Scientific Papers

Antimicrobial peptide Mastoparan X has good activity against Escherichia coli in vitro and alleviates its pathogenicity in mice | Translational Medicine Communications


Ethics statement

BALB/c mice (6–8 weeks old, 18–20 g, female) were purchased from Zhengzhou University. All animal experiments were performed in accordance with the guidelines of the Animal Welfare and Research Ethics Committee and were approved by the Animal Ethics Committee at Henan Institute of Science and Technology.

Peptide synthesis

MPX (H-INWKGIAAMAKKLL-NH2) was obtained from Shanghai Jier Biochemical Company (China) using a solid-phase N-9-fluoromethoxycarbonyl (Fmoc) strategy and high-performance liquid chromatography (HPLC) purification, and its purity was 98%.

Bactericidal activity test

E. coli was cultured in LB liquid medium to the logarithmic phase (OD600 = 1.0), and the final concentrations of MPX (31.25 µg/mL), PR39 (50 µg/mL), and Enro (50 µg/mL) were added to the bacterial solution. ddH2O was added as the negative control and incubated at 37 °C for 0 h, 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h. Then, the OD600 of the bacterial solution was measured, and the bacterial solution was diluted every 1 h. Each plate was placed on the LB plate and incubated in a 37 °C incubator for 12 h until a single colony was clearly visible, and then the plates were counted [14]. The assays were repeated three times.

Determination of BCA protein content

BCA method for protein concentration measurement principle: In an alkaline environment, proteins will form complexes with Cu2+ ions and reduce them to Cu1+ ions (biuret reaction). BCA will then react with Cu1+ to form a stable purple-blue complex, which has a high absorbance value at 562 nm and is proportional to the protein concentration. Proteins are one of the most important nitrogen-containing biomolecules in cells and are involved in various biological functions. Briefly, 25 μL of each sample and 200 μL of BCA working reagent were added to each well. The microplate was shaken for 15 s and then incubated for 30 min at 37 °C using a BioTek® Cytation 5 Cell Imaging Multi-Mode Reader. Absorbance was measured at 562 nm [15]. The assays were repeated three times.

Detection of live and dead bacteria

The LIVE/DEAD BacLight Bacterial Viability L-7012 Kit (Molecular Probes, Eugene, OR, USA), containing two component dyes (SYTO 9 and PI in a 1:1 mixture) in solution, was used for microscopy and quantitative assays according to the test instructions. A total of 3 μL of the dye mixture was added to each well, the wells were incubated at room temperature in the dark for 15 min, and the bacterial survival in the biofilm was observed by confocal laser microscopy.

MPX on the outer membrane of E. coli

E. coli were washed with a 1:1 mixture of 5 mM HEPES buffer and resuspended with the same solution. The concentration of E. coli was 1 × 108 CFU/mL. This study was performed in a Corning 96 black well plate with clear bottom containing 10 μM of N-phenyl naphthylamine (NPN) dye and 190 μL of bacterial suspension. After that, bacterial suspensions with dye in each well were treated with 10 μL of MPX at concentrations of 1 MIC, 2 MIC, and 4 MIC. Then, the fluorescence was monitored at excitation wavelength of 350 nm and emission wavelength of 420 nm. The increase in fluorescence intensity was monitored for another 10 min with an INFINITE M PLEX microplate reader [16]. The assays were repeated three times.

MPX on the cell membrane potential

E. coli were collected and separately washed with a 1:1 ratio of 5 mM glucose and HEPES buffer (pH = 7.4). Next, the bacterial plate was resuspended in a 1:1:1 ratio of 5 mM HEPES buffer and 100 mM KCl solution supplemented with 0.2 mM EDTA and 5 mM glucose. For this study, EDTA was used to enable dye uptake by permeabilizing the outer membrane of E. coli. This study was performed in a Corning 96 black well plate with a clear bottom containing 2 μM 3,3′-dipropylthiadicarbocyanine iodide [DiSC3(5)] and 190 μL of bacterial suspension. After that, 10 μL of MPX (1 MIC, 2 MIC, 4 MIC) was mixed with the suspension of bacteria and dye from each well. ddH2O was used as the control. Fluorescence intensity was measured at 622 nm excitation wavelength and 670 nm emission wavelength for 10 min using an INFINITE M PLEX microplate reader [17]. The assays were repeated three times.

MPX on the inner membrane of E. coli

Briefly, the mid-log phase of E. coli was separately centrifuged (8000 rpm/min, 5 min), washed and resuspended in 1:1 ratio of 5 mM glucose and HEPES buffer (pH = 7.4). The working concentration of E. coli was 1 × 108 CFU/mL. Next, 190 µL of bacterial suspension containing 10 µM propidium iodide (PI) was added to the well of black Corning 96-well plate with clear bottom. Next, 10 µL of MPX (1 MIC, 2 MIC, 4 MIC) was added to the wells containing dye and bacterial suspension. The control was ddH2O. An excitation wavelength of 535 nm and emission wavelength of 617 nm were used to monitor PI fluorescence for 10 min using an INFINITE M PLEX microplate reader [18]. The assays were repeated three times.

