Structural and functional insights into plant bactericidal peptides

Science against microbial pathogens: communicating current research and technological advances _______________________________________________________________________________ A. Méndez-Vilas (Ed.)

Structural and functional insights into plant bactericidal peptides E.S. Cândido1, W.F. Porto1, D.S. Amaro1, J.C. Viana1, S.C. Dias1 and O.L. Franco1 1

Centro de Análises Proteômicas e Bioquímicas, Pós-Graduação em Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, Campus Avançado Asa Norte - SGAN 916 Avenida W5 - CEP: 70790-160 - Brasilia – DF, Brazil.

Plant bactericidal peptides perform essential roles in plant survival, being directly involved in defense mechanisms against multiple pathogens. These proteins may accumulate inside plant cells, and in response to determined signals, may be used by the different plant tissues in response to pathogen attack. In order to shed some light on these remarkable compounds, bactericidal peptides found in different plant bactericidal peptides from different tissues are here discussed in depth, as well the structural-function protein relation. Amongst these proteins we will focus on cyclotides, defensins, glycine-rich proteins, and other unusual classes. Finally, the potential use of these molecules in development of drugs to control human and plant pathogens, contributing to the development of new biotechnology-based medications, will be also presented. Keywords: plant antimicrobial peptides; structure; function; biotechnological drugs

1.Introduction In 1942, Kent Arnold Balls and colleagues discovered the first plant protein fraction with antimicrobial activity. This fraction, isolated from wheat (Triticum aestivum), was named purothionin. Later, in vitro activities against human pathogens were reported. Nevertheless, the study of antimicrobial activity was dropped because its existence was not proven by using mice in in vivo experiments. In 1968, it was discovered that this fraction had several peptides, among them the ɑ- and β-thionins, which were tested separately against pathogens, showing the ability to inhibit their development [1]. After this first antimicrobial peptide had been isolated, many others were characterized and, by 2011, 271 different peptides of plants have been described in the Antimicrobial Peptide Database (http://aps.unmc.edu) [2] and PhytAMP (http://phytamp.pfba-lab-tun.org/main.php) [3], isolated from seeds [4], roots [5], fruits [6] leaves [7] and flowers [8]. Essentially, antimicrobial peptides have been classified according to their structure and composition [1-2]. Therefore, there are three main groups: (I) linear peptides, (II) cysteine stabilized peptides and (III) peptides with unusual composition, e.g. glycine- or proline-rich peptides [1-2]. Plants show several peptides in both classes, but most belong to the cysteine stabilized peptides class [3].The majority of plant peptides show between 20 and 67 amino acid residues length. From 238 plant peptides, only 61 have had antibacterial activity reported; among them, 57 are cysteine stabilized peptides, six are linear and only one is glycine-rich [3]. The vast majority possess cationic net charges of 0 to +10, on average +4.6, with the highest presenting +17. Only a few of them have overall negative charge, the most negative being -6 [20]. This could be related to the fact that more than half of these peptides have more than six basic residues, while a small number of acidic ones are observed. Another important feature observed is that most plant peptides have 4 to 13 hydrophobic residues, which contribute to insertion in biological membranes. Only 39 of 271 plant structure AMPs were resolved, a point which will be examined in this chapter, and only half have had their biological activity confirmed. Of these, half show antifungal activity and one third, antibacterial. In recent years the number of reports of new antimicrobial peptides from plants and other organisms has decreased, but a considerable increase is observed in the study of existing peptides, in order to elucidate their structure-functional relations [9] and to establish alternative activities, such as immunomodulatory and antitumor [10]. Many groups are trying to develop drugs based on templates of antimicrobial peptides [11], as an alternative to conventional antibiotics, against the problem of increased bacterial resistance.

