**2. Antimicrobial peptides**

Antimicrobial peptides (AMPs) may represent an alternative to current antibiotics in MDR *A. baumannii* ESKAPE pathogen [33]. AMPs (also known as host defense peptides) are small polycationic peptides naturally produced by living organisms with both microbicidal and immunomodulatory activities, acting as a primary barrier against pathogens, including protozoa, víruses, bacteria, archaea, fungi, plants, and animals as a part of innate immunity system [34–41]. However, the computational design of synthetic AMPs with improved activity is also being developed [42]. They interact with cell membrane through electrostatic

interactions, causing the inhibition of protein and nucleic acid synthesis and final cellular lysis by apoptosis and necrosis [43–44]. In addition to the antimicrobial properties, some AMPs have other activities, such as anticancer antioxidant, wound healing, immunoregulatory [38, 45, 46]. AMPs also play an essential role in regulating immune processes such as activating and recruiting immune system cells, angiogenesis, and inflammation [47]. AMPs are amphipathic molecules with a positive electric charge, varying molecular weight, and containing about 11–50 amino acid residues [47, 48]. AMPs are classified into α-helical, β-sheet, and extended peptide families [49–51] and interact with the membranes initially through electrostatic and hydrophobic interactions (**Figure 1**), accumulating at the surface and self-assemble on the bacterial membrane after reaching a particular concentration [52, 53].

At this stage, various models have been proposed to describe the action of AMPs. The models can be classified under two broad categories: transmembrane pore (TMP) and non-pore models (NPM), and the TMP can be further subdivided into the barrel-stave pore and toroidal pore models. In the barrel-stave model, the AMPs are initially oriented parallel to the membrane but eventually insert perpendicularly in the lipid bilayer [54] (**Figure 2A**), thus promoting lateral peptide-peptide interactions, like that of membrane protein ion channels. Peptide amphipathic structure (α and/or β sheet) is essential in this pore formation mechanism as the hydrophobic regions interact with the membrane lipids and hydrophilic residues from the lumen of the channels [55, 56]. A unique property associated with AMPs in this category is a minimum length of 22 residues (α helical) or 8 residues (β sheet) to span the lipid bilayer. Only a few AMPs, such as alamethicin [57], pardaxin [58, 59], and protegrins [55], have been shown to form barrel stave channels.

Furthermore, in the toroidal pore model, the peptides also insert perpendicularly in the lipid bilayer, but specific peptide-peptide interactions are not present [57]. Instead, the peptides induce a local curvature of the lipid bilayer with the pores partly formed by peptides and partly by the phospholipid head group (**Figure 2B).** Thus, the dynamic and transient lipid-peptide supramolecule is known as the "toroidal pore." The distinguishing feature of this model compared to the barrelstave pore is the net arrangement of the bilayer. In the barrel-stave pore, the hydrophobic and hydrophilic sequence of the lipids is maintained, whereas, in toroidal pores, the hydrophobic and hydrophilic arrangement of the bilayer is

#### **Figure 1.**

*Interaction of cationic AMPs with eukaryotic and bacterial membranes. Images were created using BioRender.com.*

#### **Figure 2.**

*Mechanisms of action of AMPs in bacteria. A) Barrel-stave model: AMPs stack into the bilayer of the cell membrane to form a channel. (B) Toroidal pore model: Accumulation of vertically and bend embedded AMPs in the cell membrane to form a pore structure, (C) carpet model: Distribution of AMPs on membrane surface that evolve to detergent-like mode, forming micelles, (D) images were created using BioRender.com.*

disrupted, thus providing alternate surfaces for the lipid tail and the lipid head group to interact with. Furthermore, as the pores are transient upon disintegration, some peptides translocate to the inner cytoplasmic leaflet entering the cytoplasm and potentially targeting intracellular components [60]. Other features of the toroidal pore include ion selectivity and discrete size [61]. Several AMPs such as magainin 2 [62], lacticin Q [62], aurein 2.2 [63], and melittin [57, 62] have been shown to form toroidal pores. In addition, the type of pore started by aurein 2.2 has been shown to depend on the lipid composition: In a 1-palmitoyl-2-oleoyl-*sn*glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-*sn*-glycerol-3-phospho- (1<sup>0</sup> -*rac*-glycerol) POPG (1:1) membrane model, the peptides induce toroidal pores, whereas in a 1,2-dimyristoyl-*sn*-glycerol-3-phosphocholine (DMPC)/1,2 dimyristoyl-*sn*-glycero-3-phospho-(1<sup>0</sup> -*rac*-glycerol) DMPG (1,1) membrane model, the peptides work in a detergent-like model (details below) indicating the importance of the hydrophobic thickness of the lipid bilayer and the membrane composition [64, 65]. Ultimately, both pore-forming models (toroidal pore and barrel) lead to membrane depolarization and eventually cell death.

