Introduction

Antibacterial resistance by multidrug-resistant (MDR) pathogens has become a global health challenge and threatens the health of societies. The emergence of resistant infections leads to existing antibacterial drugs becoming less effective or even ineffective and therefore, there is an urgent need for the development of new antibacterial agents (Khameneh et al. 2016). The death by drug-resistant bacterial infections in each year for USA, EU, and India was 23,000, 25,000, and 58,000, respectively (Chaudhary 2016). Additionally, without developing novel approaches to combat MDR pathogens, many fields of medicine such as surgery, cancer chemotherapy, and transplantation medicine will be affected severely (Worthington and Melander 2013). Whenever the antibacterial resistance has emerged, the problem has been tackled with various approaches such as repurposing non-antibiotic drugs, modification of the existing antibiotic classes with limited cross-resistance, developing new classes of antibiotics, antibiotic combinations, development of adjuvant therapy, developing a novel formulation of antibiotic, and using natural compounds (Bazzaz et al. 2019; Farhadi et al. 2019; Khameneh et al. 2015a, 2019a, b; Soheili et al. 2019; Theuretzbacher et al. 2020).

During the past years, the efforts for discovering the novel antimicrobial agents have been focused on developing groups of short polypeptides, up to 40 residues, namely antimicrobial peptides (AMPs) (Fox 2013; Javia et al. 2018). AMPs can be obtained from both natural and synthetic sources and show a broad spectrum of targeted organisms including viruses to parasites (Bahar and Ren 2013; Zasloff 2002). These peptides are categorized based on different properties such as similarities in charge, sequence, functional and 3-dimensional structure. Based on the charge of AMPs, they are grouped into anionic and cationic peptides. Anionic ones are small peptides (721.6–823.8 Da) and present in bronchoalveolar lavage, fluid surfactant extracts, and airway epithelial cells, while, cationic peptides are mainly found in all living species. These latter AMPs contain 12–50 amino acid residues with a net positive charge of + 2 to  + 7. They have an excess of basic amino acid residues such as arginine, lysine, and histidine in comparison with acidic residues. Cationic AMPs have a diverse range of cellular targets (Bahar and Ren 2013; Brogden et al. 2003; Bulet et al. 2004; Huang et al. 2010).

AMPs show their antimicrobial properties via different mechanisms. They showed weak antimicrobial actions but have wide and strong immune-modulatory properties. Taken together, they have been considered as alternative agents for traditional antibiotics. AMPs have been successfully used against resistant infections such as infectious biofilm or MDR bacteria (Boparai and Sharma 2020; Park et al. 2011).

In contrast to an antibiotic peptide that is synthesized through a specific metabolic pathway, the amino acid sequence of AMPs is encoded naturally by the genetic materials of the host organisms (Boparai and Sharma 2020). Upon pathogen invasion, transcription and translation processes are started and followed by post-translational modifications including precursor protein cleavage, C-terminus amidation, and disulfide bond formation for approximately 50% of the known AMPs. Moreover, glycosylation modification may happen in uncommon cases (Toke 2005). These AMPs were first detected in plant organisms, then in a wide range of other organisms like humans (Toke 2005). So far, the number of detected AMPs has reached more than one million (Boparai and Sharma 2020).

AMPs show a broad variety of secondary structures like β-strands, α-helices with extended structures, loops, and one or more disulfide bridges (Edwards et al. 2017; Hwang and Vogel 1998). These observed structural differences are necessary for their antimicrobial activities in a broad spectrum (Hancock 2001). Additionally, particular crucial factors like peptide self-association to biological membranes, amphipathic stereo-geometry, hydrophobicity, charge, and also size contribute to their antimicrobial activities (Boparai and Sharma 2020). Moreover, the smaller size of antimicrobial peptides facilitates the fast secretion and diffusion of peptide outside the cells that is important for inducing defense responses against pathogenic fungi and bacteria (Brogden 2005). This article aims to review the classification of AMPs based on the structure and composition and their mode of action. Moreover, the essential aspects of human AMPs that have revealed their significance will be discussed.

Classification of AMPs

Knowing about the three-dimensional structure of AMPs leads to a better understanding of their mode of actions. Nuclear magnetic resonance (NMR) has been used extensively for analyzing the structures of most of the AMPs. Because of the small size of AMPs in comparison with other biopharmaceuticals, the three-dimensional structures could be obtained by conventional two-dimensional NMR methods (Reddy et al. 2004; Zhang and Falla 2006). Based on the NMR data, AMPs could be classified into five groups.

