Peptides having antimicrobial activity

Abstract

The present invention relates to a peptide wherein the peptide has an amino acid sequence according to SEQ ID N degree 1. Compositions comprising the peptides, as well as specific uses, are also disclosed.

Description

Technical Field

[0001] The present invention relates to the technical field of antimicrobial peptides, and more specifically, to natural plant-derived peptides that exhibit antimicrobial activity against various bacterial and fungal strains. Specifically, the present invention relates to a natural, recombinant or synthetic peptide derived from plant vicilin. The present invention also relates to the use of the peptide in treating bacterial and fungal infections in plants, animals and humans. Background Art

[0002] Bacterial antibiotic resistance is a major problem for the food and agricultural industries, as well as the medical and veterinary industries. Of particular concern is the potential for bacteria with lethal antibiotic resistance to be transferred from food animals to human consumers.

[0003] Current approaches to controlling the development and spread of antibiotic-resistant bacteria include changing antibiotic use and patterns of use of different antibiotics, increased government oversight and regulation, and the continued development of new and improved antibiotics. However, the ability of most bacteria to adapt to antibiotic use and to develop resistance to existing and new antibiotics often overcomes these conventional measures, and the continued development of alternative means of controlling bacterial antibiotic resistance is required.

[0004] Antimicrobial peptides (AMPs) are a diverse class of natural compounds found in animals, plants, insects and microorganisms. These peptides are responsible for the defense against (other) microorganisms and, therefore, they can further be used as an alternative to chemical preservatives. However, their use in the treatment of microbial infections in plants, humans and / or animals has been limited so far due to poor availability in nature, high production costs, low stability and complex formulation into suitable therapeutic products. Therefore, the use of antimicrobial peptides in crop protection as well as in human and veterinary treatments remains limited.

[0005] Antimicrobial proteins exhibit a variety of three-dimensional structures, which largely determine their activity and stability. The stability of a certain protein is often a critical parameter for clinical and / or industrial applications.

[0006] After the initial interest in plant-derived AMP molecules, the first plant-derived AMP, purinethionine, was isolated. Caleya et al. reported in Appl. Microbiol, 1972, 23 (5) 998-100 that low molecular weight protein purinethionine from wheat and barley flour is an AMP that is effective against a variety of plant pathogens, such as Pseudomonas solanacearum, Xanthomonas phaseoli, and Xanthomonas campestris, Erwinia amylovora, and five strains of Corynebacterium. Since then, several major classes of AMPs have been discovered, including thioureas (type IV), defensins, cyclic peptides, 2S albumin-like proteins, and lipid transfer proteins.

[0007] Plant vicilin is a common class of plant seed storage proteins. It has been shown that some vicilins are processed to produce plant defense peptides. Among the antimicrobial peptides produced from vicilin, the most representative is the antimicrobial peptide from macadamia (Macadamia integrifolia). Macadamia kernels contain 666 amino acids (aa) of vicilin, which include a highly hydrophilic region of 212 amino acids near the N-terminal signal sequence.

[0008] WO1998027805 discloses a class of vicilin-type peptides with antimicrobial properties. The prototype proteins are of natural origin and have been isolated from macadamia nuts and certain other species including cocoa (Theobroma cacao). In particular, two sequences labeled TcAMP1 (cocoa antimicrobial protein 1) of 47 amino acids and TcAMP2 (cocoa antimicrobial protein 2) of 60 amino acids were derived from the cocoa vicilin seed storage protein gene sequence encoding 566 amino acids and recombinantly expressed in Escherichia coli. However, the isolated recombinant peptides and compositions prepared therefrom have effective antimicrobial properties at high doses (5-20 μg / ml).

[0009] Marcus et al. in Plant. Mol. Biol Rep (2008) 26, 75-87 studied three macadamia nut peptide sequences and two cocoa peptide sequences identified in the N-terminal hydrophilic region of vicilin seed proteins. The antimicrobial activity of the peptides was predicted based on the presence of the characteristic CXXXC-(10–12)XCXXXC motif in the N-terminal proximal hydrophilic region. A His-tagged version of the putative peptide was expressed in Escherichia coli. The recombinant peptides obtained showed in vitro antimicrobial activity against six plant pathogen strains, but the effective dose was very high, ranging from 9.2 μg / ml to 64 μg / ml or even higher for some pathogens.

[0010] Ecuador is the most important producer of fine cocoa, accounting for about 50% of the world's production. The fine cocoa produced in Ecuador mainly belongs to the Nacional variety (sometimes also called National or Arriba). In addition to the traditional Nacional cocoa with a strong aroma, Ecuador has been cultivating the cocoa clone CCN-51 since the 1960s. Unlike Nacional, CCN-51 is considered a bulk cocoa type because of its weaker aroma. However, it is more tolerant to changes in climatic conditions, is resistant to different pathogens and has a higher yield than other cocoa types. Therefore, the CCN-51 hybrid variety is very popular for Ecuadorian farmers to grow. US 2004 / 0172683 describes several polypeptides from cocoa beans seeds that are responsible for cocoa flavor.

[0011] The present invention describes an unknown peptide derived from cocoa that is highly effective and active against a large number of animal and plant pathogenic microorganisms. The peptide is safe to use and preferably does not contain corrosive chemicals that may be toxic and harmful to the environment. Summary of the invention

[0012] The present invention and its embodiments aim to provide a solution to one or more of the above-mentioned disadvantages. To this end, the present invention relates to a peptide having antimicrobial activity as claimed in claim 1. More specifically, the present invention provides a peptide having a sequence according to SEQ ID N° 1. Surprisingly, it was found that such a peptide obtained from cocoa or produced by recombinant or synthetic methods has antibacterial and / or antifungal activity against a broad spectrum of pathogens. Preferred embodiments of the peptide are shown in any one of claims 2 to 9.

[0013] In a second aspect, the present invention relates to a composition comprising a peptide according to claim 10. Preferred embodiments of the composition are shown in claim 11.

[0014] In a third aspect, the present invention relates to the use of a peptide as claimed in claim 12 for therapeutic purposes. Preferred embodiments of the use are shown in any one of claims 13 to 17. More particularly, the peptide is used to treat infections with Gram-positive bacteria, Gram-negative bacteria and / or fungi, preferably hyphal fungi or yeasts, in humans, animals or plants.

[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, which allows the expression of the peptide according to claim 19, the present invention also relates to the transgenic plant according to claim 20, and the method for obtaining a transgenic plant according to claim 21.

[0017] In a final aspect, the invention relates to seeds according to claim 22 coated with said peptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 A representative scheme of the detection and sequence localization of peptide mass fingerprint fragments determined by MALDI-ToF analysis is shown. The underlined amino acid sequences were identified as obtained from SDS polyacrylamide gels according to SEQ ID NO: o Tryptic digest of the recombinantly expressed peptide of 1.

[0019] Figure 2 shows the LC-MS results of a sample containing the peptide SEQ ID N°1. Figure 2A ) and restore ( Figure 2B ) The mass of the peptides detected in the samples was 8.3 kDa. DETAILED DESCRIPTION

[0020] The present invention relates to a peptide for medical or pharmaceutical use or a composition comprising said peptide as an active ingredient, wherein said peptide has an amino acid sequence according to SEQ ID N° 1. Furthermore, the present invention relates to the use of said peptide, a method for obtaining said peptide, and a vector allowing the expression of said peptide.

[0021] 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 of ordinary skill in the art to which the present invention belongs. For further guidance, term definitions are included to better understand the teachings of the present invention.

[0022] As used herein, the following terms have the following meanings:

[0023] As used herein, "a", "an" and "the" refer to both the singular and the plural, unless the context clearly dictates otherwise. For example, "a compartment" refers to one or more compartments.

[0024] As used herein, "about" refers to a measurable value, such as a parameter, amount, time interval, etc., and is intended to cover variations of + / -20% or less, preferably + / -10% or less, more preferably + / -5% or less, even more preferably + / -1% or less, and more preferably + / -0.1% or less from the specified value, as long as such variations are suitable for implementation in the disclosed invention. However, it should be understood that the value referred to by the modifier "about" itself is also specifically disclosed.

