Antibacterial defensin peptide folded variants

Abstract

Compositions and methods of using defensin peptides and proteins are provided, including folded variants of antibacterial defensin peptides and proteins (DEFPFV) that exhibit advantageous antifungal properties and desirable resistance or sensitivity to protease cleavage that can be applied directly ex vivo to plants, humans, or animals, such as, for example, plants, humans, or animals. Or can be applied in vivo to plants, humans or animals.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 498,050, filed April 25, 2023, pursuant to 35 USC § 119. The entire contents of that provisional patent application are incorporated herein by reference, including but not limited to the description, claims, and abstract, and any figures, tables, appendices, or illustrations therein.

[0003] sequence list

[0004] This application contains a sequence list that has been electronically submitted in XML format, and is incorporated herein by reference in its entirety. The XML copy was created on April 19, 2024, named “P13859WO01_SequenceListing.xml”, and is 426,679 bytes in size. Technical Field

[0005] This disclosure generally relates to antimicrobial peptides and proteins for the control of pathogenic microorganisms. This document provides compositions and methods using defensin peptides and proteins, including folded variants of antimicrobial defensin peptides and proteins (DEF_PFVs) exhibiting favorable antifungal properties and desired resistance or sensitivity to protease cleavage, which can be applied directly in vitro to plants, humans, or animals, or in vivo to plants, humans, or animals. Background Technology

[0006] Both animals and plants are susceptible to microbial infections, which, if left untreated, can lead to significant morbidity and mortality in humans and veterinary patients, and cause losses in food crops, which are particularly vulnerable to contamination and spoilage during storage. Protecting vital agricultural crops from pathogenic microorganisms is crucial for maximizing crop yields. Fungal infections are a particular problem in humid climates and can become a major issue during crop storage, where such infections can lead to spoilage and contamination of food or feed products with mycotoxins. Unfortunately, modern planting methods, harvesting, and storage systems can promote plant pathogen infections.

[0007] Controlling pathogenic fungi in humans, veterinary animals, and plants is complicated because of the need to control a variety of pathogenic microorganisms, including fungi, dimorphic fungi, yeasts, molds, and oomycetes, of different genera simultaneously. For example, plants are susceptible to infection by a variety of microorganisms, including Alternaria, Ascochyta, Botrytis, Cercospora, Elsinoe, Phaeosphaeria, Erysiphe, Fusarium, Gaeumannomyces, Helminthosporium, Sphaeropsis, Diaporthe, Guignardia, Peronospora, Phoma, Melampsora, Pythium, Plasmopara, Podosphaera, Puccinia, Pythium, Pyrenophora, Rhizoctonia, Sclerotinia, Uncinula, and Verticillium species, all of which are recognized plant pathogens.

[0008] Therefore, in the presence of multiple pathogenic fungi, an antibacterial agent that controls only a limited variety of microbial pathogens provides insufficient or ineffective protection. Furthermore, in many cases, plant pathogenic microorganisms have developed resistance to existing antibacterial agents.

[0009] Antibacterial peptides have been described for treating microbial infections in animals and plants and are reviewed in Hancock, AAC 43(6) :1317 (1999) and Yount, PNAS 101(19) :7363 (2004). Antibacterial peptides include nodule-specific cysteine-rich (NCR) peptides and defensin peptides.

[0010] Defensins are small cysteine-rich peptides of about 45 to 54 amino acids that constitute an important part of the innate immunity of plants (Sathoff, Phytopathology 109 :402 (2019)). Defensins are widely distributed in plants and vary greatly in their amino acid sequences, but they all have a compact shape stabilized by four or five intramolecular disulfide bonds. Plant defensins have been identified to contain a conserved gamma-core motif that includes the conserved GXCX3-9C sequence (where X is any amino acid) (Lacerda et al., Frontiers in Microbio. (2014) 5(116): 1-10). The three-dimensional structure of the previously characterized gamma-core motif consists of two anti-parallel beta sheets interrupted by a turn region (Id.). The antibacterial activity of certain defensins has been associated with the presence of positively charged amino acid residues in the gamma-core motif (Spelbrink et al., Plant Physiol., 2004, 135, 2055-2067; Sagaram et al., 2013, PLoS ONE, 8(12): e82485).

[0011] Certain NCR peptides exhibit antimicrobial properties and can mediate bacterial cell death and early nodule senescence when applied to free-living bacteria. Yang, PNAS 114 :6848-6853 (2017) and Wang, PNAS 114 :6854-6859 (2017). Antimicrobial NCRs (AMPs) are cationic and have a conserved cysteine residue that forms an intramolecular disulfide bond. Cysteine substitution or disulfide bond modification can affect the antimicrobial activity of certain NCR peptides. For example, substitution of cysteines with serine, changing the pair of cysteines that form the disulfide bond, and oxidation all affect the antimicrobial activity of NCRs. Haag, J. Biol. Chem. 287(14): 10791-8 (2012) and Isozumi, Nature Sci. Rep. 11: 9923 (2021).

[0012] Despite recent advances in the prevention and treatment of microbial infections, and the recognition that certain antimicrobial peptides exhibit significant antimicrobial properties and activities, there remains a large unmet need for therapeutically effective methods of preventing, treating, and controlling pathogenic microorganisms. SUMMARY

[0013] Certain cationic antimicrobial defensin (DEF) peptides can form alternative disulfide bond pairs and, as a result, can fold into a variety of different conformations, each with a unique secondary (e.g., alpha helix and beta sheet) and tertiary structure, and these defensin peptide and protein structure variants have improved antimicrobial activity and, optionally, exhibit desired resistance or sensitivity to protease cleavage as compared to other NCRs PFVs having different disulfide bond pairs. As disclosed herein, defensin peptide fold variants exhibiting desired levels of antimicrobial activity and protease resistance or sensitivity can be purified from a heterogeneous mixture containing a plurality (i.e., two or more) of defensin peptide fold variants, and the purified defensin peptide fold variants can be advantageously used in the compositions and methods disclosed herein. Accordingly, in various aspects, the present disclosure provides: cysteine-rich defensin peptide fold variant 1 (DEF PFV1), compositions comprising antimicrobial DEF PFV1, methods for making antimicrobial DEF PFV1 compositions, methods for using such antimicrobial DEF PFV1 compositions, and devices (including medical devices) comprising antimicrobial DEF PFV1.

[0014] In certain embodiments, provided herein are compositions comprising a defensin peptide fold variant 1 (DEF PFV1), wherein the defensin peptide is a cationic defensin peptide comprising, in sequential amino acid sequence from its N-terminus: (1) a first cysteine pair motif comprising C1 and C2, (2) a first intervening sequence (IS1), (3) a second cysteine pair motif comprising C3 and C4, (4) a second intervening sequence (IS2), (5) a third cysteine pair motif comprising C5 and C6, (6) a third intervening sequence (IS3), and (7) a fourth cysteine pair motif comprising C7 and C8, wherein the cysteine residues C1, C2, C3, C4, C5, C6, C7, and C8 of the DEF PFV1 form a first set of disulfide bonds, and thereby DEF PFV1 adopts a first conformation that exhibits greater antimicrobial activity, increased protease resistance, or increased protease sensitivity compared to a defensin peptide fold variant 2 (DEF PFV2) having the same amino acid sequence as DEF PFV1 but forming a different second set of disulfide bonds between cysteine residues C1, C2, C3, C4, C5, C6, C7, and C8 and adopting a different second conformation, and wherein the composition is substantially free of DEF PFV2.

[0015] In certain embodiments, provided herein are compositions comprising a defensin peptide fold variant 1 (DEF PFV1), wherein the defensin peptide is a synthetic variant of a native defensin peptide comprising, in sequential amino acid sequence from its N-terminus: (1) a first amino acid pair motif comprising X1 and C2, (2) a first intervening sequence (IS1), (3) a first cysteine pair motif comprising C3 and C4, (4) a second intervening sequence (IS2), (5) a second cysteine pair motif comprising C5 and C6, (6) a third intervening sequence (IS3), and (7) a second amino acid pair motif comprising C7 and Y1, wherein the amino acids X1 and Y1 are each independently selected from the group of amino acids other than cysteine, and wherein the cysteines C2, C3, C4, C5, C6, and C7 of DEF PFV1 form a first set of disulfide bonds, and thereby DEF PFV1 adopts a first conformation that exhibits greater antimicrobial activity, increased protease resistance, or increased protease sensitivity compared to a defensin peptide fold variant 2 (DEF PFV2) having the same amino acid sequence as DEF PFV1 but forming a different second set of disulfide bonds between cysteines C2, C3, C4, C5, C6, and C7 and adopting a different second conformation, and wherein the composition is substantially free of DEF PFV2.

[0016] In certain embodiments, provided herein are compositions comprising a defensin peptide fold variant 1 (DEF PFV1), wherein the defensin peptide is a synthetic variant of a native defensin peptide comprising, in sequential amino acid sequence from its N-terminus: (1) a first amino acid pair motif comprising X1and Y1, (2) a first intervening sequence (IS1), (3) a first cysteine pair motif comprising C3and C4, (4) a second intervening sequence (IS2), (5) a second cysteine pair motif comprising C5and C6, (6) a third intervening sequence (IS3), and (7) a second amino acid pair motif comprising X2and Y2, wherein the amino acids X1, Y1, X2, and Y2are each independently selected from the group of amino acids other than cysteine, and wherein the cysteines C3, C4, C5, and C6of DEF PFV1 form a first set of disulfide bonds, and thereby DEF PFV1 adopts a first conformation that exhibits greater antimicrobial activity, increased protease resistance, or increased protease sensitivity compared to a defensin peptide fold variant 2 (DEF PFV2) having the same amino acid sequence as DEF PFV1 but forming a different second set of disulfide bonds between cysteines C3, C4, C5, and C6and adopting a different second conformation, and wherein the composition is substantially free of DEF PFV2.

[0017] In further related embodiments, provided herein are compositions comprising a defensin peptide fold variant 1 (DEF PFV1), wherein the defensin is a cationic defensin peptide comprising a defensin gamma core peptide sequence GXC5X3-9C6 (SEQ ID NO: 455) or a variant gamma core peptide sequence GXC5X3-10C6 (SEQ ID NO: 456) and at least two additional cysteine residues selected from the group consisting of cysteine residues corresponding to C1, C2, C3, C4, C7, and C8 of a reference defensin peptide, wherein the cysteine residues of DEF PFV1 form a first set of disulfide bonds, and thereby DEF PFV1 adopts a first conformation that exhibits greater antimicrobial activity, increased protease resistance, or increased protease sensitivity compared to a defensin peptide fold variant 2 DEF_PFV2) that exhibits greater antibacterial activity, increased protease resistance, or increased protease sensitivity compared to the first conformation, the defensin peptide fold variant 2 (DEF_PFV2) having the same amino acid sequence as DEF_PFV1 but forming a different second set of disulfide bonds between the cysteine residues and adopting a different second conformation, and wherein the composition is substantially free of DEF_PFV2. In certain embodiments, the cationic defensin peptide comprises a defensin gamma core peptide sequence GXC5X3-9C6 (SEQ ID NO: 455) or a variant gamma core peptide sequence GXC5X3-10C6 (SEQ ID NO: 456) and cysteine residues C7 and C8 of a reference defensin peptide, wherein the cysteine residues of DEF_PFV1 form a first set of disulfide bonds and thereby DEF_PFV1 adopts a first conformation, and wherein DEF_PFV2 adopts a second conformation DEF_PFV2) that exhibits greater antibacterial activity, increased protease resistance, or increased protease sensitivity compared to the first conformation, the defensin peptide fold variant 2 (DEF_PFV2) having the same amino acid sequence as DEF_PFV1 but forming a different second set of disulfide bonds between the cysteine residues and adopting a different second conformation, and wherein the composition is substantially free of DEF_PFV2.

[0018] In other embodiments, provided herein are methods for making the compositions disclosed herein, comprising: (a) separating a fraction comprising DEF_PFV1 from one or more fractions comprising DEF_PFV2 or DEF_PFV2 peptide fragments thereof from a mixture comprising both defensin peptide fold variant 1 (DEF_PFV1) and defensin peptide fold variant 2 (DEF_PFV2) or DEF_PFV2 peptide fragments thereof; and (b) combining the fraction comprising DEF_PFV1 or a preparation further purified from the DEF_PFV1 fraction with an agriculturally, pharmaceutically, or veterinarily acceptable carrier, diluent, or excipient, thereby making the composition.

[0019] In further embodiments, provided are methods for preventing or reducing crop damage or post-harvest loss caused by a plant pathogenic microorganism, the method comprising contacting a plant, a plant seed, a pre-harvest or post-harvest grain, a pre-harvest or post-harvest fruit, or a pre-harvest or post-harvest vegetable with an effective amount of a composition disclosed herein and under conditions suitable for preventing or reducing crop damage or post-harvest loss.

[0020] In yet further embodiments, provided are medical devices having antibacterial properties, the medical device comprising a medical device in operable combination with a composition disclosed herein, wherein the medical device comprises at least one surface that is topically coated or impregnated with the composition.

[0021] In other embodiments, methods are provided for treating, preventing, or inhibiting a microbial infection in a subject in need thereof, the methods comprising administering to the subject a therapeutically effective amount of a composition disclosed herein under conditions and for a duration suitable for treating, preventing, or inhibiting the microbial infection.

[0022] In yet other embodiments, plant parts are provided that are at least partially coated with a composition disclosed herein.

[0023] These and other related aspects of the present disclosure will be better understood and appreciated with reference to the following drawings and detailed description, which illustrate certain aspects of various embodiments. DETAILED DESCRIPTION

[0024] In certain embodiments, the present disclosure provides compositions enriched for cationic and antimicrobial defensin peptide fold variant 1 (DEF PFV1) having a defined set of disulfide bond pairs, adopting a unique conformation and / or having a unique tertiary structure, having improved antimicrobial activity, and optionally exhibiting a desired resistance or susceptibility to proteolytic cleavage as compared to other DEF PFV2 having different disulfide bond pairs. In certain embodiments, the present disclosure provides cationic and antimicrobial defensin peptide fold variant 1 (DEF PFV1) having a defined set of disulfide bond pairs, exhibiting a desired resistance or susceptibility to proteolytic cleavage as compared to other DEF PFV2 having different disulfide bond pairs. As disclosed herein, cationic and antimicrobial DEF PFV1 can be purified from a heterogeneous mixture comprising multiple (i.e., two or more) defensin peptide fold variants, and the desired purified DEF PFV1 can be advantageously used in the compositions, methods, and devices disclosed herein.

[0025] These and other aspects of the present disclosure can be better understood with reference to the following non-limiting definitions.

[0026] Definitions

[0027] As used herein, the terms “microbe” and “microbes” and “microbial” are used to mean bacteria, fungi (including yeast), and oomycetes.

[0028] As used herein, the phrases “susceptible microbe (or microbes),” “susceptible microbial infection,” and the like refer to a microbe or microbial infection thereof that infects plants, humans, and non-human animals, the microbial growth of which is susceptible to inhibition by antimicrobial peptides (including defensin peptides), variants thereof, and multimers.

[0029] As used herein, the phrases "combating microbial damage," "combating or controlling microbial damage," or "controlling microbial damage" refer to reducing the damage caused to a plant, such as an ornamental or a crop plant, or an ornamental or crop plant product, as a result of infection by a microbial pathogen. More generally, these phrases refer to reducing the adverse effects caused by the presence of a pathogenic microbe in a crop plant. Adverse effects of microbial growth are understood to include any type of plant tissue damage or necrosis, any type of reduction in plant yield, any reduction in the value of the crop plant product, and / or the production of undesirable microbial metabolites or microbial growth byproducts, including mycotoxins.

