Construct design and cloning
The nucleotide sequences of CirA, FepA, FhuA, and IroN derived from Salmonella enterica subspecies enterica (subsp. I) serotype Enteritidis were retrieved from GenBank (Accession Number AM933172.1). To facilitate detection of the recombinant proteins, a hemagglutinin (HA) tag was added to the 5’ end and a c-Myc tag was added to the 3’ end of the genes. The genes were then synthesized by BioBasic Inc. (Mississauga, ON), subjected to NheI and NcoI digestion, and subsequently introduced into the chloroplast expression cassette vector (pCEC5)69. through direct cloning at the corresponding restriction sites. The vector contains the promoter and 5’ UTR of the tobacco psbA gene, along with the 3’ UTRs of psbC and rbcL derived from the white poplar plastome, to control gene expression. Integration of the target genes into the tobacco plastome occurred between the trnI (tRNA-Ile) and trnA (tRNA-Ala) genes (Fig. 2).
Generation of transplastomic plants
Transplastomic tobacco plants (cv. 81V9)70. were obtained by the biolistic method69. For bombardment of leaf tissue, plants were aseptically grown from seed on Murashige-Skoog (MS) medium containing 3% (w/v) sucrose and 0.7% (w/v) agar. Leaves were placed abaxial side up on RMOP medium in a Petri dish. The RMOP medium consists of MS salts, N6-benzyladenine (1 mg/liter), 1-naphthaleneacetic acid (0.1 mg/liter), thiamine (1 mg/liter), myo-inositol (100 mg/liter), agar (7 g/liter) at pH 5.8, and sucrose (30 g/liter). Leaf tissue bombardment was conducted using the PDS-1000/He Biolistic particle delivery system (BioRad). Two days following bombardment, the leaves were carefully cut into sections measuring 5 mm×5 mm. These leaf sections were then transferred to RMOP medium supplemented with 500 μg/ml of spectinomycin dihydrochloride. On the bleached leaves, green calli emerged which were subcultured onto the same selective medium. Following three rounds of regeneration on selective medium, transplastomic plants were transferred to soil for seed production. Homoplasmy of all the clones was confirmed by Southern blot analysis as previously described69. DNA samples from CirA, FepA, and IroN transplastomic plants, along with wild-type N. tabacum serving as a control for expected band patterns without transgene integration, were extracted and digested with the RsrII restriction enzyme. The digested DNA was separated on a 0.8% agarose gel. A DIG-labeled probe synthesized by Kolotilin et al (2013) was used for hybridization69. The sequences of the PCR primers used to generate the DIG-labeled probes were Probe-F (5’-caccacggctcctctcttctcg-3’) and Probe-R (5’-ttcctacggggtggagatgatgg-3’), which target the intergenic spacer region between the trnI and trnA genes in the tobacco plastome.
Recombinant protein extraction and detection
Leaf tissue samples weighing approximately 0.05 g were flash-frozen in liquid nitrogen and then pulverized in 2 ml microcentrifuge tubes containing 3 silica beads (Bio Spec Products Inc., Bartlesville, OK, USA) using a TissueLyser II (Qiagen, Venlo, Netherlands) for 2 minutes. Subsequently, 300 μl of 2x Laemmli sample buffer (65.8 mM Tris-HCl, pH 6.8, 2.1% SDS, 26.3% (w/v) glycerol, and 0.01% bromophenol blue) was added to the tubes. The samples were vortexed for 30 seconds and sonicated with a 30 second burst at 30% amplitude (Fisherbrand™ Model 120 Sonic Dismembrator, Fisher Scientific, Schwerte, Germany).
