Fundamentals on the molecular mechanism of action of antimicrobial peptides

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FUNDAMENTALS ON THE MOLECULAR MECHANISM OF ACTION OF ANTIMICROBIAL PEPTIDES ˆ , Alberto Gonc¸alves Evangelista , Jessica Audrey Feijo´ Correa Tiago de Melo Nazareth , Fernando Bittencourt Luciano PII: DOI: Reference:

S2589-1529(19)30290-X https://doi.org/10.1016/j.mtla.2019.100494 MTLA 100494

To appear in:

Materialia

Received date: Accepted date:

4 June 2019 27 September 2019

ˆ , Alberto Gonc¸alves Evangelista , Please cite this article as: Jessica Audrey Feijo´ Correa Tiago de Melo Nazareth , Fernando Bittencourt Luciano , FUNDAMENTALS ON THE MOLECULAR MECHANISM OF ACTION OF ANTIMICROBIAL PEPTIDES, Materialia (2019), doi: https://doi.org/10.1016/j.mtla.2019.100494

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Highlights 1. The mechanism of action of AMPs is of main concern when establishing possible uses; 2. The major AMPs‘ mechanisms of action involve membrane destabilization; 3. Studies regarding AMPs must be continuous to remediate the generation of resistance.

FUNDAMENTALS ON THE MOLECULAR MECHANISM OF ACTION OF ANTIMICROBIAL PEPTIDES a*

Jessica Audrey Feijó Corrêa (ORCID: 0000-0002-4741-031X) a

Alberto Gonçalves Evangelista (ORCID: 0000-0001-7445-8901) Tiago de Melo Nazareth

ab

(ORCID: 0000-0002-3052-2817) a

Fernando Bittencourt Luciano (ORCID: 0000-0003-0816-2111)

a

School of Life Sciences, Pontifícia Universidade Católica do Paraná, Rua Imaculada Conceição 1155, Prado Velho, Curitiba, Paraná 80215-901, Brazil.

b

Laboratory of Food Chemistry and Toxicology, Faculty of Pharmacy, University of Valencia, Av. Vicent Andrés Estellés s/n, 46100 Burjassot, Spain. *Corresponding author: [email protected]

Graphic abstract

ABSTRACT Antimicrobial peptides (AMPs) are produced by several organisms as their first line of defense. Constituted by amino acids, they may present different mechanisms of action. The antimicrobial activity can be used by the peptide-producing organism itself, as innate immune strategy, or in the industry, applying as natural source preservatives. Understanding the possibilities of the operation of these compounds is a prerequisite for the development of effective uses, as well as for the establishment of combinations, which can even expand their applications considering the possibilities of genetic manipulations. Thus, the objective of this article is to review the basic principles of AMPs acting. The main mechanisms of peptides are related to changes in the physiological function of membranes, but there are also alterations in cytoplasmic components. We gathered the three most described and accepted, the barrel-stave, the carpet, and the wormhole mechanisms, along with the possible microorganisms' mechanisms of resistance towards AMPs action. Such information may contribute to the process of choosing the best peptide to obtain an expected action against a given microorganism, optimizing the handling of financial and human resources in research projects. Keywords: Antimicrobials resistance; Bioactive molecules; Peptides biochemistry.

1

Introduction Antimicrobial peptides (AMPs) are small compounds formed by amino acids,

presenting biological activity as the first line of defense of several organisms. They are produced by bacteria, fungi, plants, and animals, and can be classified in numerous groups according to their structure [1–3]. Antimicrobial activity can be used by the peptide-producing organism itself in their innate immune functions or, through the isolation of these molecules, in the industry [4–7]. Some peptides, such as nisin (produced by specific strains of Lactococcus lactis lactis), polyoxin (from Streptomyces cacaoi) and lactoferricin B (isolated mainly from bovine milk), are already industrially used as a food preservative, pesticide and as controlling agent of mastitis, respectively [8–11]. Basically, these antimicrobial molecules combine with membrane and/or cytoplasmic components of the microorganisms, altering their cellular functions, which can lead to cellular

Abbreviation: AMP: Antimicrobial peptide CL: Cardiolipin LPS: Lipopolysaccharide PG: Phosphatidylglycerol