[0030] As used herein, the phrases "inhibiting the growth of a plant pathogenic microbe," "inhibiting microbial growth," and the like, refer to methods that result in any measurable reduction in the growth of a microbe, where microbial growth includes, but is not limited to, any measurable reduction in the number and / or extent of microbial cells, spores, conidia, or mycelium. As used herein, "inhibiting the growth of a plant pathogenic microbe" is also understood to include any measurable reduction in the adverse effects caused in a plant by the growth of a microbe. Adverse effects of microbial growth in a plant include any type of plant tissue damage or necrosis, any type of reduction in plant yield, any reduction in the value of the crop plant product, and / or the production of undesirable microbial metabolites or microbial growth byproducts, including but not limited to mycotoxins. As used herein, the phrase "inhibition of microbial growth" and the like, unless otherwise specified, can include inhibition in a plant, a human, or an animal.

[0031] As used herein, belonging to an "antibacterial peptide" refers to a peptide, particularly a defensin peptide, that exhibits any one or more of the following characteristics: inhibits the growth of a microbial cell, kills a microbial cell, disrupts or delays a stage of the life cycle of a microbe (such as spore germination, spore formation, or mating), and / or disrupts microbial cell infection, penetration, or spread within a plant or other susceptible subject, including a human, a livestock, a poultry, a fish, or a companion animal (e.g., a dog or a cat).

[0032] As used herein, the term "defensin peptide" refers to a peptide comprising a conserved gamma core peptide. Plant defensins have been previously characterized as comprising a conserved GXCX3-9C gamma core peptide sequence (SEQ ID NO: 455), wherein X is any amino acid residue (Lacerda et al.), or a conserved GXCX3-10C variant gamma core peptide sequence (SEQ ID NO: 456), wherein X is any amino acid residue. Thus, as used in the present disclosure, a plant defensin or a C-terminal peptide comprising a fragment thereof can comprise a conserved GXCX3-9C or GXCX3-10C gamma core peptide sequence, wherein X is any amino acid residue. Defensin peptides include proteins having antimicrobial effects, proteins that are permeable through the plasma membrane, proteins that can bind to phospholipids, proteins that can bind to sphingolipids, or proteins having any combination of these properties. Defensin peptides can be naturally occurring or non-naturally occurring (e.g., synthetic and / or chimeric).

[0033] As used herein, the term "defensin peptide variant" refers to a modified defensin peptide comprising: (i) a conserved gamma core peptide and at least one amino acid substitution in the source defensin peptide; and / or (ii) a modified gamma core variant sequence GXC5X3-9 (F / W / Y) (SEQ ID NO: 457) or GXC5X3-10 (F / W / Y) (SEQ ID NO: 458), wherein the C6 residue is substituted with F, W, or Y. In certain embodiments, the defensin peptide variants provided herein are smaller than full-length defensin peptides (e.g., peptides comprising, consisting essentially of, or consisting of: (i) 30 or fewer amino acid residues; or (ii) 15, 16, or 17 to 30 amino acid residues).

[0034] The phrase "reference defensin C-terminal peptide" is used herein to refer to a less than full-length defensin peptide comprising a conserved GXCX3-9C gamma core peptide sequence (SEQ ID NO: 455) or a conserved GXCX3-10C variant gamma core peptide sequence (SEQ ID NO: 456) and two additional conserved cysteine residues located C-terminal to the gamma core peptide sequence, wherein the cysteine located closest to the N-terminus of the reference defensin C-terminal peptide corresponds to the cysteine located closest to the N-terminus of the gamma core sequence of SEQ ID NO: 455 or SEQ ID NO: 456.

[0035] As used herein, the phrase "cation tolerant" refers to a defensin peptide or variant thereof that exhibits comparable in vitro antifungal or antibacterial activity, or a reduction in in vitro antifungal or antibacterial activity that is no more than about 1.5-fold, 2-fold, 3-fold, or 4-fold, in the presence of 100 mM KC1 or 100 mM NaCl, as compared to the antifungal activity of the defensin peptide or variant thereof in the absence of KC1 or NaCl.

[0036] As used herein, the phrase "consensus sequence" refers to an amino acid sequence that is generated by aligning two or more homologous sequences and deriving a new sequence that has a set of conserved alternative amino acid residues of the homologous sequences at each position of the peptide sequence.

[0037] As used herein, the term "peptide variant" refers to any peptide having antibacterial activity that comprises one or more non-conservative amino acid substitutions in a defensin peptide. In addition to such non-conservative substitutions, a defensin peptide variant can further comprise a peptide having conservative amino acid substitutions in the defensin peptide variants provided herein, a deletion of from 1 to 5 amino acids from the N-terminus, and an internal deletion of one or more amino acid residues.

[0038] As used herein, the phrase "percent identity" or "sequence identity" refers to the number of identical elements (i.e., amino acids or nucleotides) in a sequence within a defined length of the aligned protein segments that produce the greatest number of identical elements in the alignment, and is calculated by dividing the number of identical elements by the total number of elements within the defined length of the aligned segment and multiplying by 100.

[0039] As used herein, the term "heterologous peptide" or "heterologous protein" refers to a peptide, such as a defensin peptide, that does not occur in a cell or organism in nature. Thus, a heterologous peptide includes a peptide that is located in a subcellular location, an extracellular location, or a peptide that is expressed in a tissue other than the subcellular location, extracellular location, or a tissue in which the peptide or protein is found in a naturally occurring cell or organism.

[0040] As used herein, the term "amino acid" refers to an organic compound that contains an amino (-NH3) and carboxylic acid (-CO2) functional group, and a side chain (R group) that is unique to each amino acid. In certain instances, in polypeptides, the single letter amino acid code is used herein, as follows: G - glycine (Gly); P - proline (Pro); A - alanine (Ala); V - valine (Val); L - leucine (Leu); I - isoleucine (lie); M - methionine (Met); C - cysteine (Cys); F - phenylalanine (Phe); Y - tyrosine (Tyr); W - tryptophan (Trp); H - histidine (His); K - lysine (Lys); R - arginine (Arg); Q - glutamine (Gin); N - asparagine (Asn); E - glutamic acid (Glu); D - aspartic acid (Asp); S - serine (Ser); or T - threonine (Thr).

[0041] As used herein, the terms "acidic" or "anionic" are used interchangeably to refer to amino acids such as aspartic acid and glutamic acid.

[0042] As used herein, the terms "basic" or "cationic" are used interchangeably to refer to amino acids such as arginine, histidine, and lysine.

[0043] As used herein, the phrase "percent identity" or "sequence identity" refers to the number of amino acids in a sequence that are identical within a defined length of the two peptide or protein segments that produce the greatest number of identical elements in an alignment, and is calculated by dividing the number of identical elements by the total number of elements within the defined length of the aligned segments and multiplying by 100.

[0044] It should be understood that, when any prior definition is inconsistent with a definition provided in any patent or non-patent reference incorporated by reference herein, any patent or non-patent reference listed herein, or any patent or non-patent reference found elsewhere, the prior definition will be used herein.

[0045] The practice of the present disclosure will employ, unless otherwise indicated, conventional methods and techniques of microbiology, molecular biology, structural biology, and enzymology, which are well known and easily available to those skilled in the art. Such methods and techniques are explained fully in the laboratory manuals and scientific and patent literature. See, e.g., Sambrook, et al., “Molecular Cloning: A Laboratory Manual” (2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989); Maniatis et al., “Molecular Cloning: A Laboratory Manual” (1982); “DNA Cloning: A Practical Approach, Vols. I & II” (Glover, ed.); “Oligonucleotide Synthesis” (Gait, ed., 1984); Ausubel et al. (eds.), “Current Protocols in Molecular Biology” (John Wiley & Sons, 1994); “Nucleic Acid Hybridization” (Hames & Higgins, eds., 1985); “Transcription and Translation” (Hames & Higgins, eds., 1984); “Animal Cell Culture” (Freshney, ed., 1986); and Perbal, “A Practical Guide to Molecular Cloning” (1984). All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0046] Antimicrobial defensin peptide fold variant (DEF PFV) compositions

[0047] In certain embodiments, the present disclosure provides compositions comprising cationic antimicrobial defensin (DEF) peptide fold variant (PFV). Compositions according to these embodiments (1) include DEF peptide fold variants 1 (DEF PFV1) having desirable antimicrobial activity and / or resistance / susceptibility to proteolysis, and (2) exclude DEF peptide fold variants 2 (DEF PFV2) having undesirable reduced antimicrobial activity and / or undesirable resistance / susceptibility to proteolysis.

[0048] Representative cationic antimicrobial defensin peptides that can be advantageously used in the compositions disclosed herein are listed in Table 1. Variants of such defensin peptides comprising amino acid insertions, deletions, or substitutions can also be used in the compositions provided herein.

[0049]

[0050] Certain exemplary antimicrobial defensin peptides and C-terminal fragments thereof that can be advantageously used in the compositions disclosed herein are listed in Table 2. Variants of such defensin peptides comprising amino acid insertions, deletions, or substitutions can also be used in the compositions provided herein.

[0051]

[0052] In certain aspects of these compositions, the defensin peptide is a cationic defensin peptide having: (1) a first cysteine pair motif comprising C1 and C2, (2) a first intervening sequence (IS1), (3) a second cysteine pair motif comprising C3 and C4, (4) a second intervening sequence (IS2), (5) a third cysteine pair motif comprising C5 and C6, (6) a third intervening sequence (IS3), and (7) a fourth cysteine pair motif comprising C7 and C8. Typically, the cationic defensin peptides according to these embodiments have an isoelectric point between 8.0 and 12.0, and / or DEF PFV1 exhibits greater protease resistance or greater protease sensitivity than the corresponding DEF PFV2. Typically, these compositions are substantially free of DEF PFV2.

[0053] According to these aspects, the cysteine residues C1, C2, C3, C4, C5, C6, C7, and C8 of DEF_PFV1 form a first set of disulfide bonds, and thereby DEF_PFV1 adopts a first conformation that exhibits a desired antibacterial activity and a preferred resistance / susceptibility to proteolysis, as compared to a defensin peptide folding variant 2 (DEF_PFV2) having the same amino acid sequence as DEF_PFV1 but forming a different second set of disulfide bonds between cysteine residues C1, C2, C3, C4, C5, C6, C7, and C8 and adopting a different second conformation that has an undesired reduced antibacterial activity and / or an undesired resistance / susceptibility to proteolysis.

[0054] Exemplified herein is a defensin peptide folding variant 1 (DEF_PFV1) in which the defensin peptide is a natural defensin peptide or a synthetic variant of a natural defensin peptide having one or more amino acid insertions, deletions, and / or substitutions in the natural defensin peptide, including a substitution of one hydrophobic amino acid with another hydrophobic amino acid, a substitution of one cationic amino acid with another cationic amino acid, or a substitution of one anionic amino acid with another cationic or anionic amino acid.

[0055] In other aspects of embodiments of the present application, the defensin peptide folding variant 1 (DEF_PFV1) forms a first set of disulfide bonds including a C1-C8 disulfide bond, a C2-C5 disulfide bond, a C3-C6 disulfide bond, and a C4-C7 disulfide bond, and / or wherein the defensin peptide folding variant 2 (DEF_PFV1) forms a second set of disulfide bonds other than the C1-C8 disulfide bond, the C2-C5 disulfide bond, the C3-C6 disulfide bond, and the C4-C7 disulfide bond.

[0056] In other aspects of these embodiments, the defensin can include a first cysteine pair motif that is a C1-10AA-C2 motif flanked by an N-terminal C1 and a C-terminal C2 having ten amino acids (A1, A2, A3, A4, A5, A6, A7, A8, A9, and A10) in sequential order from its N-terminal end, wherein A1 can be an anionic amino acid selected from Asp (D) and Glu (E); wherein A4 can be a polar neutral amino acid selected from Ser (S) and Thr (T); wherein A7 can be an aromatic amino acid selected from Phe (F), Tyr (Y), and Trp (W); and / or wherein A9 can be an aliphatic amino acid selected from Gly (G), Ala (A), Val (V), Leu (L), Ile (I), and Pro (P). 10 ), wherein A1 can be an anionic amino acid selected from Asp (D) and Glu (E); wherein A4 can be a polar neutral amino acid selected from Ser (S) and Thr (T); wherein A7 can be an aromatic amino acid selected from Phe (F), Tyr (Y), and Trp (W); and / or wherein A9 can be an aliphatic amino acid selected from Gly (G), Ala (A), Val (V), Leu (L), Ile (I), and Pro (P).

[0057] In related aspects of these embodiments, the defensin can comprise a second cysteine pair motif that is a C3-3AA-C4 motif flanked by a N-terminal C3 and a C-terminal C4, having three amino acids (A 11 , A 12 , and A 13 ) in consecutive sequence from its N-terminus, wherein A 11 is an aliphatic amino acid selected from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), He (I), and Pro (P) or a cationic amino acid selected from the group consisting of His (H), Arg (R), and Lys (K), and / or wherein A 13 is an aliphatic amino acid selected from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), He (I), and Pro (P) or a polar neutral amino acid selected from the group consisting of Ser (S) and Thr (T).

[0058] In other related aspects of these embodiments, the defensin can comprise a third cysteine pair motif that is a C5-5-6AA-C6 motif flanked by a N-terminal C5 and a C-terminal C6, having five or six amino acids (A 14 , A 15 , A 16 , A 17 , A 18 , and optionally A 19 ) in consecutive sequence from its N-terminus, wherein A 14 is a cationic amino acid selected from His (H), Arg (R), and Lys (K), or an aliphatic amino acid selected from Gly (G), Ala (A), Val (V), Leu (L), He (I), and Pro (P), or a polar neutral amino acid selected from Ser (S) and Thr (T); wherein A 16 is an aliphatic amino acid selected from Gly (G), Ala (A), Val (V), Leu (L), He (I), and Pro (P) or an aromatic amino acid selected from Phe (F), Tyr (Y), and Trp (W); wherein A 18 is a cationic amino acid selected from the group consisting of His (H), Arg (R), and Lys (K).

[0059] In other related aspects of these embodiments, the defensin can comprise a fourth cysteine pair motif that is a C7-3AA-C8 motif flanked by a N-terminal C7 and a C-terminal C8, having three amino acids (A 20 , A21 and A 22 ), wherein A 20 is a polar neutral amino acid selected from the group consisting of Ser (S) and Thr (T); wherein A 21 is a cationic amino acid selected from the group consisting of His (H), Arg (R), and Lys (K); and / or wherein A 22 is an aliphatic amino acid selected from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), He (I), and Pro (P).

[0060] In further related aspects of these embodiments, the defensin can include a first intervening sequence (IS1) having five amino acids and / or a second intervening sequence (IS2) having nine to eleven amino acids, and / or a third intervening sequence (IS3) having one amino acid selected from an aliphatic amino acid selected from Gly (G), Ala (A), Val (V), Leu (L), He (I), and Pro (P) or an aromatic amino acid selected from Phe (F), Tyr (Y), and Trp (W).

[0061] In other aspects of these compositions, the defensin peptide is a synthetic variant of a native defensin peptide having: (1) a first amino acid pair motif including X1and C2, (2) a first intervening sequence (IS1), (3) a second cysteine pair motif including C3and C4, (4) a second intervening sequence (IS2), (5) a third cysteine pair motif including C5and C6, (6) a third intervening sequence (IS3), and (7) a fourth cysteine pair motif including C7and Y1. Typically, the cationic defensin peptides according to these embodiments have an isoelectric point between 8.0 and 12.0, and / or DEF_PFV1exhibits greater protease resistance or greater protease sensitivity than the corresponding DEF_PFV2. Typically, these compositions are substantially free of DEF_PFV2.

[0062] In other aspects, the cysteines C2, C3, C4, C5, C6, and C7 of DEF_PFV1may form a first set of disulfide bonds, and thereby DEF_PFV1may adopt a first conformation that exhibits greater antibacterial activity than a defensin peptide fold variant 2 (DEF_PFV2) having the same amino acid sequence as DEF_PFV1but forming a different second set of disulfide bonds between cysteines C2, C3, C4, C5, C6, and C7 and adopting a different second conformation.

