previously described [75]. To prepare the EVs, 20 mL of overnight bacterial culture was added to 1.0 L of LB broth supplemented with 1.0 mL of ampicillin (100 mg/ mL).…”