Antimicrobial Peptides: Mechanism of Action

Abstract

Antimicrobial peptides (AMPs) are a diverse class of small peptides that are found in most life forms ranging from microorganisms to humans. They can provoke innate immunity response and show activity against a wide range of microbial cells which includes bacteria, fungi, viruses, parasites, and even cancer cells. In recent years AMPs have gained considerable attention as a therapeutic agent since bacterial resistance towards conventional antibiotics is accelerating rapidly. Thus, it is essential to analyze the mechanism of action (MOA) of AMPs to enhance their use as therapeutics. The MOA of AMPs is classified into two broad categories: direct killing and immunological regulation. The direct killing action mechanism is categorized into membrane targeting and non-membrane targeting mechanisms. There are several models and biophysical techniques which determine the action mechanism of antimicrobial peptides.

1. Introduction

Antimicrobial peptides (AMPs) are a broad class of small peptide molecules that are found in most life forms ranging from microorganisms to humans. They can provoke an innate immune response in various species, thus they represent the first line of defense against foreign microbes [1]. They show antimicrobial activity against a wide range of microbial cells including bacteria, viruses, fungus, parasites, and even cancer cells. Although, Gram-negative bacteria and Gram-positive bacteria are the most studied targets of AMPs [2]. In recent years AMPs have gained significant attention as potential therapeutic agents since bacterial resistance towards conventional antibiotics is accelerating rapidly [3].

Antimicrobial peptides (AMPs) are considered as attractive alternative antimicrobial agents, as these small biological molecules have mechanism-of-action (MOA) different from the conventional antibiotics, thus they can be applied to combat against various microorganisms even drug-resistant ones [4]. Several properties of these peptides like net charge, hydrophobicity, secondary structure, etc. lead them to have therapeutic action. AMPs are positively charged amphipathic molecules that kill bacteria by using two major MOAs. In the first MOA, AMPs cause membrane disruption, leading to cell lysis and finally cell death. In the second mechanism, AMPs traverse through the cell membrane without disrupting it and inhibit critical intracellular functions by binding to DNA, RNA, or intracellular proteins [5, 6, 7]. There are several models which have been used to explain the membrane binding activity of AMPs. Based on the ability to form pores the models are divided into two groups: pore-forming models and non-pore forming models [6]. A large number of biophysical techniques are available that describe their action mechanism whether these peptides disrupt microbial membrane or they target intracellular activities [7].

2. Mechanism of action

AMPs are considered as promising antimicrobial agents due to which their mechanism of action has been explored widely. Characterization of AMPs is very crucial to enhance their utilization as therapeutic agents [6]. Antimicrobial peptides exert both bacteriostatic and bactericidal effects and they develop less resistance to microbes than conventional antibiotics [8].

These peptides are positively charged amphiphilic molecules possessing both hydrophilic and hydrophobic residues. Cationic peptides being positively charged interact with negatively charged cell membranes through electrostatic attraction then undergo membrane adsorption and conformational change. These peptides complete their activity after binding to the cell membrane through different mechanisms such as the barrel stave model, the carpet model, the toroidal pore model, etc. [4]. The mechanisms of action of AMPs differ from antibiotics. There are several hypothetical mechanisms of action of these peptides, including the plasma membrane disruption, intracellular antimicrobial mechanism, the inhibition of the synthesis of macromolecules such as protein, nucleic acids, and enzyme activity, and antimicrobial effect via participating in immune regulatory effects [9, 10].

AMPs are divided into four major types based on their secondary structure including linear α-helical peptides, β-sheet peptides, linear extended peptides, and both α-helix and β-sheet peptides [11]. According to extensive research on members of all four groups of AMPs, the permeabilization of microbial cytoplasmic membranes appears to be the main mechanism for most AMPs to kill cells [2]. The helical peptides damage membranes through the carpet or barrel-stave pore model. Their main function is to introduce amphipathic helices into bacterial cell membranes, which disrupts the structure of the membranes [12]. The β-sheet peptides can act in a variety of ways, including prevention of cell wall formation and binding to particular lipid components in membranes [2]. They translocate across lipid bilayers which are associated with the development of temporary pores. The αβ family contains both α and β structures. Elongated AMPs are linear and rich in one or more amino acids, like glycine, tryptophan, arginine, and histidine. The members of this category have a flexible structure in the aqueous environment that allows them to convert into an amphipathic structure when they come into contact with a membrane. They do not act directly on pathogen membranes, but rather permeate them and interact with cytoplasmic proteins.