[0063] In further aspects, amino acids X1and Y1are independently selected from the group of amino acids other than cysteine. For example, amino acids X1and Y1may both be Ser (S) or Thr (T).

[0064] Exemplified herein are defensin peptide fold variant 1 (DEF PFV1), which forms a first set of disulfide bonds including C2-C5 disulfide bond, C3-C6 disulfide bond, and C4-C7 disulfide bond, and / or defensin peptide fold variant 2 (DEF PFV2), which forms a second set of disulfide bonds other than C2-C5 disulfide bond, C3-C6 disulfide bond, and C4-C7 disulfide bond.

[0065] In other aspects of these embodiments, the synthetic defensin peptide includes a first cysteine pair motif that is an X1-10AA-C2 motif flanked by N-terminal X1and C-terminal C2, having ten amino acids (A1, A2, A3, A4, A5, A6, A7, A8, A9, and A10) in sequential order from the N-terminal thereof, wherein A1may be an anionic amino acid selected from Asp (D) and Glu (E); wherein A4may be a polar neutral amino acid selected from Ser (S) and Thr (T); wherein A7may be an aromatic amino acid selected from Phe (F), Tyr (Y), and Trp (W); and / or wherein A9may be an aliphatic amino acid selected from Gly (G), Ala (A), Val (V), Leu (L), He (I), and Pro (P). 10 In other aspects of these embodiments, the synthetic defensin peptide includes a first cysteine pair motif that is an X1-10AA-C2 motif flanked by N-terminal X1and C-terminal C2, having ten amino acids (A1, A2, A3, A4, A5, A6, A7, A8, A9, and A10) in sequential order from the N-terminal thereof, wherein A1may be an anionic amino acid selected from Asp (D) and Glu (E); wherein A4may be a polar neutral amino acid selected from Ser (S) and Thr (T); wherein A7may be an aromatic amino acid selected from Phe (F), Tyr (Y), and Trp (W); and / or wherein A9may be an aliphatic amino acid selected from Gly (G), Ala (A), Val (V), Leu (L), He (I), and Pro (P).

[0066] In related aspects of these embodiments, the synthetic defensin peptide includes a second cysteine pair motif that is a C3-3AA-C4 motif flanked by N-terminal C3and C-terminal C4, having three amino acids (A 11 , A 12 , and A 13 in sequential order from the N-terminal thereof, wherein A 11 is an aliphatic amino acid selected from Gly (G), Ala (A), Val (V), Leu (L), He (I), and Pro (P), or a cationic amino acid selected from the group consisting of His (H), Arg (R), and Lys (K), and / or wherein A 13 is an aliphatic amino acid selected from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), He (I), and Pro (P), or a polar neutral amino acid selected from the group consisting of Ser (S) and Thr (T).

[0067] In other related aspects of these embodiments, the synthetic defensin peptide includes a third cysteine pair motif that is a C5-5-6AA-C6 motif flanked by a N-terminal C5 and a C-terminal C6, having five or six amino acids (A 14 , A 15 , A 16 , A 17 , A 18 , and optionally A 19 ) in sequential order from its N-terminus, wherein A 14 is a cationic amino acid selected from His (H), Arg (R), and Lys (K), or an aliphatic amino acid selected from Gly (G), Ala (A), Val (V), Leu (L), He (I), and Pro (P), or a polar neutral amino acid selected from Ser (S) and Thr (T); wherein A 16 is an aliphatic amino acid selected from Gly (G), Ala (A), Val (V), Leu (L), He (I), and Pro (P), or an aromatic amino acid selected from Phe (F), Tyr (Y), and Trp (W); wherein A 18 is a cationic amino acid selected from the group consisting of His (H), Arg (R), and Lys (K).

[0068] In yet other related aspects of these embodiments, the synthetic defensin peptide includes a fourth cysteine pair motif that is a C7-3AA-Y1 motif flanked by a N-terminal C7 and a C-terminal Y1, having three amino acids (A 20 , A 21 , and A 22 ) in sequential order from its N-terminus, wherein A 20 is a polar neutral amino acid selected from the group consisting of Ser (S) and Thr (T); wherein A 21 is a cationic amino acid selected from the group consisting of His (H), Arg (R), and Lys (K); and / or wherein A 22 is an aliphatic amino acid selected from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), He (I), and Pro (P).

[0069] In further related aspects of these embodiments, the synthetic defensin peptide can have a first intervening sequence (IS1) of five amino acids, and / or a second intervening sequence (IS2) of nine to eleven amino acids, and / or a third intervening sequence (IS3) of one amino acid selected from aliphatic amino acids selected from Gly (G), Ala (A), Val (V), Leu (L), He (I), and Pro (P), or aromatic amino acids selected from Phe (F), Tyr (Y), and Trp (W).

[0070] In other aspects of these compositions, the defensin peptide is a synthetic variant of a native defensin peptide having, in sequential amino acid sequence from its N-terminus: (1) a first amino acid pair motif comprising X1and Y1, (2) a first intervening sequence (IS1), (3) a first cysteine pair motif comprising C3and C4, (4) a second intervening sequence (IS2), (5) a second cysteine pair motif comprising C5and C6, (6) a third intervening sequence (IS3), and (7) a second amino acid pair motif comprising X2and Y2. The cationic defensin peptide according to these embodiments has an isoelectric point between 8.0 and 12.0, and / or DEF_PFV1exhibits greater protease resistance or greater protease sensitivity than the corresponding DEF_PFV2. Typically, these compositions are substantially free of DEF_PFV2.

[0071] In other aspects, the amino acids X1, Y1, X2, and Y2are each independently selected from amino acids other than cysteine, and the cysteines C3, C4, C5, and C6of DEF_PFV1form a first set of disulfide bonds, and thereby DEF_PFV1adopts a first conformation that exhibits greater antimicrobial activity than a defensin peptide fold variant 2 (DEF_PFV2) having the same amino acid sequence as DEF_PFV1but forms a different second set of disulfide bonds between cysteines C3, C4, C5, and C6and adopts a different second conformation. In certain compositions, the amino acids X1, Y1, X2, Y2are each Ser (S) or Thr (T).

[0072] In further aspects, the first amino acid pair motif is an X1-10AA-Y1motif flanked by an N-terminal X1and a C-terminal Y1, having, in sequential sequence from its N-terminus, ten amino acids (A1, A2, A3, A4, A5, A6, A7, A8, A9, and A10) that are each independently selected from amino acids other than cysteine. 10), wherein A1 is an anionic amino acid selected from Asp (D) and Glu (E), and / or wherein A4 is a polar neutral amino acid selected from the group consisting of Ser (S) and Thr (T), and / or wherein A7 is an aromatic amino acid selected from Phe (F), Tyr (Y) and Trp (W), and / or wherein A9 is an aliphatic amino acid selected from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), lie (I) and Pro (P).

[0073] In other aspects, the first cysteine pair motif is a C3-3AA-C4 motif flanked by a N-terminal C3 and a C-terminal C4, having three amino acids (A 11 , A 12 and A 13 ) in consecutive sequence from its N-terminal end, wherein A 11 is an aliphatic amino acid selected from Gly (G), Ala (A), Val (V), Leu (L), lie (I) and Pro (P), or a cationic amino acid selected from His (H), Arg (R) and Lys (K), and / or wherein A 13 is an aliphatic amino acid selected from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), lie (I) and Pro (P), or a polar neutral amino acid selected from the group consisting of Ser (S) and Thr (T).

[0074] In yet other aspects, the second cysteine pair motif is a C5-5-6AA-C6 motif flanked by a N-terminal C5 and a C-terminal C6, having five or six amino acids (A 14 , A 15 , A 16 , A 17 , A 18 and optionally A 19 ) in consecutive sequence from its N-terminal end, wherein A 14 is a cationic amino acid selected from His (H), Arg (R) and Lys (K), or an aliphatic amino acid selected from Gly (G), Ala (A), Val (V), Leu (L), lie (I) and Pro (P), or a polar neutral amino acid selected from Ser (S) and Thr (T), and / or wherein A 16 is an aliphatic amino acid selected from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), lie (I) and Pro (P), or an aromatic amino acid selected from the group consisting of Phe (F), Tyr (Y) and Trp (W), and / or wherein A18 A is a cationic amino acid selected from the group consisting of His (H), Arg (R), and Lys (K).

[0075] In further aspects, the second amino acid pair motif is the X2-3AA-Y2 motif flanked by N-terminal X2 and C-terminal Y1, having three amino acids (A 20 , A 21 , and A 22 from the N-terminal in sequential order, wherein A 20 is a polar neutral amino acid selected from the group consisting of Ser (S) and Thr (T), and / or wherein A 21 is a cationic amino acid selected from His (H), Arg (R), and Lys (K), and / or wherein A 22 is an aliphatic amino acid selected from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), He (I), and Pro (P).

[0076] In yet further aspects, IS1 comprises five amino acids, and / or IS2 comprises nine to eleven amino acids, and / or IS3 comprises one amino acid that is an aliphatic amino acid selected from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), He (I), and Pro (P) or an aromatic amino acid selected from the group consisting of Phe (F), Tyr (Y), and Trp (W).

[0077] In yet further aspects, the defensin peptide comprises the defensin gamma core peptide sequence GXC5X3-9C6 (SEQ ID NO: 455) or the variant gamma core peptide sequence GXC5X3-10C6 (SEQ ID NO: 456).

[0078] In other aspects of these compositions, the defensin peptide is a cationic defensin peptide that is a natural defensin peptide or a synthetic variant of a natural defensin peptide having one or more amino acid insertions, deletions, and / or substitutions in the natural defensin peptide, and optionally, wherein the substitutions in the natural defensin peptide comprise at least one of: substitution of a hydrophobic amino acid with a hydrophobic amino acid, substitution of a cationic amino acid with a cationic amino acid, or substitution of an anionic amino acid with a cationic or anionic amino acid, and / or wherein the amino acid sequence of the defensin peptide has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% sequence identity to any one of SEQ ID NOs: 1-454 or SEQ ID NO: 459-SEQ ID NO: 472.

[0079] In further aspects, the cysteine residues of DEF_PFV1 form a first set of disulfide bonds, and thereby DEF_PFV1 adopts a first conformation that exhibits greater antimicrobial activity than a defensin peptide folding variant 2 (DEF_PFV2) having the same amino acid sequence as DEF_PFV1 but forming a different second set of disulfide bonds between the cysteine residues and adopting a different second conformation. The cationic defensin peptides according to these embodiments have an isoelectric point between 8.0 and 12.0, and / or DEF_PFV1 exhibits greater protease resistance or greater protease sensitivity than the corresponding DEF_PFV2. Typically, these compositions are substantially free of DEF_PFV2. (DEF_PFV2) having the same amino acid sequence as DEF_PFV1 but forming a different second set of disulfide bonds between the cysteine residues and adopting a different second conformation. The cationic defensin peptides according to these embodiments have an isoelectric point between 8.0 and 12.0, and / or DEF_PFV1 exhibits greater protease resistance or greater protease sensitivity than the corresponding DEF_PFV2. Typically, these compositions are substantially free of DEF_PFV2.

[0080] In other aspects, the defensin peptide includes a defensin gamma core peptide sequence GXC5X3-9C6 (SEQ ID NO: 455) or a variant gamma core peptide sequence GXC5X3-10C6 (SEQ ID NO: 456), GXC5X3-9(F / W / Y) (SEQ ID NO: 457), or GXC5X3-10(F / W / Y) (SEQ ID NO: 458) and at least two or three additional cysteine residues selected from the group consisting of cysteine residues corresponding to C1, C2, C3, C4, C7, and C8 of the reference defensin peptide. In certain embodiments, the defensin peptide comprises a C-terminal fragment of the defensin peptide comprising the defensin gamma core peptide sequence GXC5X3-9C6 (SEQ ID NO: 455) or the variant gamma core peptide sequence GXC5X3-10C6 (SEQ ID NO: 456) and the cysteine residues corresponding to C7 and C8 of the reference defensin peptide.

[0081] In related aspects, the cationic defensin peptide includes at least four additional cysteine residues selected from the cysteines corresponding to C1, C2, C3, C4, C7, and C8 of the reference defensin peptide, and / or wherein one to four of the cysteine residues corresponding to C1, C2, C3, C4, C7, and C8 of the reference defensin peptide are substituted or deleted with a different amino acid, and / or wherein one or more cysteines are substituted with a Ser (S) or Thr (T) residue.

[0082] In other related aspects, at least one of the cysteine residues corresponding to C1, C2, C3, C4, C7, and C8 of the reference defensin peptide is substituted with a different amino acid, at least one of the cysteine residues corresponding to C1, C2, C3, C4, C5, C6, C7, and C8 of the reference defensin peptide is deleted, wherein at least two of the cysteine residues corresponding to C1, C2, C3, C4, C7, and C8 of the reference defensin peptide are retained, and / or the defensin peptide comprises cysteine residues corresponding to C5, C6, C7, and C8 of the reference defensin peptide.

[0083] In other aspects, the defensin peptide comprises a C5-5-6AA-C6 motif flanked by an N-terminal C5 and a C-terminal C6, having three amino acids (A 14 , A 15 , A 16 , A 17 , A 18 and optionally A 19 in sequential order from its N-terminus, wherein: (i) A 14 is a cationic amino acid selected from His (H), Arg (R), and Lys (K), or an aliphatic amino acid selected from Gly (G), Ala (A), Val (V), Leu (L), lie (I), and Pro (P), or a polar neutral amino acid selected from the group consisting of Ser (S) and Thr (T); wherein A 16 is an aliphatic amino acid selected from Gly (G), Ala (A), Val (V), Leu (L), lie (I), and Pro (P), or an aromatic amino acid selected from Phe (F), Tyr (Y), and Trp (W); and / or (iii) wherein A 18 is a cationic amino acid selected from His (H), Arg (R), and Lys (K).

[0084] In further aspects, the defensin peptide comprises a C7-3AA-C8 motif flanked by an N-terminal C7 and a C-terminal C8, having three amino acids (A 20 , A 21 , and A 22 in sequential order from its N-terminus, wherein (i) A 20 is a polar neutral amino acid selected from Ser (S) and Thr (T); (ii) A 21 is a cationic amino acid selected from His (H), Arg (R), and Lys (K); and / or (iii) A 22an aliphatic amino acid selected from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), He (I), and Pro (P).

[0085] Defensin peptides according to these embodiments can comprise an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% sequence identity to any one of the exemplary defensin peptides shown in Table 2. Compositions according to these embodiments can comprise an agriculturally, pharmaceutically, or veterinarily acceptable carrier, diluent, or excipient, depending on the particular application contemplated.

[0086] DEF_PFV1 can be isolated from a mixture comprising the desired DEF_PFV1 and one or more undesired DEF_PFV2 to provide a composition enriched in the desired DEF_PFV1. Compositions comprising the desired DEF_PFV1 generally exhibit higher units of activity (e.g., units of antimicrobial activity per microgram or micromole of defensin protein) relative to compositions comprising the desired DEF_PFV1 and one or more DEF_PFV2 that is less active. Depending on the particular application contemplated for the DEF_PFV1 composition, it can be desirable to use a DEF_PFV1 that has increased resistance to proteases or increased sensitivity to proteases (e.g., to serine proteases, including trypsin) relative to the undesired DEF_PFV2.

