Strains and growth conditions
L. reuteri SH 23 (DSMZ 8533) was cultured in MRS Broth at 37°C with shaking, and LEP expression was carried out using E. coli BL21(DE3) competent cells (Vazyme, Nanjing, China) and the pET-28a vector in our laboratory. HT-29 cells were purchased from FuHeng BioLogy (ShangHai, China) and cultured in a humidified 5% CO2 atmosphere at 37°C. The cell culture medium used is DMEM/F12-Dulbecco's Modified Eagle Medium (Gibco, CA, USA).
Lep Recombinant Strain Construction
The LPxTG gene (A5VKB5) of L. reuteri SH 23 consists of a 2175bp open reading frame that encodes a polypeptide of 725 amino acids (NCBI: GenBank: EDX43010.1). The total DNA from bacteria was isolated using the EasyPure Quick Gel Extraction Kit method (Transgen, Pecking, China). The following PCR-amplified primers were used:
Forward: 5’- gtggtggtggtggtgctcgagGATATGACTGGGACAACAGTTAATGG;
Reverse: 5’- cagcaaatgggtcgcggatccTTAAGCATGTTTACGCTTGCG.
The primers were designed using Primer Premier 5.0 (bold in italics are digestion sites). The amplified target genes were purified using an agarose gel DNA purification kit (Transgen, Pecking, China). Thereafter, the fragments were digested with Xho Ⅰ and BamH Ⅰ restriction sites (Takara Biomedical Technology, Beijing, China) and cloned into pET-28a, expressed in E. coli BL21 (DE3) and grown in Luria-Bertani (LB) medium. The recombinant strain was given the name LEP Strain.
Expression Purification Of Lep
The LEP strain was grown in LB medium containing 50 ng/mL kanamycin. When the OD600 value reached 0.6 (25°C, 4 h), 0.4 mM of Isopropyl-beta-D-thiogalactopyranoside (IPTG) was added to induce expression. After induction, the cells were washed three times with PBS. The bacteria were then disrupted by ultrasound (30 min, 300 W), and the supernatant was retained after centrifugation (4°C, 10,000 rpm, 30 min). LEP was purified in imidazole buffer (0, 10, 50, 75, 100, 200 mM) elution using HisTrap columns of ProteinIso Ni-IDA resin (TransGen, Beijing, China). The purified LEP was verified by SDS-PAGE (10% polyacrylamide) and the concentration was determined by BCA kit (Sangon, Beijing, China). After determining the elution concentration, the protein was dialyzed (4℃, 12 h) and lyophilized.
Lep Bioinformatics Analysis
Homology comparison and analysis of LEP were performed using Geneious, SWISS, uniport, Pfam, and other websites. ProtParam (http://web.expasy.org/protparam/) was used to predict the primary structure (physicochemical property analysis, hydrophilic/hydrophobic analysis, aliphatic index, instability index, etc.) of LEP. The hydropathy plot was predicted using ProtScale (http://web.expasy.org/protscale/). Based on statistical analysis of the TMBase database, the TMpred method predicts the protein transmembrane region. The Multiple Em for Motif Elimination website (http://meme-suite.org/) was used to predict the conserved motif of LEP. The secondary structure of LEP was predicted by the SOPMA server (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html). Pfam (http://pfam.xfam.org/) prediction domain. To predict the tertiary structure of LEP, the Homology/analogY Recognition Engine V2.0 (PHYRE2) server was used.
Cytotoxicity Of Lep To Ht-29 Cells
HT-29 cells in good condition were digested, inoculated into 96-well plates, and pre-cultured for 24 hours in a cell culture incubator at 37℃ with 5% CO2. After LEP (100 µL) was added to the plates at different concentrations, with final concentrations of 0, 20, 40, 60, 80, 100, 120, 140 µg/mL, and after 4 h incubation, 10 µL of Cell Counting Kit-8 (Dojindo) reagent was added to each well. After 12 h of incubation, 10 µL of CCK-8 reagent was added to each well, and the cells were incubated in the cell incubator for 4 h before being determined for cell viability using the CCK-8 method.
Confirmation Of Lep Binding To Ht-29 Cell Surface Proteins
The protein interaction protocol and His-Tag Isolation & Pulldown technique were used to identify the cell surface proteins of LEP protein that interact with HT-29 cells. LEP samples containing histidine-tagged proteins were first prepared in a total volume of 700 µL of 1X binding/washing buffer (2 X BWB:100 mM Sodium Phosphate, pH 8.0,
600 mM NaCl, 0.02% Tween™-20) with Dynabeads™ magnetic beads adsorbed (30 min) and washed three times with BWB. Prepare the lysed HT-29 cell lysate in a total volume of no more than 700 µL of 1X Pull-down Buffer (2X PDW: 6.5 mM Sodium phosphate, pH 7.4, 140 mM NaCl, 0.02% Tween™-20).
