A Review of Antimicrobial Peptides: Structure, Mechanism of Action, and Molecular Optimization Strategies

Abstract

Antimicrobial peptides (AMPs) are bioactive macromolecules that exhibit antibacterial, antiviral, and immunomodulatory functions. They come from a wide range of sources and are found in all forms of life, from bacteria to plants, vertebrates, and invertebrates, and play an important role in controlling the spread of pathogens, promoting wound healing and treating tumors. Consequently, AMPs have emerged as promising alternatives to next-generation antibiotics. With advancements in systems biology and synthetic biology technologies, it has become possible to synthesize AMPs artificially. We can better understand their functional activities for further modification and development by investigating the mechanism of action underlying their antimicrobial properties. This review focuses on the structural aspects of AMPs while highlighting their significance for biological activity. Furthermore, it elucidates the membrane targeting mechanism and intracellular targets of these peptides while summarizing molecular modification approaches aimed at enhancing their antibacterial efficacy. Finally, this article outlines future challenges in the functional development of AMPs along with proposed strategies to overcome them.

1. Introduction

The easy accessibility and widespread utilization of conventional antibiotics have resulted in their excessive use, leading to the emergence of antimicrobial resistance in healthcare systems worldwide. Therefore, there is an urgent need to develop novel antibiotics or antibiotic alternatives. AMPs represent a class of oligopeptides that exhibit resistance against bacteria and viruses [1]. Typically composed of 12–50 amino acid residues [2], these peptides can be derived from various sources, including animals, plants, and other origins [3]. AMPs play a crucial role in the innate immune system as they are the primary defense against pathogenic infections [4]. Distinguishing themselves from traditional antibiotics, AMPs possess the ability to target multiple pathogens, thereby minimizing the formation of drug-resistant strains while exhibiting broad-spectrum antibacterial properties, so they can be widely used in food, medicine, and agriculture, as shown in Figure 1 [5]. Consequently, they hold significant potential as alternative therapeutic agents for combating bacterial infections in the future [6,7,8]. Numerous clinical cases have already demonstrated the efficacy of AMPs in treating pathogenic bacterial transmission, wound healing, and tumors [9]. However, natural AMPs are limited by origin, cost implications, and biosafety concerns; these limitations hinder their development for therapeutic purposes [10,11]. To overcome these constraints and enhance stability, activity levels, and targeting specificity while reducing cytotoxicity toward cells, it is essential to gain further insights into the structural characteristics and mechanisms underlying the action of AMPs. This review provides an extensive analysis of the structure and mechanism of AMPs, focusing mainly on molecular modification strategies aimed at enhancing antibacterial activity. These efforts aim to offer new prospects for future clinical development and application.

2. Structure and Characteristics of AMPs

AMPs are distinguished by their conserved physicochemical properties, despite the variation observed in their primary peptide sequences. Contrary to expectations, certain specific amino acids at crucial positions show high conservation across diverse AMPs, with lysine and arginine particularly abundant in most AMPs. The efficacy of these peptides does not depend on any single attribute alone; rather, it is a result of the precise and intricate integration of numerous characteristics.

2.1. Sequence and Size

Most AMPs contain fewer than 100 amino acids, and the composition and distribution of amino acids within these peptides vary across different species [12]. The length of AMPs significantly influences their antibacterial activity and specificity against microbial targets. Shorter membrane-active AMPs (e.g., Aurein 1.2 or Citropin 1.1) are expected to disrupt membranes through micellization, while longer membrane-active AMPs (e.g., Caerin) may adopt extended conformations to stabilize transmembrane pores. The antifungal activity of a series of synthetic peptides (KWn-NH2), consisting of varying lysine and tryptophan repeats, is dependent on peptide length. Peptide activity increases with increasing peptide length, with only the most extended KW5 peptide exhibiting cytotoxicity against human keratinocyte cell lines.

2.2. Charge

Most AMPs possess a net positive charge ranging from +1 to +9 and can form well-defined cationic domains [13]. Electrostatic forces primarily drive their initial interaction with the cell membrane. There is a strong correlation between the cationic charge and biological activity, where an increase in charge leads to enhanced activity and a higher concentration of bound peptides at the membrane interface [14]. However, there generally exists an optimal charge threshold; surpassing this threshold typically results in decreased antimicrobial peptide activity. For example, analogs of Magainin-2 demonstrate a dependence on the charge, with activity increasing as charges rise while hydrophobicity and structure remain unchanged. Moreover, exceeding the threshold point through additional cationic residues significantly reduces peptide selectivity toward microbial targets while promoting hemolysis [15]. Removing the cationic C-terminal tail from melittin analogs substantially reduces their hemolytic activity and binding affinity for cell membranes [16].

2.3. Hydrophobicity

Hydrophobicity plays a critical role in the interaction with cell membranes, as it regulates the peptide’s ability to partition into the membrane layer. AMPs typically contain approximately 50% hydrophobic residues [17]. Like charge, optimizing the percentage of hydrophobicity enhances activity against cell membranes. However, exceeding this optimal point leads to a decline in antimicrobial efficacy and an increase in cytotoxicity toward mammalian cells [18]. For instance, studies utilizing Magainin-2 analogs have demonstrated that even slight increases in hydrophobicity significantly enhance the ability to cleave zwitterion/anionic mixtures compared to purely anionic PG liposomes, highlighting the substantial impact of hydrophobicity on membrane interactions [19].

