Host defense peptides (HDPs), previously known as antimicrobial peptides (AMPs), are small molecular weight peptides and are present in all strata of life, i.e., prokaryotes (bacteria) to eukaryotes (fungi, plants, and animals including invertebrates and vertebrates). They play a crucial role in immunity to the host [1] by acting against bacteria, fungi, parasites, and viruses [2, 3]. AMPs exert broad-spectrum bioactivity with less toxicity and minute chances of resistance development. To date, the AMP database [data repository of antimicrobial peptides (DRAMP)] has reported a total of 22,499 entries, out of which 6,105 are natural and synthetic AMPs, 16,110 patent candidates, and 96 are in drug development (preclinical and clinical stage). AMPs from the different kingdoms are also categorized, such as 2,519 from animals, 826 from plants, 6 from fungi, 431 from bacteria, 4 from archaea, and 7 from the protozoan kingdom [4]. AMPs produced by bacteria (also called bacteriocins) become non-favorable to other’s survival as they compete for the nutrients present in the same niche. Lantibiotics and non-lantibiotics are two categories of bacteriocins. Lantibiotics contain non-natural amino acids like nisin produced by Lactococcus lactis; it was the first lantibiotic to be isolated and characterized well in 1947 [5]. Nisin has a nanomolar (nM) minimum inhibitory concentration (MIC) against a myriad of Gram-positive bacteria and has thus been used as a food preservative also for the last 6 decades [6]. Another famous lantibiotic is paenibacillin produced by Paenibacillus spp., which is tolerant to high temperature and proteinase K degradation, making it a potential candidate in the health sector at the industrial scale [7]. Mersacidin is another clinically important bacteriocin resistant to Gram-positive bacteria [8]. Eukaryotic examples are also flooded with AMPs with beneficial attributes. The hemolymph of Hyalophora cecropia (silk moth) contained AMPs named cecropins [9]. Cecropins are positively charged and amphipathic in nature and possess broad-spectrum anti-microbial activity against Gram-positive and Gram-negative bacteria and fungi. It went on to discover more eukaryotic AMPs like defensins from mammalian macrophages [10] and sacrotoxins from Sarcophaga peregrine, a flesh fly [11]. Since plants do not possess B cells and T cells as in mammals for the innate and adaptive immune response against foreign pathogens; AMPs play a key role in giving protection against bacteria and fungi. AMPs produced by plants are cysteine-rich and disulfide linkages. Plant flowers, tubers, seeds, and leaves produce AMPs. Well-characterized AMPs from plant sources come under thionins [12], plant defensins [13], and cyclotides [14]. Invertebrates are like plants in terms of immune response, i.e., they lack adaptive and innate immune responses. Nearly all invertebrates produce AMPs; however, insects and marine invertebrate AMPs (oysters, shrimps, horseshoe crabs) have been studied the most [15–17]. Horseshoe crabs produce tachyplesin and polyphemusin AMPs, which have antibacterial and antifungal activity [18]. Also, polyphemusin possesses antiviral properties, especially against the human immunodeficiency virus (HIV) [19]. Vertebrate AMPs have innate and adaptive immunity arsenal. Still, a variety of vertebrates produce AMPs like magainins from the skin of Xenopus laevis, a frog [20], crypt peptides from mouse paneth cells [21], granules of white blood cells, epithelial tissues of mouth, lungs, skin, and bodily fluids [22–25]. Cathelicidins and defensins are AMP subcategories produced by vertebrates. Regulation of AMPs occurs by posttranslational modifications (PTMs) like proteolysis, amidation, ADP-ribosylation, glycosylation, and phosphorylation. Anticancer treatment medicines pose resistance to patients undergoing pain; AMPs serve as an anticancer treatment option. AMPs may both promote and inhibit cancer growth [26]. AMPs produced by vertebrates also possess immunomodulatory and anti-inflammatory effects [27]. AMP has also a role in wound healing [28]. Pathogenic bacterial diseases have been treated by antibiotics for a long time, but drug resistance due to long-term use, extensive utilization, and single target of antibiotics is one of the most recent unsolved challenges for clinical infection management [29, 30]. Here comes the AMP, which acts against all ordeals because it acts on multiple targets on the plasma membrane and intracellular targets of drug-resistant pathogens [3, 31, 32]. A smaller number of AMPs enter the clinical stage with therapeutic efficacy because of innate defects in the natural AMPs. Further exploration of AMPs for structural characteristics, and mechanism of action of AMPs may improve stability, activity, targeting, and lesser cytotoxicity. This review elaborates on the origin, characteristics, and mechanism of action and bioactivity of AMPs so that readers have comprehensive latest knowledge and understanding of AMPs along with updates on clinical development and AMPs applications.

