Characteristics of antimicrobial peptide OaBac5mini and its bactericidal mechanism against Escherichia coli

Introduction Antimicrobial peptides (AMPs) play an important role in defending against the attack of pathogenic microorganisms. Among them, the proline-rich antibacterial peptides (PrAMPs) have been attracting close attention due to their simple structure, strong antibacterial activity, and low cell toxicity. OaBac5mini is an active fragment of the sheep-derived OaBac5 belonging to the PrAMPs family. Methods In this study, the antibacterial activity of OaBac5mini was investigated by testing the MICs against different stains of E. coli and S. aureus as well as the time-kill curve. The bactericidal mechanism was explored by determining the effect of OaBac5mini on the cell membrane. The stability and biosafety were also evaluated. Results The susceptibility test demonstrated that OaBac5mini showed potent antibacterial activity against the multidrug-resistant (MDR) E. coli isolates. It is noticeable that the absence of inner membrane protein SbmA in E. coli ATCC 25922 caused the MIC of OaBac5mini to increase 4-fold, implying OaBac5mini can enter into the cytoplasm via SbmA and plays its antibacterial activity. Moreover, the antibacterial activity of OaBac5mini against E. coli ATCC 25922 was not remarkably affected by the serum salts except for CaCl2 at a physiological concentration, pH, temperature, repeated freeze-thawing and proteases (trypsin < 20 μg/mL, pepsin or proteinase K). Time-kill curve analysis showed OaBac5mini at the concentration of 200 μg/mL (8 × MICs) could effectively kill E. coli ATCC 25922 after co-incubation for 12 h. In addition, OaBac5mini was not hemolytic against rabbit red blood cells and also was not cytotoxic to porcine small intestinal epithelial cells (IPEC-J2). Bioinformatic analysis indicated that OaBac5mini is a linear peptide with 8 net positive charges. Furthermore, OaBac5mini significantly increased the outer membrane permeability and impaired the inner membrane integrity and ultrastructure of E. coli ATCC25922. Conclusion OaBac5mini is a stable and potent PrAMP that kills E. coli by two different modes of action - inhibiting intracellular target(s) and damaging cell membrane.

Introduction: Antimicrobial peptides (AMPs) play an important role in defending against the attack of pathogenic microorganisms. Among them, the proline-rich antibacterial peptides (PrAMPs) have been attracting close attention due to their simple structure, strong antibacterial activity, and low cell toxicity. OaBac mini is an active fragment of the sheep-derived OaBac belonging to the PrAMPs family.
Methods: In this study, the antibacterial activity of OaBac mini was investigated by testing the MICs against di erent stains of E. coli and S. aureus as well as the timekill curve. The bactericidal mechanism was explored by determining the e ect of OaBac mini on the cell membrane. The stability and biosafety were also evaluated.

Results:
The susceptibility test demonstrated that OaBac mini showed potent antibacterial activity against the multidrug-resistant (MDR) E. coli isolates. It is noticeable that the absence of inner membrane protein SbmA in E. coli ATCC caused the MIC of OaBac mini to increase -fold, implying OaBac mini can enter into the cytoplasm via SbmA and plays its antibacterial activity. Moreover, the antibacterial activity of OaBac mini against E. coli ATCC was not remarkably a ected by the serum salts except for CaCl at a physiological concentration, pH, temperature, repeated freeze-thawing and proteases (trypsin < µg/mL, pepsin or proteinase K). Time-kill curve analysis showed OaBac mini at the concentration of µg/mL ( × MICs) could e ectively kill E. coli ATCC after coincubation for h. In addition, OaBac mini was not hemolytic against rabbit red blood cells and also was not cytotoxic to porcine small intestinal epithelial cells (IPEC-J ). Bioinformatic analysis indicated that OaBac mini is a linear peptide with net positive charges. Furthermore, OaBac mini significantly increased the outer membrane permeability and impaired the inner membrane integrity and ultrastructure of E. coli ATCC .

