The antimicrobial effect of a novel peptide LL-1 on Escherichia coli by increasing membrane permeability

Materials

The E. coli strain ATCC25922 was preserved by the Henan International Joint Laboratory for the Development and Application of Veterinary Biological Products (Anyang, China). Antimicrobial peptide LL-1 (EPRWKGWKKIEKAVRRVRDGIIKAGPAVAVVGQVQGIAG) was synthesized and purified by Sangon Biotech (Shanghai) Co., Ltd., China. The Ezup Column Bacteria Genomic DNA Purification Kit was purchased from Sangon Biotech. DL-15000 DNA Marker was purchased from Takara Biomedical Technology (Beijing) Co., Ltd., China. O-Nitrophenyl-β-D-Galactopyranoside (ONPG) and the ATP Assay Kit were purchased from Beytime (Shanghai, China). Electron microscope fixative solution was purchased from Wuhan Sevier Biotechnology Co., Ltd., China.

Characterizations of the LL-1 peptide

To study the LL-1 peptide, we predicted its physicochemical characteristics through the ExPASy Bioinformatics Resource Portal (http://www.expasy.org/tools/) [24].

Additionally, the hemolytic activity of LL-1 was determined using red blood cells from BALB/c mice. Blood containing anticoagulants collected from mice was washed, cells counted, and diluted as previously reported by our laboratory [25]. The red blood cell suspension was mixed with different concentrations of LL-1 (final concentrations: 50, 100, 150, 200, and 250 μg/mL). These mixtures were co-incubated at 37 °C for 1 h, and then centrifuged at 3,000 g for 10 min. The obtained supernatants were used to determine the absorbance at 405 nm. The hemolysis ratio was calculated using the following formula: hemolysis ratio = (A405peptide—A405PBS)/(A405Triton—A405PBS) × 100%.

The cytotoxicity of LL-1 was determined in porcine kidney-15 (PK-15) cells through a CCK-8 cell counting kit (Vazyme, Nanjing, China). PK-15 cells were counted, diluted and inoculated into 96-well cell-culture plates, and then cultured at 37 °C for 24 h as previously reported by our laboratory [25]. Different concentrations of LL-1 were added to the 96-well cell-culture plates at final concentrations of 50, 100, 150, 200, and 250 μg/mL, respectively. The 96-well cell-culture plates were further incubated at 37 °C for 12 h, and then 10 μL of CCK-8 reagent was added to each well. After incubation at 37 °C for 1 h, the absorbance at 450 nm was detected by an automatic multi-function microplate reader (Synergy H1, BioTek, USA).

The minimum inhibitory concentration (MIC) of LL-1 against E. coli

E. coli was cultured in the sterile LB medium for 12 h and centrifuged for 2 min at 6,000 g at room temperature to collect the bacterial pellet, which was diluted with sterile LB medium to 2 × 106 CFU/mL. LL-1 was diluted with phosphate-buffered saline (PBS) to 200 µg/mL. The MIC of LL-1 against E. coli was detected by double dilution method as previously described [25]. Kanamycin (200 µg/mL) was used as a positive control and PBS was used as a negative control. After treatment for 16 h, resazurin was used as an indicator, and 10 µL of 6 mM resazurin was added to each well. Following incubation at 37℃ for 3 h, the color change in each well was observe.

Effects of LL-1 on E. coli growth

E. coli in the logarithmic growth phase was inoculated into sterile LB liquid medium at the volume ratio of 1:100. E. coli solutions were divided into four groups. LL-1 was added into four groups with a final concentration of 0, 1, 4, and 8 MIC LL-1, respectively. The four bacterial solutions were incubated at 37℃ with shaking at 200 rpm. Next, 100 µL of bacterial solution was removed from each group after 0, 2, 4, 6, 8, 10, and 12 h of incubation and placed in 96-well microtiter plates. The absorbance values at 600 nm were detected with an automatic multi-function microplate reader (Synergy H1, BioTek, USA). Each experiment was repeated three times.

Effects of LL-1 on the permeability of E. coli cell membrane

E. coli in the logarithmic growth phase was centrifuged at 6,000 g and room temperature. The bacterial cells obtained were collected, washed three times with sterile PBS, resuspended and divided into five samples. LL-1 was added to the each sample with the final concentration of 0, 1, 4, and 8 MIC, respectively, and Triton X-100 served as the positive control with the final concentration of 0.3% (v/v). All samples were placed in a constant temperature water bath at 37℃ for 1 h, and then centrifuged at 6,000 g for 5 min. Then, 100 µL of the supernatant was removed and placed in a 96-well plate, mixed with 4.5 µL of ONPG at the concentration of 3 mmol/L, and then incubated at 37℃ for 30 min. The absorbance value at 420 nm was measured with an automatic multi-function microplate reader (Synergy H1, BioTek, USA). Each experiment was repeated three times.

Using the same method, the E. coli solutions were divided into four samples. LL-1 was added to each sample with the final concentration of 0, 1, 4, and 8 MIC, respectively. All samples were placed in a constant temperature water bath at 37℃. Then, 200 µL of bacterial solution was removed from each tube after 0, 1, 2, 3, 4, 5, and 6 h of incubation and centrifuged at 6,000 g and 4℃ for 5 min. The supernatant was removed and the absorbance was determined at 260 nm and 280 nm using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA), respectively. Three replicates were prepared for each sample.