Eukaryotes have an evolutionary conserved mechanism of AMP production in response to pathogen invasion. AMP production takes less time and energy as compared to antibody synthesis and moves to target relatively in less time as compared to immunoglobulins; also, some eukaryotes, like insects, lack lymphocyte-based immune arsenal and here, smaller peptide synthesis combat bacterial infections [33]. Very first discovered AMP was from chrysalis (silkworm) and from micro-organisms [34], plants [35], invertebrates [36], fish [37], amphibians [38], reptiles [39], birds [40], and mammals [41]. Nisin was the first AMP from bacteria to be isolated, and it is cytotoxic to other bacterial communities present in the same environment [5]. This feature of nisin is exploited at an industrial scale by using it as a food preservative [42–44]. Mammalian intestinal microbiota bacterial communities also produce AMPs [45, 46]. Mushrooms like Coprinopsis cinerea produce AMP named copsin, which is bactericidal against Enterococcus faecium (E. faecalis) and Listeria monocytogenes (L. monocytogenes) [47]. AMPs derived from plants like thionins, defensins, and cyclotides prevent pathogenic microbe invasion. Such AMPs are rich in disulfide linkages, which allows structural stabilization [48]. Since invertebrates do not possess a lymphocyte-based immune system, they possess innate immune responses present in hemolymph, skin, mucosa, etc., to resist pathogens [49]. Hyalophora cecropia’s hemolymph has cecropins, which are antibacterial against Gram-positive and harmful bacteria [50]. Drosocin from the intestine of Drosophila is effective against (Pseudomonas entomophila) P. entomophila [50]. To date, 1,770 AMPs have been reported from vertebrates (fish, amphibians, reptiles, birds, and mammals). Fishes produce hepcidins and piscidins [51]. Frog-like Paa yunnanensis’s skin has cathelicidin-PV, which is antibacterial and antifungal to even drug-resistant candidates [52, 53]. AMPs from mammals fall under cathelicidins and defensins [54]. Crotalicidin and fragments from rattlesnakes have antibacterial, antifungal, and anti-tumor activity [55]. Cathelicidins from mammals have conserved cathelin domain [56], stored in nonfunctional form encapsulated in granules of neutrophils and macrophages and they become functional upon leukocyte activation and processing [54]. LL-37 is the only cathelicidin found in humans [57] and gets accessory help from moonlighting protein GAPDH to lessen M.tb (Mycobacterium tuberculosis) burden intracellularly [25].

Another important class of AMPs belonging to mammals is defensins, which are further subdivided into α-, β-, and θ-defensins based on disulfide bond arrangement [58, 59]. β-defensins are predecessors for α- and θ-defensins [60]. Humans possess both except θ-defensins. Birds, reptiles, and cattle produce β-defensins. Non-human primates produce θ-defensins from leukocytes and bone marrow [54, 60]. Both cathelicidin and α-defensins are trimmed to C-terminal peptides possessing antimicrobial activity after getting cleaved by elastase, metalloproteinase [61]. AvBD9, a β-defensins isolated from quail, was antibacterial to 11 species of bacteria, including Gram-positive and Gram-negative [62]. Mammals have mucus layers where AMPs are abundant and prevent the colonization of bacteria, fungi, and parasites [63, 64], as well as in mast cells and granulocytes [65]. α-defensins are secreted from neutrophils, macrophages, and Paneth cells, while β-defensins are harbored in leukocytes and epithelial cells of the skin, respiratory, digestive, and genitourinary tract, urine, heart, and skeletal muscle [66]. Similar to defensins, most cathelicidins are stored in granules of epithelial and immune cells in humans [54, 67] along with mucus, blood, urine, sweat, and tears [63, 68–70]. Therefore, extensive structural and physiochemical characterization of AMPs from different sources can identify novel AMPs with substantial clinical and industrial potential. Directed evolution, machine learning, and sequence-based encoding are a few key approaches that can accelerate the AMP discovery process [71]. Various AMPs and antimicrobial proteins produced at varied sites in human tissue after contracting microbial pathogens are enlisted in Table S1.

