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Review

Plant Protease Inhibitors as Emerging Antimicrobial Peptide Agents: A Comprehensive Review

by
Mónica G. Parisi
1,
Brenda Ozón
2,
Sofía M. Vera González
2,
Javier García-Pardo
3,* and
Walter David Obregón
2,*
1
Instituto de Ecología y Desarrollo Sustentable (INEDES, CONICET-UNLu) and Departamento de Ciencias Básicas, Universidad Nacional de Luján, Ruta 5 y Avenida Constitución, Luján B6700, Buenos Aires, Argentina
2
Centro de Investigación de Proteínas Vegetales (CIProVe) and Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, 47 y 115 s/N, La Plata B1900, Buenos Aires, Argentina
3
Institut de Biotecnologia i de Biomedicina (IBB) and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain
*
Authors to whom correspondence should be addressed.
Pharmaceutics 2024, 16(5), 582; https://doi.org/10.3390/pharmaceutics16050582
Submission received: 24 February 2024 / Revised: 16 April 2024 / Accepted: 18 April 2024 / Published: 24 April 2024
(This article belongs to the Special Issue Bioactive Molecules from Plants: Discovery and Pharmaceutical Applications, 3rd Edition)
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Abstract

:
Antimicrobial peptides (AMPs) are important mediator molecules of the innate defense mechanisms in a wide range of living organisms, including bacteria, mammals, and plants. Among them, peptide protease inhibitors (PPIs) from plants play a central role in their defense mechanisms by directly attacking pathogens or by modulating the plant’s defense response. The growing prevalence of microbial resistance to currently available antibiotics has intensified the interest concerning these molecules as novel antimicrobial agents. In this scenario, PPIs isolated from a variety of plants have shown potential in inhibiting the growth of pathogenic bacteria, protozoans, and fungal strains, either by interfering with essential biochemical or physiological processes or by altering the permeability of biological membranes of invading organisms. Moreover, these molecules are active inhibitors of a range of proteases, including aspartic, serine, and cysteine types, with some showing particular efficacy as trypsin and chymotrypsin inhibitors. In this review, we provide a comprehensive analysis of the potential of plant-derived PPIs as novel antimicrobial molecules, highlighting their broad-spectrum antimicrobial efficacy, specificity, and minimal toxicity. These natural compounds exhibit diverse mechanisms of action and often multifunctionality, positioning them as promising molecular scaffolds for developing new therapeutic antibacterial agents.

1. Introduction

Natural products (NPs) have long served as a source of active ingredients for a broad array of pharmaceutical applications, owing to their extensive chemical and structural diversity and generally low toxicity [1,2]. Predominantly sourced from plants, these compounds exhibit a wide range of biological mechanisms, offering novel activities and biological properties essential for the development of nutraceuticals, pharmacologically active substances, phytosanitary agents, and other economically valuable products [3,4,5,6].
The increasing emergence of multidrug-resistant (MDR) bacteria is a critical global problem [7,8,9]. From the One Health perspective, this can be considered one of the most significant challenges for the biomedical field, animal health, and phytosanitary services [10,11,12,13]. Indeed, it has recently been estimated that antimicrobial resistance (AMR) was directly responsible for 1.27 million global deaths and contributed to 4.95 million deaths, affecting countries in all regions and at all income levels, being higher in low- and middle-income countries, thus producing significant economic costs (WHO) [14].
In this scenario, the search for natural therapeutic agents, especially as alternatives to synthetic antibiotics, remains a fervent area of research and development within the scientific community. Notably, in the last 35 years, natural compounds have been the basis for over 35% of drugs approved by the Food and Drug Administration (FDA) (https://www.fda.gov/drugs) [15]. This trend is expected to continue, with predictions suggesting that natural and biopharmaceutical products, including small chemical compounds, peptides, and proteins, could form the majority of our pharmacotherapeutic tools in the near future [16].
In the plant kingdom, antimicrobial compounds act as the primary defense mechanism against pathogenic microorganisms. In this context, numerous plant antimicrobial molecules capable of inhibiting a number of human pathogens have been isolated from various plant sources [17,18,19,20]. These natural compounds include primary metabolites essential for plant growth, substance transport, photosynthesis, respiration, nutrient assimilation, flowering, and fruit ripening (i.e., proteins, lipids, and carbohydrates) and secondary plant metabolites (i.e., terpenes, polyphenols, and carotenoids), which do not participate in fundamental physiological processes, although they exhibit diverse bioactive chemical scaffolds. In addition to these small chemical compounds, a variety of proteins and peptides with promising antimicrobial properties have also been isolated and characterized so far. These include chitinases, β-1,3-glucanases, thaumatin-like proteins, endoproteases, peroxidases, ribonuclease-like proteins, γ-thionins, plant defensins, oxalate oxidases, oxalate oxidase-like proteins, proteases, and peptide protease inhibitors (PPIs) [21,22]. During the last two decades, up to eighteen PPIs with antimicrobial activity have been described so far. Among them, several PPIs have demonstrated broad-spectrum efficacy against a wide range of pathogens, including bacteria, fungi, and viruses [23,24,25,26]. These include Kunitz- and Trypsin-type inhibitors from plants belonging to the Solanaceae, Fabaceae, and Moringaceae families.
This work aims to offer a comprehensive review of plant-derived PPIs and their potential as novel antibacterial and antifungal agents, exploring their mechanisms of action, efficacy against various microbial strains, and the potential for their application as innovative antimicrobial peptide agents. Ultimately, we also discuss future directions of this field.

2. Antimicrobial Peptides (AMPs)

The extensive research has brought many novel antimicrobial candidates into clinical and pre-clinical development [27,28]. An important number of these molecules are antimicrobial peptides (AMPs), a diverse group of small molecules synthesized and ubiquitously distributed in plants and other living organisms. Due to antimicrobial potential and immunomodulatory capacities, research on AMPs has steadily increased in the last decade. Currently, over 3940 AMPs have been reported so far, including 3146 natural AMPs of different natural sources, including 383 bacteriocins/peptide antibiotics from bacteria; 5 from archaea; 8 from protists; 29 from fungi; 250 from plants; and 2463 from animals, according to the latest update of the Antimicrobial Peptide Database (APD3) (http://aps.unmc.edu/AP/) [29] (Figure 1). While the majority of AMPs have been identified in animals and plants, those isolated from archaea and protists include a set of relevant molecules, such as halocins, that have acquired specific adaptations to extreme environments. These adaptations could translate into robust stability and efficacy under conditions that are challenging for other antimicrobial compounds. The activity and mechanism of these and other AMPs have been thoroughly reviewed elsewhere [30,31,32,33]; therefore, this review will specifically focus on the recent developments in plant-derived AMPs, particularly those with protease inhibition activity.
Plant AMPs are generally classified based on their tridimensional structure, their sequence, and the presence of disulfide bonds, such as thionins, defensins, hevein-like peptides, knottins, stable-like peptides, lipid transfer proteins, snakins, and cyclotides [24,34,35,36,37]. Thionins are essential peptides found in monocots and dicots, with sizes ranging from 45 to 48 amino acid residues and a molecular weight of around 5 kDa [36]. They are classified into two groups: α/β-thionins and γ-thionins, according to amino acid sequence homology, three-dimensional structural similarity, and disulfide bridge positions. Regarding the antimicrobial activity of thionins, antibacterial [38,39], antifungal, anti-larvicidal, and in vitro cytotoxic effects against mammal cell cultures have been reported [24,40].
Defensins constitute one of the largest groups of AMPs (Figure 2). They have been shown to disrupt cellular functions by binding extracellularly to the cell wall components, membrane targets, and/or to specific membrane lipids such as sphingolipids or phospholipids or binding to cell surface targets and membrane lipids [41]. An important aspect of AMP functionality involves the modulation of the host plant’s defense mechanisms. In this regard, some AMPs have been found to prime the plant’s immune response, preparing it for a more robust defense against subsequent pathogen attacks. This priming effect boosts the plant’s innate immune system, enhancing its responsiveness through the modulation of various signaling cascades [42]. Hevein-like peptides are basic peptides containing 29–45 amino acid residues stabilized by 3–5 disulfide bonds, with a conserved chitin-binding domain (SXFGY/SXYGY), which is associated with antifungal activity [43]. These molecules were first identified in the latex of Hevea brasiliensis and are responsible for defense against a wide range of pathogenic fungi based on the interaction of the hevein domain with fungal chitin present in the cell, causing damage to the fungal cell.
Plant knottins are peptides with approximately 30 amino acid residues, the smallest plant AMPs, involving three disulfide bonds (cysteine knot motifs) and two conformations, cyclic and linear, showing high thermal stability and resistance to chemicals and proteolytic action [44]. Knottins are known as “promiscuous peptides” based on their multiple biological functions: they can bind to cell membranes, K+ and Na+ channels in membranes, and acid-sense channels; are highly cytotoxic to human cells [45]; and have antimicrobial activity against bacteria, fungi, viruses, and insects via interacting with membranes to exert their activities.
The α-hairpinin family represents a class of Lys/Arg-rich plant defense peptides, with special Cys motifs (i.e., XnC1X3C2XnC3X3C4Xn, where X is any amino acid different from cysteine), to form a highly characteristic helix–loop–helix secondary structure with two antiparallel α-helices [46] stabilized by two disulfide bonds in the tertiary structure. This AMP family shows antibacterial, antifungal, trypsin-inactivating, and ribosome-inactivating activities [46,47].
Lipid transfer proteins (LTPs) are small cationic peptides (7 to 10 kDa), composed of a conservative pattern of eight Cys residues and four disulfide bonds that stabilize a tight tertiary fold with hydrophobic cavities. LTPs inhibit the growth of fungi and bacteria, thus participating in plant defense systems [48].
The snakin class contains 12 cysteine residues (cysteine-rich), the largest number of disulfide bonds and are constitutively or inducibly expressed via biotic or abiotic stress in different organs, such as the roots, stem, leaves, flowers, and seeds. Snakins inhibit both fungal (e.g., Magnaporthe grisea, Fusarium solani, and Botrytis cinerea) and bacterial (e.g., Dickeya dadantii, Ralstonia solanacearum, and Sinorhizobium meliloti) growth [49].
Cyclotides are long-chain cyclic peptides (28–37 amino acids) consisting of six loops with six conserved cysteine residues, stabilized via three disulfide bonds. They are anionic AMPs with high resistance to thermal and chemical denaturation as well as proteolytic degradation, making them potential therapeutic agents as antitumor, anti-HIV, insecticidal, and antibacterial agents [50,51].
In this context, it is known that around 3% of AMPs are plant PPIs (Figure 2) with proven effectiveness in inhibiting the growth of a variety of pathogenic bacterial and fungal strains [36]. Not surprisingly, many of these AMPs share common structural and functional properties with plant PPIs, including their ability to alter the integrity of microbial cell membranes, a mechanism crucial for their function [36,52,53,54]. This positions this set of natural peptides as promising molecules for developing novel therapeutics against MDR bacteria.