2. Classes of plant bactericidal peptides with activity toward human pathogens. There are currently three main families of plant bactericidal peptides: cyclotides, defensins (also known as γ-thionins), and α/β-thionins. There are other families with only one member whose structure has been clarified, which is the case of β-barrelins [12] and hevein-like ones [13]. These families will be better discussed later according their structures. Defensins are probably the most ancient antimicrobial peptides, present in the whole Eukaryote Domain [16]. Overall, defensins can have three, four or five disulfide bonds, but in the Plant Kingdom they have been found only with four or five disulfide bonds. Interestingly, most defensins that are active against bacteria have three disulfide bonds; in other words, the majority of defensins that are active against bacteria belong to animals or fungi. At the beginning of the 1990s the first plant defensins were characterized. Due to their cysteine content and size, they were named γ-thionins, in reference to the α/β-thionin family, which displays four disulfide bonds and has a molecular mass around 5 kDa. Further, structural analysis showed that γ-thionins and other thionins are different: while γ-thionins show

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Science against microbial pathogens: communicating current research and technological advances ______________________________________________________________________________ A. Méndez-Vilas (Ed.)

a cysteine stabilized αβ fold (CSαβ), α/β-thionins show a fold with two α-helices and a short antiparallel β-sheet. This structural divergence has led γ-thionins to be renamed as defensins [14]. As well as being the most ancient, defensins are among the most basic peptides, showing between 5 and 7 kDa size [14]. The mature defensin peptide has eight conserved cysteine residues that are responsible for structural stabilization [14]. In plants, they are normally found in abundance in seeds, but also appear in other tissues including leaves, pods, tubers, fruits, roots, bark and floral tissues [15]. This peptide class shows deleterious inhibitory activity against Gram-positive and –negative bacteria, but the mechanism of action is not completely understood [14]. Among defensins with bactericidal activity, Zhang and Lewis [16] demonstrated that fabatin has clear activity against the Gram-negative bacterium Pseudomonas aeruginosa, but only slight toxicity against Escherichia coli, suggesting that the action mode of this peptide is unlike that of other defensins. Numerous known defensins act as highly toxic to Gram-negative pathogens, and this is supposed to be due to the primary binding of the charged peptides to the surface lipopolysaccharide. Franco and colleagues [17] showed that the defensin Cp-thionin II acts in a lethal action against Staphylococcus aureus and E. coli. A second class of important bactericidal peptides is the cyclotides, which are globular disulfide-rich peptides, ranging in size from about 28 to 37 amino acids [18]. Cyclotides present unique structural features, with a head-to-tail cyclized backbone and a knotted arrangement of three disulfide bonds, referred to as a cyclic cystine knot (CCK) motif. This motif gives cyclotides high resistance to thermal, chemical and enzymatic degradation. This peptide class may be frequently found in the botanical families Rubiaceae, Violaceae, Apocynaceae, Curcubitaceae, and Poaceae [18-20]. Cyclotides are desirable targets for the pharmaceutical and agrochemical industries, due to their unique cyclic structural scaffold, biological activities and diversity of sequence. They are today one of the most studied plant peptide families, and comprise a huge library of natural peptides that can be used in the search for new bactericides [18, 19]. Tam and his team [21] demonstrated that kalata B1 and circulin A have a very similar profile and show high activity against S. aureus. While circulin B presented strong activity against E. coli, it was only moderately active against S. aureus. Although defensins and cyclotides represent the major families of plant antibacterial activity peptides, it is interesting to highlight other AMPs that play an important role in the discovery of potential targets for antibacterial drug development. A brief inventory of antibacterial peptides is showed in Table 1 to summarize these activities. Xiao and coworkers [22] identified a cysteine-rich AMP in Jatropha curcas (JCpep7) which shows activity against the Grampositive bacteria S. aureus, Bacillus subtilis and Streptococcus pneumonia, and the Gram-negative bacteria Salmonella typhimurium, P. aeruginosa and Shigella dysenteriae. JCpep7 seems to act by attracting the bacterial surfaces and changing membrane permeability. The authors demonstrated that the use of cell membrane affinity chromatography is an efficient method to screen AMPs from J. curcas, which suggests that this technique can be used for plant tissue AMP screening with drug discovery purposes. Furthermore, the peptides Cn-AMP1, Cn-AMP2 and Cn-AMP3, isolated from green coconut water, present activity against S. aureus, P. aeruginosa and E. coli. The peptides Cn-AMP2 and Cn-AMP 3 have acidic properties, while Cn-AMP1 is cationic, suggesting that these features can improve Cn-AMP1 antibacterial activity [23]. Impatiens balsamina isolated peptides Ib-AMP1 and Ib-AMP4 were able to inhibit the development S. aureus and Streptococcus faecalis, presenting inhibition potential tantamount to that of the Magainin I peptide. Their mechanism of action is still unknown, but it is known that these two peptides do not affect human cells, are not cytotoxic to plant and insect cells, and may act by inhibiting a specific cellular process [24]. A bactericidal peptide in guava seeds, named Pg-AMP1, was also identified. Pg-AMP1is a glycine-rich peptide with deleterious effects against Gram-negative bacteria Klebsiella sp. and Proteus sp, but little is known about its mechanism of action [25]. The sweet polypeptide brazzein presents 54 typical amino acids and no carbohydrates; it was originally purified from the fruit of Pentadiplandra brazzeana [26]. Yount and Yeaman [27] state that this peptide exerted antimicrobial activity against bacteria and, remarkably, that this activity is pH-specific. Finally, Kiba and colleagues [28] purified a hevein-like peptide from Wasabia japonica leaves, WjAMP-1, with antimicrobial activity. This peptide inhibits E. coli, presenting an IC50 of 8 ug.mL-1.

