An Approach Towards Structure Based Antimicrobial

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REVIEW ARTICLE

An Approach Towards Structure Based Antimicrobial Peptide Design for Use in Development of Transgenic Plants: A Strategy for Plant Disease Management Humaira Ilyas, Aritreyee Datta and Anirban Bhunia* Department of Biophysics, Bose Institute, P-1/12 CIT Scheme VII (M), Kolkata 700 054, India

ARTICLE HISTORY Received: October 05, 2016 Revised: December 09, 2016 Accepted: January 10, 2017 DOI: 10.2174/0929867324666170116 124558

Abstract: Antimicrobial peptides (AMPs), also known as host defense peptides (HDPs), are ubiquitous and vital components of innate defense response that present themselves as potential candidates for drug design, and aim to control plant and animal diseases. Though their application for plant disease management has long been studied with natural AMPs, cytotoxicity and stability related shortcomings for the development of transgenic plants limit their usage. Newer technologies like molecular modelling, NMR spectroscopy and combinatorial chemistry allow screening for potent candidates and provide new avenues for the generation of rationally designed synthetic AMPs with multiple biological functions. Such AMPs can be used for the control of plant diseases that lead to huge yield losses of agriculturally important crop plants, via generation of transgenic plants. Such approaches have gained significant attention in the past decade as a consequence of increasing antibiotic resistance amongst plant pathogens, and the shortcomings of existing strategies that include environmental contamination and human/animal health hazards amongst others. This review summarizes the recent trends and approaches used for employing AMPs, emphasizing on designed/modified ones, and their applications toward agriculture and food technology.

Keywords: Antimicrobial peptides, global food security, multi-drug resistance, plant protection, de novo designed peptides, transgenic development. Dedicated to Prof. Dr. Thomas Peters, University of Lübeck, Germany on the occasion of his 60th birthday 1. INTRODUCTION Ensuring global food security is the biggest challenge faced by the human race. The challenge lies in making food available to all, alongside the maintenance of environmental balance. It has been estimated that by 2050, around 9 billion people will need to be fed and the inability to meet this food demand will project itself through an increased rate of malnutrition and huge economic losses [1]. In this context, plant disease emerges to be one of the major reasons for the loss of crop yield. It is widely known that around 1.3 billion tonnes of global food produce is lost, out of which 34% is due to plant dis*Address correspondence to this author at the Department of Biophysics, Bose Institute, P-1/12 CIT Scheme VII (M), Kolkata 700 054, India; Tel: (91) (33) 2569 3336; E-mail: [email protected] or [email protected] 0929-8673/17 $58.00+.00

eases, leading to an increase in the number of people experiencing food deficit amounting to about 800 million [2, 3]. All major crop plants are threatened by infection with various kinds of organisms including bacteria, fungi, protozoa, nematodes, parasitic plants, viruses and virusoids. The biggest example of this is provided by the Great Bengal Famine of 1943 caused by an infection of rice plants with the fungus Cochliobolus miyabeanus which led to the death of 7 million people [4]. These plant pathogens become difficult to be targeted or controlled owing to the fact that their populations are highly variable in terms of genotypic [5] and environmental effect [6]. Various factors such as genetic drift, mutation, migration, and selection play an important role to bring the major changes in the genetic structure as well as to effect pathogen population [7]. Globalization of agricultural practices is another cause leading to such enormous diversity. The spaciotempo© 2017 Bentham Science Publishers