α-Helical AMPs

Abundant classes of AMPs are those with amphipathic properties that have α-helical domains. Their particularly successful structural arrangements help them for innating defense. Most of them are short (< 40 residues) and therefore can be synthesized easily. Because of the structure, they can be characterized by circular dichroism (CD) or NMR spectroscopies, simply. It should be noted that these types of AMPs are active against a wide range of pathogens, including both Gram‐positive and ‐negative bacteria, fungi, and protozoa (Giangaspero et al. 2001; Reddy et al. 2004). Some of them are illustrated in Fig. 1.

Fig. 1
figure 1

Some examples of α-helical antimicrobial peptides

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Cysteine-Rich AMPs

Cysteine-rich AMPs are diverse and widely distributed in animal and plant tissues. They have an important role in host defense systems (Dimarcq et al. 1998). The human neutrophil peptides HNP-1, -2, and -3 were the first of these AMPs which isolated from the human granules (Ganz et al. 1985). These α-defensins compose of 30 amino acid residues which are rich in cysteine and could be found in a wide variety of organisms (Reddy et al. 2004). Most of these AMPs contain six cysteine residues and all of them participate in forming three intramolecular disulfide bonds (Dimarcq et al. 1998). These disulfide bridges are mostly between C1–C4, C2–C5, and C3–C6 (Dimarcq et al. 1998; Hill et al. 1991). Based on the NMR data, α-defensin has three-stranded antiparallel β-sheets (Pardi et al. 1992). Some of them are illustrated in Fig. 2.

Fig. 2
figure 2

Some examples of cysteine-rich antimicrobial peptides

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β-Sheet AMPs

The third group of AMPs is peptides with β-sheets structure. Most of them are further stabilized by one or more disulfide bonds. Based on their cysteine content and structural properties, they can be divided into two groups: (i) β-hairpin peptides; and (ii) α-defensin peptides (Koehbach and Craik 2019). β-hairpin peptides encompass approximately 20 residues containing one or two disulfide bonds. Examples of β-hairpin peptides are Horseshoe crab (Limulus polyphemus) peptides, tachyplesins, and polyphemusin II, which are stabilized via two disulfide bridges; thanatin from insects (one disulfide bond); and protegrin-1, peptides isolated from porcine leukocytes. These molecules form an antiparallel β-sheet connected to a β-turn and are composed of disulfide bonds (Kawano et al. 1990; Tamamura et al. 1993). As shown by NMR studies, lactoferricin B, containing 25 amino acids, presents a β-sheet structure that stabilized by a single disulfide bridge (Hwang et al. 1998). Some of these AMPs are illustrated in Fig. 3.

Fig. 3
figure 3

Some examples of β-sheet antimicrobial peptides

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AMPs Rich in Regular Amino Acids

Some of the AMPs contain high numbers of regular amino acid residues and therefore their structures are different from the regular α-helical or β-sheet peptides. For example, histatin, a peptide isolated from human saliva is histidine-rich AMPs and also effective against Candida albicans (Xu et al. 1991). Cathelicidins, a family of endogenous AMPs, are proline-rich and show irregular structures (Tomasinsig and Zanetti 2005). Indolicidin, a cationic AMP that consists of only 13 amino acids, has the highest tryptophan content, and tritripticin like indolicidin is rich in tryptophan (Bacalum et al. 2017; Falla et al. 1996). Bactenecins are a class of arginine-rich AMPs of bovine neutrophil granules (Ciociola et al. 2018).

AMPs with Unnatural Amino Acids

Some of the AMPs contain unnatural amino acids such as peptides from bacteria themselves. Nisin, a lantibiotic, is a notable example of AMPs from this group. It is produced by Lactococcus lactis and contains rare amino acids like lanthionine, 3-methyllanthionine, dehydroalanine, and dehydrobutyrine (de Vos et al. 1993; Zhang et al. 2014). Nisin Q, consisting of 34 amino acids, is a ribosomally-synthesized AMP and contains post-translationally modified residues such as lanthionine and dehydroalanine (Fukao et al. 2008). Leucocin A is another peptide that composes of 37 amino acids and isolated from Leuconostoc gelidum. It can form an amphiphilic conformation and could interact with cell membranes (Fregeau Gallagher et al. 1997). These AMPs undergo post-translational modifications and after that, their conformations could not see in other classes of AMPs. For example, subtilin is a ribosomally synthesized AMP that composes of several unusual amino acids as a result of post-translational modifications (Liu and Hansen 1993). Gramicidin is another example that contains several DH-amino acids that result in forming an unusual cyclic β-hairpin (Gibbs et al. 1998). Cbf-14, derived from cathelicidin-BF, is effective against antibiotic-resistant bacteria. In this AMP, lysine or leucine was substituted with similar unnatural amino acids such as ornithine (Orn) and norleucine (Ile) to generate AMPs with enhanced antibacterial activities. The data indicated that the mutant of Cbf-14 possesses potent antibacterial activities against penicillin-resistant bacteria (Kang et al. 2017).