The samples were mixed with the beads for mixing and incubated at room temperature for 5 min. The tube was placed on the magnet for 2 min, and then the supernatant was discarded. The beads were washed four times with 300 µL of BWB. The process described above, from incubation to cleaning, was repeated twice. 100 µL His-Elution buffer (300 mM Imidazole, 50 mM Sodium phosphate, pH 8.0, 300 mM NaCl, 0.01% Tween™-20) was added and the suspension was incubated on the roller for 5 min at room temperature. After the beads were collected on the tube wall by a magnet, the supernatant containing eluting histidine-labeled protein and its interacting protein was transferred to a clean tube (1.5 mL). The protein was identified by SDS-PAGE and visualized by Coomassie brilliant blue staining.
Lep Complexed With Ht-29 Cells Protein By Lc-ms/ms Analysis
After SDS-PAGE detection, the gum strips were enzymatically digested (Wisniewski et al. 2009), desalted (Zheng et al. 2016), and dried. The analytical instrument for the subsequent LC-MS/MS analysis (Qing et al. 2017; Zhao et al. 2018) was an Ultimate 3000nano ultra-performance liquid chromatography-tandem with a Q Exactive plus high-resolution mass spectrometer. The separation was performed using a nano-HPLC liquid phase system, UltiMate 3000 RSLCnano (Thermo Fisher Scientific). Nano-HPLC buffer A was 0.1% formic acid in water and Nano-HPLC buffer B was 0.1% formic acid in acetonitrile. The Chromatography column trap column (100 µm×20 mm, RP-C18, Agilent) was equilibrated with 100% Buffer A (3 L/min). The samples were loaded from an autosampler, combined with a trap column, and then separated by an analytical column (75 µm × 150 mm, RP-C18, New Objective, USA) at a flow rate of 300 µL/min. Samples were washed through the gradient mobile phase with blank solvent for 30 min. The enzymatic digestion products were separated using capillary HPLC and analyzed by Q-Exactive plus mass spectrometry (Thermo Fisher Scientific). For the qualitative (or relatively quantitative) protein analysis in experimental samples, ProteomeDiscover 2.5 and UniProt-Human/UniProt-Lactobacillus reuteri (strain DSM 20016) were used.
The Effect Of Lep On The Expression Level Of Ht-29 Cells Epitope Protein
After co-culture of LEP and HT-29 cells in 6-well plates for 4 hours, total RNA of HT-29 cells was extracted using the Trizol method, and RNA concentration and purity were determined. Following that, sample RNA reverse transcription was performed (US EVERBRIGHT INC, SuZhou, China). The reverse transcription system (4 µL UEIris II RT MasterMix, 1 µg template RNA, 1 µL dsDNase, RNase-free water to 20 µL) was gently mixed and centrifuged (the liquid settled to the bottom). The following conditions were performed on the PCR instrument: incubation at 37°C for 2 min, 55°C for 10 min, and 85°C for 10 sec. The obtained product was stored at -40°C. Reference RT-qPCR was performed using the kit PerfectStart Green qPCR SuperMix (transgen, Pecking, China). Primers used for the experiments are described in Table S1. The 2−ΔΔCt method was used to assess mRNA expression levels.
The Adhesion Experiment Of Lep To Ht-29 Cells
In 6-well plates, HT-29 cells in good growth conditions were inoculated, and LEP (0, 20, 40, 60 µg/mL) was added for overnight growth (37°C, % CO2). L. reuteri SH 23 was cultured after being activated overnight (37°C). After centrifugation, the cells were washed three times with PBS and resuspended to OD600 = 1. L. reuteri SH-23 was stained for 30 min at 37°C in the dark with Fluorescein isothiocyanate isomer (FITC, 10 µM). Subsequently, L. reuteri was washed three times with PBS to remove excess dye, and the initial fluorescence intensity A0 (excitation wavelength 485 nm; emission wavelength 538 nm) of L. reuteri SH 23 was measured and recorded using Tecan Infinite M200 Pro (Tecan Group, Switzerland). Thereafter, the FITC-stained bacteria were added separately to 12-well plates and incubated for 2 h. Finally, the non-adherent bacteria were removed, and the 12-well plates were washed three times with PBS. The cells were observed and photographed through an inverted fluorescence microscope. The cells were then digested, and the digestive juice collected to determine the fluorescence intensity, which was recorded as the fluorescence value A1 after elution. The adhesion rate of bacteria was calculated according to the following formula:
$$\text{A}\text{d}\text{h}\text{e}\text{s}\text{i}\text{o}\text{n} \text{r}\text{a}\text{t}\text{e}\text{s} \left(\text{\%}\right)=\frac{{\text{A}}_{1}}{{\text{A}}_{0}}\times 100\text{\%}$$
Effect Of Ph And Metal Ions On Lep Esterase Activity
First, 0.63 mL of solution A (3.0 mg of p-nitrophenyl palmitate dissolved in 1.0 mL of isopropanol) was added to 5.67 mL of solution B (1000 mL of water with one drop of TritonX-100, pH = 4, 6, 7) in a centrifuge tube and thoroughly mixed. The mixed liquor was kept in a 37°C water bath for 5 min.
