The AFPs produced by microorganisms, including bacteria and archaea (both prokaryotes) as well as fungi (eukaryotes), can be secreted into extracellular surroundings and offer a competitive advantage in ecological niches. Bacteria generate a number of different AFPs (Table 1). The first example of an archaeal antimicrobial peptide with antifungal activity is VLL-28, isolated from the archaeon Sulfolobus islandicus, which showed antifungal activity against 10 clinical isolates of Candida spp. (Roscetto et al., 2018). The well-known AFP-producing bacteria include the genera Bacillus, Lactobacillus, Streptomyces, and Burkholderia. For example, the iturin A produced by Bacillus subtilis exhibits a conspicuous antifungal activity against Aspergillus spp., Fusarium spp., and Penicillium spp. (Klich et al., 1991); the peptide mixture generated by Lactobacillus plantarum TE10 suppresses Aspergillus flavus in maize seeds, displaying a considerable potential for the development of bio-control agents (Muhialdin et al., 2020); the champacyclin, a head-to-tail cyclic octapeptide obtained from Streptomyces champavatii, inhibits the growth of the yeast C. glabrata (Pesic et al., 2013); the natamycin from Streptomyces philanthi RL-1-178 possesses a fungicidal activity against A. flavus (Boukaew et al., 2023); and the AFC-BC11, a lipopeptide isolated from Burkholderia cepacia, exerts an antifungal activity toward a variety of soil fungi (Kang et al., 1998). Similarly, eukaryotic microorganisms such as filamentous fungi and yeasts also produce a variety of AFPs (Table 1). For instance, the peptide PeAfpC produced by the filamentous fungus Penicillium expansum can effectively inhibit the growth of Byssochlamys spectabilis, which is capable of causing the spoilage of pasteurized juices and canned foods (van der Weerden et al., 2013). In addition, the two peptides, namely, PAF and PAFB, generated by the filamentous fungus Penicillium chrysogenum, are capable of exerting antifungal activity against a variety of filamentous fungi, with PAFB suppressing the growth of some toxigenic molds (Kaiserer et al., 2003; Rodríguez-Martín et al., 2010; Huber et al., 2018).

Plants have developed various mechanisms in their innate immune systems to protect themselves against fungal attacks, including soluble peptides and proteins released from plants with antifungal activities (Chiu et al., 2022). These peptides/proteins that are constitutively synthesized are able to trigger defense responses in plants. On the basis of sequence, cysteine residues, and function, the plant-sourced AFPs can be divided into different families (Table 1), including chitinases, defensins, and snakins, as well as hevein-type and gly-rich peptides (Tam et al., 2015; Yan et al., 2015). Chitinases are among the best-known and most-studied plant AFPs. They display strong antifungal activity against a wide range of phytopathogenic fungi, including Botrytis cinerea (Van Baarlen et al., 2007) and Rhizoctonia solani (Shrestha et al., 2007). Plant chitinases have been used to treat fungal infections as exogenously applied pest control agents (Karasuda et al., 2003). The Dm-AMP1 is a defensin peptide found in Dahlia merckii that shows inhibitory activity against a variety of fungi, such as B. cinerea and Leptosphaeria maculans in the presence of CaCl₂ and KCl (Osborn et al., 1995). Another defensin Ace-AMP1, a potent AFP found in onion (Allium cepa) seeds, has been applied to control the tomato early blight disease caused by the pathogen Alternaria solani (Wu et al., 2011). The peptide Ee-CBP containing five disulfide bridges obtained from the bark of spindle tree (Euonymus europaeus) is a hevein-type AFP, exhibiting antifungal activity against various fungi including Alternaria brassicicola (50% growth inhibition IC₅₀ = 3 μg/mL), Phoma exigua (IC₅₀ = 33 μg/mL), and Fusarium oxysporum f.sp. cubense (IC₅₀ = 15 μg/mL) (Van den Bergh et al., 2002). The WAMP-1a, another hevein-type AFP from seeds of Triticum kiharae, shows high broad-spectrum inhibitory activity against a wide range of chitin-containing and non-chitin-containing pathogens including Bipolaris sorokiniana, Neurospora crassa, Fusarium verticillioides, and Fusarium solani (Odintsova et al., 2009). The snakins 1 and 2 are representative AFPs of snakin family identified from Solanum tuberosum, exerting antifungal activity against fungi such as B. cinerea, F. solani, A. flavus, Colletotrichum graminicola, and Bipolaris maydis (Segura et al., 1999; Berrocal-Lobo et al., 2002). The Cc-GRP is a gly-rich AFP identified from Coffea canephora that can combat fungi such as F. oxysporum and Colletotrichum lindemuthianum (Zottich et al., 2013).