death or metabolism depletion [12]. This results from several characteristics of such molecules, including their amino acids composition, amphipathicity (double polarity), electric charges and reduced size, which facilitate their insertion into lipid bilayers and consequent pore formation and cellular extravasation [13,14]. Although this general mechanism is relatively widespread, studies have countered this information, arguing that pore formation is not the only mechanism of action of AMPs. The increasing numbers of observations suggest these peptides may also inhibit cell wall, nucleic acids, and proteins synthesis processes, besides inhibiting several other enzymatic functions of the target cells [15,16]. In addition, many AMPs can be ―generated‖ through the proteolytic digestion of food, such as egg, milk, and cereals, being called "cryptic peptides", since they are found inside other larger proteins [17,18]. This fact reflects on the nutraceutical potential of these foods, since, besides the nutritional aspects, their ingestion can bring benefits to those who consume, either by the antimicrobial action against pathogens, or by other activities already verified in cryptic peptides, which include antihypertensive, antioxidative, antitumoral, hypocholesterolemic, neuroprotective, hypoglycemic and anti-inflammatory activities, among others [19,20]. This represents great potential both in treatment and prevention of diseases linked to hypertension, cancers, diabetes and autoimmune diseases, such as arthritis and neurodegenerative diseases such as Parkinson's and Alzheimer's [21–24]. For instance, βcasein and β-lactoglobulin from cow milk have several cryptic peptides within their sequences with proved antioxidative activity in biological systems (sequences AVPYPQR, KVLPVPEK, MHIRL, YVEEL, among others) [25–27]. The study of AMPs presents increasing importance both in Academia and industry since the search for natural antimicrobial alternatives gains relevance. Many sanitizers traditionally used have been reported as now ineffective against various pathogens – possibly through the acquisition of resistance –, and the development of new approaches and substances are severely encouraged [28]. Along with AMPs studies, research with essential oils, essential oil components, phenolic acids, and even active microorganisms have shown good results when applied as substitutes for traditional antimicrobials, with great potential for the industry [29–34]. However, these substitutes are also capable of inducing changes in gene expression of targeted microorganisms, leading to complex and effective responses [35,36]. Different mechanisms of resistance against AMPs have been elucidated in recent years, although much remains to be discovered in this area, majorly at genomic levels [37,38]. The understanding of the molecular mechanisms surrounding AMPs is essential for their effective application to industrial interests. The elucidation of mechanisms of action, as well as mechanisms of resistance generation, can guide the development of more effective

combinations, optimize the use and even expand possibilities – considering genetic manipulation of strains. Such data is vital, especially at the current critical time when the emergence of resistant strains to the latest developed antibiotics threatens the public health. 2

Molecular Mechanisms of AMPs Selectivity The interest in AMPs as new substances stimulated the development of several

studies in the isolation, characterization, and elucidation of the mechanisms of action related to these substances. Hundreds of peptides with these characteristics have been identified and hundreds more have been synthetically designed and produced. In general, there are dozens of classifications that are based on different characteristics – including size, structure, and chemical composition. AMPs are remarkably heterogeneous, for instance, when it comes to size. AMPs containing from less than 10 to more than 100 amino acids have already been described [39–41]. On the other hand, some features, such as net positive charge and hydrophobicity, are quite frequent among known AMPs [12,42]. A simple classification is based on the activity spectra of AMPs, which divides them into peptides toxic to microorganisms but non-toxic to mammalian cells – such as nisin; and peptides toxic to microorganisms and mammalian cells – such as melittin. Within the first group, it is possible to find selective AMPs against Gram-positive bacteria or against fungi, as well as broader-spectrum AMPs, acting against both Gram-positive and Gram-negative bacteria, and those with activity against bacteria and fungi. More specific examples and descriptions are brought in the sequence. The second group, however, because of its toxicity towards mammalian cells, might not represent a great interest in the industry at first, since it may compromise the safe use of these compounds [43–45]. However, AMPs toxic to mammalian cells represent potential in the research and development of drugs for tumors and autoimmune diseases [46–48]. For instance, melittin, a peptide of 26 amino acids major constituent of honeybee (Apis melifera) venom, and pardaxin, of 33 amino acids, derived from Moses Sole fish (Pardachirus marmoratus) venom, cause lysis of both mammalian and bacterial cells [49,50]. Also, peptide fractions of agglutinin and abrin from Abrus, a genus of flowering plants with highly toxic seeds, have shown antitumor and immunostimulatory potential, majorly due to apoptotic activity towards mammalian cells [51,52]. Also, Abrus protein extracts have shown antimicrobial activity towards enterobacteria species [53], which may be related to the AMPs present in the extracts.

