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Additional file 1. Additional Figures and Tables.

Campylobacter jejuni is the leading bacterial cause of human gastroenteritis worldwide and handling or consumption of contaminated poultry meat is the key source of infection. Glycoconjugate vaccines containing the C. jejuni N-glycan have been reported to be partially protective in chickens. However, our previous studies with subunit vaccines comprising the C. jejuni FlpA or SodB proteins with up to two or three C. jejuni N-glycans, respectively, failed to elicit significant protection. In this study, protein glycan coupling technology was used to add up to ten C. jejuni N-glycans onto a detoxified form of Pseudomonas aeruginosa exotoxin A (ExoA). The glycoprotein, G-ExoA, was evaluated for efficacy against intestinal colonisation of White Leghorn chickens by C. jejuni strains M1 and 11168H relative to unglycosylated ExoA. Chickens were challenged with the minimum dose required for reliable colonisation, which was 102 colony-forming units (CFU) for strain M1 and and 104 CFU for strain 11168H. Vaccine-specific serum IgY was detected in chickens vaccinated with both ExoA and G-ExoA. However, no reduction in caecal colonisation by C. jejuni was observed. While the glycan dose achieved with G-ExoA was higher than FlpA- or SodB-based glycoconjugates that were previously evaluated, it was lower than that of glycoconjugates where protection against C. jejuni has been reported, indicating that protection may be highly sensitive to the amount of glycan presented and/or study-specific variables.

Additional file 1: Figure S1. Exploiting G-ExoA constructs to test N-linked glycosylation in χ7122. (a) Schematic representation of carrier protein ExoA genetically detoxified and modified with either two, G-ExoA(2), or ten ,G-ExoA(10), PglB glycosylation sequons. (b) SDS-PAGE followed by western blotting showing results of a His-pulldown from χ7122 expressing G-ExoA(2) or G-ExoA(10) and the pgl locus from pACYCpgl plasmid. Glycosylation increases as the number of glycosylation sequons increases, facilitating detection of glycosylation with G-ExoA(10). The negative control (- ctrl) consists of χ7122 expressing G-ExoA(10) in absence of the pgl locus. Percentage in parenthesis indicates sample loading (v/v) per lane. Figure S2. Identification of conditions that favour protein glycosylation in the χ7122 pgl integrant. (a) SDS-PAGE followed by western blotting showing results of a His-pulldown from the χ7122pgl integrant transformed with a plasmid-encoded L-arabinose inducible G-ExoA(10) carrier protein. Differences in culture conditions are enlisted in the table below the blot. (b) SDS-PAGE followed by western blotting showing results of a His-pulldown from either glycoengineering strains SDB1 or χ7122 pgl integrant transformed with a plasmid-encoded L-arabinose inducible G-ExoA(10) carrier protein and with pACYCpgl for SDB1. Both strains were cultured in either LBB Lennox or 2YT-M9 media to assess their effect on glycosylation. The most favourable conditions for protein glycosylation in χ7122 pgl were identified as cultures setup in a closed system (20 ml in 50 ml tube), induction of carrier protein expression at late exponential phase (OD600 ~0.8), growth post induction at 28°C shaking in LBB Lennox medium. Percentages in parenthesis indicate sample loading (v/v) per lane. Figure S3. Testing functionality of χ7122 pgl integrants. SDS-PAGE followed by western blotting of periplasmic extracts shown in Figure 3b. Fluorescent signals were acquired from single channels at 680 nm wavelength for the protein (anti-His) and 800 nm for the glycan (SBA lectin). The boxes highlighted were used for semi-quantitative densitometry analysis of protein and glycan levels. Percentage in parenthesis indicates sample loading (v/v) per lane. Figure S4. Assessing G-NetB as a carrier protein. (a) Schematic representation of carrier protein NetB genetically detoxified and modified with either two, G-NetB(2), or ten, G-NetB(10), PglB glycosylation sequons. (b) SDS-PAGE followed by western blotting showing glycosylation of G-NetB(2) or G-NetB(10) by a χ7122 pgl integrant and the glycoengineering strains used as controls. Lanes 1-2 SDB1 cells expressing G-NetB(2), lane 3 CLM24 cedA::pglB cells expressing G-NetB(10), lanes 4-5 SDB1 cells expressing G-NetB(10), lanes 6-7 χ7122 pgl expressing G-NetB(2) and G-NetB(10). pACYCpglΔpglB (in lanes 2 and 5) serves as a negative control on protein glycosylation. In strain CLM24 cedA::pglB (lane 4) pACYCpglΔpglB provides pgl genes responsible for the glycan assembly, while an IPTG-inducible copy of PglB is chromosomally integrated. Figure S5. Expression of inducible and constitutive G-NetB/unG-NetB from biological triplicates of χ7122 pgl vaccine strains. (a) SDS-PAGE followed by western blotting showing results of a His-pulldown of biological triplicates of χ7122 pgl vaccine strain expressing G-NetB. Lanes 1-3 G-NetB expressed from IPTG-inducible pEXT20 backbone, lanes 4-6 from constitutive pFPV25.1 backbone; (b) SDS-PAGE followed by western blotting showing results of a His-pulldown of biological triplicates of χ7122 pgl vaccine strain expressing unG-NetB as a negative control on glycosylation. Lanes 1-3 unG-NetB expressed from IPTG-inducible pEXT20 backbone, lanes 4-6 from constitutive pFPV25.1 backbone. Percentages in parenthesis indicate samples loading (v/v) per lane. Figure S6. PCR validation of the vaccine strains. Agarose gel showing PCR amplicons validating the presence of four endogenous plasmids of strain χ7122 (lanes 1-4) and carrier protein-encoding plasmid pFPV25.1-G-NetB(10) in the G-NetB(10) vaccine strain (lane 5, left), and unG-NetB in the unglycosylatable control (lane 5, right). Figure S7. Purified G-NetB(10) and unG-NetB for use as coating antigens in serological ELISAs. Comassie (a) and SDS-PAGE followed by western blotting (b) of purified fractions of G-NetB(10) and unG-NetB. The glycoprotein and its unglycosylated control where purified by His-affinity chromatography and anion exchange on an AKTÄ purifier, followed by size exclusion chromatography using PD-10 gravity columns. Fraction 1 of G-NetB(10), which is entirely glycosylated at 1 to 10 sites and has no degradation product was used in ELISA together with unG-NetB to quantify antibody responses against the glycoprotein and protein-only, respectively. 1 µg/lane was loaded. Table S1. RNA-Seq: number of reads assigned to each gene within the pgl locus. Table S2. Oligos used in this study