[0087] Compositions comprising the desired DEF PFV1 can be substantially free of the undesired DEF PFV2. In certain embodiments, such compositions can comprise less than about 20%, 10%, 5%, 2%, 1%, or 0.5% by weight of the undesired DEF PFV2, and correspondingly at least about 80%, 90%, 95%, 98%, 99%, or 99.5% by weight of the desired DEF PFV1. In certain embodiments, compositions comprising the desired DEF PFV1 and substantially free of the undesired DEF PFV2 can comprise less than about 20%, 10%, 5%, 2%, 1%, or 0.5% by weight of the undesired DEF PFV2 in the composition than the total amount of the desired DEF PFV1 and the undesired DEF PFV2. In certain embodiments, compositions comprising the desired DEF PFV1 and substantially free of the undesired DEF PFV2 can comprise at least about 80%, 90%, 95%, 98%, 99%, or 99.5% by weight of the desired DEF PFV1 of the total defensin peptides in the composition (e.g., in the form of the desired DEF PFV and one or more undesired DEF PFV1 and one or more undesired DEF PFV2). In certain embodiments, less than 20%, 10%, 5%, 2%, 1%, or 0.5% by weight of the total defensin peptides in the composition (e.g., in the form of the desired DEF PFV and one or more undesired DEF PFV1 and one or more undesired DEF PFV2) is the undesired DEF PFV2, and correspondingly at least about 80%, 90%, 95%, 98%, 99%, or 99.5% by weight of the total defensin peptides in the composition is the desired DEF PFV1.

[0088] Without being bound by theory, it is contemplated that compositions comprising or enriched for the desired DEF PFV1 that exhibit increased protease resistance relative to the undesired DEF PFV2 can be useful in certain methods disclosed herein, where it is desirable to extend the shelf-life or half-life of the DEF PFV1. Alternatively, it is contemplated that compositions comprising or enriched for the desired DEF PFV1 that exhibit increased protease sensitivity relative to the undesired DEF PFV2 can be useful in methods where it is desirable to decrease the half-life of the DEF PFV1.

[0089] Defensin peptides suitable for generating DEF PFV1 peptides can include defensin peptides comprising the amino acid sequences set forth in Table 1 or Table 2. Variants of these defensin peptides are contemplated, wherein, for example, one or more hydrophobic, basic, and / or acidic amino acid residues are substituted with alternative hydrophobic, basic, and / or acidic amino acid residues. In the examples provided herein, defensin peptide variants comprise an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity over the entire length of one or more defensin peptides set forth in Table 1 and Table 2 and / or set forth in SEQ ID NO: 1 to 454 or 459 to 472.

[0090] Conserved cysteine residues in defensin peptides can be identified as CI, C2, C3, C4, C5, C6, C7, and C8, as set forth in Table 1. For defensin peptides, the most amino-terminal conserved cysteine residue is CI and the most carboxy-terminal cysteine residue is C8, as set forth in Table 1.

[0091] In certain aspects of these examples, defensin peptides that can adopt alternative disulfide bond pairings and constitute defensin peptide folding variants can carry a net positive charge at neutral pH. For example, certain defensin peptides carry a net positive charge of at least +4, +5, +6, +7, +8, +9, or +10 to +12, +13, +14, or +15, or at least +5, +6, +7, +8, +9, or +10 at neutral pH. It will be appreciated that such a net positive charge in a defensin peptide can be achieved by methods including (i) retaining cationic (basic) amino acid residues found in defensin peptides, including those set forth in Table 1 and Table 2, respectively, or substituting such residues with another cationic amino acid residue; (ii) substituting anionic or polar amino acid residues found in defensin peptides, including those set forth in Table 1 and Table 2, respectively, with a basic amino acid residue; or a combination of (i) and (ii). Such a net positive charge in a defensin peptide can be achieved by preferentially selecting or substituting cationic amino acid residues at variable positions in a defensin peptide corresponding to variable positions.

[0092] In other aspects of these embodiments, cathelicidin peptides that can adopt alternative disulfide bond pairings and constitute a cathelicidin peptide fold variant can include a significant proportion of hydrophobic amino acid residues. For example, a cathelicidin peptide can include at least about 25%, 26%, 28%, 30%, 32%, 34%, or 36% to 37%, 38%, 40%, 42%, or 45% hydrophobic amino acid residues, or at least about 25%, 26%, 28%, 30%, 32%, 34%, 36%, 37%, or 38% hydrophobic amino acid residues. It will be appreciated that such a percentage of hydrophobic amino acids in a cathelicidin peptide can be achieved by (i) retaining a hydrophobic amino acid residue found in a cathelicidin peptide (including the cathelicidin peptides shown in Tables 1 and 2, respectively) or substituting such a residue with another hydrophobic amino acid residue or a neutral polar amino acid residue; (ii) substituting a polar amino acid residue found in a cathelicidin peptide (including the cathelicidin peptides shown in Tables 1 and 2, respectively) with a hydrophobic amino acid residue; (iii) substituting a neutral polar amino acid with a hydrophobic amino acid; or a combination of (i), (ii), and (iii). Such a percentage of hydrophobic amino acids in a cathelicidin peptide can be achieved by preferentially selecting or substituting a hydrophobic amino acid residue at a variable position in a cathelicidin peptide corresponding to a variable position.

[0093] In further aspects of these embodiments, a cathelicidin peptide includes four, five, six, or seven of the eight conserved C1, C2, C3, C4, C5, C6, C7, and C8 cysteine residues listed in Table 1. Cathelicidin peptide fold variant 1 (DEF PFV1) can form a first set of disulfide bonds including the C1-C8 disulfide bond, the C2-C5 disulfide bond, the C3-C6 disulfide bond, and the C4-C7 disulfide bond, and / or wherein cathelicidin peptide fold variant 2 (DEF PFV2) forms a second set of disulfide bonds other than the C1-C8 disulfide bond, the C2-C5 disulfide bond, the C3-C6 disulfide bond, and the C4-C7 disulfide bond.

[0094] A defensin peptide can also include four, five, six, or seven of the eight conserved C1, C2, C3, C4, C5, C6, C7, and C8 cysteine residues. For example, one or more of the cysteinyl residues in a defensin peptide can be substituted with or deleted for a different amino acid residue to provide a defensin peptide having only four, five, six, or seven of the eight conserved C1, C2, C3, C4, C5, C6, C7, and C8 cysteine residues. Alternatively, one or more of the conserved C1, C2, C3, C4, C5, C6, C7, and C8 cysteine residues in a defensin peptide can be substituted with another amino acid residue (including a glycine, serine, threonine, cysteine, cystine, tyrosine, asparagine, or glutamine residue) to provide a defensin peptide having only four, five, six, or seven of the eight conserved C1, C2, C3, C4, C5, C6, C7, and C8 cysteine residues. For example, it can be desirable to substitute one or two of the conserved cysteine residues with a serine residue to provide a defensin peptide having only four, five, six, or seven of the eight conserved C1, C2, C3, C4, C5, C6, C7, and C8 cysteine residues. Such a defensin peptide having only four, five, six, or seven of the eight conserved cysteine residues can comprise a deletion or substitution of the C1, C2, C3, and / or C4 cysteine residues, and thus will comprise the C5, C6, C7, and C8 cysteine residues. Alternatively, a defensin peptide having only four, five, six, or seven of the eight conserved cysteine residues can comprise a deletion or substitution of the C1 and C2 cysteine residues in the defensin peptide, and thus comprise the C3, C4, C5, C6, C7, and C8 cysteine residues. 6、 C7 and C8 cysteine residues.

[0095] Alternatively, a defensin peptide can comprise the defensin gamma core peptide sequence GXC5X3-9C6 (SEQ ID NO: 455) or the variant gamma core peptide sequence GXC5X3-10C6 (SEQ ID NO: 456) and at least two additional cysteine residues selected from the group consisting of the cysteine residues corresponding to C1, C2, C3, C4, C7, and C8 of the reference defensin peptide. A cationic defensin peptide variant can include at least four additional cysteine residues selected from the cysteines corresponding to C1, C2, C3, C4, C5, C6, C7, and C8 of the reference defensin peptide, and / or one to four of the cysteine residues corresponding to C1, C2, C3, C4, C7, and C8 of the reference defensin peptide are substituted with a different amino acid or are deleted. One or more of the cysteines can be substituted with a Ser (S) or Thr (T) residue.

[0096] A defensin peptide variant can also include substitution of at least one of the cysteine residues corresponding to C1, C2, C3, C4, C7, and C8 of a reference defensin peptide, and / or deletion of at least one of the cysteine residues corresponding to C1, C2, C3, C4, C5, C6, C7, and C8 of a reference defensin peptide, with a different amino acid, wherein typically at least two of the cysteine residues corresponding to C1, C2, C3, C4, C7, and C8 of a reference defensin peptide are retained. For example, a defensin peptide variant can include cysteine residues corresponding to C5, C6, C7, and C8 of a reference defensin peptide, and substitution of C1, C2, C3, and C4 with another amino acid such as a Ser (S) or Thr (T) residue, or a defensin peptide variant can include deletion of one or more of C1, C2, C3, and C4.

[0097] Suitable defensin peptides for use in preparing compositions according to these embodiments can include an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to one or more of the defensin peptide sequences shown in Table 1 or one or more of the defensin peptide sequences shown in Table 2, wherein one or more of the hydrophobic, basic, and / or acidic amino acid residues are substituted with other hydrophobic, basic, and / or acidic amino acid residues, respectively.

[0098] Any one of the defensin peptides shown in Table 1 and Table 2 or one or more of the amino acids in a variant defensin peptide can be substituted with other amino acids that have similar charge and polarity as the original amino acid, i.e., conservative amino acid substitutions. Substitutes for an amino acid within a defensin peptide sequence can be selected from other members of the class to which the original amino acid belongs. Amino acids can be divided into four groups: (1) acidic amino acids; (2) basic amino acids; (3) neutral polar amino acids; and (4) neutral nonpolar amino acids. Representative amino acids in these different groups include, but are not limited to: (1) acidic (anionic; negatively charged) amino acids such as aspartic acid and glutamic acid; (2) basic (cationic; positively charged) amino acids such as arginine, histidine, and lysine; (3) neutral polar amino acids such as glycine, serine, threonine, cysteine, cystine, tyrosine, asparagine, and glutamine; (4) neutral nonpolar (hydrophobic) amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Conservative amino acid changes within a defensin peptide sequence can be made by substituting one amino acid within one of these groups for another amino acid within the same group.

[0099] Biological functional equivalents of defensin peptides can have 10 or fewer conservative amino acid changes, seven or fewer conservative amino acid changes, or five, four, three, two, or one conservative amino acid change. The encoding nucleotide sequence (e.g., a gene, plasmid DNA, cDNA, or synthetic DNA) will thus have the corresponding base substitutions, allowing it to encode a biologically functional equivalent of a defensin peptide.

[0100] Also provided are certain semi-conservative substitutions in defensin peptides, including: (i) substitution of a neutral, polar amino acid residue with a neutral, non-polar (hydrophobic) amino acid residue; or (ii) substitution of a neutral, non-polar (hydrophobic) amino acid residue with a neutral, polar amino acid residue. In particular, provided is a semi-conservative substitution of a neutral, polar tyrosine residue with a hydrophobic amino acid residue. Also provided is a semi-conservative substitution of a hydrophobic amino acid residue with a tyrosine residue. Biological functional equivalents of defensin peptides can have 10 or fewer semi-conservative amino acid changes, seven or fewer semi-conservative amino acid changes, or five, four, three, two, or one semi-conservative amino acid change.

[0101] A functional fragment of any of the defensin peptides or defensin peptide variants disclosed herein can include an amino-terminal deletion, a carboxy-terminal deletion, an internal deletion, or any combination thereof, leaving intact the conserved cysteine residues of the defensin, or comprising at least four, five, six, or seven of the eight conserved Cl, C2, C3, C4, C5, C6, C7, and C8 cysteine residues of the reference defensin peptide. The functional fragment can comprise a deletion of at least one, two, three, four, five, six, seven, or more amino acid residues of the amino-terminal, carboxy-terminal, internal region, or any combination thereof. For example, an antibacterial fragment of a defensin peptide can comprise at least about 10, 14, 15, 18, or 20 to about 22, 24, 25, 26, 27, or 28 amino acid residues of the C-terminal end of the defensin peptide. Chimeric defensin peptides comprising portions of any of the defensins disclosed herein or comprising variants or fragments of these defensins can also be used alone or in the defensin proteins provided herein.

[0102] Methods for making, testing, and using antimicrobial DEF PFV compositions

[0103] In related embodiments, the disclosure provides methods for making, testing, and using an antibacterial defensin (DEF) peptide fold variant (PFV) -comprising composition that (1) includes a defensin peptide fold variant 1 (DEF PFV1) having a desired antibacterial activity and / or resistance / susceptibility to proteolysis, and (2) excludes a defensin peptide fold variant 2 (DEF PFV2) having the same or reduced antibacterial activity and / or an unwanted resistance or susceptibility to proteolysis.

[0104] DEF PFV1 can comprise an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to an amino acid sequence set forth in Table 1 or Table 2, or a variant of an amino acid sequence set forth in Table 1 or Table 2, wherein one or more of a hydrophobic, basic, and / or acidic amino acid residue is substituted with a hydrophobic, basic, and / or acidic amino acid residue, respectively; a variant of an amino acid sequence set forth in Table 1 or Table 2, wherein one or more neutral polar amino acid (e.g., tyrosine) residue is substituted with a hydrophobic amino acid residue; and / or wherein one or more hydrophobic amino acid residue is substituted with a neutral polar amino acid residue (e.g., tyrosine); functional fragments thereof, and chimeras thereof.

[0105] Defensin peptides or proteins can be synthesized de novo from the defensin peptide sequences disclosed herein, or can be expressed from nucleotide sequences encoding the defensin peptides or proteins. Sequences of the peptide or nucleotide sequences encoding the proteins can be deduced from the defensin peptide sequences by reference to the genetic code. Computer programs such as "BackTranslate" (GCG™ package, Acclerys, Inc. San Diego, CA) can be used to convert a peptide sequence into the corresponding nucleotide sequence encoding the peptide.

[0106] Expression of defensin peptides in yeast and filamentous fungi to produce DEF PFV1 is specifically contemplated herein. Construction of expression vectors for production of heterologous proteins in various genera of yeast is well established. Typically, such expression vectors comprise a promoter operably linked to a sequence of interest, which is operably linked to a polyadenylation or terminator region. Examples of genera of yeast that have been used successfully to express heterologous genes include Candida, Kluyveromyces, Hansenula, Pichia, Saccharomyces, Schizosaccharomyces, and Yarrowia. A general description of expression vectors and transformation systems for Saccharomyces is found in Kingsman et al. (1985) Biotechnol Genet Eng Rev. 3 :377-416. Expression vectors and transformation systems that can be used for yeasts other than Saccharomyces are described in Reiser et al. (1990) Adv Biochem Eng Biotechnol. 43 :75-102.

[0107] Other examples of fungal systems that can be used to express defensin peptides include filamentous fungal systems such as Acremonium, Aspergillus, Bifida, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coptotrichum, Cryptococcus, Filibascula, Fusarium, Humicola, Magnusiomyces, Mucor, Myrothecium, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, and Trichoderma systems (e.g., U.S. Patent Nos. 11,046,736 and 11,180,767, both of which are incorporated by reference herein in their entireties). Other systems that can be used to express defensin peptides include Chrysosporium lucknowense systems (e.g., U.S. Patent No. 8,871,493 and U.S. Patent No. 9,175,296, both of which are incorporated by reference herein in their entireties).

[0108] It is also specifically contemplated herein to express defensin peptides in bacterial cells, including Escherichia species (e.g., Escherichia coli) to produce defensin PFVs. Systems for expressing proteins comprising disulfide bonds can be adapted for use in expressing defensin peptides in E. coli, including those disclosed in U.S. Patent Publication No. US 2020 / 0172915 (which is incorporated by reference herein in its entirety) and Berkmen, M. Protein Expr Purif. 2012; 82(1): 240-51. doi: 10.1016 / j.pep.2011.10.009. Systems for expressing proteins comprising disulfide bonds can be adapted for use in expressing defensin peptides in E. coli, including those disclosed in Kuddus et al., (2017) Biotechnol Prog 233:1520-1528. doi: 10.1002 / btpr. Protein Science 2508; Kiedzierska et al. (2008) Protein Expr Purif 60, 82-88; Chang et al., (2015) Amino Acids 47, 579-587; Buchko et al. (2018) (2018) Protein Science 27, 1611-1623; and Marques et al., (2008) J Appl Microbiol 106, 1640-1648; Pazgier, M., Lubkowski, J. (2006) Protein Expr Purif 49, 1-8.