Introduction
Escherichia coli (E. coli) is a rod-shaped Gram-negative bacterium belonging to Enterobacteriales, Enterobacteriaceae, Escherichia, which includes commensal and pathogenic strains. Escherichia coli can cause a variety of diseases in humans and animals, such as diarrhea, extraintestinal diseases (1), urinary tract infection (UTI) and bovine mastitis (2, 3). However, with the emergence and spread of resistant E. coli, treatment options are becoming more limited (4). Therefore, finding alternatives to antibiotics to treat pathogenic E. coli is becoming more important for both humans and animals.
Antimicrobial peptides (AMPs) are small molecular peptides in living organisms and serve as natural barriers against the invasion of pathogenic microorganisms (5). They are often referred to as cationic host defense peptides (HDPs) or cationic antimicrobial peptides (CAMPs) (6). Cathelicidins are one of the best characterized AMP families. Each cathelicidin includes an N-terminal signal domain, a cathelin-like domain, and an antimicrobial C-terminal domain (7). Among them, the prolinerich antibacterial peptides (PrAMPs) have been attracting the close attention of researchers due to their strong antibacterial activity against Gram-negative bacteria, remarkably low toxicity toward mammalian cells, and the lack of extensive membrane-damaging effects (8). Bac5 is the first member of the PrAMPs to be identified in cattle, sheep, and goats (9,10). Moreover, its homologous peptides have been subsequently found in another mammal (deer) (11). It has been demonstrated that mature Bac5 is a linear peptide composed of 43 amino acid residues (12) which includes a critical element for antibacterial activity containing 15 Arg-rich residues (13). In 2018, Mardirossian et al. (10) confirmed that Bac5 (1-25) and Bac5 (1-31), two active fragments of Bac5, inhibited bacterial protein synthesis by binding to ribosome after entertaining into the cytoplasm of E. coli BW25113 via an inner membrane transporter SbmA. In addition, OaBac5, a homologous PrAMP of Bac5 was first isolated from the crude neutrophil extraction from ovine blood in 2003. Based on its cDNA sequence, OaBac5 is a 51residue PrAMP (14). Research showed that OaBac5 exhibited potent activity against E. coli 0157:H7, Staphylococcus aureus 1056 MRSA, and Candida albicans 3153A (MICs, 4-10 µg/ml) (15). Likewise, two other variants (OaBac5α and OaBac5β) of OaBac5 were isolated from elastase-treated extracts of sheep leukocytes which showed potent activity against bacteria and fungi (16). In 2004, OaBac5mini was first synthesized as a truncated fragment of OaBac5, comprising the first 24 N-terminal residues. OaBac5mini exhibited potent activity against Gram-negative bacteria (MICs, 0.125-8 µg/ml) but weak activity against Gram-positive bacteria and C. albicans (MICs, 16-64 µg/ml), which was similar to that of OaBac5. However, compared to Bac5, OaBac5mini was more active against Gram-negative bacteria (17). In addition, the antibacterial activity of OaBac5mini was not significantly affected by pH or temperature. Moreover, it also showed synergistic activity with other PrAMPs (SMAP29 and OaBac7.5mini), polymyxin B, and lysozyme against E. coli 0157:H7 and S. aureus 1056 MRSA (18,19). The above-mentioned results imply OaBac5mini has the potential to become a promising lead compound for new antibiotic substitutes. However, the characteristics and bactericidal mechanism of OaBac5mini have been unknown and are the subject of this study.
In the present study, we demonstrated that OaBac5mini showed stronger antibacterial activity against MDR E. coli isolates than susceptible E. coli ATCC 25922. Its antibacterial activity was not significantly affected by in vitro conditions factors (pH, temperature, repeated freeze-thawing) and in vivo conditions factors (serum salts, pepsin and proteinase K). Moreover, the physicochemical characteristics, the cytotoxicity and the bactericidal mechanism of OaBac5mini were fully investigated in the present study to illustrate the bactericidal action and the safety.

Antimicrobial
Peptides OaBac5mini (N-RFRPPIRRPPIRPPFRPPFRPPVR-C) was prepared via solid-phase synthesis using 9-fluorenylmethoxycarbonyl (F-moc) chemistry at GL Biochem (Shanghai) Ltd. and analyzed by HPLC and MALDI-TOF MS to confirm that the purity was >91.06%.
Amoxicillin, kanamycin, florfenicol, and tetracycline were purchased from China National Institute for Drug and Biological Products Control (Beijing, China). Polymyxin B sulfate (PMB) purchased from Beijing Solarbio Science & Technology co., Ltd (Beijing, China).

