Peptides with antibacterial activity

Abstract

The present invention is directed to a peptide, wherein said peptide has an amino acid sequence according to SEQ ID NO. 1. The present invention also discloses compositions comprising said peptide and specific uses.

Description

[Technical field]

[0001] The present invention relates to the technical field of antimicrobial peptides, more particularly natural plant-derived peptides, which exhibit antimicrobial activity against various bacterial and fungal strains. In particular, the present invention relates to natural, recombinant or synthetic peptides derived from plant vicilin proteins. The present invention further relates to the use of the peptides for the treatment of bacterial and fungal infections in plants, animals and humans. [Background technology]

[0002] Antibiotic resistance is a major problem facing the food, agricultural, medical and veterinary industries, with particular concern being the transfer of potentially lethal antibiotic-resistant bacteria from food-producing animals to human consumers.

[0003] Current methods to control the emergence and spread of antibiotic-resistant bacteria include changes in antibiotic usage and different antibiotic usage patterns, increased government oversight and regulation, and the continued development of new or improved antibiotics. However, most bacteria often thrive on these traditional measures due to their ability to adapt to antibiotic usage and acquire resistance to existing and new antibiotics. Therefore, there is a need to continue to develop alternative methods to control antibiotic resistance in bacteria.

[0004] Antimicrobial peptides (AMPs) are a diverse group of natural compounds present in animals, plants, insects, and microorganisms. These peptides are responsible for the defense against (other) microorganisms and can therefore be further exploited as alternatives to chemical preservatives. However, their use in the treatment of microbial infestations in plants, humans, and / or animals has been limited to date due to their poor availability in nature, high production costs, low stability, and the complexity of their formulation into suitable treatment products. Thus, the use of antimicrobial peptides in crop protection and for therapeutic purposes in human and veterinary medicine is still limited.

[0005] Antimicrobial proteins exhibit various three-dimensional structures that determine to a large extent their activity and stability, and the stability of a particular protein is usually an important parameter for clinical and / or industrial applications.

[0006] The initial interest in plant-derived molecules that are AMPs followed the isolation of the first plant-derived AMP, purothionin. Caleya et al., Appl. Microbiol, 1972, 23(5) 998-100, reported that purothionin, a small protein from wheat and barley flour, is an effective AMP against many plant pathogenic bacteria, such as Pseudomonas solanacearum, Xanthomonas phaseoli and X. campestris, Erwinia amylovora, and five Corynebacterium strains. Since then, several major classes of AMPs have been discovered, including thionins (type IV), defensins, cyclotides, 2S albumin-like proteins, and lipid transfer proteins.

[0007] Plant vicilins are commonly found as a type of plant seed storage protein. It has been found that certain vicilins can be processed to produce plant defense peptides. The best-characterized antimicrobial peptides produced from vicilins are found in Macadamia integrifolia. The kernel of macadamia nut contains a 666 amino acid (aa) vicilin protein, which contains a 212aa highly hydrophilic region adjacent to the N-terminal signal sequence.

[0008] WO 1998 / 027805 discloses a family of vicilin-type peptides with antibacterial properties. The protein prototypes are of natural origin and have been isolated from Macadamia integrifolia and from other species, including Theobroma cacao. In particular, two sequences, designated 47-aa TcAMP1 (Theobroma cacao antibacterial protein 1) and 60-aa TcAMP2 (Theobroma cacao antibacterial protein 2), were derived from the 556-aa encoding coco vicilin seed storage protein gene sequence and recombinantly expressed in Escherichia coli. However, the isolated recombinant peptides and compositions prepared with the recombinant peptides only had effective antibacterial properties at high doses (5-20 μg / ml).

[0009] Marcus et al., Plant. Mol. Biol Rep (2008) 26, 75-87, investigated three Macadamia integrifolia peptide sequences identified as N-proximal hydrophilic regions of vicilin seed proteins, and two Theobroma cacao peptide sequences. Antibacterial activity of the peptides was predicted based on the presence of a characteristic CXXXC-(10-12)XCXXXC motif in the hydrophilic region proximal to the N-terminus. His-tagged versions of the predicted peptides were expressed in Escherichia coli. The resulting recombinant peptides showed antibacterial activity in vitro against six plant pathogen strains, with effective doses very high, ranging from 9.2 to 64 μg / ml, and even higher for some pathogens.

[0010] Ecuador is the most important producer of fine-flavored cocoa, accounting for approximately 50% of the world's production. The fine-flavored cocoa varieties produced in Ecuador mainly belong to the Nacional (often called National or Arriba) variety. In addition to the aromatic and traditional Nacional cocoa, a cocoa clone called CCN-51 has been cultivated in Ecuador since the 1960s. Unlike Nacional, CCN-51 has a weaker aroma and is therefore considered a bulk cocoa type. However, CCN-51 is more tolerant to changes in climatic conditions, is resistant to various pathogens, and produces higher yields than other cocoa varieties. Therefore, the CCN-51 hybrid is very popular among farmers in Ecuador. US Patent No. 2004 / 0172683 describes polypeptides from the seeds of the cocoa bean that are responsible for the cocoa flavor.

[0011] The present invention therefore describes a novel peptide derived from cocoa that is highly effective and highly active against a wide range of animal and plant pathogenic microorganisms, which is safe to use and preferably does not contain aggressive chemicals that are toxic and harmful to the environment. Summary of the Invention

[0012] The present invention and each of its embodiments contribute to providing a solution to one or more of the abovementioned disadvantages. To this end, the present invention relates to a peptide with antibacterial activity according to claim 1. More specifically, a peptide is provided having a sequence according to SEQ ID NO.1. This peptide is obtained from Theobroma cacao or is recombinantly or synthetically produced and has surprisingly been found to have antibacterial and / or antifungal activity against a wide range of pathogens. Preferred embodiments of this peptide are presented in any of claims 2 to 9.

[0013] In a second aspect, the present invention relates to a composition comprising a peptide according to claim 10. A preferred embodiment of this composition is presented in claim 11.

[0014] In a third aspect, the present invention relates to the use of a peptide for therapeutic purposes according to claim 12. Preferred embodiments of this use are given in any of claims 13 to 17. More particularly, the peptide is used for treating Gram-positive, Gram-negative and / or fungal infections in humans, animals or plants, said fungi being preferably hyphal fungi or yeasts.

[0015] In a fourth aspect, the present invention relates to a method for obtaining the peptide according to claim 18.

[0016] In a fifth aspect, the present invention relates to a vector comprising the sequence of said peptide and allowing the expression of said peptide according to claim 19, to a transgenic plant according to claim 20 and to a method for obtaining a transgenic plant according to claim 21.

[0017] In a final aspect, the present invention relates to seeds coated with the peptide according to claim 22. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 shows a representative scheme of detection and sequence location of peptide mass fingerprinting fragments determined by MALDI-ToF analysis. The underlined amino acid sequence was identified as the tryptic digestion product of the peptide according to SEQ ID NO. 1 obtained from an SDS polyacrylamide gel. [Diagram 2] Figure 2 shows the LC-MS results of samples containing the peptide according to SEQ ID NO. 1. The mass of the detected peptide was 8.3 kDa in both the non-reduced (Figure 2A) and reduced (Figure 2B) samples. Detailed Description of the Invention

[0019] The present invention relates to a peptide or a composition comprising said peptide, as an active ingredient for medical or pharmaceutical use, the peptide having an amino acid sequence according to SEQ ID NO.1.Furthermore, the present invention relates to the use of the peptide, a method for obtaining the peptide and a vector allowing the expression of the peptide.

[0020] Unless otherwise defined, all terms used in the description of the present invention, including technical and scientific terms, have the meanings commonly understood by one of ordinary skill in the art to which this invention belongs. As a further guide, definitions of terms are included to better understand the teachings of the present invention.

[0021] As used herein, the following terms have the following meanings.

[0022] As used herein, "a," "an," and "the" refer to singular and plural referents unless the context clearly indicates otherwise. By way of example, "a compartment" refers to one or more compartments.

[0023] As used herein, "about" is used in reference to a measurable value such as a parameter, amount, time duration, etc., and is meant to encompass, as appropriate for the practice of the present invention, a variation of the stated value of ±20% or less, preferably ±10% or less, more preferably ±5% or less, even more preferably ±1% or less, and even more preferably ±0.1% or less, however, it should be understood that the value to which the modifier "about" refers is itself specifically disclosed.