2.4. Amphipathic

The amphipathic nature of AMPs is primarily determined by two specific amino acid residues within the protein [19]. One type consists of hydrophilic residues, such as glycine, histidine, arginine, and aspartic acid. These hydrophilic residues are the main constituents of AMPs and contribute to their stability in the environment [19]. Additionally, they can bind with organic substances on cell membrane proteins to enhance adhesion to bacterial cells [20]. Another type is hydrophobic residues, such as alanine, leucine, and isoleucine. These residues are mainly located at the C-terminus of antimicrobial peptide proteins. Due to their hydrophobic properties, they interact with the hydrophobic region of bacterial cell membranes to exert their effects [21]. AMPs containing a combination of hydrophilic and hydrophobic amino acid residues play a crucial role in penetrating and sterilizing bacterial membranes. For instance, Magainin-2 possesses both residues: a hydrophilic amino acid at its N-terminus and a hydrophobic one at its C-terminus. This structural arrangement enables it to penetrate bacterial membranes through lipophilic sites effectively, disrupting them for antibacterial and bactericidal effects [22].

2.5. Secondary Structure

One of the most important features of AMPs is the diversity of their secondary structures, which are essential for their ability to interact with and disrupt microbial membranes, including α-helix, β-sheet structures, and αβ and non-αβ conformations [23]. They are not fixed and unchanging but can undergo dynamic changes when in contact with the cell membrane of microorganisms such as bacteria. This change enables AMPs to effectively interact with hydrophobic regions of the cell membrane, thereby disrupting membrane integrity [23]. The secondary structure of AMPs usually also has amphiphilicity, which means they have both hydrophilic and hydrophobic parts. This property allows them to form channels or pores on the cell membrane, leading to microbial death. In addition, the diversity and plasticity of this structure are also key factors for AMPs to resist various types of microorganisms. Table 1 shows the structures of AMPs from different sources will their mechanism of action.

α-helical AMPs have been subjected to intense investigation, owing to their prevalence, extensive occurrence across different species, and potent activity against a wide array of microbial threats, as shown in Figure 2a [24]. Nature is replete with α-helical AMPs, such as Cecropin, Pleurocidin, melittin, Magainin, and Moricin, which are sourced from a diverse range of organisms including aquatic vegetation, insects, amphibians, teleosts, and mammals [25,26]. Studies have shown that the α-helical configuration of these peptides is predominantly modulated by their engagement with the target membrane [27]. This interaction induces conformational recombination leading to the separation of hydrophilic and hydrophobic amino acid residues within the peptide, which is crucial for developing amphiphilic structures that are favorable for membrane rupture [28]. As alpha helices gradually accumulate within the lipid bilayer, their hydrophobic nuclei bind to the lipid matrix, while their hydrophilic surfaces align internally, ultimately forming membrane pores [29].

β-sheet AMPs are constructed from at least two β-strands, with numerous linear constructs adopting a β-hairpin-like conformation, as shown in Figure 2b [30]. Members of this group commonly feature well-preserved cysteine residues and disulfide bridges that are instrumental in defining their structure and function [31]. These disulfide bonds contribute to the enhanced stability of AMPs, decreasing their vulnerability to proteolytic degradation [32]. The antimicrobial efficacy of these peptides is typically ascribed to the cationic residues and hydrophobic side chains presented on the antiparallel β-sheets. Notable examples include protegrin-1 (PG-1) [33], thanatin [34], tachyplesin [35], polyphemusinI [36], and gomesin [37]. Defensins represent the predominant class within the β-sheet AMP family, further subdivided into subgroups according to the placement of the disulfide bond that forms.

The secondary structure of αβ AMPs is characterized by the presence of both α-helices and β-sheets, endowing them with a pronounced affinity for cellular membranes [23]. Plant and insect defensins, in particular, are distinguished by their antifungal capabilities, which arise from interactions with fungal membrane sphingolipids or microsomal membranes, as shown in Figure 2c [38]. For instance, the antifungal plant-derived peptide pea defensin 1 (Psd1) disrupts cyclin F in Neurospora crassa, interfering with the fungal cell cycle [39]. RsAFP2, a defensin isolated from radish, engages with glucosylceramide in yeast and fungi, initiating signaling pathways linked to reactive oxygen species in Candida albicans, which ultimately leads to cell death [40]. Moreover, the plant defensin Nad1 binds to membrane phosphatidylinositol 4,5-bisphosphate prior to interacting with intracellular targets, leading to the accumulation of reactive oxygen species [41]. These interactions highlight the intricate mechanisms by which αβ AMPs exert their antifungal effects, underscoring the role of their secondary structures in targeting and disrupting microbial membranes.