3. Mechanism of Antimicrobial Action of Plant AMPs

Typically, AMPs are characterized by their low molecular weight, displaying less than 100 amino acids with a positive net charge at physiological pH, due to the presence of lysine and arginine residues and a high proportion (≥30%) of hydrophobic residues [32,55]. These characteristics render them highly cationic molecules that can interact with the phospholipid and lipopolysaccharide layers found in many types of bacteria, leading to pore formation, membrane destabilization, and rapid cell death [25,32,56,57,58]. At present, several mechanisms have been reported on cationic AMPs that cause cell membrane damage by interacting with the negatively charged phospholipid layer and lipopolysaccharides of the bacterial membrane, causing pores, membrane destabilization, and rapid cell death [25,56,57,58]. For Gram-negative bacteria, the AMP needs to permeabilize the outer membrane before it can access the cytoplasmic membrane. Conversely, for Gram-positive bacteria, the AMP can directly diffuse through the peptidoglycan layer via nano-sized pores.
AMPs can also mediate their action by acting as lipid transfer proteins. Through this mechanism, these proteins directly interact with fungal membrane lipids, effectively blocking pathogen penetration into the host cell and, thus, preventing infection [59]. Furthermore, many AMPs can specifically bind to the sphingolipid and phospholipid bilayers of the cell wall and/or the cell membrane of fungi, being able to enter or remain on the outside of the fungal cell. Regardless of their possible absorption, antifungal peptides can affect intracellular targets, causing, among other actions, the production of ROS, programmed cell death, mitochondrial dysfunction, the alteration of cation homeostasis, ATP efflux, cell cycle impairment, and autophagy, among others [29]. They can penetrate the target cell by breaking the membrane and interfering with protein or nucleic acid synthesis or cell division or by inhibiting protease activity [53,60]. For instance, heveins bind chitin in fungi [43] and some defensins interact with eukaryotic target proteins to target intracellular functions [41]. Another mechanism of action is priming the plant by inducing defense responses against pathogens due to the modulation of different cascades in their immune system (Figure 3) [61].

4. Peptide Protease Inhibitors (PPIs) Derived from Plants

Protease inhibitors (PIs) are regulatory molecules of a proteinaceous or non-proteinaceous nature, ubiquitously distributed in animals, plants, and across microbial species. Those of a proteinaceous nature are typically known as PPIs. Based on the type of protease they inhibit, PPIs can be categorized into six main classes: serine protease inhibitors (SPIs), cysteine protease inhibitors (CPIs), aspartyl protease inhibitors (APIs), metalloprotease inhibitors (MPIs), glutamate protease inhibitors (GPIs), and threonine protease inhibitors (TPIs) [62,63,64].
PPIs play a central role in controlling the activity of target proteases, often inhibiting their excessive and uncontrolled activity in both normal and pathological conditions [65]. This regulation is essential for activating coenzymes and releasing biologically active polypeptides [66,67]. In the case of mammals, the presence of PPIs in plasma is closely associated with the regulation of critical proteolytic cascades, including blood coagulation and complement activation [66,68,69,70]. Moreover, proteolysis is involved in many vital biological functions, such as immunity, blood coagulation, cell cycle regulation, and tissue morphogenesis [71,72]. In this context, PPIs have been exploited by the pharmaceutical industry for different applications, including drug discovery and diagnosis [22].
Notably, several low-molecular PPIs from plants contain a high content of cysteine residues that form disulfide bridges that confer resistance to heat treatment, extreme pH or ionic forces, and proteolysis [62,73,74,75,76,77]. Owing to their multifunctionality and remarkable physicochemical properties, PPIs have found a wide array of applications in biotechnology and biomedicine, including their use as antimicrobial agents [21,78,79,80] (Figure 4). Despite their high stability, the presence of multiple cysteine residues involved in disulfide bridges often complicates the production of these molecules, requiring a comprehensive understanding of their folding mechanisms and three-dimensional structure [81,82].

5. Plant-Derived Peptide Protease Inhibitors with Antimicrobial Properties

Over the last 15 years, there has been a notable increase in the reports on novel PPIs with antimicrobial properties isolated from various plant sources [22,83,84,85]. An important number of these PPIs have been linked to antimicrobial roles in a physiological context. This is the case of the systemin hormone, an 18-amino acid peptide that plays a critical role in the defense against pathogens and is a key part of the jasmonic acid signaling pathway that triggers PPI expression [73]. In the Solanaceae family, the prosystemin mechanism involves the expression of genes predominantly associated with metallo carboxypeptidase inhibitors, cysteine PPIs, and trypsin PPIs, including Kunitz-type PPIs [74,86].
PPIs isolated from a variety of plants have shown potential in inhibiting the growth of pathogenic bacteria, protozoans, and fungal strains (Table 1) [21]. As introduced above, the antimicrobial activity of many plant PPIs is typically mediated through the ability of these molecules to alter the permeability of bacterial cytoplasmic membranes. This toxic action is facilitated via the cationic nature of these peptides, which bind to cell membranes through interactions between positive and negative charges, causing destabilization and the formation of temporary cavities or disruptions. Such damage leads to the leakage of cellular contents and ultimately results in bacterial death [67,75,85]. In addition to their antimicrobial actions, it is also common that these molecules simultaneously target diverse metabolic processes, which has fueled interest in applying these molecules for diverse biomedical applications.