Many animal-sourced AFPs have been found to be part of the innate immune responses of both invertebrates and vertebrates (Tables 2, 3). As invertebrates lack adaptive immunity, AMPs play a significant role in their immune response and comprise an essential source of AFPs (Table 2). The AFPs obtained from marine invertebrates include penaeidins, Cm-p1, and tachystatins. The penaeidin family originates from shrimp, and currently, there are nine members available for this family in the ADP3 database (Destoumieux et al., 2000; Cuthbertson et al., 2002; An et al., 2016). The members of the penaeidin family show broad-spectrum fungicidal activity. For example, both penaeidins 2 and 3a are fungicidal against filamentous fungi and shrimp pathogen F. oxysporum (Destoumieux et al., 2000). Cm-p1 is a 10-mer short peptide isolated from marine snails. Cm-P1 has the ability to inhibit the growth of yeasts and filamentous fungi, while it shows little toxic effects on mammalian cells (López-Abarrategui et al., 2012). The tachystatin family consists of four members: tachystatin A, tachystatin B1, tachystatin B2, and tachystatin C, all of which have been identified in horseshoe crabs. All four members of the tachystatin family contain three disulfide bridges and have sequences similar to spider neurotoxins. Since horseshoe crabs are close relatives of spiders, tachystatins and neurotoxins may have evolved from a common ancestral peptide gene. Tachystatins are capable of binding to chitin and then exert their antifungal activity against C. albicans and Pichia pastoris (Osaki et al., 1999). AFPs are also found in insects. Representative AFPs from insects include melittin and thanatin. Melittin was isolated from bee venom by Neuman et al. (1952), which existed in hemolysin phospholipase A (Habermann, 1972). Melittin shows strong antifungal activity against various strains of fungi, including Aspergillus sp., Candida sp., Malassezia sp., Penicillium sp., and Trichoderma sp. (Memariani and Memariani, 2020). It is found that melittin exerts inhibitory activity against fungi via a series of combined mechanisms of inhibition of (1,3)-β-D-glucan synthase, membrane permeabilization, apoptosis induction by reactive oxygen species (ROS), and alterations in gene expression (Memariani and Memariani, 2020). Thanatin, a 21-residue peptide, was first isolated from the insect Podisus maculiventris (Fehlbaum et al., 1996). It is highly potent in inhibiting the growth of fungi at considerably low concentrations (Fehlbaum et al., 1996; Dash and Bhattacharjya, 2021). Protonectin was originally isolated from the venom of the neotropical social wasp Agelaia pallipes pallipes (Mendes et al., 2004). Later, protonectin was shown to have potent antifungal and fungicidal activity against C. glabrata, C. albicans, C. parapsilosis, C. tropicalis, and C. krusei by disturbing membrane integrity and inducing ROS production in yeast cells (Wang et al., 2015). Recently, a 41-amino acid peptide called blapstin, isolated from the Chinese medicinal beetle Blaps rhynchopetera, was shown to possess antifungal activity against C. albicans and Trichophyton rubrum. Cryo-scanning electron microscope (Cryo-SEM) observations showed that blapstin directly resulted in disruption in the cell structure of C. albicans and T. rubrum (Zhang et al., 2023).

Spiders and centipedes are also known to produce AFPs, such as juruin, gomesin, and lactoferricin B-like peptide (LBLP), especially in venoms. Juruin isolated from the venom of the Amazonian pink toe spider Avicularia juruensis has antifungal activity against filamentous fungi and yeasts, including Aspergillus niger, Beauveria bassiana, C. albicans, C. krusei, C. glabrata, C. parapsilosis, C. tropicalis, and Candida guilliermondii (Ayroza et al., 2012). Gomesin, an 18-amino acid AMP isolated from the hemolymph of the tarantula spider Acanthoscurria gomesiana, inhibits the development of filamentous fungus and yeast (Silva et al., 2000). LBLP, a 23-mer AMP derived from the centipede Scolopendra subspinipes mutilans, has been found to have antifungal and fungicidal activity against C. albicans, C. parapsilosis, Malassezia furfur, and Trichosporon beigelii by forming pores in the membrane, eventually leading to fungal cell death (Choi et al., 2013). Recently, LBLP has been reported to trigger mitochondrial disruption-mediated apoptosis by inhibiting respiration under nitric oxide accumulation in C. albicans (Kim et al., 2020).

