Antimicrobial Peptides: Classification, Design, Application and Research Progress in Multiple Fields

Antimicrobial peptides (AMPs) are a class of small peptides that widely exist in nature and they are an important part of the innate immune system of different organisms. AMPs have a wide range of inhibitory effects against bacteria, fungi, parasites and viruses. The emergence of antibiotic-resistant microorganisms and the increasing of concerns about the use of antibiotics resulted in the development of AMPs, which have a good application prospect in medicine, food, animal husbandry, agriculture and aquaculture. This review introduces the progress of research on AMPs comprehensively and systematically, including their classification, mechanism of action, design methods, environmental factors affecting their activity, application status, prospects in various fields and problems to be solved. The research progress on antivirus peptides, especially anti-coronavirus (COVID-19) peptides, has been introduced given the COVID-19 pandemic worldwide in 2020.

The diversity of natural AMPs causes difficulty in their classification. AMPs are classified based on (1) source, (2) activity, (3) structural characteristics, and (4) amino acid-rich species ( Figure 1 ). FIGURE 1 Classification of antimicrobial peptides. Classification of AMPs Based on Sources The sources of AMPs can be divided into mammals (human host defense peptides account for a large proportion), amphibians, microorganisms, and insects according to statistical data in APD3. The AMPs found in oceans have also attracted widespread attention. Mammalian Antimicrobial Peptides Mammalian antimicrobial peptides are found in human, sheep, cattle, and other vertebrates. Cathelicidins and defensins are the main families of AMPs. Defensins can be divided into α-, β-, and θ-defensins depending on the position of disulfide bonds ( Reddy et al., 2004 ). Human host defense peptides (HDPs) can protect human from microbial infections but show different expressions in every stage of human growth. For example, cathelicidin LL-37, a famous AMP derived from the human body, is usually detected in the skin of newborn infants, whereas human beta-defensin 2 (hBD-2) is often expressed in the elderly instead of the young ( Gschwandtner et al., 2014 ). HDPs can be identified in many parts of the body such as skin, eyes, ears, mouth, respiratory tract, lung, intestine, and urethra. Besides, AMPs in human breast milk also play an important role in breastfeeding because it can decrease the morbidity and mortality of breast-feeding infants ( Field, 2005 ). What’s interesting is that Casein201 (peptide derived from β-Casein 201–220 aa), identified in colostrum, shows different levels in preterm human colostrum and term human colostrums ( Zhang et al., 2017 ). Dairy is an important source of AMPs, which are generated through milk enzymatic hydrolysis. Several AMPs have been identified from α-lactalbumin, β-lactoglobulin, lactoferrin, and casein fractions, and the most famous peptide obtained is lactoferricin B (LfcinB) ( Sibel Akalın, 2014 ). Furthermore, whether the AMPs derived from dairy products can be used for dairy preservation is also an interesting subject to develop. In addition to antimicrobial activity, HDPs, such as cathelicidins and defensins, also affect immune regulation, apoptosis, and wound healing ( Wang, 2014 ).

Antimicrobial peptides are mainly synthesized in fat bodies and blood cells of insects, which is one of the main reasons for insects’ strong adaptability to survival ( Vilcinskas, 2013 ). Cecropin is the most famous family of AMPs from insects, and it can be found in guppy silkworm, bees, Drosophila . Cecropin A shows activity against different inflammatory diseases and cancers ( Dutta et al., 2019 ). What should be known is that the number of AMPs varies greatly between species, for example, invasive harlequin ladybird ( Harmonia axyridis ) and black soldier fly ( Hermetia illucens ) have up to 50 AMPs, while pea aphid ( Acyrthosiphon pisum ) lacks AMPs ( Shelomi et al., 2020 ). Jellein, a peptide derived from bee royal jelly, shows promising effects on several bacteria and fungi, and its lauric acid-conjugated form can inhibit the parasite Leishmania major ( Zahedifard et al., 2020 ).

The activity of AMPs can be divided into 18 categories according to the statistics of the ADP3 database. These categories can be summarized as antibacterial, antiviral, antifungal, antiparasitic, anti-human immunodeficiency virus (HIV), and anti-tumor peptides ( Figure 2 ). FIGURE 2 Statistics of the main functions of antimicrobial peptides. Antibacterial peptides account for the largest proportion, approximately 60%, followed by antifungal peptides, which account for 26%, and antiviral, antiparasitic, anticancer, anti-HIV peptides account for almost the same about 2–5% (the figure is drawn based on data in APD3). Antibacterial Peptides Antibacterial peptides account for a large part of AMPs and have a broad inhibitory effect on common pathogenic bacteria, such as VRE, Acinetobacter baumannii , and MRSA in clinical medicine and S. aureus , Listeria monocytogenes , E. coli in food and Salmonella , Vibrio parahaemolyticus in aquatic products. Many natural and synthetic AMPs like nisin, cecropins and defensins have shown good inhibition activity to Gram-positive bacteria and Gram-negative bacteria. In recent research, AMPs P5 (YIRKIRRFFKKLKKILKK-NH 2 ) and P9 (SYERKINRHFKTLKKNLKKK-NH 2 ), which are designed based on Aristicluthys nobilia interferon-I, inhibit MRSA and show a low cytotoxicity ( Li C. et al., 2019 ).

