Bacterial strains and growth conditions

The bacterial strains and plasmids used in this study are listed in Table 1. and cells were grown aerobically in a LB medium at 37C. Antibiotics were added appropriately: ampicillin at 100 µg/mL for , chloramphenicol at 10 µg/mL, and neomycin at 25 µg/mL for . Competent cells of and followed the previously described procedures15, 16.

Plasmids and strains construction

To anchor the heterologous protein on the spore surface, we used two plasmids of which design at the expression cassettes was similar to pCotB-CL and pCotG-CL9, named pHT2304 and pHT2305. The Hla gene was amplified from synthesized plasmid pHT2328 with primers ON2345 ON2336. After being cleaved with restriction enzymes I and II, the target fragment was inserted into template pHT2304 and pHT2305, which had been double-digested with the same enzymes to result in recombinant plasmids pHT2331 and pHT2334, respectively. colonies containing recombinant plasmids were selected on ampicillin LB-agar plates. The methods used to confirm the target sequences included colony PCR and sequencing.

Next, pHT2331 and pHT2334 were introduced into the strain WB80013and WB800N14, respectively. Double cross-over integration occurred during the natural transformation process to insert into the chromosome at homologous loci (for pHT2331) and (for pHT2334). colonies were selected on LB-agar plates with neomycin (for pHT2331) or chloramphenicol (for pHT2334). The correct integration of the target gene was confirmed by the PCR method with three pairs of primers per colony. All primer details are presented in Table 2. The new strains BsHT2331 and BsHT2334 were stored at —80C for the following experiments.

Preparation of spores

The sporulation was prepared according to the previously described method17. strains were pre-cultured in 5 mL LB medium to OD value at around 2 – 3, and then sub-cultured in 40 mL LB medium at 37C (200 rpm) to reach OD of 0.8. When the bacterial growth started entering the log phase, the cells were centrifuged at 13,000 x g for 1 minute and washed with PBS 1X. The pellet was then resuspended in 20 mL Difco Sporulation Medium (DSM) and cultivated at 37C (200 rpm) to induce sporulation. After 48 hours, the vegetative cells in the culture were eliminated by lysozyme treatment (15 mg/mL, Serva) and the spores were washed six times with PBS 1X. The spores were confirmed by Schaeffer & Fulton’s method and stored in glycerol 10% at —80C.

Schaeffer & Fulton’s method

20 µL of 48-hour cultured broth and washed spores were smeared and heat-fixed on a glass slide for 5 minutes with 5% Malachite green. The slide was cooled and washed with water to discard the dye. After that, the sample had 2.5% Safranin O applied for 30 seconds, then it was rinsed with water and dried. The results can be observed under a light microscope.

Determination of spore number

The spores' suspension at a concentration of OD value as 2 per mL was put under a heat shock at 80°C for 10 minutes to remove vegetative cells. The viable spores were counted by ten-fold serial dilution in distilled sterile water. 100 μL of each dilution was incubated on a LB agar plate overnight at 37°C. The experiment was performed in triplicate.

sporeELISA

Spores anchoring the protein on the surface, acting as an antigen, were resuspended in a 200 µL coating buffer (100 mM NaHCO; pH 9,6), and then coated onto a 96-well plate (Thermo Scientific™ Nunc™ MicroWell™ 96-Well Microplates) with a volume of 50 μL per well. After incubation at 4°C overnight, the wells were washed with PBS-Tween and blocked with a blocking buffer (PBS-Tween with 5% skim milk) for 1 hour at room temperature. After washing, 50 μL of the Hla-antibody, which was developed in Swiss mice by our research group, was added at a dilution ratio of 1/10000 incubated in 2 h at room temperature, and then washed again. The second antibody was anti-mouse IgG - peroxidase antibody produced in rabbits (whole molecule) (Sigma, A9044 – 2 mL), used at a ratio of 1/40000 at the same incubation condition. 50 μL TMB Liquid Substrate for ELISA (Sigma) was added after washing, then 50 μL HCl 1N to stop the reaction. The absorbance values were measured in a CLARIOstar plate reader at a wavelength of 450 nm. The experiment was replicated three times for each sample and presented as mean ± SD. Statistical significance was analyzed using ANOVA-one way and the Graphpad 7.0 software.

Oral immunization in mice

Each group of five 6-week-old female Swiss mice were immunized by oral gavage on days 0, 14, and 28 with 250 µL of spores BsHT2331 and BsHT2334 at a spore number corresponding to ODof 60 diluted in PBS 1X. The control groups were mice orally administrated with BsHT2304, BsHT2305, or pre-immunized mice. Serum and fecal samples were collected on day -1 (pre-immune samples), 21, and 42. Every 0.6 gram of fecal was treated with 500 uL of PBS 1X containing 0.2 mg/mL PMSF, homogenized and centrifuged at 10,000 x g in 10 minutes to collect the supernatant. All samples were stored at —20C.

Indirect ELISA

50 μL of Hla protein (5 g/mL), which was expressed in by our research group, was loaded into a 96-well plate (Thermo Scientific™ Nunc™ MicroWell™ 96-Well Microplates). The primary antibody was used as 50 µL of serum at a dilution ratio of 1/250 or fecal extract at a dilution ratio of 1/50 for overnight incubation at 4°C. Next, the plates were incubated for 2 hours with anti-mouse IgG (whole molecule) — peroxidase antibody produced in rabbits (Sigma, A9044 — 2 mL) (1/40000) or anti-mouse IgA (α-chain specific) — peroxidase antibody produced in goats (A4789 — 1 mL, Sigma) (1/10000) according to the serum or fecal sample. The IgG measurement was analyzed at room temperature, while the IgA level analysis was performed at 37°C. All samples were assayed in triplicate and read at 450 nm in a CLARIOstar plate reader. Statistical significance was analyzed using ANOVA-one way and Graphpad 7.0.