The Mode of AMPs Action

It was shown that the main mechanism of AMPs is presumably to include the establishment of membrane pore or ion channel, without stereo-special interactions with chiral receptors (Toke 2005). These observations were also confirmed by others that showed D enantiomers of melittin, magainin 2 amide, and cecropin A were assayed and discovered to show the identical hemolytic and antibacterial efficacies as their L counterparts that naturally occurred. Furthermore, the compounds all generated one channel conductance in lipid bilayers, with the L, as well as D analogs, inducing the identical extent of conductivity (Boparai and Sharma 2020; Wade et al. 1990). AMPs attain dynamic interchanges in their topologies and structures upon interacting with cell membranes in microorganisms (Sansom 1998).

The outer surface of eukaryotic cells is composed of sphingomyelin phospholipids and zwitterionic phosphatidylcholine, whereas the outer surface of prokaryotic cells is negatively charged, because of the existence of teichoic acid or lipopolysaccharides (Dolis et al. 1997). The electrostatic interactions of AMPs with the negatively charged molecules on membranes seem to be the preliminary mechanism responsible for antimicrobial activities (Boparai and Sharma 2020). AMPs apply their activities in host cells through translocating across the cell membranes and prevent fundamental cellular processes like cell wall synthesis, enzymatic activities, as well as nucleic acid and protein synthesis (Brogden 2005). Particular other factors like outer membrane fluidity, molecular architecture, the concentration of negatively charged molecules, as well as outer membrane charge and magnitude also are important for the transport of AMPs across the biological membranes (Kondejewski et al. 1999). The membrane fluidity was determined to regulate the insertion and adsorption of antimicrobial peptides into the cell membranes.

According to the mechanisms of action, AMPs are classified widely into non-membrane acting and membrane acting peptides. Three mechanisms were suggested for pore-forming by membrane acting peptides (Boparai and Sharma 2020). The barrel-stave mechanism refers to insert AMPs into the biological membrane hydrophobic substance that flip inward and create pores through producing trans-membrane helical bundles (Peters et al. 2010). In the carpet-like mechanism, AMPs destruct the cell membranes assembly via their collaborative activity. In this path, AMPs self-associate onto the acidic phospholipids-rich areas located at lipid bilayer, and as soon as their concentrations approach specific thresholds, they can permeate into biological membranes. This phenomenon is facilitated via escalating in the positive potential of lipid bilayers (Mookherjee and Hancock 2007). Toroidal refers to build toroidal pore in a lipid bilayer by the AMPs. Pore constructions are administrated through the helix bundles and lipid polar head groups, which vertically orient to the external section of membranes. In other words, the AMPs cumulate and impel the lipid monolayers for continuous bending through the pore so that both lipid head groups as well as inserted peptides line the water core (Rahnamaeian 2011). The related mechanisms were illustrated in Fig. 4.

Fig. 4
figure 4

The pore-forming mechanisms of antimicrobial peptides

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The permeabilizing non-membrane peptides are mostly represented by the capability for translocating across the cell membranes without permeabilizing the membrane, whereas the permeabilizing membrane peptides are cationic peptides with the ability to form the transient pores on the biological membranes (Pushpanathan et al. 2013). Particular AMPs inducing trans-membrane pores on the membrane of target cells comprise LL-37 (Harder et al. 2007), magainins (Hallock et al. 2003), melittin (Yang et al. 2001), and defensin (Schneider et al. 2005a). AMPs like mersacidin (Brotz et al. 1995), pyrrhocidin (Kragol et al. 2001), indolicidin (Friedrich et al. 2001), pleurocidin (Patrzykat et al. 2002), HNP-1 (Lee et al. 2002), dermaseptin (Patrzykat et al. 2002), and buforin II (Park et al. 2000) get translocated across the biological membranes and suppress important cellular processes that result in cells death. Besides, some AMPs like lactoferrin (Patrzykat et al. 2002), histatin (Kavanagh and Dowd 2004), melittin (Park and Lee 2010), and papiliocin (Hwang et al. 2011) apply their antimicrobial activity by the formation of reactive oxygen species.