The MOA of AMPs can be broadly classified into two categories –first is direct killing and second is immunological regulation as shown in Figure 1. The direct killing MOA is further classified into two classes – membrane targeting and non-membrane targeting. Membrane permeabilizing peptides are capable to create transient pores on the membrane, mostly recognized by cationic peptides, such as defensin, LL37, melittin. Non-membrane targeting peptides can pass through the cell membrane and interfere with crucial cellular processes that ultimately lead to the death of cells without permeabilizing the membrane such as pleurocidin, pyrrhocidin, and mersacidin [9].

Figure 1.

Mechanism of action of antimicrobial peptides.

2.1 Direct killing: membrane targeting mechanism of action

AMPs bind through electrostatic and hydrophobic interactions to negatively charged membranes, such as bacterial outer membrane lipids with anionic head groups, like phosphatidylglycerol and cardiolipin, thereby disrupting the membranes. AMPs can interact with negatively charged membranes of microbes and display their antimicrobial activity due to the positive charge present on their α-helix surface, which play important role in killing microbes. The hydrophobic regions of AMPs only have weak interaction with the zwitterionic phospholipids in mammalian membranes. These peptides show less cytotoxicity towards eukaryotic cells since membranes of eukaryotic cells are generally neutral and composed of uncharged neutral phospholipids (like phospholipids comprising of phosphatidylcholine or phosphatidylethanolamine), sphingomyelins, and a huge concentration of cholesterol (Figure 2). Cholesterol decreases AMPs binding to mammalian cell membranes. The amino acid composition of AMPs decides their net charges, amphiphilic properties, and hydrophobicity, which is responsible for their crucial effects on the selective action to microbes [10]. For cellular communication, the electrostatic interaction between anionic phospholipids and cationic AMPs, as well as negatively charged bacterial membranes, is critical. In contrast, phospholipids having phosphatidylcholine head groups and sphingomyelin with a minor part of some ganglioside make up the outer surface of eukaryotic cell membranes, hence the hydrophobic contact between cationic AMPs and mammalian membranes is comparatively weak. Due to the presence of negatively charged phospholipids, there is significant contact between the hydrophobic portion of AMPs and the outer surface of bacterium membranes [13].

Figure 2.

AMPs’ interactions with mammalian membrane or bacterial membrane [6].

The membrane targeting AMPs interact through two ways: receptor-mediated mechanism or non-receptor-mediated mechanism.

2.1.1 Receptor-mediated mechanism

This is mediated by a small group of AMPs i.e., receptor-mediated peptides that consists of a receptor-binding domain and pore-forming domain [14]. They usually resist microbes in vitro at micromolar or nanomolar concentrations and works by interacting with membrane components.

This mechanism is found in the majority of AMPs generated by bacteria, viruses, and tumor cells, for example – nisin, Lacto-coccin, and mesentericin [14]. Nisin tends to be a decent example of antimicrobial activity at low concentration assisted by a definite receptor-like interaction with lipid II as a membrane-bound element concerned with peptidoglycan synthesis. Hence, nisin is reasonably more effective against peptidoglycan-rich gram-positive organisms than others [6]. It mainly comprises of two domains: the first attaches to a cell wall precursor contained in the membrane, the lipid II molecule, with high affinity and the second one is a membrane-anchored pore-forming domain. Alike Nisin, mersacidin is another AMP synthesized by Bacillus species that affiliates with the lantibiotics group. According to previous researches, mersacidin straightforwardly targets lipid II and causes interference with transglycosylation and peptidoglycan synthesis in gram-positive bacteria.

PR-39 is another example that shows a receptor-mediated mechanism to the membrane receptor SbmA. PR-39 is a cathelicidin AMP that is linear in nature and rich in proline-arginine [12]. This AMP is unable to form the pores in the bacterial membrane, although is known to possess multi-functional activities like wound healing by repressing syndecan expression, anti-inflammation via NADPH oxidase inhibition, chemoattraction for neutrophil leucocyte, and intervening protein and DNA synthesis by swift induction of proteolytic activity, prompting degeneration of some proteins involved in DNA replication [12, 15].