AMPs that deviate from the conventional αβ-structure classification are known as non-αβAMPs, encompassing extended or cyclic peptides. These unique molecules do not possess the typical α-helix or β-sheet conformations and are divisible into three groups: those enriched in tryptophan, proline, or glycine, as shown in Figure 2d [42]. Tryptophan-enriched peptides frequently adopt an amphipathic shape, exemplified by indolicidin, which boasts a tryptophan-centric core essential for its binding affinity [43]. Studies involving the interaction of indocyanine with dodecyl phosphocholine micelles have elucidated the engagement of aromatic rings from Trp6 and Trp9 with Pro7 and Pro10, respectively [44]. Similarly, the B2 domain of lactoferrin undergoes a transition to a non-αβ structure with an irregular backbone when engaging with SDS micelles [45]. Proline-rich Insect KingdomAMPs, consisting of 15 to 39 amino acid residues, are notable for their intracellular targeting capabilities [46]. Emerging evidence suggests that these peptides assume non-αβ conformations, thereby obstructing the ribosomal tunneling process by inhibiting the entry of aminoacyl tRNA into the Amide site [47]. On the other hand, glycine-rich peptides are prevalent in the insect kingdom and typically exhibit molecular weights between 8 and 30 kDa. A notable example is hymenoptaecin, a glycine-rich antimicrobial peptide derived from Hymenoptera with a typical αβ antimicrobial peptide structure that significantly inhibits both Gram-negative and Gram-positive bacteria [48].

Table 1. Examples of structures of AMPs and their mechanisms of action.

AMP	Origin	Length	Secondary Structure	Mechanism	Ref.
Magainins	Amphibian	27	α-helical AMPs	Forms toroidal pores in lipid bilayers	[49]
LL-37	Human	37	α-helical AMPs	Pore-forming carpet model	[50]
β-defensin2	Human	36	Three antiparallel β-strands and an α-helical domain	Biofilm inhibition	[51]
Protegrins	Porcine	18	β-sheet Amps	Forms octameric transmembrane pores	[52]
Indolicidin	Bovine	13	Linear peptides	Lyses membranes; binds DNA	[53]
Drosocin	Drosophila	19	Linear peptides	Inhibits protein translation via binding to 50S or 70S ribosomal subunit	[54]
Rattusinb	Rodent	62	β-sheet AMPs	Inhibits RNA polymerase; membranolytic	[55]
Nisin	Lactococcus lactis	34	α-β AMPs	Inhibits cell wall biosynthesis via binding to lipid II	[56]
Phormicin	Insect	40	αβ insect defensin	Inhibits gene expression in vivo	[57]
NaD1	Plant	105	αβ plant defensin	Binds to phosphatidic acid	[41]
TO17	Sciaenops ocellatus	17	α-helical AMPs	Induces DNA and RNA degradation	[58]
Buforin2	Sphaenorhynchus lacteus	21	α-helical AMPs	Inhibits the synthesis of DNA, RNA, and proteins	[59]
MBP-1	Marze kernels	33	α-helical AMPs	Forms small pores in the lipid bilayer, induces membrane permeability, and disrupts potassium balance	[60]
Microcin J25	Escherichia coli	58	Lasso peptide	Binds RNA polymerase and inhibits its activity	[61]
Bac7	Bovine	35	α-helical AMPs	Inhibits protein synthesis	[62]
Apidaecin	Pimpla disparis	20	Linear peptides	Participates in the termination process of translation	[63]
Histone H2A	Mammal	39	α-helical AMPs	Disrupts bacterial proton gradient and chromosome tissue	[64]
Plectasin	Pseudoplectania nigrella	95	α-β AMPs	Inhibits the biosynthesis of peptidoglycan, thereby suppressing cell wall synthesis	[65]
HNP1	Human	94	β-sheet Amps	Interacts with the essential precursor of cell wall synthesis lipid II to inhibit bacterial cell wall synthesis	[66]
Gramicidin S	Bacillus pumilus	10	Cyclic AMPs	Disrupts the integrity of the inner membrane lipid bilayer	[67]

Table 1. Examples of structures of AMPs and their mechanisms of action.

AMP	Origin	Length	Secondary Structure	Mechanism	Ref.
Magainins	Amphibian	27	α-helical AMPs	Forms toroidal pores in lipid bilayers	[49]
LL-37	Human	37	α-helical AMPs	Pore-forming carpet model	[50]
β-defensin2	Human	36	Three antiparallel β-strands and an α-helical domain	Biofilm inhibition	[51]
Protegrins	Porcine	18	β-sheet Amps	Forms octameric transmembrane pores	[52]
Indolicidin	Bovine	13	Linear peptides	Lyses membranes; binds DNA	[53]
Drosocin	Drosophila	19	Linear peptides	Inhibits protein translation via binding to 50S or 70S ribosomal subunit	[54]
Rattusinb	Rodent	62	β-sheet AMPs	Inhibits RNA polymerase; membranolytic	[55]
Nisin	Lactococcus lactis	34	α-β AMPs	Inhibits cell wall biosynthesis via binding to lipid II	[56]
Phormicin	Insect	40	αβ insect defensin	Inhibits gene expression in vivo	[57]
NaD1	Plant	105	αβ plant defensin	Binds to phosphatidic acid	[41]
TO17	Sciaenops ocellatus	17	α-helical AMPs	Induces DNA and RNA degradation	[58]
Buforin2	Sphaenorhynchus lacteus	21	α-helical AMPs	Inhibits the synthesis of DNA, RNA, and proteins	[59]
MBP-1	Marze kernels	33	α-helical AMPs	Forms small pores in the lipid bilayer, induces membrane permeability, and disrupts potassium balance	[60]
Microcin J25	Escherichia coli	58	Lasso peptide	Binds RNA polymerase and inhibits its activity	[61]
Bac7	Bovine	35	α-helical AMPs	Inhibits protein synthesis	[62]
Apidaecin	Pimpla disparis	20	Linear peptides	Participates in the termination process of translation	[63]
Histone H2A	Mammal	39	α-helical AMPs	Disrupts bacterial proton gradient and chromosome tissue	[64]
Plectasin	Pseudoplectania nigrella	95	α-β AMPs	Inhibits the biosynthesis of peptidoglycan, thereby suppressing cell wall synthesis	[65]
HNP1	Human	94	β-sheet Amps	Interacts with the essential precursor of cell wall synthesis lipid II to inhibit bacterial cell wall synthesis	[66]
Gramicidin S	Bacillus pumilus	10	Cyclic AMPs	Disrupts the integrity of the inner membrane lipid bilayer	[67]