[0025] As used herein, "includes," "comprising," "containing," and "consisting of" are synonymous with "including" and its variations or "containing" and its variations, and are inclusive or open-ended terms that specify the presence of what follows (e.g., components), and do not exclude or exclude the presence of additional, unlisted components, features, elements, members, steps known in the art or disclosed herein.

[0026] In addition, unless otherwise indicated, the terms first, second, third, etc. in the specification and claims are used to distinguish similar elements and not necessarily to describe a sequential or chronological order. It should be understood that the terms so used are interchangeable under appropriate circumstances, and that the embodiments of the invention described herein are capable of operation in a sequence different from that described or illustrated herein.

[0027] The recitation of numerical ranges by endpoints includes all numbers and fractions within that range, as well as the recited endpoints.

[0028] Unless otherwise defined, the expressions "weight %", "weight percent", "% wt" or "wt %" here and throughout the specification refer to the relative weight of each component based on the total weight of the formulation.

[0029] Although the term "one or more" or "at least one", such as one or more or at least one member of a group of members, is clear in itself, by way of further example, the term specifically covers reference to any one of the members, or to any two or more of the members, for example, any ≥3, ≥4, ≥5, ≥6 or ≥7, etc., of the members, and up to all of the members.

[0030] The term "peptide" as used herein refers to any compound containing two or more amino acid residues, which are connected by an amide bond formed by the carboxyl group of one amino acid residue and the amino group of the adjacent amino acid residue. The amino acid residues can have L-form as well as D-form, and can be naturally occurring or synthetic, linear and cyclic.

[0031] The term "therapeutically effective amount" refers to an amount that is effective in ameliorating symptoms of a disease. A therapeutically effective amount can be a "prophylactically effective amount" because prevention can be considered treatment.

[0032] Unless otherwise defined, all terms used in the disclosure of the present invention, including technical terms and scientific terms, have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. As a further guide, the terms used in the specification are defined to better understand the teachings of the present invention. The terms or definitions used herein are only used to help understand the present invention.

[0033] References to "one embodiment" or "an embodiment" throughout this specification mean that the specific features, structures, or characteristics described in conjunction with the embodiment are included in at least one embodiment of the present invention. Therefore, the phrases "in one embodiment" or "in an embodiment" appearing throughout this specification do not necessarily all refer to the same embodiment, but may also be. In addition, it will be clear to those skilled in the art from this disclosure that specific features, structures, or characteristics may be combined in one or more embodiments in any suitable manner. In addition, although certain embodiments described herein include certain features included in other embodiments but not other features, the combination of features of different embodiments is intended to be within the scope of the present invention and to form different embodiments, as understood by those skilled in the art. For example, in the following claims, any claimed embodiment may be used in any combination.

[0034] In a first aspect, the present invention relates to a peptide, wherein the peptide has an amino acid sequence according to SEQ ID N°1.

[0035] In another embodiment, the present invention also discloses a peptide having an amino acid sequence that exhibits at least 95%, 96%, 97%, 98%, 99%, 99.5% sequence identity to SEQ ID N°1, and still has the antimicrobial activity observed for the peptide having the sequence according to SEQ ID N°1.

[0036] The term "sequence identity" as used herein refers to the degree to which a sequence is identical on an amino acid-by-amino acid basis within a comparison window. Therefore, "sequence identity percentage" is calculated as follows: compare two optimally aligned sequences over a comparison window, determine the number of positions where identical amino acids occur in the two sequences to obtain the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window (i.e., window size), and multiply the result by 100 to obtain the sequence identity percentage. Gaps, i.e., positions where residues are present in one sequence but not in another sequence in the comparison, are considered to have positions with non-identical residues. Determining the percentage of sequence identity can be done manually, or using a computer program available in the art to complete. PILEUP is an example of a useful algorithm. Software for performing BLAST analysis can be publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0037] In another embodiment, the peptide has a sequence that differs from the sequence SEQ ID N°1 by at most 4, more preferably 3, even more preferably 2, even more preferably 1 amino acid residues, while still exhibiting the antimicrobial activity as disclosed herein.

[0038] The amino acid sequence variants of the peptides contemplated herein can be substitution, insertion or deletion variants. Deletion variants lack one or more peptide residues that may not be important for function. Substitution variants typically contain alternative amino acids at one or more sites within the peptide and can be designed to modify one or more properties of the polypeptide, such as stability against proteolytic cleavage. The substitution is preferably conservative, i.e. an amino acid is replaced by an amino acid with a similar size and side chain or functional group. Conservative substitutions are well known in the art and include, for example, alanine to glycine, valine or leucine; arginine to lysine; asparagine to glutamine; aspartic acid to glutamic acid; cysteine ​​to methionine; glutamine to asparagine; glutamic acid to aspartic acid; glycine to proline; histidine to glutamine, tyrosine, arginine, lysine, asparagine or cysteine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to phenylalanine; tyrosine to tryptophan or phenylalanine; valine to isoleucine or leucine.

[0039] Preferably, the peptides of the present invention are derived from or isolated from cocoa. More specifically, the peptides of the present invention are derived from the N-terminal region of vicilin from cocoa. Vicilins are storage proteins present and characterized in a variety of plant species, such as, but not limited to, pineapple (Ananas comosus), peanut (Arachis hypogaea), sugar beet (Betavulgaris), Mexican pepper (Capsicum annuum), Chinese pepper (Capsicum chinense), American pecan (Carya illinoinensis), quinoa (Chenopodium quinoa), long-capsule jute (Corchorus olitorius), winter squash (Cucurbita maxima), wild strawberry (Fragaria vesca), soybean (Glycine max), cottonwood (Gossipumarboreum), Colombian mallow (Herrania umbratica), barley (Hordeum vulgare), jatropha (Jatropha curcas), walnut (Juglans regia), macadamia (Macadamia integrifolia), Macleaya cordata, Musa acuminata, opium poppy (Papaver somniferum, castor (Ricinus communis), tomato (Solanum lycopersicum), spinach (Spinacia oleracea), fire wheel wood (Stenocarpus sinuatus), cocoa (Theobroma cacao) and corn (Zea mays). Such proteins usually contain extremely hydrophilic N-proximal and C-terminal regions. In addition, they carry a hydrophobic N-terminal signal sequence, which is usually removed during protein maturation. The N-proximal region of the precursor protein is particularly interesting because it contains at least two pairs, preferably four pairs of cysteine ​​motifs (CXXXC) with the same spacing pattern.

[0040] The peptide according to the invention was identified in experiments carried out with recombinant peptides derived from cocoa vicilin. Without being bound by theory, it is speculated that the peptide is a degradation product of one of those recombinant peptides.

[0041] While the peptide disclosed herein can be obtained by recombinant expression, it can also be purified from a cocoa variety, more preferably variety CCN-51. Surprisingly, the cocoa variety CCN-51 or "lavados", known to be highly resistant to microbial infection, was shown to be particularly rich in vicilin-derived N-terminal peptides.

[0042] In another or additional embodiment, the peptide comprises a signal peptide at its N-terminus. Preferably, the signal peptide has a sequence according to SEQ ID N°2. In another embodiment, the signal peptide has at least 95%, 96%, 97%, 98%, 99% or more preferably 100% sequence identity with SEQ ID N°2. Alternatively, the peptide has a signal peptide sequence that differs from the sequence SEQ ID N°2 by at most 3 amino acid residues, more preferably at most 2 amino acid residues, even more preferably by 1 amino acid residue.

[0043] In one embodiment, the peptide of the invention containing a signal peptide has a sequence according to SEQ ID N°3.

[0044] In another embodiment, the peptide is a recombinant peptide or a synthetic peptide. A suitable expression system, such as E. coli, or another suitable system known in the art, produces the peptide after inserting a vector containing the coding sequence of the peptide having SEQ ID N° 1. Any suitable protein / peptide synthesis method known in the art can be used to synthesize the peptide of the present invention.

[0045] In other or another embodiment, the peptides described herein are provided with affinity tags at their N and / or C termini. Affinity tags are used for affinity purification of recombinant proteins and peptides expressed in E. coli and other systems. The tag may be a polyhistidine tag, which is an amino acid motif consisting of at least six histidine (His) residues.