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Table 1. Bactericidal plant peptides with activity toward human pathogens.

AMP Class

AMP Name

Plant Source

Defensin

Cp-thionin-2

V. unguiculata

Defensin

Fabatin-1

V. faba

Defensin

Fabatin-2

V. faba

Cyclotide

Circulin-A

C. parviflora

Cyclotide

Circulin-B

C. parviflora

Cyclotide

Cyclopsychotride-A

P. longipes

Cyclotide

Kalata-B1

O. affinis

Hevein-like

WjAMP-1

E. wasabi

Activity (MIC) S. aureus (128 µg.ml-1) and E. coli (64 µg.ml-1 ) E. coli (100 µg.ml-1) and P. aeruginosa (30 µg.ml-1) E. coli (100 µg.ml-1) and P. aeruginosa (30 µg.ml-1) S. aureus (0.59 µg.ml-1) and P. vulgaris (172.04 µg.ml-1) S. aureus (44.32 µg.ml-1), E. coli (1.35 µg.ml-1), P. aeruginosa (83.7 µg.ml-1), P. vulgaris (22.3 µg.ml-1) and K. oxytoca (26.92 µg.ml-1) S. aureus (125.97 µg.ml-1), E. coli (5.01µg.ml-1), P. aeruginosa (43.60 µg.ml-1), P. vulgaris (42.63 µg.ml-1) and K. oxytoca (18.73 µg.ml-1 ) S. aureus (0.75 µg.ml-1) and K. oxytoca (158.37 µg.ml-1) E. coli (N.D.) -1

References [15] [14] [14] [19]

[19]

[19]

[19] [26]

Snakins

Snakin-1

S. tuberosum

L. monocytogenes (10µg.ml )

[27]

Shepherin

Shepherin I

C. bursapastoris

S. aureus (N.D.), S. mutans (N.D.), E. coli (N.D.), S. typhirium (N.D.) and Serratia sp. (N.D.)

[5]

Shepherin

Shepherin II

C. bursapastoris

S. aureus (N.D.), S. mutans (N.D.), E. coli (N.D.), S. typhirium (N.D.) and Serratia sp. (N.D.)

[5]

Hairpin

MBP-1

Z. mays

E. coli (30µg.ml-1)

[28]

-

Brazzein

P. brazzeana

S. aureus (N.D.) and E. coli (N.D.)

[25]

-

Ib-AMP1

I. balsamina

S. aureus (N.D.) and S. faecalis (N.D.)

[22]

-

Ib-AMP4

I. balsamina

S. aureus (N.D.) and S. faecalis (N.D.)

[22]

-

-1

E. coli (72 µg.ml ) and Klebsiella sp. (32 µg.ml-1) S. aureus (80 µg.ml-1), Cn-AMP1 C. nucifera E. coli (82 µg.ml-1) and P. aeruginosa (79 µg.ml-1) S. aureus (170µg.ml-1), E. coli (170 µg.ml-1) and Cn-AMP2 C. nucifera P. aeruginosa (169 µg.ml-1) S. aureus (274 µg.ml-1), -1 N.D. corresponds to non determined; MIC corresponds minimal inhibitory concentration Cn-AMP3 C. nucifera E. to coli (302 µg.ml ) and P. aeruginosa (259 µg.ml-1) Pg-AMP1

P. guajava

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3. Structural and functional insights into plant bactericidal peptides Essentially, antimicrobial peptides have been classified according to their structure and composition as previously described [29, 30]. Up to 2011, 238 plant peptides have been listed in the Antimicrobial Peptides Database (APD http://aps.unmc.edu/AP/). However, only 61 have had antibacterial activity reported; among them, 57 are cysteine stabilized peptides, six are linear and only one is glycine-rich [2]. On the other hand, the three-dimensional structure of only nine peptides has been evaluated by NMR or Crystallography (Figure 1) [12, 13, 31-37]. The lack of information on structure of plant bactericidal peptides prevents more detailed classification of plant peptides.