[0109] In general, the promoters and polyadenylation regions are selected based on their operability in a given bacterial, yeast, or fungal host. For example, the AOXl or AOX2 promoter of Pichia pastoris can be used in conjunction with the AOXl, AOX2, p40, or p76 polyadenylation sequences of P. pastoris to express a heterologous protein, such as a defensin peptide. Both the AOXl and AOX2 promoters are particularly useful in P. pastoris because both promoters allow for high level expression of a linked heterologous gene under induction by the addition of methanol to the growth medium.

[0110] The use of these P. pastoris promoters and polyadenylation sequences is described in U.S. Patent No. 4,855,231, which is expressly incorporated by reference herein in its entirety. Similarly, the H. polymorpha MOX, DHAS, or FMDH promoters can be used to express a heterologous protein, such as a defensin, in H. polymorpha. The MOX, DHAS, or FMDH promoters are particularly useful in H. polymorpha because these promoters allow for high level expression of a linked heterologous gene under induction by the addition of methanol to the growth medium. The use of the MOX and DHAS promoters in H. polymorpha is described in U.S. Patent No. 5,741,672, while the use of the FMDH promoter in H. polymorpha is described in U.S. Patent No. 5,389,525, each of which is expressly incorporated by reference herein in its entirety. For K. lactis, the lactase promoter and polyadenylation sequences can be used to express a heterologous gene, such as a defensin. Expression of a heterologous gene operably linked to the lactase promoter and polyadenylation sequences is achieved by culturing K. lactis in the presence of galactose. The use of the lactase promoter and polyadenylation sequences in K. lactis is described in U.S. Patent No. 6,602,682, which is expressly incorporated by reference herein in its entirety.

[0111] Yeast, bacterial, or fungal expression vectors that secrete a heterologous protein, such as a defensin, into the growth medium by the transformed yeast or fungus are also contemplated. Secretion of a mature defensin peptide is typically achieved by operably linking a signal peptide sequence or a signal peptide and propeptide sequence to the sequence encoding the mature defensin protein or peptide. Examples of useful signal peptides for secreting a heterologous protein in yeast include, but are not limited to, the alpha factor signal peptide, invertase signal peptide, and PHOl signal peptide, all of which are derived from yeast. The alpha factor signal peptide is typically derived from S. cerevisiae, K. lactis, or Candida, while the PHOl signal peptide is derived from P. pastoris.

[0112] A particularly useful signal peptide sequence for secretion of a protein in yeast is derived from the Saccharomyces cerevisiae alpha factor and is described in U.S. Patent Nos. 4,546,082; 4,588,684; 4,870,008; and 5,602,034, each of which is expressly incorporated herein in its entirety. The Saccharomyces cerevisiae alpha factor signal peptide and propeptide sequence consists of amino acids 1-83 of the primary, unprocessed translation product of the Saccharomyces cerevisiae alpha mating factor gene (GenBank Accession No. P01149). In certain embodiments, the signal peptide sequence of the alpha mating factor, comprising amino acids 1 to about 19 to 23 of the alpha mating factor protein precursor, can be directly linked to the N-terminus of the mature defensin protein to provide secretion of the mature defensin protein. In this case, the signal peptide is cleaved from the mature defensin protein during secretion. Alternatively, the signal peptide and propeptide of the alpha mating factor can be operably linked to the mature defensin coding sequence by a cleavage site sequence. The cleavage site sequence can comprise a variety of sequences for proteolytic processing of the leader sequence and the gene of interest.

[0113] In the native Saccharomyces cerevisiae alpha mating factor gene, the cleavage site sequence corresponds to amino acid residues 84 to 89 and is represented by the sequence Lys84-Arg85-Glu86-Ala87-Glu88-Ala89 (SEQ ID NO: 473). The Lys-Arg sequence corresponds to a KEX2 protease recognition site, while the Glu-Ala-Glu-Ala sequence corresponds to a duplicated dipeptidyl amino peptidase or STE13 recognition site. In certain embodiments, a DNA fragment encoding the 89 amino acid Saccharomyces cerevisiae alpha factor signal, propeptide coding region and the entire native spacer coding region (i.e., the N-terminal 89 amino acid residues of the alpha mating factor precursor protein, including the Lys-Arg KEX2 protease cleavage site at residues 84 and 85 and the Glu-Ala-Glu-Ala dipeptidyl amino peptidase or STE13 recognition site at residues 86 to 89) is operably linked to a sequence encoding a mature defensin protein.

[0114] When the N-terminal 89 amino acids of the alpha mating factor precursor protein are fused to the N-terminus of a heterologous protein, such as a defensin, the pro-peptide sequence is typically separated from the heterologous protein by cleavage at the KEX2 or STE13 recognition site by an endogenous yeast protease. In other embodiments, a DNA fragment encoding a smaller 85 amino acid S. cerevisiae alpha factor signal peptide, pro-peptide, and KEX2 spacer element (i.e., the N-terminal 85 amino acid residues of the alpha mating factor precursor protein containing only the Lys-Arg KEX2 protease cleavage site at residues 84 and 85) is operably linked to a sequence encoding a mature defensin protein. When the N-terminal 85 amino acids of the alpha mating factor precursor protein are fused to the N-terminus of a heterologous protein, such as a defensin, the pro-peptide sequence is typically separated from the heterologous protein by cleavage at the KEX2 recognition site by an endogenous yeast protease. Thus, a defensin protein can be expressed without the glu-ala repeat sequence.

[0115] To obtain a transformed yeast expressing a defensin peptide, the yeast defensin expression cassette (e.g., a yeast promoter, a yeast signal peptide coding sequence, a mature defensin protein sequence, and a polyadenylation sequence) is typically combined with other sequences for selection of the transformed yeast. Examples of useful selectable marker genes include genes encoding ADE proteins, HIS5 proteins, HIS4 proteins, LEU2 proteins, URA3 proteins, ARG4 proteins, TRP1 proteins, LYS2 proteins, proteins that confer resistance to the antibiotics bleomycin or phleomycin, proteins that confer resistance to chloramphenicol, proteins that confer resistance to G418 or geneticin, proteins that confer resistance to hygromycin, proteins that confer resistance to methotrexate, AR04-OFP proteins, and FZF1-4 proteins. Similar selectable marker cassettes that confer antibiotic resistance or rescue auxotrophic traits can be used in bacterial or fungal systems.

[0116] DNA molecules comprising a yeast defensin expression cassette and a selectable marker gene are introduced into a yeast cell by techniques such as transfection into yeast protoplasts or electroporation. In certain embodiments, DNA molecules comprising a yeast defensin expression cassette and a selectable marker gene are introduced as a linear DNA fragment that integrates into the genome of the transformed yeast host cell. Integration can occur at random sites in the genome of the yeast host cell or at specific sites in the genome of the yeast host cell. Integration at specific sites in the genome of the yeast host cell is typically achieved by homologous recombination between sequences contained in the expression vector and sequences in the genome of the yeast host cell.

[0117] Homologous recombination is typically achieved by linearizing the expression vector within the homologous sequence (e.g., when integrating the expression vector into the endogenous AOX1 gene of a Pichia host cell, within the AOX1 promoter sequence of the Pichia expression vector). In other embodiments, the yeast expression cassette can also include additional sequences, such as an autonomously replicating sequence (ARS), for replication of DNA containing the expression cassette as an extrachromosomal (non-integrated) element. Such extrachromosomal elements are typically maintained in the yeast cell by the presence of a selectable marker gene linked in series, through continuous selection. Yeast artificial chromosomes (YACs) containing sequences for replication and mitotic segregation are another type of vector that can be used to maintain DNA constructs in yeast hosts.

[0118] Yeast, bacterial, or fungal cells transformed with a yeast or fungal defensin expression cassette can be used to produce defensin PFV mixtures, which can be used to obtain preparations enriched in a desired DEF PFV. The desired DEF PFV can be used as an antimicrobial agent to produce an antimicrobial composition that can be applied to a plant or food, or to produce an antimicrobial composition that can be administered to a human or non-human subject. The method of producing a mixture of DEF PFVs generally first includes the step of culturing a yeast, bacterial, or fungal cell transformed with a defensin expression cassette under conditions in which the yeast cell expresses a mature defensin molecule.

[0119] In general, the conditions under which the yeast, bacterial, or fungal cell expresses a mature defensin molecule are conditions that allow or specifically induce expression of the yeast promoter operably linked to the defensin-encoding sequence in the defensin expression cassette. When the yeast is Pichia and the signal peptide / MD gene is controlled by an AOX1 or AOX2 promoter, the addition of methanol to the growth medium will result in expression of the mature defensin protein. Similarly, when the yeast is Hansenula and the signal peptide / MD gene is controlled by an MOX, DHAS, or FMDH promoter, the addition of methanol to the growth medium will result in expression of the mature defensin protein. Alternatively, when the yeast is Kluyveromyces and the signal peptide / MD gene is controlled by a lactase promoter, the addition of galactose to the growth medium will result in expression of the mature defensin protein.

[0120] After the transformed yeast, bacterial, or fungal culture has been incubated under conditions in which the mature defensin peptide is expressed for a sufficient time, the desired defensin molecule PFV can be separated from any undesired DEF PFV molecules isolated from the culture as a mixture. The sufficient time can be determined by periodically collecting portions or aliquots of the culture and assaying for the presence of the desired and / or undesired DEF PFV. Analytical methods such as HPLC can be used to monitor the production of the desired DEF PFV and the undesired DEF PFV. In certain embodiments, the incubation period of the culture can be optimized to produce the desired DEF PFV, resulting in a culture enriched in the desired DEF PFV.

[0121] The methods according to these embodiments comprise (a) separating a fraction comprising DEF_PFV1 from one or more fractions comprising DEF_PFV2 or DEF_PFV2 peptide fragments thereof from a mixture comprising DEF_PFV1 and DEF_PFV2 or DEF_PFV2 peptide fragments thereof; and (b) combining the fraction comprising DEF_PFV1 or a preparation further purified from the DEF_PFV1 fraction with an agriculturally, pharmaceutically, or veterinarily feasible carrier, diluent, or excipient, thereby making the composition.

[0122] The mixture comprising DEF_PFV1 and DEF_PFV2 or DEF_PFV2 peptide fragments thereof can be prepared by (a) culturing a microorganism expressing a recombinant polynucleotide comprising a transcriptional promoter operably linked to a polynucleotide encoding a signal peptide that is in frame with and upstream of a polynucleotide encoding DEF_PFV1 or a variant thereof in a fermentation broth; (b) separating the microorganism from the fermentation broth comprising the mixture (e.g., by size exclusion chromatography); (c) disrupting the microorganism to obtain a cell lysate, and (d) separating insoluble debris from the cell lysate in an aqueous fraction comprising the mixture.

[0123] The isolation of the desired DEF_PFV from the culture can be partial or complete. For yeast, bacterial, or fungal signal peptide-defensin expression vectors in which a signal peptide is operably linked to a sequence encoding a mature defensin protein, a mixture comprising the desired DEF_PFV and any DEF_PFV or other undesired DEF_PFV (e.g., increased or decreased protease resistance) can be recovered from the yeast cell culture medium, bacterial periplasm, or fungal cell culture medium. In certain embodiments, yeast or fungal cell culture medium comprising the mature defensin protein in the form of the desired DEF_PFV and undesired DEF_PFV can be separated from the yeast or fungal cells by centrifugation or filtration, thereby providing a composition comprising a mixture of the desired and undesired DEF_PFV. In certain embodiments, a preparation of bacterial periplasmic protein comprising the mature defensin protein in the form of the desired DEF_PFV and undesired DEF_PFV can be obtained by a method comprising physical force, pressure, osmotic shock, or chemical (e.g., EDTA + heat or a detergent) treatment, thereby providing a composition comprising a mixture of the desired and undesired DEF_PFV.

[0124] Such methods for isolating bacterial periplasmic proteins can be adapted from published methods for isolating other periplasmic proteins (e.g., Schimek et al. (2020) Biotechnology progress, 36(5), e2999. doi.org / 10.1002 / btpr.2999). The yeast or fungal cell culture media or preparation of bacterial periplasmic proteins containing mature defensin proteins can be further processed by any combination of dialysis and / or concentration techniques (e.g., precipitation, lyophilization, filtration) to produce a composition containing one or more DEF PFVs (e.g., a mixture of desired DEF PFVs and undesired DEF PFVs). In certain embodiments, a filtration size exclusion membrane can be used to enrich a fraction containing a mixture of desired DEF PFVs and undesired DEF PFVs.

[0125] A combination of various separation techniques can also be used to produce a mixture of DEF PFVs. For example, the cell culture media can be separated from the cells by centrifugation, followed by dialysis or adjustment. In certain embodiments, the buffer used for dialysis or adjustment is a 25 mM sodium acetate buffer at about pH 4.5 to pH 6.0. The dialysate is then subjected to ion exchange chromatography. For example, a cation exchange resin such as CM-Sephadex C-25 equilibrated with a 25 mM sodium acetate buffer at about pH 6.0 can be used. The defensin proteins bound to the cation exchange resin are washed and then eluted. For example, the column described above is washed with a 25 mM sodium acetate buffer at about pH 6.0 and subsequently eluted in 1 M NaCl, 50 mM Tris, pH 7.6. Fractions containing defensin proteins are identified by assay or UV absorbance and subsequently concentrated by size cutoff filtration membranes. The concentrated defensin proteins are then dialyzed to obtain substantially or essentially pure defensin proteins in a buffer. Buffers include, for example, 10 mM Tris, pH 7.6.

[0126] Production of a composition comprising or enriched for a desired DEF PFV can be obtained by methods that separate the desired DEF PFV from the undesired DEF PFV. In certain embodiments, such methods can include standard protein separation techniques that enrich for the desired DEF PFV based on different chemical properties other than mass (e.g., the hydrodynamic radius of different DEF PFVs and / or the charge characteristics of different DEF PFVs). In certain embodiments, chromatographic techniques, such as sedimentation, size exclusion chromatography (SEC), ion exchange chromatography, and / or affinity chromatography can be used to produce a composition enriched for the desired DEF PFV. Published methods for separating proteins having different conformations by SEC can be adapted to separate a desired DEF PFV from one or more undesired DEF PFVs (La Verde et al., Bio Protoc. 20 April 2017; 7(8): e2230. doi: 10.21769 / BioProtoc.2230).

[0127] In certain embodiments, the method can provide a composition comprising the desired DEF PFV, wherein the composition comprises less than 5%, 2%, 1%, 0.5%, 0.25%, or 0.1% by weight of one or more undesired DEF PFVs. In certain embodiments, the method can provide a composition comprising the desired DEF PFV, wherein less than 5%, 2%, 1%, or 0.5% by weight of the total of the desired DEF PFV and one or more undesired DEF PFVs in the composition is an undesired DEF PFV. In certain embodiments, the method can provide a composition comprising the desired DEF PFV, wherein at least about 80%, 90%, 95%, 98%, 99%, or 99.5% by weight or moles of the total defensin peptide in the composition is the desired DEF PFV.

[0128] In certain embodiments, the method can provide a composition in which less than 20%, 10%, 5%, 2%, 1%, or 0.5% of the total defensin peptides in the composition by weight are undesired DEF_PFVs, or in which at least about 80%, 90%, 95%, 98%, 99%, or 99.5% of the total defensin peptides in the composition by weight are desired DEF_PFVs. The total defensin peptides in the composition by weight can be determined by quantifying the combined total of desired and undesired DEF_PFVs in the composition. Quantification of total defensin peptides and / or isolated DEF_PFVs can be accomplished by a variety of methods, including immunoassay (e.g., ELISA, RIA when purified defensin peptides are used to establish a reference curve) or mass spectrometry (Zhang et al. Methods Mol Biol. 2010; 673:211-222. doi:10.1007 / 978-1-60761-842-3_13).