Viruses cause serious harm to human life and huge economic losses to the animal husbandry. The COVID-19, which is the recent outbreak, has caused great loss of lives and properties. Furthermore, foot-and-mouth disease virus, avian influenza virus (AIV), and HIV are long-term threats to human life. So, it is extremely urgent to solve these problems, and antiviral peptides provide new ways. Antiviral peptides show a strong killing effect on viruses mainly by (1) inhibiting virus attachment and virus cell membrane fusion, (2) destroying the virus envelope, or (3) inhibiting virus replication ( Jung et al., 2019 ) (shown in Figure 3 ). A recent report has shown that AMP Epi-1 mediates the inactivation of virus particles and has good inhibitory activity against foot-and-mouth disease virus ( Huang et al., 2018 ). Moreover, infectious bronchitis virus (IBV) is the pathogen of infectious bronchitis and the inoculation of swine intestinal AMP (SIAMP)–IBV mixed solution remarkably reduced the mortality of chicken embryos compared with the IBV infection group, showing the good inhibitory activity of SIAMP on IBV ( Sun et al., 2010 ). Anti-HIV peptides are a subclass of anti-viral peptides. The most important examples of these peptides include defensins (including α- and β-defensins, which have different mechanisms), LL-37, gramicidin D, caerin 1, maximin 3, magainin 2, dermaseptin-S1, dermaseptin-S4, siamycin-I, siamycin-II, and RP 71955 ( Madanchi et al., 2020 ) and antiviral peptide Fuzeon TM (enfuvirtide) has been commercialized as an anti-HIV drug ( Ashkenazi et al., 2011 ). FIGURE 3 Examples of specific targets for Antiviral peptides. Due to the global spread of the COVID-19 ( Figure 4A ), the antiviral peptides against the coronavirus will be discussed in more detail. Coronaviruses (CoVs) belong to the family Coronaviridae; they are enveloped viruses with a positive-sense single-stranded RNA genome and have a helical symmetry ( Franks and Galvin, 2014 ). CoVs, including severe acute respiratory syndrome CoV (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) ( Mustafa et al., 2018 ), and the recent outbreak of COVID-19 have caused serious threats to human life and property. CoVs can cause life-threatening respiratory diseases and the viral particle is formed by spike glycoprotein (S), the envelope (E), the membrane (M), and the nucleocapsid (N) ( Vilas Boas et al., 2019 ). It should be noted that their infectivity requires viral spike (S) protein. Fusion inhibitor peptides combine with the S protein to interfere with its folding and prevent infection. Besides, the S2 domain of the SARS-CoV S protein contains heptad repeat HR1 and HR2 sequences. Peptide HR2 (HR2: SLTQINTTLLDLTYEMLSLQQVVKALNESYIDLKEL) and its lipid-binding peptide is highly similar or even identical to the near-membrane portion of S protein ferredoxin, which interferes with refolding into post-fusion fusion-catalyzing domains (FDs) ( Du et al., 2009 ; Park and Gallagher, 2017 ). According to recent research, the lipopeptide EK1C4, derived from EK1 (SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL), is the most effective fusion inhibitor against COVID-19 S protein-mediated membrane fusion ( Xia et al., 2020 ). Homology modeling and protein-peptide docking showed that temporin has potential therapeutic applications against MERS-CoV ( Marimuthu et al., 2019 ). Two AMPs from the non-structural protein nsp10 of SARS-CoV, K12, and K29, can inhibit SARS-CoV replication ( Ke et al., 2012 ). Furthermore, rhesus theta-defensin 1 (RTD-1) treated animals have a marked reduction in mortality in the presence of SARS-CoV while the peptide alone shows airway inflammation and the one possible mechanism of action for RTD-1 is immunomodulatory ( Wohlford-Lenane et al., 2009 ). In general, AMPs against coronavirus can be roughly classified as i) peptides derived from HR1, HR2 and RBD subunits of the spike protein, ii) peptides derived from other AMPs, iii) Peptides derived from non-structural protein ( Mustafa et al., 2018 ). Furthermore, molecular docking analysis indicated that peptides were employed to disrupt the interaction between COVID-19 and ACE2 (angiotensin-converting enzyme 2) to inhibit COVID-19 entrance in cells ( Figure 4B ) ( Souza et al., 2020 ). Finally, it should be noted that this therapy lacks clinical trials and the main method of animal experiments is an intranasal administration. This reminds us that nasal drug delivery (NDD) is a potential therapy for AMPs as anti-coronavirus drugs. Besides, the antiviral database AVPdb 2 includes numerous antiviral peptides. FIGURE 4 Information of COVID-19. (A) C-F13-nCoV Wuhan strain 02, Strain Number: CHPC 2020.00002; NPRC 2020.00002, Source: National Pathogen Resource Collection Center (National Institute for Viral Disease Control and Prevention under Chinese Center for Disease Control and Prevention). (B) Structure of novel coronavirus spike receptor-binding domain complexed with its receptor ACE