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ral and genetic diversity allows the pathogens to maintain polymorphism and thus regulates their resistance and infectivity towards the host plant [8]. Similarly, the environmental effect also plays a very important role in the incidences of plant diseases. The disease triangle model, formulated by George Lee McNew, very well explains such correlation between the host susceptibility, pathogen’s ability, and the impact of environment on infection and pathogenesis [6]. The potato blight, which affected Europe in the mid-1800s due to an infestation of potato plants provides a very good example for the effect of environment on pathogenesis of Phytophthora infestans. Several authors already have reported that the weather and humidity conditions of Europe were responsible for the development of potato late blight infection. In recent times, diverse policies and approaches are being devised to overcome these problems, and to prevent plant pathogenicity. The development of strategies and methods to protect plants by preventing or limiting pathogen introduction is crucial. Plant disease management involves both curative and preventive methods. These are majorly based on several important parameters that involve quick identification of the causal organism/s, their mechanism of action, and severity of the disease [5]. Although various methods for plant protection exist since ancient times and plants themselves develop systemic resistance towards some common phytopathogens like aphids [9], most common approach is the use of chemical compounds like copper-based chemicals exerting toxic effects on the pathogen [10]. Despite the available naturally occurring or designed chemical compounds, many of them are effective only at the initial stages of disease establishment and fail to protect after the initiation of disease [11]. These compounds belong to various different classes, such as organochlorine, organophosphate, pyrethroid, sulfonylurea, and antibiotics like streptomycin, tetracycline, and griseofulvin [12]. Some of these are highly specific for a particular class of pathogen, while others have more diverse functions. However, the use of chemical pesticides have been largely debated due to their negative impacts on the environment and human health [13]. Most of these chemicals accumulate in the soil and water causing environmental pollution. Some of these, especially, the copper-based compounds are carcinogenic and evoke neurological disorder in the biotic system. Moreover, many of these compounds generate pathogen strains with properties of drug resistance [14]. Multi-drug resistance is an emerging problem in medicine and agriculture [15]. Some well-known plant

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pathogens exhibiting multidrug resistance include the strains of Pseudomonas syringae that infect Arabidopsis and tomato [16], Erwinia amylovora which causes fire blight in various members of Rosaceae family [17]. Additionally, various fungal pathogens including Mycosphaerella graminicola (Fückel) Schroter, and Botrytis cinerea Pers also show resistance to antimicrobial agents [18]. All these ill-effects have severely affected the frequent and long-term use of chemical pesticides and antibiotics, thus making plant disease management difficult. Several other techniques for plant protection also exist including use of pathogenfree seed, pathogen eradication, quarantine of diseased plants, crop rotation, cultural management practices, and integrated pest management amongst others [19]. However, they do not provide a long-term solution for curbing plant pathogens. Hence, there is an urgent need for alternative methods to offer plant protection. This necessitates the search for novel compounds with new and wide range of targets that do not evoke pathogen resistance and have negligible negative impact on the host plant, environment, and public health. In this context, plant breeding and development of diseaseresistant transgenic varieties of important crop plants have gained huge importance [20]. Traditional plant breeding techniques, involving artificial mating or cross-pollinating plants to obtain desirable plant characteristics have proved to be a boon during the 20th century leading to a remarkable increase in both productivity and quality [21]. This has been further improved with the advent of techniques like molecular breeding and marker-assisted selection [22]. However, the increase in global food demand and the current environmental status including the dearth of land and water for agricultural practices along with limited scope of some of these approaches have necessitated the development of transgenic plants capable of resisting disease [23]. Antimicrobial peptides from both plant and animal sources have received immense attention in this regard, to confer disease resistance to plants [24]. Additional approaches based on the the usage of phage therapy and genetically engineered bateriocins are also being considered as potential antimicrobial agents [25, 26]. Antimicrobial peptides (AMPs) owing to their highly potent nature have been used in recent decades for the prevention and treatment of large numbers of plant diseases. Several reports showing the effective use of AMPs have been published either in the form of spray peptides or through generation of transgenic expressing natural and/or designed AMPs.

An Approach Towards Structure Based Antimicrobial Peptide Design

2. ANTIMICROBIAL PEPTIDES (AMPs) Antimicrobial peptides (AMPs), also called host defense peptides (HDPs), are highly diverse group of biologically active molecules which have been found and characterized in almost all forms of life including vertebrates, invertebrates, and plants [27]; where they form an evolutionarily conserved component of innate immune system [28] and help provide defense against the majority of pathogenic species [29, 30]. Most AMPs are gene encoded and can be of either ribosomal or non-ribosomal origin having a wide range of structures [31]. They share many important characteristics including short sizes ranging up to 50 amino acids, being mostly cationic in nature and showing heat stability [32]. A large number of AMPs occur naturally and many have been synthesized through rational designing. They possess antimicrobial activity against an extensive range of pathogenic organisms that include bacteria, viruses, fungi, protozoa, while some even show toxicity towards tumour cells [33]. Several AMPs have also been found to possess good immunemodulatory activity and thus help to maintain the immune response [34]. Unique characteristics including low cytotoxicity towards host organism, high specificity, and unique mode of action/s make AMPs amazingly different from that of the conventional antibiotics [32]. Their primary mode of action involves interaction with microbial membrane, mainly through electrostatic interaction, that culminates in generating physical damage to the membrane and further downstream effects. This membrane permeabilizing mode of activity makes resistance development against AMPs less likely as cell membrane is a highly conserved structural component of cell which cannot be changed or altered easily [35]. This is the most striking difference of AMPs from antibiotics, which are mostly specific in their action and target a definite cell module, thus giving the pathogens a chance to become resistant through slight mutational and functional changes [36]. Moreover, eukaryotic cells with a different cell membrane makeup comprising of cholesterol and lesser negative charge usually remains unaffected [37]. AMPs are thus looked upon as a new generation of biological molecules having huge potential application in plant disease prevention and control [38]. AMPs owe their net positive charge to the presence of high proportion of positively charged amino acids like lysine, arginine, and/or in some cases histidine (though negatively charged AMPs also exist) [39], and adopt an amphipathic structure with segregation of hydrophobic and hydrophilic amino acid residues allowing them to interact with the negatively charged lipid bilayer membrane. However, even with