Bacterial strains and cells
The strains of E. coli ATCC 25922 and S. aureus ATCC 25923 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, United States). The strains of E. coli W13, E. coli IF4, E. coli 2, and E. coli 17 were individually isolated from the livers of diseased chickens infected by E. coli. The strains of S. aureus B5, S. aureus B9, S. aureus B54, and S. aureus B63 were individually isolated from the milk of cows with mastitis. These clinical isolates were all identified by the VITEK-32 system (bioMérieux, France) and PCR amplification sequencing.

Construction of E. coli ATCC sbmA
The method described by Datsenko and Wanner was used for chromosomal gene deletion (20 GTGTAGGCTGGAGCTGCTTC-3 ′ and LBMAR: 5 ′ -CTCGCGTACCGTAGGCGGCGTCGCGCGCGTGGCATCGTCT TCACCCATATGAATATCCTCCTTAG-3 ′ , which consisted of 20 nucleotides (nt) of the helper plasmid pKD4 and 45 nt on the 5 ′ and 3 ′ ends of the inactivated sbmA. The PCR fragment (1,567 bp) was purified, digested with DpnI, repurified and transferred into E. coli ATCC 25922 by electroporation, in which the Red recombinase expression plasmid pKD46 was previously transformed and induced by L-arabinose. Transformants were selected on Luria-Bertani (LB) agar containing 50 µg/ml of kanamycin at 37 • C. The inserted sequence was amplified from the kanamycin-resistant strains by using the primers SBMF: 5 ′ -GCACGGCAGAAAAAAGCA-3 ′ and SBMR: 5 ′ -GACGGAAACAGCAAGAACAAA-3 ′ which is located outside of inactivated gene. The length of the PCR product of sbmA using the primers set SBMF and SBMR in E. coli ATCC 25922 is 1,404 bp. When sbmA is successfully inactivated, the PCR product (2,198 bp) was amplified and sequenced for the verification of the gene deletion.

Antibacterial activity
MICs of OaBac5mini, amoxicillin, kanamycin, florfenicol and tetracycline were determined using the two-fold broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) guidance (21). MIC values of the tested antibacterial agents were determined on three independent occasions. Escherichia coli ATCC 25922 was used as the quality control in all the susceptibility tests.

Salt sensitivity assay
To determine the effect of different salt ions on the antibacterial activity of OaBac5mini, MIC values of OaBac5mini were tested at different physiological salt concentrations (150 mM NaCl, 4.5 mM KCl, 6 µM NH 4 Cl, 1 mM MgCl 2 , 2.5 mM CaCl 2 , and 4 µM FeCl 3 ) as described by Maisetta et al. (22).

In vitro and in vivo conditions factors sensitivity assay
The sensitivities of OaBac5mini to different pH, temperature, digestive enzymes, and repeated freeze-thawing conditions were determined using the inhibition zone assay as previously described (23). For pH sensitivity, OaBac5mini was diluted with each solution whose pH was individually adjusted to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 with HCl or NaOH, and incubated for 2 h, followed by adjusting the pH to 7 to end the reaction. For temperature sensitivity, the diluted peptide was incubated at 30, 40, 50, 60, 70, 80, 90, and 100 • C for 30 min. To determine the digestive enzymes sensitivity, OaBac5mini was incubated with 100 µg/ml pepsin (in pH 2 sterile water, sigma, USA), 5-100 µg/ml trypsin (in pH 6.8 KH 2 PO 4 buffer, sigma, USA) and 100 µg/ml protease K (Solarbio, China) at 37 • C for 3 h, respectively. To end the reaction after incubation, the pepsin buffer was adjusted to pH 8, the trypsin solution and protease K solution were incubated at 95 • C for 10 min. For repeated freeze-thawing sensitivity, OaBac5mini was frozen and then thawed at room temperature 0-12 times. All the final concentrations of OaBac5mini in sensitivity tests were 50 µg/ml. All experiments were repeated three times.