2.1 Mechanisms of Binding and Targeting AMPs can interact with the target for subsequent cell death by nonreceptor-mediated or receptor-mediated mechanisms [54,55]. AMPs which mechanisms are nonreceptor-mediated are more abundant and include most up-to-date known peptides and, regarding their compositions, are the most diverse in amino acids. This is associated with the fact that these peptides do not have specific receptors, but rather a more general target: cell membranes [56,57]. This is the case of the AMPs alamethicin – a 20-amino acid ionophore antibiotic produced by the fungus Trichoderma viride [58]– and magainin-2 – a 23-amino acid peptide isolated from Xenopus and Rana frog skin [59]. In general, cell surface of microorganisms presents net negative charge – both Grampositive and Gram-negative bacteria contain negatively charged phospholipids and, in addition, Gram-positive bacteria possess teichoic acid while Gram-negative present lipopolysaccharides (LPS), which are negatively charged substances [60]. This feature is essential for the AMPs‘ activity since the molecules present net positive charge. In addition, AMPs are usually amphipathic, presenting hydrophobic and hydrophilic regions in their structure. This amphipathicity also influences the antimicrobial action, affecting the interactions with cellular membranes [61]. However, some nonreceptor-mediated peptides – like CF-14 from the epidermal mucus of catfish, which presents antimicrobial activity towards both Gram-positive and negative bacteria – appear to have intracellular targets, usually DNA or enzymes. In the case of CF-14, it penetrates the cell after interaction of proline hinges with the membrane. Anyhow, the AMP penetration through the membrane is a mandatory step for the action, despite in such cases no membrane lysis is verified [62]. On the other hand, receptor-mediated AMPs are mainly peptides produced by bacteria, such as bacteriocins. Important AMPs in this category are nisins, produced by the Gram-positive bacteria Lactococcus lactis lactis and widely used as preservatives in dairy products, since they are the only bacteriocins allowed for application in food by the Food and Drug Administration [29,63,64]. In addition, the first described receptor-mediated AMP was nisin Z, which uses as receptor the Lipid II, a membrane-anchored cell wall precursor. The affinity to the receptor along with the ability to build pores makes nisin Z highly effective – especially towards Gram-negatives – at very low concentrations [65]. Another example of receptor-mediated AMP is polymyxin B, which uses LPS, phosphatidylglycerol (PG) and cardiolipin (CL) specific receptors for the peptide action on microbial membranes [36,66]. Receptor-mediated AMPs have greater specificity due to the presence of specific domains in their structures that promote their binding to the receptor. The loss of these

domains leads to loss of specificity, yet the peptide usually remains active, acting through nonreceptor-mediated mechanisms and, therefore, demanding higher concentrations to produce the desired antimicrobial effect [67]. For instance, mesentericin Y, which uses a receptor exclusively found in Listeria sp., loses its selectivity after removal of the receptorbinding domain, becoming active toward other bacteria genera [68].

2.1.1

Differentiation Between Target Microorganisms and “Self”-Cells Considering that AMPs are produced by living organisms as components of their

innate defense, it is essential that these molecules differentiate invading microbial cells from the organism‘s cells. Membrane characteristics such as composition, hydrophobicity, and charge are important in this differentiation. Although all biological membranes are essentially composed of amphipathic phospholipids, there are significant differences between prokaryotic and eukaryotic membranes. Most of the phospholipids in eukaryotic membranes are considered zwitterionic, which means that they have opposite charges on different atoms, resulting in a more

neutral

net

charge

[69,70].

This

is

the

case

of

phosphatidylcholine,

phosphatidylethanolamine, and sphingomyelin. On the other hand, prokaryotic membranes have mostly negatively charged phospholipids, such as PG, CL, and phosphatidylserine. It is noteworthy that these lipids can be found in both types of membranes, but in different amounts, resulting in eukaryotic membranes electrically closer to neutrality, and very electronegative prokaryotic membranes [71,72]. The charge difference between microbial and mammalian membranes is exposed in the transmembrane potential values, which is very influential in cationic AMPs targeting processes. Thus, microbial membranes – which present potentials varying from -130 to -150 mV – are more electronegative than mammalian cell membranes (-90 to -110 mV). It is believed that this difference in transmembrane potential also assists the selective toxicity of AMPs [36,73,74]. In addition, the activity of streptolysin O, listeriolysin, perfringolysin, and pneumolysin, toxins from the pathogens Streptococcus sp., Listeria monocytogenes, Clostridium perfringens and Streptococcus pneumoniae, respectively, are related to cholesterol presence in mammalian cells [75,76], which endorses the postulation that cholesterol and also ergosterol influence the differentiation between eukaryotic cells and prokaryotes in the targeting process of AMPs [36,77]. However, the exact mechanisms remain unclear. Thereon, peptides produced by microorganisms have shown greater selectivity against mammalian cells when compared to other microbial cells [78], which is related to the cholesterol-dependent targeting process [75,79].