[0129] In certain embodiments, a composition enriched for desired DEF_PFVs can be obtained by methods that exploit the relative susceptibility of the desired DEF_PFVs to proteases (e.g., serine proteases such as trypsin). In embodiments in which the desired DEF_PFVs exhibit enhanced resistance to proteases (e.g., at a given protease concentration), a mixture comprising desired and undesired DEF_PFVs can be treated with a concentration of protease that will cleave the undesired DEF_PFVs, but leave the desired DEF_PFVs intact. The intact DEF_PFVs can then be separated from fragments of defensin peptides generated by cleavage of the undesired DEF_PFVs by methods including size exclusion membrane filtration, SEC, and / or IEC. Proteases suitable for such methods can include trypsin family serine proteases. In certain embodiments, the serine protease will cleave the undesired DEF_PFVs at the carboxy terminus of arginine or lysine residues of the undesired DEF_PFVs under non-denaturing conditions. In certain embodiments, the trypsin family serine protease is recombinant bovine, porcine, human, or microbial trypsin, optionally wherein the microbial trypsin is Streptomyces trypsin or a variant thereof. In certain embodiments, about 6.25, 10, or 12.5 to about 15, 20, or 25 BAEE units / mL of trypsin (e.g., Sigma-Aldrich T1426, St. Louis, MO) can be incubated with the DEF_PFVs in 60 mM sodium dihydrogen phosphate buffer, pH 7.5, at 37°C for about 1, 2, or 3 to about 4, 5, or 6 hours to cleave the undesired DEF_PFVs, but leave the desired DEF_PFVs intact. One BAEE (N-benzoyl-L-arginine ethyl ester) unit is defined as the amount of enzyme required to hydrolyze 1 μmol of BAEE per minute at pH 7.5 and 25°C. α- Benzoyl-L-arginine ethyl ester hydrochloride) Trypsin Substrate Units will produce 0.001 change in A253 (absorbance change at 253 nm) per minute at pH 7.6, 25°C, in a reaction volume = 3.2 ml (1 cm path length) with BAEE as substrate.

[0130] In certain aspects of these methods, a mixture comprising DEF_PFV1 and DEF_PFV2 or DEF_PFV2 peptide fragments is treated with a protease under non-denaturing conditions sufficient to produce DEF_PFV2 peptide fragments but insufficient to produce DEF_PFV1 peptide fragments. Suitable proteases include trypsin family serine proteases, which cleave defensin peptides at the carboxy-terminal end of Arg (R) or Lys (K). Trypsin family serine proteases include recombinant bovine, porcine, human, or microbial trypsin, and optionally, wherein the microbial trypsin is Streptomyces trypsin or a variant thereof.

[0131] According to the present embodiments, DEF_PFV1 can exhibit increased phospholipid binding compared to the corresponding DEF_PFV2. A desired DEF_PFV1 provided herein can exhibit a lower IC50 value against one or more microbial pathogens, enhanced binding to phospholipids, or any combination thereof compared to either (i) a mixture comprising the desired DEF_PFV1 and one or more corresponding undesired DEF_PFV2, or (ii) the DEF_PFV2 alone. A desired DEF_PFV1 that exhibits a lower IC50 value against one or more microbial pathogens can be identified and / or selected by selecting a DEF_PFV1 that provides enhanced phospholipid binding compared to either (i) a mixture comprising the desired DEF_PFV1 and one or more corresponding undesired DEF_PFV2, or (ii) the DEF_PFV2 alone.

[0132] Suitable assays for determining enhanced phospholipid binding include protein-lipid overlay assays (e.g., Dowler et al., 2002, Sci STKE. 2002 Apr 23; 2002(129): 16), surface plasmon resonance assays (e.g., Baron and Pauron, 2014, Bio-protocol 4(18): e1237), biotin capture lipid nucleophilic assays (e.g., Davidson et al., 2006, Lipid Research, 47, 440-449), titration calorimetry assays (e.g., Miller and Cistola, 1993 Molecular and Cellular Biochemistry, 123(1): 29-37), and the like.

[0133] Generally, the permeability of a microbial plasma membrane treated with DEF_PFV1 described herein can be increased compared to the permeability of a microbial plasma membrane treated with a corresponding DEF_PFV2. Membrane permeability can be measured by a variety of techniques including dye uptake. A convenient dye uptake assay that can be used to assess changes in membrane permeability includes the uptake assay of Hoechst 33342 (H0342), Rhodamine 123, SYTOX™ Green, and the like. These dyes only enter microbial cells when the plasma membrane of the microbial cell is permeabilized by a defensin peptide, defensin, or other membrane permeabilizing agent. Without being bound by theory, in certain embodiments, it is believed that a defensin protein comprising a defensin peptide joined by a spacer peptide and another antifungal peptide can provide improved microbial inhibition by increasing the permeability of the microbial membrane being treated compared to a microbial membrane treated with a defensin peptide.

[0134] It has been described that potato plants expressing transgenic MsDefl were identified for resistance to P. cinnabarinus using a microbial disease severity assay and colony forming assay in combination with an expression assay (U.S. Patent No. 6,916,970 and Gao et al., 2000, Nature Biotechnology 18, 1307-1310). Similarly, it is envisioned that various DEF_PFV1 treated plants that are resistant to or control microbial pathogens can be identified by scoring treated plants for resistance to infection by a microbial pathogen that infects these plants. Examples of microbial resistance conferred by DEF_PFV1 that can be determined by observing a reduction in disease symptoms or microbial growth include; resistance of treated corn to P. teres, P. series, P. graminicola, P. narrow of and / or P. maydis; resistance of treated wheat to scab (F. graminearum), powdery mildew (Blumeria graminis f. sp. tritici), stripe rust, stem rust or leaf rust (Puccinia triticina); resistance of treated cotton to F. oxysporum and P. cinnabarinus; resistance of treated rice to M. grisea and R. solani, and resistance of treated soybean to Asian rust (P. pachyrhizi), Pythium root rot (Pythium sp.), white mold (Sclerotinia sp.), sudden death syndrome (F. verticillioides) and / or brown stem rot (Diaporthe phaseolorum).

[0135] The amount of DEF_PFV1 required to inhibit a pathogenic microorganism in a given plant can also be determined by measuring a reduction in adverse effects caused by microbial growth in such plants. This reduction can be determined by comparing the degree of adverse effects in DEF_PFV1 -treated plants relative to otherwise identical control plants treated with a control composition lacking DEF_PFV1 or comprising a corresponding DEF_PFV2. Adverse effects of microbial growth in plants that can be measured include any type of plant tissue damage or necrosis, any type of reduction in plant yield, any reduction in the value of crop plant products, and / or production of undesirable microbial metabolites or microbial growth byproducts, including but not limited to mycotoxins. Mycotoxins include a variety of toxic molecules produced by microbial species, including but not limited to polyketides (including aflatoxins, deoxynivalenol, O-methylsterigmatocystin, etc.), fumonisins, alpepoxins (e.g., Als A2, Bls B2), sphingolipid fungi (A, B, C, and D), trichothecene mycotoxins, fusaricidins, etc. Methods for quantifying mycotoxin levels have been well documented. In addition, commercial kits for measuring mycotoxins such as aflatoxins, fumonisins, deoxynivalenol, and zearalenone are also available (VICAM, Watertown, MA, USA).

[0136] Infection of certain plants with certain plant pathogenic microorganisms can have a pronounced effect on plant growth that is easily observable. Thus, plants treated with a desired DEF_PFV1 can be distinguished from plants treated with a control lacking a defensin peptide or comprising a corresponding DEF_PFV2 by infecting these plants with a pathogenic plant microorganism and observing a reduction in symptoms typically associated with such infection. Such observations can be facilitated by co-infecting otherwise identical control plants treated with a control lacking a defensin peptide or comprising a corresponding undesired DEF_PFV2. Identification of treated plants that prevent or combat microbial infection can be based on observing a reduction in disease symptoms, measuring a reduction in microbial growth in infected plants (e.g., by determining the number of colony forming units per gram of infected tissue), and / or measuring the amount of mycotoxins in infected plant tissue.

[0137] Target plants include food crop plants and biofuel or energy crop plants. Plants, parts thereof, and harvested portions thereof (e.g., grains, fruits, and vegetables) to which the methods and compositions disclosed herein can be applied can refer to plants, parts thereof, and harvested portions thereof that are directly edible or that produce edible products (e.g., typically for direct or indirect feeding of humans, either directly or through animals). As used herein, plants, parts thereof, and harvested portions thereof include cereal crops (e.g., wheat, rice, corn, barley, oat, sorghum, rye, and millet), legumes (e.g., peanut, chickpea, lentil, bean, soybean, lima bean); root or tuber crops (e.g., potato, sweet potato, and cassava); oil crops (e.g., rapeseed, wheat, peanut, palm, coconut, safflower, cottonseed, sunflower, flax, and olive); sugar crops (e.g., sugarcane and sugar beet); fruit crops (e.g., banana, orange, apple, pear, breadfruit, pineapple, strawberry, grape, and cherry); vegetable crops and tubers (e.g., tomato, lettuce, carrot, melon, asparagus, Brassica species); nut crops (e.g., cashew, peanut, walnut, pistachio, almond); pasture and turf grasses; forage legumes (e.g., alfalfa, clover); medicinal crops (e.g., Cannabis species, coffee, cocoa, cola, poppy); spice and flavor crops (e.g., vanilla, sage, thyme, anise, saffron, menthol, mint, spearmint, coriander), fiber crops (e.g., cotton or industrial hemp), and biofuel crops (e.g., castor, pennycress, switchgrass, miscanthus, and jatropha) plants, parts thereof, and harvested portions thereof.

[0138] Different DEF_PFV can be analyzed by mass spectrometry-based methods to assign specific disulfide bonds (Weinfurtner, in Oxidative Folding of Proteins: Basic Principles, Cellular Regulation and Engineering, 2018, pp. 81-98 DOI: 10.1039 / 9781788013253-00081; Tang and Speicher, Current protocols in protein science Volume 96, Issue 1 (2019): e86. doi: 10.1002 / cpps.86).

[0139] Also provided are antimicrobial compositions for agricultural, pharmaceutical, or veterinary use comprising an antimicrobial plant or antimicrobial human or veterinary pathogenic microorganism inhibiting amount ("antimicrobial effective amount") of one or more of the isolated, purified antimicrobial DEF PFV peptides of the application or biologically functional equivalents thereof. Such compositions can comprise one or any combination of the defensin peptides disclosed herein, as well as an agriculturally, pharmaceutically, or veterinarily practically acceptable carrier, diluent, or excipient. Other components relevant in the agricultural and therapeutic contexts can also be included in such compositions, as shown below. The antimicrobial compositions can be used to inhibit the growth of or kill defensin protein or defensin peptide sensitive pathogenic microorganisms associated with plant, human, or animal microbial infection. Such antimicrobial compositions can be formulated for topical application and applied topically to plants, plant environments (including soil), or humans or animals.

[0140] Agricultural compositions comprising any of the defensin molecules of the application, alone or in any combination, can be formulated as described, for example, in Winnacker-Kuchler (1986) Chemical Technology, Fourth Edition, Volume 7, Hanser Verlag, Munich; van Falkenberg (1972-1973) Pesticide Formulations, Second Edition, Marcel Dekker, N.Y.; and K. Martens (1979) Spray Drying Handbook, Third Edition, G. Goodwin, Ltd., London. Formulation aids such as carriers, inert materials, surfactants, solvents, and other additives are also well known in the art and are described, for example, in Watkins, Handbook of Insecticide Dust Diluents and Carriers, Second Edition, Darland Books, Caldwell, N.J. and Winnacker-Kuchler (1986) Chemical Technology, Fourth Edition, Volume 7, Hanser Verlag, Munich. Using these formulations, the desired DEF PFV can also be combined with other pesticidally active substances, fertilizers and / or growth regulators, etc., to prepare compositions (e.g., finished formulations or tank mixes).

[0141] The antibacterial defensins of the present application, alone or in combination with other active agents, can be applied at concentrations ranging from about 0.1 pg / ml to about 100 mg / ml, or from about 5 pg / ml to about 5 mg / ml, at a pH ranging from about 3.0 to about 9.0. Such compositions can be buffered using, for example, a phosphate buffer of between about 1 mM and 1 M, about 10 mM to about 100 mM, or about 15 mM to about 50 mM. In cases where the buffer concentration is low, salts can be added to increase the ionic strength. In certain embodiments, NaCl can be added ranging from about 1 mM to about 1 M, or about 10 mM to about 100 mM.

[0142] Many conventional microbial antibiotics and chemical fungicides that can be combined with the defensin peptides of the present application are described in Worthington and Walker (1983) The Pesticide Manual, 7th Edition, British Crop Protection Council. These include, for example, polyoxin, solanin, carboxamide, aromatic carbohydrate, iprodione, morpholines, sterol biosynthesis inhibitors, and organophosphorus compounds. In addition, azole, triazole, and echinocandin fungicides can also be used. Other active ingredients that can be combined with the antimicrobial peptides and proteins of the present application include, for example, insecticides, attractants, sterilants, acaricides, nematicides, and herbicides. U.S. Patent No. 5,421,839, which is incorporated by reference herein in its entirety, contains a comprehensive summary of many active agents that can be formulated with materials such as the antibacterial defensin peptides and proteins of the present application.

[0143] The compositions provided herein can be applied in an antibacterially effective amount, which will vary depending on such factors as the particular fungal pathogen to be controlled, the particular plant (and plant parts or soil) to be treated, and the method of application of the composition comprising the desired defensin peptide.

[0144] The desired defensin peptides and their biological functional equivalents provided herein, and compositions containing the same (e.g., in the numbered examples below), can be used to inhibit the growth of a variety of susceptible microorganisms in plants. In certain embodiments, the growth of microorganisms of the following genera or species can be inhibited: Alternaria (e.g., A. brassicicola; A. solani); Ascochyta (e.g., A. pisi); Aspergillus (e.g., A. flavus; A. fumigatus); Botrytis (e.g., B. cinerea); Cercospora (e.g., C. kikuchii; C. zeae); Colletotrichum (e.g., C. dematium); Erysiphe (e.g., E. graminis tritici; E. graminis hordei); Fusarium (e.g., F. oxysporum; F. oxysporum; F. graminearum; F. culmorum; F. solani; F. verticillioides; F. semitectum); Gaeumannomyces (e.g., G. graminis); Helminthosporium (e.g., H. kikuchii; H. maydis); Phoma (e.g., P. candida); Podosphaera (e.g., P. leucotricha); Puccinia (e.g., P. kuehnii; P. graminum; P. graminum tritici; P. recondita; P. arachidicola); Pythium (e.g., P. aphanidermatum; P. ultimum); Pyrenophora (e.g., P. teres); Rhynchosporium (e.g., R. solani); Sclerotinia (e.g., S. sclerotiorum); Septoria (e.g., S. nodorum); Sphaerotheca (e.g., S. fuliginea); Uncinula (e.g., U. necator); Venturia (e.g., V. inaequalis); and Verticillium (e.g., V. albo-atrum; V. dahliae).