2.1.2 Non-receptor mediated mechanism

The non-receptor mediated action mechanism mostly includes in most vertebrate and invertebrate AMPs who exert their activity by interacting with membrane components [6]. The outer surface of the membrane of Gram-negative bacteria contains lipopolysaccharide and Gram-positive bacteria contains teichoic acid, each leads to a net negative charge on membrane surface binds with cationic AMPs through electrostatic attraction [16, 17]. The membrane permeability is the most researched mechanism to understand the MOA of AMPs. AMPs bind to microbial membranes and then destruct the membrane structures of bacteria or cancer cells, leading to the release of cell contents and resulting in cell death [12, 18, 19, 20].

In Gram-negative bacteria, the extra-cellular membrane is composed of negatively charged lipopolysaccharide (LPS). The cationic AMPs cause breakage or a cavity on the outer membranes of bacteria and finally translocate through extracellular membranes by replacing the ions such as Mg²⁺ and Ca²⁺ bound to LPS.

In contrast, Gram-positive bacteria are bounded by a single bilayer membrane which is surrounded by a cell wall containing a thick coating of peptidoglycan and lipoteichoic acid (LTA), thus creating a thick matrix that maintains the bacterial cell’s stiffness. AMPs can diffuse through nano-sized pores that permeate the peptidoglycan layers [21]. LTA is a key component of cell wall of Gram-positive bacteria. It’s a negatively charged molecule with a diacylglycerol moiety bound to the peptidoglycan. The presence of anionic teichoic acids in Gram-positive bacterial cell walls can potentially enhance AMP penetration by providing an extra site to interact with AMPs [14]. After penetrating through the outer membrane and single layer of peptidoglycan in Gram-negative bacteria and thick layers of peptidoglycan in Gram-positive bacteria, AMPs bind to the phospholipids which are present on the inner cellular membranes, causing the formation of a cavity on the cell membranes, thereby resulting in the destruction or permeability of cell membranes, and eventually releasing the contents of the bacteria, further bacterial cell lysis and death [22].

The mechanism of cell membrane damage comprises two steps. First, the cationic AMPs selectively bind onto the surface of the negatively charged bacterial cell membranes and then destroy bacterial membranes by either perforation or non-perforation mode. The hypothetical models which come under membrane perforation mode can be classified into four models including the barrel-stave model, the carpet model, the toroidal-pore model, and the aggregated channel model. In the non-perforation mechanism, it predicts that AMPs bind to the surface of the bacterial cell membranes to cause cell death by disrupting the normal cellular processes of the cells, such as DNA replication, RNA transcription, or protein synthesis [4].

2.2 Direct killing: non-membrane targeting mechanism of action

The non-membrane targeting MOA is broadly grouped into two major categories: AMPs that target intracellular components of bacteria and those that target the cell wall of bacteria [6].

2.2.1 Peptides who target cell wall

AMPs inhibit the synthesis of cell walls similar to traditional antibiotics via interacting with a variety of precursor molecules that are essential for cell wall formation. An example of such precursor molecule which is a main target of AMPs is lipid II [23]. For example, Peptides like defensins bind to the lipid II molecule’s anionic pyrophosphate sugar moiety [24]. Due to this binding, pores formation can occur and further leads to membrane disruption [23]. Human α defensin 1 [16] and human β defensin 3 [24] are AMPs that bind to lipid II to show their bactericidal action mechanism.

2.2.2 Peptides who target Intracellular components

Many research studies indicate that AMPs can traverse the bacterial cell membranes and interact with intracellular targets such as DNA and RNA, disturbing bacterial physiological activity. This can cause interference in proteins and cell wall synthesis [17]. AMPs first interact with the cytoplasmic membrane before attacking intracellular components by inhibiting crucial cellular processes. Mechanisms that involve intracellular targets include inhibit cell-wall synthesis, inhibit the synthesis of macromolecules such as protein or nucleic acids, or inhibit enzymatic activity. Some AMPs, for example, buforin II, indolicidin translocate through and enter inside the bacterial membrane and bind to nucleic acids (DNA or RNA) and inhibit nucleic acid synthesis [6]. This mode of action is still not clear but it is assumed that the cationic amino acids of the peptides interact with the negatively charged phosphate groups of the nucleic acids electrostatically or other synthesized proteins [19]. Some AMPs now target intracellular components, as they do not produce membrane permeabilization at the minimum optimal dose yet nevertheless induce the death of bacteria [17].