Human AMPs

Human AMPs protect the human from microbial infection by various mechanisms. They have been identified in a variety of tissues or surfaces such as eyes, skin, ears, mouth, lungs, intestines, and also the urinary tract (Wang 2014). Some of the most important ones with more details are summarized here. Some important features of these AMPs are summarized in Table 1.

Table 1 Some important features of human antimicrobial peptides
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Human Defensins

α-Defensins were the first human group of AMPs to be characterized which were isolated from human blood. They are mostly cationic peptides containing between 29 and 35 amino acid residues. They have six cysteine residues that form three disulphide bonds. The cysteine bridges are between C1–C6, C2–C4, and C3–C5 that form cyclic peptides with a typical structure of a triple-stranded β-sheet and a β-hairpin loop (Ryley 2001; Wang 2014). Based on the source, property, and size, these peptides α-defensins are categorized into four groups: (I) HNP-1, HNP-2, and HNP-3; these three AMPs have almost identical amino acid sequences. In comparison with HNP-2, HNP-1 and HNP-3 contain only one additional residue at the N-terminus: alanine in HNP-1 and aspartate in HNP-3. (II) The fourth human neutrophil defensin, HNP-4, has a distinct peptide sequence with 33 amino acid residues. (III) HD-5 was obtained from human Paneth cells and present in the female reproductive tract. (IV) HD-6 ones are only expressed in the Paneth cells of human intestines (Schneider et al. 2005; Wang 2014). The human β-defensin family (hBD) was different from α-defensins. The disulfide bonds of β-defensins are between C1–C5, C2–C4, and C3–C6. Also, these AMPs have a slightly longer sequence than α-defensins that leads to form an additional helical region. The related structures are illustrated in Fig. 5.

Fig. 5
figure 5

The related structures of human defensins

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Spectrum of Activity

They are active against a wide spectrum of both Gram-positive and -negative bacteria, mycobacteria, fungi, and some enveloped viruses (Winter and Wenghoefer 2012). For example, at concentrations higher than 100 μg/ml, HNP-1 and HNP-2 could kill the bacteria, including both intracellular and extracellular microorganisms (Lehrer et al. 1993). HNP-1 exerts potent in vitro microbicidal activity against a wide range of human pathogens, such as Staphylococcus aureus. Based on the evidence, HNP-1 could target and disrupt the bacterial membrane (Xiong et al. 1999). HNP1–4 and HD-5 show antibacterial activities against Gram-positive bacteria, such as S. aureus, and also Gram-negative bacteria as Enterobacter aerogenes and Escherichia coli (Ericksen et al. 2005). HNP1 was also highly effective against Mycobacterium tuberculosis (Sharma et al. 2000, 2001). HNP1–3 by binding to the lethal factor of the anthrax pathogen, Bacillus anthracis, was able to inhibition of its enzymatic activity (Verma et al. 2007). HD-5 shows potent antimicrobial activities against a wide range of pathogens such as E. coli, Listeria monocytogenes, Salmonella typhimurium, and C. albicans, whereas minimal inhibitory concentration (MIC) values are in the nanomolar range (Porter et al. 1997). hBD-1, -2, -3 showed antibacterial activities against E. coli and S. aureus in a dose-dependent manner (Chen et al. 2005). hBD-3 showed a broad spectrum of antimicrobial activities against different pathogenic microbes such as multiresistant S. aureus, vancomycin-resistant Enterococcus faecium, Pseudomonas aeruginosa, Klebsiella pneumonia, Streptococcus pneumoniae, and Burkholderia cepacia. This AMP has also antifungal activities against Candida glabrata and synergistic effects with fluconazole were observed (Dhople et al. 2006; Harder et al. 2001; Inthanachai et al. 2020; Pazgier et al. 2006b). The in vitro activities of hBD-3 alone or combined with other antimicrobial agents were investigated and the results showed that it was effective against Streptococcus mutans, Streptococcus sanguinis, Streptococcus sobrinus, Lactobacillus acidophilus, and Porphyromonas gingivalis. The bactericidal activities were enhanced in combination with the antimicrobial agents (Maisetta et al. 2003). Antimicrobial effect of hBD-4 on Fusobacterium nucleatum and P. gingivalis has been studied and the results showed antibacterial activity (Zhai et al. 2019). hBD‐4 exhibits potent antibacterial activity against P. aeruginosa (MIC = : 4.1 μg/ml) (Garcia et al. 2001). These AMPs also showed the anti-viral activities and at natural levels they could provide a minimal level of defense against viral infections (Park et al. 2018).