The toxic selectivity of AMPs is, as previously cited, associated to the binding mechanism in its targeting process – whether or not it is receptor-mediated. These receptors are usually specific lipids in the target membrane, such as Lipid II for lantibiotics targeting. It is essential that these receptors are absent or present at low concentrations in ―self‖-cells, avoiding adverse effects [80,81]. The structure and conformation of AMPs are determinant in their antimicrobial function [15]. There is evidence that the peptide ability to bind to its receptor or to the nonspecific regions of the membrane in the target cell depends directly on its inherent or dynamic conformation – in case there are transitional steps prior or during binding, such as associations or oligomerizations [82]. These conformational changes influence the AMP‘s selective toxicity since the correct ligand is required to promote these changes, directly reflecting in a successful targeting process [36,83,84]. Regarding peptide characteristics, charge and hydrophobicity differences between regions of the molecule also play major role. A higher amount of basic amino acids contributes to a positive net charge [85]. The amino acid composition of AMPs determines their electrostatic and hydrophobic/hydrophilic affinities, culminating in the correct binding to the target membranes and avoiding undesired interactions with ―self‖ membranes. Along with the electrical characteristics of membranes, this differentiation mechanism is of significant relevance in the targeting process [86,87]. Such event had been reported in the biological activity of some nutraceutical peptides, like lactostatin, peptide derived from bovine milk βlactoglobulin [88], endorsing that an analogous process might occur in specific AMPs. Considering the in vivo process, compartmentalization of peptides is also an effective mechanism to restrict the exposure of possibly vulnerable ―self‖ cells, due to physiological and electrical differences between cells from different tissues [89]. This is the case of some mammalian AMPs with immunomodulator activity, such as cathelicidin LL-37 which plays a major role in the inflammation processes [90,91].

3

Molecular Action Mechanisms of AMPs In general, AMPs are quite conserved in terms of structure and charges among

different phyla, usually forming amphipathic structures in their chains and having a cationic character (affinity for negative charges) at physiological pH [92]. This high conservation explains the importance of structure in the determination of the antimicrobial activity [93]. The structural determinants of the activity of the AMPs will be presented, followed by the involved mechanisms and the interactions of the peptides with their targets that will lead to the microorganism death. Some AMPs, their mechanisms of action, producers and targets are described in Table 1.

Table 1. Examples of antimicrobial peptides (AMPs), mechanisms of action, producers and targets. Antimicrobial peptide

Mechanism of action Inhibits bacterial growth and causes cell lysis, acting on cytoplasmic membrane, where it forms pores that disrupt the membrane integrity

Producer/Source

Target

Lactococcus lactis

Gram-positive bacteria, spores of Bacilli and Clostridia

C18G

Binds anionic lipid bilayers and induces membrane disruption in both Grampositive and Gram-negative bacteria

C-terminus of the human platelet factor IV protein (Derived)

Antibacterial activity

Cathelicidin

Immunomodulatory properties, including modulating the expression of immune related genes, activation, differentiation and chemoattraction of immune cells

Isolated from human neutrophils

Pseudomonas aeruginosa and Staphylococcus epidermidis

[97,98]

Magainins

Membrane-crossing capability and disturbance of the normal membrane integrity of the bacteria

Xenopus laevis and Rana temporaria

Gram-positive and Gramnegative bacteria

[97,99]

Gramicidin

The peptide forms prototypical ion channels in the membrane, specific for monovalent cations

Bacillus brevis

Gram-positive and Gramnegative bacteria

[97,100]

KLR

ND

C-terminal residues of human β-Defensin 3 (Derived)

Escherichia coli and Staphylococcus aureus

Increase the membrane permeability, resulting in loss of potassium, leading to membrane depolarization. Cessation of

Produced mainly by leukocytes and

Nisin

Human Defensin

Gram-positive and Gramnegative bacteria, and yeasts

Ref. [94,95]

[96]

[101]

[102,103]

DNA, RNA and protein synthesis and respiration is observed

ND = not determined

epithelial cells

3.1 Structural Determinants of AMPs Activity Different aspects contribute to the determination of the structure and consequent activity of AMPs. Some parameters such as conformation, charge, amphipathicity, and hydrophobicity, which, together and interdependently, influence the mechanisms of action of AMPs [104]. The conformation is related to the three-dimensional topology of the sequences that form the peptide. The most relevant groups, in this case, are α-helix and β-sheets (Figure 1), although peptides rich in one or more specific amino acid residues are also considered in the literature as responsible for specific folds related to the targeting process [36,105]. This is the case of the porcine cathelicidin PR-39, rich in proline and arginine, which exhibits different folds depending on the ligand receptor [106]. Possibly the most studied class of AMPs, which contains those that form amphipathic and cationic α-helix, is also the class with greater activity and industrial interest [107]. However, there are also peptides that contain hydrophobic and even anionic α-helixes, which may lead to loss of selectivity to microbial cells, and the mammalian cells also become targets [14,108], such as alamethicin and gramicidin, AMPs which also exert cytotoxicity towards mammalian cells [58,109]. On the other hand, β-structured peptides are aggregated or curved monomers that allow the formation of antiparallel β-sheets. They are usually cyclic or longer chain peptides, which enable a smaller loss of entropy in their formation, since they are significantly more complex than the helix. In addition to the hydrogen bonds inherent to this type of structure, there are also disulfide bonds keeping it cohesive [110]. The mechanisms of action of this type of peptide are not completely elucidated yet. It is believed that there are different ways of insertion of these peptides into the membrane, directly related to the concentration and nature of the AMP. The most accepted is the promotion of membrane permeabilization and subsequent translocation of the peptide through the bilayer, leading to pore formation, similar to the action of α-helix [111,112]. Some β-structured AMPs characterized up to date are the porcine cathelicidin protegrin-1 and the bovine milk lactoferricin B [3,113,114].