The ACPs show anticancer mechanisms by (1) recruiting immune cells (such as dendritic cells) to kill tumor cells, (2) inducing the necrosis or apoptosis of cancer cells, (3) inhibiting angiogenesis to eliminate tumor nutrition and prevent metastasis, and (4) activating certain regulatory functional proteins to interfere with the gene transcription and translation of tumor cells ( Wu D. et al., 2014 ; Ma et al., 2020 ). Tritrpticin and its analogs induce considerable toxicity toward Jurkat cells in vitro , whereas indolicidin and puroindoline A can also act as ACPs ( Arias et al., 2020 ). It should be noted that both net charge and hydrophobicity play important roles in optimizing the anticancer activity of ACPs and they can constrain and influence each other. Thus, achieving a balance between net charge and hydrophobicity is important for better anticancer activity. Besides the peptide mentioned above, anti-inflammatory, anti-diabetic peptides, spermicidal peptides etc. have been noticed, but they are not the same as antimicrobial peptides. Simply put, anti-inflammatory peptides decrease the release of inflammatory mediators and inflammatory cytokines (nitric oxide, interleukin-6, and interleukin-1β) and some of them also inhibit inflammatory signals like NF-κB, MAPK, and JAK-STAT pathways ( Meram and Wu, 2017 ; Gao et al., 2020 ). Anti-diabetic peptides play their function by modulating the G protein-coupled receptor kinase (GRK 2/3) or activating glucagon-like peptide-1 (GLP-1), glucagon receptors ( Marya et al., 2018 ; Graham et al., 2020 ). However, it is not accurate to classify these types of peptides as AMPs and bioactive peptides may be more convincing.

Tryptophan (Trp), as a non-polar amino acid, has a remarkable effect on the interface region of the lipid bilayer, whereas Arg, as a basic amino acid, confers peptide charge and hydrogen bond interactions, which are essential properties to combine with the bacterial membrane’s abundant anionic component. And it seems that Trp residues play the role of natural aromatic activators of Arg-rich AMPs by ion-pair-π interactions ( Walrant et al., 2020 ), thereby promoting enhanced peptide-membrane interactions ( Chan et al., 2006 ). In addition to indolicidin and Triptrpticin which both are famous AMPs that rich in Arg and Trp residues. Octa 2 (RRWWRWWR) is also a typical Trp- and Arg-rich AMP that inhibits Gram-negative E. coli and Pseudomonas aeruginosa and Gram-positive S. aureus . And short Trp- and Arg-rich AMPs designed based on bovine and murine lactoferricin have also shown strong inhibitory action against bacteria ( Strøm et al., 2002 ; Bacalum et al., 2017 ).

The R group of glycine is generally classified as a non-polar amino acid in biology. Glycine-rich AMPs, such as attacins and diptericins, widely exist in nature ( Lee et al., 2001 ; Kwon et al., 2008 ). These peptides contain 14% to 22% glycine residues, which have an important effect on the tertiary structure of the peptide chain. A glycine-rich AMP derived from salmonid cathelicidins activates phagocyte-mediated microbicidal mechanisms, which differ from the mechanism of conventional AMPs ( D’Este et al., 2016 ). Furthermore, the glycine-rich central–symmetrical GG3 is an ideal commercial drug candidate against clinical Gram-negative bacteria ( Wang et al., 2015 ).

Membrane Targeting Mechanism The membrane-targeting mechanisms of AMPs can be described through models, including the pole and carpet models and the pole model can be further divided into the toroidal pore and barrel-stave models ( Figure 6 ). FIGURE 6 Models of action for extracellular AMP activity. (A) Carpet model: accumulation of AMPs on the surface and then destroy the cell membrane in the manner of “detergent”. (B) Barrel stave model: AMPs aggregate with each other and are inserted into the bilayer of the cell membrane in the form of multimers and arrange parallel to the phospholipids, then form a channel. (C) Toroidal pore model: accumulation of AMPs vertically embed in the cell membrane, and then, bend to form a ring hole. The Toroidal Pore Model The toroidal pore model is also known as the wormhole model. In this model, AMPs vertically embedded in the cell membrane accumulate and then bend to form a ring hole with a diameter of 1–2 nm ( Matsuzaki et al., 1995 , 1996 ). The typical examples of this model are magainin 2, lacticin Q, and arenicin. Furthermore, cationic peptides, including TC19, TC84, and BP2, compromise the membrane barrier by creating fluid domains ( Omardien et al., 2018 ).