[0024] As used herein, "comprise," "comprising," and "comprises" as well as "comprised of" are synonymous with "include," "including," "includes," or "contain," "containing," or "contains," and are inclusive or open-ended terms that specify the presence of subsequent components, e.g., but do not exclude or preclude the presence of additional, unrecited components, features, elements, members, or steps that are known or disclosed in the art.

[0025] Moreover, in this specification and claims, the terms first, second, third, etc., unless otherwise specified, are used to distinguish between similar elements and not necessarily to describe a sequential or chronological order. The terms so used may be used interchangeably under appropriate circumstances, with the understanding that the embodiments of the invention described herein are capable of operating in sequences other than those described or illustrated herein.

[0026] The recitation of numerical ranges by endpoints includes all values ​​and fractions subsumed within that range, as well as the recited endpoints.

[0027] The terms "% by weight," "weight percent," "%wt," or "wt%" throughout this specification, unless otherwise defined, refer to the relative weight of each component based on the weight of the total formulation.

[0028] The term "one or more" or "at least one," e.g., one or more elements in a group of elements, or at least one element, is itself explicit, and by way of further illustration, the term specifically encompasses reference to any one of the elements, or any two or more of the elements, e.g., any three or more, four or more, five or more, six or more, seven or more, etc., of the elements, up to all of the elements.

[0029] The term "peptide" as used herein refers to a compound containing two or more amino acid residues joined by an amide bond formed between the carboxyl group from one amino acid residue and the amino group from an adjacent amino acid residue. The amino acid residues may be in the D- or L-form, naturally occurring or synthetic, and linear or cyclic.

[0030] The term "therapeutically effective amount" refers to an amount effective to ameliorate any symptom of a disease. Since prevention can be considered as treatment, a therapeutically effective amount can also be referred to as a "prophylactically effective amount."

[0031] Unless otherwise defined, all terms used in the disclosure of the present invention, including technical and scientific terms, have the meanings commonly understood by those skilled in the art to which the present invention belongs. As a further guide, definitions of terms used herein are included to better understand the teachings of the present invention. Terms or definitions used herein are provided solely to aid in the understanding of the present invention.

[0032] Throughout this specification, references to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the present invention. Thus, the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification may refer to the same embodiment, although not necessarily all of them. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments, as would be apparent to one of ordinary skill in the art from this disclosure. Furthermore, although some embodiments described herein include some features and do not include other features included in other embodiments, it is meant that combinations of features of different embodiments are within the scope of the present invention and form different embodiments, as would be understood by one of ordinary skill in the art. For example, in the following claims, any of the claimed embodiments may be used in any combination.

[0033] In a first aspect, the present invention relates to a peptide, said peptide having an amino acid sequence according to SEQ ID NO.1.

[0034] In another embodiment, peptides having an amino acid sequence that exhibits greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, greater than 99.5% sequence identity with SEQ ID NO.1 and having antibacterial activity as observed in peptides having a sequence according to SEQ ID NO.1 are also disclosed herein.

[0035] The term "sequence identity" as used herein refers to the amino acid-by-amino acid sequence identity over a window of comparison. Thus, "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over a window of comparison to determine the number of amino acid positions that match in both sequences, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the size of the window), and multiplying the result by 100 to obtain the percentage of sequence identity. Gaps, i.e., positions in the alignment where a residue is present in one sequence but not in the other, are considered to be positions of non-identical residues. The determination of percentage of sequence identity can be performed manually or with the aid of computer programs available in the art. An example of a useful algorithm is PILEUP. Software for performing BLAST analysis is publicly available at the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0036] In another embodiment, the peptide differs from the sequence of SEQ ID NO.1 by up to four, even more preferably three, even more preferably two, and even more preferably one amino acid residue and exhibits the antibacterial activity disclosed herein.

[0037] The amino acid sequence variants of the peptides contemplated herein can be substitution, insertion, or deletion variants. Deletion variants lack one or more residues in the peptide that may not be important for function. Substitution variants generally contain alternative amino acids at one or more sites in the peptide and can be designed to adjust one or more properties of the polypeptide, such as stability against proteolytic cleavage. Substitutions are preferably conservative, i.e., an amino acid is replaced with an amino acid of similar size and similar side chain or similar substituent. Conservative substitutions are known in the art and include, for example, an alanine to glycine, valine, or leucine; an arginine to lysine; an asparagine to glutamine; a cysteine ​​to methionine; a glutamine to asparagine; a glutamic acid to aspartic acid; a glycine to proline; a histidine to glutamine, tyrosine, arginine, lysine, asparagine, or cysteine; an isoleucine to leucine or valine; a leucine to valine or isoleucine; a lysine to arginine; a phenylalanine to tyrosine, leucine, or methionine; a serine to threonine; a threonine to serine; a tryptophan to phenylalanine; a tyrosine to tryptophan or phenylalanine; a valine to isoleucine or leucine, and the like.

[0038] Preferably, the peptide of the present invention is derived from or isolated from Theobroma cacao. More particularly, the peptide of the present invention is derived from the N-terminal region of the vicilin protein of Theobroma cacao. Vicilins are storage proteins present and characterized in several plant species, including, but not limited to, Ananas comosus, Arachis hypogaea, Beta vulgaris, Capsicum annuum, Capsicum chinense, Carya illinoinensis, Chenopodium quinoa, Corchorus olitorius, Cucurbita maxima, Fragaria vesca, Glycine max, Gossipum arboreum, Herrania umbratica, Hordeum vulgare, Jatropha curcas, Juglans regia, Macadamia integrifolia, Macleaya cordata, Musa acuminata, Papaver somniferum, Ricinus communis, Solanum lycopersicum, Spinacia oleracea, Stenocarpus sinuatus, Theobroma cacao, and Zea mays. Such proteins typically contain highly hydrophilic N-proximal and C-terminal regions. In addition, they have a hydrophobic N-terminal signal sequence, which is usually removed during protein maturation. The N-proximal region of the precursor protein is of particular interest because it contains at least two, and preferably four, pairs of cysteine ​​motifs (CXXXC) in an evenly spaced pattern.

[0039] The peptides according to the invention were identified in experiments with recombinant peptides derived from vicilin from Theobroma cocoa, and without wishing to be bound by theory, it is hypothesized that the peptides are degradation products of one of these recombinant peptides.

[0040] The peptides described herein can be obtained by recombinant expression, but can also be purified from Theobroma cacao cultivars, more preferably from the CCN-51 cultivar. Surprisingly, the cocoa cultivar CCN-51 or "lavados", known to be highly resistant to microbial invasion, has been shown to be particularly rich in vicilin-derived N-terminal peptides.

[0041] In another or further embodiment, the peptide comprises a signal peptide at its N-terminus. Preferably, the signal peptide has a sequence according to SEQ ID NO.2. In another embodiment, the signal peptide exhibits 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or more preferably 100% sequence identity to SEQ ID NO.2. Alternatively, the peptide has a signal peptide sequence that differs from the sequence SEQ ID NO.2 by at most three, more preferably at most two, and even more preferably by one amino acid residue.

[0042] In one embodiment, a peptide of the invention including a signal peptide has a sequence according to SEQ ID NO.3.

[0043] In another embodiment, the peptide is a recombinant or synthetic peptide. Insertion of a vector containing the coding sequence of the peptide having SEQ ID NO.1 into a suitable expression system, such as E. coli or another suitable expression system known in the art, results in the peptide. Any suitable protein / peptide synthesis method known in the art can be employed to synthesize the peptides of the present invention.

[0044] In further or alternative embodiments, the peptides described herein comprise an affinity tag at their N-terminus and / or C-terminus for use in affinity purification of recombinant proteins and peptides expressed in E. coli and other systems. The tag can be a polyhistidine tag, an amino acid motif consisting of at least six histidine (His) residues.

[0045] Alternatively, but not limited to, the peptide may be an HQ tag (HQHQHQ) in which histidine and glutamine residues are alternated, an HN tag (HNHNHNHNHNHN) in which histidine and aspartate are alternated, a HAT tag (KDHLIHNVHKEEHAHAHNK), an ALFA tag (SRLEEELRRRLTE), an Avi tag (GLNDIFEAQKIEWHE), a C tag (EPEA), a calmodulin tag (KRRWKKNFIAVSAANRFKKISSSGAL), a poly(Asp) tag (PAT) tag (PAT), a β-glutamine ... Tagging can be achieved with a glutamic acid tag, polyarginine tag, E tag (GAPVPYPDPLEPR), FLAG tag (DYKDDDDK), HA tag (YPYDVPDYA), Myc tag (EQKLISEEDL), NE tag (TKENPRSNQEESYDDNES), Rho1D4 tag (TETSQVAPA), S tag, SBP tag (DEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP), Strep tag (WSHPQFEK), etc.