Fig. 1 Structures of plant bactericidal peptides. The first line displays the structures of cyclotides circulin A (PDB 1BH4) [4], circulin B (PDB 2ERI) [32] and kalata B1 (PDB 1NB1) [33]. The second line displays the structures of β-barrelin MiAMP1 (PDB 1C01) [12]; hevein-like Ac-AMP2 (PDB 1MMC) [13] and defensin brazzein (PDB 1BRZ) [34]. The third line displays the structures of thionins, α-1-purothionin (PDB 2PLH) [35], β-purothionin (PDB 1BHP) [36] and viscotoxin A3 (PDB 1ED0) [37]. Disulfide bonds are represented in ball and stick. For cyclotides, proposed active residues are also represented in ball and stick.

The establishment of a relationship between structure and bactericidal activity is made more difficult by the lack of any structural or sequential similarities within peptide families. Furthermore, these classes of plant defence compounds show other activities beyond the antibacterial, such as insecticidal, antifungal or hemolytic ones [21, 37, 38]. Cyclotides represent this paradox very well. Cybase (a database dedicated only to cyclic peptides http://www.cybase.org.au/) [39] shows 139 natural cyclotide sequences, but not all have antimicrobial activity. This family is composed of cyclic peptides with ~29 amino acid residues with a characteristic cysteine knot motif showing