[0046] Alternatively and without limitation, the peptide can be tagged with an HQ tag (HQHQHQ) of alternating histidine and glutamine residues, an HN tag (HNHNHNHNHNHN) of alternating histidine and asparagine, a HAT tag (KDHLIHNVHKEEHAHAHNK), an ALFA tag (SRLEEELRRRLTE), an Avi tag (GLNDIFEAQKIEWHE), a C tag (EPEA), a calmodulin tag (KRRWKKNFIAVSAANRFKKISSSGAL), a polyglutamic acid tag, a polyarginine tag , E tag (GAPVPYPDPLEPR), FLAG tag (DYKDDDDK), HA tag (YPYDVPDYA), Myc tag (EQKLISEEDL), NE tag (TKENPRSNQEESYDDNES), Rho1D4 tag (TETSQVAPA), S tag (KETAAAKFERQHMDS), SBP tag (DEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP) or Strep tag (WSHPQFEK).

[0047] Analysis of the peptides of the present invention showed that the peptides have antibacterial and / or antifungal activities.

[0048] Antimicrobial peptides themselves have a specific three-dimensional structure that can be determined using X-ray crystallography or nuclear magnetic resonance techniques. Although not wishing to be bound by theory, it is believed that this structure plays a role in the observed activity of the peptides. The alpha helical plant-derived antimicrobial peptides (AMPs) to which the peptides of the present invention belong generally have an amphipathic helix, wherein one side of the helix is ​​primarily hydrophilic, and the other side is primarily hydrophobic. This structure is generally found in AMPs that damage the cell membrane, causing leakage and cleavage of cell contents, as well as in AMPs that enter cells to attack other structures within the cell. Alpha-helical AMPs are generally disordered in structure in solution, which helps to pass through the dense network of the cell wall. After binding to the hydrophobic and charged cell membrane below the lipid head, a secondary structure (alpha-helix) is formed, which enables the protein to penetrate the cell membrane. This order / disorder transition is mainly regulated by the length of the hydrophobic domain within the alpha helix. Excessively long hydrophobic helices can lead to ordered structures in solution, which have a negative impact on AMP activity.

[0049] In one embodiment, the peptides are active against Gram-positive and Gram-negative bacteria and fungi, preferably hyphal fungi or yeasts. Without limitation, the peptides are active against bacteria selected from the group consisting of Acinetobacter spp., Bartonella spp., Bordetella spp., Borrelia spp., Brucella spp., Campylobacter spp., Chlamydia spp., Clostridium spp., Corynebacterium spp., Enterococcus spp., Enterobacter spp., Erwinia spp., Escherichia spp., Francisella spp., Haemophilus spp., Helicobacter spp. spp.), Klebsiella spp., Legionella spp., Leptospira spp., Listeria spp., Mycobacterium spp., Mycoplasma spp., Neisseria spp., Rickettsia spp., Salmonella spp., Shigella spp., Staphylococcus spp., Streptococcus spp., Treponema spp., Ureaplasma spp., Vibrio spp., Yersinia spp., Acidovorax spp.), Agrobacterium spp., Arthrobacter spp., Bacillus spp., Burkholderia spp., Clavibacter spp., Cronobacter spp., Curtobacterium spp.), Lefisonia spp., Pantoea spp., Paenibacillus spp., Pectobacterium spp., Phytoplasma spp., Proteus spp., Pseudomonas spp., Ralstonia spp., Rhizobacter spp., Rhizomonas spp., Rhodococcus spp., Serratia spp., Sphingomonas spp., Spiroplasma spp., Streptomyces spp., Xanthomonas spp. spp.), Xylella spp. or Xylophilus spp.

[0050] Without limitation, the peptides of the present invention are active against fungi selected from the group consisting of Ajellomyces spp., Aspergillus spp., Basidiobolus spp., Blastomyces spp., Candida spp., Coccidioides spp., Conidiobolus spp., Cryptococcus spp., Emmonsia spp., Histoplasma spp., Hanseniaspora spp., Lacazia spp., Paracoccidioides spp., Pneumocystis spp., Sporothrix spp. spp.), Stachybotrys spp., Talaromyces spp., Acrocalymma spp., Aecidium spp., Albonectria spp., Allodus spp., Alternaria spp., Amphobotrys spp., Apiosporina spp., Armillaria spp., Blumeria spp., Botryotinia spp., Botrytis spp., Ceratosystis spp., Colletotrichum spp., Cryptosporiopsis spp. spp.), Exobasidium spp., Fusarium spp., Hypocrea spp., Leptosphaeria spp., Magnaporthe spp., Melampsora spp., Meyerozyma spp., Monilinia spp., Mycospharella spp., Microsphaera spp.), Mucor spp., Penicillium spp., Pichia spp., Phytophtora spp., Saccharomyces spp., Sporobolomyces spp., Plasmodiophora spp., Podosphaera spp., Puccinia spp., Pythium spp., Rhizoctonia spp., Sclerotinia spp., Septoria spp., Taphrina spp., Thanatephorus spp., Torulasporas spp., Uromyces spp.), Ustilago spp., Venturia spp., or Verticillium spp.

[0051] In a second aspect, the present invention provides a composition comprising a peptide as described in the above paragraph. Said composition is particularly suitable for pharmaceutical and veterinary applications, and / or for crop protection.

[0052] In one embodiment, the composition is liquid, semisolid, solid or gas, and / or is the dosage form of tablet, capsule, powder, granule, aerosol, paste, syrup, suspension, emulsion or solution. The non-limiting examples of the composition are soluble powder, soluble granule, wettable granule, tablet formulation, dry flow agent, water flow agent, wettable dispersible granule, oil dispersion, suspension concentrate, dispersible concentrate, emulsifiable concentrate, aqueous suspension, fertilizer granule or sprayable composition. The composition can be formulated to be suitable for oral, injection, intravenous, intramuscular, skin, suction, local or intranasal administration. In other embodiments, the composition is formulated to allow coating one or more objects, spraying, spray coating, evaporation, atomization, atomization, suspension, dilution, etc.

[0053] In certain embodiments, the composition comprises a pharmaceutically acceptable carrier, excipient or diluent. Preferably, the excipient is selected from a filler, a binder, a disintegrant, a sweetener, a coating agent, a lubricant and / or a glidant. A diluent or filler can increase the volume of a solid composition and can make a dosage form containing the composition easier for patients and caregivers to handle. Diluents suitable for tablets of the present invention include, for example, microcrystalline cellulose (e.g. ), microcellulose, lactose, starch, pregelatinized starch, calcium carbonate, calcium sulfate, sugar, dextrates, dextrin, glucose, calcium hydrogen phosphate dihydrate, calcium phosphate trihydrate, kaolin, magnesium carbonate, magnesium oxide, maltodextrin, mannitol, polymethacrylates (e.g. ), potassium chloride, powdered cellulose, sodium chloride, sorbitol, and talc.

[0054] Solid compositions compressed into dosage forms (e.g., tablets) may contain excipients, the functions of which include helping to contain the active ingredient and other excipients after compression. Suitable binders include acacia, alginic acid, carbomers (e.g., carbopol), sodium carboxymethylcellulose, dextrin, ethylcellulose, gelatin, guar gum, hydrogenated vegetable oils, hydroxyethylcellulose, hydroxypropylcellulose (e.g., ), hydroxypropyl methylcellulose (e.g. ), liquid glucose, magnesium aluminum silicate, maltodextrin, methylcellulose, polymethacrylate, povidone (e.g. ), pregelatinized starch, sodium alginate and starch.

[0055] The dissolution rate of a compacted solid composition can be increased by adding a disintegrant to the composition. Suitable disintegrants include alginic acid, calcium carboxymethylcellulose, sodium carboxymethylcellulose (e.g. ), colloidal silicon dioxide, cross-linked sodium carboxymethyl cellulose, cross-linked polyvinylpyrrolidone (e.g. ), guar gum, magnesium aluminum silicate, methylcellulose, microcrystalline cellulose, polacrilin potassium, powdered cellulose, pregelatinized starch, sodium alginate, sodium starch glycolate (e.g. ) and starch.

[0056] Glidants may be added to improve the flowability of non-compacted solid compositions and to increase dosage accuracy. Excipients that may be used as glidants include colloidal silicon dioxide, magnesium trisilicate, magnesium stearate, powdered cellulose, starch, talc, and tricalcium phosphate.