Crystal violet staining

The ability of MPX to inhibit E. coli biofilm formation was tested according to the reference. In a 96-well polystyrene microtiter plate, an overnight culture of E. coli was inoculated into 100 µL of LB liquid medium at a proportion of 1%, and different concentrations of MPX (0.5 MIC, 1 MIC, 2 MIC, 4 MIC) were applied; ddH2O was used as a negative control. The culture plate was placed in a 37 °C incubator for 24 h. The culture supernatant was aspirated and discarded. Each well was washed 3 times with 200 µL of sterile PBS and fixed with 70% methanol for 30 min. The fixative was aspirated and discarded, and the wells were dried at 37 °C in the incubator for 30 min. Then, 100 µL of 1% Hucker crystal violet staining solution was added to each well and stained for 5 min at room temperature. The staining solution was removed, and the culture plate was rinsed under a slow stream of water until the flowing water was colourless. After the oven was dried, the sample was placed under a microscope for observation. Then, 100 µL of 70% ethanol solution was added to each well for decolorization, vortexed and mixed, and quickly placed in a multifunctional microplate reader to determine the absorbance value of OD570 [19].

Scanning electron microscopy observation of biofilm formation

After culturing the E. coli overnight, it was diluted by a factor of 100 with fresh LB liquid medium and transferred to 6-well cell plate with sterile glass slides. Then, 500 µL of bacterial solution was added to each well, and MPX (1 MIC) and ddH2O were added as negative control. After 24 h of incubation in a constant-temperature incubator at 37 °C, the cell culture solution was slowly removed and rinsed with sterile saline 3 times to wash away floating bacteria. Then, 300 µL of 2.5% glutaraldehyde solution was added to each well. After fixation at room temperature for 30 min, the samples were rinsed with pH = 7.4 phosphate buffer 3 times, with an interval of 10 min each time. Next, 30%, 50%, 70%, 80%, 90%, 95%, and 100% alcohol were used for gradient dehydration, and each time interval was 15 min. After the slides were dried, the formation of biofilms was observed under a scanning electron microscope [13].

IPEC-J2 cells adhesion and invasion detection

IPEC-J2 cells were cultured in a 6-well plate and 1 × 106 cells were added to each well. After 24 h of culture, the medium was replaced with fresh DMEM:F12 blank medium, 50 µM Rac1 inhibitor NSC23766 or the same volume of 0.1% DMSO was added, and then 10 µg/mL MPX or sterile water was added. The cells were placed in the cell incubator and cultured for 12 h, washed with PBS 3 times, and then added at an MOI of 10. The E. coli suspension was incubated at 37 °C for 1 h, and then unadhered and contaminant bacteria were washed away with PBS, 200 µL of 0.5% Triton X-100 was added to each well for 5 min, followed by 800 µL of precooled PBS. The cells were collected and subjected to multiple dilution and LB medium coating, with inverted culture in a biochemical incubator at 37 °C for 16 h. The assays were repeated three times.

Animal experiments

A total of 20 BALB/c mice (6–8 weeks old, 18–20 g, female) were purchased from Zhengzhou University. All mice were housed in individual cages at a constant humidity (40–70%) and temperature (21 ± 1 °C) under a 12-h light/dark cycle for 3 days to acclimate to the environment. The animals were randomly divided into four experimental groups (control, E. coli, E. coli + MPX, and E. coli + enrofloxacin (Enro); five mice per group) and challenged with an intraperitoneal injection of E. coli (4.5 × 107 CFU/mL). The mice were treated with an intraperitoneal injection of MPX (20 mg/kg) or Enro (20 mg/kg) for 3 days after E. coli infection for 2 h. Ethics Approval and Consent to Participate is Henan Institute of Science and Technology. The ethical approval No. 2022HIST022. The assays were repeated three times.

qRT–PCR detection

IPEC cells were cultured to form a monolayer in a 6-well plate and then divided into four groups: IPEC + E. coli, IPEC + E. coli + MPX, IPEC + E. coli + MPX + NSC 23766, and IPEC + E. coli + NSC 23766. Infection was carried out at an MOI of 10. After 12 h, IPEC-J2 cells were collected for mRNA expression detection of the tight junction proteins ZO-1, Occludin, and Claudin-1. Total cell RNA was extracted by adding 1 mL of RNAio Plus to each well, followed by the addition of 200 μL of chloroform and centrifugation at 12,000 rpm, 4℃ for 10 min. The supernatant was slowly aspirated, and 500 μL of isopropanol was added and mixed, followed by centrifugation at 12,000 rpm at 4 °C for 10 min. Then, 1 mL of 75% ethanol was added to each tube, which was then centrifuged at 12,000 rpm/min at 4 °C for 5 min. Finally, 20–30 μL of DEPC water was added for reverse transcription using a Takara kit (Cat. No. DRR047A) under the following reaction conditions: 95 °C for 5 min, 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 20 s, for a total of 40 cycles. The relative expression levels were determined using the 2−ΔΔCT method [20]. The primer sequences were shown in Table 1. The assays were repeated three times.

Table 1 The primers for this study

H&E staining

After being sterilized with alcohol wipes, mouse organs and intestines were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with H&E to observe the pathological changes in the duodenum, ileum, colon, liver, spleen, and lungs. The specific procedures were carried out following the previously described methods [21].

Statistical analyses

GraphPad Prism 8.0 software was used to perform statistical analysis of the experimental results and identify significant differences (one-way ANOVA). P < 0.05 was considered a significant difference (*P < 0.05; **P < 0.01; * ** P < 0.001).



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