Mechanism of Action

It was assumed that the antibacterial activities the defensins are composed of a two-stage mode of action. It should be noted that there is the same mechanism of action for both types of defensins. At first, they bond to the outer membrane of the bacteria that leads to access to the inner (cytoplasmic) membrane. Then, by internalization into the cytoplasmic membrane, they form channels in the bacterial surfaces (Ryley 2001).

In more detail, these AMPs interact with the divalent cationic binding sites such as Ca2+ and Mg2+ in the lipopolysaccharide of bacterial surface and displacing these cations. In the case of Gram-positive bacteria, AMPs could interact with the anionic lipoteichoic acid of the cell resulting in access to the cytoplasmic membrane (Malanovic and Lohner 2016). It should be noted that the size of AMPs has direct influences on the distortion of the outer layer and then access to the underlying cytoplasmic layer (White et al. 1995).

The second stage is similar for both Gram-positive and -negative bacteria. The cationic residues interact with the negatively charged membrane and then because of high electrical potential, the AMPs being inserted into the membrane. Following the aggregation of AMPs in the membrane, they could form the channels, and finally, leads to membrane permeabilization and disruption (Ryley 2001).

Mechanism of Bacterial Resistance

The mechanisms of antibacterial resistance are poorly understood, however, it was assumed that changing in LPS structure leads to make it less susceptible to AMPs, and therefore binding of peptides is restricted and antibacterial resistance will be developed (Ryley 2001).

Cathelicidins

Cathelicidins, along with defensins belong to the group of cationic AMPs with amphipathic properties and recognize as an integral part of the immune system (De Smet and Contreras 2005). They are mainly stored in neutrophil and macrophage granules and show a direct antimicrobial activity against a wide range of microbial pathogens via the oxygen-independent activities (Bals and Wilson 2003; Tomasinsig and Zanetti 2005). Despite a large number of cathelicidin family members in animals, to date, only a single cathelicidin, LL-37, has been known in humans (Ryley 2001). This AMP is encoded by the cathelicidin gene (CAMP) which is composed of 39 residues, two leucine residues at N-terminal, and 37 residues long and has a molecular weight of 18 kDa (Agerberth et al. 1995). Subsequently proteolysis of the precursor, possibly by elastase digestion, in the neutrophil and missing the terminal residues, a 37 amino acid peptide was released and consequently termed LL-37 (Gudmundsson et al. 1996).

LL-37 has an α-helix structure that lacks cysteine. Therefore, it has a linear structure without disulphide bonds. It is expressed in leukocytes such as neutrophils, monocytes, NK cells, T cells, and B cells, and also, like hBD-2, in human epithelial tissue such as testis, skin, and the gastrointestinal and respiratory tracts in the presence of inflammation and is possibly related to the interleukin-6 production (De Smet and Contreras 2005; Frohm Nilsson et al. 1999).