In vertebrates (Table 3), the immune system is divided into innate immunity and adaptive immunity. Adaptive immunity provides an effective and specific immune response against pathogens, while innate immunity consisting of the first line of defense is much quicker to respond to initial attacks. AMPs produced in response to pathogenic attacks form part of the first line of defense and are a source of AFPs. AFPs derived from vertebrates are usually produced on the skin, mucous membranes, and other areas that are easily exposed to microbial environments (López-Meza et al., 2011). It has been shown that the piscidins synthesized in the epithelia of gills, skin, stomach, and gut of a variety of teleost species exhibit antifungal activity against C. albicans, M. furfur, and T. beigelii through membrane disruption mode (Asensio-Calavia et al., 2023; Rakers et al., 2013). In addition, pleurocidin secreted by the skin of winter flounder inhibits the growth of Alternaria spp., C. albicans, F. Oxysporum, and A. niger (Cole et al., 1997; Souza et al., 2013). Recently, we have shown that AP10W, a short peptide derived from AP-2 complex subunit mu-A of zebrafish, displays conspicuous antifungal activities against the main fungal pathogens of human infections C. albicans and A. fumigatus. We also show that AP10W inhibits fungal biofilm formation and decrease pre-established fungal biofilms (Gong et al., 2022).

Similarly, the skin and secretory glands of amphibian frogs are also a rich source of AFPs, such as magainins and peptide glycine-leucine-amide (PGLa) identified in the clawed frog Xenopus laevis (Zasloff, 1987; Soravia et al., 1988), and temporins A, B, and L identified in the red frog Rana temporaria (Simmaco et al., 1996; Marcocci et al., 2018; Roscetto et al., 2021). Temporin G, recently isolated from the skin of Rana temporaria, is demonstrated to exert fungicidal ability against C. neoformans, Candida spp., and Aspergillus spp. In addition, temporin G reduces the metabolic activity of C. albicans cells, induces moderate membrane perturbation, and is effective against virulence factors of C. albicans (D'Auria et al., 2022).

In mammals, both neutrophils and epithelial cells are known to produce AFPs, including defensins, cathelicidins, histatins, and lactoferricins. Defensins are widely present in eukaryotes (fungi, plants, and animals), with four types of human defensins, known as HNP-1, HNP-2, HNP − 3, and HNP-4. All human defensins have been shown to possess candidacidal ability and are capable of influencing the ionic environment and the metabolic state of C. albicans cells (Selsted et al., 1985; Lehrer et al., 1988; Wilde et al., 1989). Cathelicidins have been identified in both humans and chimpanzees. LL-37, a 37-mer peptide derived from the N-terminal 37 residues of human cathelicidins, inhibits the growth of fungi including Aspergillus, Candida, Colletotrichum, Fusarium, Malassezia, Pythium, and Trichophyton. LL-37 exerts its fungal inhibition through several mechanisms, including cell wall integrity disruption, membrane permeabilization, and intracellular effects such as formation of autophagy-like structures, disturbance of endoplasmic reticulum homeostasis, induction of oxidative stress, inhibition of cell cycle progression, and alterations in gene expression (Memariani and Memariani, 2023). Histatins are histidine-rich peptides abundantly present in human saliva and the oral cavity (Oppenheim et al., 1988). Histatins exhibit a broad spectrum of antifungal activities and play an important role in controlling periodontal and oral fungal infections (Kavanagh and Dowd, 2004; Pólvora et al., 2018). Human histatins are composed of nine classes (human histatins 1–9), and histatin 5 is found to have strong fungicidal activity against C. albicans, C. glabrata, C. krusei, C. neoformans, and Saccharomyces cerevisiae (Sharma et al., 2021). Lactoferrin is a glycoprotein with AMP activity, found in saliva, milk, vaginal secretions, tears, and other exocrine secretions of mammals (cows, pigs, mice, and humans) (Moreno-Expósito et al., 2018; Rascón-Cruz et al., 2021; Drago-Serrano et al., 2018). Lactoferricin is found to have antifungal activity against a variety of fungi including Clavispora lusitaniae, Pichia kudriavzevii, Kluyveromyces marxianus, Meyerozyma guilliermondii, S. cerevisiae, Candida spp., and Cryptococcus spp.(Fernandes et al., 2020). Moreover, lactoferricin also shows inhibitory activity against the fungal biofilm formed by keratitis-associated fungal pathogens, such as A. fumigatus, F. solani, and C. albicans (Sengupta et al., 2012).