Antimicrobial peptides are arranged parallel to the cell membrane. Their hydrophilic end faces the solution, and their hydrophobic end faces the phospholipid bilayer. AMPs will cover the membrane surface that similar to a carpet and destroy the cell membrane in a ‘detergent’-like manner ( Oren and Shai, 1998 ). However, this pore-forming mechanism requires a certain concentration threshold and the required concentration of AMPs is high. Human cathelicidin LL-37 exhibits its activity through this mechanism, and AMPs with β-sheet structure also play a role in this model ( Shenkarev et al., 2011 ; Corrêa et al., 2019 ). Polarized light-attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) was used to study the effect of AMP cecropin P1 on the bacterial cell membrane and found that it was an applied flat on the surface of the pathogen’s cell membrane to destabilize and eventually destroy the cell membrane ( Lyu et al., 2019 ). Membrane targeting mechanisms (the cell membrane composition differences of bacteria and fungi shown in Figure 7 ) can be further refined to address the large differences in the lipid composition of the cell membranes of bacteria, fungi, and mammals. The main lipids in cell membranes include glycerophospholipids (GPLs), lysolipids, sphingolipids, and sterols. Phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cardiolipin (CL) are the most common anionic lipids in bacteria, whereas phosphatidylcholine (PC), phosphatidylinositol (PI), PE, and phosphatidic acid (PA) are the main GPLs in fungal cell membranes ( Ejsing et al., 2009 ; Singh and Prasad, 2011 ; Li et al., 2017 ). Furthermore, fungal cell membranes are more anionic than mammalian cell membranes and have higher PC content. Meanwhile, ergosterol is the sterol found in the plasma membrane of lower eukaryotes, such as fungi, whereas that of animals contains cholesterol ( Faruck et al., 2016 ). Many AMPs take advantage of differences in membrane components to exert their effects. FIGURE 7 Comparison of Gram-negative bacteria, Gram-positive bacteria and fungi cell walls. Antimicrobial peptides are promising to be anti-biofilm agents but it should be noticed that they are different from the cell penetrating peptides (CPPs) which typically comprise 5–30 amino acids and can translocate across the cell membrane. CPPs could be categorized according to physicochemical properties into three classes: Cationic, amphipathic, and hydrophobic, but anti-biofilm peptides have stricter requirements for these physicochemical properties. Anti-biofilm peptides target the biofilms by different mechanisms including (1) degradation of signals within biofilms; (2) permeabilize within cytoplasmic membrane/EPS; (3) modulating EPS production etc. and then can address chronic multi-resistant bacterial infections ( Pletzer et al., 2016 ; Ribeiro et al., 2016 ; Guidotti et al., 2017 ; Derakhshankhah and Jafari, 2018 ; Rajput and Kumar, 2018 ). For instance, SAAP-148, synthesized based on LL-37, showed activity to prevent biofilm formation by S. aureus and A. baumannii ( Crunkhorn, 2018 ).

Antimicrobial peptides can affect key enzymes or induce the degradation of nucleic acid molecules to inhibit nucleic acid biosynthesis. Indolicidin, a C-terminal-amidated cationic Trp-rich AMP with 13 amino acids, specifically targets the abasic site of DNA to crosslink single- or double-stranded DNA and it can also inhibit DNA topoisomerase I ( Subbalakshmi and Sitaram, 1998 ). TFP (Tissue factor pathway inhibitor)1-1TC24, which is an AMP from tongues, enters the cytoplasm of target cells after the rupture of cell membrane and then degrades DNA and RNA ( He et al., 2017 ).

