1. Introduction
As multi-drug-resistant bacteria emerge, especially “superbugs,” antimicrobial peptides (AMPs) are increasingly recognized as a promising therapeutic alternative to conventional antibiotics [
1]. Numerous AMPs that act as components of the innate immune system have been isolated from living organisms [
2]. Currently, more than 2000 AMPs originating from natural sources have been characterized, emphasizing the importance of expanding AMPs research [
3]. Antibacterial peptides have many advantages compared to antibiotics, such as a broad antibacterial spectrum, good stability, minimal side effects, and minimal drug resistance. Their principal mechanism of action involves binding to the conserved structural components of the bacterial envelope (e.g., lipopolysaccharide and lipoteichoic acid of Gram-negative and Gram-positive bacteria, respectively) [
4]. This allowed AMPs to interact with the bacterial cells membrane, which was lethal to the bacteria. Some AMPs also bonded to intracellular targets and inhibited essential biological processes, including cell wall formation and the synthesis of DNA, RNA, and proteins [
5]. Compared with conventional antibiotics, this mechanism could kill bacteria quickly and reduce the chances of bacteria producing resistance [
6].
Bovine lactoferricin (LFcinB) is a 3.1 kDa protein and corresponds to residues 17–41 of bovine lactoferrin (LFB). The majority of these 25 amino acids are basic amino acids with isoelectric point (pI) >12. Researchers have shown that LFcinB has a broad range of antimicrobial activities against Gram-positive and Gram-negative bacteria [
7,
8]. It also displayed candidacidal activity [
9]. Residues 19 and 36 of LFcinB are cysteines, which form a disulfide bond. While some studies had suggested that the disulfide bond was not essential for antimicrobial activities [
10], several forms of LFcinB derived from peptide fragments (LFcinB17–31, LFcinB20–30, and LFcinB20–25) clearly exhibited antimicrobial activities. In fact, the antimicrobial activity was comparable to that of intact LFcinB [
11]. Residues 4–9 (RRWQWR) of LFcinB have been termed the “antimicrobial center” of LFcinB, and when the C-terminal carboxyl group was amidated, the hexapeptide has been shown to display antimicrobial activity similar to full-length LFcinB [
12]. Because of technical difficulties, extracting endogenous antimicrobials from organisms has low efficiency, and there was a high cost for chemical synthesis. Furthermore, the natural amino acid polypeptides are often rapidly hydrolyzed into shorter peptides as a result of enzyme action in the human body, thus greatly reducing or eliminating their antimicrobial activity [
13]. Therefore, designing and screening synthetic peptides with greater stability, high antibacterial activity, and safety is needed. There are many ways to design antibacterial peptides, but the most widely used method is based on naturally occurring antimicrobial peptides that have been modified by the substitution of optimized amino acids [
14].
For an antimicrobial peptide, its biological activity is closely related to many different structural parameters, including chain length, number of α-helices, hydrophilicity, cationicity, hydrophobicity, and other structural parameters [
15]. In this paper, we mainly consider hydrophobicity and cationicity, due to hydrophobicity and cationicity being directly affected the affinity between AMPs and cell membrane [
16,
17]. We used the peptide LFcinB18–28 (KCRRWQWRMKK), which has asymmetrical amino acid sequence, as a template; the hydrophobicity amino acids included tryptophan (W), proline (P), phenylalanine (F) and the positively charged amino acids lysine (K) and arginine (R) were used to substitute the original amino acids at the N-terminus and C-terminus, making a peptide with a symmetrical amino acid sequence. Recent studies have also shown that AMPs with a symmetrical amino acid sequence have enhanced antibacterial activities [
18]. The AMPs with symmetrical amino acid sequence were more likely to produce translocation to destroy the cell membrane when the AMPs interacted with the cell membrane. We engineered a total of four different AMPs, keeping the active center RRWQWR unchanged. The molecular characteristics of the four engineered peptides were greatly changed compared to the original peptide. By observing the biological activities of these peptides, we determined the effects of the symmetrical amino acid sequence and amino acid substitution. This furthered our understanding of the mechanism of bacteriostasis.
3. Discussion
LFcinB has a broad spectrum of activity against Gram-negative and Gram-positive bacteria [
9]. Because of a unique bacteriostatic mechanism, it significantly reduces the likelihood of bacterial resistance [
24]. However, extracting natural LFcinB from organisms is expensive, and it is unstable in many circumstances. The design and modification of AMPs can solve this problem. The sequential template method means that a natural antimicrobial peptide can be studied by inserting selected amino acid in the peptide sequence to alter the net positive charge, in addition to other features, such as α-helices, hydrophilicity and hydrophobicity. Antimicrobial peptides with more robust activity and a wider antimicrobial spectrum can be obtained. Many of the physiological activities associated with LFcinB have been localized to the cationic N-terminal lobe of this iron-binding protein [
9]. The peptide LFcinB18–28 is an 11-residue peptide with the sequence KCRRWQWRMKK. It contains the active center RRWQWR [
12]. We used the method of amino acid substitution to obtain four engineered peptides with symmetrical amino acid sequences.