5.1. Plant Protease Inhibitors with Antibacterial Activity

Potatoes (Solanum. tuberosum spp.) have historically been a rich source of plant PPIs with antimicrobial (antifungal and antibacterial) activities against a wide variety of agricultural and clinical pathogens [100]. A prototypical example is the Kunitz-type serine PPI, Potide-G, isolated from the tubers of S. tuberosum L. cv. Golden Valley, which has been shown to be effective in inhibiting the growth of diverse human pathogens, including S. aureus and L. monocytogenes, through the regulation of extracellular enzymes related to nutrition. This inhibitor showed a strong potency with minimum inhibitory concentration (MIC) values lower than 30 µg/mL, demonstrating a potency comparable to established antibiotics [26]. Similarly, PG-2, a peptide isolated from the potato tubers of cv. Gogu Valley, exhibited antibacterial activity against S. aureus and other pathogens, displaying minimal cytotoxic effects against human red blood cells [92].
A significant number of studies have reported on the action mechanisms of trypsin inhibitors on the bacterial membrane. A trypsin PPI called API was purified from A. amara seeds, showing antimicrobial activity against P. aeruginosa and B. subtilis, being useful as a potential antibacterial component [87]. In 2018, Martins et al. [88] discovered a Bowman–Birk PPI (called LzaBBI) from L. auriculata seeds that exhibits robust antibacterial activity against S. aureus. This 14.3 kDa thermostable protein, similar to other Bowman–Birk inhibitors, demonstrates mixed-type inhibitory activity against trypsin and chymotrypsin. Scanning electron microscopy experiments unequivocally revealed that LzaBBI compromises S. aureus membrane integrity, possibly through oxidative stress and reactive oxygen species (ROS) production, leading to cell lysis. In a previous study, Satheesh and Murugan [83] identified a 14.3 kDa trypsin PPI from C. grandis leaves that effectively inhibits several pathogenic bacteria, including S. aureus. Similarly, a 4 kDa trypsin inhibitor from C. fistula leaves, called Fistulin, demonstrated significant antibacterial activity, paralleling the efficacy of streptomycin sulfate by inhibiting microbial proteases [89]. Additionally, a new Kunitz-type inhibitor was isolated from R. frangula leaves. This inhibitor displayed a potent inhibition against several proteases from Bacillus sp, such as sd B. licheniformis [94,95].
In 2020, Almeida and coworkers [90] synthesized Adepamycin, a peptide based on a trypsin inhibitor from A. pavonina seeds, showing antimicrobial activity by damaging the membrane integrity of various pathogens without displaying toxicity against human red blood cells. Additionally, Costa et al. [96] explored the action of a peptide from J. curcas seeds, JcTI-I, on proteases from S. enterica and S. aureus, suggesting an alternative antimicrobial mechanism of trypsin inhibitors. Further studies, such as those by Mehmood et al. [62], Rodrigues et al. [91], and Wang et al. [97], have further uncovered the bacteriostatic properties of trypsin inhibitors and their efficacy against bacterial pathogens.
Notably, two recent studies have unveiled novel PPIs with significant antimicrobial potential from Moringa and Salvia plants. In 2021, Cotabarren et al. [98] reported the purification and characterization of the first phytocystatin isolated from M. oleifera seeds (MoPI). MoPI, with a molecular mass of 19 kDa, shows higher physicochemical stability against acidic pHs and high temperatures. The study highlighted MoPI as one of the most potent cysteine PPI identified to date, exhibiting Ki and IC50 values within the nanomolar range. Additionally, MoPI has been recognized as a multifunctional molecule, showing robust antimicrobial activity against human pathogens, including E. faecalis and S. aureus, as well as significant anticoagulant activity. More recently, a trypsin inhibitor named ShTI was isolated and characterized from mucilage and fat-free chia seeds (S. hispanica L.). This inhibitor demonstrated exceptional thermostability and wide pH tolerance, likely due to its disulfide bridge structure [99]. ShTI exhibited antibacterial activity against S. aureus, including strains resistant to methicillin. However, it showed no significant effects against the Gram-negative bacteria tested. As such, ShTI presents itself as a promising candidate for standalone use or in combination therapy with oxacillin for managing S. aureus infections.

5.2. Plant Protease Inhibitors with Antifungal Activity

Phytopathogenic fungi produce extracellular proteinases that play an active role in the development of plant diseases [101]. In response to the attack, plants synthesize inhibitors to inactivate these proteinases [21]. This phenomenon was observed for the first time in tomatoes infected by Phytophthora infestans [102], in which it was found that high levels of trypsin and chymotrypsin inhibitors correlated with the resistance of the plants to the pathogen [103]. A similar strategy is also used by S. tuberosum spp., which produce various PPIs with strong antifungal activity [100]. Importantly, these bioactive molecules extend their fungicidal efficacy beyond phytopathogenic strains to include numerous human pathogens, highlighting their broad-spectrum antimicrobial potential.
Among the inhibitors mentioned in the previous section, Potide-G and PG-2, both Kunitz-type serine PPIs, have been shown to effectively inhibit the growth of several pathogenic fungal strains, such as C. albicans [26,92]. Similarly, AFP-J, isolated from S. tuberosum cv. L. Jopung [63], targets chymotrypsin, pepsin, and trypsin, exhibiting significant antifungal activity against C. albicans, T. beigelii, and S. cerevisiae. Additional Kunitz-type trypsin and chymotrypsin inhibitors with antifungal properties have been isolated and characterized. Among these, ApTIA, ApTIB, and ApTIC were extracted from the seeds of the Brazilian plant A. plumosa, demonstrating notable antifungal activity against pathogenic fungi, including A. niger, T. paradoxa, and Colletotrichum spp. Their mechanism of action is believed to involve the inhibition of serine proteases secreted by these fungi [77]. Moreover, Kunitz-type inhibitors such as RfIP-1 and PDInhibitor have been identified from R. frangula and Conyza dioscoridis, respectively. These inhibitors have exhibited strong antifungal activity against various fungal strains, including the commercial aerobic filamentous fungus A. oryzae. Furthermore, the protease inhibitor from Coccinia grandis has demonstrated an antifungal effect on C. albicans, Mucor indicus, Penicillium notatum, Aspergillus flavus, Aspergillus oryzae, and Cryptococcus neoformans [83].
From the Fabaceae family, the tropical trees I. edulis and I. laurina have also been explored as sources of novel bioactive molecules with antifungal properties. Two distinct Kunitz-type trypsin inhibitors, ILTI and IETI, exhibiting anticandidal activity, were isolated, and their activities characterized [76,84]. ILTI demonstrated potent inhibitory enzymatic activity against trypsin and significantly inhibited the growth of C. tropicalis and C. buinensis, causing changes in the membrane and permeabilization of yeast cells, as well as the production of reactive oxygen species (ROS). However, this inhibitor did not show efficacy against human pathogenic bacteria such as E. coli, S. aureus, and K. pneumoniae. IETI, characterized by a single trypsin-reactive site stabilized by a disulfide bridge and notable pH and thermal stability, displayed growth-inhibitory activity towards Candida spp., including C. buinensis and C. tropicalis. The yeast inhibitory action of these inhibitors involves multiple mechanisms, including protease inhibition, plasma membrane disruption affecting cell viability, oxidative stress targeting mitochondria, and inducing apoptosis in yeast by blocking critical serine peptidases (e.g., metacaspases) and a nuclear apoptosis mediator [104]. It was also observed that API from Albizia amara had an inhibitory effect on C. albicans [87], and Adepamycin from Adenanthera pavonina inhibited the growth of C. albicans and C. tropicalis [87,88]. Only recently, Cotabarren et al. [93] reported the purification and characterization of YBPTI, a thermostable trypsin inhibitor from yellow pepper (C. annuum L.) seeds with dual antifungal and hypoglycemic properties. The purified inhibitor showed potent and specific in vitro activity against C. albicans and showed no cytotoxicity against human Hela cells (ATCC CCL-2). Furthermore, the inhibitor also exhibited α-1,4-glucosidase inhibition activity, positioning this inhibitor as one of the first plant-derived molecules with such a particular dual combination of biological activities, demonstrating that this new inhibitor could be potentially used as an antifungal agent in pharmaceutical preparations to prevent invasive Candida infections.
In summary, plant-derived PPIs represent a valuable resource for the development of novel antimicrobial agents. The inherent stability, broad-spectrum antimicrobial efficacy, multifaceted functionality, and minimal toxicity to human cells position these natural compounds as promising candidates for advancing the treatment of microbial infections. Additionally, some of these inhibitors combine both strong antifungal activity and antibacterial properties, potentially serving as dual-purpose antimicrobial molecules.

6. Concluding Remarks and Future Perspectives

The worldwide rise of AMR and its impacts on plant, animal, and human health represent a formidable challenge to global health, with the rate of resistance development outpacing the introduction of new antibiotics. The widespread and sometimes indiscriminate use of antimicrobials in medicine, agriculture, and industry has accelerated this process, driving the emergence and spread of AMR as one of the world’s most pressing public health issues. Currently, infections by AMR pathogens are causing over 700,000 deaths per year. It is estimated that by 2050, AMR infections will be responsible for 10 million deaths annually worldwide, significantly impacting the global economy [13,105,106]. Due to its great complexity, it must be approached from different disciplines to frame it within the One Health approach. Thus, several countries have implemented national action plans to combat antibiotic-resistant microbes, following the guidelines of the FAO [107] and the WHO [14]. In this context, natural products, particularly from plant sources, offer a vast repository of chemical diversity that could lead to the discovery of novel antimicrobial agents [2].
Over the last 20 years, a number of natural PPIs with antibacterial and antifungal properties have been isolated from diverse plant sources. In this review, we aim to provide a comprehensive analysis of these molecules and insight into their specific activities, with a special focus on exploring PPIs as relevant agents against biomedical pathogens. The review has described the classification and the diverse mechanisms of action of these proteinaceous molecules, which exhibited a wide range of activities, ranging from disrupting microbial cell membranes to inhibiting vital enzymatic pathways. Taken together, these mechanisms provide novel opportunities for combating pathogens that have evolved resistance to current treatments.
However, to date, only a limited number of antimicrobial peptides (AMPs), such as nisin, gramicidin, polymyxins, and daptomycin have been adopted for clinical use [27]. There remains a significant need for extensive research to identify and enhance potential plant-derived PPIs with therapeutic significance. In this context, efforts have been made to develop novel computational tools aimed at predicting antimicrobial peptide sequences [108]. Moreover, such in silico approaches can also be utilized to improve molecules already known for their antimicrobial activity. Structure-based drug discovery strategies, for instance, might employ the known three-dimensional structures of target proteins to design molecules with enhanced biological activities [109]. This rational design approach will facilitate the development of novel, engineered molecules with superior antimicrobial properties.
In summary, this review illustrates the enormous potential of the PPIs from plant species in medicine. Their broad-spectrum antimicrobial activities, combined with their specificity and minimal toxicity towards non-target organisms, highlight their value as a sustainable and effective alternative to conventional synthetic chemicals. This, coupled with their multifunctionality, positions these molecules as integral components of the next generation of antimicrobial agents.