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all these similarities, the secondary structure of AMPs is not conserved. A large number of different secondary conformations exist and this forms the basis of their classification. Such varied structural forms of AMPs play a very important role in providing them with a broad range of activity [40]. On the basis of their secondary structure, four major groups of AMPs have been recognised. These include α-helices, β-sheets, extended, and looped peptides [47]. There also occur certain αβ peptides [48] and a large majority of unclassified peptides. Most naturally occurring AMPs are linear, however, polycyclic peptides like lantibiotics occur in bacteria and circular peptides like cyclotides from plants, bacteriocins from bacteria, and defensin (theta) from animals also exist. Structural insights into these AMPs using NMR spectroscopy have shown that there exists a large correlation between the structure and biological properties of different classes of AMPs, a fact which is also being utilised for designing of newer peptide-based antibiotics [49]. 2.1. AMPs from Plant Sources Upon encountering phytopathogens, plants upregulate various set of genes involved in systemic resistance development [50]. Majority of the compounds involved in resistance development includes secondary metabolites like phenolic compounds, phytoalexins, and PRproteins, etc. Pathogenesis-related (or PR-) proteins are one class of AMPs that have gained immense importance due to their large diversity including as many as 17 different families possessing diverse immune defense functions [51]. Plant AMPs are finely tuned being either constitutively expressed or induced when faced with challenging situations. A large collection of AMPs mainly active against phytopathogens are synthesized and used by plants (Fig. 1) and are mainly found at sites which are highly susceptible to encroaching pathogens like the leaves, flowers, seeds, or pods. Their major role is to protect these very important and vulnerable sites to allow proper development and growth. All the major classes of plant AMPs exhibit antimicrobial activity, though to different extents, while some can also be directed against insects and pests. In addition to the cationic residues, and amphipathic arrangement, the plant AMPs have high percentage of cysteine residues, which are involved in multiple disulfide bond formation and structure stabilisation. Most plant AMPs have a globular β-sheet structure stabilised by disulfide bridges which imparts them a compact


Fig. (1). Three-dimensional model structure of different classes of naturally-occurring plant antimicrobial peptides taken from the RCSB Protein Data Bank. (a) Thionin like peptide corresponding to Viscotoxin A3 from Viscum album L. (PDB ID: 1ED0), (b) Defensin-type peptide corresponding to Vignaradiata Defensin-2 (PDB ID: 1TI5), (c) Nonspecific (ns) LTP corresponding to ns LTP1 from Vigna radiata (PDB ID: 1SIY), (d) Hevein-like peptide from Hevea brasiliensis (PDB ID: 1HEV), (e) Knottin-type peptide corresponding to amaranthus α-amylase inhibitor (AAI) from Amaranthus hypochondriacus (PDB ID: 1QFD), and (e) Cyclotide peptide corresponding to Kalata B1 from Oldenlandia affinis (PDB ID: 2MN1). All chains are held together by intra-strand disulfide bridges, a common feature of plant AMPs.

structure and resistance to various physical and chemical environments, usually encountered by the AMPs in the cell wall and vacuoles respectively [52]. Plant AMPs have mostly been defined on the basis of their sequence, their cysteine residues and hence disulfide patterns. Among the many different AMPs expressed in plants, thionins and defensins are two major and most studied classes as they well represent the forms and