Our results indicated that the structure of the three engineered peptides (KW-WK, FW-WF, and KK-KK) in the simulated membrane environment would be transformed into an α-helical structure. Studies have shown that an α-helical structure can enhance the activity of antimicrobial peptides because the structure of the water-lipid is favorable to the combination and penetration of bacterial cell membranes by antimicrobial peptides [
25]. This may explain why the antimicrobial activity of these peptides was higher than that of LFcinB18–28. Peptide FP-PF had an irregular structure in SDS, probably because it contained proline. Studies have shown that the extent of helicity decreases as the number of proline residues increases [
26]. The original LFcinB18–28 peptide was a β-fold structure. This is likely because the amino acid sequence contained a cysteine, which is a necessary amino acid for forming the β-fold structure [
27].
The antimicrobial assays proved that engineered peptides were much more active than LFcinB18–28. This showed that the symmetrical amino acid sequence could improve the antimicrobial activities of AMPs. The transformation of LFcinB18–28 was successful. When we analyzed the molecular characteristics of each peptide, we found that the results were consistent with previous findings that the hydrophobicity of KW-WK, FP-PF and FW-WF was enhanced compared with LFcinB18–28, suggesting that hydrophobic amino acid substitutions increased the hydrophobicity of AMPs and, thus, increased the antimicrobial activity [
28]. Hydrophobic groups enabled the peptide chains to form aggregates in solution via hydrophobic interactions, increasing the affinity for eukaryotic membranes [
17]. The ability of AMPs to form an α-helix was also strengthened with enhanced antimicrobial activity [
29]. However, the increased hydrophobicity was not necessarily better. If hydrophobicity was too high, this would result in self-aggregation and precipitation, reducing the antimicrobial activity of AMPs [
30]. The hydrophobic moment represented the cumulative hydrophobicity of a peptide and reflected the structural characteristics of the interaction between peptides and membranes. It could be used to express the hydrophilicity of AMPs. The higher the hydrophobic moment, the stronger the ability of AMPs to break the membrane [
31]. Cationic properties are essential for most AMPs with antimicrobial activity because positive charge promotes the activity of antimicrobial peptides at low concentrations to enrich the surface of the film to kill bacteria. This directly affected antimicrobial activity [
32]. The net charge and hydrophobic moment of peptide KW-WK were relatively higher, which enabled it to have better bacteriostasis than FP-PF and FW-WF, even though they were more hydrophobic than peptide KW-WK. The net charge of peptide KK-KK was 9, but the inhibition was still very weak. These results indicated that if the net charge was too high, bacteriostasis would be restricted [
16]. This may be because too much positive charge can enable the AMPs to firmly bind the head of the phospholipid. Thus, the film-penetrating efficiency is reduced. At the same time, when the number of positive charges exceeds a certain critical value, this leads to electrostatic repulsion between the AMPs, which is stronger than their electrostatic attraction to the membrane. This prevents the accumulation of AMPs molecules and the formation of transmembrane pores, reducing membrane lysis and antimicrobial activity [
33]. Therefore, the antimicrobial activities of AMPs were likely related to hydrophobicity, hydrophobic moment, and net charge.
The hemolytic activity of the peptides against human erythrocytes and the cytotoxicity of the peptides against HEK 293 cells have been suggested to be major parameters determining peptide toxicity in higher eukaryotic cells. Melittin is one of the most biologically active polypeptides, accounting for 50% of the dry weight of bee venom. It has high hemolytic and cytotoxic effects, and is commonly used as a control peptide [
34]. Compared with melittin, we observed in this study that the hemolytic activity and cytotoxicity of the five peptides were not significant. Compared with the other four peptides, FW-WF showed a slightly higher hemolytic effect, probably because both ends of the peptide contained the hydrophobic amino acids tryptophan and phenylalanine, which increased the overall hydrophobicity [
35]. Frecer et al. [
36] also showed that hydrophobic amino acids affected hemolytic. The treatment index (TI) was defined as the ratio of MHC to MIC, showing antimicrobial specificity of antimicrobials, the larger the value of TI, the greater the antimicrobial specificity [
37]. As shown in
Table 2, the cell selectivity of peptides KW-WK and FP-PF was better and safer. Thus, they can be widely used in the fields of food, medicine, and feed.