Antimicrobial peptides inhibit cell division by inhibiting DNA replication and DNA damage response (SOS response), blocking the cell cycle or causing the failure of chromosome separation ( Lutkenhaus, 1990 ). For instance, APP (GLARALTRLLRQLTRQLTRA), which is an AMP with 20 amino acid residues, can efficiently kill C. albicans because of its cell-penetrating efficiency, strong DNA-binding affinity, and ability to induce S-phase arrest in intracellular environment ( Li L. et al., 2016 ). MciZ, which has 40 amino acid residues, is an effective inhibitor of bacterial cell division, Z-ring formation, and localization ( Cruz et al., 2020 ). Moreover, it has been reported that several AFPs have damaging effects on the organelles of fungi. For example, Histintin 5 can interact with mitochondria, causing the production of ROS, and inducing cell death ( Helmerhorst et al., 2001 ). In addition to intracellular targets, differences in cell wall composition, such as lipopolysaccharide (LPS), lipid A and mannoproteins, are potential targets for AMPs. Specifically, Gram-positive and Gram-negative bacteria are classified based on their bacterial cell wall structure. Gram-positive bacteria have a layer of cross-linked peptidoglycan, whereas Gram-negative bacteria have an additional outer membrane with an inner leaflet containing only phosphatidic acid and an outer leaflet made of LPS. LPS has numerous negatively charged phosphate groups, which combine with a salt bridge with a divalent cation (e.g., Ca 2+ and Mg 2+ ) to form an electrostatic network ( Nikaido, 2003 ). This electrostatic zone is the main barrier against hydrophobic antibiotics and causes the low permeability of Gram-negative bacteria. The main components of the fungal cell wall are mannoprotein, β-glucans and chitin (polymers of 1,4-β- N -acetylglucosamine) and the mutations in the relevant genes of the LPS pathway and phospholipid trafficking provide resistance to the AMPs ( Cabib, 2009 ; Spohn et al., 2019 ). Mannoproteins in fungal cell walls include a variety of proteins, including structural proteins, cell adhesion proteins (floccrin and lectin) and enzymes involved in cell wall synthesis and remodeling (hydrolytic enzymes and transglycosylase). These proteins differ from human cell membrane proteins and are potential targets of AFPs ( Rautenbach et al., 2016 ). Furthermore, teichoic acid and lipoteichoic acid in the cell wall are also potential targets of AMPs and these theories could support the design of AMPs with low cytotoxicity.

The de novo design of peptides attaches importance to the design of amphiphilic AMPs ( Guha et al., 2019 ). For example, GALA is a well-known de novo -designed AMP. Amphipathic α-helical peptide GALA is created by placing protonatable glutamic acid residues in most positions with the spacing of i to i + 4 ( Goormaghtigh et al., 1991 ). The repeated sequence (XXYY)n, where X 1 and X 2 are hydrophobic amino acids, Y 1 and Y 2 are cationic amino acids, and n is the number of repeat units, is designed based on the hydrophobicity cycle that mimics natural α-helical AMPs and successfully designs broad-spectrum α-helical AMPs. Sequences (LKKL) 3 and (WKKW) 2 . 5 have the highest selectivity ( Khara et al., 2017 ). Moreover, L l K m W 2 model peptides are also de novo -designed peptides. Amphipathic helical properties were conferred by using leucines and lysines, and two tryptophan residues were positioned at the amphipathic interface between the hydrophilic ending side and the hydrophobic starting side. Among the model peptides, L 4 K 5 W 2 has good anti-MRSA activity ( Lee et al., 2011 ).

Peptides can form nanostructures, such as micelles, vesicles, nanotubes, nanoparticle nanobelt, and nanofibre nanotube, and can increase or impart antibacterial activity to AMPs during the self-assembly of peptides. For example, KLD-12 (KLD) is a self-assembling peptide with 12 amino acid residues that can adopt nanostructures and are known for their tissue engineering properties. The addition of Arg residues in KLD shows no remarkable change in its β-sheet secondary structure and the self-assembly characteristics of the forming nanostructures ( Tripathi et al., 2015 ). Dimer structure can also be used to enhance the antimicrobial activity of AMPs and reduce toxicity, but membrane-destabilizing effects are reduced after dimer formation ( Malekkhaiat Häffner and Malmsten, 2018 ).

Three modes of cyclisation, including cyclisation via disulfide bonds, head-to-tail cyclisation and internal bonding between side chains, have been found in natural AMPs. The synthesis of disulfide bonds often complicates the development of synthetic peptides. The circularisation of the main chain of arenicin-1 molecule resulted in increased activity against drug-resistant clinical isolates but caused no substantial effect on cytotoxicity ( Orlov et al., 2019 ). The HDPs tachyplesins I, II, and III and their cyclic analogs cTI, cTII, and cTIII, respectively, have similar structures and activities and can resist bacterial and cancer cells. The cyclisation of the backbone reduces the hemolytic activity and improves the stability of the peptides whilst maintaining effective anticancer and antibacterial activities ( Vernen et al., 2019 ).

Peptide conjugation has been the goal of most research in recent years to produce active and stable AMPs with high selectivity. Different side chains or AMP fragments can be used aside from the repetition of the same amino acid motifs. For example, conjugating fatty acids with a length of 8–12 carbon atoms to the 4th or 7th side chain of the D-amino acids of Ano-D 4 , 7 improves antibacterial selectivity and anti-biofilm activity. In addition, the new peptide exhibits high stability against trypsin, serum, salt, and different pH environments ( Zhong et al., 2020 ). The conjugation of different AMPs can also be performed. For example, the hybrid peptide (PA2–GNU7) constructed by the addition of PA2 to GNU7 has a high activity and specificity to P. aeruginosa ( Kim et al., 2020 ).