The tubes were heated at 100°C for 10 minutes, then centrifuged for 10 minutes at 14,000 x g at room temperature. The resulting supernatant was loaded onto Express Plus 4-20% gradient polyacrylamide gels (Genscript Inc., Piscataway, NJ, USA) and electrophoresed at 100 V for 100 minutes. The proteins were subsequently transferred from the gels to polyvinylidene difluoride (PVDF) membranes using the Trans-Blot Turbo transfer system (Bio-Rad Laboratories Inc., Hercules, CA, USA). Following transfer, the membranes were blocked overnight at 4°C in 5% (w/v) skimmed milk dissolved in Tris-buffered saline (TBS; 20 mM Tris, 150 mM NaCl, pH 7.5). Western blotting involved probing the blots with mouse anti-hemagglutinin (HA) primary antibody (Sigma-Aldrich Cat. No. H3663), diluted 1:5,000 in 0.5% blocking solution for 1 hour at room temperature. After three washes in TBS pH 7.5, the membranes were incubated with goat anti-mouse horseradish peroxidase (HRP)-conjugated antibodies (Bio-Rad #1706516), diluted 1:5,000 in 0.5% blocking solution. Chemiluminescent signals were detected using Enhanced Chemiluminescent detection solution (Biorad Laboratories Inc., Hercules, CA, USA) and imaged with a MicroChemi imaging system using GelCapture acquisition software (DNR Bio-Imaging Systems Ltd., Jerusalem, Israel).
Preparation of chloroplast sub-compartments from transplastomic plants for the analysis of protein localization by immunoblotting
Chloroplast sub-compartments from transplastomic plants were prepared as described by32. Briefly, in a cold room, 400 to 500 g of leaf material from transplastomic plants were harvested and homogenized in a grinding buffer containing 20 mM Tricine-KOH (pH 8.4), 0.4 M sorbitol, 10 mM EDTA (pH 8), 10 mM NaHCO₃, and 0.1% (w/v) bovine serum albumin (BSA) at a 1:4 (w/v) sample-to-buffer ratio. The homogenate was blended, filtered through four layers of Miracloth (Calbiochem), and the liquid extract was collected. The crude cell extract was then centrifuged at 2,070 x g for 2 minutes at 4 °C. The supernatant discarded to obtain concentrated crude chloroplast pellets.
The washing medium (2x), pH 7.6, was prepared with 20 mM Tricine-KOH (pH 7.6), 0.8 M sorbitol, 5 mM MgCl₂, and 2.5 mM EDTA. The pH was adjusted to 7.6 using NaOH pellets. The concentrated chloroplasts were gently resuspended in 1x washing medium. The final chloroplast suspension volume was 36 ml. For chloroplast purification, 200 ml of Percoll gradient was prepared by mixing equal volumes of Percoll and washing medium (2x), resulting in a final concentration of 50% (v/v) Percoll/0.4 M sorbitol. Thirty ml of this preparation were dispensed into 6 tubes and centrifuged at 38,700 x g for 55 min at 4 °C. Six ml of the chloroplast suspension was loaded onto each of the six preformed gradients and centrifuged at 13,300 x g for 10 minutes at 4 °C using a swinging-bucket rotor. The upper phase was discarded, and intact chloroplasts from the lower phase were collected, diluted 3-4-fold with 1x washing buffer, and centrifuged at 2,070 x g for 2 minutes at 4 °C. The pellet of intact chloroplasts was kept on ice.
The intact chloroplasts were lysed using up to 12 ml of hypotonic buffer containing 10 mM MOPS (pH 7.8), 4 mM MgCl₂, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM benzamidine hydrochloride hydrate, and 0.5 mM ε-amino caproic acid. Three milliliters of the lysed chloroplasts were loaded onto step sucrose gradients (0.3 M/0.6 M/0.93 M) and ultracentrifuged at 70,000 x g for 1 hour at 4 °C. Fractions were then collected from the tubes and pooled.
The presence of recombinant protein was determined using western blot analyses. Samples from each step of the experiment were mixed with 2x Laemmli sample buffer, heated at 100 °C for 10 min, and then loaded onto Express Plus 4−20% gradient polyacrylamide gels (Genscript Inc., Piscataway, NJ, USA). The remaining steps of the western blot procedure were carried out following the aforementioned protocol.