3. Mechanism of Action of AMPs

The exploration of the mode of action of AMPs has always been rigorous, using various membrane models to reveal their effects. However, few studies have utilized membrane potential sensitive dyes and fluorescently labeled peptides to investigate the direct effects of these peptides on intact microbial cells. The results of these studies elucidate the main interaction modes between AMPs and cell membranes, which are typically classified into two categories: those involving membrane disruption and those leading to non-destructive membrane permeability [68]. The contemporary conceptual model of the mechanism of action of AMPs now includes not only the classical view of gradually forming pores but also considerations for structural reorganization, such as changes in molecular conformation or models based on interfacial activity [69]. Given that several proposed mechanisms may be applicable to different AMPs, it is crucial to understand the continuous mechanism steps leading to membrane rupture.

Research indicates that traditional antibiotics primarily target metabolic enzymes, which can selectively induce bacteria to develop resistance mechanisms. In contrast, AMPs employ distinct mechanisms of action and mainly rely on gradually forming pores on the cell membrane to eradicate microorganisms [70]. This makes it challenging for microorganisms to develop resistance against them. AMPs selectively inhibit bacterial growth and effectively kill bacteria [71,72]. Bacterial cell membranes are rich in negatively charged phospholipid head groups such as phospholipids glycerol, cardiolipin, or serine phospholipids. On the other hand, mammalian cell membranes consist primarily of zwitterionic phospholipids like cephalin, phospholecithin, and sphingomyelin. Additionally, cholesterol—an essential component in mammalian cell membranes—can bind to AMPs and diminish their activity [25,73]. Therefore, differences in cell membrane composition serve as the primary factor contributing to the selective eradication of bacteria by AMPs [74,75]. Although ion channels, transmembrane pores, and extensive membrane disruption ultimately lead to microbial cell lysis, evidence suggests that AMPs also possess additional intracellular target sites.

3.1. Cell Membrane Action Mechanism

The structure of antimicrobial peptide (AMP) molecules in solution is usually disordered. The initial binding between AMP and bacterial membrane is driven by the electrostatic affinity between the cationic component of AMP and the negatively charged lipid head group [68]. In particular, lysine and arginine residues show a clear tendency to bind to the phosphate portion within the lipid bilayer [76]. Bacterial cells typically maintain a significant electrochemical gradient, coupled with strong electrostatic interactions extending over longer distances, which facilitate the aggregation and membrane-bound accumulation of AMP, thereby improving its targeting efficiency [77]. Once close to the target membrane, most AMPs transition from a coil conformation to a helical structure [78]. Conversely, β-sheet AMPs tend to exhibit more excellent structural stability when solubilized. They maintain their structure through disulfide bonds or the cyclization of the peptide backbone [79]. For instance, the cyclic β-sheet AMP tachyplesin undergoes minimal structural changes when transitioning from an aqueous environment to a membrane analog [79]. Due to their stable structure in solution, these types of AMPs may retain their conformation during interactions with the membrane surface. These structures often display amphiphilic properties which have been found crucial for cleavage activity [80]. Moreover, throughout the binding and destruction processes, activity relies more on maintaining a balance between electrostatic interactions at early stages and hydrophobic interactions at subsequent stages.

After binding to the cell membrane surface, the antimicrobial peptide proceeds to the second stage known as the idle concentration stage. This stage is initiated by accumulating AMPs on the cell membrane surface [81]. Several factors can impede antimicrobial peptide concentration, including their concentration, their self-aggregation tendency, the composition and fluidity of phospholipid membranes, and their size. Another crucial factor that may hinder concentration is the bacterial cell membrane’s membrane potential (ΔΨ) [82]. Presumably, ΔΨ facilitates cationic peptides’ migration toward nonpolar membrane regions and effectively reduces energy barriers for pore formation. The activity of certain AMPs such as nisin is significantly influenced by ΔΨ; when N-terminal lysine is replaced with leucine in nisin, its voltage dependence diminishes [83].

Upon binding to cell membranes, AMPs undergo conformational changes. Studies have demonstrated that α-helical AMPs exhibit a random conformation in an aqueous solution environment but rapidly adopt a well-defined amphipathic α-helical conformation upon interaction with phospholipid bilayers [84]. However, some AMPs only undergo conformational transitions when bound to negatively charged bilayers. For instance, ergot PGLa displays an irregular conformation when bound to a lecithin- and sphingomyelin-composed membrane but gradually forms an α-helical conformation when interacting with a phosphatidic acid glycerol- and cephalin-composed membrane [85]. Some studies suggest that after binding to cell membranes, AMPs may progressively form complex structures; however, their potential for forming quaternary structures fundamentally depends on the composition and conformation of their monomers [86].