2.3 Immunological regulation mechanism of action

AMPs not only directly target and destroy bacteria but may exert their antimicrobial activity by immune modulatory mechanism [20]. AMPs display their immune-modulatory effects in different ways, like reducing the endotoxin-induced inflammatory response, provoking synthesis of pro-inflammatory factors and cytokines, controlling adaptive immunity, and finally recruiting macrophages to show immune modulatory effects [25, 26, 27]. These peptides enhance the body’s ability to fight microbes rather than directly killing bacteria [4].

AMPs are among the innate immunity components and they represent “the first line of defense” being one of the first molecules that fight with foreign microbes as they are produced by immunological cells like macrophages and neutrophils [28]. Some AMPs display various immune reactions like activation and differentiation of white blood cells (WBCs); reduction of expression of inflammatory chemokines; and expression management of chemokines and reactive nitrogen/oxygen species [29, 30, 31, 32, 33]. AMPs stimulate the immune system through various methods in mammals, viz. (i). T cell activation; (ii). Stimulation of Toll-like receptors; (iii). Elevation of phagocytosis; (iv). Dendritic cells activation; (v). chemoattraction of neutrophils (Figure 3) [34].

Figure 3.

AMPs affect gene expression in different cells which include macrophages, neutrophils, monocytes, and epithelial cells, and cause these cells to release chemokines and cytokines, which cause leukocytes to return to the infection site, induce cell differentiation, activate certain cells, and block or activate the Toll-like receptor signaling cascade. Infection prevention, inflammation management, healing of wounds, and provoking the defense of adaptive immunity are all aided by their actions [12].

AMPs are produced by a variety of cells in the body, including epithelial cells, lymphocytes, phagocytes, neutrophils, and keratinocytes in places including the lymphatic system, genitourinary tract, gastrointestinal tract, and immune systems.

With the advancement in research studies of AMPs, it became quite evident that AMPs are produced either constitutively (frequently) or triggered by inflammation [35].

Certain immune cells including neutrophils and macrophages generate AMPs constitutively, whereas other cells like epithelial cells, produce them as a result of mucosal surface stimulation [35]. Most β-defensins are produced due to induction and AMPs that are generated frequently include α-defensins [36]. The human AMPs e.g., LL-37 and β defensins are capable of attracting immune cells like leukocytes [37], dendritic cells [38], and mast cells [25].

3. Models to describe MOA

The activity of AMPs must be considered at the cytoplasmic membrane since most of the AMPs pass through the cell membrane [26]. MOA of peptides depends upon the number of properties including the amino acids sequence, net charge, secondary structure, amphipathicity, hydrophobicity, etc. [27]. There are different mechanisms by which AMPs cause membrane disruption [39]. The capability of AMP’s to bind with bacterial membrane leads to their significant development [40, 41]. There are various models hypothesized by scientists, used to describe the mechanisms of binding AMPs on a membrane including the barrel-stave, toroidal pore wormhole, carpet model as depicted in Figure 4 [6].

Figure 4.

Mechanism of interaction of the antimicrobial peptide with microbial membrane. (a) ATP independent cellular uptake mechanism: barrel stave model, carpet model, toroidal pore wormhole model. (b) ATP-dependent cellular uptake mechanism: macropinocytosis [9].

The models that describe structurally less-defined mechanisms are interfacial activity, segregation of lipids into domains, non-lamellar phases formation, and the transient pore mechanism. Antimicrobial peptides’ mechanism of action has been described using a variety of models. Models which are significantly applied to MOA of AMPs on the membrane are barrel stave, toroidal pore wormhole, and carpet mechanism among all the various models as depicted in Table 1 [30, 41]. The two main pore formation models are barrel-stave and toroidal pore. The non-pore formation model is the carpet mechanism. The mechanism can be divided further into two based on cellular absorption processes: ATP dependent and ATP independent uptake process. The barrel-stave model, carpet model, or toroidal model are all ATP-independent uptake mechanisms, while macropinocytosis is an energy-dependent uptake mechanism [9]. Example of AMPs that follows energy independent cell-penetration mechanism is MMG, alamethicin and gramicidin S [9]. Example of AMP that acts through energy-dependent endocytic pathway CGA-N9 [42].