Author Contributions

Conceptualization, M.G.P., J.G.-P. and W.D.O.; writing—review and editing, M.G.P., B.O., S.M.V.G., J.G.-P. and W.D.O. All authors have read and agreed to the published version of the manuscript.

Funding

W.D.O. (Researcher Career Program) and B.O. (Doctoral Fellow) are members of CONICET, Argentina. The authors acknowledge the support from Universidad Nacional de La Plata (Argentina) under grants UNLP I+D 2020 project Code X-920, and the ANPCyT under Project PICT-2021-00955. J.G.-P. was supported by the Spanish Ministry of Science and Innovation with a Juan de la Cierva Incorporacion (IJC2019-041039-I).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Lorsbach, B.A.; Sparks, T.C.; Cicchillo, R.M.; Garizi, N.V.; Hahn, D.R.; Meyer, K.G. Natural products: A strategic lead generation approach in crop protection discovery. Pest Manag. Sci. 2019, 75, 2301–2309. [Google Scholar] [CrossRef]
  2. Bergonzi, M.C.; Heard, C.M.; Garcia-Pardo, J. Bioactive Molecules from Plants: Discovery and Pharmaceutical Applications. Pharmaceutics 2022, 14, 2116. [Google Scholar] [CrossRef] [PubMed]
  3. Harvey, A.L. Natural products in drug discovery. Drug Discov. Today 2008, 13, 894–901. [Google Scholar] [CrossRef] [PubMed]
  4. Harvey, A.L.; Edrada-Ebel, R.; Quinn, R.J. The re-emergence of natural products for drug discovery in the genomics era. Nat. Rev. Drug Discov. 2015, 14, 111–129. [Google Scholar] [CrossRef] [PubMed]
  5. Haider, R. harmaceutical and Biopharmaceuticals Industries: Revolutionizing Healthcare. Asian J. Nat. Sci. 2023, 2, 11. [Google Scholar]
  6. Clemons, P.A.; Bodycombe, N.E.; Carrinski, H.A.; Wilson, J.A.; Shamji, A.F.; Wagner, B.K.; Koehler, A.N.; Schreiber, S.L. Small molecules of different origins have distinct distributions of structural complexity that correlate with protein-binding profiles. Proc. Natl. Acad. Sci. USA 2010, 107, 18787–18792. [Google Scholar] [CrossRef] [PubMed]
  7. Despotovic, M.; de Nies, L.; Busi, S.B.; Wilmes, P. Reservoirs of antimicrobial resistance in the context of One Health. Curr. Opin. Microbiol. 2023, 73, 102291. [Google Scholar] [CrossRef] [PubMed]
  8. Tang, K.W.K.; Millar, B.C.; Moore, J.E. Antimicrobial Resistance (AMR). Br. J. Biomed. Sci. 2023, 80, 11387. [Google Scholar] [CrossRef] [PubMed]
  9. Mudenda, S.; Daka, V.; Matafwali, S.K. World Health Organization AWaRe framework for antibiotic stewardship: Where are we now and where do we need to go? An expert viewpoint. Antimicrob. Steward. Healthc. Epidemiol. 2023, 3, e84. [Google Scholar] [CrossRef] [PubMed]
  10. Collignon, P.J.; McEwen, S.A. One Health-Its Importance in Helping to Better Control Antimicrobial Resistance. Trop. Med. Infect. Dis. 2019, 4, 22. [Google Scholar] [CrossRef] [PubMed]
  11. McEwen, S.A.; Collignon, P.J. Antimicrobial Resistance: A One Health Perspective. Microbiol. Spectr. 2018, 6. [Google Scholar] [CrossRef] [PubMed]
  12. White, A.; Hughes, J.M. Critical Importance of a One Health Approach to Antimicrobial Resistance. Ecohealth 2019, 16, 404–409. [Google Scholar] [CrossRef] [PubMed]
  13. Velazquez-Meza, M.E.; Galarde-Lopez, M.; Carrillo-Quiroz, B.; Alpuche-Aranda, C.M. Antimicrobial resistance: One Health approach. Vet. World 2022, 15, 743–749. [Google Scholar] [CrossRef] [PubMed]
  14. WHO. Antimicrobial Resistance. Available online: https://www.who.int/news-room/fact-sheets/detail/antimicrobial-resistance (accessed on 2 April 2024).
  15. FDA. Drugs. Available online: https://www.fda.gov/drugs (accessed on 2 April 2024).
  16. Newman, D.J.; Cragg, G.M. Natural Products as Sources of New Drugs over the Nearly Four Decades from 01/1981 to 09/2019. J. Nat. Prod. 2020, 83, 770–803. [Google Scholar] [CrossRef] [PubMed]
  17. Boman, H.G. Antibacterial peptides: Basic facts and emerging concepts. J. Intern. Med. 2003, 254, 197–215. [Google Scholar] [CrossRef] [PubMed]
  18. Bulet, P.; Stocklin, R.; Menin, L. Anti-microbial peptides: From invertebrates to vertebrates. Immunol. Rev. 2004, 198, 169–184. [Google Scholar] [CrossRef] [PubMed]
  19. van Loon, L.C.; Rep, M.; Pieterse, C.M. Significance of inducible defense-related proteins in infected plants. Annu. Rev. Phytopathol. 2006, 44, 135–162. [Google Scholar] [CrossRef] [PubMed]
  20. Parisi, M.G.; Moreno, S.; Fernandez, G. Isolation and characterization of a dual function protein from Allium sativum bulbs which exhibits proteolytic and hemagglutinating activities. Plant Physiol. Biochem. 2008, 46, 403–413. [Google Scholar] [CrossRef] [PubMed]
  21. Kim, J.Y.; Park, S.C.; Hwang, I.; Cheong, H.; Nah, J.W.; Hahm, K.S.; Park, Y. Protease inhibitors from plants with antimicrobial activity. Int. J. Mol. Sci. 2009, 10, 2860–2872. [Google Scholar] [CrossRef]
  22. Shamsi, T.N.; Parveen, R.; Fatima, S. Characterization, biomedical and agricultural applications of protease inhibitors: A review. Int. J. Biol. Macromol. 2016, 91, 1120–1133. [Google Scholar] [CrossRef]
  23. Srivastava, S.; Dashora, K.; Ameta, K.L.; Singh, N.P.; El-Enshasy, H.A.; Pagano, M.C.; Hesham, A.E.; Sharma, G.D.; Sharma, M.; Bhargava, A. Cysteine-rich antimicrobial peptides from plants: The future of antimicrobial therapy. Phytother. Res. 2021, 35, 256–277. [Google Scholar] [CrossRef] [PubMed]
  24. Lima, A.M.; Azevedo, M.I.G.; Sousa, L.M.; Oliveira, N.S.; Andrade, C.R.; Freitas, C.D.T.; Souza, P.F.N. Plant antimicrobial peptides: An overview about classification, toxicity and clinical applications. Int. J. Biol. Macromol. 2022, 214, 10–21. [Google Scholar] [CrossRef] [PubMed]
  25. Zhang, Q.Y.; Yan, Z.B.; Meng, Y.M.; Hong, X.Y.; Shao, G.; Ma, J.J.; Cheng, X.R.; Liu, J.; Kang, J.; Fu, C.Y. Antimicrobial peptides: Mechanism of action, activity and clinical potential. Mil. Med. Res. 2021, 8, 48. [Google Scholar] [CrossRef] [PubMed]
  26. Kim, M.H.; Park, S.C.; Kim, J.Y.; Lee, S.Y.; Lim, H.T.; Cheong, H.; Hahm, K.S.; Park, Y. Purification and characterization of a heat-stable serine protease inhibitor from the tubers of new potato variety “Golden Valley”. Biochem. Biophys. Res. Commun. 2006, 346, 681–686. [Google Scholar] [CrossRef] [PubMed]
  27. Dijksteel, G.S.; Ulrich, M.M.W.; Middelkoop, E.; Boekema, B. Review: Lessons Learned From Clinical Trials Using Antimicrobial Peptides (AMPs). Front. Microbiol. 2021, 12, 616979. [Google Scholar] [CrossRef] [PubMed]
  28. Mazurkiewicz-Pisarek, A.; Baran, J.; Ciach, T. Antimicrobial Peptides: Challenging Journey to the Pharmaceutical, Biomedical, and Cosmeceutical Use. Int. J. Mol. Sci. 2023, 24, 9031. [Google Scholar] [CrossRef]