Figure 1. The secondary structure of peptides confers a conformation to the molecules. A. αhelix is characterized by a spiral structure caused by hydrogen bonds between the amino group of an amino acid to the carboxyl group of other located three or four residues earlier B. In the β-sheets structure, proximal regions of the chain are associated laterally with hydrogen bonds, forming a flat and rigid structure. In relation to the charge, as mentioned previously, most of the AMPs characterized to date have positive net charges, usually result of the presence of cationic domains. This property allows the initial electrostatic attractions between peptide and target membrane. In addition, it is observed that the increase of the AMP cationicity leads to an increase in its antimicrobial potential, although this relation has limits – very large increases in the peptide charge can lead to loss of antimicrobial function. This is caused because the interaction between the peptides and the polar head of the membrane phospholipids may strong enough to prevent subsequent interactions with the cell interior [115,116]. This limitation was observed before in optimization studies with charge alterations in magainin 2, demonstrating that in cationic charges ranging between +3 and +7, +5 is the optimal value for antimicrobial activity and selectivity [117]. Hydrophobicity is determined by the percentage of hydrophobic residues in a peptide, being responsible for AMP ability to cleave the lipid bilayer of the target membrane [118]. Amphipathicity reflects the abundance and polarization of the hydrophobic and hydrophilic domains within the peptide [119]. The properties of hydrophobicity and amphipathicity are related. Theoretically, all AMPs form amphipathic structures in the interaction with membranes. AMPs with α-helix conformation are the ones that most easily acquire the amphipathic character and present interactions with microbial membranes, although those of β-sheet conformation are also amphipathic since their strands are organized in a way to

polarize the hydrophilic regions [112,120,121]. For instance, the α-helical magainin-2 and βstructured protegrin-1 have distinct amphipathical characters, cited previously in this review. There are AMPs which chains are considered of irregular composition, which means their amino acids do not form defined conformational structures. Some have a high amount of one or more amino acid residues – such as the proline- and arginine-rich PR-39 – while others have other smaller structures such as thioether rings – such as lantibiotics, including nisin – and cyclic regions [122–125]. The mechanisms of action in these cases vary widely, usually depending on additional interactions between the peptides themselves or receptor interactions, but in general the mode of action of these AMPs remain not fully elucidated up to date. The targeting and action mechanism of nisin, an important agent for food preservation, has been briefly described in this review (items 2.1 and 3.3, respectively).

Figure 2. The antimicrobial activity of peptides is determined by several parameters in their structure. First, the amino acids composition will determine the conformation of the molecule, establishing hydrophobic and hydrophilic regions, as well as domains and charges. The net charge, amphipathic character, and size of the molecule also influence the AMP ability to penetrate bacterial membranes.

3.2 Membrane Destabilization As mentioned before, most of the peptides of industrial interest are those of conformation α-helix, which present the formation of transmembrane pores as the main mode of action [3,126]. Different mechanisms have been proposed for this, being that the

three most accepted by the scientific community and better postulates will be treated here – the barrel-stave, the carpet, and the wormhole mechanisms. The barrel-stave mechanism (Figure 3) is responsible for the formation of transmembrane pores in the channel format [127,128]. Considering the amphipathicity of the AMPs, the hydrophobic region of the chains – which can be in α-helix or β-sheet conformation – interacts with the core of the membrane while the hydrophilic region is turned outwards, thus forming an aqueous pore [128,129]. In this mechanism, the molecules of the AMPs are recognized so that the insertions are made in the same place progressively, like the staves of a barrel. The more monomers inserted, the larger the pore size, initiating the pore with at least 4 monomers [3,129]. The first step of the mechanism is to position the AMP on the membrane surface prior to its insertion. This is necessary to make the process energetically favorable since, when in quantity, the hydrophilic regions of AMPs will be facing each other (light of the pore), aggregating and energetically facilitating the complex insertion in the membrane [36,126]. Currently, the only peptide described to perform its pore-forming activity through the barrel-stave mechanism is alamethicin [130]. Previously classified peptides as possessing this mechanism, such as magainin 2 [3,14], were later known to perform their activities through the wormhole model. Such model will be brought in more details later.

Figure 3. In the barrel-stave mechanism, AMPs accumulate on the membrane surface and their successive insertions form an aqueous pore in the membrane. The carpet mechanism (Figure 4) is a membrane permeabilization model that resembles the mode of action of detergents and is currently accepted for several AMPs. Unlike the barrel-stave mechanism, there is no insertion of the peptides in the membrane, but rather an accumulation on the surface, forming a "carpet" [129,131,132]. The electrostatic interactions in the membrane at a given moment are so many – because of the

high density of AMPs – that the bilayer is destabilized by fluidity changes and thus collapses in several micelles [42,126,133]. The human cathelicidin LL-37 is an example of AMP which expresses its activity through this mechanism, disrupting the structure of membranes of bacteria [134–136].

