Fish and feeding conditions
Two-month-old zebrafish with similar weight (0.082 g) were randomly dispersed into four tanks for each group (26.5×14.5×18 cm) at a density of 20 fish after acclimated two weeks, all of which were cultured in the recirculating water system (at 60 L/h). During the feeding period, the fish were given an experimental diet twice a day (9:00 and 17:00), the temperature was maintained at approximately 28°C, the dissolved oxygen was >6.0 mg/L, the pH was 7.0-7.2, the nitrite content was <0.005 mg/L, and the ammonia nitrogen was <0. 2 mg/L.
Bacterial strains and growth conditions
B. subtilis wt55 strains isolated from sturgeon and kept in China General Microbiological Culture Collection Center (CGMCC No. 1.19039) were directed at using in combination with AiiO-AIO6 and constructing recombinant strains which could display QQ enzyme AiiO-AIO6 on the surface of spores and secrete this protein by vegetative cells. Proliferation of B. subtilis were routinely in Luria-Bertani (LB) broth at 37 ℃, and the spores were produced in Difco-Sporulation medium (DSM) using the exhaustion method. Construction, transformation, screening and sequencing of recombinant plasmid were performed in Escherichia coli strain DH5α, it grows well in LB. The pathogenic bacteria A. veronii Hm091 also kept in China General Microbiological Culture Collection Center (CGMCC No. 22536) could be cultured in LB and has ampicillin resistant through identify. This pathogen was used to infect zebrafish and determine the disease resistance of the host, also used for co-culture with B. subtilis wt55 or AiiO-AIO6 to test the antimicrobial properties of them.
Construction and confirmation of recombinant expression B. subtilis strain
A recombinant B. subtilis strain expressing AiiO-AIO6 protein on the surface of B. subtilis spore and constitutive secretion of vegetative cells was constructed, the construction diagram was shown in Fig. 2. The anchoring protein selected for spore surface display was CotC, and the gene of AiiO-AIO6 was linked to its N terminal. The target genes by gene synthetic are respectively BamH I cutting site, CotC promoter, His tag, AiiO-AIO6, linker with alpha-helix structure, CotC and EcoR I cutting site, which string together in sequence. This gene segment was digested by BamH I and EcoR I and then inserted into the vector pDG364 which go through the same double digestion. The resulted recombinant plasmid named pDG364-N-AIO6 was transferred into the wild strain of B. subtilis wt55 by chemical transformation and homologous recombination was carried out at the homologous arm (amylase gene). Chloramphenicol resistance of 10 μg/mL was used to screen the positive clones. Chromosomal integration of exogenous gene was verified using amylase activity and genomic PCR (primers are listed in Table 1). After inducing spore generation, the AiiO-AIO6 displayed on the surface of wt55 spore was confirmed through western blot (WB) and immunofluorescence microscopy. The positive clone was named wt55-N-AIO6(SD).
The plasmid pWB-AIO6BS which could secrete AiiO-AIO6 in B. subtilis was constructed in our laboratory before (Pan et al. 2016), and the extracted plasmid was convert to transform competent cells of wt55-N-AIO6(SD) strain by chemical method. The concentration of kanamycin to screen positive clones was 25 μg/mL, and the clones were verified by plasmid extraction and WB of secreted AiiO-AIO6 in supernatant. The enzyme activity on spore surface and secretion in supernatant were determined by high performance liquid chromatography (HPLC) according to the method previously reported (Xia et al. 2020). The final bifunctional recombinant strain was named wt55-AIO6. The information of plasmids and strains used in this paper are shown in Table 3.
Diets and rearing experiments
Feeding trial firstly evaluated the effect of simple combination of B. subtilis and AiiO-AIO6. Four experimental diets were prepared: basal diet (ck), diets with each gram of feed contains 5U QQ enzyme AiiO-AIO6 (AIO6), AiiO-AIO6 with the same enzyme activity paired with B. subtilis wt55 (5U AiiO-AIO6+108 cfu B. subtilis /g feed, wt55+AIO6) or the same number of wt55 strain (wt55). Protein and bacteria were added directly during feed preparation, the feed was air-dried at room temperature and homogenized with a grinder. Fish were fed 6% of body weight per day, increase by 1% after a week, turn off the circulating water during feeding, and the experiment lasted for two weeks.