Then the mixture (0.9 mL) and the enzyme solution (200g / mL, 0.1 mL) were added into the centrifuge tube and reacted at 37°C for 30 min. Finally, 95% ethanol (0.5 mL) was added to terminate or stop the reaction, and the absorbance was measured at 410 nm. The average value was taken three times for each sample. Metal ions (K+, Ca2+, Cu2+, Mn2+, Zn2+, 1 mM) were added to solution B at pH = 7 to determine the effect of metal ions on enzyme activity.
Effect Of Lep On Hydrolysis Of Milk Fat
In the experimental group, milk (30 mL, Ningbo Dairy Group) was sterilized at 80°C for 15 min, then LEP was added to make the final protein concentration 100 µg/mL and hydrolyzed at 37°C for 1 hour. Without changing other conditions, inactivated protein was added to the control group. The hydrolyzed milk (30 mL) was transferred to a flask (250 mL) and mixed with pyrogallic acid (100 mg), 95% ethanol (2 mL), water (4 mL) and ammonia (5 mL). Place the flask in a 75°C water bath for 20 min. Every 5 min, the flask was shaken to mix the particles adhered to the flask wall into the solution. After the hydrolysis, the flask was taken out and cooled to room temperature.
For the hydrolysate extraction, the sample was mixed with 95% (10 mL) ethanol, and the hydrolysate in the flask was transferred to the separating funnel. The flask and the plug or stopper were washed with an ether-petroleum ether mixture (1:1 v/v, 50 mL), and the rinse solution was added to the separatory funnel. After shaking for 5 min, the sample was stored for 10 min. The extract of the ether petroleum ether mixture was collected in a flask (250 mL). The hydrolysate was repeatedly extracted three times using the steps outlined above. Finally, the separation funnel was washed with the ether-petroleum ether mixture, and the liquid was collected in a flask (250 mL). The sample bottle was blown with nitrogen and kept at 4°C overnight.
In the saponification experiment, the above sample bottle was filled with KOH (6%) dissolved in methanol and water (4:1 v/v). Saturated salt water (1 mL), hydrochloric acid (0.5 mL,12 moL/L), and a chloroform hexane mixture (2 mL, 1:4 v/v) were centrifuged for 2 min in a centrifuge tube (10 mL). The upper liquid was transferred to a new centrifuge tube (10 mL), and a mixture of chloroform and hexane (2 mL, 1:4 v/v) was added to shake for 2 min. Repeat the above steps twice. The centrifuge tube was washed once with distilled water (2 mL) and the supernatant was transferred to the sample bottle (4 mL) after shaking for 2 min. The sample bottle was blown with nitrogen and kept at 4°C overnight.
Thereafter, the samples were methylated, BF3 (0.5 mL) methanol solution was added to the sample, which was then methyl esterified in a water bath at 60°C for 1 h, and then cooled in a centrifuge tube (10 mL). Add saturated NaCl solution (2 mL) and hexane (2 mL), transfer the organic phase to a new centrifuge tube, and repeat once. After that, centrifuge with chloroform: hexane (1:4 v/v) and remove the upper organic phase. The water was removed from the organic phase using Na2SO4, nitrogen was blown until the organic phase was evaporated, and 0.5 mL of hexane (chromatographic purity) was used to reconstitute the solution.
The GC-MS analysis conditions were as follows: column TG-5MS (30 m×0.25 mm×0.25 µm), heating program was 100°C for 13 min, 10°C/min to 180°C for 6 min, 1°C/min to 200°C for 20 min, 4°C/min to 230°C for 10.5 min, inlet temperature is 290°C ; carrier gas flow rate was 1.2 mL / min, shunt ratio was no shunt, mass spectrometry conditions were ion source temperature 280°C, transmission line temperature was 280°C, scanning range 30–400 amu, ion source EI 70 eV.
Statistical analysis
All data were presented as means ± standard deviations (SD) and analyzed using GraphPad Prism 8.0 program (GraphPad Software, San Diego, Canada). One-way ANOVA was used to compare data from more than two groups, followed by Tukey’s multiple comparison tests. The adjusted p < 0.05 was considered statistically significant. The Figures were created and edited using CorelDRAW 2020 and the Biorender website.