[0145] Also provided are pharmaceutical or veterinary compositions comprising an antimicrobially effective amount of a desired defensin peptide and a pharmaceutically acceptable or veterinarily feasible carrier. Such pharmaceutical or veterinary compositions can be used to inhibit the growth of or kill susceptible pathogenic microorganisms that infect humans or animals, i.e., to treat such fungal infections by administration to a patient or other subject in need thereof. In certain embodiments, compositions comprising defensin peptides can be formulated by methods adapted from those described in Remington: The Science and Practice of Pharmacy (2005), 21stEd., University of the Sciences in Philadelphia, Lippincott Williams & Wilkins. In certain embodiments, compositions can have a pH in the range from about 3.0 to about 9.0, comprising a defensin peptide at a concentration in the range from about 0.1 pg / ml to about 100 mg / ml, or about 5 pg / ml to about 5 mg / ml. Such compositions can be buffered using, for example, a phosphate buffer at a concentration of about 1 mM to about 1 M, about 10 mM to about 100 mM, or about 15 mM to about 50 mM. In cases where the concentration of the buffer is low, a salt can be added to increase the ionic strength. In certain embodiments, NaCl can be added in the range from about 1 mM to about 1 M, or about 10 mM to about 100 mM.

[0146] Defensin peptides can be formulated alone or in combination with other conventional antimicrobial treatment compounds, such as, for example and without limitation, polyene antimicrobial agents; imidazole, triazole, and thiazole antimicrobial agents; allylamines; and echinocandins, which are conventionally used in human and veterinary medicine.

[0147] Compositions comprising defensin peptides can be administered to a human or animal subject in need thereof by a variety of routes, including topical application, enterally, parenterally, and / or intravenously.

[0148] In further embodiments, the present disclosure provides a method for preventing or reducing crop damage or post-harvest loss caused by a plant pathogenic microorganism, the method comprising contacting a plant, a plant seed, a pre-harvest or post-harvest grain, a pre-harvest or post-harvest fruit, or a pre-harvest or post-harvest vegetable with an effective amount of a composition comprising a cationic antimicrobial defensin peptide fold variant 1 (DEF PFV1) and under conditions suitable for preventing or reducing crop damage or post-harvest loss.

[0149] In other embodiments, the present disclosure provides a medical device in operable combination with a composition comprising a cationic antimicrobial defensin peptide fold variant 1 (DEF PFV1), wherein the medical device comprises at least one surface that is topically coated or impregnated with the composition.

[0150] In yet further embodiments, provided herein are methods for treating, preventing, or inhibiting a microbial infection in a subject in need thereof, including a plant, a human, or a non-human animal, comprising administering (ex vivo or in vivo) to the subject a therapeutically effective amount of a composition comprising a cationic antimicrobial defensin peptide fold variant 1 (DEF PFV1) for a duration and under conditions suitable to treat, prevent, or inhibit the microbial infection.

[0151] In related embodiments, provided herein are compositions comprising a cationic antimicrobial defensin peptide fold variant 1 (DEF PFV1) for use in a method of treating, preventing, or inhibiting a microbial infection in a subject in need thereof.

[0152] In other related embodiments, provided herein are plant parts at least partially coated with a composition comprising a cationic antimicrobial defensin peptide fold variant 1 (DEF PFV1).

[0153] Examples Definitions

[0154] The following numbered embodiments form part of the disclosure.

[0155] 1a. A composition comprising a defensin peptide fold variant 1 (DEF PFV1), wherein the defensin peptide is a cationic defensin peptide comprising, in sequential amino acid sequence from its N-terminus: (1) a first cysteine pair motif comprising C1 and C2, (2) a first intervening sequence (IS1), (3) a second cysteine pair motif comprising C3 and C4, (4) a second intervening sequence (IS2), (5) a third cysteine pair motif comprising C5 and C6, (6) a third intervening sequence (IS3), and (7) a fourth cysteine pair motif comprising C7 and C8, wherein the cysteine residues C1, C2, C3, C4, C5, C6, C7, and C8 of the DEF PFV1 form a first set of disulfide bonds, and thereby the DEF PFV1 adopts a first conformation that exhibits greater antimicrobial activity, increased protease resistance, or increased protease sensitivity compared to a defensin peptide fold variant 2 (DEF PFV2) having the same amino acid sequence as the DEF PFV1 but forming a different second set of disulfide bonds between the cysteine residues C1, C2, C3, C4, C5, C6, C7, and C8 and adopting a different second conformation, and wherein the composition is substantially free of DEF PFV2, optionally wherein at least about 80%, 90%, 95%, 98%, 99%, or 99.5% of the total defensin peptide in the composition by weight is DEF PFV1.

[0156] 1b. A composition comprising a defensin peptide fold variant 1 (DEF PFV1), wherein the defensin peptide is a C-terminal fragment of a cationic defensin peptide comprising, in sequential amino acid sequence from its N-terminus: (1) a third cysteine pair motif comprising conserved C5 and C6 of a full-length defensin peptide, (2) a third intervening sequence (IS3) of a full-length defensin peptide, and (3) a fourth cysteine pair motif comprising C7 and C8 of a full-length defensin protein, wherein cysteine residues C5, C6, C7, and C8 of the DEF PFV1 form a first set of disulfide bonds, and whereby the DEF PFV1 adopts a first conformation that exhibits greater antimicrobial activity, increased protease resistance, or increased protease sensitivity compared to a defensin peptide fold variant 2 (DEF PFV2) having the same amino acid sequence as the DEF PFV1 but forming a different second set of disulfide bonds between cysteine residues C5, C6, C7, and C8 and adopting a different second conformation, and wherein the composition is substantially free of DEF PFV2, optionally, wherein at least about 80%, 90%, 95%, 98%, 99%, or 99.5% of total defensin peptide in the composition by weight is DEF PFV1.

[0157] 2. The composition of embodiment 1a or 1b, wherein the defensin peptide is a native defensin peptide.

[0158] 3. The composition of embodiment 1a or 1b, wherein the defensin peptide is a synthetic variant of a native defensin peptide comprising one or more amino acid insertions, deletions, and / or substitutions in the native defensin peptide.

[0159] 4. The composition of embodiment 3, wherein the substitutions in the native defensin peptide comprise at least one of: substitution of a hydrophobic amino acid with a hydrophobic amino acid, substitution of a cationic amino acid with a cationic amino acid, or substitution of an anionic amino acid with a cationic or anionic amino acid.

[0160] 5. The composition of any one of embodiments 1a or 1b to 4, wherein the first set of disulfide bonds comprises a C1-C8 disulfide bond, a C2-C5 disulfide bond, a C3-C6 disulfide bond, and a C4-C7 disulfide bond.

[0161] 6. The composition of embodiment 5, wherein the second set of disulfide bonds comprises a disulfide bond other than a C1-C8 disulfide bond, a C2-C5 disulfide bond, a C3-C6 disulfide bond, and a C4-C7 disulfide bond.

[0162] 7. The composition according to any one of embodiments 1a or 1b to 6, wherein the first cysteine pair motif is a C1-10AA-C2 motif flanked by an N-terminal C1 and a C-terminal C2, comprising in sequential order from its N-terminal end ten amino acids (A1, A2, A3, A4, A5, A6, A7, A8, A9, and A10) wherein A1, A2, A3, A4, A5, A6, A7, A8, A9, and A10 are independently selected from the group consisting of C, H, and S, and wherein A1, A2, A3, A4, A5, A6, A7, A8, A9, and A10 are not all H. 10 ].

[0163] 8. The composition according to any one of embodiments 1a or 1b to 7, wherein the second cysteine pair motif is a C3-3AA-C4 motif flanked by an N-terminal C3 and a C-terminal C4, comprising in sequential order from its N-terminal end three amino acids (A 11 , A 12 , and A 13 ].

[0164] 9. The composition according to any one of embodiments 1a or 1b to 8, wherein the third cysteine pair motif is a C5-5-6AA-C6 motif flanked by an N-terminal C5 and a C-terminal C6, comprising in sequential order from its N-terminal end three amino acids (A 14 , A 15 , A 16 , A 17 , A 18 , and optionally A 19 ].

[0165] 10. The composition according to any one of embodiments 1a or 1b to 9, wherein the fourth cysteine pair motif is a C7-3AA-C8 motif flanked by an N-terminal C7 and a C-terminal C8, comprising in sequential order from its N-terminal end three amino acids (A 20 , A 21 , and A 22 ].

[0166] 11. A composition comprising a defensin peptide fold variant 1 (DEF PFV1), wherein the defensin peptide is a synthetic variant of a native defensin peptide comprising, in sequential amino acid sequence from its N-terminus: (1) a first amino acid pair motif comprising X1 and C2, (2) a first intervening sequence (IS1), (3) a first cysteine pair motif comprising C3 and C4, (4) a second intervening sequence (IS2), (5) a second cysteine pair motif comprising C5 and C6, (6) a third intervening sequence (IS3), and (7) a second amino acid pair motif comprising C7 and Y1, wherein the amino acids X1 and Y1 are each independently selected from the group of amino acids other than cysteine, and wherein the cysteines C2, C3, C4, C5, C6, and C7 of DEF PFV1 form a first set of disulfide bonds and thereby DEF PFV1 adopts a first conformation that exhibits greater antimicrobial activity, increased protease resistance, or increased protease sensitivity compared to a defensin peptide fold variant 2 (DEF PFV2) having the same amino acid sequence as DEF PFV1 but forming a different second set of disulfide bonds between the cysteines C2, C3, C4, C5, C6, and C7 and adopting a different second conformation, and wherein the composition is substantially free of DEF PFV2, optionally wherein at least about 80%, 90%, 95%, 98%, 99%, or 99.5% of total defensin peptide in the composition by weight is DEF PFV1.

[0167] 12. The composition of embodiment 11, wherein the amino acids X1 and Y1 are each Ser (S) or Thr (T).

[0168] 13. The composition of embodiments 11-12, wherein the first set of disulfide bonds comprises a C2-C5 disulfide bond, a C3-C6 disulfide bond, and a C4-C7 disulfide bond.

[0169] 14. The composition of any one of embodiments 11-13, wherein the second set of disulfide bonds comprises a disulfide bond other than a C2-C5 disulfide bond, a C3-C6 disulfide bond, and a C4-C7 disulfide bond.

[0170] 15. The composition of embodiments 11-14, wherein the first amino acid pair motif is an X1-10AA-C2 motif flanked by an N-terminal X1 and a C-terminal C2, comprising ten amino acids (A1, A2, A3, A4, A5, A6, A7, A8, A9, and A10) in sequential order from its N-terminus. 10 )。

[0171] 16. The composition of embodiments 11-15, wherein the first cysteine pair motif is a C3-3AA-C4 motif flanked by an N-terminal C3 and a C-terminal C4, comprising three amino acids (A 11 , A 12 , and A 13 ) in sequential order from its N-terminus.

[0172] 17. The composition of embodiments 11-16, wherein the second cysteine pair motif is a C5-5-6AA-C6 motif flanked by an N-terminal C5 and a C-terminal C6, comprising three amino acids (A 14 , A 15 , A 16 , A 17 , A 18 , and optionally A 19 ) in sequential order from its N-terminus.

[0173] 18. The composition of embodiments 11-17, wherein the second amino acid pair motif is a C7-3AA-Y1 motif flanked by an N-terminal C7 and a C-terminal Y1, comprising three amino acids (A 20 , A 21 , and A 22 ) in sequential order from its N-terminus.

[0174] 19. A composition comprising a defensin peptide fold variant 1 (DEF PFV1), wherein the defensin peptide is a synthetic variant of a native defensin peptide, comprising in sequential amino acid order from its N-terminus: (1) a first amino acid pair motif comprising X1 and Y1, (2) a first intervening sequence (IS1), (3) a first cysteine pair motif comprising C3 and C4, (4) a second intervening sequence (IS2), (5) a second cysteine pair motif comprising C5 and C6, (6) a third intervening sequence (IS3), and (7) a second amino acid pair motif comprising X2 and Y2, wherein the amino acids X1, Y1, X2, and Y2 are each independently selected from the group of amino acids other than cysteine, and wherein the cysteines C3, C4, C5, and C6 of DEF PFV1 form a first set of disulfide bonds and thereby DEF PFV1 adopts a first conformation that exhibits greater antimicrobial activity compared to a defensin peptide fold variant 2 (DEF PFV2) having the same amino acid sequence as DEF PFV1 but forming a different second set of disulfide bonds between cysteines C3, C4, C5, and C6 and adopting a different second conformation, and wherein the composition is substantially free of DEF PFV2, optionally wherein at least about 80%, 90%, 95%, 98%, 99%, or 99.5% of total defensin peptide in the composition by weight is DEF PFV1.

[0175] 20. The composition of embodiment 19, wherein each of the amino acids X1, Y1, X2, Y2 is Ser (S) or Thr (T).

[0176] 21. The composition of embodiments 19-20, wherein the first pair of amino acid motifs is an X1-10AA-Y1 motif flanked by an N-terminal X1 and a C-terminal Y1, comprising ten amino acids (A1, A2, A3, A4, A5, A6, A7, A8, A9, and A10) in sequential order from the N-terminus thereof. 10 )。

[0177] 22. The composition of embodiments 19-21, wherein the first pair of cysteine motifs is a C3-3AA-C4 motif flanked by an N-terminal C3 and a C-terminal C4, comprising three amino acids (A1, A2, and A3) in sequential order from the N-terminus thereof. 11 , A 12 , and A 13 ).

[0178] 23. The composition of embodiments 19-22, wherein the second pair of cysteine motifs is a C5-5-6AA-C6 motif flanked by an N-terminal C5 and a C-terminal C6, comprising three amino acids (A1, A2, and A3) in sequential order from the N-terminus thereof. 14 , A 15 , A 16 , A 17 , A 18 , and optionally A 19 ).

[0179] 24. The composition of embodiments 19-23, wherein the second pair of amino acid motifs is an X2-3AA-Y2 motif flanked by an N-terminal X2 and a C-terminal Y2, comprising three amino acids (A1, A2, and A3) in sequential order from the N-terminus thereof. 20 , A 21 , and A 22 ).

[0180] 25. The composition of any one of embodiments 19-24, wherein the defensin peptide comprises a defensin gamma core peptide sequence GXC5X3-9C6 (SEQ ID NO: 455) or a variant gamma core peptide sequence GXC5X3-10C6 (SEQ ID NO: 456).

[0181] 26. The composition of any one of embodiments 19-25, wherein A1 is an anionic amino acid selected from the group consisting of Asp (D) and Glu (E).

[0182] 27. The composition according to any one of embodiments 19 to 26, wherein the A4 is a polar neutral amino acid selected from the group consisting of Ser (S) and Thr (T).

[0183] 28. The composition according to any one of embodiments 19 to 27, wherein the A7 is an aromatic amino acid selected from the group consisting of Phe (F), Tyr (Y) and Trp (W).

[0184] 29. The composition according to any one of embodiments 19 to 28, wherein the A9 is an aliphatic amino acid selected from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), lie (I) and Pro (P).

[0185] 30. The composition according to any one of embodiments 19 to 29, wherein the second cysteine pair motif is a C3-3AA-C4 motif flanked by a N-terminal C3 and a C-terminal C4, comprising three amino acids (A 11 , A 12 and A 13 ) in consecutive sequence from its N-terminal end.

[0186] 31. The composition according to any one of embodiments 19 to 30, wherein the A 11 is an aliphatic amino acid selected from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), lie (I) and Pro (P) or a cationic amino acid selected from the group consisting of His (H), Arg (R) and Lys (K).

[0187] 32. The composition according to any one of embodiments 19 to 31, wherein the A 13 is an aliphatic amino acid selected from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), lie (I) and Pro (P) or a polar neutral amino acid selected from the group consisting of Ser (S) and Thr (T).

[0188] 33. The composition according to any one of embodiments 19 to 32, wherein the third cysteine pair motif is a C5-5-6AA-C6 motif flanked by a N-terminal C5 and a C-terminal C6, comprising three amino acids (A 14 , A 15 , A 16 , A 17 , A 18 and optionally A 19 ) in consecutive sequence from its N-terminal end.

