**4. Molecular mechanisms of antimicrobial peptides action**

Antimicrobial peptides can alter bacterial membrane properties by different mechanisms. Alteration of the bulk physical properties of the membrane is one way (**Figure 2**) [8]. AMPs can modify bulk properties while not having a specific target on the membrane. Changes in the spatial distribution of cell membrane molecules within or modification of a bulk physical property as intrinsic curvature or fluidity are examples of these alterations. Contrary to this, altering the bulk biophysical properties by AMPs can occur by targeting a class of particular lipids. Specific phospholipids are the potential targets which AMPs being effective against both Gram-negative and Gram-positive bacteria. These mechanisms are not completely independent of each other. For example, membrane clustering will lead to packing defects at the boundary between domains and physical curvature can drive clustering. In addition, directly targeting lipids can lead to any of the three phenomena that are mentioned.

Antimicrobial agents also can target membrane phospholipids. Cardiolipin; CL, phosphatidylglycerol; PG, and phosphatidylethanolamine; PE, are the three main phospholipids in most bacteria. PG is the most abundant of them. It is an anionic lipid, and therefore, attracts cationic antimicrobial peptides. Modifying the PG head group by adding lysine and or alanine and reducing the negative charge on the membrane is one of the ways that bacteria use to protect themselves from these peptides and thus will be more resistant to the cationic antimicrobial peptides. In Gram-negative bacteria, PE is generally abundant. Several cyclic peptides are able to specifically bind to PE, and therefore, can be used to target these bacteria. In addition, PE and anionic lipid mixtures can create segregated clusters when the anionic

#### **Figure 2.**

*Different outcomes of AMPs on properties of the bacterial cell membrane. AMPs can affect the physical properties of the cellular membrane, such as (A) induction of membrane physical curvature [44], (B) lipid clustering, (C) prompting packing defects resulting in complete or partial loss of the permeability barrier, and (D) directly targeting components of a membrane such as lipids leading to a variety of consequences.*

lipid is bound to an AMP. The activity of antimicrobial agents that bind to cardiolipin is also based on a clustering mechanism [9]. Some AMPs can bind to either CL or PE and target specific bacteria. So, the cell membrane is a multipurpose target for AMPs that serves as targets or wards them off in resistance and provides a crucial site for toxic activities [8].

However, the head group structure of phospholipids is the same in bacteria and eukaryotes, the acyl chains in bacteria are shorter and more saturated [45]. In addition, while anionic lipids and PE are sequestered to the cytoplasmic surface of eukaryotic membranes, they are exposed to the external surface of bacterial membranes. These differences provide the feasibility of designing antibacterial agents that target specific bacterial lipids [8].

The antimicrobial activities of AMPs against various types of pathogens, including Gram-positive and Gram-negative bacteria, viruses, and fungi occur through a wide range of mechanisms, for example, membrane disruption, intracellular penetration, and immunomodulation [46]. Although AMPs may have different mechanisms of action, it is thought that their ability to act against such diverse cellular organisms is related to membrane activity [7]. The positive charge of cationic amino acid AMPs enables electrostatic interaction to the negatively charged microbial membranes [47, 48] and that the hydrophobic region is involved in the penetration of the cells [49]. The nature of the cell surface, in particular, the composition of the OM of Gram-negative bacteria has a major impact on the antimicrobial activity and efficacy of antimicrobial agents including cationic AMPs [50]. Antimicrobial peptides penetrate the bacterial membranes through several different mechanisms [51]. Briefly, AMPs binding to cell membrane break down the membrane potential, lead to alteration membrane permeability and metabolite leakage, and finally cause bacterial cell death [41].

Structural antimicrobial peptide studies have strongly suggested that the physicochemical properties of AMPs are responsible for their microbiological activities, rather than any specific amino acid residues. Since the amphiphilic topology is fundamental for insertion into the cytoplasmic membrane and disruption of cells, AMPs and their mimics have been considered as attractive targets for drug development. In particular, they are able to kill bacteria quickly and development of the bacterial antibiotic resistance is relatively difficult [7].

There is a clear phenomenological link between anionic lipid clustering and the bacterial species specificity of a number of antimicrobial agents. Direct activity on the bacterial cell membrane is the most prevalent mechanism of antimicrobial peptides [52]. Antimicrobial peptides can interact with the bacterial membranes due to their amphipathic nature. Most AMPs have a net positive charge, and therefore, are named cationic antimicrobial peptides. The binding of cationic antimicrobial peptides to the bacterial membranes is stabilized through electrostatic interactions between the cationic parts of AMPs and anionic compounds on bacterial membranes. Consequently, the bacterial membrane integrity is disrupted, causing antimicrobial peptides penetration into the membranes, and in most cases, finally forming the pores [53].

The clustering of anionic lipids to a region of the bacterial membrane would concentrate negative charge in a domain to which cationic peptides would congregate, possibly leading to the formation of a pore. After increasing the concentration of cationic antimicrobial agents on the anionic surface of the membrane, the rest of the membrane will surround the domain of anionic lipids and lead to less membrane stability under line tension. It seems that there are always domains with phase boundary defects in bacterial membranes [54] and those that would form in the presence of lipid clustering AMPs would appear suddenly [55]. Under these conditions, bacteria would not have enough time to repair this rearrangement and would

be damaged as a result of the redistribution of membrane's lipids. Consequently, disruption of functional natural domains or decreasing the availability of anionic lipids that may be necessary for the specific protein function in the cytoplasmic membrane would happen [8].