[0046] Analysis of the peptides of the present invention has shown that they have antibacterial and / or antifungal activity.

[0047] The antimicrobial peptides themselves have a specific three-dimensional structure, which can be determined using X-ray crystallography or nuclear magnetic resonance spectroscopy techniques. Without wishing to be bound by theory, it is believed that this structure plays a role in the activity observed in the peptides. The α-helical plant-derived antimicrobial peptides (AMPs), to which the peptides of the present invention belong, often have an amphipathic helix, with one face of the helix being predominantly hydrophilic and the other face being predominantly hydrophobic. This structure is common in AMPs that disrupt cell membranes, causing leakage of cellular contents and lysis, as well as AMPs that enter cells and attack other structures within the cell. α-helical AMPs are usually structurally disordered in solution, which allows them to easily pass through the dense meshwork of cell walls. Upon binding to cell membranes that are hydrophobic and charged below the lipid heads, a secondary structure (α-helix) is formed, allowing the protein to penetrate the cell membrane. This order / disorder transition is mainly controlled by the length of the hydrophobic region in the α-helix. If the hydrophobic helix is ​​too long, it will form ordered structures in solution, adversely affecting the activity of the AMP.

[0048] In certain embodiments, the peptides are active against gram-positive and gram-negative bacteria and fungi, preferably mycelial fungi and yeasts. Without being limited thereto, the peptides are active against bacteria selected from the group including: Acinetobacter, Bartonella, Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia, Clostridium, Corynebacterium, Enterococcus, Enterobacter, Erwinia, Escherichia, Francisella, Hae mophilus, Helicobacter, Klebsiella, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Treponem A, Ureaplasma, Vibrio, Yersinia, Acidovorax, Agrobacterium, Arthrobacter, Bacillus, Burkholderia, Clavibacter, Cronobacter, Curtobacterium, Refisonia, Pantoea, Paenibacillus, Pecto Bacterium, Phytoplasma, Proteus, Pseudomonas, Ralstonia, Rhizobacter, Rhizomonas, Rhodococcus, Serratia, Sphingomonas, Spiroplasma, Streptomyces, Xanthomonas, Xylella, Xylophilus, etc.

[0049] Without being limited thereto, the peptides of the present invention are active against fungi selected from the group including: Ajellomyces, Aspergillus, Basidiobolus, Blastomyces, Candida, Coccidioides, Conidiobolus, Cryptococcus, Emmonsia, Histoplasma, Hanseniaspora, Lacazia, Paracoccidioides, Pneumocystis, Sporothri x species, Stachybotrys species, Talaromyces species, Acrocalymma species, Aecidium species, Albonectria species, Allodus species, Alternaria species, Amphobotrys species, Apiosporina species, Armillaria species, Blumeria species, Botryotinia species, Botrytis species, Ceratosystis species, Colletotrichum species, Cry ptosporiopsis, Exobasidium, Fusarium, Hypocrea, Leptosphaeria, Magnaporthe, Melampsora, Meyerozyma, Monilinia, Mycospharella, Microsphaera, Mucor, Penicillium, Pichia, Phytophtora, Saccharomy ces, Sporobolomyces, Plasmodiophora, Podosphaera, Puccinia, Pythium, Rhizoctonia, Sclerotinia, Septoria, Taphrina, Thanatephorus, Torulaspora, Uromyces, Ustilago, Venturia, Verticillium, etc.

[0050] In a second aspect, the present invention provides a composition comprising a peptide as described in the above paragraph, said composition being particularly suitable for pharmaceutical and veterinary applications and / or for crop protection applications.

[0051] In some embodiments, the composition is a liquid, semi-solid, solid, or gaseous composition, and / or the composition is in the form of a tablet, capsule, powder, granule, aerosol, paste, syrup, suspension, emulsion, or solution. Non-limiting examples of the composition are soluble powders (SP), soluble granules (SG), wettable granules, tablet formulations, dry flowables, aqueous flowables, water dispersible granules, oil dispersions (OD), suspension concentrates, dispersible concentrates (DC), emulsifiable concentrates, aqueous suspensions, fertilizer granules, sprayable compositions, and the like. The composition can be formulated for oral, injectable, intravenous, intramuscular, dermal, inhalation, topical, or nasal administration. In other embodiments, the composition can be formulated for coating, spraying, spray coating, evaporation, nebulizing, atomizing, suspension, dilution, and the like, of one or more objects.

[0052] In some embodiments, the composition comprises a pharma- ceutically acceptable carrier, excipient, or diluent. Depending on preference, the excipient may be selected from fillers, binders, disintegrants, sweeteners, coating agents, lubricants, and / or glidants. Diluents or fillers can increase the bulk of a solid composition and make dosage forms containing the composition easier for patients and caregivers to handle. Suitable diluents for tablets according to the present invention include, for example, microcrystalline cellulose (e.g., Avicel®), microfine cellulose, lactose, starch, pregelatinized starch, calcium carbonate, calcium sulfate, sugar, dextrates, dextrin, dextrose, dibasic calcium phosphate dihydrate, tricalcium phosphate, kaolin, magnesium carbonate, magnesium oxide, maltodextrin, mannitol, polymethacrylates (e.g., Eudragit®), potassium chloride, powdered cellulose, sodium chloride, sorbitol, talc, and the like.

[0053] Solid compositions to be compressed into tablets or the like may contain excipients. Excipients have the function of helping to hold the active ingredient and other excipients together after compression. Suitable binders include acacia, alginic acid, carbomers (e.g., Carbopol), sodium carboxymethylcellulose, dextrin, ethylcellulose, gelatin, guar gum, hydrogenated vegetable oils, hydroxyethylcellulose (e.g., Klucel®), hydroxypropylcellulose (e.g., Methocel®), liquid glucose, magnesium aluminum silicate, maltodextrin, methylcellulose, polymethacrylates, povidone (e.g., Kollidon®, Plasdone®), pregelatinized starch, sodium alginate, starch, and the like.

[0054] Disintegrants can be added to the composition to increase the dissolution rate of the compacted solid composition.Suitable disintegrants include alginic acid, carboxymethylcellulose calcium, carboxymethylcellulose sodium (e.g., Ac-Di-Sol®, Primellose®), colloidal silicon dioxide, croscarmellose sodium, crospovidone (e.g., Kollidon®, Polyplasdone®), guar gum, magnesium aluminum silicate, methylcellulose, microcrystalline cellulose, polacrilin potassium, powdered cellulose, pregelatinized starch, sodium alginate, sodium starch glycolate (e.g., Explotab®), starch, etc.

[0055] The addition of a lubricant can improve the flowability of a non-compacted solid composition and increase the accuracy of dosing. Excipients that function as lubricants include colloidal silicon dioxide, magnesium trisilicate, magnesium stearate, powdered cellulose, starch, talc, tricalcium phosphate, etc.

[0056] When a dosage form such as a tablet is produced by compression of a powder composition, the composition is subjected to pressure from a punch and die. Some excipients and active ingredients tend to adhere to the punch and die, which can result in pitting and surface irregularities in the product. A lubricant can be added to the composition to reduce adhesion and facilitate release of the product from the die. Lubricants include magnesium stearate, calcium stearate, glyceryl monostearate, glyceryl palmitostearate, hydrogenated castor oil, hydrogenated vegetable oil, mineral oil, polyethylene glycol, sodium benzoate, sodium lauryl sulfate, sodium stearyl fumarate, stearic acid, talc, zinc stearate, and the like. Preferably, the lubricant is present at 0.25-1% by weight (w / w).

[0057] Flavoring agents and flavor enhancers make the dosage form more palatable to the patient. Common flavoring agents and flavor enhancers for pharmaceuticals that can be included in the compositions disclosed herein include maltol, vanillin, ethyl vanillin, menthol, citric acid, fumaric acid, ethyl maltol, tartaric acid, etc.

[0058] The solid composition may also be dyed using any pharma- ceutically acceptable coloring agent to improve its appearance and / or to facilitate product and unit dosage identification.