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functions of plant AMPs [53]. Both the classes exhibit strong antibacterial and antifungal activity although they differ in their tertiary structures [54]. Apart from these there exist several other classes of plant AMPs which include lipid transfer proteins (LTPs), snakins, knottins, hevein-like peptides, vicillin like peptides, cyclotides, etc [53]. Other than LTPs, thionins and defensins are the only two classes which have the specific ability to interact with lipids including phospholipid for thionins and sphingolipid for defensins, which is responsible for their biological activity [55]. On the other hand, hevein-like peptides mostly target chitin, while knottin-like petides inhibit proteases [56]. Though LTPs are majorly involved in shuttling lipids between membrane, hydrophobic cavity, a conserved structural feature found in all LTPs, imparts them with antimicrobial potency as well. Membrane mode interaction via the hydrophobic hub causes pore formation and efflux of intracellular ions, causing cell death due to lysis. Additionally, plant LTPs also exhibit synergism with thionins [57]. Similarly, thionins and defensins also have well conserved tertiary structural features including the presence of well-conserved tyrosine residue, which helps them in interacting with membrane, leading to cell death. The mode of action of defensins is still unclear, though receptor-mediated membrane interaction leading to membrane destabilization, disruption, and ion efflux is most common [58]. Apart from these major classes of plant AMPs, there are several other classes of α-hairpin peptides as well as unclassified CRP-AMPs and non-CRP AMPs. In recent years, 2S storage albumins, which are storage proteins important for plant growth and development, have also been shown to possess widespread functional importance. Duan and co-worker reported a 2S albumin, 2S1, from peanut cotyledons, showing antifungal activity against Aspergillus flavus [59] while others have been implicated in plant defense [60]. They have been isolated from seeds of a wide variety of plants and are reported to synergistically increase activity of thionins [61]. Plant AMPs not only help in innate defense response of the body through direct recognition of the pathogen (PAMP-assisted immunity) but they also interact with other molecules, including other AMPs, signalling molecules like MAP kinase, sugars, hormones like salicylic acid and reactive oxygen species (ROS) which are all part of the defense response, thus helping in the signalling cascade leading to effectorassisted immunity [62]. This latter function is mostly mediated by defensins and LTPs as other classes like cyclotides and thionins are known to have toxic proper-

An Approach Towards Structure Based Antimicrobial Peptide Design

ties as well. In addition to their immune defense and antimicrobial activity various classes of plant AMP like cyclotides also possess a wide array of different functional importance including anti-cancer [63], antihypertensive and anti-HIV [64] activity, a reason for their use in generation of various bioactive substances. A classic example of this is provided by various classes of Angiotensin Converting Enzyme (ACE)-inhibitory peptides derived from a variety of plant sources. This class of peptides have proven very successful for the treatment of hypertension. Though majority has been synthesized chemically but increased attention is being paid to search for dietary compounds that can be used as food peptidomics. Among these, plant derived ACEinhibitor peptides like, VY (from oat, and wheat, IC50: 7.1 µM) and GGY (from rice, IC50: 1.3 µM) represent a promising class of molecules [65]. As shown in Table 1, the major classes of plant AMPs, except knottins, have been used for transgenic generation with positive outcomes. However, the problem with such natural products is that they are mostly weak antimicrobial agents and also exhibit limited spectrum of activity. Only transgenic tobacco expressing radish defensin, Rs-AFP2 shows some resistance property toward its host Alternaria longipes [66]. Therefore, emphasis has to be put on designed antimicrobial peptides from diverse sources which help to overcome such limitations. 3. USE OF AMPS FOR PLANT DISEASE MANAGEMENT 3.1. Background Since a long time in history, agricultural scientists have tried to modify crop plants, improving them for cultivation. However, only recently has this field established a much intense and rigorous outlook. The extensive upsurge in disease resistant phytopathogens has led to an increase in the demand for novel and advanced approaches in plant protection. The approach of genetic engineering has made it possible to manipulate and modify genetic makeup of almost all organisms. It offers a very positive approach in which modern technology could be used in the form of a seed, representing a traditional agricultural practice. The first transgenic plant was created less than a few years ago and since then this methodology has been applied to up to 50 plant species [23]. Plant AMPs from various different host plants have been expressed in newer hosts to reduce disease severity as well as to generate disease resistance. Several reports are available for such transgenics showing resistance to bacterial pathogens, while