Figure 4. The total collapse of the membrane provoked by the carpet mechanism is the main consequence of the high density of AMPs accumulated on the surface. One of the most well-known mechanisms in the literature is the wormhole (Figure 5), also called the toroidal pore, both denominations that refer to the pore type originated by this mechanism [3,59]. The essential difference between the pore formed by the barrel-stave mechanism and the wormhole is the presence of membrane lipids intercalated with the AMPs in the formed channel. Thus, the "light" of the pore shows the polar surfaces of the AMPs and the heads of the phospholipids, also polar [59,137,138]. In the wormhole, the peptides also initially accumulate oriented parallelly to the membrane, as in the mechanisms already mentioned. The hydrophobic amino acid residues then displace the polar heads of the lipids, which induces bending stress in the membrane. This tension leads to the destabilization of the membrane surface and consequent cytoplasmic extravasation and lysis [139,140]. As cited previously, magainin 2 is an AMP which exerts its activity through this mechanism. Some authors present the wormhole mechanism as a carpet stage corresponding to the initial destabilization of the membrane

before its total collapse [126,141], which explains why classification of AMPs regarding the mechanism of action can differ between works. For instance, cathelicidin LL-37, presented previously as exhibiting the carpet mechanism, can also be found in the literature as a toroidal pore-forming AMP [3,142].

Figure 5. In the wormhole mechanism, AMPs are inserted in the membrane forming a pore with intercalated lipids, causing a bending tension leading to lysis. Also, as approached earlier, the interactions between the peptides themselves, such as self-association and multimerization [36,143,144] should be considered. These interactions lead to the formation of complexes that influence and allow the mechanisms used by AMPs to bind and act on their target microorganisms, representing initial stages of the activity towards membranes and other cellular targets. Other proposed mechanisms can be found in the literature and, although the three mentioned are the most well developed and that present extensive work that corroborates the theories, there is nothing to prevent these mechanisms from being modified or totally discarded in the future, with a clearer and definitive elucidation of the AMPs mechanisms of action in membranes.

3.3 Inhibition of Intracellular Components Although the ability of AMPs to cause cell lysis through membrane rupture has been widely confirmed, it has been speculated that there are other mechanisms involved in the antimicrobial activity of these molecules. Evidence such as the absence of membrane permeabilization, but confirmed antimicrobial activity, in assays with pathogenic bacteria

against AMPs, support the theory that there are other intracellular targets for peptide action, along with the pore formation [145,146]. In addition, many microorganisms have the ability to remain viable even after extensive damage to their membranes, suggesting that other mechanisms that do not involve membrane lysis may influence cell death [35]. Among the lantibiotics, a class of bacteriocins produced by Gram-negative bacteria, such as nisin, there are some AMPs that act as enzymatic inhibitors, interfering majorly in the cell wall synthesis of competing microorganisms. The mechanism is based on the AMP combination to Lipid II, which leads to the prevention of polymerization of peptidoglycans precursors – the main component of cell walls [147,148]. Such inhibition may also be caused by the AMPs binding to other enzymes of the wall synthesis process, such as those related to the peptidoglycan translocation. In general, this is a more effective mechanism against Gram-positive bacteria, because of the greater number of peptidoglycans in their walls [140,149]. Other mechanisms such as binding to DNA and RNA have been observed, which lead to inhibition of the processes involving these nucleic acids, especially replication, transcription, and translation – essential for cell growth and multiplication. In other words, DNA, RNA and protein syntheses are inhibited. Inhibition of DNA gyrase, a major replication enzyme, is a very common mechanism in peptides produced by bacteria [150,151]. This is observed in the CF-14 peptide antimicrobial action, which presents high affinity to DNA, leading to alterations to microbial functions [62]. Other ligands were observed in proline-rich peptides, which bind specifically to DnaK, a major heat shock protein, and non-specifically to GroEL, a bacterial chaperone. These bounds inhibit the activity of these proteins, damaging essential cellular processes such as protein folding [83,152]. The insect AMPs drosocin, pyrrhocoricin, and apidaecin action occurs through this mechanism, entering in Gramnegative cells after interaction with LPS in the outer membrane [153,154]. Some AMPs may bind to receptors on the membrane of fungal species, promoting their insertion into the cytoplasm and then inducing ATP loss in a non-lytic way. These events disrupt the cell cycle leading to reactive oxygen species production, extremely toxic and harmful to the cell [43,155]. The inhibition of fungal cell wall synthesis – composed of chitin – is also a mechanism led by some AMPs, in addition to lytic interactions with mitochondrial membranes [156]. Potent antifungal peptides had been isolated from plants and insect. For instance, IWF4 from sugar beet (Beta vulgaris L.), a 30-amino acid peptide rich in cysteines and glycines, exhibits a domain with chitin-binding properties [157]. Similar amino acid profile is found in drosomycin, a 44-amino acid antifungal peptide isolated from Drosophila insects [158]. It is important to emphasize that, for the interaction between the AMPs and the target intracellular