AMPs can also act without forming specific pores in the membrane. One of these models is designated as the carpet model [61, 62, 66]. In this case, the AMPs adsorb parallel to the lipid bilayer and reach a threshold concentration to cover the surface of the membrane, thereby forming a "carpet" (**Figure 2C**) and leading to unfavorable interactions on the membrane surface. Consequently, the membrane integrity is lost, producing a detergent-like effect, which eventually disintegrates the membrane by forming micelles. The final collapse of the membrane bilayer structure into micelles is the detergent-like model **(Figure 2D).** The carpet model does not require specific peptide-peptide interactions of the membrane-bound peptide monomers; it also does not require the peptide to insert into the hydrophobic core to form transmembrane channels or specific peptide structures [67]. Many peptides act as antimicrobial agents despite their specific amino acid composition or the length of the sequence. Such AMPs typically act using the carpet model [66] at high

concentrations because of their amphiphilic nature. Examples of AMPs acting by the carpet model are cecropin [68], indolicidin [69], aurein 1.2 [67], and LL-37 [66].

Overall, there are many models to describe the MOA of AMPs. In addition to those given above, other related models include the interfacial activity model, the electroporation model, and the Shai-Huang-Matsazuki model [62]. Some models do not make the specific distinctions shown in **Figure 2.** For example, it has been suggested that the carpet-like mechanism is a prerequisite step for the toroidal pore model [62]. Most studies to elucidate the MOA of AMPs involve the use of model membranes. The mode of action of only a few AMPs has been investigated with whole bacterial cells using imaging techniques [70, 71]. Different results may be obtained using other membrane models or assay conditions; for example, more than one MOA is possible for certain AMPs such as BP100 as the peptide-to-lipid ratio changes [72], indicating that the models described here may or may not translate directly to what is occurring in bacteria.

An online antimicrobial peptide database, APD3, list examples of AMPs, including both synthetically synthesized and compounds produced by living organisms [37]. In addition, many AMPs are currently being studied to elucidate their therapeutic efficacy against *A. baumannii* strains (**Table 1**).

#### **2.1 Cathelicidins**

Cathelicidins are a group of cationic AMPs (CAMPs) (with more than 30 members) detected in the immune system of some vertebrates that have in their structure two domains involved in antimicrobial activity [145]. Compared with carbapenems (imipenem and meropenem), which are considered the drugs of choice for infections caused by MDR *A. baumannii* (MIC = 16–32 mg/L) [146], these peptides exhibit excellent activity.

#### *2.1.1 LL-37*

The most studied member of the cathelicidins family is LL-37 (Human cathelicidin) with an α-helical structure. It is produced by many cell types as a part of innate immunity and exhibits broad-spectrum microbicidal activities against Gram-positive and Gram-negative bacteria by plasma-membrane disruption [147]. Other properties were also described, like immunomodulation properties such as chemoattraction and activation of various immune cells, neutralizing the lipopolysaccharide (LPS), regulating the inflammatory response, wound closure, and chemotaxis [38, 148–151]. Feng et al. Investigated the anti-*A. baumannii* activity of LL-37 and fragments KS-30 and KR-12 against one sensitive and four MDR *A. baumannii* clinical isolates [73]. The minimum inhibitory concentration (MIC) for three pieces of KS-30, KR-20, and KR-12 was 8–16, 16–64, and 128–256 μg/ml, respectively. At the same time, LL-37 inhibited all sensitive and drug-resistant strains at the concentration of 16–32 μg/ml. Furthermore, LL-37 and the fragment KS-30 have been found to significantly inhibited and dispersed the *A. baumannii* biofilm in abiotic surfaces at 32 and 64 μg/ml, respectively [73]. A panel of synthetic peptides based on human LL-37 AMP shows potent microbicidal activity against several ESKAPE pathogens without selecting resistance and can also eliminate persister cells and biofilms of *P. aeruginosa*, *A. baumannii*, and *S. aureus* in the micromolar scale [74]. SAAP-148 is an α-helical AMP, able to suppress MDR *A. baumannii* without causing resistance and prevents biofilm formation. Studies showed that this peptide could inhibit the growth of *A. baumannii* MDR at a concentration of 6 μg/m. Treatment with this peptide (animal model) appointment has been shown to eliminate acute and biofilm-related infections by *A. baumannii* in


*Insights on Antimicrobial Peptides*