[0059] In some embodiments, particularly when the composition is for agricultural use, the excipient is an agriculturally compatible excipient. The "agriculturally compatible carrier" or "agriculturally compatible excipient" can be considered a solvent and is generally inert, but must be agriculturally acceptable. Thus, the phrase "agriculturally compatible" refers to a substance that can be routinely used in cultivated areas without interfering with the grower's planting equipment and without adversely affecting the development of the crop or the desired ecological balance in the cultivated area.

[0060] The agriculturally compatible carrier or excipient may be a solid. The solid carrier or excipient may include, but is not limited to, clay, natural or synthetic silicates, silica, resins, waxes, solid fertilizers, polymers, granular mass, perlite, perlite granules, peat, peat pellets, soil, vermiculite, charcoal, carbonated press mud of sugar mills, rice husk, carboxymethylcellulose, fine sand, calcium carbonate, flour, alum, starch, talc, polyvinylpyrrolidone, or combinations thereof. The agriculturally compatible carrier or excipient may be a liquid. The liquid carrier or excipient may include, but is not limited to, water, alcohols, ketones, petroleum fractions, oils, aromatic or paraffinic hydrocarbons, chlorinated hydrocarbons, liquefied gases, or combinations thereof. More specifically, the agriculturally compatible carrier or excipient may include dispersants, surfactants, additives, thickeners, anti-caking agents, residue breakers, composting formulations, granular application agents, diatomaceous earth, colorants, stabilizers, preservatives, polymers, coatings, or combinations thereof. One skilled in the art can easily determine the appropriate carrier or excipient to be used, taking into consideration factors such as the particular compound, the plant to which the inoculant is applied, the type of soil, the weather conditions, whether the inoculant is in liquid, solid, or powder form, and the like. The additive may include oils, gums, resins, clays, polyoxyethylene glycols, terpenes, viscous organics, fatty acid esters, sulfated alcohols, alkyl sulfonates, petroleum sulfonates, alcohol sulfates, sodium alkylbutane diamino acids, polyesters of sodium thiobutan dioate, benzeneacetonitrile derivatives, proteinaceous materials, or combinations thereof. Proteinaceous materials include dairy products, wheat flour, soybean meal, blood, albumin, gelatin, or combinations thereof. The thickening agent may include a long chain alkyl sulfonate of polyethylene glycol, polyoxyethylene oleate, or a combination thereof.The surfactants may include heavy petroleum oils, heavy petroleum distillates, polyol fatty acid esters, polyethoxylated fatty acid esters, aryl alkyl polyoxyethylene glycols, alkyl amine acetates, alkyl aryl sulfonates, polyhydric alcohols, alkyl phosphates, or combinations thereof. Anti-caking agents include sodium sulfite, sodium sulfate, sodium salts such as monomethyl naphthalene sulfonate sodium salt, or combinations thereof; calcium salts such as calcium carbonate, diatomaceous earth, or combinations thereof, and the like.

[0061] In some embodiments, the composition further comprises one or more selected from water, other nutritional substances, weak acids, vegetable oils, essential oils, metabolic enhancers, emulsifiers, viscosity agents, colorants, suspending agents, dispersing agents, preservatives, complexing agents, stabilizers, carriers, solvents or wetting agents, or any combination thereof. In some embodiments, the composition further comprises at least one oil, surfactant, and polymer.

[0062] The composition can be prepared by conventional methods.In certain embodiments, the composition provided herein comprises one or more preservatives that inhibit microbial activity.Suitable preservatives include mercury-containing substances such as merphen and thiomersal; stabilized chlorine dioxide; and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide and cetylpyridinium chloride.

[0063] The peptides described herein may be administered at a concentration of 0.1 to 50 μg / ml, preferably 0.1 to 45 μg / ml, preferably 0.1 to 40 μg / ml, preferably 0.1 to 35 μg / ml, preferably 0.1 to 30 μg / ml, preferably 0.1 to 25 μg / ml, preferably 0.1 to 20 μg / ml, preferably 0.1 to 15 μg / ml, preferably 0.1 to 10 μg / ml, preferably 0.1 to 9 μg / ml, preferably 0.1 to 8 μg / ml, preferably 0.1 to 7 μg / ml, preferably 0.1 It can be incorporated in the composition in an amount of 0.1 to 6 μg / ml, preferably 0.1 to 5 μg / ml, preferably 0.1 to 4 μg / ml, preferably 0.1 to 3 μg / ml, preferably 0.1 to 2 μg / ml, preferably 0.1 to 1 μg / ml, preferably 0.1 to 0.9 μg / ml, preferably 0.1 to 0.8 μg / ml, preferably 0.1 to 0.7 μg / ml, preferably 0.1 to 0.6 μg / ml, preferably 0.1 to 0.5 μg / ml, or preferably 0.1 to 0.4 μg / ml.

[0064] Alternatively, the peptide is administered at a concentration of 0.2 to 50 μg / ml, preferably 0.3 to 50 μg / ml, preferably 0.4 to 50 μg / ml, preferably 0.5 to 50 μg / ml, preferably 0.6 to 50 μg / ml, preferably 0.7 to 50 μg / ml, preferably 0.8 to 50 μg / ml, preferably 0.9 to 50 μg / ml, preferably 1 to 50 μg / ml, preferably 2 to 50 μg / ml, preferably 3 to 50 μg / ml, preferably 4 to 50 μg / ml, preferably 5 to 50 μg / ml. The amount of the compound can be blended in the composition in an amount of 0.5 to 20 μg / ml, preferably 25 to 50 μg / ml, preferably 30 to 50 μg / ml, preferably 35 to 50 μg / ml, preferably 40 to 50 μg / ml, and preferably 45 to 50 μg / ml.

[0065] In another embodiment, the composition comprises 0.1% or more by weight of the peptide, more preferably 0.5% or more, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, 20% or more, 21% or more, 22% or more by weight. Above, 23 weight% or more, 24 weight% or more, 25 weight% or more, 26 weight% or more, 27 weight% or more, 28 weight% or more, 29 weight% or more, 30 weight% or more, 31 weight% or more, 32 weight% or more, 33 weight% or more, 34 weight% or more, 35 weight% or more, 36 weight% or more, 37 weight% or more, 38 weight% or more, 39 weight% or more, 40 weight% or more, 41 weight% or more, 42 weight% or more, 43 weight% or more, 44 weight% or more, 45 weight% or more, 46 weight% or more, 47 weight% or more, 48 weight% or more, 49 weight% % or more, 50 wt% or more, 51 wt% or more, 52 wt% or more, 53 wt% or more, 54 wt% or more, 55 wt% or more, 56 wt% or more, 57 wt% or more, 58 wt% or more, 59 wt% or more, 60 wt% or more, 61 wt% or more, 62 wt% 63 weight% or more, 64 weight% or more, 65 weight% or more, 66 weight% or more, 67 weight% or more, 68 weight% or more, 69 weight% or more, 70 weight% or more, 71 weight% or more, 72 weight% or more, 73 weight% or more, 74 weight% or more, 75 weight% or more , 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more or 99.5% or more by weight of peptides.

[0066] In a third aspect, the peptides or compositions described herein are suitable for therapeutic use. "Therapeutic use" refers to the use of the peptides or compositions thereof to improve symptoms of disease in humans or non-human animals. In carrying out the methods of treatment or use provided herein, a therapeutically effective amount of the pharmaceutical composition described herein is administered to a subject, such as a mammal or non-mammalian, having a disease, disorder, or condition to be treated. In some embodiments, the subject is a human. The therapeutically effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the therapeutic agent used, and other factors. The therapeutic agents, and optionally compositions, described herein can be used alone or in combination with one or more therapeutic agents, as components of a mixture.

[0067] In some embodiments, the peptides described herein are used in pharmaceutical compositions for the treatment or prevention of infections caused by a wide variety of microorganisms, including gram-positive bacteria, gram-negative bacteria, and / or fungi, in a subject in need thereof, where the fungi are preferably mycelial fungi or yeasts, and the subject can be a human or an animal. The pharmaceutical composition can include a therapeutically effective amount of the antimicrobial peptide and a suitable carrier.

[0068] The peptides or compositions described herein can be administered to a subject by any suitable route of administration, including, but not limited to, intravenous, intraarterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, inhalation, intraperitoneal, etc. Compositions described herein include, but are not limited to, aqueous dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast dissolve formulations, tablets, capsules, pills, delayed release formulations, sustained release formulations, pulsatile release formulations, multiparticulate formulations, and mixed formulations of immediate release and controlled release formulations.