Spectrum of Activity

LL-37 shows a wide spectrum of antibacterial activities against both Gram-positive and negative bacteria, with the MIC values lower than those of defensins (Turner et al. 1998). However, unlike defensins, it presents little activity against C. albicans or herpesviruses. This AMP is also inactive against some bacteria, like B. cepacia, that is naturally resistant to cationic peptides (Ryley 2001). In vitro antibacterial activities, LL-37 against Legionella pneumophila was tested and the results indicated that the AMP displayed broad-spectrum and in vitro activity against L. pneumophila (Birteksoz-Tan et al. 2019). Synergic enhancement of activity was observed between LL-37 and alpha-defensin against both E. coli and S. aureus (Nagaoka et al. 2000). In a study, the antibacterial effects of this peptide were evaluated against methicillin-resistant S. aureus (MRSA) and multidrug-resistant P. aeruginosa. Additionally, the antipseudomonal activities of colistin or imipenem combined to LL-37 were also studied. The results of the study revealed the rapid antibacterial effects of LL-37 against both antibiotic susceptible and resistant bacterial strains. When antibiotics were combined with LL-37, the MIC values of colistin and imipenem decreased up to eight-fold and four-fold, respectively (Geitani et al. 2019). In a study, the in vitro anti-Helicobacter pylori activity of LL-37 in simulated gastric juice was assessed and the results showed that after incubation the antibacterial activity was not retained (Leszczynska et al. 2009). It was shown that LL-37 and its fragments exerted antibacterial activities against drug-resistant Acinetobacter baumannii strains. This AMP and two of its fragments also could inhibit biofilm formation by A. baumannii (Feng et al. 2013). Microscopy studies show that the treatment of fluconazole-resistant Candida strains with LL-37 causes Candida cells to undergo surface changes that indicating surface membrane damage (Durnas et al. 2016). LL-37 is also effective against mycobacteria and can kill Mycobacterium smegmatis, Mycobacterium bovis BCG, and Mycobacterium tuberculosis under in vitro growth conditions, additionally, this AMP reduced the intracellular survival of the mycobacteria remarkably (Sonawane et al. 2011).

Mechanism of Action

They act as antimicrobial agents by either directly killing the pathogen or indirectly by binding to the bacterial exopolysaccharides, outer bacterial cell wall components such as the lipopolysaccharide (LPS) layer in the Gram-negative or the teichoic acids in the Gram-positive bacteria (Ramanathan et al. 2002; Xhindoli et al. 2016). Amphipathicity is an important factor for effective antibacterial properties. This attitude is more pronounced for α-helical cathelicidins such as LL-37. These peptides can form ion channels or aqueous pores in the bacterial membrane and rapidly permeabilize the membranes of microbial pathogens (Gennaro et al. 1998; Oren et al. 1999). Additionally, it was observed that LL-37 could strongly bind to zwitterionic or acidic phospholipid membranes of the vesicles and leads to leakage of vesicular content (Oren et al. 1999). Then the peptide forms quite sizeable toroidal pores. In more detail, at the first step, LL-37 is electrostatically attracted by membranes followed by assembly and partial integration. In the next step and after full integration into the lipid bilayer, the peptide forms channels based on peptide-peptide and peptide-lipid interactions. These interactions initiate the conformational changes and the formation of fiber-like oligomers on the inner membrane. The fibers lead to increasing the local concentration of the peptide that finally will interfere with the bacterial membrane stability. These transient pores allow the peptide to translocate into the bacterium. At this site, the peptide may interfere with internal targets such as DNA and also vital processes such as transcription (Brogden 2005; Wang 2008; Xhindoli et al. 2016). Moreover, in Gram-negative bacteria, LPS may be translocated apart from the bacterial cell wall to build holes for the LL-37 translocation into the periplasmic space (Vandamme et al. 2012).

Mechanism of Bacterial Resistance

It was assumed that the changes in the structure of LPS in Gram-negative bacteria affect the binding properties of LL-37. For example, surface structures containing phosphorylcholine, a component of eukaryote cell membranes, have been found in some respiratory pathogens such as Haemophilus influenza. The Mutants that do not express this structure were 1000-fold more sensitive to LL-37 than those having this lipid in their membrane (Lysenko et al. 2000). It was also shown that Neisseria gonorrhoeae has a type of efflux pump system that can reduce the susceptibility of the bacteria to LL-37 (Miyasaki et al. 1990). In Bordetella pertussis, d-alanine incorporation induces resistance to the AMPs. It was demonstrated that the dra operon of B. pertussis is responsible for the d-alanylation of the outer membrane component. Mutation in this operon results in increasing sensitivity of bacteria to LL-37, and other AMPs (Taneja et al. 2013).

Additionally, the antibacterial resistance against AMPs might be due to the production of degradation enzymes such as proteases AMPs. Bacterial strains can promote bacterial resistance to the AMPs by their proteolytic degradation properties (Abdi et al. 2019).

Histatin

Histatins are a family of cationic small AMPs that are largely composed of histidine-rich repeats. They have a molecular weight of about 3–4 kDa and are produced by the submandibular, sublingual, and parotid glands and secreted into human saliva (De Smet and Contreras 2005).

