Bacillus Subtilis Expressing Epidermal Growth Factors Induces Intestinal Repair and Growth-Promoting in Mice

Background: Probiotics are widely acknowledged for their pro-health attributes, but the ecacy of traditional probiotics is quite limited. This limitation can be overcome using a gene engineered to enhance the ecacy of existing probiotics. In this study, a strain of Bacillus subtilis (WB800) expressing the eukaryotic protein porcine epidermal growth factor (pEGF) was generated via genetic modication, and mice with intestinal injury were used as a model to evaluate the potential of this bioengineered probiotic in preventing or treating intestinal damage. Integration of the pEGF gene into the B. subtilis WB800 genome using an integrated expression vector pDG1730 resulted in stable expression of pEGF in B. subtilis (dubbed WB-EGF). Results: Female the Institute for Cancer Research (ICR) mice with intestinal damage received recombinant WB-EGF (1×10 8 –8×10 8 CFU/mL) for 10 d before collection of blood and intestinal tissues. Mice receiving WB-EGF had signicantly higher body weight and longer intestinal villi than those of mice treated with Luria-Bertani (LB) broth or B. subtilis transformed with an empty vector. Cell proliferation assays conrmed enhanced intestinal cell proliferation in mice receiving WB-EGF. Conclusions: This study provides evidence that WB-EGF may have use as a novel therapy for early prevention or treatment of intestinal damage and for promoting intestinal development. The recombinant B. subtilis strain developed here can expected to provide protection when used as a feed additive in animals with gastrointestinal infections. study demonstrates that WB-EGF repairs intestinal injury, inhibits inammatory factor secretion, and promotes intestinal development and weight gain in early-weaned mice. This study provides baseline data for optimizing the performance of the early-weaning transition phase in animals. The utilization of probiotics to deliver EGF provides a basis for the further development of microecological animal dietary supplements.


Background
Antibiotics play a crucial role in reducing infections and intestinal diseases and promoting gastrointestinal development in animals, but overuse or misuse of antibiotics can result in the development of microbial resistance. Therefore, nding antibiotic alternatives to repair the damaged gastrointestinal tract as well as to promote growth in animals is crucial for animal husbandry and food security. Among various options, probiotic therapy appears to be the most feasible, with a long history of consumption and veri ed bene cial effects [1] . Although probiotics have numerous potential bene ts in gastrointestinal disorders, there are certain limitations as well. The mechanisms of action by which probiotics affect host health are not completely understood, limiting their use in a practical setting. In recent years, engineered probiotic strains have become increasingly common. Bioengineering technology can be used to impart new attributes to existing probiotics or enhance their bene cial characteristics, which will expand their promotion and application [2,3] . Bacillus has emerged as a promising feed additive because of its able to form stable dormant spores and its tolerance to low pH and high temperature that allow it to survive and maintain viability under harsh production conditions [4] . B. subtilis, a member of the Bacillus genus, is metabolically active in the gut and is therefore considered an excellent candidate for the delivery of recombinant proteins in real time [5] . B. subtilis has been demonstrated to express and secrete biologically active eukaryotic proteins [6,7] .
Epidermal growth factor (EGF) is a 53 amino acid peptide that is important for the regulation of cell growth, proliferation, and differentiation [8,9] . Many studies have reported that porcine EGF (pEGF), similar in function and structure to its the homologs in humans and mice, regulates intestinal epithelial cell growth and differentiation during the early stages of animal intestinal development [9,10] . Growing evidence has indicated that exogenous EGF can be effectively absorbed by early-weaned animals, including mice [6] , pigs [8] , and humans [11] . EGF has received considerable attention; however, the production and puri cation of recombinant proteins using conventional methods are expensive and timeconsuming. Therefore, use of probiotics to deliver pEGF to the gut has become an increasingly popular strategy for reducing production costs. This study investigated the feasibility of expressing pEGF in the food-grade bacterium B. subtilis and its effect on intestinal damage and immune function in earlyweaned mice. The results show that the EGF-expressing B. subtilis promoted the growth of intestinal villi in mice and increased the depth of crypts in the jejunal intestine, promoting the growth of mice.