Mouse immunization
All mouse immunization procedures were conducted at Cedarlane Labs (4410 Paletta Court, Burlington, ON, Canada) under the approved Animal Use Protocol AUP117AB. This protocol was reviewed and approved by Cedarlane’s institutional Animal Care Committee and was developed in accordance with the Canadian Council on Animal Care (CCAC) guidelines for the ethical use of animals in research.
Twenty-four female BALB/c mice, aged six to eight weeks, were assigned to four groups. Pre-immune blood samples of 50 µl were collected from each mouse. Each mouse was subcutaneously immunized with 100 µl of chloroplast extract from transplastomic plants containing 30 µg of recombinant protein. Chloroplasts were isolated from 30 g of transplastomic plant leaves using 120 mL of chloroplast isolation buffer supplemented with 0.1% (w/v) bovine serum albumin (BSA). The homogenate was filtered through six layers of Miracloth and subjected to differential centrifugation—initially at 200 × g for 3 minutes to remove debris, followed by 1000 × g for 7 minutes at 4 °C to pellet intact chloroplasts. The resulting green pellet was resuspended in 1 mL of PBS and sonicated on ice (amplitude 30%; 30 sec on/30 sec off cycle for 1 minute) to disrupt the chloroplast membrane. Post-sonication, samples were centrifuged at 20,000 × g for 10 minutes at 4 °C. The supernatant was then passed through a 0.22 μm filter to obtain purified chloroplast components. The primary immunization was emulsified with 100 µl of Freund’s complete adjuvant, resulting in a total injection volume of 200 µl (100 µl antigen + 100 µl adjuvant). Two booster immunizations were administered on days 21 and 42. For each boost, 100 µl of chloroplast extract from transplastomic plants containing 30 µg of recombinant protein was emulsified with 100 µl of Freund’s incomplete adjuvant. In all injections, the resulting 200 µl mixture was administered subcutaneously, with 50 µl injected at four different sites, two injections at the front legs and two at the back legs. The mice were euthanized on day 49 for final bleeds.
Whole-cell Salmonella ELISA
Following the methodology outlined by Levinson et al. (2015), the whole-cell Salmonella ELISA was performed with some modifications71. The S. Enteritidis strain used in this study was provided by Dr. Ed Topp (London Research and Development Centre, London, Ontario, Canada). In the initial step of coating, 50 µl of a poly-L-lysine solution, diluted at a 1:10 ratio from a 0.01% concentration, was added to each well of ELISA MaxiSorp1 plates (Thermo Fisher Scientific Inc., Waltham, MA, USA). The plates underwent an overnight incubation at 4 °C. For subsequent plate preparation, the wells were emptied without washing. Subsequently, 50 µl of S. Enteritidis, washed twice with phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, 1.47 mM KH₂PO₄, pH 7.4) at 4000 × g for 10 minutes at room temperature, was added following a 1:10 dilution. The plate underwent centrifugation at 1500 x g for 3 minutes, followed by a reversal of the plate and a repetition of the process. Following this, 50 µl of a 2% paraformaldehyde solution was added, and the plate underwent a 15 minute incubation at room temperature. After a single wash with PBS-T (0.1% Tween 20), 100 µl of 0.1 M glycine was added, and the plate was incubated at room temperature for 30 minutes. Another wash with PBS-T was conducted, followed by the addition of 100 µl of blocking solution (2% BSA-PBS-T) overnight at 4 °C.