Based on amino acid sequences, peptide net charge, protein structure, and origins, AMPs are separated into multiple subgroups. Most anti-microbial peptides have a net charge of +2 to +9 and are made up of 10–100 amino acids [72]. Data on the bioactivities, toxicities, chemical modifications, amino acid sequences, and three-dimensional structures of peptides having antimicrobial properties can be found in the vast, publicly accessible Database of Antimicrobial Activity and Structure of Peptides (DBAASP, https://dbaasp.org/). Over 15,700 entries (8,000 more than the previous version) are included in the most recent version 3.0 (DBAASP v3) [73]. Anionic AMPs, with a net charge range of −1 to −8, comprise the first class of AMPs and have five to seventy amino acid residues [74]. While tiny molecules encoded by genes make up a minor portion of anionic AMPs, most anionic AMPs are peptide fragments following proteolysis. Among their structural characteristics are cyclic cysteine knots and α-helical peptides seen in certain amphibians [75]. Similar to bigger proenzymes’ capacity to neutralize charges, they appear to use metal ions and the negatively charged elements of the microbial membrane to create salt bridges and interact with bacteria [76, 77]. For instance, it was shown that in the presence of zinc ions, the ovine pathogen Mannheimia haemolytica was susceptible to the antibacterial effects of ovine pulmonary surfactant-associated anion peptide (SAAP), the first anionic AMP ever identified with 5−7 aspartate residues [78]. The surfactant solution’s bactericidal activity was significantly reduced when 0.14 mol/L NaCl and EDTA were added, but it was recovered when ZnCl₂ was added again. Furthermore, the α-helical anionic AMP maximin H5’s amidated C-terminal fragment establishes an intra-peptide hydrogen bond with the peptide’s N-terminal portion, which is crucial for maintaining the tipped α-helix shape [75]. The cationic α-helical AMPs comprise the second subgroup. These short peptides, typically less than 40 amino acids long, exhibit amidation at the C-terminus and have a net charge ranging from +2 to +9 [79]. In aqueous solutions, the structure of these peptides is disordered; however, in the presence of liposomes or liposomes A, phospholipid vesicles, sodium dodecyl sulfate (SDS) micelles, and trifluoroethanol, all or parts of the molecules convert into α-helical structure [80]. Moreover, these AMPs usually contain more than 50% hydrophobic amino acids, which encourages the development of an amphiphilic structure during target-cell interaction [81]. The majority of cathelicidins are amphiphilic α-helical AMPs [82], among which LL-37, cecropins, and magainins have all been thoroughly investigated. The only human cathelicidin of an active fragment that is liberated from hCAP18 in neutrophils with a neutral pH and a net charge of +6 is LL-37, which is produced by serine protease 3 [83, 84]. When LL-37 is exposed to water at a concentration of 15 mmol/L, its circular dichroism displays a disordered structure; but, in the presence of HCO₃⁻, SO₄²⁻, or CF₃CO₂⁻, it changes into an α-helical structure [85]. The peptide concentrations directly correlate with the structural transformation efficiency. Furthermore, there exists a correlation between the degree of α-helix and LL-37’s antibacterial efficacy against both Gram-positive and Gram-negative bacteria. The cationic β-sheet AMPs comprise the third subgroup. Usually, the peptides have two to eight cysteine residues that form one to four intramolecular disulfide bond pairs [86]. These peptides’ biological activities and structural stability depend on their disulfide links. For instance, they stay active when they mutate to hydrophobic amino acids (alanine and leucine excepted) but become inactivated when cysteines are substituted with acidic amino acids [87]. Nevertheless, the antibacterial action and cytotoxicity of mouse defensins, hBD-3 (human beta-defensin 3), and human neutrophilic peptide 1 (HNP 1) are not dependent on their disulfide links or structure [61, 74]. Defensins make up the majority of the β-sheet AMPs [86]. As previously stated, the characteristic intervals between the six cysteine and disulfide bond types are used to categorize the mammalian defensins into two groups: α-defensins and β-defensins [88]. Mammalian defensins exhibit remarkably comparable tertiary structures despite differences in their covalent structures [89]. Regarding α-defensins, they create a cyclic structure by combining cysteine with disulfide bonds near the amino terminus and a three-stranded chain by hydrogen bonding with the β-hairpin [89]. Amphipathic α-defensins’ ability to destroy bacterial membranes by interaction with phosphatidyl chains is dependent on hydrophobic amino acids and their positive charge [90]. Moreover, the contact between cationic α-defensin residues and negatively charged molecules on the bacterial surface, or between hydrophobic residues and the bacterium, maybe the main mediator of membrane breakdown and bacterial killing. The chicken Gga-AvBD11’s N-terminal domain primarily mediates and amplifies its antibacterial and antiparasitic properties, although the full-length protein is necessary for antiviral activity [91]. The end-to-end cyclized tetracyclic peptides known as θ-defensins are connected by three disulfide bridges between their antiparallel β-sheets [92]. The ability of β-defensins to maintain their activity at high salt concentrations is attributed to their cyclic structure, which also contributes to their antibacterial capabilities by reducing the microbicidal activities that occur when the cyclic structure is lost [93]. According to recent research, defensins’ stability and structure are mostly determined by the quantity and location of disulfide bonds, while their capacity to bind membranes and inhibit bacteria is dependent on their cyclic backbone [94]. The fourth grouping consists of extended cationic amino acid polymers (AMPs) that do not have typical secondary structures and contain specific amino acids such as arginine, proline, tryptophan, glycine, and histidine [81]. Only hydrogen bonding and the van der Waals force, which interacts with the membrane lipids, can stabilize their structures. Tryptophan (38%) and proline (23%) are abundant in indolicidin [95], histatin-8 (33.3%) and histidine are abundant in histatin-8 [96], and PR-39 (typically 24%) and proline (49%) are abundant in PR-39 [97]. Proline (53.2%) and phenylalanine (19%) are abundant in prophenin-1 [98]. Antimicrobial protein fragments make up the fifth subgroup. There are proteins found in nature that, when broken down, offer a broad range of bactericidal properties. An essential part of the innate immune system’s defense against foreign infections is lysozyme, the first antimicrobial protein ever found [99–101]. Its extracellular portion has an α-helix and β-sheet structure and 130 amino acids. Other membrane-active and DNA-binding proteins have also been shown to have a helix-loop-helix (HLH) region, as observed in the lysozyme of humans and chickens [102]. When applied to both Gram-positive and Gram-negative bacteria as well as the fungus Candida albicans (C. albicans), the HLH peptide exhibits potent bactericidal properties. A sleep-inducing gene encoding the NEMURI protein, which has an arginine-rich region and immunomodulatory properties as well as a potent bactericidal activity similar to kanamycin, was discovered by Toda et al. [103] in fruit flies. The amino terminal copper and nickel (ATCUN) binding motif is notably present in certain AMPs. The sequence H₂N-XXH is found at the N-terminus, where any amino acid other than proline can be the XX [104]. The motif is highly affinized for Cu²⁺ and Ni²⁺ [105]. The Cu²⁺-ATCUN complex has the ability to create reactive oxygen species (ROS), which can damage proteins, lipids, and nucleic acids [106, 107].