Construction of the PDG-PpEGF expression vector
In this study, we rst constructed the plasmid PDG-PpEGF containing the strong B. subtilis promoter P43, the SacB signal peptide coding sequence from Bacillus amyloliquefaciens, and the pEGF mature peptide coding sequence, as shown in Fig. 1. After con rming the sequence identity, PDG-PpEGF was transformed into WB800 to obtain the recombinant B. subtilis strain WB-EGF. Growth of the transformed WB-EGF during the 80-h fermentation period was measured, with an observed growth peak at 20 h; after 20-24 h, the transformants began to plateau (Fig. 2). Culture supernatants from WB-EGF and the empty vector-transformed control WB-BLANK were collected at different time points and analyzed by western blotting. As shown in Fig. 3A, EGF (≈ 6 kDa) was detected in the bacterial cell culture supernatant, indicating that EGF was secreted by the recombinant strain. To further determine the expression levels of pEGF secreted by the recombinant strain, pEGF content in the supernatant was determined by indirect ELISA. Figure 3B and 3C shows pEGF content in the culture supernatants of the recombinant strain during the 20-h fermentation period; after 12 h of fermentation, pEGF peaked at 292 ng/mL.

In vitro activity of EGF protein expressed by recombinant B. subtilis
To investigate the growth-promoting effect of pEGF expressed by recombinant B. subtilis (WB-EGF), we performed CCK8 cell proliferation assays. MODE-K cells were cultured in the presence and absence of fermented supernatant from WB-EGF for 48 h, and changes in cell numbers were detected with CCK8. To quantify MODE-K cells, we constructed a standard curve based on the optical density of the cell concentration gradient (Fig. 4). Cells were quanti ed using linear regression. When compared with the control group ((1.27387 ± 0.00737) × 10 5 cells)), 100 µL supernatant from the WB-EGF culture signi cantly (P < 0.001) stimulated the proliferation of MODE-K cells ((1.32187 ± 0.00473) × 10 5 cells)), suggesting that the EGF protein secreted by B. subtilis affects cell proliferation in vitro.

Effects of EGF-expressing B. subtilis on growth performance and intestinal morphology of early-weaned mice
To further determine the biological characteristics of EGF expressed by the recombinant B. subtilis strain WB-EGF, we used early-weaned mice to observe the effects of administration of oral fresh bacterial fermentation broth on the growth and repair of intestinal cells in mice. During the 10-d experimental period, no death or abnormal behavior was observed in mice in the CON and EGF groups, whereas mice in the BLANK and ETEC groups were disheveled with poor mental status. On day 10, mice fed WB-EGF showed signi cantly greater weight gain (58.45%±1.98, P < 0.05) than mice fed WB-BLANK (53.73% ±1.88) or ETEC (52.89%±2.47), but were comparable to the CON group (60.88%±3.07) fed LB (Fig. 5).
The structures of the duodenal villi and crypts from mice administered WB-EGF, WB-BLANK, and ETEC are shown in Fig. 6A. The effect of WB-EGF on intestinal growth and repair was assessed by measuring the villus height and crypt depth in the duodenum, jejunum, and ileum. As shown in Fig. 6B and 6C, the villi of the duodenum, jejunum, and ileum of mice fed WB-EGF were longer (P < 0.05) than those of BLANK and ETEC, but there was no signi cant difference compared with those of the CON group (P > 0.05). For comparison of crypt depth, there was no signi cant difference in the duodenum and ileum crypt depth between the CON, BLANK, and EGF groups, but the crypt depth was signi cantly higher compared to the ETEC group. Interestingly, the jejunal crypt depth of mice in the CON and EGF groups was greater (P < 0.01) than that in the BLANK and ETEC groups. These results indicate that pEGF-expressing B. subtilis promoted the growth of intestinal villi in mice and increased the depth of crypts in the small intestine, thus promoting the growth of mice.

