**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].

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.

**Figure 2.**

*AMPs' interactions with mammalian membrane or bacterial membrane [6].*

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 grampositive 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

#### *Antimicrobial Peptides: Mechanism of Action DOI: http://dx.doi.org/10.5772/intechopen.99190*

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, 18–20].

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 [25–27]. 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

#### **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].*

#### *Antimicrobial Peptides: Mechanism of Action DOI: http://dx.doi.org/10.5772/intechopen.99190*

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 [29–33]. 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].

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].