components

to

happen,

peptides

need

to

be

transported

into

the

microorganisms, which occurs usually through translocation processes [159]. The mechanism behind this process has not yet been elucidated, but it is believed that the pores in the membrane, promoted by the AMPs, allows the translocation of some peptides to the cellular interior, where they will act in the intracellular targets. In the case of AMPs that do not promote apparent cellular permeabilization, such as CF-14, the formation of pores would occur in a discrete way and, with the internalization, the peptides would act more actively in their intracellular targets, justifying the observed antimicrobial activity [160,161].

4

Molecular Mechanisms of Resistance to AMPs The generation of antimicrobial resistance by microorganisms is considered one of

the biggest public health problems nowadays. Over the years, numerous antibiotics and antimicrobial molecules – highly efficient in the past – have fallen into disuse because they no longer promote the desired effects [162]. Although this phenomenon is natural to the evolution of microorganisms, it is intensified by the indiscriminate and uncontrolled use of antimicrobial substances [163]. Microorganisms use numerous mechanisms to acquire resistance to molecules that threaten their survival. Some of the most important strategies against the mechanisms of action of AMPs are related to changes in the membrane that influence the fixation of the peptides and/or their insertion; and membrane permeability [164,165]. Fundamentally there are two resistance strategies the microorganism can apply: constitutive resistance and induced resistance (Figure 6). The constitutive resistance, also called passive resistance, presents mechanisms that utilize inherent properties of the organism, which are present independently of the concomitant presence of AMPs. On the other hand, induced or adaptive resistance results from mechanisms that are triggered in response to the presence of AMPs or their effects [166]. Some mechanisms of constitutive resistance include energetic changes in the membrane and in an electrostatic shield formation. The energetic alteration of the membrane is a mechanism that consists in the translocation of basic amino acids to the surface of the membrane, which leads to changes in its liquid charge. In this way, the AMPs have less affinity with the membrane, modifying the mechanisms of their connection. This is also the outcome of the electrostatic shield formation, although in this mechanism the charge change essentially occurs in Gram-negative capsules (outer membrane) [167,168]. An example of constitutive resistance is the activation of the dlt operon in Staphylococcus aureus. The dltA, dltB, dltC and dltD genes promote the reduction of the net negative charge of the membrane by transporting the basic amino acid D-alanine to the surface, influencing the charge of the teichoic acid. This change in membrane composition

decreases the affinity of membrane AMPs, disrupting the initial binding and subsequent antimicrobial action. Similar mechanisms are found in other Gram-positive bacteria [169,170]. In Gram-negative, two main mechanisms of the constitutive resistance can be found. The decrease in the net negative charge of the membrane, in this case, is accomplished by changes in the lipids A of the LPS of the outer membrane in the electrostatic shield mechanism [80,171]. The other mechanism is due to the first, since there is an increase in hydrophobic interactions between the lipids A that alters the membrane permeability, reducing or totally preventing the AMPs insertion and the consequent formation of pores and translocation of molecules [77]. In addition, the capsule presentation also assists in Gramnegative resistance, with a mechanism analogous to that of other chemical classes of antimicrobials: limiting the interactions between molecules and their targets in the membrane [12,79]. Among the mechanisms of induced resistance, we highlight the mobilization of proteases and peptidases, the activation of efflux pumps, and the mutational modifications of targets. The first mechanism refers to the use of enzymes that degrade proteins and peptides aiming the inactivation of AMPs that threaten cell viability. These enzymes can be excreted to the extracellular environment as well as acting on the cytoplasm of the underattack cells, degrading the translocated peptides and preventing them from acting on intracellular targets [172]. The activation of efflux pumps also acts in this case, returning translocated peptides to the extracellular environment and protecting the interior of the cell [173,174]. This efflux pumps activation had been identified in Neisseria gonorrhoeae after exposure to protegrin-1 and LL-37 [175]. LL-37 is also inactivated by the proteinase aureolysin S. aureus [176]. Finally, the mutational modification of targets is a mechanism that need to be better elucidated, but it is proposed to be based on transient mutations in the membrane and intracellular targets of AMPs, in order to decrease or even prevent the peptide binding to its receptors [177,178]. For instance, the pathogen Yersinia enterocolitica alters the production of outer membrane proteins to prevent AMPs interactions [179]. These mechanisms are regulated by a set of extracellular signals, such as pH and salt concentration changes, such as magnesium and calcium, that indicate phagocytosis occurrence [165].

Figure 6. Resistance strategies of microorganisms can be classified as constitutive or induced mechanisms. Such strategies are responsible for limiting the interaction or even inactivating AMPs.