Motifs with specific functions have been reported increasingly. These motifs can be repeated units for combining into new antimicrobial peptides, or specific amino acid combination units appearing at the end (such as capping) of or even in the peptide chain. Motif at the End of the Chain ATCUN motif This motif includes two tripeptide structures, including Gly–Gly–His or Val–Ile–His, which are added at the end of the peptide chain. ATCUN-containing AMPs in the presence of hydrogen peroxide and ascorbic acid combine with Cu 2+ to induce the valence of copper ions between +2 and +3 oxidation states and form an ATCUN–Cu (II) complex, generating ROS by Fenton-like reactions. Extracellular polymeric substances (EPS) are important for biofilms and can enhance the resistance of cells to antibacterial agents ( Flemming, 2016 ). ATCUN–AMPs have been used to degrade environmental DNA, which is one of the major components of EPS. Several related practical applications have been reported. For example, the biological activity against carbapenem-resistant Enterobacteriaceae is increased by adding this motif to the N-terminus of an alpha-helical AMP (such as CM15). Besides, the Cu–ATCUN derivative of OV-3 containing a C-terminal GGC sequence showed high levels of membrane permeation and lipid peroxidation. The concept of catalytic metal drugs has attracted widespread attention although the concept is still in its infancy because of the role of metal ions ( Alexander et al., 2017 ; Agbale et al., 2019 ).

The LPS binding motif (G-WKRKRF-G) can produce a broad spectrum of antibacterial activity when introduced into the C-terminus of temporin-1 Ta and temporin-1 Tb (close isoforms of temporin) ( Mohanram and Bhattacharjya, 2016 ).

If replace d-Phe1-Pro2 sequence in peptide chain with d-Phe-2-Abz turn motif (2-Abz is an abbreviation of 2-aminobenzoic acid D -amino acid) in AMP Tyrc A, and nuclear magnetic resonance shows that this change retains the β-hairpin structure. Unlike the traditional β-turn motif, the D-Phe-2-Abz motif can be used as a tool for β-hairpin libraries. The hydrophobic peptide can be formed into the nucleated β-hairpin formation by adding the D-Phe-2-Abz motif. Moreover, the inclusion of this part in two designed cationic amphiphilic peptides can produce broad-spectrum antibacterial activity and low hemolysis rate ( Cameron et al., 2017 ; Cameron et al., 2018 ). NGR motif The NGR motif is composed of Asn–Gly–Arg, and AMPs with this structure have strong cytotoxicity ( Table 1 ). The data indicate that the new AMPs containing NGR may bind to CD13+ or αvβ3+ tumor cells by binding to CD13 or αvβ3, respectively, to exert anti-tumor activity, especially on CD13+ tumor cells ( Zhang et al., 2015 ). TABLE 1 Examples of AMPs with NGR motif. Name Sequence 3D structure Activity Reference CORTICOSTATIN I ICACRRRFCPNSERFSGY CRVNGARYVRCCSRR Bridge Anti-Gram+ and Gram− Zhu et al., 1988 NP-3a GICACRRRFCPNSERFSGY CRVNGARYVRCCSRR Bridge Anti-Gram+ and Gram−, Antiviral, and Antifungal Selsted et al., 1985 Corticostatin VI GICACRRRFCLNFEQFSG YCRVNGARYVRCCSRR Bridge Anti-Gram+ and Gram− Fuse et al., 1993 Pediocin PA-1/AcH KYYGNGVTCGKHSCSVDWGKA TTCIINNGAMAWATGGHQGNHKC Combine Helix and Beta structure Anti-Gram+, Spermicidal Henderson et al., 1992 Lacticin 3147 CSTNTFSLSDYWGNNGA WCTLTHECMAWCK Helix Anti-Gram+, Spermicidal Martin et al., 2004 As-CATH5 TRRKFWKKVLNGALKIAPFLLG Helix Anti-Gram+ and Gram−, Antifungal, anti-sepsis Chen Y. et al., 2017

Bovine lactoferrin B is an AMP composed of 25 amino acid residues and has antibacterial, antifungal, and antiparasitic activities. The multivalent molecules LfcinB (20–25) 2 and LfcinB (20–25) 4 contain the LfcinB (20–25) motif (RRWQWR) and show inhibition activity against E. coli , P. aeruginosa , and S. aureus . Chimeric peptide chimera 3 containing two motifs, namely, the RRWQWR of LfcinB (20–25) and the RLLR of BFII (32–35), shows high antibacterial activity against E. coli ATCC 25922 and S. aureus ATCC 25923 ( Vargas-Casanova et al., 2019 ; Pineda-Castañeda et al., 2020 ).