These AMPs consist of several members, however, among them, histatin 1, 3, and 5 are the most important ones. They have linear structures and composed of 38, 32, and 24 amino acid residues for histatin 1, 3, and 5, respectively and each of them contains seven histidine residues. Histatin 1 and 3 are encoded by two highly related genes, HIS1 and HIS2, respectively and others are produced by the cleavage of these two peptides. For example, histatin 5 was proteolytic digestion of histatin 3 (Wang 2014). Histatin 5 has a unique secondary structure. This AMP forms a random coil structure in aqueous solvents while in non-aqueous solvents the structure converts to α-helix (Helmerhorst et al. 1997; Raj et al. 1998).

Spectrum of Activity

These peptides possess some bactericidal activities and, more importantly, fungicidal properties. It should be noted that of all histatins, histatin 5 has the strongest antimicrobial activity, and most of the research on histatins has focused on this peptide (Khurshid et al. 2016; Oppenheim et al. 1988). The in vitro study indicated that histatin 5 could inhibit Candida species (C. albicans, C. glabrata, C. krusei) at a certain concentration (15–30 μM). It was also demonstrated that the analog of histatin 3 with the shorter amino acid sequences has the same candidacidal activity with respect to the full-length molecule (Raj et al. 1990).

These AMPs possess their anti-fungal activities in different ways such as growth inhibitory actions on C. albicans or inhibition of the conversion of C. albicans yeast growth into hyphal growth (Moffa et al. 2015).

The antimicrobial activities against other pathogens Cryptococcus neoformans and Aspergillus fumigatus has also been reported (Helmerhorst et al. 1999b). Also, histatin 5 virucidal activity and only histatin 5 derived peptides affect HIV-1 (Wang 2014). Inhibitory and bactericidal activities of histatins have also been reported against various bacteria, including S. mutans, P. gingivalis, P. aeruginosa, E. coli, and S. aureus (Gusman et al. 2001; MacKay et al. 1984; Sajjan et al. 2001).

Mechanism of Action

Unlike other AMPs, it is well-known that all targets of histatins are intracellular and the primary mode of actions is not related to lyse lipid membranes (Puri and Edgerton 2014; Ryley 2001; Wang 2014). It has been found that the massive non-lytic release of ATP along with the decrease in intracellular ATP levels is the main mechanism of actions of histatins (Helmerhorst et al. 1999a; Koshlukova et al. 1999). It was shown that histatin 5 could bind to the cell wall proteins and glycans of C. albicans and then is taken up by the fungal polyamine transporters in the cells in an energy-dependent manner. Inside the fungal cells, the AMP may affect the mitochondrial functions and leads to oxidative stress. However, cell death is the result of other processes such as volume dysregulation and ion imbalance caused by osmotic stress. Additionally, the metal-binding ability of histatin 5 is the other mentioned mechanism (Edgerton and Koshlukova 2000; Khurshid et al. 2017a; Komatsu et al. 2011; Puri and Edgerton 2014). Taken together, the key steps in the histatin 5 antifungal activity involve a bioenergetic collapse of C. albicans, consequently decreasing the mitochondrial ATP synthesis.

The other reported fungistatic and fungicidal mechanisms include disruption of the plasma membrane, which leads to the loss of intracellular constituents (Oppenheim et al. 1988). It was also found that these AMPs were also effective in killing the yeast cells by damaging their membranes and releasing potassium ions. These effects were related to the binding to the Trk1 potassium transporter and then the loss of intracellular components (Swidergall and Ernst 2014). The other modifications caused by histatin 5 in C. albicans included organelles in disarray, intracellular membrane disarrangement, and central cavities with deformed structures displaced to the cell periphery (Isola et al. 2007).

It was found that the histatins, like other AMPs, could interact with extracellular actin and the extracellular actin might regulate histatin anti-fungal activities in the oral cavity. In a study, it was demonstrated that both histatin 3 and 5 interact with actin, however, and affect the actin structure (Blotnick et al. 2017).

Mechanism of Microbial Resistance

It was shown that cellular accumulation of the peptides is necessary for anti-fungal activities and also that accumulation of histatins depends on the availability of cellular energy. Therefore, respiratory mutants of the microorganisms which have undergone mutations in mitochondrial DNA exhibited resistance against histatins (Gyurko et al. 2000). Therefore, it was assumed that the altered membrane energetics is an important factor for developing resistance (Yeaman and Yount 2003). Additionally, histatin 5 is transported out of C. albicans cells by the Flu1 and Mrr1 efflux pumps (Hampe et al. 2017; Li et al. 2013). The multidrug resistance transporters CgTpo1_1 and CgTpo1_2 have critical roles in the virulence of C. glabrata infections such as resistance to histatin 5 (Santos et al. 2017). It was demonstrated that the upregulation of CgCDR efflux pumps could develop the resistance to histatin 5 in C. glabrata (Helmerhorst et al. 2006).