The fungal cell wall, the first barrier of the cell to effectively resist the influence of the external environment, is mainly composed of chitin, mannans, glycoproteins, and glucans (β − 1,3-glucan, β − 1,6-glucan, α-1,3-glucan, α-1,4-glucan, and mixed β-1,3−/β-1,4-glucan) (Bowman and Free, 2006). It protects the fungal cell from the pressure of the external environment and maintains normal cell metabolism, ion exchange, and osmotic pressure (Cabib and Arroyo, 2013).

Chitin and β-1,3-glucan are both the major structural components of the cell walls of many fungi and play a key role in maintaining the structural integrity of fungal cell walls (Bowman and Free, 2006; Lenardon et al., 2010; Fleet, 1991). Some AFPs are inhibitors of chitin synthase or (1–3)-β-D-glucan synthase, capable of blocking the synthesis of cell wall components, which then disrupts the normal cell morphology and diminishes the cell’s capacity to regulate osmotic pressure. For example, the fungus-sourced AgAFP suppresses the activity of chitin synthases III and V, which are important for chitin biosynthesis (Hagen et al., 2007). The echinocandin, a cyclic hexapeptide isolated from several species of Aspergillus, inhibits β-1,3-D-glucan synthase necessary for glucan biosynthesis (Ciociola et al., 2016; Thorn et al., 2024). Neopeptins have been reported against some plant pathogenic fungi by inhibiting proteoheteroglycan and β-1,3-glucan synthesis, which could affect cell wall biosynthesis (Ubukata et al., 1984). The nikkomycins (a complex of nucleoside-peptides), an analog of chitin synthase natural substrate N-acetylglucosamine, also block the synthesis of chitin in C. albicans (McCarthy et al., 1985). Analogously, the aureobasidin A, a cyclic depsipeptide produced by Aureobasidium pullulans, interferes with fungal cell wall integrity by affecting actin assembly, chitin delocalization, and synthesis of sphingolipids (Endo et al., 1997; Nagiec et al., 1997).