	Models of antimicrobial action	Antimicrobial peptides
Non-permeabilizing models
1	Transient pore model	Buforin II
2	Sinking raft model	δ-lysin
Permeabilizing models
1	Barrel-Stave model	Alamethicin, Pardaxin 1
2	Toroidal pore model	Melittin,LL-37
3	Huge toroidal pore model	Lacticin Q
4	Disordered toroidal pore model	Melittin
5	Aggregate model	Magainins, Dermaseptin
6	Interfacial activity	Magainin 2
7	Chaotic activity	Magainin 2
8	Carpet mechanism	Aurein 1.2,Cecropin,Indolicidin
9	Membrane discrimination model	V13KL
10	Shai-Huang-Matsazuki (SHM) model	PMAP-23
11	Membrane thinning/thickening model	LL-37
12	Charged clustering of lipids	Magainin analogues
13	Sand in a gearbox model	Synthetic α-AMPs
14	Oxidized phospholipid targeting	Temporin L
15	Electroporation model	NK-lysin
16	Tilted peptide mechanism	Aurein 1.2
17	Amyloid formation model	Temporin B
18	Inhibition of synthesis of macromolecules	Indolicin, PR-39
19	Inhibition of metabolic activities	Histatins

Table 1.

Various Models for the interaction of AMPs with membrane [29, 30].

3.1 Barrel-Stave model

In the barrel-stave model, AMPs bind with the membrane outer surface via electrostatic interaction following it then undergo a conformational change attaining an amphipathic structure. Peptides with a special direction are placed between the membrane and they laterally interact with each other to form an ion channel [31]. When peptide concentration reaches a critical threshold, the peptide monomers form an aggregate on the surface of the membrane, then they create a structure that is made up of a huge concentration of peptides inserted inside the membrane to form a ring just like a “barrel” pore. “Stave” here indicates the spokes which are contained inside the barrel [43]. The hydrophobic residues of the aggregated peptides face outward towards the hydrophobic region of the membrane, while the hydrophilic regions of the peptides face inward, forming an aqueous transmembrane pore that triggered exudation of intracellular contents and resulting death of cells [9]. Some examples of peptides that work through the barrel stave model mechanisms are alamethicin and gramicidin S [13, 44, 45]. Bioinformatic analysis of protegrin 1 conformed that the calculated energy of peptide insertion in artificial membranes was most congruent with this model (Figure 4(a)) [26, 33].

3.2 Toroidal pore wormhole model

The “toroidal pore wormhole” model works similarly to the “barrel stave” mechanism. The peptides are first attracted to the membrane in parallel orientation and then go through secondary structural modifications that are equivalent to those seen in the barrel stave model. The hydrophilic head of peptides faces the hydrophilic region of lipids in this arrangement, and the aqueous phase is outside of the membrane, whereas the hydrophobic portion is located in the membrane’s hydrophobic core. The hydrophobic region of the peptides attach to the phospholipid head regions and displace them. This generates a rupture in the hydrophobic part of the membrane, resulting in a strain. The strain, as well as membrane thinning, creates the surface of bilayer fragile to the AMPs by destructing the composition of the membrane [33].

When the critical threshold concentration of peptides is achieved, the peptides form a self aggregate and thus create the toroidal pore complex, directing themselves in a perpendicular direction to the bilayer surface with the hydrophobic residues not accessible to the phospholipid head groups. The peptides still have an interaction with the phospholipid head regions and are not localized inside the hydrophobic region of the membrane, which distinguishes this from the barrel stave pore. Since this configuration is less stable than a barrel stave pore, thus it is more transitory. Peptide charge appears to alter the stability of pores, with a significant amount of positive side chain residues inducing repulsion and resulting in transitory pores with very short half-lives [43]. The peptides interact through electrostatic attraction with the membrane and following it undergo the same conformational alterations in the same way as the barrel stave model.

In this model, the peptides can orient themselves in a perpendicular direction too in the bilayer membrane [46], also this model does not need specific peptide–peptide interactions to occur. Instead, the peptides cause pore formation within a local curvature of the membrane which is partially formed by the phospholipid head regions. One feature which differentiates the toroidal-pore model from the barrel-stave is the complete arrangement of the lipid membrane. The hydrophobic and hydrophilic arrangement of the bilayer is kept intact in the barrel-stave mechanism, while in toroidal pores this arrangement of the lipids is disrupted, due to which a lipid head and lipid tail groups start interacting with each other. Some peptides traverse through the cytoplasmic membrane enter inside the cytoplasm and start attacking intracellular components as the pores are transient upon the destruction of the membrane [47]. Various AMPs act by toroidal pore models like magainin 2, lacticin Q, and melittin (Figure 4(a)) [6].