Hemolysis assay
The fresh blood was collected from rabbit hearts, and then centrifuged and rinsed with phosphate-buffered saline (PBS) three times. The 1:100 diluted rabbit blood cells (RBCs) solution was mixed with an equal volume of OaBac5mini solution (3.125-400 µg/ml final concentrations), PBS (negative control) and 0.1% Triton X-100 (Solarbio, China, positive control), respectively, followed by incubating at 37 • C for 1 h. The mixed solution was centrifuged and 100 µl of the supernatant was added into a 96well plate. The optical density (OD) of samples was monitored immediately using INFINITE 200 PRO (Infinite R 2000, TECAN, Austria) at an absorption wavelength of 413 nm. The experiment was conducted in triplicate. The OD value at 413 nm was used to calculate the hemolysis ratio with the formula below: AS is the OaBac5mini-treated sample, AN is the negative control, AP is the positive control.

Real-time cell assay
Real time cellular analysis (RTCA) was used to evaluate the proliferation and viability of IPEC-J2 cells. Cells were seeded in 16-well E-plates (xCELLigence, ACEA biosciences, USA) at the concentration of 20,000 cells per well, allowing attachment overnight, and then high glucose MEM was replaced by fresh complete MEM with different concentrations of OaBac5mini (25, 50, 100, 200, and 400 µg/ml). Real time cellular analysis was monitored using xCELLigence RTCA DP system (xCELLigence, ACEA biosciences, USA) for 100 h at 15 min intervals. The cell index was normalized by RTCA software 2.0 (xCELLigence, ACEA biosciences, USA).

Permeability of the outer membrane
The permeability of OaBac5mini to E. coli ATCC 25922 cell membranes was determined by N-Phenyl-1-naphthylamine (NPN, sigma, India), which is a fluorescent dye that only can pass through the damaged cell membrane and exhibit an increase of fluorescence. The NPN uptake assay was conducted following the instructions of Helander and Mattila (25)

Integrity of the inner membrane of cell
The integrity of the inner membrane of E. coli ATCC 25922 treated by OaBac5mini was investigated by 3,3 ′ -Dipropylthiadicarbocyanine iodide [DiSC 3 (5), Sigma, USA], which is a fluorescent probe that can be concentrated in the integrated cytoplasmic membrane, leading to fluorescence selfquenching. If the membrane is depolarized or disrupted, DiSC 3 (5) is released into the aqueous medium and leads to increased fluorescence. The DiSC 3 (5) assay was conducted following the instruction of Sautrey et al.   , and µg/ml) in MH broth medium for h. Aliquots were collected at , , , , , , , and h to count the bacteria. Error bars represent means ± SEM. Assays were performed in triplicate.

Statistical analysis
The statistical analyses were performed using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA) and shown with MEAN ± SEM. One-way ANOVA was used to test the statistical significance Frontiers in Veterinary Science frontiersin.org . /fvets. .

FIGURE
The hemolysis rate of antimicrobial peptide OaBac mini. All data were measured at the maximum absorption wavelength ( nm) at least three times. Error bars represent means ± SEM. There was no significant di erence between OaBac mini treated groups and the negative control group (P > . ). of the differences, and then Dunnett's multiple comparison test or Tukey's multiple comparison test was performed using SPSS 20 (SPSS, Chicago, USA). Statistically significance defined as * P < 0.05, * * P < 0.01, and * * * P < 0.001.
Frontiers in Veterinary Science frontiersin.org . /fvets. . E. coli ATCC 25922 to OaBac5mini increase four-fold, suggesting that OaBac5mini can enter into the cytoplasm via SbmA and plays its antibacterial activity. The antibacterial activity of OaBac5mini against E. coli ATCC 25922 was investigated in the presence of different salt ions with similar concentrations to that in human serum. As shown in Table 2, the presence of increased CaCl 2 decreased susceptibility to OaBac5mini compared to MH broth alone. Other salt ions (MgCl 2 , NH 4 Cl, FeCl 3 , NaCl and KCl) did not significantly affect the antibacterial activity of OaBac5mini. This suggests that OaBac5mini will show a comparable stability in serum.
As shown in Figure 1, OaBac5mini exhibits stable antibacterial activity against E. coli ATCC 25922 in acid-base environments ( Figure 1A), high-temperature environments ( Figure 1B), repeated freeze-thawing ( Figure 1C) or digestive enzymes pepsin and proteinase K as well as trypsin with low concentrations ( Figure 1D). However, OaBac5mini completely lost activity after it was treated by trypsin with concentrations ranging from 20 to 100 µg/ml.