AFPs, as part of AMPs, are usually small (< 100 amino acids) cationic polypeptides, with amphiphilic structures with hydrophobic domains capable of binding to lipids and positively charged hydrophilic domains capable of binding to water or negatively charged residues (Hollmann et al., 2018). This property of AFPs renders them able to form strong binding to the amphiphilic part of the cell membrane, which lays the structural basis for the interaction of AFPs with fungal cell membranes. Several models have been proposed to explain the action of membrane disruption caused by AFPs, such as the barrel-stave, toroidal pore, and carpet models (Zhang et al., 2021; Yeaman and Yount, 2003). According to the barrel-stave model, AFPs bind to lipid membranes and recognize each other to form a transmembrane pore (Theis and Stahl, 2004; Soltani et al., 2007). Ultimately, the cell membrane components are penetrated by AFPs, resulting in cell collapse and death. The toroidal model suggests that AFPs insert into the hydrophobic center of the cell membrane (Soltani et al., 2007; Le et al., 2017), triggering the phospholipid molecular layer to curve inward and form a mixed cavity randomly (Yang et al., 2001). As a consequence, the membrane structures are disordered, eventually leading to cell death. In the carpet model, AFPs interact only with the lipid head groups and are oriented parallel to the surface, forming a carpet-like pattern. Once AFPs on the membrane surface accumulate to a certain concentration threshold, they act as detergents by distorting the phospholipid bilayer, reducing the stability of the cell membrane and causing cell membrane disintegration and cell lysis (Lohner and Prenner, 1999; Zhang et al., 2020). For example, plant defensins Dm-AMP1 from dahlia (Dahlia merckii), RsAFP2 from radish (Raphanus sativus), and HsAFP1 from coral bells (Heuchera sanguinea) target specific binding sites on fungal cells (e.g., mannosyl diinositolphosphoryl ceramide from S. cerevisiae for Dm-AMP1 and glucosylceramide from P. pastoris for RsAFP2), leading to membrane permeabilization and eventually decreasing viability (Thevissen et al., 2003; Thevissen et al., 2004; Aerts et al., 2008). Similarly, protonectin, isolated from the venom of the neotropical social wasp Agelaia pallipes pallipes, exerts antifungal/fungicidal activities against the tested fungal cells via interaction with lipid membranes, disruption of the membrane integrity, and induction of the production of intracellular ROS (Wang et al., 2015). MAF-1A, a linear 26-amino acid peptide, is derived from the carboxy-terminal functional domain of the antifungal peptide-1 (MAF-1) isolated from the hemolymph of Musca domestica larvae (Zhou et al., 2016). It has been shown that MAF-1A disrupts the cell membrane of C. albicans and then enters the cell where it binds and interacts with nucleic acids (Cheng et al., 2021). However, it must be pointed out that although the interaction of AMPs (including AFPs) with biological membranes plays an important role in the entire process of their antimicrobial actions, membrane disruption is a rather complex and dynamic process, which still needs detailed study to refine the specific mechanisms (Haney et al., 2019).

AFPs can also interact with fungal intracellular targets, including nucleotides (DNA and RNA), proteins, and organelles. For example, CGA-N9, an antifungal peptide derived from human chromogranin A (CGA), has been found to exert antifungal activity toward C. tropicalis by attenuating mitochondrial function (Li et al., 2019). Psd1 defensin from pea (Pisum sativum) has been found to enter N. crassa cells, localize to the nuclei, and interfere with the cell cycle, probably via interacting with cyclin F (Lobo et al., 2007). Similarly, histatin 5 binds to mitochondria after crossing fungal membranes via transmembrane potentials or receptors (without damaging the plasma membrane) and then induces non-lytic release of adenosine triphosphate (ATP) into the cytoplasm. The released ATP binds to purinergic receptors on the cell surface, leading to the inhibition of mitochondrial respiration and the generation of ROS, which then causes damage to nucleic acids and organelles (Kavanagh and Dowd, 2004; Helmerhorst et al., 2001). The antifungal peptide EcAMP1 isolated from barnyard grass shows strong antifungal action toward species of the Fusarium genus. It has been found that EcAMP1 first binds to one or several abundant components of the fungal cell surface and is then internalized by the fungal cell and accumulates in a vesicular structure in the cytoplasm without disturbing the integrity of the membrane (Nolde et al., 2011).

AFPs are naturally isolated from different species of organisms. Currently, only a limited number of AFPs are obtained from their natural sources for clinical use as the isolation of these natural peptides is time-consuming and expensive due to their relatively low abundance (Vriens et al., 2014). The methods commonly used now for industrial-scale AFP production from natural sources are microbial fermentation and proteolysis.

The natural echinocandins, echinocandin B, pneumocandin B0, and FR901379, are produced for commercial purposes from Aspergillus rugulosus, Glarea lozoyensis, and Coleophoma empetri, respectively. The production of echinocandins is an exceptional example of AFPs produced by microbial fermentation and further chemical modification in the case of the semisynthetic variants. The fermentation process is critical to obtain a competitive product and thus needs optimizing to increase the amount of natural echinocandins and control their purification costs. Temperature is a key factor in fermentation production and influences the overall production costs of AFPs (Emri et al., 2013). The optimal temperatures for the production of echinocandin B, pneumocandin B 0, and FR901379 are similar, all below 30°C. However, the optimal growth temperatures of the strains of A. rugulosus, G. lozoyensis, and C. empetri exhibited considerable variation. The optimal growth temperatures of G. lozoyensis and C. empetri were observed to be lower than 30°C, whereas the optimal growth temperatures of A. rugulosus were found to be higher than 30°C, reaching 37°C; it exhibited poor growth at the temperatures utilized for the production of echinocandin B. The addition of complex nitrogen sources and plant oils also influences the generation of echinocandins (Emri et al., 2013).