5

Application of AMPs and Prospects Currently, the application of AMPs in human and animal health are increasing, as well

as in the food industry, considering the growing search for alternatives to traditional synthetic chemicals which have been losing effectiveness or consumer acceptability. For instance, the application of peptides KG18 and VR18 with or without tungsten disulfide (WS2) against the pathogens Pseudomonas aeruginosa and Candida albicans has shown destabilization of the cell membrane and extravasation of cytoplasmic content [180]. The conjugation of peptides to other organic or even inorganic compounds represents an interesting approach to enhance effects while reducing costs. The P5 peptide has also been reported to inhibit carbapenem-resistant P. aeruginosa – one of the major pathogens related to hospital infections by resistant microorganisms – by increasing plasma membrane permeability. In addition, P5 has demonstrated the ability to inhibit biofilm production, an important factor related to persistent and repeated contaminations [181]. Similar studies have demonstrated the antimicrobial action of the

peptide chionodracine-m3 against Escherichia coli ESBL, Klebsiella pneumoniae KPC, Acinetobacter baumanni XDR, P. aeruginosa MDR, S. aureus MRSA, Staphylococcus epidermidis MRSE and Enterococcus spp. VRE, important pathogens associated with antibiotic resistant infections [182]. Such studies demonstrate the huge potential of AMP application to therapeutics. Although several functionalities for AMPs are studied, their application is still restricted due to the lack of research on the feasible mode of obtention and use. Very few AMPs have been approved for clinical use – such as polymyxin B, which is used to treat Gram-negative bacterial infections [183]. Most studies focus on the incorporation of AMPs to surfaces such as packaging and hospital supplies. For instance, modified human defensins incorporated in metal surfaces for hospital use led to a more effective microbial decrease than standard sanitation techniques, reducing the populations of Methicillin resistant-S. aureus, -E. coli, -S. epidermidis, -Enterococcus spp. and -P. aeruginosa [184]. Regarding bioactive modified packaging with AMPs, a potential consumer market with high technology application and effectiveness. Studies have shown the superiority of the application of nisin Z in packages compared to direct application in food, with a decrease of production costs and improvement of the antimicrobial action [185]. Nisin Z, when applied in packaging, has the potential to reduce populations of Listeria monocytogenes, Salmonella Typhimurium, Leuconostoc mesenteroides, S. aureus, Alicyclobacillus acidoterrestris and Bacillus cereus [185–187], related to food poisoning and/or spoilage. Several other peptides are being studied for the food industry, such as Dip KK-14 and KK-14 R10, however, matrix interactions, as well as techniques for package application and delivery to the product, shall be further studied [188]. The use of AMPs stands out as one of the main alternatives for the control of pathogens in the future, considering the current issue of resistance to traditional sanitizing and therapeutic substances. Thus, since there is a growing need for studies in the area, a deep knowledge on the molecular principles of function of AMPs shall allow researchers to better design projects in the field, preventing human and financial resources waste and mastering results. 6

Conclusion Understanding the mechanisms involved in the AMPs activity represents major

importance to enable successful applications in the industry. A better comprehension of the macro to micro aspects allows researchers to develop better approaches to explore and evaluate AMPs potential. Despite basic, the information gathered here may contribute to the choice of molecules according to the intended purpose, to de novo construction of peptides

with higher efficiency or even capable of dribbling the resistance mechanisms of target microorganisms. Currently, there are several studies on synthetic AMPs [57,189,190], and with greater knowledge of the mechanisms, the optimization of these engineered peptides is imminent. Although most of the binding, action, and resistance mechanisms have not yet been proven and/or fully elucidated, new studies and techniques will soon be able to determine exactly what steps are being taken by AMPs. It is important to note that understanding the in vivo mechanisms provide the basis for the application of the isolated AMPs, since many of these molecules act together and there are several other complementary factors that influence the antimicrobial effect. The occurrence of synergistic effects between different peptides is a growing topic within AMPs studies and has shown great potential. In short, although essential, the molecular mechanisms of AMPs action are many times neglected. Basic studies on the elucidation of mechanisms underlying the action of AMPs are needed to avoid resource waste and optimize applicable outcomes. Authors’ Contribution JAFC and AGE wrote the manuscript and collected information regarding antimicrobial peptides; TMN and FBL provided assistance and relevant commentaries during the manuscript development and revision; JAFC leaded the alterations in the manuscript in order to attend the reviewer and editor comments; JAFC, AGE, and TMN have conceived the figures and their concepts; FBL proposed the idea; All authors have read and accepted the final revised version of the manuscript. Acknowledgments The authors thank the Coordination of Improvement of Higher-Level Personnel (Capes – Financial Code 001), the Brazilian National Council for Scientific and Technological Development (CNPq Process Nº 437728/2018-8), and Pontifícia Universidade Católica do Paraná (Curitiba, Brazil) for financial support. Conflict of Interests The authors declare there are no conflicts of interest regarding the submission, peerreview and/or publication of this paper.

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