3.2. Membrane Permeabilization Mechanism

Many models of membrane permeabilization have been proposed. Considering the variations in microorganism ultrastructures, the same antimicrobial peptide can exert distinct antibacterial mechanisms on different microorganisms. Moreover, depending on factors such as growth status, tissue location, and the presence of other immune mechanisms, AMPs can employ diverse mechanisms to eliminate the same species.

The “barrel stave” model involves binding a limited number of cationic AMPs to the cell membrane surface, where they aggregate due to electrostatic forces and gradually form a pore or groove on its side. This pore acts as an ion channel and allows an inward rectifying current under the influence of an external electric field [68]. Once formed, the ion channel permits water from outside to enter the cell while also allowing cytoplasmic penetration from the exterior [87]. In severe cases, energy loss leads to cell membrane disintegration, resulting in cell death [88]. On the other hand, in the barrel stave model, transmembrane peptides tightly assemble with each other and create a rigid circular pore structure, as shown in Figure 3a [89]. Human picotin does not directly interact with membranes but instead forms a hexameric structure in solution that follows the barrel stave model.

The “Toroidal pore” model proposes that the extracellular antimicrobial peptide gradually adopts an α-helical conformation upon interaction with the charged and hydrophobic bacterial cell membrane. The displacement of polar heads of hydrophobic residues in the peptide bound to the cell membrane leads to the formation of a hydrophobic structure. This induces cracks in the domain, causing the bending and stretching of the cell membrane in a positive direction. A key distinction from the “barrel wall” model is that lipids and AMPs collaboratively form transmembrane channels, as shown in Figure 3c [90]. These cracks and stretches further compromise membrane integrity. When the ratio of AMPs to lipids reaches a critical value, the vertical orientation of AMPs within the cell membrane begins, accompanied by the self-assembly of each ring, ultimately forming a dynamic supramolecular peptide–lipid complex [91]. The formation and displacement of cyclic pores depend on this ratio. However, when there is an excessive concentration of AMPs, pore stability decreases due to electrostatic repulsion caused by positively charged side chains [92].

The “Carpet” model proposes that cationic AMPs arrange themselves in a parallel fashion on the cell membrane surface, gradually forming a structure resembling felt. Once reaching a critical threshold, the energy of the cell membrane deteriorates, leading to the loss of integrity and sharp bending followed by rupture, as shown in Figure 3b [84]. In this model, the hydrophobic portion of cationic AMPs does not insert into or progressively form grooves on the cell membrane. For instance, Cecropin Pl initially aligns parallel to the membrane surface. As time progresses and antibacterial agent concentration increases, a peptide layer with gradually increasing monomer concentration forms on the membrane surface until it eventually causes a rupture of the cell membrane [93].

The “Aggregate” model suggests that after insertion into the cell membrane, AMPs and lipids form micelle complexes that traverse through aggregates and dynamically create pores within the cell membrane. This mechanism also allows for the entry of AMPs into cells, as shown in Figure 3d [94]. Unlike in the wormhole model, there is no specific orientation for AMPs in this particular model [95].

3.3. Intracellular Mechanism of Action

Changes in membrane permeability alone cannot fully account for the potent bactericidal effects of AMPs. Inducing cell membrane permeabilization only inhibits bacterial growth, while the complete destruction of functional tissue cells significantly impacts cell death [88]. Many AMPs accumulate within tissue cells by traversing cell membranes and disrupt the normal functions of signaling cells, ultimately leading to the demise of bacteria and fungi. However, studies suggest that AMPs possess universal targets for intracellular action [96]. AMPs can bind to nucleic acids and proteins; impede replication, transcription, and translation processes; impair cellular organelles; or affect enzyme systems to disturb cell cycle progression and energy metabolism, as shown in Figure 4.

The binding mechanisms of AMPs to nucleic acids and proteins are diverse, depending on the specific AMPs and target molecules. Certain AMPs can physically interact with DNA or RNA through electrostatic interactions, hydroxyl hydrophobic interactions, etc., thereby inhibiting their replication and transcription [97]. Moreover, AMPs can also bind to protein targets such as cell membrane receptors and microbial membrane proteins, leading to structural damage and the functional impairment of the targets, ultimately compromising bacterial survival. Additionally, some AMPs can interact with nucleic acids and proteins based on their structural characteristics, including terminal charge, hydrophilicity, and hydrophobicity, to establish binding [98]. For instance, LL-37 is an antimicrobial peptide in humans with an arginine-rich region consisting of 11 amino acids at its N-terminus. This region can bind to bacterial DNA/RNA through electrostatic interaction resulting in the loss of normal replication and transcription capabilities [99]. BPI (bactericidal permeability-increasing protein), an antibacterial peptide present in bovine serum, binds to lipose sugar A on the bacterial membrane, allowing it to enter the cell, where it accelerates bacteria death with the assistance of intracellular amylase and nuclease enzymes, among others [83].

AMPs can damage bacterial mitochondria through various mechanisms. They can directly bind to ribosomes and inhibit their function: certain AMPs, upon binding to bacterial ribosomes, can hinder mRNA translation or induce premature termination, thereby impeding protein synthesis. As the concentration of AMPs increases, their affinity for bacterial ribosomes intensifies, ultimately leading to the complete disruption of ribosomal activity [100]. Additionally, AMPs can provoke an antibiotic exception response in ribosomes: specific AMPs are recognized by bacterial ribosomes, triggering an adaptive mechanism that renders them less susceptible to antibiotics [101]. For example, the antimicrobial peptide BMAP-28, an antimicrobial peptide isolated from bovine sources, is able to induce changes in mitochondrial membrane potential and release cytochrome C, thereby promoting mitochondrial outer membrane permeabilization [102].