  29. Wang, G.; Li, X.; Wang, Z. APD3: The antimicrobial peptide database as a tool for research and education. Nucleic Acids Res. 2016, 44, D1087–D1093. [Google Scholar] [CrossRef]
  30. Kundu, R. Cationic Amphiphilic Peptides: Synthetic Antimicrobial Agents Inspired by Nature. ChemMedChem 2020, 15, 1887–1896. [Google Scholar] [CrossRef] [PubMed]
  31. Kumar, V.; Singh, B.; van Belkum, M.J.; Diep, D.B.; Chikindas, M.L.; Ermakov, A.M.; Tiwari, S.K. Halocins, natural antimicrobials of Archaea: Exotic or special or both? Biotechnol. Adv. 2021, 53, 107834. [Google Scholar] [CrossRef] [PubMed]
  32. Huan, Y.; Kong, Q.; Mou, H.; Yi, H. Antimicrobial Peptides: Classification, Design, Application and Research Progress in Multiple Fields. Front. Microbiol. 2020, 11, 582779. [Google Scholar] [CrossRef] [PubMed]
  33. Saeed, S.I.; Mergani, A.; Aklilu, E.; Kamaruzzman, N.F. Antimicrobial Peptides: Bringing Solution to the Rising Threats of Antimicrobial Resistance in Livestock. Front. Vet. Sci. 2022, 9, 851052. [Google Scholar] [CrossRef] [PubMed]
  34. Ramazi, S.; Mohammadi, N.; Allahverdi, A.; Khalili, E.; Abdolmaleki, P. A review on antimicrobial peptides databases and the computational tools. Database 2022, 2022, baac011. [Google Scholar] [CrossRef] [PubMed]
  35. Bin Hafeez, A.; Jiang, X.; Bergen, P.J.; Zhu, Y. Antimicrobial Peptides: An Update on Classifications and Databases. Int. J. Mol. Sci. 2021, 22, 11691. [Google Scholar] [CrossRef] [PubMed]
  36. Li, J.; Hu, S.; Jian, W.; Xie, C.; Yang, X. Plant antimicrobial peptides: Structures, functions, and applications. Bot. Stud. 2021, 62, 5. [Google Scholar] [CrossRef] [PubMed]
  37. Dos Santos-Silva, C.A.; Zupin, L.; Oliveira-Lima, M.; Vilela, L.M.B.; Bezerra-Neto, J.P.; Ferreira-Neto, J.R.; Ferreira, J.D.C.; de Oliveira-Silva, R.L.; Pires, C.J.; Aburjaile, F.F.; et al. Plant Antimicrobial Peptides: State of the Art, In Silico Prediction and Perspectives in the Omics Era. Bioinform. Biol. Insights 2020, 14, 1177932220952739. [Google Scholar] [CrossRef] [PubMed]
  38. Taveira, G.B.; Carvalho, A.O.; Rodrigues, R.; Trindade, F.G.; Da Cunha, M.; Gomes, V.M. Thionin-like peptide from Capsicum annuum fruits: Mechanism of action and synergism with fluconazole against Candida species. BMC Microbiol. 2016, 16, 12. [Google Scholar] [CrossRef] [PubMed]
  39. Taveira, G.B.; Mello, E.O.; Carvalho, A.O.; Regente, M.; Pinedo, M.; de La Canal, L.; Rodrigues, R.; Gomes, V.M. Antimicrobial activity and mechanism of action of a thionin-like peptide from Capsicum annuum fruits and combinatorial treatment with fluconazole against Fusarium solani. Biopolymers 2017, 108, e23008. [Google Scholar] [CrossRef] [PubMed]
  40. Hong, K.; Austerlitz, T.; Bohlmann, T.; Bohlmann, H. The thionin family of antimicrobial peptides. PLoS ONE 2021, 16, e0254549. [Google Scholar] [CrossRef] [PubMed]
  41. Fu, J.; Zong, X.; Jin, M.; Min, J.; Wang, F.; Wang, Y. Mechanisms and regulation of defensins in host defense. Signal. Transduct. Target. Ther. 2023, 8, 300. [Google Scholar] [CrossRef]
  42. Tiwari, P.; Srivastava, Y.; Sharma, A.; Vinayagam, R. Antimicrobial Peptides: The Production of Novel Peptide-Based Therapeutics in Plant Systems. Life 2023, 13, 1875. [Google Scholar] [CrossRef]
  43. Odintsova, T.; Shcherbakova, L.; Slezina, M.; Pasechnik, T.; Kartabaeva, B.; Istomina, E.; Dzhavakhiya, V. Hevein-Like Antimicrobial Peptides Wamps: Structure-Function Relationship in Antifungal Activity and Sensitization of Plant Pathogenic Fungi to Tebuconazole by WAMP-2-Derived Peptides. Int. J. Mol. Sci. 2020, 21, 7912. [Google Scholar] [CrossRef] [PubMed]
  44. Molesini, B.; Treggiari, D.; Dalbeni, A.; Minuz, P.; Pandolfini, T. Plant cystine-knot peptides: Pharmacological perspectives. Br. J. Clin. Pharmacol. 2017, 83, 63–70. [Google Scholar] [CrossRef] [PubMed]
  45. Postic, G.; Gracy, J.; Perin, C.; Chiche, L.; Gelly, J.C. KNOTTIN: The database of inhibitor cystine knot scaffold after 10 years, toward a systematic structure modeling. Nucleic Acids Res. 2018, 46, D454–D458. [Google Scholar] [CrossRef] [PubMed]
  46. Slavokhotova, A.A.; Rogozhin, E.A. Defense Peptides From the alpha-Hairpinin Family Are Components of Plant Innate Immunity. Front. Plant Sci. 2020, 11, 465. [Google Scholar] [CrossRef] [PubMed]
  47. Tam, J.P.; Wang, S.; Wong, K.H.; Tan, W.L. Antimicrobial Peptides from Plants. Pharmaceuticals 2015, 8, 711–757. [Google Scholar] [CrossRef] [PubMed]
  48. Souza, A.A.; Costa, A.S.; Campos, D.C.O.; Batista, A.H.M.; Sales, G.W.P.; Nogueira, N.A.P.; Alves, K.M.M.; Coelho-de-Souza, A.N.; Oliveira, H.D. Lipid transfer protein isolated from noni seeds displays antibacterial activity in vitro and improves survival in lethal sepsis induced by CLP in mice. Biochimie 2018, 149, 9–17. [Google Scholar] [CrossRef] [PubMed]
  49. Berrocal-Lobo, M.; Segura, A.; Moreno, M.; Lopez, G.; Garcia-Olmedo, F.; Molina, A. Snakin-2, an antimicrobial peptide from potato whose gene is locally induced by wounding and responds to pathogen infection. Plant Physiol. 2002, 128, 951–961. [Google Scholar] [CrossRef]
  50. Gould, A.; Camarero, J.A. Cyclotides: Overview and Biotechnological Applications. Chembiochem 2017, 18, 1350–1363. [Google Scholar] [CrossRef]
  51. Jacob, B.; Vogelaar, A.; Cadenas, E.; Camarero, J.A. Using the Cyclotide Scaffold for Targeting Biomolecular Interactions in Drug Development. Molecules 2022, 27, 6430. [Google Scholar] [CrossRef]
  52. Slezina, M.P.; Odintsova, T.I. Plant Antimicrobial Peptides: Insights into Structure-Function Relationships for Practical Applications. Curr. Issues Mol. Biol. 2023, 45, 3674–3704. [Google Scholar] [CrossRef]
  53. Sharma, P.; Kaur, J.; Sharma, G.; Kashyap, P. Plant derived antimicrobial peptides: Mechanism of target, isolation techniques, sources and pharmaceutical applications. J. Food Biochem. 2022, 46, e14348. [Google Scholar] [CrossRef] [PubMed]
  54. Kumar, P.; Kizhakkedathu, J.N.; Straus, S.K. Antimicrobial Peptides: Diversity, Mechanism of Action and Strategies to Improve the Activity and Biocompatibility In Vivo. Biomolecules 2018, 8, 4. [Google Scholar] [CrossRef] [PubMed]
  55. Mahlapuu, M.; Hakansson, J.; Ringstad, L.; Bjorn, C. Antimicrobial Peptides: An Emerging Category of Therapeutic Agents. Front. Cell Infect. Microbiol. 2016, 6, 194. [Google Scholar] [CrossRef] [PubMed]
  56. Monroc, S.; Badosa, E.; Feliu, L.; Planas, M.; Montesinos, E.; Bardaji, E. De novo designed cyclic cationic peptides as inhibitors of plant pathogenic bacteria. Peptides 2006, 27, 2567–2574. [Google Scholar] [CrossRef] [PubMed]
  57. Guha, S.; Ghimire, J.; Wu, E.; Wimley, W.C. Mechanistic Landscape of Membrane-Permeabilizing Peptides. Chem. Rev. 2019, 119, 6040–6085. [Google Scholar] [CrossRef] [PubMed]