Production of pharmaceutical-grade AFPs can be achieved through enzymatic hydrolysis of proteins, resulting in the release of encrypted peptides. The process generally involves three steps, i.e., acquisition of raw materials, protein hydrolysis, and fractionation and isolation. By-products from dairy, fish, and meat industries are all suitable sources of proteins (Sibel Akalin, 2014; Ryder et al., 2016). For proteolysis, the utilization of immobilized enzymes possesses several advantages over the conventional soluble enzymes, such as milder and controlled conditions and recycling of enzymes used (Sewczyk et al., 2018). The final step of fractionation and isolation of AFPs includes ultrafiltration, precipitation with solvents, and liquid chromatography techniques, which are usually expensive (Brady et al., 2008). Fortunately, an alternative and cost-effective method, electro-membrane filtration, which combines electrophoresis with conventional membrane filtration, has been established (Bazinet and Firdaous, 2013) and is increasingly being applied for the fractionation and isolation of AFPs.

Recombinant expression presents a solid option for producing AFPs at low cost and high efficiency. In addition, sequencing technologies have generated a vast amount of genomic and transcriptomic data, providing valuable resources for discovering and designing new and more active AFPs (Amaral et al., 2012; Porto et al., 2012; Tracanna et al., 2017). Bacteria (mainly Escherichia coli), yeasts (mainly P. pastoris), and plants (e.g., tobacco plant Nicotiana tabacum) are the most common expression platforms for recombinant proteins.

E. coli BL21 (DE3), deficient in proteases that may lead to protein degradation, is by far the most commonly used bacterial species as a host for recombinant production of proteins (Li, 2011). Many E. coli strains are unable to export proteins across their outer membrane. As a result, in the majority of cases, proteins are secreted into the cytoplasm or periplasm, leading to the formation of inclusion bodies (Singh et al., 2015). Examples of AFPs produced in E. coli include lactoferricin B, nikkomycin, magainin-2, and cecropin (Fernández de Ullivarri et al., 2020).

In contrast to prokaryotic E. coli, eukaryotic yeasts are capable of implementing certain post-translational modifications to heterologous recombinant proteins. Yeasts commonly used as hosts for recombinant proteins include S. cerevisiae, P. pastoris, Kluyveromyces lactis, Yarrowia lipolytica, Schizosaccharomyces pombe, and Hansenula polymorpha. For example, the recombinant antifungal proteins serum albumin and hen lysozyme were produced by K. lactis and S. cerevisiae, respectively (Vieira Gomes et al., 2018). In addition, filamentous fungi such as A. pullulans, P. Chrysogenum, and P. digitatum have also been used to generate AFPs. Some examples of AFPs produced by yeasts or filamentous fungi are protegrin-1, porcine lactoferrin, aureobasidin A, and NFAP2 (Fernández de Ullivarri et al., 2020; Slightom et al., 2009).

Plants have been explored as hosts for recombinant expression of AFPs due to their capacity for large-scale production and their cost-effectiveness. The advantages of plants as expression systems are their capability to perform appropriate glycosylation, folding, and disulfide bond formation of recombinant AFPs. Different genetic approaches have been employed to produce AMPs in plants including using whole plants, tissue-specific expression, tissue culture, or transient expression (Holaskova et al., 2015). Whole tobacco plants have been utilized to produce lactoferrin and dermaseptin with a higher yield (Chahardoli et al., 2018; Shams et al., 2019).

The chemical synthesis of peptides allows scientists to design and produce specific sequences of AFPs on demand. Ideally, an AFP should be short. De novo peptide design may help reduce production costs, potential toxicity, and lability and increase bioactivity in vivo (Steckbeck et al., 2014). Peptide chemical synthesis is divided into two types: solid- (SPPS) or liquid (solution)-phase peptide synthesis (LPPS). Currently, fluorenylmethyloxycarbonyl (Fmoc) SPPS is the preferred method for chemical synthesis of AFPs due to the versatility and low cost of very high-quality building blocks. We have recently designed and synthesized a peptide named AP10W that shows increased antifungal activity (Gong et al., 2022).