Time-kill curve of OaBac mini
The time-kill curves of OaBac5mini against E. coli ATCC 25922 are shown in Figure 2. Results showed that 200 µg/ml OaBac5mini (8 × MIC) effectively killed E. coli ATCC 25922 after co-incubation for 12 h. When the concentration of OaBac5mini was 50 µg/ml (2 × MIC), the number of bacteria remained stable in the first coincubation for 8h before it increased, attaining an amount similar to that of E. coli ATCC 25922 without OaBac5mini after co-incubation for 24 h. At the same time, 100 µg/ml OaBac5mini also caused the numbers of bacteria to decrease after co-incubation for 12 h and increase in further co-incubation for 12 h.

Hemolysis and cytotoxicity of OaBac mini
The hemolysis of OaBac5mini against RBCs was decided by testing the OD of every sample at an absorption wavelength of 413 nm. As shown in Figure 3, the mean hemolysis rate of RBCs was −1.26 to 1.59% after co-incubating with different concentrations of OaBac5mini (3.125-400 µg/ml) at 37 • C for 1 h, which indicates that OaBac5mini does not cause hemolysis.
The results of RTCA showed that OaBac5mini was not cytotoxic to IPEC-J2 cells, but seemed to increase the proliferation of cells (Figure 4). Overall, the cell indexes of OaBac5mini treated groups showed a significant increase (P < 0.01) compared with the control group ( Figure 4B). The lower the concentration of OaBac5mini, the stronger the promotion effect on cell proliferation was observed ( Figure 4A). Even though the concentration of OaBac5mini attained 400 µg/ml, the cell indexes didn't change significantly compared to the control group (P > 0.05; Figure 4B).

Physicochemical properties analysis and structure prediction
To illustrate the antimicrobial mechanism of OaBac5mini, the physicochemical properties and structure were predicted. As  (Table 3 and Figure 5A). The predicted secondary structure showed that OaBac5mini is rich in random coils without other secondary structures ( Figure 5B). The 3D modeling indicated that OaBac5mini is a linear antimicrobial peptide ( Figure 5C).

E ect of OaBac mini on the cell membrane
The permeabilization of the outer membrane of E. coli ATCC 25922 was determined by detecting the fluorescence changes of NPN. As shown in Figure 6A, the fluorescence intensity increased in bacteria treated with OaBac5mini for 20 min and exhibited a slight decrease until 60 min. The average fluorescence intensity of NPN in bacteria treated with OaBac5mini for 60 min was significantly increased (P < 0.01) compared with the negative control ( Figure 6B), which indicated that the outer membrane of the bacteria was damaged by OaBac5mini.
The inner membrane integrity of E. coli ATCC 25922 was investigated by detecting the fluorescence changes of DiSC 3 (5). As shown in Figure 6C, the fluorescence intensity increased when bacteria were treated with OaBac5mini for 10 min, and gradually leveled off for the next 20 min. The average fluorescence intensity of DiSC 3 (5) of all OaBac5mini treated groups was significantly increased within 30 min (P < 0.05, Figure 6D), which indicates that the inner membranes were damaged by OaBac5mini.
. /fvets. . The morphology and ultrastructure of E. coli ATCC 25922 treated with concentrations of 50 and 200 µg/ml OaBac5mini were investigated by SEM. As shown in Figure 7, E. coli treated with PBS (negative control) exhibited smooth and regular surfaces of the membranes. But both of the bacteria treated with OaBac5mini or 50 µg/ml PMB (positive control) exhibited atrophy, corrugation, and pore formation on the surface of the cell membranes, as well as the leakage of the intracellular contents. The results of SEM directly reflect the membrane damage, and also verify the results of NPN and DiSC 3 (5).