Another evaluation research about the recombinant wt55-AIO6 strain on the disease resistance of the host was carried out. The experiment was divided into four groups, fish were respectively fed with basal diet as control (ck), diets supplemented with AiiO-AIO6 which enzyme activity was 5 U/g feed (AIO6), recombinant spores with the same enzyme activity (about 2×109 cfu/g feed, wt55-AIO6) or the same number of B. subtilis wt55 strain (wt55). Feed preparation, breeding method and time were the same as above.
Sampling and Quantitative PCR testing
After the end of the breeding experiment, all fish were starved for 24h before sampling. Intestines were mainly taken, samples from 2-3 fish were randomly taken and mixed into the one. As for zebrafish larvae, about 15 fish would be used as a sample, and each group all had six replicates. Total RNA was extracted by TRIZOL Reagent (Invitrogen) after homogenate, cDNA was synthesized using fastKing gDNA dispelling RT superMix (TianGen) to eliminate contaminated genomic DNA. Ribosomal protein S11 (RPS11) was used as reference gene for the expression of all detected genes, quantitative real-time PCR (qPCR) reaction was performed using the SYBR Green Supermix (TianGen) on the Light Cycler 480 (Roche 480). The final results show was relative mRNA expression compared to the value of the ck control group. The primers for each gene were given in Table 1.
Co-culture of A. veronii Hm091 in vitro
In order to investigate the effect of B. subtilis wt55, AiiO-AIO6 or wt55 combination with AIO6 on the growth of A. veronii Hm091, mixed culture on liquid medium was done. A. veronii and wt55 were separately cultured overnight, and OD600 was adjusted to be the same before transfer. Four culture groups were set up: PBS + A. veronii as control, AiiO-AIO6 + A. veronii, wt55+AIO6 + A. veronii and wt55 + A. veronii. Then 100 μL of overnight cultured bacteria or sterile enzyme fluid were inoculated in 100 mL LB medium, each culture group has three flask replicates. The inoculated flasks were incubated at 37 ℃ with shaking at 200 rpms in an orbital incubator. At 4, 8, 12, 24 h, 1 mL of cultures were sampled to do gradient dilution and count the number of A. veronii. Because of the different colony morphology, A. veronii and B. subtilis could be distinguished during plate counting.
Gut alkaline phosphatase activity and A. veronii counts in gut after challenge
Multiple clones of A. veronii on the plate were selected into 1 mL LB to make their OD600=0.45, then transferred 200 μL to 60 mL fresh LB medium and cultured for 18 h at 37 ℃ with shaking at 200 rpms. Thirty fish from each group were randomly assigned to three tanks, A. veronii with final concentration of about 5×108 cfu/mL were added to each tank, fish were fasting during pathogen infection.
Closely monitor the state of the fish after challenge, remove the dead fish in time and record the number of deaths. When fish state was stable at 24 h post challenge, three fish from each tank were chosen for detecting intestinal alkaline phosphatase (IAP) using Alkaline Phosphatase Assay Kit (Beyotime). Separate intact intestines were homogenized in sterile PBS and then applied on a plate containing ampicillin to count the number of A. veronii which infected the gut, counts from unchallenged fish were used as background values.
Germ free zebrafish production and experimental treatment
Germ free (GF) zebrafish were produced according to reported protocols (He et al. 2017; Oyarbide et al. 2015). At the third days post fertilization (dpf), sterility was tested, after that 80% gnotobiotic zebrafish media (GZM) was changed, the egg membranes and unhatched embryos were sucked away.
GF-zebrafish were treated on 5 dpf, and two experiments were carried out. Experiment 1 was about sterile AiiO-AIO6 (final concentration 5 U/mL), collocation of B. subtilis wt55 (106 cfu/mL) with AiiO-AIO6 (5 U/mL) or wt55 (106 cfu/mL) directly immersed GF-zebrafish, the untreated group served as control. Experiment 2 was gut microbes from four groups of rearing experiments (ck, AIO6, wt55+AIO6 and wt55) to transfer GF-zebrafish. Simply, intestinal contents of five fish were sampled 4 h after the last feeding at the end of the breeding experiment. After fully mixed, the impurities were removed by low-speed centrifugation and the bacteria with the final concentration of 106 cfu/mL were added to the GF-zebrafish culture flask which with the same fixed volume of GZM (30 mL). Every group had six replicates, and zebrafish larvae were collected after three days of immersion treatment.
Statistical analysis
Data were analyzed by one way analysis of variance (ANOVA) with Duncan’s post hoc test. Data figures were made in GraphPad Prism Version 8 software. In all cases, differences were considered statistically significant when p < 0.05.