[0057] When making dosage forms such as tablets by compressing powdered compositions, the compositions are subject to pressure from the punch and die. Certain excipients and active ingredients tend to adhere to the surface of the punch and dye, which can cause the product to have pits and other surface irregularities. Lubricants can be added to the compositions to reduce adhesion and simplify the release of the product from the die. Lubricants include magnesium stearate, calcium stearate, glyceryl monostearate, palmitic acid stearyl glyceride, hydrogenated castor oil, hydrogenated vegetable oil, mineral oil, polyethylene glycol, sodium benzoate, sodium lauryl sulfate, sodium stearyl fumarate, stearic acid, talc and zinc stearate. Preferably, the lubricant is present in 0.25 % by weight to 1 % by weight (w / w).

[0058] Flavoring agents and flavor enhancers make the dosage form more palatable to the patient. Common flavoring agents and flavor enhancers for pharmaceutical products that can be included in the compositions disclosed herein include maltol, vanillin, ethyl vanillin, menthol, citric acid, fumaric acid, ethyl maltol, and tartaric acid.

[0059] The solid composition may also be dyed using any pharmaceutically acceptable colorant to improve its appearance and / or facilitate patient identification of the product and unit dosage level.

[0060] In one embodiment, particularly when the composition is intended for agricultural use, the excipient is an agriculturally compatible excipient. The "agriculturally compatible carrier" or "agriculturally compatible adjuvant" can be considered a carrier, which is generally inert and must be agriculturally acceptable. Therefore, the term "agriculturally compatible" means a substance that can be routinely used under field conditions without interfering with the grower's planting equipment and does not adversely affect the growth of crops or the desired ecological balance in the planting area.

[0061] Agriculturally compatible carriers or excipients can be solid.Solid carriers or excipients can include but are not limited to clay, natural or synthetic silicates, silicon dioxide, resins, waxes, solid fertilizers, polymers, granular materials, perlite, perlite particles, peat, peat particles, soil, vermiculite, charcoal, sugar mill carbonization squeezing mud, rice husks, carboxymethyl cellulose, fine sand, calcium carbonate, flour, alum, starch, talcum, polyvinyl pyrrolidone or its combination.Agriculturally compatible carriers or excipients can be liquid.Liquid carriers or excipients can include but are not limited to water, alcohol, ketones, petroleum fractions, oils, aromatic hydrocarbons or paraffin hydrocarbons, chlorinated hydrocarbons, liquefied gas or its combination.More specifically, agriculturally compatible carriers or excipients can include dispersants, surfactants, additives, thickeners, anti-caking agents, residue decomposition agents, composting formulas, particle applications, diatomaceous earth, coloring agents, stabilizers, preservatives, polymers, coatings or or its combination. Those skilled in the art can consider various factors, such as specific compounds, plants to be applied inoculants, soil types, climatic conditions, whether the inoculants are liquid, solid or powdered forms, etc., and easily determine the appropriate carrier or excipient to be used. Additives can include oils, gums, resins, clays, polyoxyethylene glycols, terpenes, viscous organic matter, fatty acid esters, sulfated alcohols, alkyl sulfonates, petroleum sulfonates, alcohol sulfates, alkyl butane dioic acid sodium, polyesters of thiobutane dioic acid sodium, benzyl cyanide derivatives, protein materials or combinations thereof. Protein materials can include dairy products, wheat flour, soybean meal, blood, albumin, gelatin or combinations thereof. Thickeners can include long-chain alkyl sulfonates of polyethylene glycol, polyoxyethylene oleate or combinations thereof. Surfactants can contain heavy petroleum, heavy petroleum fractions, polyol fatty acid esters, polyethoxylated fatty acid esters, aryl alkyl polyoxyethylene glycols, alkylamine acetates, alkyl aryl sulfonates, polyols, alkyl phosphates or combinations thereof. The anti-caking agent may include a sodium salt, such as sodium sulfite, sodium sulfate, sodium monomethylnaphthalene sulfonate, or a combination thereof; or a calcium salt, such as calcium carbonate, diatomaceous earth, or a combination thereof.

[0062] In one embodiment, the composition further comprises one or more of the following: water, other nutrients, weak acid, vegetable oil, essential oil, metabolic promoter, emulsifier, viscosity agent, coloring material, suspending agent, dispersant, preservative, complexing agent, stabilizer, carrier, excipient or wetting agent or any combination thereof. In one embodiment, the composition further comprises at least one oil, surfactant and polymer.

[0063] The composition can be manufactured in a conventional manner. In certain embodiments, the composition provided herein comprises one or more preservatives to inhibit microbial activity. Suitable preservatives include mercury-containing substances such as metfen and thimerosal; stabilized chlorine dioxide; and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide and cetylpyridinium chloride.

[0064] The peptides described herein can be used at 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 to 8 μg / ml, preferably 0.1 to 9 μg / ml, preferably 0.1 to 10 μg / ml, preferably 0.1 to 15 μg / ml, preferably 0.1 to 10 ... The 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 is formulated in the composition.

[0065] Alternatively, the peptide can be used at 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, preferably 6 to 50 μg / ml, preferably 7 to 50 μg / ml, preferably 8 to 50 μg / ml, preferably 9 to 50 μg / ml, preferably 10 to 50 μg / ml, preferably 15 to 50 μg / ml, preferably 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, preferably 45 to 50 μg / ml is formulated in the composition.

[0066] In another embodiment, the composition contains at least 0.1% by weight of the peptide, more preferably at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% %, 23 wt %, 24 wt %, 25 wt %, 26 wt %, 27 wt %, 28 wt %, 29 wt %, 30 wt %, 31 wt %, 32 wt %, 33 wt %, 34 wt %, 35 wt %, 36 wt %, 37 wt %, 38 wt %, 39 wt %, 40 wt %, 41 wt %, 42 wt %, 43 wt %, 44 wt %, 45 wt %, 46 wt %, 47 wt %, 48 wt %, 49 wt%, 50 wt%, 51 wt%, 52 wt%, 53 wt%, 54 wt%, 55 wt%, 56 wt%, 57 wt%, 58 wt%, 59 wt%, 60 wt%, 61 wt%, 62 wt%, 63 wt%, 64 wt%, 65 wt%, 66 wt%, 67 wt%, 68 wt%, 69 wt%, 70 wt%, 71 wt%, 72 wt%, 73 wt%, 74 wt%, 75 %, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% peptide by weight.

[0067] In a third aspect, the peptides or compositions described herein are suitable for therapeutic use. "Therapeutic use" as described herein refers to the use of a peptide or its composition to improve the symptoms of a disease in a human or non-human animal. When practicing the treatment or use methods provided herein, a therapeutically effective amount of the pharmaceutical composition described herein is administered to a subject, such as a mammal or non-mammal suffering from a disease, disorder or condition to be treated. In certain embodiments, the subject is a human. The therapeutically effective amount may vary greatly depending on the severity of the disease, the age and relative health of the subject, the efficacy of the therapeutic agent used, and other factors. The therapeutic agents described herein and, in some cases, the compositions can be used alone or in combination with one or more therapeutic agents as a component of a mixture.

[0068] In one embodiment, peptides as described herein are used in pharmaceutical compositions to treat or prevent infections caused by various microorganisms (including gram-positive bacteria, gram-negative bacteria and / or fungi, preferably hyphae fungi or yeast) in a subject in need. The subject can be a human or an animal. The pharmaceutical composition can comprise a therapeutically effective amount of the antimicrobial peptide and a suitable carrier.

[0069] The peptides or compositions described herein can be administered to a subject by an appropriate route of administration, including but not limited to intravenous, intraarterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, inhalation or intraperitoneal administration. The compositions described herein can include but are not limited to aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast dissolving formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsed release formulations, multi-particulate formulations, and mixed immediate and controlled release formulations.

[0070] In one embodiment, the peptides or compositions described herein can be added to animal feed to reduce potential microbial infections in livestock. In another embodiment, the peptides or compositions described herein are formulated for oral, topical or parenteral use to treat microbial infections in veterinary medicine.

[0071] In another embodiment, the peptide or composition described herein is suitable for use in crop protection, as a feed or food additive, for pharmaceutical use, as a preservative, as a detergent and / or for cosmetic applications.