Changes in in ammatory cytokine levels in mouse sera
To explore the effects of EGF-expressing B. subtilis on in ammatory cytokines in mice, the serum from mice were assayed for ve different in ammatory factors after ten days of treatment with WB-EGF. As shown in Table 1, the mice in the ETEC group had signi cantly higher (P < 0.05) levels of proin ammatory factors (IFN-γ, IL-1β, TNF-α) than those in the other groups, indicating that the treatment of mice with ETEC was able to successfully elicit in ammatory responses. Interestingly, administration of WB-EGF suppressed the secretion of in ammatory factors in mice. The levels of pro-in ammatory factors IFN-γ and TNF-α were signi cantly decreased (P < 0.05) in mice treated with WB-EGF compared to those treated with WB-BLANK. IL-1β levels were also substantially reduced, but this difference was not signi cant (P > 0.05). Compared with the CON group, there were no signi cant differences in the levels of pro-in ammatory factors in the EGF group (P > 0.05), but the level of anti-in ammatory factor IL-10 was signi cantly increased (P < 0.05). These results suggest that administration of WB-EGF may alleviate the rising trend of in ammatory factors caused by Escherichia. coli. B. subtilis has become an increasingly popular host for recombinant protein expression due to several advantages, including its ability to secrete proteins directly into culture media, ease of large-scale fermentation, and designation as a Generally Regarded As Safe organism by the U.S. Food and Drug Administration [12,13] . In the current study, we cloned the pEGF gene into an integrated vector with a strong promoter p43 and signal peptide SacB, resulting in a vector that enabled rapid and e cient secretion of soluble pEGF by B. subtilis. The presence of pEGF in the supernatant was con rmed via ELISA and western blot analyses. ELISA results showed that the pEGF secretion peaked at 12 h while the yield of the recombinant protein did not increase with prolonged incubation of the cultures, which may be related to the stability of EGF in the fermentation broth. Furthermore, the growth curve of the transformed WB-EGF was largely consistent with that of host WB800, indicating that the addition of a plasmid for expressing exogenous proteins into the genome of WB800 had no effect on its growth trend.
Cheung et al. [6] reported that the addition of exogenous pEGF expressed by Lactococcus lactis improved the growth performance and intestinal development of early-weaned mice. Our study demonstrated that the addition of EGF-expressing recombinant B. subtilis promotes daily weight gain and maintenance of normal intestinal morphology in weaned mice. Most of previous studies used EGF at concentrations of 50 to 500ng/mL, which are similar to those of early mouse milk [14] . The amount of EGF actually received by mice treated with WB-EGF was approximately 180 ng/d, and although it was slightly lower than the effective dose of EGF used by Cheung et al. (≈ 600 ng/d) [6] , our study provides evidence that this dose of EGF is capable of promoting intestinal development in mice. EGF is a key growth factor regulating the proliferation of intestinal epithelial cells, which has a positive effect on the intestinal microstructure. In our study, the villus height and crypt depth (jejunum) in the small intestine of mice in the EGF group differed signi cantly from those of the control group, which is generally consistent with previous studies [6,15] . The increase in villus height and crypt depth provided the intestine with a larger absorption surface area and more active intestinal development potential, which improved the overall digestion and absorption function in the EGF-treated mice, ultimately leading to an increase in body weight.
The addition of EGF to the diet of piglets or rats can help the intestine recover from intestinal diseases, such as infection and diarrhea, and inhibit the colonization of intestinal pathogens [16,17] . Our results indicated that WB-EGF could slow the trend of weight loss and secretion of in ammatory factors IFN-γ and TNF-α caused by E. coli K88ac, and maintain the normal morphology of the intestine in mice. This may be related to the fact that EGF protein secreted by WB-EGF has a role in regulating in ammatory factors [17] . Notably, the administration of WB-BLANK could inhibit the secretion of pro-in ammatory factors (IL-1β) to some extent, which may be associated with the immunomodulatory effects of B. subtilis itself. However, WB-BLANK had little effect on the growth of intestinal villi, indicating that EGF expressed by B. subtilis is important for promoting intestinal development. Regarding the regulation of anti-in ammatory factor IL-10, the ETEC and EGF groups did not differ signi cantly, while the mice in the EGF group had higher IL-10 level compared with the BLANK group. This indicates that EGF can upregulate the secretion of anti-in ammatory IL-10, thereby inhibiting the secretion of pro-in ammatory factors. In the future, the fermentation conditions require further optimization for EGF expression; including such dimensions as temperature, culture time, and pH, control proteolysis, and ultimately achieve e cient EGF production.

Conclusions
This study demonstrates that WB-EGF repairs intestinal injury, inhibits in ammatory factor secretion, and promotes intestinal development and weight gain in early-weaned mice. This study provides baseline data for optimizing the performance of the early-weaning transition phase in animals. The utilization of probiotics to deliver EGF provides a basis for the further development of microecological animal dietary supplements.