In this way, a variety of AMP variants can be obtained. If combined with high-throughput screening, it can effectively obtain the desired AMP. For instance, some new AMPs are designed by the combinatorial peptide library of melittin and show higher activity and lower cytotoxicity ( Krauson et al., 2015 ).

Many AMPs are stable and retain their antimicrobial activity in a wide pH range. AMPs have enhanced activity at low pH because of their basic properties. This condition is related to the protonation of histidine at acidic pH, which promotes electrostatic interactions with anionic surfaces, including LPS and the anions of phospholipids, and subsequently enhances antibacterial properties. The effect of pH on the antibacterial activity of AMPs varies. For example, thanatin’s activity at neutral pH is slightly higher than that under acidic conditions. By contrast, the activity of xylan on E. coli , Listeria , and C. albicans is remarkably higher at pH 5.5 than at pH 7.4 ( Holdbrook et al., 2018 ). The inactivation of the histidine-containing AMP C18G-His under low pH conditions involves pH-dependent changes in the state of the aggregates in the solution, because the aggregates, which are sensitive to pH and lipid composition, may be affected by binding and conformation. Peptides can also enhance bacterial membrane permeability at low pH ( Hitchner et al., 2019 ). Thrombin-derived C-terminal peptides (TCPs) will also change the mode of CD14 (a protein that is abundant in human plasma) from anti-inflammatory mode to bacterial elimination mode from pH 7.4 to pH 5.5 ( Holdbrook et al., 2018 ). A dimer (e.g., P-113) can be created to provide AMPs with resistance to a higher pH range. The sensitivity of this pH-sensitive AMP can be used to achieve a certain targeting effect in practical applications. In addition, charge interaction is one of the most important factors in peptide self-assembly. pH affects the charge state of amino acid and substituent functional groups. Therefore, adjusting the pH is the most common method for controlling peptide assembly and disassembly ( Tian et al., 2015 ).

Medicine Antimicrobial peptides can regulate pro-inflammatory reactions, recruit cells, stimulate the proliferation of cells, promote wound healing, modify gene expression and kill cancer cells to participate in the immune regulation of human skin, respiratory infections, and inflammatory diseases ( de la Fuente-Núñez et al., 2017 ). For example, α-defensins HNP-1, HNP-2, and HNP-3 showed effective antibacterial activity against adenovirus, human papilloma virus, herpes virus, influenza virus and cytomegalovirus. Pulmonary diseases, such as idiopathic pulmonary fibrosis, alveolar proteinosis, and acute respiratory distress syndrome, show elevated levels of AMPs ( Guaní-Guerra et al., 2010 ). Likewise, AMPs secreted by the Paneth cells in the mammalian gut are important to shape the gut microbiota ( Bevins and Salzman, 2011 ). The application of AMPs in medicine, such as dental, surgical infection, wound healing and ophthalmology is developing now. But there are only three AMPs that have been approved by FDA including gramicidin, daptomycin, and colistin. Dental caries, endodontic infections, candidiasis, and periodontal disease are common diseases in the human oral cavity. Dental caries is a prevalent oral disease and some acidogenic bacteria like Streptococcus sp. are the main caries-associated pathogens ( Izadi et al., 2020 ). Several AMPs have good application potential. For instance, peptide ZXR-2 (FKIGGFIKKLWRSLLA) has shown potent activities against pathogenic bacteria of dental caries, Streptococcus mutans , Streptococcus sobrinus , and Porphyromonas gingivalis and peptide PAC-113 (Clinical trial identifier: NCT00659971 ) that has been sold over the counter in Taiwan for treating oral candidiasis ( Chen L. et al., 2017 ). In surgical infection and wound healing: surgical infection occurs after surgery, burns, accidental injury, skin disease, and chronic wound infections have a serious hazard to human life ( Thapa et al., 2020 ). Several AMPs have shown the therapeutic potential of these diseases. For example, AMP PXL150 shows pronounced efficacy as an anti-infective agent in burn wounds in mice and AMP D2A21 has been in the third phase of clinical trials for treating burn wound infections ( Björn et al., 2015 ). In ophthalmology: Human eyes are prone to be infected by several organisms including bacteria and fungi in which S. aureus , Streptococcus pneumoniae , P. aeruginosa , Aspergillus spp., and C. albicans are the most relevant pathogens ( Silva et al., 2013 ). Although AMPs such as Lactoferricin B, Protegrin-1 exhibited antimicrobial activity against these pathogenic bacteria, their application in the field of ophthalmology is only at the theoretical stage. With the popularity of contact lenses and the increase in cases of related eye infections, antimicrobial peptides have shown good application prospects in ophthalmology ( Khan and Lee, 2020 ). Additional methods need to be performed for the application of AMPs as drugs in medicine. The main strategies include (1) constructing precursors to reduce cytotoxicity and improve protease stability, (2) using AMPs in combination with existing antibacterial agents, (3) inducing the correct expression of AMPs with appropriate drugs and using engineering probiotics as vectors to express AMPs. For example, in the field of wound repair, different formulation strategies, such as loading AMPs in nanoparticles, hydrogels, creams, gels, ointments, or glutinous rice paper capsules, have been developed to effectively deliver AMPs to the wound ( Borro et al., 2020 ; Thapa et al., 2020 ). In recent research, the sponges developed from modified starch and HS-PEG-SH are covalently immobilized with AMP showed effective antibacterial activity ( Yang et al., 2019 ). More technical means, including pheromone-labeled AMPs, local environment-triggered AMPs (enzyme precursor drug release system, pH-activated AMPs, etc.), have been developed to improve the targeting mechanism of AMPs. Furthermore, nanotubes, quantum dots, graphene, and metal nanoparticles have been proposed to be a potential method to enhance drug delivery of AMPs ( Magana et al., 2020 ). Hybrid peptides have also been used to build targeting peptides. For example, PA2, which is a P. aeruginosa -targeting peptide, was combined with GNU7 (a broad-spectrum AMP) to construct a hybrid peptide (PA2–GNU7) that targets OprF protein and has good bactericidal activity and specificity ( Kim et al., 2020 ). Furthermore, some antibiotics, for instance, daptomycin (a lipopeptide), lugdunin which is a 21-membered cyclic peptide consists of 6 amino acid residues plus a thiazolidine moiety and telavancin (a glycopeptide) have been widely used for the clinic ( Durand et al., 2019 ; Lampejo, 2020 ). Although they are antibiotics, they have provided broader ideas for the design of AMPs.

