Bacterial strains and reagents
L. monocytogenes strain ATCC 19115 (L95) was cultured in trypticase soy broth (TSB) with various concentrations of morin (purity ≥ 98 %) that was purchased from Dalian Meilun Biotech Co., Ltd.
Anti-L. monocytogenes activity testing
The minimum inhibitory concentration (MIC) of morin for L95 was evaluated as described in previous work [14]. In brief, L95 was cultured to the stage of logarithmic growth in TSB and adjust the density to 1× 108 CFUs/ml. The morin was diluted continuously with TSB medium and then adding bacterial culture to 5× 105 CFUs/ml, respectively. The growth of bacteria under different concentration of morin was observed every 12 hours for 48 hours. For the determination of bacterial growth, the absorbance at OD600 nm of L95 cultured in TSB with the indicated concentrations of morin at 37°C was measured every 30 min for 5 h.
Hemolysis assay
L95 was cultured in TSB with various concentrations of morin (0, 2, 4, 8,16 μg/mL) at 37°C for 5 h. Following centrifugation (10,000 rpm, 2 min), 100 μL of the supernatant of each co-culture sample was incubated with sheep erythrocytes and PBS a in a system of 1 mL for 20 min. Then, the system was centrifuged and the absorption value at OD543nm of supernatant was detected to determine the hemolytic activity by comparing each sample with the control sample (sheep erythrocytes treated with PBS supplemented with 2% Triton X-100), which was set as 100% hemolysis activity.
The purified protein was incubated with morin (0, 1, 2, 4, 8 μg/mL) and the detection of hemolytic activity in these samples were measured as described above.
Western blotting assay
L95 was cultured in TSB with morin at 37°C for 5 h. Following centrifugation, the supernatants of each sample were treated with Laemmli sample buffer, boiled for 10 min and separated on SDS-PAGE gel. After electrophoretic transfer, the interest protein on the polyvinylidene fluoride membrane was blocked at room temperature for more than 2 h, and then the polyvinylidene fluoride membrane was in a incubation with a primary rabbit anti-LLO antibody (Abcam, 1:2000) for 2 h and a corresponding secondary antibody (Proteintech, 1:3000) for 1h. The ECL detection reagents were used to visualize the signals on the PVDF membranes with a Tanon-4200 imager.
Purification and the induction of oligomerization of LLO protein were performed as previously described[44]. In brief, the LLO incubated with morin (64 μg/mL) for 20 min and the oligomerization was induced under high salt conditions in vitro. Firstly ,LLO protein was incubated with morin for 30 minutes under acidic PBS conditions, then saturated potassium chlorid was added to the concentration of 0.3 mg/mL and incubated for another 10 min. Next, sheep erythrocytes were added to the reaction system and treated on ice for 5 minutes. The reaction samples were boiled with SDS-PAGE loading buffer lacking 2-hydroxy-1-ethanethiol. And the oligomerization was detected by western blotting described above.
Cell-line infections
L95 was cultured to mid-logarithmic growth at OD600 about 1.0 and the cells were washed with sterile PBS and then resuspended in complete DMEM (HyClone) containing no fetal bovine serum (FBS)
J774 macrophage-like cells grew in high-glucose DMEM at 5% CO2 and we plated the cells in corning 96-well plates ( about 2×104 cells per well) to culture for 16 h and then the cells co-cultured with L95 at 37°C for 5 h at a MOI of 8 or treated with LLO protein (12 ng per well) that pre-incubated with morin (0, 2, 4, 8,16 μg/mL) for 30 min at 37°C for the detection of lactate dehydrogenase. And the co-culture supernatant was diluted continuously and the extracellular bacteria determined by colony counting. In order to assess the intracellular bacteria, the supernatant of the culture medium was removed, washed with PBS for three times, and gentamicin was added to kill the residual extracellular bacteria. After that, the cells were lysed with saponins, and the colony count was carried out by continuous dilution.
Cells grew in corning 6-well plates (1×106 cells per well) were infected with L95 in the presence of morin treatment at an MOI of 10 at 37°C for 4 h for inflammation assays.
Inflammation assays
Following centrifugation (1,000 rpm, 10 min), inflammatory mediator (IL-1β, IL-6, and TNF-α) contained in each co-infection sample were determined using the corresponding ELISA kit (Biolegend ,CA, USA).
Cytotoxicity assays
Following centrifugation (1,000 rpm, 10 min), the lactate dehydrogenase level in samples of cell infection was assessed by a Cytotoxicity Detection Kit. The samples treated with Triton or DMEM were set as controls.
Animal experiments
Female BALB/c mice (6-8 weeks) were bought from the Experimental Animal Center of Jilin University, and the animal experiments were in accordance with the Jilin University institutional animal care committee guidelines. All of the animals were euthanized by cervical dislocation at the end of the experiment.
L95 cultured to mid-logarithmic growth stage (OD600=0.8-1.0), followed by centrifugation and collection of the bacteria, which were then washed 3 times with sterilized PBS and resuspended to a density of 1×108 CFU/mL. The mice that were intraperitoneally injected with 1×107 CFU received 100 μg/g of morin or DMSO (25μL) at 2 h after infection and then at 12-h intervals to assess the protective effect of morin. The mortality rate of infected mice (30 mice per group) was observed for 96 h. In addition, the mice intraperitoneally injected with the L95 suspension (2×106CFUs/mouse) were used to detect the bacterial burden and inflammation in vivo, morin was administered subcutaneously (100 mg/kg) 2 h after infection, then at 12-h intervals. DMSO was administered as the control. The liver and spleen were dissected and ground in PBS after sacrificing by euthanasia 48 h postinfection. The ground tissue supernatant was collected by centrifugation and then diluted onto a solid TSB medium to calculate the number of bacteria. The pro-inflammatory cytokines in the ground tissue supernatant were determined by ELISA.
Statistical analysis
All experimental data presented as the mean ± SD and repeated independently at least for three times. The statistical analysis was conducted by GraphPad Prism 5.0. P value < 0.05 and P value < 0.01 are marked in the figures (Student’s t-test).