Antibacterial activity: AMPs work against bacteria through membrane- or non-membrane-mediated mechanisms. As was previously mentioned, the presence of particular anionic components in the plasma membranes of bacteria and fungus, such as mannan in fungi, lipoteichoic acid in Gram-positive bacteria, and LPS in Gram-negative bacteria, gives cationic AMPs a greater affinity for microbial pathogens. AMPs can permeate the membrane to perform intracellular functions or produce membrane permeability or perforation to cause intracellular contents to seep out. The development of bacterial resistance to AMPs is prevented by the AMPs’ quick killing time, broad membrane and intracellular effects, and lack of targeting of particular molecules or pathways. For this reason, the use of AMPs in managing antimicrobial resistance is appealing. A broad class of cationic small molecules (AMPs) with between 30 and 60 amino acids that are extracted from bacteria are called bacteriocins. They were divided into two groups based on peptide synthesis mechanisms. Peptides produced by non-ribosomes with broad antibacterial action and peptides synthesized by ribosomes with relatively narrow antibacterial activity against bacteria and fungus make up the first group [108]. In the culture broth of Streptomyces albofaciens, Wang et al. [109] identified a novel short non-ribosomal AMP called albopeptide 6, which exhibited a narrow spectrum action against vancomycin-resistant E. faecalis. Belonging to the bacteriocins family, nisin exhibits strong antibacterial properties against a diverse array of Gram-positive and even Gram-negative bacteria [110]. Penicillin or chloramphenicol coupled with nisin enhanced the antibacterial impact against E. faecalis, although the single antibiotic did not significantly affect the bacterium, according to Tong et al. [111]. AMPs, therefore, serve as a novel therapeutic alternative for treating antibiotic-resistant bacteria, either in isolation or in combination with other agents. According to research, the forkhead box transcription factor O (FoxO) directly controls the production of AMPs in shrimp, including CrusI-3 and Alf-E1, but it is independent of the Imd signaling pathway [112]. Notably, resistance can be induced by prolonged exposure to low concentrations of AMPs. Therefore, it is advised to use a high concentration of AMPs to sustain the bactericidal activity [113].