If AMPs become widely used in production, they will be subject to a variety of destabilizing factors, such as high-temperature environments, many kinds of proteases, and a variety of cations [
38]. Therefore, it is necessary to determine the stability of each peptide. Interestingly, when cations with physiological concentrations were added to the medium, only a few cations had a slightly inhibitory effect on specific peptides. Some inhibitory effects were even increased. It is believed that the electrostatic interactions between the cationic portions of the peptide and the negative charge on the surface of the bacteria facilitated the interaction between the lipophilic regions of the peptide with the cell membrane. This led to the destruction of the cell membrane, which caused the cells to die. Generally, most cationic AMPs are salt-sensitive, displaying reduced or absent antimicrobial activity at high salt concentrations. However, at relatively low physiological concentrations, this is not always the case. At high concentrations, divalent cations progressively increased membrane rigidity through electrostatic interactions with negatively charged phospholipids, which slowly hinders pore formation [
34]. Previous studies established that the antimicrobial activity of LFcinB was modulated by the presence of monovalent and divalent cations [
39]. Na
+ reduced the antimicrobial activity of the peptide FP-PF. This may because cations affected the activity of AMPs by interfering with their ability to bind membranes [
40]. The fact that small amounts of divalent cations can contribute to peptide membrane binding could explain why NH
4+ increased the activity of FW-WF. Fe
3+ increased the activity of KK-KK and LFcinB. After heating at 100 °C for 1 h, we found that the antimicrobial activity of the peptides was still high, except for peptide KK-KK. This was consistent with a previous report that most AMPs were thermally stable [
41]. After treatment with four different proteases, it was found that the effects of these proteases were diverse. Only trypsin significantly decreased the antimicrobial activity of the peptides. Research has shown that the cleavage sites of trypsin involve the peptide bond formed by lysine and arginine [
42], which the five AMPs all contained. Thus, their activities were inactivated. Papain is a sulfhydryl protease, and its cleavage site is the peptide bond formed by arginine, lysine and glycine, which peptide KK-KK has. Thus, it was unstable after treatment with papain.
Based on the antimicrobial activity test for these antimicrobial peptides, we knew that peptides KW-WK, FP-PF and FW-WF had strong antimicrobial effects. Therefore, we studied their bacteriostatic mechanism. Phosphatidyl glycerol, cardiolipin and phosphatidylserine are predominant components of the bacterial membrane [
43]. Usually, in the first step of bacteriostasis, cationic peptides are selectively combined with negatively charged composition of the outer membrane, such as lipopolysaccharide [
44,
45]. Then, these peptides are readjusted and inserted into the cytoplasmic membrane lipid bilayer, causing the disruption of membrane permeabilization and integrity or pore/ion channel formation. At the same time, the membrane potential disappears [
46]. In this study, we found that three peptides (KW-WK, FP-PF, and FW-WF) had a concentration-dependent effect on the permeabilization of the outer membrane of
E. coli UB1005. All peptides had the ability to permeabilize the inner membrane to ONPG at 1× MIC and 1/2× MIC, which was indicative of strong, concentration-dependent membrane permeabilizing ability. From the flow cytometric analysis, we found that all peptides killed bacteria by damaging cytoplasmic membrane integrity. Our SEM and TEM results further confirmed that all peptides had potent interactions with the membrane structure, disrupted the cell membrane, and allowed the intracellular content of the bacterial cells to leak out through the membrane. Taken together, these results demonstrated that three peptides (KW-WK, FP-PF, and FW-WF) caused damage to the cytoplasmic membrane.
In conclusion, this study advocated an alternative approach to the design of AMPs according to the principles of symmetrical amino acid sequence. We used LFcinB18–28 as a template to synthesize four novel antimicrobial peptides with symmetrical amino acid sequences using the method of amino acid substitution. Two peptides, KW-WK and FP-PF, displaying high antimicrobial activity, low toxicity, and good stability had been devised successfully. These peptides might be useful as new antimicrobial drugs in the fields of food, medicine, and livestock feed. Although peptide FW-WF was relatively hemolytic, it still had good antimicrobial activity. Throughout the experiment, we observed the bacteriostatic mechanism for the three engineered peptides: KW-WK, FP-PF, and FW-WF. Unlike conventional antibiotics, which usually kill cells by inhibiting the synthesis of some substance in the cells, including cell wall, proteins, DNA or RNA, most AMPs share a cationic and amphipathic character that facilitates interaction with the anionic microbial membranes, leading to disruption of membrane integrity, permeabilizing microbial membranes, affecting the membrane potential, and resulting in cell death. The symmetrical amino acid sequence improved the antimicrobial activity to some extent, but the effect was closely related to the hydrophobicity, hydrophilicity, and charge of each AMP. Cumulatively, this work provides a theoretical basis for the design of more effective antimicrobial peptides.