[0069] In some embodiments, the peptides or compositions described herein can be added to animal feed to reduce potential microbial infections in livestock, hi another embodiment, the peptides or compositions described herein can be formulated for oral, topical, or parenteral administration to treat microbial infections in veterinary medicine.

[0070] In another embodiment, the peptides or compositions described herein are suitable for use in crop protection as feed or food additives, for pharmaceutical applications as preservatives, as decontaminants, and / or for cosmetic applications.

[0071] When used for crop protection, the peptides or compositions described herein are formulated with agriculturally compatible carriers or excipients according to the above embodiments. Those skilled in the art will determine the appropriate dose, formulation, and application method of the composition in function of the crop to be treated and the pathogen to be targeted.

[0072] In some embodiments, the peptides or compositions described herein are incorporated into feed or food additives to prevent the spread of pathogens through feed and food. Feed is known to carry pathogens that are harmful to the health and welfare of animals. It is common to disinfect feed using preservative products such as formaldehyde, but these products can have adverse effects. The antimicrobial peptides described herein are a safe alternative.

[0073] In another embodiment, the peptide or composition described herein is suitable for use as a decontaminant or disinfectant for surfaces, tools, instruments, devices, objects, or body parts.In some embodiments, such devices or objects include, but are not limited to, linen, fabric, plastics, latex fabrics, natural rubber, implantable devices, surfaces, or storage containers.In certain embodiments, the peptide or composition disclosed herein is incorporated into cleaning agents, detergents, soaps, or sprays.

[0074] In certain embodiments, the peptides described herein can also be incorporated into various health care products, particularly cosmetics. For example, the peptides can be incorporated into toothpaste, mouthwash, shampoo, soap, cream, or antiperspirant to reduce or prevent microbial colonization or recolonization in the oral cavity or on the skin.

[0075] In another embodiment, the peptides and compositions described herein can be used as food, feed, cosmetic or pharmaceutical preservatives, or in treating foods to control, reduce or remove potential pathogens or contaminants.

[0076] In another embodiment, the peptide or composition described herein is used to control pathogens in plants, preferably crops. Wherein the pathogens are gram-positive and gram-negative bacteria and / or fungi, the fungi are preferably mycelial fungi or yeast. The plants may be monocotyledonous or dicotyledonous plants, including forage or forage legumes, ornamental plants, food crops, trees, or shrubs. In one embodiment, the plants are preferably crops. Preferably, the plants belong to the following groups: Acer, Actinidia, Abelmoschus, Agave sisalana, Agropyron, Agrostis stolonifera, Allium, Amaranthus, Ammophila arenaria, Ananas comosus, Annona, Apium graveolens, Arachis, Artocarpus, Asparagus. officinalis, Avena sp., Averrhoa carambola, Bambusa sp., Benincasa hispida, Bertholletia excelsea, Beta vulgaris, Brassica sp., Cadaba farinosa, Camellia sinensis, Canna indica, Cannabis sativa, Capsicum sp., Carex elata, Carica papaya, Carissa macrocarpa, Carya sp., Carthamus tinctorius, Castanea spp., Ceiba pentandra, Cichorium endivia, Cinnamomum sp., Citrullus lanatus, Citrus sp., Cocos sp., Coffea sp., Colocasia esculenta, Cola sp., Corchorus sp., Coriandrum sativum, Corylus sp., Crataegus sp., Crocus sativus, Cucurbita sp., Cucumis sp., Cynara sp., Daucus carota, Desmodium sp., Dimocarpus longan, Dioscorea spp., Diospyros spp., Echinochloa spp., Elaeis spp., Eleusinecoracana, Eragrostis tef, Erianthus, Eriobotrya japonica, Eucalyptus, Eugenia uniflora, Fagopyrum, Fagus, Festuca arundinacea, Ficus carica, Fortunella, Fragaria, Ginkgo biloba, Glycine, Gossypium hirsutum, Helianthus, Hemerocallis fulva, Hibiscus, Hordeum, Ipomoea batatas, Juglans, Lactuca sativa, Lathyrus, Lens culinaris, Linum usitatissimum, Litchi chinensis, Lotus, Luffa acutangula, Lupinus, Luzula sylvatica, Lycopersicon, Macrotyloma, Malus, Malpighia emarginata, Mammea americana, Mangifera indica, Manihot, Manilkara zapota, Medicago sativa, Melilotus, Mentha, Miscanthus sinensis, Momordica, Morus nigra, Musa, Nicotiana, Olea, Opuntia, Ornithopus, Oryza, Panicum miliaceum, Panicum virgatum, Passiflora edulis, Pastinaca sativa, Pennisetum, Persea, Petroselinum crispum, Phalaris arundinacea, Phaseolus, Phleum pratense, Phoenix, Phragmites australis, Physalis, Pinus, Pistacia vera, Pisum, Poa, Populus, Prosopis, Prunus, Psidium, Punica granatum, Pyrus communis, Quercus, Raphanus sativus, RheumThe genera include: rhabarbarum, Ribes, Ricinus communis, Rubus, Saccharum, Salix, Sambucus, Secale cereale, Sesamum, Sinapis, Solanum, Sorghum bicolor, Spinacia, Syzygium, Tagetes, Tamarindus indica, Theobroma cacao, Trifolium, Tripsacum dactyloides, Triticosecale rimpaui, Triticum, Tropaeolum minus, Tropaeolum majus, Vaccinium, Vicia, Vigna, Viola odorata, Vitis, Zea mays, Zizania palustris, Ziziphus, and their descendants and hybrids.

[0077] The peptides or compositions described herein are suitable for introduction into a plant, part of a plant, or a substrate that constitutes or is the host of the plant, thereby conferring pathogen resistance or disease control to the plant, which introduction may be artificial.

[0078] Methods for introducing a peptide or a composition thereof into a plant or the like include treating the plant and / or a part of the plant and / or the growth medium in which the plant is grown, inoculating seeds, coating seeds, directly inoculating the plant or a part of the plant, spraying or wetting the plant or a part of the plant (e.g., panicles), etc. An appropriate method can be selected depending on the type of plant to which the peptide or composition is to be introduced.

[0079] As another non-limiting example, the peptide or composition described herein can be applied in the form of a coating. The coating can be applied to the seed by spraying the seed or by immersing the seed in a solution containing the peptide or composition. In another example, a binder, such as a binder made of carbide (calcium carbonate), can be added to coat the seed with the solution described in the previous example.

[0080] In some embodiments, the coating can be applied to bare, untreated plant parts. In other embodiments, the coating can be applied as an overcoat to previously treated plant parts. Seed coatings are particularly preferred in the treatment of soil-borne fungal diseases. In some embodiments, the seed coating can be applied to bare, untreated seeds. In other embodiments, the seed coating can be applied as a seed overcoat to previously treated seeds.

[0081] In one embodiment, the peptides or compositions described herein can be applied to the soil or any other substrate in which the plant grows, thereby removing pests and / or pathogens from the substrate.

[0082] Inoculation of the substrate comprising the plant or plant part or hosting the plant or plant part can be performed by, by way of example and not by way of limitation, applying a powder, granule, pellet, plug, or soil drench to the substrate. Inoculation can also be performed by liquid application, such as foliar spray or liquid composition. The application can be applied to the growing plant or to the plant substrate. The plant, particularly an agricultural plant, can be grown in a substrate. In one embodiment, the substrate is soil, sand, gravel, polysaccharides, mulch, compost, peat moss, straw, logs, clay, or a combination thereof. In another embodiment, the substrate can also include a hydroculture system or an in vitro culture system. In some embodiments, a combination of different application methods is applied, as described herein.

[0083] In certain embodiments, the peptides or compositions described herein are suitable for use in the treatment or prevention of a pathogenic infection, the pathogen being selected from the group consisting of gram-positive bacteria, gram-negative bacteria, and / or fungi as described in any of the previous embodiments.

[0084] The invention disclosed herein also relates to a plant seed coated with a peptide or composition described in any of the previous embodiments.

[0085] In one embodiment, the methods disclosed herein are used to obtain peptides that exhibit 95% or more sequence identity with SEQ ID NO.1 or SEQ ID NO.2, or that have an amino acid sequence that differs from SEQ ID NO.1 or SEQ ID NO.2 by up to 5 amino acids, where the peptide is a recombinant peptide, a synthetic peptide, or derived from Theobroma cacao, more preferably the CCN-51 variety. These methods are known in the art.