Binding of LL-1 to E. coli DNA

E. coli in the logarithmic growth phase was centrifuged at 6,000 g and room temperature to collect the bacterial cells. The E. coli genomic DNA was extracted according to the Ezup Column Bacteria Genomic DNA Purification Kit instructions, and the DNA concentration was determined using an ultra-micro spectrophotometer (NanoDrop 2000, Thermo Scientific, USA). Seven samples with final DNA concentration of 25 ng/µL in 20 µL system were then prepared according to a mass ratio of LL-1 to E. coli DNA of 0, 0.25, 0.5, 1, 2.5, 5 and 10, respectively. The samples were gently inverted to allow mixing and then placed in a constant temperature water bath at 37℃ for 30 min. The mixed solution was analysed by nucleic acid gel electrophoresis and observed using a nucleic acid gel imager (G:BOX, Syngene, UK).

Effects of LL-1 on the intracellular ATP of E. coli

E. coli in the logarithmic growth phase was centrifuged at 6,000 g and room temperature. The bacterial cells were collected, washed three times with sterile PBS, resuspended and divided into four samples. LL-1 was added to a final concentration of 0, 1, 4, and 8 MIC, respectively. All samples were placed in a constant temperature water bath at 37℃ for 1 h, and then centrifuged at 6,000 g for 5 min to collect the bacterial cells. The concentration of ATP in each sample was determined according to the instructions of the ATP Assay Kit. Each experiment was repeated three times.

Effects of LL-1 on E. coli observed by scanning and transmission electron microscopy

E. coli in the logarithmic growth phase was centrifuged at 6,000 g and room temperature to collect the bacterial cells, which were washed three times with sterile PBS, resuspended and divided into four samples. LL-1 was added into two samples with the final concentration of 2 MIC, while PBS was added to the other samples as the negative controls. All samples were placed in a constant temperature water bath at 37℃ for 1 h, and then centrifuged at 6,000 g for 5 min to collect the bacterial cells, which were washed three times with sterile PBS and resuspended in electron microscopy fixative solution. The samples were fixed at room temperature for 2 h, and sent to Wuhan Sevier Biotechnology Co., Ltd. Two groups of samples (PBS and LL-1 treated groups) were fixed by 1% (v/v) osmic acid for 2 h and dehydrated by different concentrations of ethanol and isoamyl acetate at 4 °C. The samples were dried, then closely attached to the conductive carbon film double-sided tape and placed on the sample stage of the ion sputtering instrument for gold spraying for approximately 30 s, observed, and images were obtained by scanning electron microscopy (Regulus 8100, Hitachi, Japan).

The another samples were pre-embedded in 1% agarose gel, and then fixed with 1% (v/v) osmic acid for 2 h at room temperature. The samples were dehydrated by different concentrations of ethanol and acetone at room temperature, permeabilized with acetone embedding reagents, and then polymerized in an oven at 60 °C for 48 h. The samples were placed in a microtome and 60–80 nm ultra-thin sections were obtained through a 150-mesh Fanghua film copper mesh. The copper mesh was dyed in 2% uranyl acetate saturated alcohol solution for 8 min in the dark, washed with 70% alcohol three times and then ultrapure water three times, dyed by 2.6% lead citrate solution in the absence of carbon dioxide for 8 min, washed with ultrapure water three times, and then slightly dried with filter paper. The copper mesh slices were placed in a copper mesh box to dry overnight at room temperature, observed and images obtained by transmission electron microscopy (HT7700, Hitachi, Japan). The bacterial death rate was evoluated by Image-Pro Plus 6.0 software (Media Cybernetics, USA).

Effects of LL-1 on E. coli determined by PI staining experiment

E. coli in the logarithmic growth phase was centrifuged at 6,000 g and room temperature to collect the bacterial cells, which were washed three times with sterile PBS, resuspended and divided into five groups, and each group had 1 mL of bacterial solution. LL-1 was added to four groups at final concentration of 0, 1, 4, and 8 MIC, respectively. Triton X-100 served as the positive control with the final concentration of 0.3% (v/v). All of samples were gently inverted to allow mixing, and then placed in a constant temperature water bath at 37℃ for 1 h. Then, these samples were centrifuged at 10,000 g for 1 min. The supernatants were discarded, and the bacterial precipitations were resuspended by sterile PBS. PI was added to the bacterial solution at a final concentration of 10 nmol/L, which was gently inverted to allow mixing and incubated in the dark at 37℃ for 30 min. The fluorescence intensity was measured using a multi-function microplate reader (Synergy H1, BioTek, USA) at the excitation wavelength of 535 nm and the emission wavelength of 615 nm.

Statistical analysis

Each experiment was repeated three times, and the results are presented as the mean and standard deviation. Data analysis and graphs were performed using GraphPad Prism software (version 6.0, La Jolla, CA, USA). *, P < 0.05 represents a significant difference; **, P < 0.01 represents an extremely significant difference.

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