[0072] When used for crop protection, the peptides or compositions described herein are formulated with an agriculturally compatible carrier or agriculturally compatible excipient according to the above embodiments. Those skilled in the art will determine the correct dosage, form and method of application of the composition according to the crop being treated and the effect of the target pathogen.

[0073] In one embodiment, the peptides or compositions described herein are incorporated into feed or food additives to prevent pathogens from spreading through feed and food. It is well known that feed may carry pathogens that are harmful to animal health and welfare. Preservative products such as formaldehyde are commonly used to sterilize feed, but these products can have a negative impact in turn. The antimicrobial peptides disclosed herein are a safe alternative.

[0074] In another embodiment, the peptides or compositions described herein are suitable for use as a detergent or disinfectant for surfaces, tools, instruments, devices, objects, or body parts. In one embodiment, such devices or objects include, but are not limited to, linens, cloths, plastics, latex fabrics, natural rubber, implantable devices, surfaces, or storage containers. In a specific embodiment, the peptides or compositions disclosed herein are incorporated into a cleaning agent, detergent, soap, or spray.

[0075] In certain embodiments, the peptides described herein can also be added to various health products. For example, the peptides can be added to toothpaste, mouthwash, shampoo, soap, cream or antiperspirant to reduce or prevent the colonization or establishment of microorganisms in the oral cavity or skin.

[0076] In another embodiment, the peptides and compositions described herein can be used as food, feed, cosmetic or pharmaceutical preservatives or for treating food to control, reduce or eliminate potential pathogens or contaminants.

[0077] In another embodiment, the peptides or compositions described herein are used to control pathogens in plants, preferably pathogens in crops, wherein the pathogens are Gram-positive and Gram-negative bacteria and / or fungi, preferably hyphal fungi or yeast. The plants may be monocots or dicots, including forage or forage legumes, ornamental plants, food crops, trees or shrubs. In one embodiment, the plants are preferably crops. Preferably, the plant belongs to the group consisting of Acer spp., Actinidia spp., Abelmoschus spp., Agave sisalana, Agropyron spp., Agrostis stolonifera, Allium spp., Amaranthus spp., Ammophila arenaria, Ananascomosus, Annona spp., Apium graveolens, Arachis spp., Artocarpus spp., Asparagus officinalis, Avena spp., Averrhoa carambola, Bambusa spp., Benincasa hispida), Brazil chestnut (Bertholletia excelsea), beet (Beta vulgaris), mustard (Brassica spp.), pink leaf leech (Cadaba farinosa), tea tree (Camellia sinensis), canna (Canna indica), hemp (Cannabis sativa), pepper (Capsicum spp.), lip grass (Carex elata), papaya (Carica papaya), large flowered tiger thorn (Carissa macrocarpa), pecan (Carya spp.), safflower (Carthamus tinctorius,), chestnut (Castanea spp.), gibbey (Ceiba pentandra), chicory (Cichorium endivia), camphor (Cinnamomum spp.), watermelon (Citrullus lanatus), citrus (Citrus spp.), coconut (Cocosspp.), coffee (Coffea spp.), Colocasia esculenta, Cola spp., Jute (Corchorus spp.), Coriandrum sativum, Corylus spp., Crataegus spp., Crocus sativus, Cucurbita spp., Cucumis spp., Cynara spp., Daucus carota, Desmodium spp., Dimocarpus longan, Dioscorea spp., Diospyros spp., Echinochloa spp., Elaeis spp., Eleusine coracana, Eragrostistef, Erianthus spp.), loquat (Eriobotrya japonica), eucalyptus (Eucalyptus spp.), red fruit (Eugenia uniflora), buckwheat (Fagopyrum spp.), beech (Fagus spp.), tall fescue (Festuca arundinacea), fig (Ficus carica), kumquat (Fortunella spp.), strawberry (Fragaria spp.), ginkgo (Ginkgo biloba), soybean (Glycine spp.), upland cotton (Gossypium hirsutum), sunflower (Helianthus spp.), daylily (Hemerocallis fulva), hibiscus (Hibiscus spp.), barley (Hordeum spp.), sweet potato (Ipomoea batatas), walnut (Juglans spp.), lettuce (Lactuca sativa), Lathyrus spp.), Lens culinaris, Linumusitatissimum, Litchi chinensis, Lotus spp., Luffaacutangula, Lupinus spp., Luzula sylvatica, Lycopersicon spp., Macrotyloma spp., Malus spp.), Malpighia emarginata, Mammea americana, Mangifera indica, Manihot spp., Manilkara zapota, Medicago sativa, Melilotus spp., Mentha spp., Miscanthus sinensis, Momordicaspp., Morus nigra, Musa spp., Nicotiana spp., Olea spp., Opuntia spp., Ornithopus spp., Oryza spp., Panicum miliaceum, Panicum virgatum, Passiflora edulis, Pastinaca sativa), Pennisetum spp., Avocado spp., Parsley (Petroselinumcrispum), Phalaris arundinacea, Phaseolus spp., Phleum pratense, Phoenix spp., Phragmites australis, Physalis spp., Pinus spp., Pistacia vera, Pisum spp., Poa spp., Populus spp., Prosopis spp., Prunus spp., Guava (Psidium spp.), Pomegranate (Punica granatum), Pyrus communis, Quercus spp.), radish (Raphanus sativus), rhubarb (Rheum rhabarbarum), Ribes spp., castor (Ricinus communis), Rubus spp., Saccharum spp., willow (Salix spp.), elderberry (Sambucus spp.), Secale cereale, Sesamum spp.), Sinapis spp., Solanum spp., Sorghum bicolor, Spinacia spp., Syzygium spp., Tagetes spp., Tamarindus indica, Theobroma cacao, Trifolium spp., Tripsacum dactyloides, Triticosecalerimpaui, Triticum spp., Tropaeolum minus, Tropaeolum majus, Vaccinium spp., Vicia spp., Vigna spp., Viola odorata, Vitis spp., Zea mays, mays), Zizania palustris, Ziziphus spp.; including their descendants and hybrids.

[0078] The peptides or compositions described herein are suitable for introduction into a plant, a plant part or a matrix comprising or carrying the plant, thereby conferring pathogen resistance or pathogen control to the plant. The introduction may be artificial.

[0079] The method of introducing the peptide or its composition into the plant may include: treating the plant and / or plant part and / or the growth medium in which the plant grows, inoculating seeds, coating seeds, directly inoculating the plant or plant part, spraying or wetting the plant or plant part (e.g., ear of corn). The appropriate method can be selected according to the type of plant into which the peptide or composition is to be introduced.

[0080] As another non-limiting example, peptide as herein described or composition can be applied in the form of coating.Coating can be applied to seed by spraying on seed or soaking the seed in a solution containing peptide or composition.In another example, in order to coat seed with the solution as described in the previous example, an adhesive can be added, for example an adhesive comprising carbide (calcium carbonate).

[0081] In embodiments, the coating can be applied to exposed and untreated plant parts. In other embodiments, the coating can be applied as an outer coating to previously treated plant parts. Seed coatings are particularly preferred in the treatment of soil fungal diseases. In embodiments, the seed coating can be applied to exposed and untreated seeds. In other embodiments, the seed coating can be applied as a seed coat to previously treated seeds.

[0082] In one embodiment, the peptides or compositions described herein may be applied to the soil or any other substrate in which the plant grows to remove pests and / or pathogens from the substrate.

[0083] Inoculation of the matrix containing or carrying the plant or plant part can be carried out by, for example, but not limited to, using a powder, granule, pellet, plug or soil soak applied to the matrix. Inoculation can also be carried out by liquid application, such as foliar spray or liquid composition. Application can be applied to growing plants or to the matrix. Plants, particularly crops, can grow in the matrix. In one embodiment, the matrix is ​​soil, sand, gravel, polysaccharide, mulch, compost, peat moss, straw, log, clay or a combination thereof. In another embodiment, the matrix can also include a hydroponic system or an in vitro culture system. In some embodiments, a combination of different application methods as described herein has been employed.

[0084] In one embodiment, the peptide or composition as described herein is suitable for treating or preventing infection by a pathogen, wherein the pathogen is selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria and / or fungi as described in any of the preceding embodiments.

[0085] The invention disclosed herein also relates to plant seeds coated with the peptide or composition described in any of the preceding embodiments.