[0086] In certain embodiments, the desired peptide can be dried by lyophilization.

[0087] In some embodiments, the peptides are concentrated. Preferably, the peptides are concentrated by standard salt or organic solvent precipitation. Alternatively, the peptides are concentrated by dialysis against a volatile buffer (e.g., ammonium carbonate), by using a filter-based concentrator, or by ion exchange chromatography.

[0088] Quantification of peptide content is performed by spectrophotometric methods such as the Lowry or Bradford assay, or any other method known in the art. In another embodiment, quantification of peptides is performed by any suitable chromatographic method known in the art. In a preferred embodiment, LC-MS methods are used to quantitate peptide content. In a preferred embodiment, MALDI-TOF analysis is performed on fragments generated via tryptic digestion of proteins obtained from cocoa extracts. In one embodiment, data obtained from MALDI-MS tryptic digest analysis can be further corroborated by preparing and performing immunochemical assays to detect specific differences between samples.

[0089] In a further preferred embodiment, the peptide is substantially free from other peptides. The term "substantially free" as used herein means 95 wt% or more free from other peptides, preferably 99 wt% or more free from other peptides. In a preferred embodiment, the peptide is present in crystalline and / or solid form.

[0090] In another aspect, the present invention relates to a vector comprising a coding sequence for a peptide having a sequence according to SEQ ID NO.1, optionally including a sequence for an N-terminal signal peptide, said signal peptide sequence having a sequence according to SEQ ID NO.2, wherein said vector is designed to allow expression of said peptide in an expression system. In another preferred embodiment, said vector comprises a purification tag as disclosed in the previous embodiment.

[0091] In one embodiment, DNA extracted from cocoa beans is used to amplify the coding sequence of the peptide, which is cloned into an appropriate expression vector, such as any plasmid or virus designed for gene expression in cells.

[0092] Thus, the peptides described herein can be expressed in any suitable system to produce the peptide for further use.Suitable hosts for protein expression include E. coli, fungal cells, insect cells, mammalian cells and plants.Standard methods for protein expression in such hosts are described in various textbooks, including Current Protocols in Molecular Biology (supra) Chapter 16 (Protein Expression).

[0093] Generally, DNA encoding the amino acid sequence of interest is contained in an expression vector, optionally linked in frame at the 5' or 3' end to another coding sequence, such that it encodes a peptide having a sequence according to SEQ ID NO.1, and optionally containing SEQ ID NO.2 at the N-terminus. The entire coding sequence is operably linked to a promoter such that the promoter drives expression of the coding sequence. The coding sequence is also referred to herein as a "target gene."

[0094] In certain embodiments, the promoter is either a promoter native to the microorganism (e.g., E. coli trpE promoter), a synthetic promoter such as the Tac promoter, or a promoter obtained from a bacteriophage, such as phage lambda or T7, that can function in a heterologous organism, e.g., a virus, a bacterium, or a microorganism. The promoter may be constitutive or, more preferably, inducible. The expression vector may also contain a selectable marker gene, which may be an antibiotic resistance gene, such as an ampicillin, tetracycline, chloramphenicol, or kanamycin resistance gene.

[0095] Many promoter systems suitable for expression of the peptides of the invention in E. coli are commercially available. For example, the PBAD promoter from the araBAD (arabinose) operon has favorable inducible properties and is inducible 1,200-fold above background (Guzman et al., 1995). LAC , P TAC , P TRC , PL, PR, etc. TAC The promoter is a hybrid derived from the E. coli trp and lac promoters and is one of the strongest known E. coli-based promoter systems. Like the lac promoter, it is inducible by IPTG.

[0096] A preferred host cell of the present invention is the E. coli M15 host strain. In another preferred embodiment, Pichia pastoris is used as a host cell for the expression of the peptides of the present invention.

[0097] In a further preferred embodiment, clones are selected and the overexpressed peptides are extracted via their affinity tags using Ni-NTA agarose. The recombinant peptides are isolated and purified using the same methods as those applied in the previous embodiment to peptides isolated from natural sources.

[0098] Expression of novel peptides in genetically engineered cells usually results in products with the same three-dimensional structure as the naturally occurring peptides isolated from cocoa plant material. This three-dimensional structure includes correctly formed intramolecular disulfide bonds between cysteine ​​residues. However, even when proteins are chemically synthesized, methods are known in the art for further processing of the proteins to cleave undesired disulfide bonds and form bonds between desired cysteine ​​residues to give the desired three-dimensional structure.

[0099] In another embodiment of the invention, the peptides can be obtained by chemical synthesis using known peptide synthesis techniques, such as solid phase synthesis on instruments such as room temperature peptide synthesizers, microwave peptide synthesizers, parallel peptide synthesizers, etc.

[0100] In one embodiment of the present invention, the peptides described herein can be expressed in transgenic plants. Thus, the present invention also relates to transgenic plants expressing one or more peptides described herein.

[0101] In one embodiment, the transgenic plant expresses a peptide having an amino acid sequence that exhibits 95% or more sequence identity to SEQ ID NO.1 or that differs by at most 3 amino acids from SEQ ID NO.1. In a further embodiment, the peptide comprises an N-terminal signal peptide sequence, wherein the signal peptide sequence has 95% sequence identity to SEQ ID NO.2.

[0102] In further embodiments, the transgenic plant expresses a peptide having 96%, more preferably 97%, more preferably 98%, more preferably 99%, or more preferably 100% identity to SEQ ID NO. 1. In one embodiment, the peptide comprises a purification tag as disclosed in the previous embodiment.

[0103] In some embodiments, the peptide comprises a purification tag as disclosed in the previous embodiment.

[0104] According to the present invention, plant cells can be transformed with DNA constructs by various known genetic engineering methods (Agrobacterium tumefaciens transformation, Ti plasmids, electroporation, microinjection, microprojectile gun, PEG-mediated transformation, etc.) As used herein, a genetic engineering method is to be understood as any method used for the introduction of foreign DNA sequences into plant cells to regenerate transgenic plants expressing a desired trait or characteristic.

[0105] In one embodiment of the present invention, DNA sequences encoding peptides having a sequence according to SEQ ID NO.1, or homologues from other plant species, can be used in combination with DNA sequences encoding preproteins to produce mature proteins. The preproteins contain the native peptide sequence and target the protein to a specific cellular compartment (e.g., apoplast or vacuole). These coding sequences can be ligated to plant promoter sequences ensuring strong expression in plant cells. The promoter sequences can be promoters that ensure strong constitutive expression of the protein in most or all plant cells, promoters that ensure expression in specific tissues or cells susceptible to microbial infection, and promoters that ensure strong induction of expression during the infection process. These types of gene cassettes also contain transcription termination and polyadenylation sequences 3' of the region encoding the antimicrobial protein to ensure efficient production and stabilization of the mRNA encoding the antimicrobial protein. Efficient expression of the antimicrobial peptides disclosed herein can be facilitated by incorporating their individual DNA sequences into sequences encoding much larger peptides.

[0106] The gene cassette encoding the peptide is expressed in plant cells using methods known in the art. First, the gene cassette is ligated into a binary vector that contains: i) left and right border sequences flanking the T-DNA of Agrobacterium tumefaciens Ti plasmid; ii) a suitable selection marker gene for the selection of antibiotic-resistant plant cells; iii) an origin of replication that functions in both Agrobacterium tumefaciens and E. coli; and iv) an antibiotic resistance gene that allows the selection of plasmid-bearing cells of Agrobacterium tumefaciens and E. coli. The DNA sequence of the peptide disclosed herein can be cloned into any binary vector known in the art, including, but not limited to, plasmids (pEXA128, pBR322, pUC19), bacteriophages (lambda phage, M13 phage), cosmids, BACs or YACs. The binary vector carrying the DNA sequence of the peptide can be introduced into Agrobacterium tumefaciens strains carrying a released Ti plasmid, such as LBA4404, GV3101, AGL1, or Agrobacterium rhizogenes strains, such as A4 or NCCP1885, either by heat shock, electroporation, or triparental mating. These Agrobacterium strains can then be co-cultured with appropriate plant excisions or intact plant tissues, and transformed plant cells and / or regenerants can be selected using antibiotic resistance. Alternatively, the binary vector carrying the DNA sequence of the peptide can be overexpressed by bacterial or fungal cells.