  58. Lee, H.; Lim, S.I.; Shin, S.H.; Lim, Y.; Koh, J.W.; Yang, S. Conjugation of Cell-Penetrating Peptides to Antimicrobial Peptides Enhances Antibacterial Activity. ACS Omega 2019, 4, 15694–15701. [Google Scholar] [CrossRef]
  59. Montesinos, E. Functional Peptides for Plant Disease Control. Annu. Rev. Phytopathol. 2023, 61, 301–324. [Google Scholar] [CrossRef] [PubMed]
  60. Le, C.F.; Fang, C.M.; Sekaran, S.D. Intracellular Targeting Mechanisms by Antimicrobial Peptides. Antimicrob. Agents Chemother. 2017, 61, e02340-16. [Google Scholar] [CrossRef] [PubMed]
  61. Tiwari, M.; Pati, D.; Mohapatra, R.; Sahu, B.B.; Singh, P. The Impact of Microbes in Plant Immunity and Priming Induced Inheritance: A Sustainable Approach for Crop protection. Plant Stress 2022, 4, 100072. [Google Scholar] [CrossRef]
  62. Mehmood, S.; Imran, M.; Ali, A.; Munawar, A.; Khaliq, B.; Anwar, F.; Saeed, Q.; Buck, F.; Hussain, S.; Saeed, A.; et al. Model prediction of a Kunitz-type trypsin inhibitor protein from seeds of Acacia nilotica L. with strong antimicrobial and insecticidal activity. Turk. J. Biol. 2020, 44, 188–200. [Google Scholar] [CrossRef] [PubMed]
  63. Park, Y.; Choi, B.H.; Kwak, J.S.; Kang, C.W.; Lim, H.T.; Cheong, H.S.; Hahm, K.S. Kunitz-type serine protease inhibitor from potato (Solanum tuberosum L. cv. Jopung). J. Agric. Food Chem. 2005, 53, 6491–6496. [Google Scholar] [CrossRef] [PubMed]
  64. Clemente, M.; Corigliano, M.G.; Pariani, S.A.; Sanchez-Lopez, E.F.; Sander, V.A.; Ramos-Duarte, V.A. Plant Serine Protease Inhibitors: Biotechnology Application in Agriculture and Molecular Farming. Int. J. Mol. Sci. 2019, 20, 1345. [Google Scholar] [CrossRef] [PubMed]
  65. Bode, W.; Huber, R. Natural protein proteinase inhibitors and their interaction with proteinases. Eur. J. Biochem. 1992, 204, 433–451. [Google Scholar] [CrossRef] [PubMed]
  66. Laskowski, M., Jr.; Kato, I. Protein inhibitors of proteinases. Annu. Rev. Biochem. 1980, 49, 593–626. [Google Scholar] [CrossRef] [PubMed]
  67. Cotabarren, J.; Lufrano, D.; Parisi, M.G.; Obregon, W.D. Biotechnological, biomedical, and agronomical applications of plant protease inhibitors with high stability: A systematic review. Plant Sci. 2020, 292, 110398. [Google Scholar] [CrossRef] [PubMed]
  68. Dzik, S. Complement and Coagulation: Cross Talk Through Time. Transfus. Med. Rev. 2019, 33, 199–206. [Google Scholar] [CrossRef] [PubMed]
  69. Bonturi, C.R.; Silva Teixeira, A.B.; Rocha, V.M.; Valente, P.F.; Oliveira, J.R.; Filho, C.M.B.; Fatima Correia Batista, I.; Oliva, M.L.V. Plant Kunitz Inhibitors and Their Interaction with Proteases: Current and Potential Pharmacological Targets. Int. J. Mol. Sci. 2022, 23, 4742. [Google Scholar] [CrossRef] [PubMed]
  70. Varkoly, K.; Beladi, R.; Hamada, M.; McFadden, G.; Irving, J.; Lucas, A.R. Viral SERPINS-A Family of Highly Potent Immune-Modulating Therapeutic Proteins. Biomolecules 2023, 13, 1393. [Google Scholar] [CrossRef] [PubMed]
  71. Udvardy, A. The role of controlled proteolysis in cell-cycle regulation. Eur. J. Biochem. 1996, 240, 307–313. [Google Scholar] [CrossRef] [PubMed]
  72. Heutinck, K.M.; ten Berge, I.J.; Hack, C.E.; Hamann, J.; Rowshani, A.T. Serine proteases of the human immune system in health and disease. Mol. Immunol. 2010, 47, 1943–1955. [Google Scholar] [CrossRef] [PubMed]
  73. Pearce, G. Systemin, hydroxyproline-rich systemin and the induction of protease inhibitors. Curr. Protein Pept. Sci. 2011, 12, 399–408. [Google Scholar] [CrossRef]
  74. Coppola, M.; Corrado, G.; Coppola, V.; Cascone, P.; Martinelli, R.; Digilio, M.C.; Pennacchio, F.; Rao, R. Prosystemin Overexpression in Tomato Enhances Resistance to Different Biotic Stresses by Activating Genes of Multiple Signaling Pathways. Plant Mol. Biol. Rep. 2015, 33, 1270–1285. [Google Scholar] [CrossRef] [PubMed]
  75. Huang, Y.; Huang, J.; Chen, Y. Alpha-helical cationic antimicrobial peptides: Relationships of structure and function. Protein Cell 2010, 1, 143–152. [Google Scholar] [CrossRef]
  76. Dib, H.X.; de Oliveira, D.G.L.; de Oliveira, C.F.R.; Taveira, G.B.; de Oliveira Mello, E.; Verbisk, N.V.; Chang, M.R.; Correa Junior, D.; Gomes, V.M.; Macedo, M.L.R. Biochemical characterization of a Kunitz inhibitor from Inga edulis seeds with antifungal activity against Candida spp. Arch. Microbiol. 2019, 201, 223–233. [Google Scholar] [CrossRef] [PubMed]
  77. Lopes, J.L.; Valadares, N.F.; Moraes, D.I.; Rosa, J.C.; Araujo, H.S.; Beltramini, L.M. Physico-chemical and antifungal properties of protease inhibitors from Acacia plumosa. Phytochemistry 2009, 70, 871–879. [Google Scholar] [CrossRef] [PubMed]
  78. Cid-Gallegos, M.S.; Corzo-Rios, L.J.; Jimenez-Martinez, C.; Sanchez-Chino, X.M. Protease Inhibitors from Plants as Therapeutic Agents—A Review. Plant Foods Hum. Nutr. 2022, 77, 20–29. [Google Scholar] [CrossRef] [PubMed]
  79. de Freitas, M.A.G.; Amaral, N.O.; Alvares, A.; de Oliveira, S.A.; Mehdad, A.; Honda, D.E.; Bessa, A.S.M.; Ramada, M.H.S.; Naves, L.M.; Pontes, C.N.R.; et al. Blood pressure-lowering effects of a Bowman-Birk inhibitor and its derived peptides in normotensive and hypertensive rats. Sci. Rep. 2020, 10, 11680. [Google Scholar] [CrossRef] [PubMed]
  80. Gitlin-Domagalska, A.; Maciejewska, A.; Debowski, D. Bowman-Birk Inhibitors: Insights into Family of Multifunctional Proteins and Peptides with Potential Therapeutical Applications. Pharmaceuticals 2020, 13, 421. [Google Scholar] [CrossRef] [PubMed]
  81. Bronsoms, S.; Villanueva, J.; Canals, F.; Querol, E.; Aviles, F.X. Analysis of the effect of potato carboxypeptidase inhibitor pro-sequence on the folding of the mature protein. Eur. J. Biochem. 2003, 270, 3641–3650. [Google Scholar] [CrossRef] [PubMed]
  82. Arolas, J.L.; Ventura, S. Protease inhibitors as models for the study of oxidative folding. Antioxid. Redox Signal. 2011, 14, 97–112. [Google Scholar] [CrossRef] [PubMed]
  83. Satheesh, L.S.; Murugan, K. Antimicrobial activity of protease inhibitor from leaves of Coccinia grandis (L.) Voigt. Indian J. Exp. Biol. 2011, 49, 366–374. [Google Scholar] [PubMed]
  84. Macedo, M.L.; Ribeiro, S.F.; Taveira, G.B.; Gomes, V.M.; de Barros, K.M.; Maria-Neto, S. Antimicrobial Activity of ILTI, a Kunitz-Type Trypsin Inhibitor from Inga laurina (SW.) Willd. Curr. Microbiol. 2016, 72, 538–544. [Google Scholar] [CrossRef] [PubMed]
  85. de Souza Nascimento, A.M.; de Oliveira Segundo, V.H.; Felipe Camelo Aguiar, A.J.; Piuvezam, G.; Souza Passos, T.; Florentino da Silva Chaves Damasceno, K.; de Araujo Morais, A.H. Antibacterial action mechanisms and mode of trypsin inhibitors: A systematic review. J. Enzyme. Inhib. Med. Chem. 2022, 37, 749–759. [Google Scholar] [CrossRef] [PubMed]