[0086] In another aspect, the invention relates to methods for obtaining the peptides disclosed herein, wherein the peptides are recombinant or synthetic peptides, or are derived (e.g. by extraction or precipitation) from the cocoa tree (more preferably variety CCN-551). These methods are known in the art.

[0087] In one embodiment, the target peptide can be dried by lyophilization.

[0088] In one embodiment, the peptide is concentrated. Preferably, the peptide is concentrated using standard salt or organic solvent precipitation. Preferably, the peptide is concentrated by dialysis against a volatile buffer (e.g., ammonium carbonate), using a filter-based concentrator or by ion exchange chromatography.

[0089] The quantification of peptide content is performed by spectrophotometry (such as the Lowry method or Bradford assay) or any other method known in the art. In another embodiment, the quantification of peptides is performed by any suitable chromatographic method known in the art. In a preferred embodiment, the LC-MS method is used to quantify the peptide content. In a preferred embodiment, the fragments produced by the protein obtained from the cocoa extract by trypsin digestion are subjected to MALDI-TOF analysis. In one embodiment, the data obtained from the MALDI-MS trypsin digestion analysis are further confirmed by preparing and performing immunochemical analysis to detect specific differences between samples.

[0090] In other preferred embodiments, the peptide is substantially free of other peptides. As used herein, the term "substantially free" means at least 95% by weight free of other peptides; preferably at least 99% by weight free of other peptides. In a preferred embodiment, the peptide is present in a crystalline and / or solid form.

[0091] In another aspect, the present invention relates to a vector comprising a coding sequence for a peptide having a sequence according to SEQ ID N°1 and optionally comprising a sequence of an N-terminal signal peptide having a sequence according to SEQ ID N°2, wherein the vector is designed to allow expression of the peptide in an expression system. In another preferred embodiment, the vector comprises a purification tag as disclosed in the previous embodiment.

[0092] In one embodiment, DNA extracted from cocoa beans is used to amplify the coding sequence of the peptide, which is cloned into a suitable expression vector, such as any plasmid or virus designed for gene expression in cells.

[0093] Therefore, the peptides described herein can be expressed in any suitable system for the purpose of producing peptides for further use. Suitable hosts for expressing the protein include Escherichia coli, fungal cells, insect cells, mammalian cells and plants. Standard methods for expressing proteins in these hosts are described in a variety of texts, including Section 16 (Protein Expression) of Current Protocols in Molecular Biology (above).

[0094] Typically, the DNA encoding the amino acid sequence of interest is contained in an expression vector, in some cases linked to another coding sequence in frame at the 5' or 3' end, thereby encoding a peptide having a sequence according to SEQ ID N°1, and optionally comprising SEQ ID N°2 at the N-terminus. The overall 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".

[0095] According to one embodiment, the promoter is a promoter natural to the microorganism (e.g., E. coli trpE promoter), a synthetic promoter such as a Tac promoter, or a promoter obtainable from a heterologous organism, such as a virus, a bacterium, or a bacteriophage, such as a bacteriophage lambda or T7 that can function in a microorganism. The promoter can be constitutive, or more preferably inducible. The expression vector can also contain a selective marker gene, which can be an antibiotic resistance gene, such as an ampicillin, tetracycline, chloramphenicol, or kanamycin resistance gene.

[0096] There are many promoter systems available, usually from commercial sources, that are suitable for expressing the peptides of the invention in E. coli. For example, P of the araBAD (arabinose) operon BAD The promoter has good induction properties, with an induction rate of 1,200-fold above background (Guzman et al., 1995). Other promoters include P LAC , P TAC , P TRC , PL and PR. TAC The promoter is a hybrid of the E. coli trp and lac promoters and is one of the most efficient E. coli-based promoter systems known. It is inducible by IPTG, like the lac promoter.

[0097] The 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 expressing the peptides of the present invention.

[0098] In other preferred embodiments, selection of clones is done and the overexpressed peptide is extracted via its affinity tag using Ni-NTA agarose.The recombinant peptide is isolated and purified using the same methods as the peptide isolated from natural sources in the previous embodiments.

[0099] Expression of the novel peptide in genetically engineered cells will generally produce a product having the same three-dimensional structure as the native peptide isolated from the cocoa plant material. The three-dimensional structure includes correctly formed intramolecular disulfide bonds between cysteine ​​residues. However, even if the protein is chemically synthesized, methods are known in the art for further processing of the protein to break undesirable disulfide bonds and form bridges between desired cysteine ​​residues to obtain the desired three-dimensional structure, if this is necessary.

[0100] In another embodiment of the present invention, the peptide can be obtained by chemical synthesis using known peptide synthesis techniques, such as solid phase synthesis on a room temperature peptide synthesizer, a microwave peptide synthesizer, a parallel peptide synthesizer, or the like.

[0101] In one embodiment of the present invention, the peptides as described herein may be expressed in transgenic plants. Therefore, the present invention also relates to transgenic plants expressing one or more peptides as described herein.

[0102] In one embodiment, the transgenic plant expresses a peptide that exhibits at least 95% sequence identity to SEQ ID N° 1 or that differs from the sequence according to SEQ ID N° 1 by at most 3 amino acids. In other embodiments, the peptide comprises the sequence of an N-terminal signal peptide, the signal peptide sequence having 95% sequence identity to SEQ ID N° 2.

[0103] In other 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 N° 1. In one embodiment, the peptide comprises a purification tag as disclosed in the previous embodiment.

[0104] In one embodiment, the peptide comprises a purification tag as disclosed in the previous embodiment.

[0105] According to the present invention, plant cells can be transformed with DNA constructs according to a variety of known genetic engineering methods (Agrobacterium tumefaciens transformation, Ti plasmid, electroporation, microinjection, microgun, PEG-mediated transformation, etc.). As used herein, genetic engineering methods can be understood as any method for introducing foreign DNA sequences into plant cells to regenerate transgenic plants expressing desired traits or characteristics.

[0106] In one embodiment of the present invention, the DNA sequence encoding the peptide according to the sequence of SEQ ID N ° 1 or the DNA encoding the homologue from other plant species can be used in combination with the DNA sequence encoding the precursor protein that produces the mature protein. The precursor protein contains a natural peptide sequence that targets the protein to a specific cell compartment (e.g., apoplast or vacuole). These coding sequences can be connected to a plant promoter sequence to ensure strong expression in plant cells. The promoter sequence can ensure strong constitutive expression of the protein in most or all plant cells, it can be a promoter that ensures expression in a specific tissue or cell susceptible to microbial infection, and it can also be a promoter that ensures strong induction of expression during infection. These types of gene cassettes will also include transcription termination and polyadenylation sequences at the 3' of the antimicrobial protein coding region to ensure effective production and stability of the mRNA encoding the antimicrobial protein. By incorporating their respective DNA sequences into the sequence encoding a larger peptide, the effective expression of the antimicrobial peptide disclosed herein may be promoted.

[0107] The gene cassette encoding the peptide is expressed in plant cells using methods known in the art. First, the gene cassette is connected to a binary vector carrying the following: i) the left border sequence and the right border sequence of the T-DNA of the Ti plasmid of Agrobacterium tumefaciens; ii) a suitable selection marker gene for selecting antibiotic-resistant plant cells; iii) a replication origin that is functional in both Agrobacterium tumefaciens and Escherichia coli; and iv) an antibiotic resistance gene that allows selection of Agrobacterium tumefaciens and Escherichia coli cells carrying plasmids. Without wishing to be limited, the DNA sequence of the peptide disclosed herein can be cloned into any binary vector known in the art, such as plasmids (pEXA128, pBR322, pUC19), phages (λ phage, M13 phage), cosmids, BACs or YACs. The binary vector carrying the DNA sequence of the peptide can be introduced into the Agrobacterium tumefaciens strain (such as strain LBA4404, GV3101 and AGL1) or Agrobacterium rhizogenes strain (such as A4 or NCCP1885) carrying the disarmed Ti plasmid by heat shock, electroporation or triparental mating. These Agrobacterium strains can then be co-cultured with suitable plant explants or intact plant tissues, and the transformed plant cells and / or regenerated bodies can be selected by antibiotic resistance. As an alternative, the binary vector carrying the DNA sequence of the peptide can be overexpressed in bacteria or fungal cells.