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the bonding pattern of (Cys1-Cys4, Cys2-Cys5 and Cys3-Cys6) [21]. Among cyclotides, there are two main sub-families, Möbius and Bracelet, and a third but lesser cyclotide subfamily, the trypsin inhibitors, not examined here since any antibacterial activity in this sub-family was observed until now. The key difference between the first two subfamilies is a cis-proline in the 5th loop, present only in Möbius, which generally shows three -sheets in its scaffold; members of the Bracelet subfamily, generally, show two -sheets and one 310-helix in their structures (Figure 1). Despite these differences, the two subfamilies show members with activity against bacteria. While the cysteine knot motif is important in stabilizing these molecules, bactericidal activity is more related to cationicity [21]. Among cyclotides, there are three members with deleterious activity against bacteria which have seen their structures solved by NMR: circulin A and B (bracelet subfamily) and kalata B1 (Möbius subfamily). Interestingly, despite the high similarity between the sequences of circulins A and B, they have distinct structures and activities: the structure of Circulin B is more similar to kalata B1, with three antiparallel β-sheets. On the other hand, the activity of kalata B1 is more similar to that of circulin A, as both cyclotides are active against Gram-positive bacteria, but have no activity against Grampositive ones. In contrast, circulin B shows activity against Gram-positive and negative bacteria. Given the exceptions to every apparent rule or link between structure and activity, some questions are inevitable: does activity have any connection with sequence or structure in cyclotides? If structure and sequence are separately analyzed in relation to activity, no valuable information about function could be obtained. However, sequence and structure need to be analyzed together. Despite the sequence similarity between circulin A and B, the orientations of residues in their structures are sufficiently different to provide different activities. circulin B has a valine residue plus circulin A, which changes the conformation of cationic clusters, making the cationic residues in circulin B more exposed at the edge of the molecule, while circulin A has an arginine residue folded back to its backbone, reducing the cationicity of the structure; this conformation makes the cationicity of circulin A more similar that of kalata B1 [21]. Defensins, like cyclotides, show no clear relationships between structure and activity. Overall, defensins show a similar scaffold, possessing one α-helix and two or three β-sheets (Figure 1), forming a typical cysteine stabilized αβ structural motif (CSαβ). A study with mutants of Anopheles gambiae defensin A (DEF-AAA) shows that the activity is related to disordered loops in the structure [40], which are reduced in plant defensins due to the presence of the fourth disulfide bond, stabilizing the N- and C-terminals. Therefore, although there are plant defensins that are active against bacteria, the majority of them are active against fungi [41]. Cp-thionin, a defensin from cowpea seeds (Vigna unguiculata), has been described by Franco and co-workers [17]. This defensin shows bactericidal activity against Gram-positive and -negative bacteria. Cp-thionin shares around 73% of structural identity with VrD1, a defensin from Vigna radiata, which has no reports of bactericidal activity, but instead shows α-amylase inhibitory activity, absent in Cp-thionin. In this case, structure and sequence similarity are insufficient to explain these different activities. However, it has not yet been possible to determine which residues are essential for antimicrobial activity in Cp-thionin. Another report of plant defensin with bactericidal activity is the sweet-tasting protein from Pentadiplandra brazzeana, brazzein. Its activity had been predicted and confirmed through of detection of a γ-core in its structure [27] and, until now, this is the only plant defensin that is active against bacteria to have had its structure solved. Given the success of the γ-core prediction in brazzein, novel peptides or sequence motifs might be identified, providing new information about the bactericidal activity of other defensins. This may be carried out, perhaps, by applying computational models, such as a linguistic model [42] or a support vector machine classifier [43]. The third main class, α/β-thionins, also shows a wide range of biological activities, besides bactericidal activity against phytopathogens. These proteins also have antifungical, insecticidal and immunomodulatory function and can inhibit protein synthesis in cell-free systems [1, 37]. The overall shape of α/β-thionins resembles a capital letter L, where the α-helixes define the long arm and the short antiparallel β-sheet the short arm (Figure 1) [37]. For these peptides, two modes of action have been proposed: (I) membrane disruption by electrostatic interactions through charged residues and (II) DNA binding through the structural helix-turn-helix motif, common in prokaryotic generegulatory proteins and homeodomain-containing proteins. Moreover, there are two lesser families with only one structure solved. In the case of the hevein-like family, only Ac-AMP2 from Amaranthus caudatus has its structure defined. The overall structure of Ac-AMP2 comprises a twisted antiparallel β-sheet composed of two strands, showing essentially the same fold as that observed in hevein-like domains (Figure 1) [13]. The activity against Gram-positive bacteria of this peptide may have a secondary character, since this hevein-like domain is associated with sugar binding. Ac-AMP2 binds specifically to chitin, providing antifungal activity. No correlations between bactericidal activity and its structure were established, and the absence of similar structures with similar bactericidal activity obstructs a more accurate conclusion. In cases where there are no similar structures, the main explanation of antimicrobial activity relies on the amphipathic surface of protein. An example of this is MiAMP1 from Macadamia integrifolia, the only peptide which have the structure solved into in the β-barrelins family; its structure is unique among bactericidal peptides, being composed of a Greek key β-barrel (Figure 1) [12], and its activity is restricted to acting against plant pathogens. Preliminary analysis suggests that this activity involves interactions with negative membranes, due to its amphipathic surface. Again, the absence of other structures obstructs a more definite conclusion. It is interesting to note that membrane interactions are the most widely proposed mechanism of action for bactericidal peptides; however, not all amphipathic surfaces have bacterial activity. Plant defensins are the most emblematic example, where bactericidal

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peptides are exceptions, but all members have an amphipathic surface. For a better understanding of relations between structure and activity, it is necessary not only to explain more structures, but also to explore new methods for detecting similarity, such as the multi-dimensional method employed in isolating the γ-core in brazzein. In future, when more structures of plant bactericidal peptides may be available, a more careful and systematic classification of these peptides must be created, avoiding the perpetuation of such errors as classifying defensins with  and thionins. Besides achieving classification, a better understanding may be gained of their mechanisms of action, not limited only to the classical explanation of amphipathic surfaces interacting with membranes, but also identifying the role of each residue in the structure, as observed in cyclotides. Perhaps, when we reach that point, we will be able to make a rational design of novel multifunctional drugs, based on these plant peptides and predicting their activity against plant and human pathogens.