Subsequently, the plate underwent a triple wash with PBS-T. Serial dilutions of Mouse serum (pre-immune and final bleed) were added and incubated for 1 hour at 37 °C. The plate underwent another triple wash with PBS-T, followed by the addition of goat anti-mouse IgG-HRP-conjugated secondary antibody (1:5,000) for 1 hour at room temperature. Subsequently, three washes with PBS-T were performed and antigen-bound antibodies were detected using ABTS (2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic) acid). The colorimetric reaction was measured at 415 nm using an ELISA plate reader (iMark™ Microplate Absorbance Reader, Bio-Rad, Hercules, California, USA).
Salmonella binding assay
Three millilitres of LB broth (Miller Formulation, Difco, Thermo Fisher Scientific, Ottawa, ON, Canada) were inoculated with 100 μl of a 5 ml overnight culture of S. Enteritidis grown at 37 °C. Subsequently, this 3 ml culture was incubated at 37 °C unitl reaching an OD600 of 0.7–0.9. Cells were harvested from 1 ml of the culture by centrifugation at 13,000 × g for 5 min, followed by three 5 minute rinses in PBS. The bacterial pellet was then resuspended in 1 ml of 2.5% paraformaldehyde (PFA) and incubated for 30 minutes at room temperature. After removing excess PFA following centrifugation at 13,000 × g for 5 min, bacterial permeabilization was achieved using 0.1% Triton X-100 in PBS (permeabilization buffer) for 5-10 minutes. The pellet was subsequently resuspended in blocking buffer (2% BSA in PBS) for 1 to 2 hours. Twenty-microliter aliquots of the cell suspension were prepared in separate tubes, centrifuged at 13,000 × g for 5 min, resuspended in serum collected from mice immunized with recombinant proteins (final bleed), diluted in 1% BSA-PBS, and incubated overnight at 4 °C. Serum collected from mice immunized with PBS (final bleed) served as a negative control. Diluted serum was removed by centrifugation at 13,000 × g for 5 min, followed by three washes in PBS for 5 min each.
The cells were then resuspended in Alexa Fluor® 594-conjugated goat anti-mouse IgG (H + L) (Abcam, Cat. No. ab150116) in 1% BSA-PBS for 1-2 hours at room temperature and incubated at 37 °C for 1 h. After washing and rinsing three times in PBS as described previously, and one final rinse in dH2O, the cells were stained by resuspending them for 2 min in 20 μl aliquots of DAPI (10 mg/ml solution diluted 1:1,000 in dH2O, Thermo Fisher Scientific Cat. No. D1306), followed by centrifugation at 13,000 × g for 5 min and resuspension in 20 μl of dH2O. The prepared cells were transferred onto poly-L-lysine coated coverslips (Millipore Sigma Cat. No. S1815) in a 24-well plate and centrifuged at 450 × g for 10 min. Coverslips were then dried and mounted onto glass slides with Aqua-Poly/Mount (Polyscience Inc., Warrington, PA, United States, Cat. No. 18606).
To visualize the binding of antibodies generated in immunized mice to S. Enteritidis cells, imaging was performed using an Olympus LSM FV 1200 microscope. Images were captured with a 60× water objective lens. DAPI, employed for staining the DNA, was imaged with excitation at 350 nm and emission at 455–465 nm. The Alexa Fluor® 594-conjugated goat anti-mouse IgG (H + L) was visualized with excitation at 590 nm and emission at 617 nm.
Statistical analysis
Statistical analysis of ELISA results was conducted using R 4.0.3 (R Core Team, 2020). The initial assessment involved checking the data for outliers, zero inflation, balance of categorical covariates, and collinearity. While a linear model was initially employed, it became evident that the assumption of equal variance was violated. Consequently, a generalized least squares (GLS) model was utilized, as it does not require equal variance assumptions. Additionally, a variance of identity residual structure was implemented to allow for distinct variance for each protein. To validate the GLS model, the residual distribution, residuals versus fitted values, and residuals against covariates were thoroughly examined. Subsequently, a two-factor ANOVA was carried out, followed by a Games Howell post hoc test (utilizing the PMCMRplus package), which does not assume equal variance.