Additional file 1 of Multivalent poultry vaccine development using Protein Glycan Coupling Technology
Creators: name: Mauri, Marta
name: Sannasiddappa, Thippeswamy H.
name: Vohra, Prerna
name: Corona-Torres, Ricardo
name: Smith, Alexander A.
name: Chintoan-Uta, Cosmin
name: Bremner, Abi
name: Terra, Vanessa S.
name: Abouelhadid, Sherif
name: Stevens, Mark P.
name: Grant, Andrew J.
name: Cuccui, Jon
name: Wren, Brendan W.
Publisher: name: figshare
Year: 2021
Subjects: subject: Microbiology
subject: FOS: Biological sciences

description: 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