4. The use of plant bactericidal peptides in biotechnology Historically, plants have provided a source of novel drug compounds, as plant-derived medicines have made substantial contributions to human health [44]. The use of medicinal plants as a biotechnological source to relieve illness can be traced back over five millennia to written documents from early civilizations such as in China and India [45]. The first generation of plant medicines was used by traditional societies from many different parts of the World. Even today, the crude form of medicines such as cinchona and aloe are still used in their natural state based on empirical evidence of their clinical application. The second generation of plant-based medicines arose following the Industrial Revolution and was based on scientific processing of plant extracts to isolate their possible active constituents. A notable example of this generation is quinine from Cinchona succirubra. This alkaloid naturally occurs in the bark of the Cinchona tree. Apart from its continued usefulness in the treatment of malaria, it can be also used to relieve nocturnal leg cramps [46]. In the development of the third generation, phytotherapeutic agents are usually adopted. This generation is characterized by previously conducted clinical trials which, when applicable, include cytotoxicity evaluation. The next step focuses particularly on formulation and trial production of the dosage forms. This step is essential to structure and mimic traditional use, while giving higher stability to finished products. A number of pharmaceutical companies are engaged in the development of ‘natural’ plant product drugs [47]. However, although drugs from plant sources continue to occupy an important niche in modern medicine, new technologies for the development of the most powerful antibiotics from plants are also being urgently explored. These technologies include all single chemical entities extracted from higher plants, or synthetically modified, although some of the chemical entities are now being made synthetically for economic reasons [48]. Manipulation of chemical structure to create synthetic peptides represents a promising strategy for the development of AMPs as a new class of drugs to prevent and treat systemic and topical infections. Amongst the advantages of chemical manipulation of the peptide structure, the possibility of increasing the antimicrobial effects of these molecules is of particular importance, decreasing side effects. However, as far as we know there is no research taking place at the moment into plant antimicrobial peptides and the use of chemical modifications. For this reason, here we illustrate an example of the use of a modified insect defensin named tenecin 1 [49]. Tenecin 1 is capable of presenting antimicrobial and antifungal activity without necessarily having increasing its hemolytic activity. However, loading a positive cationic group modified its antimicrobial activity, suggesting the importance of charged residues for this kind of action. This strategy might be extended to other peptides, including those from plants. Another interesting route was taken by Chou et al. [50] in designing and synthesizing small antimicrobial peptides with 20 amino acid residue length. By using four structural parameters - the charge, polarity angles, hydrophobicity and hydrophobic moment – this study evaluated their influence on the selectivity and antimicrobial activity against Gram-positive and -negative pathogenic bacteria. After structural and biological analyses, it was confirmed that high hydrophobicity and the hydrophobic moment may be correlated with an increase in hemolytic activity and also with antimicrobial activity. The structural and synthetic modifications reported here suggest that peptide action might well be improved by the switch of exposed amino acid residues, but maintaining structural scaffold. Although chemical peptide synthesis is a promising field for the development and improvement of bactericidal compounds, this method could be limited due to high production costs and also because of difficulties in constructing the correct fold in the presence of disulfide bounds, as is observed in defensins and cyclotides. Furthermore, since the isolation of plant peptides is sometimes inefficient and time-consuming [51, 52], different strategies have been developed to produce heterologous antimicrobial peptides from plant sources in prokaryotic (E. coli) and eukaryotic (yeasts, plants and animal cells) systems [15]. Otherwise, procedures to express antibacterial plant peptides in prokaryotic systems have encountered difficulties because E. coli is not capable of producing eukaryotic posttranslational modifications, which can be critical for the production of folded active plant AMPs [53]. Additionally, in the case of antibacterial peptides, it is not possible to achieve their expression directly in the E. coli system due to toxicity of AMPs to host cells. In order to solve those problems, several approaches in biological expression systems have been utilized, such as the fusion of an antibacterial peptide with an anionic partner protein and also the use of special E. coli strains [51]. The aim of fusion strategy is to avoid AMP toxicity to host cells, improving, at same time, the stability of the target proteins. Partners proteins with anionic features may neutralize peptides’ cationicity, allowing