AMPs can exert various effects on bacterial enzyme systems. Certain AMPs have the ability to directly bind to bacterial RNA polymerase, thereby inhibiting its activity and resulting in the impairment of gene transcription and protein synthesis [103]. Alternatively, AMPs can interact with extracellular adenylase in bacteria, disrupting its tertiary structure. This subsequently hampers the utilization of extracellular adenylate as an energy source, consequently affecting energy metabolism [104]. Microcin J25 is a ribosome-synthesized and post-translationally modified antimicrobial peptide that binds to the secondary channel of RNA polymerase. By obstructing the folding process of the trigger loop, it effectively prevents substrates from entering through this channel and inhibits RNA polymerase activity [105,106]. LL-37 exerts antibacterial effects on Escherichia coli by suppressing the palmitoyltransferase PagP activity responsible for repairing membrane permeability [107].

Several AMPs, including pyrrolomycin, streptomycin, and apigenin, inhibit the function of DnaK by targeting it to interfere with protein folding assisted by chaperone proteins [108]. When θ-defensins interact with bacterial membranes, they promote the secretion of cell-wall-degrading enzymes, breaking down sugar chains and peptide bonds in cell wall proteins, ultimately leading to the lysis of Staphylococcus cells [109]. Moreover, Mel4 initiates the cell death of Staphylococcus aureus by activating the release of bacterial autolysin [110]. Another type of AMP, PFR, induces necrotic cell death by inducing endoplasmic reticulum stress, increasing cytoplasmic calcium levels, and reactive oxygen species (ROS) in mitochondria [100].

3.4. Mechanism of Directed Co-Aggregationn

Directed co-aggregation is a novel antibacterial mechanism that involves the interaction between amyloidogenic AMPs (AAMPs) and bacterial target proteins, forming aggregates that cause the target proteins to lose their function. This process is also known as “protein silencing” [111]. In the directed confocal mechanism, AAMP is designed to recognize and bind to specific regions of bacterial proteins, which are typically critical areas for the formation of amyloid fibers. Through this binding, AAMP can prevent or interfere with the normal function of bacterial proteins, thereby exerting antibacterial effects. The key to this mechanism lies in the specific interaction between AAMP and bacterial proteins, which can lead to protein aggregation and prevent its participation in normal biological processes. The implementation of the directed confocal mechanism first involves selecting the target protein sequence, which typically involves analyzing the bacterial proteome to identify potential amyloid forming regions (APRs). Subsequently, AAMP is designed to bind to these APRs. This may involve the synthesis and optimization of AAMP sequences to ensure that they can effectively interact with the APRs of the target protein. Finally, the binding of AAMP to bacterial proteins leads to protein aggregation, which may be achieved through the formation of amyloid fibers or other forms of protein–protein interactions. The innovation of this mechanism lies in its provision of a different mode of action from traditional antibiotics, offering new possibilities for combating antibiotic resistance. Through the directed confocal mechanism, AAMP can specifically target bacterial proteins, reducing adverse effects on host cells and minimizing the development of drug resistance.

In summary, the mechanism of action of AMPs varies depending on their target cells’ specificity, the antimicrobial peptide concentration, and the microbial cell membranes’ biophysical properties. Moreover, when targeting the same microorganism, AMPs can employ various mechanisms to eliminate them; these include disrupting cell membrane permeability in combination with inhibiting one or multiple crucial substances’ normal functions within cells.

4. Molecular Modification Strategies for AMPs

Molecular modification technology can enhance the stability, activity, and targeting of antimicrobial peptide (AMP) engineering. Some techniques for achieving this include isomerization, biomimetic end modifications, and multimerization, as shown in Figure 5 [112,113,114]. Several factors, such as peptide length, net charge, hydrophobicity, and secondary structure influence the antibacterial activity of AMP. Alterations in peptide length can impact the antibacterial effectiveness of AMP since the peptide needs to span the lipid bilayer to stabilize pores [115]. However, changes in peptide length also result in variations in net positive charge and hydrophobicity. Increasing the positive charge of AMP can improve its binding affinity toward anionic bacterial membranes [116]. Nevertheless, highly charged peptides experience a significant reduction in biological activity under high-ionic-strength conditions. Peptide chains with hydrophobic groups have the ability to form polymers when dissolved, which allows them to insert themselves into the hydrophobic membrane core, thereby enhancing AMP stability. Additionally, hydrophobic residues improve the propensity for AMPs to adopt α-helical structures. Although amphipathicity serves as an essential structural basis for AMPs, some studies have indicated that high amphipathicity reduces their antibacterial activity while increasing hemolytic activity [117,118]. Due to the complexity associated with these factors mentioned above, no standardized solution is currently available for simultaneously optimizing various aspects of AMP engineering. However, it is possible to enhance the antibacterial activity of AMP by focusing on individual factors using different molecular engineering techniques.

