Neomycin

Broad-spectrum ABX was administered to mice in their drinking water for four weeks with renewal every other day. The ABX comprised of ampicillin (500 mg/L), metronidazole (500 mg/L), neomycin (500 mg/L), and vancomycin (250 mg/L) (Sangon Biotech, Shanghai, China) and with 5% sucrose in pure distilled water. This treatment eliminated most of the fecal bacteria, as has been shown in many studies [23 (link), 24 (link)].

Feces were collected from Con mice, Dith mice, and Dith+Lyz mice. Processing of the fecal microbiota transplantation (FMT) suspension was done within 2 h. One hundred milligrams of feces was resuspended in 1 mL of saline and centrifuged for 5 min. The supernatant was used as the FMT suspension. The mice receiving antibiotics were treated with vancomycin (0.5 mg/mL), neomycin (1 mg/mL), ampicillin (1 mg/mL), and metronidazole (1 mg/mL) (Sangon Biotech, China) in their drinking water for 4 weeks. Mice were divided into four groups: the Con group, the ABX+Con group, the ABX+Dith group, and the ABX+Dith+Lyz group. The Con group received no treatment. The ABX+Con group was gavaged with feces from a control mouse for 1 week, the ABX+Dith group was gavaged with 200 μL of an FMT suspension from Dith mice for 1 week, and the ABX+Dith+Lyz group was gavaged with 200 μL of an FMT suspension from Dith+Lyz mice for 1 week. AP was induced in mice from the ABX+Con group, the ABX+Dith group, and the ABX+Dith+Lyz group.

The predicted antibiotic resistance gene information in the genome was obtained by comparing the amino acid sequences of the strains with the comprehensive antibiotic research database (CARD, http://arpcard.mcmaster.ca, accessed on 24 May 2021) [51 (link)]. The strains were clustered using HemI software [50 (link)].
The microbroth dilution method was used to determine antibiotic resistance of L. sakei according to ISO 10932:2010 [52 ]. The following 11 antibiotics were detected: chloramphenicol, rifampicin, streptomycin, kanamycin, gentamycin, tetracycline, clindamycin, neomycin, erythromycin, ciprofloxacin, and vancomycin (all purchased from Sangon Biotech Co., Ltd., Shanghai, China). OD₆₂₅ was determined using an enzyme-labeled instrument (Varioskan Lux, Thermo, Waltham, MA, USA) to determine the MIC of strain to antibiotics.

Curcumin (purity ≥ 95.0%) was purchased from Shanghai Yuanye Biotech (Shanghai, China). Carboxymethylcellulose sodium was obtained from Sigma-Aldrich (Saint Louis, MO, USA). Neomycin, streptomycin, penicillin, vancomycin, metronidazole, bacitracin, ciprofloxacin, ceftazidime and gentamycin were purchased from Sangon Biotech (Shanghai, China). TRIzol® Reagent was from Invitrogen (Carlsbad, CA, USA). High-Capacity cDNA Reverse- Transcription Kits were purchased from Applied Biosystems (Foster City, CA, USA). SYBR Green PCR Master Mix was obtained from Promega (Fitchburg, MI, USA). Proteinase inhibitor cocktail and phosphorylase inhibitor were purchased from Roche (Basel, Switzerland). Bicinchoninic acid (BCA) Protein Assay Kits were purchased from Genstar Technologies (Beijing, China). Antibodies against Akt (9272), pAkt (9271) were purchased from Cell Signaling Technology (Danvers, MA, USA). The antibody against FGF15 (ab229630) was obtained from Abcam. The antibody against Tubulin (CW0098) was from Cwbiotech (Beijing, China).

Coding sequences of the target genes were amplified using specific primers (see Table S3) and cloned into pYF11 vector with fusion of the GFP tag at C terminus or pHZ126 vector with fusion of 3×FLAG tag (97 (link)). The fusion constructs were confirmed by sequencing and desired combinations of constructs were introduced into WT. Transformants were selected on PDA supplemented with Hyg (Roche Diagnostics, Shanghai, China) and neomycin (Sangon Biotech, Shanghai, China). Putative transformants expressing two fusion constructs were verified by PCR and Western blot analyses. Total proteins were extracted and incubated with GFP-Trap beads (ChromoTek, Planegg-Martinsried, Germany) at 4°C for 4 h. After being washed three times, proteins were eluted from the GFP-Trap beads. The eluted samples were detected by Western blotting with anti-GFP antibody (Abcam, Cambridge, MA, USA) or anti-FLAG antibody (Sigma-Aldrich, St. Louis, MO, USA).

Antibiotics treatment was performed according to Refs.^{20 (link),53 (link)} and divided into two stages. In the first stage, mice were gavaged with amphotericin B (Solarbio Life Science, Beijing, China) at a dose of 1 mg/kg body weight every 12 h for three days. The second stage was from the 4th day to the 40th day, when the water was supplemented with 1 g/L ampicillin (Sangon Biotech, Shanghai, China). Furthermore, an antibiotic combination consisting of 50 mg/kg vancomycin (Sangon Biotech, Shanghai, China), 100 mg/kg neomycin (Sangon Biotech, Shanghai, China), 100 mg/kg metronidazole (Sangon Biotech, Shanghai, China), and 1 mg/kg amphotericin B was administered by gavage every 12 h in the second stage.