Strains and plasmids used in this study; Primers used in this study for reverse transcriptase analysis of RNA extracts from A. pleuropneumoniae HS143, complementation of ngt and agt mutants and construction of A. pleuropneumoniae deletion mutants; Expression analyses of ngt and sodC. Reverse transcriptase PCR results of RNA extracted from A. pleuropneumoniae serotype 15 at various time points and in various mutants. 1, 1.5 hr cDNA template; 2, 1.5 hr RNA template; 3, 3.0 hr cDNA template; 4, 3.0 hr RNA; 5, 5.0 hr cDNA template; 6, 5.0 hr RNA template; 7, Δngt overnight culture cDNA template; 8, Δngt overnight culture RNA template; 9, Δngt complemented with pMKExpressNGT overnight culture cDNA template; 10, Δngt complemented with pMKExpressNGT overnight culture RNA template; 11, genomic DNA template (positive PCR control).; Predicted transcriptional promoter and Rho-independent terminators identified by bioinformatics analysis. V. Solovyev, A Salamov (2011) Automatic Annotation of Microbial Genomes and Metagenomic Sequences. In Metagenomics and its Applications in Agriculture, Biomedicine and Environmental Studies (Ed. R.W. Li), Nova Science Publishers, p. 61-78

Strains and plasmids used in this study; Primers used in this study for reverse transcriptase analysis of RNA extracts from A. pleuropneumoniae HS143, complementation of ngt and agt mutants and construction of A. pleuropneumoniae deletion mutants; Expression analyses of ngt and sodC. Reverse transcriptase PCR results of RNA extracted from A. pleuropneumoniae serotype 15 at various time points and in various mutants. 1, 1.5 hr cDNA template; 2, 1.5 hr RNA template; 3, 3.0 hr cDNA template; 4, 3.0 hr RNA; 5, 5.0 hr cDNA template; 6, 5.0 hr RNA template; 7, Δngt overnight culture cDNA template; 8, Δngt overnight culture RNA template; 9, Δngt complemented with pMKExpressNGT overnight culture cDNA template; 10, Δngt complemented with pMKExpressNGT overnight culture RNA template; 11, genomic DNA template (positive PCR control).; Predicted transcriptional promoter and Rho-independent terminators identified by bioinformatics analysis. V. Solovyev, A Salamov (2011) Automatic Annotation of Microbial Genomes and Metagenomic Sequences. In Metagenomics and its Applications in Agriculture, Biomedicine and Environmental Studies (Ed. R.W. Li), Nova Science Publishers, p. 61-78