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less in the case of fungus [67]. Many important plants have been engineered by expression of AMPs (Fig. 2) from varied sources including plants themselves to develop disease resistant transgenic varieties (Table 2). The table depicts a large number of transgenic plants, which shows resistance to bacterial pathogens that have been generated till date using naturally occurring AMPs. However, such studies may be strengthened further using designed peptides that can be modified based on structural information to obtain increased potency and improved stability. The first fungus resistance transgenic plant showing endurance to Rhizotonia solani was developed by constitutive expression of chitinase gene in Nicotiana tabacum and Brasica napus [68]. In the same year, Carmona and co-workers reported Pseudomonas syringae resistant transgenic tobacco plant expressing α-hordothionin gene [69], while Terras and co-worker generated enhanced resistance in tobacco against fungal leaf pathogen Alternaria longipes by expressing radish defensin [70]. More recently, heterologous expression of natural/modified antimicrobial peptides from diverse sources have also largely contributed to the development of disease resistant plants. Plants like tomato [71], tobacco [72] and rice [73] have been subsequently modified to express natural/modified AMPs from animal sources. Animal defensins like Cecropins A and B have been expressed in rice that conferred protection against X. oryzae [74] and Magneporthe grisea [75]. Insect defensins like heliomycin, drosomycin, etc. have been able to provide resistance against B. cinerea, P. syringae and E. caratovora in tobacco [76]. The use of natural or modified AMPs from diverse sources represent an environmentally favourable strategy over chemical microbicidal compounds [72]. Designed, synthetic new molecules, selected via high throughput screening, combinatorial chemistry and data processing are also used in the form of aerosol spray for protecting crop plants [78]. Zietlar and co-workers reported the designing of a library consisting of four groups of peptides, differing in their size and structure. Each group also showed unique site of charged and hydrophobic patches, ensuring different extent of interaction. Several peptides showed potent activity against a stretch of plant pathogens, both bacterial and fungal along with low hemolyitc and phytotoxic activity even at higher concentration. They subsequently went for peptide application via spraying, where the designed peptides remained active even upon direct spraying on surface of plant tissues. Such designed peptides can also be used for the generation of disease resistant transgenic plants. In addition, however, peptide susceptibility to prote-


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Table 1. Major classes of naturally occurring plant antimicrobial peptides. Class

Amino acid length

Structural features

No. of disulfide bonds

Occurrence and distribution

Antimicrobial activity

Molecular breeding

Thionin

~ 45 to 47

Characterized by a global gamma fold, having a long arm formed by two antiparallel αhelices, and the short arm formed by two short antiparallel β- strand

Two groups consisting of 6 and 8 disulfide bonds respectively

Expressed in various different by organ-specific gene/s

Inhibits both bacteria and fungi except some gram negatives like Pseudomonas and Erwinia

Arabidopsis Thi2.1 has been used for creating transgenic tomato showing resistance to Fusarium wilt and bacterial wilt [41]

Defensin

~45 to 54

Characterized by triple stranded antiparallel β-sheet and a single αhelix lying paralle to it forming a βαββ motiff

Typically 4

Mostly in seeds but can occur in other tissues as well

Mostly antifungal but few are active against bacterial strains as well

Dm-AMP1 and RsAFP2 fusion peptide to create transgenic rice resistance to Magnaporthe oryzae and Rhizoctonia solani [42]

Non-specific LTPs

LTP1: ~90-95

A bundle of 4 αhelices linked through flexible loops, forming a hydrophobic cavity

Typically 4

Expressed in multiple plant organs, especially, leaves, stems, embryos, etc.

Activity varies between nsLTPs depending on the plant species. Mostly antifungal but few show antibacterial activity

Constitutive expression of CALTP I & CALTP II genes in transgenic tobacco generated resistance to Phytophthora nicotianae and Pseudomonas syringae pv. Tabaci[43]

Snakins

~63

No 3-dimensional structure elucidated. Snakin1 predicted to have 2 α-helices and a short small helix

Typically 6

Multiple sites of occurrence

Active against some bacterial and multiple fungal strains

Transgenic potato expressing high levels of StSN1 mRNA exhibited significant resistance to Rhizoctonia solani and Erwinia carotovora infection[44]

Hevein-like

~40

Mostly dominated by triple stranded β-sheet and a short single α-helix connecting the βstrand (strand 2 to 3)

Between 3-4

Mostly in leaves and stem

Shows broad and potent antifungal activity and low anti-gram negatives activity

Pn-AMP2 cDNA has been used to develop transgenic tomato resistant to fungus Phytophthora capsici & Fusarium oxysporum[45]

Knottins