AMPs Challenges

Although AMPs often present antibacterial activities, own several unfavorable features for clinical applications, including (I) the pharmacokinetic profiles of antimicrobial peptides are not well known, and only a few clinical research have been performed with AMPs; (II) sensitivity to proteolysis process derived by bacterial proteases; and (III) cytotoxicity to the eukaryotic cell, which could result in neurotoxicity, nephrotoxicity, and hemolysis (Craik et al. 2013; Grassi et al. 2017). Nevertheless, several types of research have been conducted for improving our knowledge of the above-mentioned challenges (Chou et al. 2008). In comparison to conventional antibiotics, AMPs are also expensive for being synthesized on mass scales (Otvos and Wade 2014). Various strategies have been persuaded to overcome these limitations. For this scenario, employing nanocarriers, developing pegylated AMPs, and covalent immobilization of AMPs on surfaces have been mentioned (Long et al. 2017; Manteghi et al. 2020).

Covalent attachment of the polyethylene glycol polymer to peptides and proteins (PEGylation) could improve the bioavailability of AMPs and enhance their pharmacokinetic properties such as bio-distribution and rate of clearance. Additionally, the proteolytic degradation of AMPs might be decreased by protecting their C- and N-terminus (Gong et al. 2015; Gonzalez-Valdez et al. 2012; Khameneh et al. 2015b).

The progress in nanotechnology has permitted various nanoparticles to work as a favorable approach for minimizing the unfavorable features of synthetic as well as natural AMPs (Umerska et al. 2017). The researchers have suggested that AMPs in nanoparticles represent increased efficiency, decreased degradation, and lower toxicity (Wang et al. 2017). As a result, nanoparticles could contribute to the manufacturing of AMPs and their applications in the industry. Nano-carriers are considered as a drug delivery system that presents many profits, like improvement of drug pharmacokinetic profile, treatment selectivity, and the protection of AMPs against extracellular degradations (Sadat et al. 2016). Over the last 50 years, multiple drug delivery systems have been used for encapsulating drugs and other biomolecules, including polymeric nanoparticles, dendrimers, micelles, and liposomes (Malaekeh-Nikouei et al. 2020).

Nanobiotechnology has proposed two major procedures to encapsulate AMPs. The passive delivery, as non-directed procedures, includes traditional nano-delivery systems that do not own surface modifications for guiding the nano-carriers; this could be manipulated by control the nano-carriers shape as well as size (Makowski et al. 2019). Active targeting, as directed delivery, includes surface modifications of the nano-carriers with various ligands and/or other moieties for permitting interaction of nano-carriers and the target sites (Biswaro et al. 2018). However, developing viable drug delivery systems for clinical trials remains a challenge (Wimley and Hristova 2011). There are two procedures of nano-delivery systems that have some advantages and disadvantages (Makowski et al. 2019). Passive systems attend to own fewer agents in their combination in contrast to active systems (Biswaro et al. 2018). As passive systems possess fewer agents in their combination, this causes it easier for preparing them (Makowski et al. 2019). Besides, active systems contain modified surface-carrying ligands and/or other moieties for facilitating its interactions with infected cells and increase the drug transports at such particular sites (Biswaro et al. 2018), while passive systems involve encapsulating peptides without making available extra surface modifications (Hunter et al. 2012). Given the above-mentioned, the terms nano-delivery system as well as nano-carriers have been suggested (Makowski et al. 2019).

Conclusion

Clinical overuse of antibiotics unavoidably results in the growing emergence of drug-resistant strains of microbial pathogens. Consequently, the development of a new class of antibiotics is an urgent need. During the last decades, considerable efforts have been conducted for investigating the possible use of AMPs or synthetic derivatives as therapeutic antibacterial agents. They are implicated in several biological processes, and also have an important role in the innate immune system. The AMPs protect the host against both systemic and topical infections either alone or in combination with conventional antibiotics. Human AMPs are an important class of these peptides which attract attention for the treatment of various disease and some of them have entered clinical trials for use in clinical applications such as the treatment of diabetic ulcers as topical anti-infective agents for the treatment of microbial infections. The ongoing discovery and development of new AMPs are promising approaches in the fight against increasingly resistant pathogens.