[0108] The present disclosure also relates to a method for obtaining a transgenic plant, which expresses a sequence that exhibits at least 95% sequence identity with SEQ ID N°1 or that differs from the sequence according to SEQ ID N°1 by a maximum of 3 amino acids and optionally comprises an N-terminal signal peptide, said signal peptide sequence having 95% sequence identity with SEQ ID N°2.

[0109] The method comprises the following steps:

[0110] (a) optionally isolating or synthesizing a nucleic acid sequence encoding a peptide that exhibits at least 95% sequence identity to SEQ ID N° or that differs from the sequence according to SEQ ID N° 1 by a maximum of 3 amino acids;

[0111] and incorporating the nucleic acid sequence into a suitable expression vector;

[0112] (b) introducing the nucleic acid into at least one plant cell by genetic engineering methods to produce a transformed plant cell expressing the peptide;

[0113] (c) regenerating the transformed plant cells into intact transgenic plants; and

[0114] (d) selecting and identifying transgenic plants expressing the peptide using a suitable screening method.

[0115] In another embodiment of the method disclosed herein, the DNA sequence of the peptide is introduced into the plant cell by gene gun bombardment. Gene gun bombardment involves the use of a gene gun to introduce foreign DNA sequences into plant cells. The DNA sequence encoding the antimicrobial peptide disclosed herein is attached to metal particles such as gold particles and then bombarded into the plant cell.

[0116] In another embodiment, the DNA sequence of the peptide is introduced into the plant cell by electroporation, which involves the use of electric pulses to produce pores on the cell membrane, thereby making the DNA sequence encoding the antimicrobial peptide enter the cell. As an alternative, the DNA sequence encoding the antimicrobial peptide can be directly injected into the plant cell (microinjection).

[0117] In another alternative embodiment, the DNA sequence encoding the peptide is introduced into the plant cell by PEG-mediated transformation, which involves the use of polyethylene glycol (PEG) to create temporary pores in the cell membrane, thereby allowing the DNA sequence to enter the cell.

[0118] Any plant organ, tissue or explant can be genetically engineered to obtain the transgenic plants disclosed herein. Non-limiting examples include: single cells, callus, seeds, leaf explants, stem explants, root explants, embryonic tissue, meristem or pollen.

[0119] In general, the specific genetic engineering methods and explants used to obtain transgenic plants expressing the peptides disclosed herein depend on factors such as the plant species, the efficiency of the transformation method, and the desired level of antimicrobial peptide expression.

[0120] In other embodiments of the methods disclosed herein, the transformed cells are regenerated into whole plants expressing the peptide.

[0121] Identify and select transgenic plants expressing the peptide. This step is critical to eliminate non-transformed plants. Antibiotic or herbicide resistance selection is one of the most commonly used selection methods for transgenic plants. Transgenic plants are transformed with genes that confer resistance to antibiotics or herbicides and then grown in a medium containing the antibiotics or herbicides, which would kill or inhibit the growth of non-transformed plants. In certain embodiments, transgenic plants are transformed with a gene encoding a reporter protein, which can then be easily identified and selected based on the expression of the reporter gene.

[0122] In other embodiments, transgenic plants are selected by nutritional selection. This method is based on the ability of transgenic plants to grow in a medium lacking specific nutrients necessary for the growth of non-transformed plants. Transgenic plants are transformed with genes that give the plant the ability to synthesize the missing nutrients.

[0123] Marker-free selection involves the use of selectable markers that can be removed from transgenic plants after selection. This can be achieved by using site-specific recombination systems, such as the Cre-lox system, which allows the removal of selectable marker genes without leaving any traces of foreign DNA.

[0124] The present invention is further described by the following non-limiting examples, which further illustrate the present invention but are not intended to, nor should they be construed to, limit the scope of the present invention.

[0125] Example

[0126] Example 1: Production of peptides

[0127] 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'.

[0128] The amplified DNA fragment was ligated to the BamH1 / Kpn1 site of the cloning vector pEXA128. The plasmid carrying the peptide DNA and the pQE30 overexpression plasmid were both transformed into E. coli DH5α cells for amplification. The plasmids were extracted from the overnight culture, digested with BamHI and KpnI for 2 hours at 37°C, and then separated on a 1% agarose gel. The insert fragments of the linearized pQE30 plasmid and the pEXA128 plasmid were extracted from the gel and ligated to the pQE30 plasmid. The pQE30 overexpression plasmid encodes a HIS tag, and overexpression of the peptide of the present invention will produce 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 plasmid extracted from the overnight culture was sequenced, and only clones matching the expected sequence were used for overexpression. 16 ml of the overnight culture was inoculated into 800 ml of liquid LB medium at 37°C. When the OD 600When it reached between 0.6 and 0.8, IPTG was added to a final concentration of 1 mM to induce overexpression. Cells overexpressing the peptide were incubated at 37°C for 5 hours. The cells were lysed and the supernatant was subjected to protein extraction, followed by affinity chromatography using a NiNTA agarose column. After eluting the HIS-tagged protein with imidazole, the purified peptides were separated according to 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 minutes and then 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 minutes. After electrophoresis, the gel was washed with Blue (45% (v / v) methanol, 10% acetic acid, 2.93 x 10 -3 M Brilliant Blue G-250) staining for 20 minutes, or blotted onto a PVDF membrane by electrophoresis, as previously described by Towbin in Proc. Natl. Acad. Sci. USA, 1979, 76, 4350-4354. The blot was treated with 5% (w / v) milk in PBST at 4°C overnight. Subsequently, the protein blot was probed with a polyclonal antibody diluted 1:2,000 in PBST containing 5% (w / v) milk powder and incubated for 2 hours (h) at room temperature. In addition, the blot was washed 6 times, each time gently shaking at room temperature for 5 minutes. Subsequently, the blot was incubated for 1 hour at room temperature with an HRP-conjugated goat anti-rabbit IgG antibody, which was diluted 1:10,000 in PBST containing 5% (w / v) milk powder. After incubation, the blot was washed 6 times with PBST, each time gently shaking at room temperature for 5 minutes. Finally, indirectly bound HRP was detected by enzymatic activity using luminol as substrate in the presence of hydrogen peroxide as described by Mruk and Cheng in Spermatogenesis, 2011, 1, 121-122.

[0129] Results: Separation of SEQ ID NOs by SDS polyacrylamide gel electrophoresis o The recombinant peptide of 1 was excised from the gel and digested with trypsin to obtain Figure 1 The MALDI-TOF-MS peptide fingerprints shown in . In both the reduced and non-reduced samples containing the peptide according to SEQ ID N°1, the mass detected using the intact mass fingerprinting method was 8.3 kDa. The LC-MS results of the sample according to SEQ ID N°1 are shown in FIG2 .

[0130] Example 2: Synthesis of peptides

[0131] The peptide having the same sequence as SEQ ID N° 1 was chemically synthesized by Seramun Diagnostica GmbH (Heidesee, Germany).

[0132] Example 3: Minimum Inhibitory Concentration (MIC) Determination

[0133] MIC assays determine the lowest concentration at which a compound inhibits the growth of a microbial population. The antimicrobial potential of the peptides described herein was compared with the activity of two other AMPs (TcAMP1 and TcAMP2) discussed in WO1998027805, as well as antibiotics or fungicides. Comparative MIC assays were performed against several bacteria and fungi (Table 1).

[0134] The peptides according to SEQ ID N°1, SEQ ID N°4 and SEQ ID N°5 were serially diluted to create a concentration sequence ranging from 0.39 μg / ml to 303 μg / ml.

[0135] Pantoea, Staphylococcus, and Listeria were cultured in Tryptic Soy Broth (TSY) medium consisting of 1.7% casein peptone, 0.3% soy peptone, 0.25% glucose, 0.5% NaCl, 0.25% K2HPO4, and optionally 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 optionally 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 optionally 1.5% agar.

[0136] For MIC determination, bacteria and fungi were grown on agar plates with the respective culture medium. Precultures were prepared by inoculating a single bacterial colony or a small piece of fungus in 5 ml of the respective liquid medium. The optical density (OD) at 600 nm was measured after one to five days and the OD 600 Adjust to 1. This cell suspension is used to prepare a 1:500 dilution, equivalent to approximately 2x10 6 microbial cells / ml.