Discussion
OaBac5 is a sheep-derived PrAMP consisting of a 6-residue N terminus followed by two copies of a 16-residue repeat and a 5-residue C terminus. Within the 16-residue repeat sequence is a combination of four repeats of the smaller 4-residue (XRRP), which is a shared characteristic in the amino acid sequences of Bac5 from different animals. Compared to that of bovine-derived Bac5, only five residues differences occur, which makes OaBac5 less hydrophobic and more cationic. Research demonstrated that OaBac5 showed potent activity against E. coli, S. aureus, and C. albicans (15). However, Bac5 only exhibited antibacterial activity against Gram-negative bacteria (27). Although OaBac5 is a potent broad-spectrum PrAMP, its complete amino acid sequence has not been obtained or synthesized in vitro. In 2004, OaBac5mini, a truncated fragment of OaBac5, was synthesized to contain the 6 N-terminal residues, one copy of the 16-residue repeat, and the first two residues of the second repeat. The susceptible tests against different strains showed that OaBac5mini had antibacterial activity similar to that of OaBac5, which implies that the full-length  , and µg/ml. Error bars represent means ± SEM, n = . *, **, and *** represent significant, very significant and extremely significant, respectively (P < . , P < . , and P < . by One-way ANOVA).
sequence is not required for the antibacterial activity of OaBac5 (17). In this study, we found that OaBac5mini showed stronger activity against four strains of MDR E. coli isolates (MICs, 1.95-3.26 µg/ml) than E. coli ATCC 25922 (MICs, 25 µg/ml). However, the MICs of OaBac5mini against S. aureus (MICs, 208.33 to >250 µg/ml) are far greater than those reported (MICs, 16-64 µg/ml) (17). We think that the main reasons are probably due to the different MIC test methods and strains used. Wu et al. performed the MICs experiment using a modified twofold microtiter broth dilution method in which the antibacterial peptide was first diluted by a two-fold decrease in the buffer containing 0.2% bovine serum albumin and 0.01% acetic acid in Eppendorf tubes. At the same time, the LB medium used to culture the bacteria and test MICs did not contain NaCl (28). In this study, OaBac5mini was dissolved in purified water and bacteria were cultured in LB broth. It was reported that the antibacterial activity of OaBac5mini against E. coli O157:H7 increased with the decreasing concentrations of NaCl (18).
Salt ions in serum are important factors affecting the antibacterial activity of AMPs in vivo because they can bind to the negatively charged groups of lipopolysaccharide (LPS), a monolayer molecule on the outer membrane of Gramnegative bacteria (29). In this study, OaBac5mini exhibits similar antibacterial activity against E. coli ATCC 25922 in the presence of physiological concentrations of NaCl, KCl, NH 4 Cl, MgCl 2 or FeCl 3 . But in the presence of physiological concentrations of CaCl 2 , the MIC of OaBac5mini increased four-fold and attained 100 µg/ml (Table 2). It has been reported that the presence of Ca 2+ and Mg 2+ caused the antibacterial activity of Bac5 against E. coli to significantly decrease. Moreover, Bac5 could kill E. coli more effectively by decreasing the concentration of NaCl (30). In addition, the antibacterial activity of OaBac5mini against E. coli O157:H7 was also affected by salt and metal ions. The presence of 100 mM NaCl significantly decreased the activity of OaBac5mini and its MIC attained about 30 µg/ml, which is similar to that of OaBac5mini against E. coli ATCC 25922 in the presence of Frontiers in Veterinary Science frontiersin.org . /fvets. .