[0107] The present disclosure also relates to a method for obtaining a transgenic plant that expresses a peptide having an amino acid sequence that exhibits 95% or more sequence identity with SEQ ID NO.1 or that differs from SEQ ID NO.1 by up to three amino acids, and optionally includes an N-terminal signal peptide sequence, wherein the signal peptide sequence has 95% sequence identity with SEQ ID NO.2.

[0108] The method comprises the steps of: (a) optionally isolating or synthesizing a nucleic acid sequence encoding a peptide having a sequence that exhibits 95% or more sequence identity with SEQ ID NO.1 or that differs from SEQ ID NO.1 by up to three amino acids; and incorporating the nucleic acid sequence into a suitable expression vector; (b) introducing the nucleic acid into at least a plant cell by a genetic engineering method to produce a transformed plant cell expressing the peptide; (c) regenerating the transformed plant cells into whole transgenic plants; and (d) using a suitable screening method to select and identify transgenic plants which express the peptide.

[0109] In another embodiment of the method disclosed herein, the DNA sequence of the peptide is introduced into the plant cell by biological bombardment. Biological bombardment includes using a gene gun to introduce a foreign DNA sequence into the plant cell. The DNA sequence encoding the antimicrobial peptide disclosed herein is attached to a metal particle, such as a gold particle, and then bombarded into the plant cell.

[0110] In yet another embodiment, the DNA sequence of the peptide is introduced into the plant cell by electroporation, which uses an electric pulse to create holes in the cell membrane, allowing the DNA sequence encoding the antimicrobial peptide to enter the cell. Alternatively, the DNA sequence encoding the antimicrobial peptide may be directly injected into the plant cell (microinjection).

[0111] In yet another embodiment, the DNA sequence encoding the peptide is introduced into plant cells by polyethylene glycol (PEG)-mediated transformation, in which PEG is used to form temporary pores in the cell membrane, allowing the DNA sequence to enter the cell.

[0112] Any plant organ, tissue or excision can be used with the genetic engineering methods to obtain the transgenic plants disclosed herein, including, but not limited to, individual cells, callus, seeds, leaf excisions, stem excisions, root excisions, embryonic tissue, meristem tissue, and pollen.

[0113] Generally, the particular genetic engineering method and excision used to obtain a transgenic plant expressing the peptides disclosed herein will depend on factors such as the plant species, the efficiency of the transformation method, and the desired expression level of the antimicrobial peptide.

[0114] In a further embodiment of the methods disclosed herein, the transformed cells are regenerated into whole plants that express the peptide.

[0115] Transgenic plants expressing the peptide are identified and selected. This step is essential to eliminate non-transgenic plants. Antibiotic or herbicide resistance selection is one of the most commonly used selection methods for transgenic plants. Transgenic plants are transformed with a gene that confers resistance to an antibiotic or herbicide, and then grown on a medium containing the antibiotic or herbicide, killing or inhibiting the growth of non-transgenic plants. In some embodiments, transgenic plants are transformed with a gene encoding a reporter protein, allowing them to be easily identified and selected based on the expression of the reporter gene.

[0116] In another embodiment, transgenic plants are selected by nutritional selection. This method is based on the ability of transgenic plants to grow on media lacking a particular nutrient that is essential for the growth of non-transformed plants. The transgenic plants are transformed with a gene that confers the ability to synthesize the missing nutrient.

[0117] Marker-free selection uses a selectable marker that can be removed from the transgenic plants after selection. This can be achieved using a site-specific recombination system, such as the Cre-lox system, which allows the selection marker gene to be removed without leaving any trace of foreign DNA.

[0118] The present invention is further illustrated by the following non-limiting examples which further illustrate the invention but are not intended, and should not be construed as limiting the scope of the invention. EXAMPLES

[0119] Example 1: Production of peptides Total DNA was extracted from CCN-51 cocoa bean embryos, and a 171-bp DNA fragment (target DNA) was amplified using primers Forward_BamH1-5'CGCGGATCCTATGGCAGAAAACAATAT3' and Reverse_Kpn1-5'GGTACCTCATTGCCTTTGAAGCTCTTCTTCTT3'.

[0120] The amplified DNA fragment was ligated into the BamH1 / Kpn1 sites of the cloning vector pEXA128. Both the plasmid carrying the peptide DNA and the pQE30 overexpression plasmid were transformed into E. coli DH5α cells for amplification. Plasmids were extracted from overnight cultures, digested with BamHI and KpnI for 2 hours at 37°C, and separated on a 1% agarose gel. The linearized pQE30 plasmid and the insert from the pEXA128 plasmid were extracted from the gel, and the insert was ligated into the pQE30 plasmid. The pQE30 overexpression plasmid encodes a HIS tag, and overexpression of the peptide of the invention results in a fusion peptide consisting of the peptide and an N-terminal HIS tag. The resulting plasmid was transformed into E. coli M15 overexpression cells. The nucleotide sequence of the plasmid extracted from the overnight culture was determined, and only clones matching the expected sequence were used for overexpression. 16 ml of the overnight culture was used to inoculate 800 ml of liquid LB medium at 37°C. After the OD600 reached 0.6–0.8, IPTG was added to a final concentration of 1 mM to induce overexpression. Cells overexpressing peptides with signal peptides were incubated at 20°C for 24 h, and cells overexpressing peptides without signal peptides were incubated at 37°C for 5 h. Cells were lysed and the supernatant was subjected to protein extraction followed by affinity chromatography using Ni NTA agarose columns. After elution of HIS-tagged proteins with imidazole, purified peptides were separated by molecular weight using SDS-PAGE. Briefly, 25 μl of protein sample was mixed with 5 μl of 6X sample buffer containing bromophenol blue as a tracking dye. The mixture was heated at 95 °C for 5 min and loaded onto an SDS-PAGE gel (83 mm × 65 mm × 1 mm) containing 12.5% ​​or 15% (w / v) acrylamide. Electrophoresis was performed at 130 V for 90 min. After electrophoresis, the gels were stained with Coomassie® Blue (45% (v / v) methanol, 10% acetic acid, 2.93 x 10-3 M Coomassie® Brilliant Blue G-250) for 20 min or electrophoresed onto PVDF membranes as previously described by Towbin et al. in Proc. Natl. Acad. Sci. USA, 1979, 76, 4350-4354. Blots were treated with 5% (w / v) milk in PBST overnight at 4°C. Protein blots were then probed with polyclonal antibodies diluted 1:2,000 in PBST containing 5% (w / v) milk powder and incubated for 2 hours (h) at room temperature. Blots were further washed six times at room temperature for 5 min each with gentle shaking. The blots were then incubated with HRP-conjugated goat anti-rabbit IgG antibody, diluted 1:10,000 in PBST containing 5% (w / v) milk powder, for 1 h at room temperature. After incubation, the blots were washed six times with PBST, each for 5 min, with gentle shaking at room temperature. Finally, the bound HRP was indirectly detected by its enzymatic activity in the presence of hydrogen peroxide using luminol as a substrate, as described by Mruk and Cheng in Spermatogenesis, 2011, 1, 121-122.

[0121] (result) The recombinant peptide of SEQ ID NO.1 was separated by SDS polyacrylamide gel electrophoresis, excised from the gel, and subjected to trypsin digestion, resulting in the MALDI-TOF-MS peptide fingerprint shown in Figure 1. A mass of 8.3 kDa was detected using the intact mass fingerprint in both reduced and non-reduced samples containing the peptide according to SEQ ID NO.1. The LC-MS results for the sample according to SEQ ID NO.1 are shown in Figure 2.

[0122] Example 2: Synthesis of peptides Peptides of sequence identical to SEQ ID NO.1 can be chemically synthesized by Seramun Diagnostica GmbH (Heidesee, Germany).

[0123] Example 3: Minimum inhibitory concentration (MIC) assay MIC assays determine the lowest concentration at which a compound inhibits the growth of a microbial population.The antibacterial activity of the peptides described herein was compared with that of two other AMPs, TcAMP1 and TcAMP2, discussed in WO 1998 / 027805, and the activity of antibiotics or bactericides.Comparative MIC assays were performed against several bacterial and fungal species (Table 1).

[0124] Peptides according to SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, and SEQ ID NO.7 were serially diluted to generate a concentration array ranging from 0.39 μg / ml to 303 μg / ml.