4.1. Change the Number and Distribution of Net Positive Charges

The presence of basic amino acid residues, such as Arg, Lys, and His, in cationic AMPs primarily influences the electrostatic adsorption force with negatively charged phospholipids in the cell membrane [119]. Substituting Arg and Lys for amino acid residues in the α-helical region of the Tenecin1 structure can increase the overall charge and significantly enhance antibacterial activity [120]. Replacing Gln at position 39 of Rr-AFP2 with Lys can improve antibacterial activity; however, substituting Lys at position 44 with Gln decreases antibacterial activity [121]. Jiang et al. converted LV13K into a series of AMPs with net charges ranging from −5 to +10. They observed that the antibacterial activity increased over time for a range of net charges from −5 to +8 [122]. Studies have shown that AMPs containing His residues exhibit pH-dependent bactericidal activity. Under acidic conditions, protonated His residue mimics metal ion-like effects, significantly improving antibacterial activity [123]. Zhang et al. modified the net charge and distribution of positively charged residues in AR-23 antimicrobial peptide by replacing them and found that increasing the net charge affected antibacterial activity but was not correlated with the number of positively charged residues [124].

4.2. Isomerization

The conversion from L to D amino acid isomers is a strategy widely utilized to bolster the resistance of peptides against degradation by both host and microbial proteases. Enhancing the stability of peptides in serum involves several strategies, including the isomerization of AMPs, integration of non-natural amino acids, and the use of D-amino acids [125,126,127]. For example, replacing L-amino acids with D-amino acids in chicken cathelicidin-2 leads to an increase in serum stability and a decrease in cytotoxicity, while still retaining its antibacterial and LPS-neutralizing capabilities [127]. The replacement of select D-amino acid residues in the AMP W3R6 enhances its protease resistance without affecting its antibacterial efficacy. In a similar manner, a mammalian HBcARD peptide, when modified with D-amino acids, demonstrated greater stability, enhanced antibacterial activity, and reduced hemolytic effects [128]. Similarly, mammalian HBcARD peptide substituted with D-amino acids exhibited improved stability, stronger antibacterial activity, and minimal hemolytic effects [129]. Furthermore, the incorporation of disubstituted β-amino acids into the peptide sequence has been shown to not only enhance stability but also to increase lipophilicity and improve the peptide’s ability to penetrate cellular barriers [130], offering a promising route for the development of next-generation antimicrobial agents.

4.3. Change Hydrophobicity and Average Hydrophobic Moment Size

The strength of hydrophobicity is an important structural basis for determining the entry of AMPs into the phospholipid layer of the cell membrane and membrane permeability. The penetrating power is weak, but it is easy to cause the self-aggregation of AMPs, further improving the cytotoxic and hemolytic activities on the original basis [19]. Hydrophobicity exhibits a positive correlation with antibacterial activity within a certain range. Introducing hydrophobic groups can broaden the antibacterial spectrum and enhance antibacterial efficacy; however, excessive hydrophobicity may compromise cell selectivity by increasing mammalian cytotoxicity and should not exceed 50% [21]. Chen et al. demonstrated that antimicrobial peptide V13KL possesses optimal antibacterial activity with low hemolytic effects on human red blood cells. Altering its sequence to increase or decrease hydrophobicity significantly reduces its antibacterial potency, possibly due to enhanced hydrophobicity facilitating easier penetration into the hydrophobic core of red blood cell membranes [131]. The average hydrophobic moment plays a more significant role in antibacterial activity compared to helicity and hydrophobicity. The average hydrophobic moment plays a greater role in antibacterial activity than helicity and hydrophobicity. The reasonable adjustment of the average hydrophobic moment will help to further improve antibacterial activity [20]. Lee et al. substituted Gln at position 16 and Asp at position 18 in antimicrobial peptide HP with Trp, resulting in improved hydrophobicity and positively correlated enhancement in antibacterial activity [132]. Decreasing the average hydrophilic moment reduces antibacterial potency and hemolytic ability [133].

4.4. Cyclization

The cyclization of peptides has become an efficient strategy to enhance the stability and biological activity of AMPs [134]. These cyclic peptides exhibit significant affinity for bacterial membranes, attributed to the formation of β-folding structures at the membrane interface [135]. For example, Dathe and his colleagues developed a series of concise cyclic hexapeptides that showed enhanced antibacterial activity against Bacillus subtilis and Escherichia coli, surpassing their linear versions of activity [136]. To further demonstrate the benefits of cyclization, a study showed that cyclic AMPs analogs equipped with flexible linkers exhibited superior activity against Staphylococcus aureus and Pseudomonas aeruginosa compared to linear precursors [137]. In addition, the formation of intramolecular disulfide bonds between cysteine residues represents an alternative cyclization method to enhance protein hydrolysis resistance, thereby expanding the therapeutic potential of these peptides.

4.5. Change the Position of Active Amino Acid Residues and the Length of the Peptide Chain

The majority of AMPs start with a Gly amino acid residue. AMPs that are rich in Arg and Val exhibit strong antibacterial activity due to their robust electrostatic adsorption onto the lipid layer of cell membranes. Among them, an abundance of Arg facilitates membrane perforation, while an abundance of Val promotes the formation of β-sheet spatial conformation and enhances antibacterial activity. Additionally, Pro and Gly abundances play a crucial role in stabilizing the structure and antibacterial activity [46,138]. Li et al. systematically removed each C-terminal and N-terminal amino acid from antimicrobial peptide P1 individually and discovered that Pro is the primary hindrance to antibacterial activity [139]. On the other hand, Mishra et al. investigated Try-rich AMPs and observed that positional changes can impact antibacterial activity; however, quantitative changes have minimal effect on such activity [140]. Furthermore, substituting Lys for His residue within the colistin sequence diminishes its antibacterial efficacy [141].