3.3 Carpet model

The carpet model, originally described by Shai [40], is the widely studied model for destabilization of the membrane by AMPs. AMPs can also perform the antimicrobial activity without pores formation in the membrane. Carpet model is one such model [17, 41, 48]. Similar to the other two models the mechanism occurs when cationic AMPs are initially attracted with strong electrostatic interaction to a negatively charged phospholipid membrane. AMPs are oriented in a parallel direction to the lipid bilayer membrane surface. Peptides accumulate themselves until they reach to critical threshold concentration, to form a “carpet” on the membrane, leading to unnecessary binding interactions on the outer surface of the membrane, thus rupture of the membrane occurs by creating an effect just like detergent, which leads to micelle formation [23]. There are a few models which can’t be distinguished. The carpet model is one of them and it has been proposed as a necessary step for the toroidal pore model [30]. The membrane bilayer is broken into micelles is referred to as a detergent-like model. The carpet mechanism does not need peptide–peptide interactions of the peptide individuals bound to the membrane; nor does it need the peptide to embed itself into the hydrophobic region to create transmembrane channels [33]. Some peptides’ antimicrobial activity is independent of their amino acid or sequence length; such peptides use the carpet model to demonstrate their action [41], and they perform their action when they are in large amounts because of their amphiphilicity [28]. AMPs performing their activity with the mechanism of carpet model are e.g., cecropin [49] and aurein 1.2 (Figure 4(a)) [50].

3.4 Other models

The models which have ATP-independent cellular uptake mechanisms involve the barrel-stave model, carpet model, or toroidal model, which we have already discussed. ATP-dependent uptake mechanism involves macropinocytosis. Macropinocytosis is the ATP-dependent uptake method of action of AMPs, where the target cell’s plasma membrane folds inward along with the peptide to generate macropinosomes. Furthermore, the AMPs in the vesicles are exudated inside the cytoplasm and show the antibacterial effect (Figure 4(b)) [18, 45]. There are several other models by which peptides perform their antimicrobial action. In models like sinking raft and electroporation, unstable holes emerge in the membrane, altering the charge on both sides of the membrane and eventually developing holes [44].

4. Mechanism of action against other targets

Mechanism of AMPs is widely studied against other targets as well like viruses, fungi, and cancer. Gram-positive bacteria and Gram-negative bacteria are the most commonly studied targets though [2].

4.1 Anticancer antimicrobial peptides

Cancer cells are moderately anionic because of the negatively charged molecules present on their membrane-like phosphatidylserine, O-glycosylated mucins, sialylated gangliosides, and heparin sulfate [51]. In cancer cells, the asymmetry between the inner and outside membranes in terms of negatively charged phospholipids is lost, leads to an increase in negatively charged phosphatidylserine (PS) on the outer leaflet, which improves interactions with AMPs [52]. Due to these anionic molecules present on the cancer cells, electrostatic attraction occurs between cationic AMPs and anionic cancerous cells leading to membrane disruption through mechanisms like carpet or barrel-stave [53, 54].

Anticancer peptides also display anticancer activity through non-membrane targeting mechanisms (i) recruitment and activation of dendritic or macrophage cells to kill tumor cells (ii) obstructing angiogenesis to prevent tumor nutrition and metastasis (iii) inducing cancer cell necrosis or apoptosis (iv) activation of some functional proteins which interfere with tumor cell gene transcription and translation. It’s worth noting that both net charge and hydrophobicity play key roles in anticancer activity optimization, and they are interdependent. For greater anticancer activity, maintaining a balance between net charge and hydrophobicity is crucial [11]. Examples of AMPs that exhibit anticancer activity are magainins and defensins [2].

4.2 Antiviral antimicrobial peptides

AMPs have been found to have inhibitory effects on a variety of DNA and RNA viruses, including HIV and influenza virus, herpes virus, and the hepatitis B virus.

AMPs have been discovered to have antiviral properties in various ways [22]. Antiviral peptides block viruses at various life cycle stages which include entry, attachment, penetration, uncoating, biosynthesis, assembly, and release. AMPs display antiviral mechanisms broadly through three ways: (i) hindering virus attachment and virus-cell membrane fusion; (ii) disrupting the virus envelope; and (iii) inhibition of virus replication by interacting with viral polymerase [12]. AMPs can potentially have an indirect antiviral effect, by altering the host immunological response. They can stimulate the synthesis of cytokines and chemokines, displaying both normal pro-inflammatory activity and triggering the infection-induced inflammatory response. AMPs may also operate as a chemoattractant, attracting immune cells to the infection site and aiding viral clearance [55]. Examples of AMPs showing antiviral activity include α-defensins interfere with the ability of the human immunodeficiency virus (HIV) to multiply within CD4 cells by directly inactivating viral particles. Retrocyclin 2 is a synthetic θ-defensin, capable of preventing influenza virus infection. Human β-defensins can prevent HIV-1 replication [26].