[0137] In order to assess MIC, 96 well plates were prepared with the test antimicrobial peptides. Each well contained 100 μl microbial cell cultures, 90 μl culture medium and 10 μl corresponding dilutions of peptide or control. The plates were incubated overnight (ON) at the optimal growth temperature of every kind of tested organism. Visual assessment results were determined, and MIC was determined according to growth or no growth.

[0138] Table 1. Organisms tested in MIC assays. Species name, classification, and growth conditions are given. DSM refers to the order number of the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ, Braunschweig, Germany). SF refers to organisms obtained from the Jena Microbiological Resources Collection (JMRC, Jena, Germany).

[0139]

[0140] MIC determinations were performed in at least two independent replicates using different peptide starting concentrations. Preliminary tests showed that any steps to purify the tested proteins did not affect the MICs obtained.

[0141] Results: Table 2 shows the MIC values ​​obtained for the peptides according to SEQ ID N°1, SEQ ID N°4 and SEQ ID N°5. The peptides having the sequence according to SEQ ID N°1 had an inhibitory effect on all fungal species tested. In addition, in most of the tested species, the peptides of the present invention inhibited fungal growth more effectively at lower concentrations than the peptides of the prior art.

[0142] Table 2. Results of MIC determinations using fungal species as test organisms and peptides of the invention (SEQ ID N°1) and prior art peptides (SEQ ID N°4 and N°5). Clotrimazole was used as reference.

[0143]

[0144] Table 3 shows the MIC values ​​obtained for the peptides according to SEQ ID N°1, SEQ ID N°4 and SEQ ID N°5 against bacterial species. The peptides having the sequence according to SEQ ID N°1 had an inhibitory effect on all fungal species tested. In addition, in almost all tested species, the peptides of the invention inhibited fungal growth more effectively at lower concentrations than the peptides of the prior art, with the exception of Mycosphaeria cruciferae, which was most effectively inhibited by the peptide having SEQ ID No 5.

[0145] Table 3. Results of MIC assays using bacterial species as test organisms and peptides of the invention (SEQ ID N°1) and prior art peptides (SEQ ID N°4 and N°5). Gentamycin and streptomycin were used as references.

[0146] Sequence Listing

[0147] SEQ ID N°1 (peptide according to one embodiment of the present invention)

[0148] YGRKQYERDPRQQYEQCQRRCSEATEEREQEQCEQRCEREYKEQQRQQEEE LQRQ

[0149] SEQ ID N°2 (signal peptide according to one embodiment of the present invention)

[0150] MVISKSPFIVLIFSLLLSFALLCSGVSA

[0151] SEQ ID N°3 (peptide according to one embodiment of the present invention)

[0152] MVISKSPFIVLIFSLLLSFALLCSGVSAYGRKQYERDPRQQYEQCQRRCSEATEEREQEQCEQRCEREYKEQQRQQEEELQRQ

[0153] SEQ ID N°4 (prior art peptide)

[0154] YERDPRQQYEQCQRRCSEATEEREQEQCEQRCEREYKEQQRQQEEE

[0155] SEQ ID N°5 (prior art peptide)

[0156] LQRQYQQCQGRCQEQQQGQREQQQCQRKCWEQYKEQERGEHENYHNHKK NRSEEEEGQQR

[0157] SEQ ID N°6 (Forward_BamH1 forward DNA primer)

[0158] CGCGGATCCTATGGCAGAAAACAATAT

[0159] SEQ ID N°7 (Reverse_Kpn1 forward DNA primer)

[0160] GGTACCTCATTGCCTTTGAAGCCTCTTCTTCTT

[0161] SEQ ID N°8 (HQ tag)

[0162] HQHQHQ

[0163] SEQ ID N°9 (HN tag)

[0164] HNHNHNHNHNHNH

[0165] SEQ ID N°10 (HAT tag)

[0166] KDHLIHNVHKEEHAHAHNK

[0167] SEQ ID N°11 (ALFA tag)

[0168] SRLEEELRRRLTE

[0169] SEQ ID N°12 (Avi tag)

[0170] GLNDIFEAQKIEWHE

[0171] SEQ ID N°13 (C-tag)

[0172] EPEA

[0173] SEQ ID N°14 (Calmodulin-Tag)

[0174] KRRWKKNFIAVSAANRFKKISSSGAL

[0175] SEQ ID N°15 (E-tag)

[0176] GAPVPYPDPLEPR

[0177] SEQ ID N°16 (FLAG-tag)

[0178] DYKDDDDK

[0179] SEQ ID N°17 (HA-tag)

[0180] YPYDVPDYA

[0181] SEQ ID N°18 (Myc-tag)

[0182] EQKLISEEDL

[0183] SEQ ID N°19 (NE-tag)

[0184] TKENPRSNQEESYDDNES

[0185] SEQ ID N°20 (Rho1D4 tag)

[0186] TETSQVAPA

[0187] SEQ ID N°21 (S-tag)

[0188] KETAAAKFERQHMDS

[0189] SEQ ID N°22 (SBP tag)

[0190] DEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP

[0191] SEQ ID N°23 (Streptomycin-tag)

[0192] WSHQ

Claims

1. A peptide having a sequence which has at least 95% sequence identity to SEQ ID N°1 or which differs from the sequence according to SEQ ID N°1 by a maximum of 3 amino acids.

2. The peptide according to claim 1, wherein The peptide has a sequence according to SEQ ID N°1.

3. The peptide according to any one of claims 1 to 2, wherein The peptide comprises a signal peptide at its N-terminus.

4. A peptide as claimed in any one of claims 1 to 3, wherein The signal peptide has at least 95% sequence identity to the sequence according to SEQ ID N°2 or is the sequence according to SEQ ID N°2.

5. A peptide as claimed in any one of claims 1 to 4, wherein The peptides have antibacterial and / or antifungal activity.

6. A peptide as claimed in any one of claims 1 to 5, wherein The activity is against Gram-positive bacteria, Gram-negative bacteria and / or fungi, preferably hyphal fungi and yeasts.

7. A peptide as claimed in any one of the preceding claims, wherein The peptide is derived from or isolated from Theobromacacao, preferably the cocoa variety CCN-51.

8. A peptide as claimed in any one of the preceding claims, wherein The peptide is a recombinant peptide or a synthetic peptide.

9. A peptide as claimed in any one of the preceding claims, wherein The peptide is provided with an affinity tag.

10. A composition comprising: the peptide according to any one of claims and an excipient.

11. The composition according to claim 10, wherein The composition is in liquid, semi-solid, solid or gaseous form, and / or wherein The composition is in the form of tablets, capsules, powders, granules, aerosols, pastes, syrups, suspensions, emulsions or solutions.

12. A peptide as claimed in any one of claims 1 to 9 or a composition as claimed in any one of claims 10 to 11 for use in therapy.

13. Use of a peptide as claimed in any one of claims 1 to 9 or a composition as claimed in claims 10 to 11 for treating a bacterial and / or fungal infection in a subject in need thereof.

14. The peptide for use according to claims 12 to 13, wherein The subject is a human or an animal.

15. Use of a peptide as claimed in claims 1 to 9 or a composition as claimed in claims 10 to 11 in crop protection, as a feed or food additive, as a preservative, as a detergent or in cosmetic applications.

16. The use according to claim 15, wherein The peptide or composition is used for controlling pathogens in plants, preferably crops.

17. The use according to claim 16, wherein The pathogen is selected from Gram-positive bacteria, Gram-negative bacteria and / or fungi, preferably hyphal fungi or yeast.

18. A method for obtaining the peptide according to any one of claims 1 to 9, wherein The peptide is a recombinant peptide or a synthetic peptide.

19. A vector comprising a coding sequence for a peptide having a sequence according to SEQ ID N°1 and optionally comprising a sequence of an N-terminal signal peptide having 95% sequence identity with a sequence according to SEQ ID N°2, wherein: The vector is designed to allow expression of the peptide in an expression system.

20. A transgenic plant expressing the peptide of any one of claims 1 to 9.

21. A method for producing the transgenic plant of claim 20, wherein: The method comprises the steps of introducing a nucleic acid encoding the peptide into a plant cell, thereby producing a transformed plant cell expressing the peptide, and regenerating the transformed plant cell into a transgenic plant.

22. A plant seed coated with the peptide or composition of any one of claims 1 to 11.

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