4.6. Construction of Hybrid AMPs

Protein fusion refers to the modification method of using genetic engineering technology to fuse protein or peptide molecules with some functional proteins to produce new molecules, which is an effective means of prolonging the half-life of peptide drugs. By integrating specific residues from two to three peptides with distinct modes of action into a singular sequence, it is possible to engineer novel hybrid AMPs [142,143]. Li et al. utilized this method to generate broad-spectrum antibacterial fusion peptides (BPI21/LL-37) by fusing BPI21 and LL-37, which were then used to modify human umbilical cord mesenchymal stem cells (hUC MSCs). Research has shown that the BPI21/LL-37 fusion peptide not only has an extended half-life, but also exhibits broader antibacterial activity. In addition, this modification enhances the antibacterial and endotoxin neutralizing abilities of hUC MSCs without compromising their fundamental functions in stem cell-based tissue repair, regeneration, and anti-inflammatory responses, which is crucial for the prevention and treatment of sepsis [144].

Furthering the innovation in this field, Alzoubi’s research team designed H4, a hybrid peptide that merges alpha-helical segments from BMAP-27 and OP-145, showcasing broad-spectrum antibacterial activity and reduced toxicity [143]. Additionally, a “triple hybrid” peptide composition involving sericin-A, melittin, and LL-37 has demonstrated a significant boost in antibacterial efficacy against both Gram-positive and Gram-negative bacteria, while concurrently diminishing hemolytic activity [145]. The development of antibiotic peptide conjugates (APCs) represents another frontier in antimicrobial therapy, wherein antibiotics are linked to peptides via specific connectors. These APCs leverage the synergistic antibacterial effects of both components, while simultaneously overcoming limitations associated with standalone antibiotics or peptides. Such limitations include challenges with cell permeability, serum stability, cytotoxicity, hemolysis, and resilience under high salt concentrations [146]. This approach holds the promise of providing a novel strategy for addressing the complexities of bacterial infections and the urgent need for new antimicrobial agents.

5. Summary and Prospect

AMPs are a class of natural antimicrobial agents with broad-spectrum bioactivity and potent bactericidal effects. They play a significant role in the endogenous antimicrobial systems of organisms and are gradually emerging as potential alternatives to traditional antibiotics in the medical field. Although their clinical application is currently limited by challenges in biosynthesis and uncertainties regarding human safety, the rapid advancement of genetic and biotechnological techniques is opening up vast prospects for the application of AMPs.

Firstly, the progress of modern biotechnology has made the large-scale production of AMPs possible. The development of gene sequencing technologies allows us to isolate and screen antimicrobial peptide genes from a variety of organisms, which can then be introduced into expression hosts such as bacteria, yeast, and plants through genetic engineering techniques, enabling the mass production of AMPs. This method has already been successfully applied in the production of numerous AMPs. In this process, fermentation technology plays a crucial role. Due to the high production costs of AMPs, traditional chemical synthesis methods struggle to meet the demands of large-scale production, making microbial fermentation an ideal solution. In recent years, significant progress has been made in the application of fermentation technology in the production of AMPs. Researchers have selected microbial strains with high antimicrobial peptide production capabilities and optimized fermentation conditions, such as temperature, pH, and dissolved oxygen, to enhance the yield of AMPs. Additionally, genetic engineering techniques for genetically modifying microbial strains to improve their antimicrobial peptide synthesis capacity are also an important means of increasing fermentation efficiency. During the fermentation process, both solid-state and liquid fermentation methods are widely used, with solid-state fermentation typically employing natural materials such as grains and soybean meal as carriers, which not only yield higher antimicrobial peptide production but also lower production costs. Beyond optimizing fermentation techniques, the extraction and purification of AMPs are also critical steps. Researchers have developed a series of efficient extraction and purification methods, such as centrifugation, filtration, gel filtration chromatography, and high-performance liquid chromatography, to ensure the purity and activity of AMPs. Fermentation technology has significantly improved the production efficiency of AMPs. For example, nisin, an antimicrobial peptide produced by lactic acid bacteria fermentation, has been widely used in food preservation [147]. Furthermore, AMPs produced through fermentation technology, such as plant-derived AMPs, animal-derived antimicrobial polypeptides, and fungal-derived AMPs, show broad application prospects in the fields of pharmaceuticals, cosmetics, and feed additives.

Secondly, the specific mechanisms of AMPs can be harnessed to develop safer antimicrobial drugs. Compared with traditional antibiotics, AMPs have different killing mechanisms, making them less prone to resistance, more adaptable, and less toxic to host cells. Therefore, they are considered a safer and more reliable class of antimicrobial agents.

Lastly, modifying the structure of AMPs can yield new functionalities. Many AMPs have a wide range of applications in the field of life sciences. For instance, tachyplesin, an antimicrobial peptide, has been used in permanent pacemakers to prevent device infections [148]. Additionally, combining AMPs with other biological properties can lead to a range of new antimicrobial products, such as antimicrobial sutures and bandages.

Overall, AMPs hold tremendous potential in the fields of medicine and biology. With improvements in production methods and further research progress, it is anticipated that the importance and effectiveness of AMPs as disinfectant tools will be enhanced, which is a development that will contribute to creating healthier, safer, and more satisfying lives for individuals.