4.3 Antifungal antimicrobial peptides

Antifungal antimicrobial peptides attack either intracellular components or cell walls, causing fungal cell membrane integrity to be disrupted and permeability to be altered due to pore creation in the membrane structure [12]. Antifungal peptides have several recognized mechanisms of action including, (i) direct membrane disruption, (ii) inhibition of cell wall formation, primarily of components like (1,3)- β- d-glucan or chitin, and (iii) interaction with fungal mitochondria [2]. A classic example of an AFP that inhibits 1,3- β-glucan synthase is the echinocandin family. This enzyme is crucial for fungi to maintain cell wall stability. The cell wall is destabilized and the cells become vulnerable to osmotic pressure when the function of this enzyme is blocked. The β-glucan synthase enzyme is broadly found in Aspergillus, Cryptococcus, Candida, and Pneumocystis species. Inhibitors like nikkomycin and polyoxins are known to block chitin synthase in species like C. albicans [12].

5. Biophysical techniques to determine MOA

Various biophysical methods can be used to explain the MOA of AMPs. These methods depend upon bacterial components (e.g., DNA, lipid extracts, nucleotides, etc.). They provide significant insights on the MOA in-depth specifically when the MOA does not include membrane disruption.

5.1 Membrane disruption

The membrane damaging MOA has been the focal point of many researchers for many years [6]. Following are the techniques to determine membrane disrupting mechanisms.

5.1.1 Pyranine leakage assay

Pyranine (Trisodium 8-hydroxypyrene-1,3,6-trisulfonate) is a pH-sensitive hydrophilic polyanionic molecule used as a fluorescent dye to detect the quantity of internal aqueous proton in phospholipid vesicles. Pyranine and anionic phospholipid vesicles have no considerable interaction, due to the anionic nature of pyranine. These properties utilize the use of pyranine molecules to examine the transport of hydrogen ions and counterion across phospholipid vesicle membranes even in the presence of AMPs that disrupt membranes [56, 57]. The accessory information provided through this method is that leakage is affected by membrane composition or ions which are very crucial for activity, like Ca²⁺ [2].

5.1.2 Calcein leakage assay

Calcein leakage assay can be used to study the ability of AMPs to disrupt the lipid bilayers like large unilamellar vesicles (LUVs) or small unilamellar vesicles (SUVs). Calcein or carboxyfluorescein is an aqueous soluble fluorescent dye that is entrapped into LUVs and a gel filtration mechanism is used to eliminate the non-entrapped calcein after self-quenching at critical extreme concentrations [7]. If peptides cause membrane disruption or create large pores in the bilayer, this can lead to leakage of entrapped calcein out of the vesicle lumen thus relieving the self-quenching and resulting in an increase in fluorescence emission intensity [58]. This assay can also be utilized to understand the effect of AMP on bacterial cytoplasmic membrane integrity [59].

5.2 Membrane interaction

Following techniques are used to explain the mechanism of how antimicrobial peptides interact with the plasma membrane.

5.2.1 Oriented circular dichroism

It is significantly crucial to understand the interaction of AMPs with model membranes since many AMPs show their activity by traversing through the membranes. Oriented circular dichroism (OCD) is the method by which we can study the interaction. Oriented lipid bilayers are employed in OCD to gain insights into the peptide membrane alignment [7]. A clear difference between parallel vs. perpendicular localization of a peptide concerning the bilayer membrane can be observed through the signal [60]. This method is mostly studied on α-helical peptides. A change in the CD signal is observed when peptides form well-defined pores with respect to increase in peptide concentration [7].