Construction of vectors pHT2331 and pHT2334

The target gene was amplified through PCR using template pHT2328 with two specific primers, ON2345 and ON2336 (Figure 1C). was fused with the C-terminal region of in pHT2304 and in pHT2305 to result in two plasmid vectors pHT2331 and pHT2334, respectively (Figure 1A & B). colonies carrying the target plasmids were selected on the LB agar plate with ampicillin and by the colony PCR method which yielded the expected band of 943 bp on agarose gel (Figure 1 D & E). We confirmed via DNA sequencing that the correct sequence was inserted into the recombinant vectors (data not shown).

Generation of recombinant strains carrying integrated into the chromosomes

To generate recombinant strains carrying mutant gene, two vectors pHT2331 and pHT2334 were extracted through alkaline lysis from cells and introduced into WB800 or WB800N via natural transformation. Throughout the process, competent cells took up DNA from the environment and integrated heterologous genes into its genome at homologous loci and . These are the coding sequences of the non-essential genes of . The chromosomal integration was first confirmed via the colonies’ growth on LB agar with appropriate antibiotics. To verify that the integration of the fusion andinto chromosome occurred through a double-crossover recombination eventthree pairs of primers were used for each strain in the PCR performed. This was one pair for checking the presence of a target gene and two pairs for checking the accuracy of the integration sites, specifically and (Figure 2 A&B). The gel electrophoresis results showed that colonies with visible bands with sizes of three pairs of primers as predicted were successfully integrated strains (Figure 2 C & D). The new strains were named BsHT2331 and BsHT2334.

Display of Hla on the surface of the spores using the CotB and CotG anchor proteins

In Schaeffer-Fulton’s method, the spore coat was loosened due to the high temperature, meaning that the primary stain can penetrate the spore. The spore coat is impermeable while the cell wall has a low affinity with the water-soluble dye. Malachite green can be easily removed from vegetative cells but not the spore. Safranin is applied to re-colorize cells. In the end, the vegetative cells will color in pink and the endospores will be rod-shaped and green. Sporulation was observed under a light microscope at 100X magnification. Pictures of both BsHT2331 and BsHT2334 strains revealed an assembly consisting of pink cells, green endospores, and pink cells containing green endospores in the middle or at the end of the cell (Figure 3 A & B). This demonstrates that the sporulation reached the desired ratio and quality. The mature and free spores were then purified and their quantity determined. The density of BsHT2331 and BsHT2334 was 1.37 x 10 spores/mL and 3.83 x 10spores/mL, respectively.

To verify the presence of the target protein on the spore surface, we performed the sporeELISA experiment. Recombinant spores were considered as an antigen to interact with the polyclonal anti-Hla antibody. The spores coat was stable that allowed them to retain its original shape throughout the process. Thus, the signal released from the HRP enzyme and the substrate reaction can demonstrate the expression on the surface. The results showed that there was a significant detection of antigen Hla anchored by CotB (p-value < 0.0001) and CotG (p-value < 0.05) on the spore surface compared with each of the control strains Figure 3C). These signals reveal the presence of the target protein on the surface of the spore.

Anti-spore IgG and IgA level response by oral administration

spores can safely pass through the acidic condition of the stomach and reach the upper part of the intestine during ingestion18. Throughout the process, recombinant spores have been shown to induce both mucosal (IgA) and systemic (IgG) responses. To evaluate the antigen-delivery effect of spores displaying Hla in genetically heterogeneous animals, Swiss mice were subjected to three doses of spores on days 0, 14, and 28. Blood and fecal samples were collected on days 0, 21, and 42 for the analysis of the serum IgG and fecal IgA.

The serum IgG level of the mice orally delivered with BsHT2331 showed a slight development but not significant after the first and second doses (0.084 ± 0.009). There was a dramatic increase after the third dose (0.32 ± 0.028, p-value < 0.0001), which was 3.4-fold higher than the control strain BsHT2304 on day 42 (0.095 ± 0.001) and 5.5-fold higher than the serum sample before subjection (Figure 4A). The systemic response to BsHT2334 spore in mice also rose continuously from day 21 (0.1883 ± 0.017) to day 42 (0.2087 ± 0.036) with a 99% confidence interval compared to the mice that received BsHT2305 at the corresponding time (Figure 4B). Similarly, the mucosal immune response was determined via IgA detection in feces. Mice immunized with the BsHT2331 spore elicited a significant growth in the anti-Hlafecal IgA level on day 21 (0.2233 ± 0.014, p-value < 0.0001) compared with BsHT2305 (0.1587 ± 0.015). After three doses, the immune stimulation in mice caused by spores displaying CotB-Hla resulted in a high IgA level (0.353 ± 0.014, p-value < 0.0001) that was 2.2-fold greater than the control group BsHT2304 and 2.5-fold greater than the feces sample from day 0 (Figure 4C). Nevertheless, a moderate decrease in the IgA titer appeared from 0.093 ± 0.009 on day 21 to 0.067 ± 0.005 on day 42 in the mice that took up the CotG-Hla expression strain, BsHT2334. The ODvalue of this group also revealed no significant difference from the BsHT2305 strains (Figure 4D). The results indicate that spores expressing Hla on the surface through the CotB and CotG protein could evoke a systemic immune response in mice via the oral route. There is a difference in the IgA antibody production in the animals between the spores strains using CotB (BsHT2331) or CotG (BsHT2334) to anchor the protein.