Bacterial strains and cells, plasmids, and culture conditions
B. subtilis WB800 was used as the host strain for the EGF expression. ETEC strains (strain K88ac, O139, LT+, and ST+) were kindly provided by the Veterinary Pharmacology Laboratory of Huazhong Agricultural University, China. The recombinant plasmid was transformed and ampli ed using E. coli DH5α (Invitrogen, Carlsbad, CA, USA). Both E. coli and B. subtilis cells were routinely cultured in Luria-Bertani (LB) broth (Oxoid, Wesel, Germany) at 37 °C. B. subtilis competent cell preparation was performed following the nutrient downshifting method described by Anagnostopoulos and Spizizen in 1960 [5] and modi ed by Yasbin et al. in 1975 [18] . MODE-K cells (Bioleaf Biotech Co., Shanghai, China) were cultured in RPMI-1640 medium (GE Healthcare, Chicago, IL, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin. pDG1730 (Genbank U46199), a generous gift from Prof. Ming Sun (Huazhong Agricultural University, Wuhan, China), was used to integrate pEGF into the amyE locus in B. subtilis WB800. We used restriction endonucleases (see Section 5.2) to digest fragments and vectors according to standard molecular biology procedures. For selective media, the following antibiotics were added: 100 μg/mL ampicillin (E. coli) and 100 μg/mL spectinomycin (B. subtilis).

Construction of integrated expression vector PDG-PpEGF
Based on the study by Chomczynski and Sacchi [19] , total RNA was extracted from porcine kidney cortical cells using TRIzol reagent (Invitrogen) according to the manufacturer's instructions and reverse transcription was performed. The pEGF cDNA (GenBank NM_214020) sequence was ampli ed by polymerase chain reaction using forward primers (5'-CCCAAGCTTATGAATAGTTACTCTGAATGCCC-3') and reverse primers (5'-GGAATTCTTAGCGCAGCTCCCACCATTTCAA-3'). Underlined bold letters indicate the restriction enzyme sites adding HindIII and EcoRI to the forward and reverse primers, respectively. After the fragments were veri ed by 0.8% agarose gel electrophoresis, they were cloned into the pMD-18T vector using a T/A cloning kit (Takara Inc., Mountain View, CA, USA) and sequenced (TsingKe, Beijing, China). Both the correctly sequenced pEGF fragment and the pDG1730 vector were double-digested with HindIII and EcoRI, followed by gel extraction and ligation to construct the vector PDG-pEGF.

Recombinant B. subtilis growth curve and fermentation test
Overnight cultures of WB-EGF and WB-BLANK were inoculated into 50 mL LB broth containing spectinomycin with 1% inoculum and cultured at 37 °C, 200 r/min. Cultures (0.5 mL) were taken at 0 h, 2 h, 4 h , 6 h, 8 h, 10 h, 12 h, 14 h, 16 h, 18 h, 24 h, 30 h, 48 h, 54 h, 60 h, 72 h, and 80 h and diluted to a suitable concentration with PBS. The biomass of the recombinant strain was determined using a plate pour. The determination of growth curves was independently carried out in triplicate, and the data are presented as the mean ± SD. The supernatants of the transformants at different time points were collected and stored at -80 °C for subsequent research.

Western blot and ELISA analysis of pEGF
Proteins were separated on 16.5% SDS-PAGE at 30 V for 1 h, followed by voltage adjustment to 100 V for another 4 h. Proteins were transferred to a PVDF membrane at 4 °C for 1 h. The membranes were blocked in 5% skim milk in Tris buffered saline with Tween-20 (TBST) for 2 h, followed by incubation with primary rabbit anti-EGF antibody (1:1000; ABclonal, Wuhan, China) overnight at 4 °C. The PVDF membranes were washed three times in TBST and incubated with goat anti-rabbit IgG horseradish peroxidase-labeled secondary antibody (1:10000 dilution; ABclonal) for 1 h at 25 ℃. The pEGF protein band was detected using a Western ECL blot assay kit (Bio-Rad Inc., Hercules, CA, USA), following the manufacturer's instructions, and the blots were imaged using the ChemiDoc [TM] MP system (BioRad).
Secretion of pEGF by B. subtilis was assayed using an indirect enzyme-linked immunosorbent assay (ELISA) kit (mlbio, Shanghai, China), following the manufacturer's instructions. Brie y, a standard curve was prepared using serial dilutions of pEGF. Culture supernatants (10 µL) were placed in 96-well plates, and pEGF in the samples was developed by adding HRP-labeled detection antibody and TMB substrate. The absorbance of the samples at 450 nm was determined, and the content of pEGF was subsequently calculated using a standard curve. LB was used as a negative control.