FIGURE
Scanning electron microscopy images of E. coli ATCC treated with OaBac mini and PMB for h. The bacteria treated with mM PBS is considered as negative control; the bacteria treated with µg/ml PMB is considered as positive control; the bacteria were treated by OaBac mini with the concentration of µg/ml ( × MICs) or µg/ml ( × MICs). The back arrows indicate the broken membrane.
150 mM NaCl described in this study. Furthermore, the divalent ions Mg 2+ and Ca 2+ inactivated OaBac5mini at a concentration of 10 and 5 mM, respectively, which is higher than its corresponding physiological concentration (1 mM for Mg 2+ and 2.5 mM for Ca 2+ ) in human serum (18). Taken together, OaBac5mini will keep antibacterial activity against E. coli in human serum. Apart from its stability in the presence of physiological concentrations of ions, we first investigated the effect of some gut factors on the antibacterial activity of OaBac5mini. The results showed that the activity of OaBac5mini against E. coli ATCC 25922 was not significantly affected by pH and pepsin, suggesting OaBac5mini may remain stable in the stomach. Furthermore, at concentrations of ≤10 µg/ml, proteinase K and trypsin also had no significant effect on the activity of OaBac5mini (Figure 1). Physicochemical toxicity is an important limitation during the application of various antibacterial agents. In the present study, OaBac5mini showed no hemolysis, whose mean value of hemolysis ratio was −1.26 to 1.59% (Figure 3). In addition, all the concentrations of OaBac5mini rapidly promoted the proliferation of IPEC-J2 cells when it was added ( Figure 4A) and IPEC-J2 cells showed increased cell indexes when treated with OaBac5mini ( Figure 4B). The results demonstrate that OaBac5mini has no physicochemical toxicity, but can promote cell proliferation and survival. Cathelicidins are multifunctional peptides with diverse functions such as proliferation and migration of cells, immunoregulation, wound healing, angiogenesis and the release of cytokines (31). We previously found that PrAMP BSN-37 also promotes the proliferation of IPEC-J2 cells and Vero cells (13). Another cathelicidin derived from human LL-37 stimulated the proliferation of airway epithelial cells and wound closure (32). This may be because cathelicidins stimulate the expression of cytokines such as growth factors (33) and growth factor receptors (34), or activate the growth factor receptor-related signaling pathways (35).
The cell membrane is the first barrier of bacterial defense against external adverse factors. Most cathelicidin family AMPs contain amphiphilic α helix structures and are rich in positively .
/fvets. . charged amino acids. These kinds of AMPs accumulate on the surface of the negatively charged moieties in the cell wall through electrostatic attraction, such as LPS in the outer membranes of Gram-negative bacteria, and then disrupt the cytoplasmic membrane followed by cell lysis (36). However, some PrAMPs have been confirmed to kill Gram-negative bacteria mainly by binding with intracellular targets without apparent membrane damage (10,37). Furthermore, the crystal structures of Bac7 (1-16), Bac7 (1-35) and Onc112 binding to the 70S ribosome from Thermus thermophilus showed that a common mechanism of action used by these PrAMPs is to inhibit protein synthesis (38,39). In this study, OaBac5mini is a positively-charged hydrophilic linear peptide ( Figure 5) with a net charge of 8, which is higher than the net charges of Bac5 and other truncated fragments (10). Furthermore, the significantly increased fluorescence intensity ( Figure 6) and incomplete ultrastructure (Figure 7) indicated that OaBac5mini inhibited E. coli ATCC 25922 by damaging the cell membrane. However, when the transporter sbmA mainly responsible for the internalization of antibacterial peptides was inactivated in E. coli ATCC 25922, the sbmA deletion mutant showed a 4-fold increase of the MIC, suggesting OaBac5mini can enter into the cytoplasm and play its antibacterial activity. On the other hand, the time-kill curve showed that OaBac5mini could effectively kill E. coli ATCC 25922 at a concentration of 200 µg/ml. When the concentration of OaBac5mini is 50 or 100 µg/ml, the growth of bacteria was firstly inhibited and then, increased step by step ( Figure 2). Therefore, the minimal inner membrane depolarization of bacteria treated by OaBac5mini with a low concentration was not enough to cause cell death. As a result, OaBac5mini plays its bacteriostatic role against E. coli by lytic and nonlytic modes of action. At present, some AMPs such as Bac7(1-35), Cathelicidin-BF, and Bac5(1-17) derivatives have been confirmed to kill Gram-negative bacteria by two different modes of action (40)(41)(42). Furthermore, Bac7(1-35) could not only bind to the ribosome to inhibit protein synthesis, but it can also bind to DnaK, which participates in purine metabolism and enriched kinase activity (43). It is evident that different action modes and multiple targets in the cytoplasm will make clinical strains unlikely to develop resistance against PrAMPs. Recently, Dolzani et al. (44) found that Acinetobacter baumannii AB5075 induced by sub-MICs of Bac7(1-35) for 16 passages did not produce resistance to Bac7(1-35). Taken together, PrAMPs are promising lead compounds for the development of the next antibiotics for treating clinical MDR bacterial infections.

Conclusion
OaBac5mini is a potential and promising lead compound for the development of antibiotic substitutes with potent antibacterial activity against MDR E. coli isolates. Its activity was not seriously affected by in vitro condition factors (pH, temperature, repeated freeze-thawing) and in vivo condition factors (the serum salts, pepsin and proteinase K). Moreover, OaBac5mini was not hemolytic or cytotoxic. Furthermore, it kills E. coli by two different modes of action: inhibiting intracellular target(s) and damaging cell membranes. The next work is to search for the intracellular target(s) of OaBac5mini and illustrate its (their) antibacterial mechanism (s).

Data availability statement
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.