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efficient AMP expression. On the other hand, although these molecules could act on increasing of peptide stability, problems like low expression yield and difficulties in purification strategies are often described [54]. Such fusion partners include glutathione S-transferase (GST), maltose-binding protein (MBP), thioredoxin (Trx), ubiquitin and chaperone [52]. The use of the partner proteins is efficient not only for plant proteins, but also for AMPs from multiple sources such as insects, amphibians or marine invertebrates [52, 55] For example, the AMP isolated from the insect Bombyx mori, CM4, was fused with TrxA, a fusion protein, clearly improving peptide expression. Moreover, the expression cassette was constructed with several tandem multimeric CM4 genes fused to TrxA using the vector pET32, forming pET32-nCM4 (n = 1, 2, 3, …, 8), n being the number of times that CM4 is present in the cassette. This vector was cloned and expressed in E.coli BL21 DE3 under the control of the T7 promoter. After expression, the results sshowed that the methodology used is an efficient procedure for producing a large quantity of recombinant AMPs, overcoming problems like the inherent toxicity and low yield of antimicrobial peptides to host cells without losing their antimicrobial activity [56]. Although there are no studies about antimicrobial peptides from plants fused with partner proteins, this strategy might be extended to peptides extracted from plants with antimicrobial activity. Another way to increase therapeutic effectiveness of peptides from plants may involve fusing peptides with different functions for the development of a single compound with higher efficiency toward human pathogenic bacteria. Viscotoxins are antifungal proteins isolated from Viscum album L. that could provide resistance to transgenic plants. Due the toxic effect of viscotoxins, two isoforms of viscotoxins were fused with different partner proteins in order to achieved high expression level and low cytotoxicity. The researchers found differences in cytotoxicity using this method, where one isoform showed higher cytotoxicity than another. Moreover, the expression levels were clearly improved by using this strategy [57]. The search for another way to solve the problem of E. coli expression led researchers to use eukaryotic expression systems such as methylotrophic yeasts, Pichia pastoris and plants [58, 59], obtaining a large amount of functional recombinant Pisum sativum defensin1 (rPsd1), by expressing it in P. pastoris. Other plant defensins were expressed in P. pastoris, producing promising results when evaluated by in vitro activities against bacteria [41]. The use of plants for expression and studies of AMPs has become a reality and is established as a valuable biotechnological approach. AMPs may also be used in transgenic plants of different crop species. Moreover, plants could be used as bio-factories, producing large quantities of antibiotics with activity toward pathogenic bacteria that show enhanced resistance to classic antibiotics. Recent studies show that using plants to produce recombinant proteins offers considerable advantages over the prokaryote and other conventional systems, such as mammalian cell culture and microbial fermentation. In addition, the recombinant protein produced in plants can perform most of the post-translational modifications required for protein stability, bioactivity and favorable pharmacokinetics [60]. To this end, some plant peptides have been expressed in tobacco, Arabidopsis, rice, potato, barley, tomato and potato, producing transgenic plants to control plant-pathogenic bacteria and fungi [14, 61, 62]. Additionally, a promising strategy for producing the next generation of antibiotics in plants can be the use of plastids strategy. This approach was designed to allow the expression of proteins that are toxic to E. coli, leading to high expression levels. In plastids, production reached up to 30% of the plant’s total soluble protein, and recombinant antimicrobial protein maintained deleterious activity when evaluated against Streptococcus pneumonia, the causal agent of pneumonia [63]. Another approach that could be applied to improve and modify the delivery profile of plant peptides is nanotechnology. This relatively new branch of technology studies the development and use of materials, devices and systems in nano-scale, and it has been particularly effective in the health area, improving diagnosis and treatment of disease. The use of nanotechnology for peptide research consists of developing medicine and molecule carrier systems on a nano-scale, aiming to improve peptide stability and absorption and also to control drug release [64]. Several reports show nano capsules containing vaccines in drug delivery system aiming to increase stability and bioavailability. Furthermore, Santos-Magalhães et al. [65] evaluated the efficient loading of antibiotic rifampicin in nanoparticles, aiming to obtain an intelligent drug release. After nanoformulation, the rifampicin underwent continuous release for up to 30 days, demonstrating that nanoparticles associated to rifampicin may be considered an effective drug delivery system for therapy. The use of nanotechnology for developing new biotechnology products with antimicrobial properties may be an alternative to improve and achieve desirable pharmacokinetic features. In summary, there is still a great deal of research to be done with antimicrobial peptides, especially with those obtained from plants. Over the next few years, novel classes will probably be elucidated and new methods of expression evaluated. The combination of multiple peptides and molecules, including nanoparticles, could bring real benefits to this research field. It is a difficult task to discover novel antibiotics, and the transformation of these compounds into therapeutic drugs is a real challenge in many parts of the world. Indeed, researchers must continue to try to find better and more efficient strategies to help to solve the problem of bacterial infections, bringing immeasurable benefits to society.

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