The European Union banned the use of animal growth promoters in animal feed in 2006. Thus, a new antibacterial strategy is needed. Many AMPs are the potential to be used in poultry, swine, and ruminants breeding and aquaculture because they can improve production performance ( Liu et al., 2008 ; Bao et al., 2009 ), immunity and promote intestinal health and some of them have a stronger inhibitory effect on bacterial inflammation if used with antibiotics ( Wang et al., 2019 ; Cote et al., 2020 ). For example, SIAMP has a good effect on the treatment of IBV in chicken ( Sun et al., 2010 ). By adding swine gut intestinal antimicrobial peptides (SGAMP), broilers showed higher average daily gain and feed efficiency under chronic heat stress conditions ( Hu et al., 2017 ). Frog caerin 1.1, European sea bass dicentracin and NK-lysine peptides (NKLPs) have good inhibitory effects on Nodavirus , Septicaemia haemorrhagic virus , Infectious pancreatic necrosis virus and Spring viremia carp virus , which are devastating to fish farming ( León et al., 2020 ). The AMP in soybean meal fermented by B. subtilis E20 effectively inhibits V. parahaemolyticus and Vibrio alginolyticus and enhances the resistance level of Litopenaeus vannamei against V. parahaemolyticus when added to feeds ( Cheng et al., 2017 ).

Antimicrobial peptides constitute a global research hotspot, but many key issues in design and application need to be solved urgently. Several restrictive factors hinder the application of AMPs. The interaction of multidisciplinary subjects, such as biology, materials science, chemistry, bioinformatics, molecular informatics and pharmacy can further develop prospective AMPs. Computer molecular dynamics simulation, cell membrane simulation, and more methods are being applied to study the mechanism of AMPs. How to further understand the correlation between AMPs and various targets instead of conducting one-sided experimental research might improve experimental designs to obtain stronger systemic and scientific demonstrations. On this basis, further animal experiments are required instead of simple cell-level experiments to test the effect of AMPs under complex physiological conditions. Several complicated methods, such as the chemical method of peptidomimetics and non-natural amino acid modifications, have been applied in designing AMPs to solve the problem of protease hydrolysis. Most methods use chemical substrates, but the cost of these methods cannot be ignored in practice. In addition, chemical synthesis and the use of engineered bacteria are currently the mainstream for such procedures. Finding a better biological preparation method, reducing the cost and increasing the yield is important problems in practical application. Furthermore, studying the AMP expression of the organism itself and finding a better expression vector are necessary for mass production in the future as more AMPs in nature are discovered. Further research is needed on the reported AMPs to solve the problem on structure–function relationship. As a branch of peptide drugs, AMPs need to progress with the advancement of medical science against the background of the current low success rate of the clinical application of AMPs. More attention can be focused on food, agriculture, and animal husbandry.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.