In vitro proliferation assay of mouse small intestinal cells
The MODE-K murine jejunal epithelial cell line was seeded in 96-well plates at an initial cell density of approximately 1.0×10 3 cells/well. Next, the cells were co-incubated with 100 μL serum-free RPMI-1640 media and 100 μL lter-sterilized WB-EGF or WB-BLANK supernatant containing empty vector, respectively, at 37 °C for 48 h. Then, the media was removed, cells were washed twice with 1× PBS, and cultured in 100 μL RPMI-1640 media containing 10% CCK-8 (Biosharp, Hefei, China) at 37 °C for 4 h. Absorbance was measured at 450 nm for the evaluation of cell proliferation.

Recombinant pEGF-expressing B. subtilis in early weaning mice
We assessed the in vivo effectiveness of the WB-EGF strain in 60 pathogen-free 19-to 21-d-old ICR female mice purchased from the Experimental Animal Centre of Huazhong Agricultural University (Wuhan, China). Mice were randomly assigned to four groups: EGF, BLANK, CON, and ETEC, with fteen mice per group and ve mice per cage in the same treatment group. All mice were housed in controlled environment (temperature 22 ± 2℃ and 12 h dark/light cycle). Food and water were provided ad libitum. The mice in the CON group received 300 μL LB via intragastric gavage for 10 d, twice daily. Mice in the ETEC group received LB-only in the same manner. Mice in the EGF and BLANK groups received 300 μL of either WB-EGF or WB-BLANK (1~8×10 8 CFU/mL for both treatments) of fresh bacterial broth twice daily for 1-7 d and 9-10 d, respectively. On day 7, all mice were fasted overnight (free water access). After fasting, mice in the EGF, BLANK, and ETEC groups were orally infected with 300 μL E. coli K88ac at a dose of 10 9 CFU/animal. The body weight (BW) of the mice was recorded every 2 d, and diarrhea, behavioral abnormalities, or death were monitored throughout the experiment. The nal BW change was calculated as a percentage of the initial BW of each mouse. On day 11, all mice were anaesthetized with diethyl ether and approximately 0.4 mL of blood was collected by puncturing the orbital plexus of the mice. After the end of blood collection, all mice were sacri ced by cervical dislocation, and intestinal tissues were removed. All animal treatments were performed in accordance with the China Animal Protection Association and ARRIVE guidelines, the study was approved by the Institutional Animal Care and Use Committee of Huazhong Agricultural University.

Histological analysis of intestinal morphology and determination of serum in ammatory factors
Approximately 1-cm long intestinal segments were isolated at the same sites in the duodenum, jejunum, and ileum of each mouse, washed with 0.9% saline, and xed with 4% paraformaldehyde. The xed tissue was embedded in para n, sectioned at 5 µm, and stained with hematoxylin and eosin (H&E) for light microscopy (Nikon ECLIPSE Ci, Tokyo, Japan). For each mouse, at least three cross-sections per small intestinal segment were examined in which ten intact villus-crypt structures were observed per cross-section, and the villus length and crypt depth were measured using Image-Pro Plus 6.0. The identities of all tissue sections were disguised and measurement of villus height and crypt depth were performed in a blinded fashion.
Collected blood samples were placed at 25 ℃ for 2 h and centrifuged at 1000×g for 15 min to separate the serum. The pro-in ammatory factors IFN-γ, IL-1 β, TNF-α, IL-12, and the anti-in ammatory factor IL-10 in the isolated serum were determined using the MSD kit (Meso Scale Diagnostics, Rockville, MD, USA), following the manufacturer's instructions.

Statistical analysis
Western blot, ELISA, in vitro cell proliferation assays, and determination of in ammatory factors were performed in triplicate, with data representing the mean values with standard deviations of all repeats  Growth curves of WB-EGF (black) and WB800 (blue) during the 80-h fermentation period. The determination of growth curves was performed independently in triplicate, and data are presented as the mean ± SD.   Effect of pEGF-expressing B. subtilis (WB-EGF) on growth in mice. Body weight is expressed as the percentage of the initial body weight for each animal. Data are expressed as the mean ± SD (n=15). Different lowercase letters indicate statistically signi cant differences, P<0.05.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download.