[0125] Pantoea, Staphylococcus, and Listeria were cultured in Trypticase Soy Broth (TSY) medium, consisting of 1.7% casein peptone, 0.3% soy peptone, 0.25% glucose, 0.5% NaCl, 0.25% K2HPO4, and optional 1.5% agar. Candida cells were cultured in Yeast Extract Peptone Dextrose (YPD) broth medium, consisting of 1% yeast extract, 2% peptone, 2% glucose, and optional 2% agar. Ceratocystis, Botrytis, Leptosphaeria, Mycosphaerella, Sclerotinia, and Verticillium were cultured in Potato Dextrose Agar (PDA) medium, consisting of 2.4% Difco premix broth and optional 1.5% agar.

[0126] For the MIC assays, bacteria and fungi were grown on agar plates in the respective media. Precultures were prepared by inoculating 5 ml of the respective liquid medium with a single bacterial colony or a small piece of fungus. The optical density (OD) at 600 nm was measured after 1–5 days and adjusted to an OD600 of 1. A 1:500 dilution was prepared with this cell suspension, corresponding to approximately 2 × 106 microbial cells per ml.

[0127] To assess MICs, 96-well plates were prepared with the test antimicrobial peptides. Each well contained 100 μl of microbial cell culture, 90 μl of culture medium, and 10 μl of peptide or corresponding dilution of control. Plates were incubated overnight (ON) at the optimal growth temperature for each test organism. Results were visually assessed and MICs were determined based on the presence or absence of growth.

[0128] Table 1 lists the organisms tested in the MIC assay. Species names, taxonomy, and growth conditions are shown. DSM is the order number of the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ, Braunschweig, Germany). SF refers to organisms obtained from the Jena Microbial Resource Collection (JMRC, Jena, Germany).

[0129] [Table 1] MIC assays were performed in at least two independent replicates and with different starting concentrations of peptides. Preliminary testing showed that none of the steps performed to purify the tested proteins affected the MICs obtained.

[0130] (result) The MIC values ​​obtained for peptides according to SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO.7 against fungal species are shown in Table 2. All peptides of the present invention inhibited fungal growth more efficiently at lower concentrations than prior art peptides.

[0131] Table 2 shows the results of MIC assays using the peptide of the invention (SEQ ID NO.1) and the prior art peptides (SEQ ID NO.4, NO.5) with fungi as test organisms. Clotrimazole was used as a reference.

[0132] [Table 2]

[0133] The MIC values ​​for the peptides according to SEQ ID NO.1, SEQ ID NO.4 and SEQ ID NO.5 against bacterial species are shown in Table 3. The peptide having the sequence according to SEQ ID NO.1 had an inhibitory effect against all fungal species tested. Furthermore, the peptides of the present invention inhibited fungal growth more effectively at lower concentrations than the prior art peptides in almost all of the species tested, with the sole exception of Leptosphaeria maculans, which was most effectively inhibited by the peptide having SEQ ID NO.5.

[0134] Table 3 shows the MIC results using the peptide of the present invention (SEQ ID NO.1) and the prior art peptides (SEQ ID NO.4, NO.5) with bacterial species as tester organisms. Gentamicin and streptomycin were used as references.

[0135] [Table 3] [Sequence table] SEQ ID NO.1 (a peptide according to an embodiment of the present invention) YGRKQYERDPRQQYEQCQRRCESEATEEREQEQCEQRCEREYKEQQRQQEEELQRQ SEQ ID NO.2 (signal peptide of one embodiment of the present invention) MVISKSPFIVLIFSLLLSFALLCSGVSA SEQ ID NO.3 (a peptide according to an embodiment of the present invention) MVISKSPFIVLIFSLLLSFALCSGVSAYGRKQYERDPRQQYEQCQRRCESEATEEREQEQCEQRCEREYKEQQRQQEEELQRQ SEQ ID NO.4 (prior art peptide) YERDPRQQYEQCQRRCESEATEEREQEQCEQRCEREYKEQQRQQEEE SEQ ID NO.5 (prior art peptide) LQRQYQQCQGRCQEQQQGQREQQQCQRKCWEQYKEQERGEHENYHNHKKNRSEEEEGQQR SEQ ID NO.6 (Forward_BamH1 forward DNA primer) CGCGGATCCTATGGCAGAAAACAATAT SEQ ID NO.7 (Reverse_Kpn1 forward DNA primer) GGTACCTCATTGCCTTTGAAGCTCTTCTTCTT SEQ ID NO.8 (HQ tag) HQHQHQ SEQ ID NO.9 (HN tag) HNHNHNHNHNHN SEQ ID NO.10 (HAT tag) KDHLIHNVHKEEHAHAHNK SEQ ID NO.11 (ALFA tag) SRLEEELRRRLTE SEQ ID NO.12 (Avi tag) GLNDIFEAQKIEWHE SEQ ID NO.13 (C-tag) EPEA SEQ ID NO.14 (Calmodulin-tag) KRRWKKNFIAVSAANRFKKISSSGAL SEQ ID NO.15 (E-tag) GAPVPYPDPLEPR SEQ ID NO.16 (FLAG-tag) DYKDDDDK SEQ ID NO.17 (HA-tag) YPYDVPDYA SEQ ID NO.18 (Myc-tag) EQKLISEEDL SEQ ID NO.19 (NE-tag) TKENPRSNQEESYDDNES SEQ ID NO.20 (Rho1D4 tag) TETSQVAPA SEQ ID NO.21 (S-tag) KETAAAKFERQHMDS SEQ ID NO.22 (SBP tag) DEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP SEQ ID NO.23 (Strep-tag) WSHPQFEK

Claims

1. A peptide having a sequence that exhibits 95% or more sequence identity with SEQ ID NO. 1, or a sequence that differs from SEQ ID NO. 1 by up to three amino acids.

2. The peptide according to claim 1, wherein the peptide has the sequence according to SEQ ID NO.

1.

3. The peptide according to claim 1, wherein the peptide is fused with a signal peptide at its N-terminus.

4. The peptide according to claim 3, wherein the signal peptide has a sequence that exhibits 95% or more sequence identity with SEQ ID NO. 2, or a sequence according to SEQ ID NO.

2.

5. The peptide according to claim 1, wherein the peptide has antibacterial activity and / or antifungal activity.

6. The peptide according to claim 5, wherein the antibacterial activity and / or antifungal activity is activity against Gram-positive bacteria and Gram-negative bacteria, and / or activity against fungi, wherein the fungi are preferably mycelial fungi and yeast.

7. The peptide according to claim 1, wherein the peptide is derived from or isolated from theobroma cacao, preferably derived from or isolated from theobroma cacao species CCN-51.

8. The peptide according to claim 1, wherein the peptide is a recombinant peptide or a synthetic peptide.

9. A composition comprising the peptide and excipient described in claim 1.

10. The composition according to claim 9, wherein the composition is a liquid, semi-solid, solid, or gaseous composition, and / or the composition is in the form of a tablet, capsule, powder, granules, aerosol, paste, syrup, suspension, emulsion, or solution.

11. A peptide or composition for therapeutic use, which is the peptide according to any one of claims 1 to 8 or the composition according to claim 9.

12. A peptide or composition for use in subjects requiring treatment of bacterial and / or fungal infections, the peptide according to any one of claims 1 to 8 or the composition according to claim 9.

13. The peptide or composition according to claim 12, wherein the subject is a human or an animal.

14. Use of the peptide according to any one of claims 1 to 8 or the composition according to claim 9 as a feed additive or food additive for crop protection, as a preservative, as a decontamination agent, or for cosmetic purposes.

15. The use according to claim 14, wherein the peptide or composition is used for controlling pathogens in plants, and the plant is preferably a crop.

16. The use according to claim 15, wherein the pathogen is selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, and / or fungi.

17. A method for producing the peptide described in any one of claims 1 to 8, wherein the peptide is produced by recombinant technology or synthesis.

18. A vector comprising a coding sequence for a peptide having a sequence according to SEQ ID NO. 1, wherein the vector optionally has a signal peptide sequence fused to its N-terminus, wherein the signal peptide sequence preferably has 95% or more sequence identity with SEQ ID NO. 2, and the vector is designed to enable the expression of the peptide in an expression system.

19. A transgenic plant expressing the peptide described in any one of claims 1 to 8.

20. A method for producing a transgenic plant according to claim 19, the method comprising the steps of introducing a nucleic acid encoding the peptide into plant cells to produce transformed plant cells expressing the peptide, and regenerating the transformed plant cells into a transgenic plant.

21. Plant seeds coated with the peptide according to any one of claims 1 to 8 or the composition according to claim 9.

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