5.2.2 Differential quenching

Differential quenching is the method that is utilized to gain insights into the localization of peptides in the lipid bilayer membrane [61]. The method takes advantage of simple diffusional quenching notions to the bilayer membrane’s constrained dimensions. The membrane bilayer serves as a slab in which fluorophores and quenchers are distributed uniformly. The quenchers’ distribution has been described using simulations of single-molecule Brownian dynamics, whereas the fluorophores’ distribution has been determined using quenchers’ pairs in phospholipids that are generally in different orientations of the acyl chain in the phospholipids [62]. Because the relative degree of quenching between quenchers and fluorophores is dependent on their propinquity, and the information on the peptides’ current location in the membrane will be provided by fluorescence intensity [61]. A water-soluble quencher, like acrylamide, can also be employed to detect whether the fluorophore is not affected by the aqueous conditions, which is a useful addition to this assay [63].

5.2.3 Other methods

Several other methods like differential scanning calorimetry (DSC) and ²H or ³¹P solid-state NMR can be used to examine how antimicrobial peptides affect lipid arrangement. The DSC thermogram does not show any alterations in AMPs that traverse through the membrane. Alteration in phosphorus nuclei orientation in the membrane depends upon alteration in ³¹P chemical shift, and this also gives accessory information on AMP mode of action. ²H NMR analyses the effect of the AMP on the order of the acyl chain. Currently, DSC is utilized to examine the binding of the AMP MSI-78 with bacteria. Similarly, ³¹P NMR is used to detect the whole bacterial cells [7].

5.3 Nucleic acid interaction

Gel electrophoresis can be used to examine the interaction of AMPs with nucleic acids. The mobility of the nucleic acid bands (Deoxyribonucleic acid (DNA) or Ribonucleic acid (RNA)) is typically measured with respect to AMP concentration. When AMP binds to DNA, the band migration rate is reduced or completely inhibited [64]. Ethidium bromide is used to stain the bands, it is an intercalating agent which can insert itself between nitrogenous base pairs in the DNA, thus used as nucleic acid fluorescent tag and detected by Ultraviolet light. For instance, the nucleic acid binding activity of AMP such as buforin II kills E. coli cells quickly without lysis, detected by the agarose gel electrophoresis technique [7].

5.4 Nucleotide interaction

AMPs show interaction with nucleotides like Adenosine triphosphate (ATP) [65]. In biofilms, one such crucial mechanism for AMPs is to interact with the alarmone nucleotides (p) ppGpp which can be detected by a co-precipitation assay. This interaction results in ppGpp degradation in bacteria, blocking the stress reaction, which further results in the prevention of biofilms or removal of already formed biofilms [7].

5.5 Other methods

Several other methods include determining different types of interactions, for instance, the capacity of AMPs to interact with protein molecules to prevent the formation of biofilm [66]. The co-precipitation method can be used, to detect the capability of AMPs to interact with proteins. For example, the ribosomal protein binding activity of Bac71–35 was examined by measuring the activity of co-sedimentation of ribosomes that have been purified with Bac71–35. After incubating E. coli 70S ribosomes with various doses of the peptide, the peptide bound to the ribosome was isolated using ultracentrifugation. Immunoblotting was used to validate the existence of Ba71–35 and ribosomal protein interaction in the ribosomal pellets [56].

Alternatively, the presence of peptide can be detected by labeling it with rhodamine whether the peptide is on the membrane surface of bacteria or inside the bacteria or on a solid attachment [66] or fluorescent dyes [57]. It is crucial to confirm that the label should not cause any interference with the activity and composition of peptides. Finally, several other kinds of interactions (for instance, lipid II or LPS) can be detected by the use of techniques such as Nuclear Magnetic Resonance (NMR) or surface plasmon resonance (SPR) [7].

6. Conclusion

Antimicrobial peptides are an essential component of innate immunity. They have the potential to be a viable alternative to antibiotics. It is critical to comprehend the MOA used by AMPs to kill bacteria to increase their development as therapeutics. The selectivity and activity of these peptides are influenced by a variety of parameters. Properties such as net charge, hydrophobicity, secondary structure, and amphipathicity are all important for function and are so interconnected that changing one attribute generally causes alterations in others.

The AMPs aggregate at the membrane surface following the hydrophobic and electrostatic attraction, forming self-aggregate on the bacterial membrane after they reach a particular concentration. The MOA of peptides can be broadly divided into two classes including direct killing and immunological regulation, wherein direct killing is further categorized into membrane targeting and non-membrane targeting. Different models have been proposed to explain the mechanism of interaction of peptides with membranes. Some biophysical techniques are used to determine their action mechanism whether these peptides disrupt microbial membrane or they target intracellular activities. Therefore, it is very crucial to know about the diverse biological features of AMPs for their clinical development.