  86. Coppola, M.; Lelio, I.D.; Romanelli, A.; Gualtieri, L.; Molisso, D.; Ruocco, M.; Avitabile, C.; Natale, R.; Cascone, P.; Guerrieri, E.; et al. Tomato Plants Treated with Systemin Peptide Show Enhanced Levels of Direct and Indirect Defense Associated with Increased Expression of Defense-Related Genes. Plants 2019, 8, 395. [Google Scholar] [CrossRef] [PubMed]
  87. Dabhade, A.R.; Mokashe, N.U.; Patil, U.K. Purification, characterization, and antimicrobial activity of nontoxic trypsin inhibitor from Albizia amara Boiv. Process Biochem. 2016, 51, 659–674. [Google Scholar] [CrossRef]
  88. Martins, T.F.; Vasconcelos, I.M.; Silva, R.G.G.; Silva, F.D.A.; Souza, P.F.N.; Varela, A.L.N.; Albuquerque, L.M.; Oliveira, J.T.A. A Bowman-Birk Inhibitor from the Seeds of Luetzelburgia auriculata Inhibits Staphylococcus aureus Growth by Promoting Severe Cell Membrane Damage. J. Nat. Prod. 2018, 81, 1497–1507. [Google Scholar] [CrossRef] [PubMed]
  89. Arulpandi, I.; Sangeetha, R. Antibacterial Activity of Fistulin: A Protease Inhibitor Purified from the Leaves of Cassia fistula. ISRN Pharm. 2012, 2012, 584073. [Google Scholar] [CrossRef] [PubMed]
  90. Almeida, L.H.O.; Oliveira, C.F.R.; Rodrigues, M.S.; Neto, S.M.; Boleti, A.P.A.; Taveira, G.B.; Mello, E.O.; Gomes, V.M.; Santos, E.L.D.; Crusca, E., Jr.; et al. Adepamycin: Design, synthesis and biological properties of a new peptide with antimicrobial properties. Arch. Biochem. Biophys. 2020, 691, 108487. [Google Scholar] [CrossRef] [PubMed]
  91. Rodrigues, M.S.; de Oliveira, C.F.R.; Almeida, L.H.O.; Neto, S.M.; Boleti, A.P.A.; Dos Santos, E.L.; Cardoso, M.H.; Ribeiro, S.M.; Franco, O.L.; Rodrigues, F.S.; et al. Adevonin, a novel synthetic antimicrobial peptide designed from the Adenanthera pavonina trypsin inhibitor (ApTI) sequence. Pathog. Glob. Health 2018, 112, 438–447. [Google Scholar] [CrossRef] [PubMed]
  92. Kim, J.Y.; Gopal, R.; Kim, S.Y.; Seo, C.H.; Lee, H.B.; Cheong, H.; Park, Y. PG-2, a Potent AMP against Pathogenic Microbial Strains, from Potato (Solanum tuberosum L. cv. Gogu Valley) Tubers Not Cytotoxic against Human Cells. Int. J. Mol. Sci. 2013, 14, 4349–4360. [Google Scholar] [CrossRef] [PubMed]
  93. Cotabarren, J.; Ozon, B.; Claver, S.; Geier, F.; Rossotti, M.; Garcia-Pardo, J.; Obregon, W.D. A Multifunctional Trypsin Protease Inhibitor from Yellow Bell Pepper Seeds: Uncovering Its Dual Antifungal and Hypoglycemic Properties. Pharmaceutics 2023, 15, 781. [Google Scholar] [CrossRef] [PubMed]
  94. Bacha, A.B.; Jemel, I.; Moubayed, N.M.S.; Abdelmalek, I.B. Purification and characterization of a newly serine protease inhibitor from Rhamnus frangula with potential for use as therapeutic drug. 3 Biotech 2017, 7, 148. [Google Scholar] [CrossRef] [PubMed]
  95. Manojlovic, N.T.; Solujic, S.; Sukdolak, S.; Milosev, M. Antifungal activity of Rubia tinctorum, Rhamnus frangula and Caloplaca cerina. Fitoterapia 2005, 76, 244–246. [Google Scholar] [CrossRef] [PubMed]
  96. Costa, H.P.; Oliveira, J.T.; Sousa, D.O.; Morais, J.K.; Moreno, F.B.; Monteiro-Moreira, A.C.; Viegas, R.A.; Vasconcelos, I.M. JcTI-I: A novel trypsin inhibitor from Jatropha curcas seed cake with potential for bacterial infection treatment. Front. Microbiol. 2014, 5, 5. [Google Scholar] [CrossRef] [PubMed]
  97. Wang, C.; Shao, C.; Fang, Y.; Wang, J.; Dong, N.; Shan, A. Binding loop of sunflower trypsin inhibitor 1 serves as a design motif for proteolysis-resistant antimicrobial peptides. Acta Biomater. 2021, 124, 254–269. [Google Scholar] [CrossRef] [PubMed]
  98. Cotabarren, J.; Claver, S.; Payrol, J.A.; Garcia-Pardo, J.; Obregon, W.D. Purification and Characterization of a Novel Thermostable Papain Inhibitor from Moringa oleifera with Antimicrobial and Anticoagulant Properties. Pharmaceutics 2021, 13, 512. [Google Scholar] [CrossRef] [PubMed]
  99. de Souza, A.A.; Lima, A.M.; Dede Oliveira BezerraSousa, D.; Nogueira, F.C.; do Sacramento Neto, J.C.; Dias, L.P.; Araujo, N.M.S.; Nagano, C.S.; Junior, H.V.N.; da Silva, C.R.; et al. Chia (Salvia hispanica L.) Seeds Contain a Highly Stable Trypsin Inhibitor with Potential for Bacterial Management Alone or in Drug Combination Therapy with Oxacillin. Probiotics. Antimicrob. Proteins 2023, 15, 1221–1233. [Google Scholar] [CrossRef] [PubMed]
  100. Bartova, V.; Barta, J.; Jarosova, M. Antifungal and antimicrobial proteins and peptides of potato (Solanum tuberosum L.) tubers and their applications. Appl. Microbiol. Biotechnol. 2019, 103, 5533–5547. [Google Scholar] [CrossRef] [PubMed]
  101. Jashni, M.K.; Mehrabi, R.; Collemare, J.; Mesarich, C.H.; de Wit, P.J. The battle in the apoplast: Further insights into the roles of proteases and their inhibitors in plant-pathogen interactions. Front. Plant Sci. 2015, 6, 584. [Google Scholar] [CrossRef]
  102. Woloshuk, C.P.; Meulenhoff, J.S.; Sela-Buurlage, M.; van den Elzen, P.J.; Cornelissen, B.J. Pathogen-induced proteins with inhibitory activity toward Phytophthora infestans. Plant Cell 1991, 3, 619–628. [Google Scholar] [CrossRef] [PubMed]
  103. Vernekar, J.V.; Tanksale, A.M.; Ghatge, M.S.; Deshpande, V.V. Novel bifunctional alkaline protease inhibitor: Protease inhibitory activity as the biochemical basis of antifungal activity. Biochem. Biophys. Res. Commun. 2001, 285, 1018–1024. [Google Scholar] [CrossRef] [PubMed]
  104. Carmona-Gutierrez, D.; Frohlich, K.U.; Kroemer, G.; Madeo, F. Metacaspases are caspases. Doubt no more. Cell Death Differ. 2010, 17, 377–378. [Google Scholar] [CrossRef] [PubMed]
  105. Aslam, B.; Khurshid, M.; Arshad, M.I.; Muzammil, S.; Rasool, M.; Yasmeen, N.; Shah, T.; Chaudhry, T.H.; Rasool, M.H.; Shahid, A.; et al. Antibiotic Resistance: One Health One World Outlook. Front. Cell Infect. Microbiol. 2021, 11, 771510. [Google Scholar] [CrossRef] [PubMed]
  106. Woods, M.; McAlister, J.A.; Geddes-McAlister, J. A One Health approach to overcoming fungal disease and antifungal resistance. WIREs Mech. Dis. 2023, 15, e1610. [Google Scholar] [CrossRef] [PubMed]
  107. FAO. Antimicrobial Resistance. Available online: https://www.fao.org/antimicrobial-resistance/key-sectors/animal-production/en/ (accessed on 2 April 2024).
  108. Barcenas, O.; Pintado-Grima, C.; Sidorczuk, K.; Teufel, F.; Nielsen, H.; Ventura, S.; Burdukiewicz, M. The dynamic landscape of peptide activity prediction. Comput. Struct. Biotechnol. J. 2022, 20, 6526–6533. [Google Scholar] [CrossRef] [PubMed]