Humans include three main AMP groups: histatins, cathelicidins, and defensins. Defensins are categorized as either β- or α-defensins according to the type of disulfide bond arrangement. Neutrophils, lymphocytes, and epithelial cells of the skin and mucous membranes produce α- and β-defensins. Epithelial cells, neutrophils, and lymphocytes present in mucosal membranes and the skin are the primary producers of α- and β-defensins [151]. Since neutrophils produce human α-defensins 1−4, also known as HNPs 1−4, these proteins can make up as much as 50% of the total protein content in these cells [152]. These peptides help break down bacterial pathogens in conjunction with other proteins, including lysozyme, proteases, and RNases. This makes them crucial for both local and systemic innate immunity [153]. For HNPs 1−3, the normal concentration in human blood plasma is 254.8 ± 7.1 pg/μL; in bacterial sickness, this can increase by 4.2 times, and in non-bacterial infection, by 3.2 times.

Numerous insect AMPs have been found to have cytotoxic effects on various malignant cell lines, including breast, lung, melanoma, leukemia, and lymphoma [160]. Anticancer peptides (ACPs) are cationic low-molecular-weight AMPs that exhibit both antibacterial and anticancer properties. AMPs have several characteristics, such as amphipathic structure, high hydrophobicity, and cationic (positive net charge), which enhance their affinity for cell membranes. Because of these qualities, ACPs can be thought of as a valuable resource with a low tendency to develop cancer cell resistance. The outer membranes of cancer cells have more negatively charged molecules than those of normal cells. Through electrostatic interactions, this feature facilitates the ACPs’ adhesion to cancer cells, leading to selective membrane disruption in the cancer cells and necrosis or death [161]. A brief overview of AMPs in combatting cancer types is highlighted in Table 1.

As AMPs are thought to be promising antibacterial agents, their mechanisms of action have been extensively studied. AMPs have both bacteriostatic and bactericidal properties, and they cause less germ resistance than traditional antibiotics. Electrostatic interaction between the positively charged cationic peptides and the negatively charged cell membranes causes membrane adsorption and conformational change. Peptides can connect to the cell membrane in a variety of ways, including the ones listed below.

Since many fungi, yeasts, and other pathogenic micro-organisms are to blame for the ongoing spread of illnesses and the development of medication resistance against bacteria, microbial infections continue to rank among the leading public health concerns. The growth of antibiotic resistance is making it more important to create novel defenses against superbugs that are resistant to drugs. Small peptide-based compounds with a length of 5–100 amino acids that possess strong and all-around antibacterial capabilities are known as antimicrobial peptides or AMPs for short. Easy access to cutting-edge carriers made possible by nanomedicine eventually makes it possible to design and build focused delivery systems for the most effective medications, ones with lower toxicity and greater efficacy.

In multicellular organisms, AMPs, commonly called HDPs, are significant effector immune defense molecules. AMPs use various methods to carry out their antimicrobial actions; it has been thought improbable that AMPs might induce drug resistance. They, therefore, have much potential as antibacterial agents of the next generation. Currently, many medications related to AMP are undergoing clinical trials [182]. Recent research indicates that certain AMPs and traditional antibiotics work well together. Drug-resistant infections can be eliminated, drug resistance can be avoided, and the therapeutic benefits of antibiotics can be significantly enhanced when AMPs and antibiotics are used together. Much research demonstrated the advantages of combining AMPs with traditional antibiotics, which frequently result in increased activity against strains resistant to drugs and an extended range of action for antibiotics [183]. Table 2 enumerates the peptide and antibiotic combinatorial approach in various research projects against microbial pathogens.

Altogether, AMPs are candidates for broad-spectrum anti-microbial, immune-modulatory, and anti-cancerous properties. AMPs possess potent MDR bacterial and anticancer activity. Hereby, AMPs offer researchers and industries a promising target for addressing emerging antibiotic resistance of already available drugs and chemotherapy resistance of cancer cells. However, the short half-life of these peptides, their toxicity to some extent, and their few side effects may limit clinical and therapeutic development. There is a requirement for new technological interventions to transform the natural AMPs into efficacious, less toxic, and more potent in action modulation to make these amphiphilic and lipophilic with bare minimum side effects and prevent drug resistance. There is a long road ahead for the potential FDA-approved AMP candidates to enter clinical trials. We look forward to the development of AMP-based curing cancer and microbial infections in the human race with enhanced specificity, safety, and efficacy to the coming generations for a better life.