## 3 Antibacterial Peptides and Their Mechanism of Action  
Many factors can influence membrane perturbation and disruption by AMPs, i.e., amino acids sequence, the lipid composition of the membrane, peptide concentration as well as differences in membrane composition between eukaryotic and bacterial cells allow the AMPs to distinguish a microbial target from the host. Bacterial membranes are negatively charged due to the presence of anionic phospholipids groups, e.g., phosphatidylcholineylglycerol, phosphatidylcholineyline, while eukaryotic cells possess groups with a neutral charge, e.g., phosphatidylcholine and phosphatidylcholineylethanolamine [54]. Moreover, the presence of cholesterol, a common feature in eukaryotic cells, is able to interact with AMPs either neutralizing or reducing their activity or stabilizing the phospholipid bilayer [55]. In Gram-positive bacteria, AMPs have to cross first the cell wall composed of crosslinked peptidoglycan with lipoteichoic acid prior to reaching the membrane whereas in Gram-negative they face a coat of lipopolysaccharide-aschardile (LPS) followed by a phospholipidic outer membrane and a less cross-linked peptidoglycan layer [56]. Electrostatic interactions between the cationic peptide and the negatively charged components, e.g., lipopolysaccharide-aschardile in Gram-negative and teichoic acid in Gram-positive, are the first steps to contribute to bacterial membrane affinity [57]. However, while AMPs seem to traverse the peptidoglycan layer with ease and access to the cytoplasmic membrane of the Gram-positive, they need to disrupt or perturb both outer and cytoplasmic membrane in Gram-negatives. Impedance in crossing or permeabilization results in loss of antimicrobial activity (Figure 1a (A,B)) [58]. In order to explain the perturbation of the phospholipidic membranes operated by the AMPs, three main models have been proposed: _carpet-like, barrel-stave_ and _toroidal pore_ (Figure 1a (D)). Generally, when the ratio of peptide/lipids is low, AMPs interact with the phospholipidic layer of the membrane in a parallel manner, defined as _carpet-like_ model, and interaction among the peptides or penetration in the hydrophobic core of the bilayer are not taking place [59]. Membrane integrity is disrupted and micelles are formed as in a detergent-like process [60]. With increasing AMPs ratio, they move to a perpendicular orientation until reaching such a concentration that they can cross the membrane forming pores (1:50-1:500 and more) [61; 62]. A minimum length of -22 amino acid for \(\alpha\)-helix peptides is required to span the phospholipid layer, while \(\beta\)-sheet structures necessitate a minimum of 8 [63]. In the _barrel-stave_, interaction among peptides is a prerequisite as they mimic a transmembrane pore, whereas, in the case of the _toroidal_ model, peptides are loosely arranged [64; 65]. Despite the perturbation of the membrane seems to vary depending on the peptides, actually, the mechanisms of action are not completely well-defined and they are partially overlapping [66]. Moreover, all these models are based on the membrane perturbation but, then, the killing effect is not always enough to provide antimicrobial activity [67]. Besides membrane disruption, recent studies showed how peptides could act on other targets as well (Figure 1b) [68]. Some AMPs have shown their efficacy by binding some components and receptors on the extracellular side of the membrane and wall, thus destabilizing the permeability and/or activating intracellular signaling pathways that have, as a response, the inhibition or the activation of several functions. An interesting example is represented by the binding of Toll-like receptors and the consequential amplification of the inflammatory response via NFkB cascade followed by activation of the immune system towards microbiological pathogens [69; 70]. Other antimicrobial peptides manage to enter the cytosol through direct penetration, endocytosis (bothropinocytosis and receptor-mediated) [71], or the exploit of delivery systems [72].