  109. Yue, L.; Song, L.; Zhu, S.; Fu, X.; Li, X.; He, C.; Li, J. Machine learning assisted rational design of antimicrobial peptides based on human endogenous proteins and their applications for cosmetic preservative system optimization. Sci. Rep. 2024, 14, 947. [Google Scholar] [CrossRef] [PubMed]
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Figure 1. Composition and Diversity of Antimicrobial Peptides (AMPs). (A–C) These panels summarize the data from the Antimicrobial Peptide Database (APD3) (http://aps.unmc.edu/AP/) [29]. As of January 2024, APD3 has cataloged 3940 AMPs, categorized by (A) biological origin, (B) secondary structural characteristics, and (C) the spectrum of biological activity. In panel (B), Unknown 3D structure refers to AMPs for which the three-dimensional structures have not yet been determined.
Figure 1. Composition and Diversity of Antimicrobial Peptides (AMPs). (A–C) These panels summarize the data from the Antimicrobial Peptide Database (APD3) (http://aps.unmc.edu/AP/) [29]. As of January 2024, APD3 has cataloged 3940 AMPs, categorized by (A) biological origin, (B) secondary structural characteristics, and (C) the spectrum of biological activity. In panel (B), Unknown 3D structure refers to AMPs for which the three-dimensional structures have not yet been determined.
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Figure 2. Relative abundance of AMPs available on the Antimicrobial Plant Database (https://aps.unmc.edu/) [29].
Figure 2. Relative abundance of AMPs available on the Antimicrobial Plant Database (https://aps.unmc.edu/) [29].
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Figure 3. Mechanisms of action of antimicrobial peptides. (a) Damage of cell membranes, causing cell lysis. (b) Interference with internal cellular processes. (c) Inhibition of biofilm formation. (d) Interaction with external cell structures such as lipopolysaccharides, fimbriae, or flagella in bacteria or chitin in fungi. (e) Inhibition of virus attachment or replication [59].
Figure 3. Mechanisms of action of antimicrobial peptides. (a) Damage of cell membranes, causing cell lysis. (b) Interference with internal cellular processes. (c) Inhibition of biofilm formation. (d) Interaction with external cell structures such as lipopolysaccharides, fimbriae, or flagella in bacteria or chitin in fungi. (e) Inhibition of virus attachment or replication [59].
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Figure 4. Plant peptide protease inhibitors and their potential applications in biomedicine.
Figure 4. Plant peptide protease inhibitors and their potential applications in biomedicine.
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Table 1. Peptide protease inhibitors derived from plants with antimicrobial activity.
Table 1. Peptide protease inhibitors derived from plants with antimicrobial activity.
Plant Family Plant Source Name Type of PPIMW (kDa)ActivityTarget MicroorganismsRef.
Fabaceae
(Leguminosae)		Inga edulisInga Edulis Trypsin Inhibitor (IETI)Kunitz19.7Antifungal Candida tropicalis and Candida buinensis[76]
Inga laurinaInga Laurina Trypsin Inhibitor (ILTI)Kunitz20AntifungalC. tropicalis and C. buinensis[84]
Acacia plumosaAcacia plumosa Trypsin Inhibitor (ApTI A, B, C)Kunitz20Antifungal Aspergillus niger, Thielaviopsis paradoxa, and Colletotrichum spp.[77]
Acacia niloticaAcacia nilotica Trypsin Inhibitor (AnTI)Trypsin inhibitor21AntibacterialG+: Staphylococcus aureus and Bacillus subtilis,
G−: Escherichia coli, and Pseudomonas aeruginosa		[62]
Albizia amaraProteinaceous protease inhibitor (API)Unknown49AntibacterialG+: B. subtilis
G−: P. aeruginosa		[87]
AntifungalC. albicans
Luetzelburgia auriculataLuetzelburgia auriculata Bowman-Birk protease inhibitor (LzaBBI)Bowman-Birk14.3Antibacterial G+: S. aureus[88]
Cassia fistulaFistulinTrypsin inhibitor4AntibacterialG+: S. aureus, and B. subtilis
G−: Klebsiella pneumoniae, E. coli, and P. aeruginosa		[89]
Adenanthera pavoninaAdepamycinTrypsin inhibitor2.4AntibacterialG+: S. aureus
G−: E. coli, Klebsiella oxytoca, K. pneumoniae, and
P. aeruginosa		[90,91]
AntifungalC. albicans and C. tropicalis
SolanaceaeSolanum tuberosum L. Cv. Golden ValleyPotide-GKunitz5.57AntibacterialG+: S. aureus, and Listeria monocytogenes
G−: E. coli		[26]
AntifungalC. albicans and
Rhizoctonia solani
S. tuberosum L. Cv. Gogu ValleyPeptide G2 (PG-2)Kunitz3.2AntibacterialG+: S. aureus[92]
AntifungalC. albicans
S. tuberosum L. Cv. L. JopungAntifungal protein J (AFP-J)Kunitz13.5AntifungalC. albicans,
Trichosporon beigelii, and Saccharomyces cerevisiae		[63]
Capsicum annuum L. Yellow Bell Pepper Trypsin Inhibitor
(YBPTI)		Trypsin inhibitor AntifungalC. albicans[93]
CucurbitaceaeCoccinia grandis (L.) Voigt.Coccinia grandis (L.) inhibitorProtease inhibitor14.3AntibacterialG+: S. aureus, and B. subtilis
G−: K. pneumoniae, Proteus vulgaris, and E. coli		[83]
AntifungalC. albicans, Mucor indicus, Penicillium notatum, Aspergillus flavus Aspergillus oryzae, and Cryptococcus neoformans
RhamnaceaeRhamnus frangulaRflP-1Kunitz22.5AntibacterialG+: Bacillus sp. and
Bacillus licheniformis		[94,95]
EuphorbiaceaeJatropha curcasJatropha curcas Trypsin inhibitor I
(JcTI-I)		Trypsin inhibitor10.2AntibacterialG+: S. aureus
G−: Salmonella enterica		[96]
AsteraceaeHelianthus annuusSunflower Trypsin Inhibitor 1 (SFTI1)Trypsin inhibitor AntibacterialG+: Staphylococcus epidermidis, S. aureus, and Enterococcus faecalis
G−: E. coli, P. aeruginosa, and Salmonella typhimurium		[97]
MoringaceaeMoringa oleiferaMoringa oleifera Protein Inhibitor
(MoPI)		Phytocystatin19AntibacterialG+: S. aureus, and E. faecalis[98]
LamiaceaeSalvia hispanicaSalvia hispanica Trypsin Inhibitor (ShTI)Trypsin inhibitor11AntibacterialG+: Methicillin-resistant
S. aureus		[99]
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Parisi, M.G.; Ozón, B.; Vera González, S.M.; García-Pardo, J.; Obregón, W.D. Plant Protease Inhibitors as Emerging Antimicrobial Peptide Agents: A Comprehensive Review. Pharmaceutics 2024, 16, 582. https://doi.org/10.3390/pharmaceutics16050582

AMA Style

Parisi MG, Ozón B, Vera González SM, García-Pardo J, Obregón WD. Plant Protease Inhibitors as Emerging Antimicrobial Peptide Agents: A Comprehensive Review. Pharmaceutics. 2024; 16(5):582. https://doi.org/10.3390/pharmaceutics16050582

Chicago/Turabian Style

Parisi, Mónica G., Brenda Ozón, Sofía M. Vera González, Javier García-Pardo, and Walter David Obregón. 2024. "Plant Protease Inhibitors as Emerging Antimicrobial Peptide Agents: A Comprehensive Review" Pharmaceutics 16, no. 5: 582. https://doi.org/10.3390/pharmaceutics16050582

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