[0189] 34. The composition according to any one of embodiments 19-33, wherein the A 14 is a cationic amino acid selected from the group consisting of His (H), Arg (R), and Lys (K), or an aliphatic amino acid selected from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), lie (I), and Pro (P), or a polar neutral amino acid selected from the group consisting of Ser (S) and Thr (T).

[0190] 35. The composition according to any one of embodiments 19-34, wherein the A 16 is an aliphatic amino acid selected from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), lie (I), and Pro (P), or an aromatic amino acid selected from the group consisting of Phe (F), Tyr (Y), and Trp (W).

[0191] 36. The composition according to any one of embodiments 19-35, wherein the A 18 is a cationic amino acid selected from the group consisting of His (H), Arg (R), and Lys (K).

[0192] 37. The composition according to any one of embodiments 19-36, wherein the A 20 is a polar neutral amino acid selected from the group consisting of Ser (S) and Thr (T).

[0193] 38. The composition according to any one of embodiments 19-37, wherein the A 21 is a cationic amino acid selected from the group consisting of His (H), Arg (R), and Lys (K).

[0194] 39. The composition according to any one of embodiments 19-38, wherein the A 22 is an aliphatic amino acid selected from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), lie (I), and Pro (P).

[0195] 40. The composition according to any one of embodiments 19-39, wherein the IS1 comprises five amino acids.

[0196] 41. The composition according to any one of embodiments 19-40, wherein the IS1 consists of five amino acids.

[0197] 42. The composition according to any one of embodiments 19-41, wherein the IS2 comprises nine to eleven amino acids.

[0198] 43. The composition according to any one of embodiments 19 to 42, wherein the IS2 consists of nine amino acids.

[0199] 44. The composition according to any one of embodiments 19 to 43, wherein the IS3 comprises one amino acid that is an aliphatic amino acid selected from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), lie (I), and Pro (P), or an aromatic amino acid selected from the group consisting of Phe (F), Tyr (Y), and Trp (W).

[0200] 45. The composition according to any one of embodiments 19 to 44, wherein the IS3 consists of one amino acid.

[0201] 46a. A composition comprising a defensin peptide fold variant 1 (DEF PFV1), wherein the defensin is a cationic defensin peptide comprising a defensin gamma core peptide sequence GXC5X3-9C6 (SEQ ID NO: 455) or a variant gamma core peptide sequence GXC5X3-10C6 (SEQ ID NO: 456), GXC5X3-9(F / W / Y) (SEQ ID NO: 457), or GXC5X3-10(F / W / Y) (SEQ ID NO: 458) and at least two or three additional cysteine residues selected from the group consisting of cysteine residues corresponding to C1, C2, C3, C4, C7, and C8 of a reference defensin peptide, wherein the cysteine residues of DEF PFV1 form a first set of disulfide bonds and thereby DEF PFV1 adopts a first conformation that exhibits greater antimicrobial activity, increased protease resistance, or increased protease sensitivity compared to a defensin peptide fold variant 2 (DEF PFV2) having the same amino acid sequence as DEF PFV1 but forming a different second set of disulfide bonds between the cysteine residues and adopting a different second conformation, and wherein the composition is substantially free of DEF PFV2, optionally wherein at least about 80%, 90%, 95%, 98%, 99%, or 99.5% of total defensin peptide in the composition by weight is DEF PFV1. (DEF PFV2) having the same amino acid sequence as DEF PFV1 but forming a different second set of disulfide bonds between the cysteine residues and adopting a different second conformation, and wherein the composition is substantially free of DEF PFV2, optionally wherein at least about 80%, 90%, 95%, 98%, 99%, or 99.5% of total defensin peptide in the composition by weight is DEF PFV1.

[0202] 46b. A composition comprising a defensin peptide fold variant 1 (DEF PFV1), wherein the defensin is a carboxy-terminal (C-terminal) fragment of a cationic defensin peptide comprising a defensin gamma core peptide sequence GXC5X3-9C6 (SEQ ID NO: 455) or a variant gamma core peptide sequence GXC5X3-10C6 (SEQ ID NO: 456) and cysteine residues C7 and C8 of a reference defensin peptide, wherein the cysteine residues of DEF PFV1 form a first set of disulfide bonds and thereby DEF PFV1 adopts a first conformation that exhibits greater antibacterial activity, increased protease resistance, or increased protease sensitivity compared to a defensin peptide fold variant 2 (DEF PFV2) having the same amino acid sequence as DEF PFV1 but forms a different second set of disulfide bonds between the cysteine residues and adopts a different second conformation, and wherein the composition is substantially free of DEF PFV2, optionally wherein at least about 80%, 90%, 95%, 98%, 99%, or 99.5% of total defensin peptide in the composition by weight is DEF PFV1. (DEF PFV2) having the same amino acid sequence as DEF PFV1 but forms a different second set of disulfide bonds between the cysteine residues and adopts a different second conformation, and wherein the composition is substantially free of DEF PFV2, optionally wherein at least about 80%, 90%, 95%, 98%, 99%, or 99.5% of total defensin peptide in the composition by weight is DEF PFV1.

[0203] 47. The composition of embodiment 46a or b, wherein the cationic defensin peptide comprises at least four additional cysteine residues selected from the group consisting of cysteines corresponding to C1, C2, C3, C4, C5, C6, C7, and C8 of the reference defensin peptide.

[0204] 48. The composition of embodiment 46a or b to 47, wherein one to four of the cysteine residues corresponding to C1, C2, C3, C4, C7, and C8 of the reference defensin peptide are substituted with a different amino acid or are deleted, optionally wherein the cysteine is substituted with a Ser (S) or Thr (T) residue.

[0205] 49. The composition of embodiment 46a or b to 48, wherein at least one of the cysteine residues corresponding to C1, C2, C3, C4, C7, and C8 of the reference defensin peptide is substituted with a different amino acid, at least one of the cysteine residues corresponding to C1, C2, C3, C4, C5, C6, C7, and C8 of the reference defensin peptide is deleted, and wherein at least two of the cysteine residues corresponding to C1, C2, C3, C4, C7, and C8 of the reference defensin peptide are retained.

[0206] 50. The composition of any one of embodiments 46a or b to 49, wherein the defensin peptide comprises cysteine residues corresponding to C5, C6, C7, and C8 of a reference defensin peptide.

[0207] 51. The composition of embodiment 46a, wherein the defensin peptide is a native defensin peptide.

[0208] 52. The composition of any one of embodiments 46a or b to 51, wherein the defensin peptide is a synthetic variant of a native defensin peptide comprising one or more amino acid insertions, deletions, and / or substitutions in the native defensin peptide.

[0209] 53. The composition of any one of embodiments 46a or b to 52, wherein the substitutions in the native defensin peptide comprise at least one of: substitution of a hydrophobic amino acid with a hydrophobic amino acid, substitution of a cationic amino acid with a cationic amino acid, or substitution of an anionic amino acid with a cationic or anionic amino acid.

[0210] 54. The composition of any one of embodiments 46a or b to 53, wherein the defensin peptide comprises a C5-5-6AA-C6 motif flanked by an N-terminal C5 and a C-terminal C6, comprising in sequential order from its N-terminus three amino acids (A 14 , A 15 , A 16 , A 17 , A 18 , and optionally A 19 ).

[0211] 55. The composition of any one of embodiments 46a or b to 54, wherein: (i) said A 14 is a cationic amino acid selected from the group consisting of His (H), Arg (R), and Lys (K), or an aliphatic amino acid selected from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), lie (I), and Pro (P), or a polar neutral amino acid selected from the group consisting of Ser (S) and Thr (T); (ii) said A 16 is an aliphatic amino acid selected from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), lie (I), and Pro (P), or an aromatic amino acid selected from the group consisting of Phe (F), Tyr (Y), and Trp (W); and / or (iii) wherein said A 18 is a cationic amino acid selected from the group consisting of His (H), Arg (R), and Lys (K).

[0212] 56. The composition of any one of embodiments 46a or b to 55, wherein the defensin peptide comprises a C7-3AA-C8 motif flanked by a N-terminal C7 and a C-terminal C8, comprising three amino acids (A 20 , A 21 , and A 22 ) in sequential order from its N-terminus.

[0213] 57. The composition of any one of embodiments 46a or b to 56, wherein: (i) the A 20 is a polar neutral amino acid selected from the group consisting of Ser (S) and Thr (T); (ii) the A 21 is a cationic amino acid selected from the group consisting of His (H), Arg (R), and Lys (K); and / or (iii) the A 22 is an aliphatic amino acid selected from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), lie (I), and Pro (P).

[0214] 58. The composition of any one of embodiments 46a or b to 57, wherein the cationic defensin peptide has an isoelectric point between 8.0 and 12.0.

[0215] 59. The composition of any one of embodiments 46a or b to 58, wherein the DEF PFV1 exhibits greater protease resistance than the DEF PFV2.

[0216] 60. The composition of any one of embodiments 46a or b to 59, wherein the DEF PFV1 exhibits less protease resistance than the DEF PFV2.

[0217] 61. The composition of any one of embodiments 46a or b to 60, wherein the defensin peptide comprises an amino acid sequence having at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% sequence identity to any one of SEQ ID NOs: 1 to 454 or SEQ ID NO: 459 to SEQ ID NO: 472.

[0218] 62. The composition of any one of embodiments 46a or b to 61, wherein the composition further comprises an agriculturally, pharmaceutically, or veterinarily feasible carrier, diluent, or excipient.

[0219] 63. A method for making a composition according to any one of embodiments 1a or b to 62, the method comprising: (a) separating a fraction comprising DEF_PFV1 from one or more fractions comprising DEF_PFV2 or DEF_PFV2 peptide fragments thereof from a mixture comprising DEF_PFV1 and DEF_PFV2 or DEF_PFV2 peptide fragments thereof; and (b) combining the fraction comprising DEF_PFV1 or a preparation further purified from the DEF_PFV1 fraction with an agriculturally, pharmaceutically, or veterinarily feasible carrier, diluent, or excipient, thereby making the composition.

[0220] 64. The method of embodiment 63, further comprising obtaining the mixture comprising DEF_PFV1 and DEF_PFV2 or DEF_PFV2 peptide fragments thereof by: (a) culturing a microorganism expressing a recombinant polynucleotide comprising a transcriptional promoter operably linked to a polynucleotide encoding a signal peptide that is in-frame with and upstream of a polynucleotide encoding DEF_PFV1 or a variant thereof in a fermentation broth; and (b) isolating the microorganism from the fermentation broth comprising the mixture, wherein the microorganism is optionally selected from the group consisting of Candida, Kluyveromyces, Hansenula, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia, Myceliopthora, and Escherichia.

[0221] 65. The method of embodiments 63 to 64, further comprising obtaining the mixture comprising DEF_PFV1 and DEF_PFV2 or DEF_PFV2 peptide fragments thereof by: (a) culturing a microorganism expressing a recombinant polynucleotide comprising a transcriptional promoter operably linked to a polynucleotide encoding DEF_PFV1 or a variant thereof in a fermentation broth, and (b) isolating the microorganism comprising the mixture from the fermentation broth, (c) disrupting the microorganism to obtain a cell lysate, and (d) separating insoluble debris in the cell lysate from an aqueous fraction comprising the mixture, wherein the microorganism is optionally selected from the group consisting of Candida, Kluyveromyces, Hansenula, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia, Myceliopthora, and Escherichia.

[0222] 66. The method of embodiments 63 to 65, further comprising treating the mixture comprising DEF_PFV1 and DEF_PFV2 with a protease under non-denaturing conditions sufficient to produce DEF_PFV2 peptide fragments but insufficient to produce DEF_PFV1 peptide fragments.

[0223] 67. The method of embodiments 63-66, wherein the protease is a trypsin family serine protease that cleaves DEF_PFV2 at the carboxy-terminal end of DEF_PFV2 Arg (R) or Lys (K).

[0224] 68. The method of embodiments 63-67, wherein the trypsin family serine protease is a recombinant bovine, porcine, human, or microbial trypsin, and optionally, wherein the microbial trypsin is Streptomyces trypsin or a variant thereof.

[0225] 69. The method of embodiments 63-68, further comprising separating the DEF_PFV1 and DEF_PFV2 peptide fragments by size exclusion chromatography.

[0226] 70. A method for preventing or reducing crop damage or post-harvest loss caused by a plant pathogenic microorganism, the method comprising contacting a plant, a plant seed, a pre-harvest or post-harvest grain, a pre-harvest or post-harvest fruit, or a pre-harvest or post-harvest vegetable with an effective amount of a composition according to any one of embodiments la or lb-62 and under conditions suitable for preventing or reducing crop damage or post-harvest loss.

[0227] 71. The method of embodiment 70, wherein the plant pathogenic microorganism is selected from the group consisting of a Fusarium species, an Alternaria species, a Verticillium species, a Phytophthora species, a Colletotrichum species, a Erysiphe species, a Cercospora species, a Phakopsora species, a Rhizoctonia species, a Sclerotinia species, a Pythium species, a Phoma species, an Elsinoe species, an Oidiopsis species, a Puccinia species, a Septoria species, a Penicillium species, a Cladosporium species, a Pseudocercospora species, a Mycosphaerella species, a Erysiphe graminis species, a Uncinula species, a Albugo species, a Leveillula species, a Microsphaera species, a Catenaspora species, a Pyrenochaeta species, a Helminthosporium species, a Cylindrocarpon species, or a Brachysporium species.

[0228] 72. The method of embodiments 70-71, wherein the plant, plant seed, pre-harvest or post-harvest grain, pre-harvest or post-harvest fruit, or pre-harvest or post-harvest vegetable is selected from the group consisting of a cereal crop, a legume crop, a root or tuber crop, an oil crop, a fruit crop, a vegetable crop, a nut crop, a forage or turf grass crop, a forage legume, a medicinal crop, a spice or flavor crop, a fiber crop, and a biofuel crop, a plant seed, a pre-harvest or post-harvest grain, a pre-harvest or post-harvest fruit, or a pre-harvest or post-harvest vegetable.

[0229] 73. The method of any one of embodiments 70-72, wherein the plant, plant seed, pre- or post-harvest grain, pre- or post-harvest fruit, or pre- or post-harvest vegetable is selected from the group consisting of wheat, rice, maize, barley, oat, sorghum, rye, millet, peanut, chickpea, pigeon pea, lentil, bean, soybean, lima bean, potato, sweet potato, cassava, oilseed rape, peanut, palm, coconut, safflower, cotton, sunflower, flax, olive, sugar cane, sugar beet, banana, orange, apple, pear, breadfruit, pineapple, strawberry, grape, cherry, coffee, cocoa, tomato, lettuce, carrot, melon, asparagus, Brassica species, cashew, walnut, pistachio, almond, alfalfa, clover, castor, camelina, borage, switchgrass, miscanthus, and jatropha plant, plant seed, pre- or post-harvest grain, pre- or post-harvest fruit, or pre- or post-harvest vegetable.

[0230] 74. A medical device having antimicrobial properties, comprising a medical device operatively combined with a composition according to any one of embodiments la or b to 62, wherein the medical device comprises at least one surface that is topically coated or impregnated with the composition.

[0231] 75. The medical device of embodiment 74, wherein the medical device is selected from the group consisting of a stent, a catheter, a contact lens, a condom, a patch, and a diaphragm.

[0232] 76. A method for treating, preventing, or inhibiting a microbial infection in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition according to any one of embodiments 1 to 62 under conditions and for a duration suitable for treating, preventing, or inhibiting the microbial infection.

[0233] 77. The method of embodiment 76, wherein the administering comprises introducing the composition to the subject topically, enterally, parenterally, or intravenously.

[0234] 78. The method of embodiments 76 to 77, wherein the subject is selected from the group consisting of a human, a livestock, a poultry, a fish, and a companion animal.

[0235] 79. The method of embodiments 76 to 78, wherein the microbial infection is a microbial infection of a mucosa, an eye, a skin, or a nail, and the composition is applied to the mucosa, the eye, the skin, or the nail.

[0236] 80. The method of embodiments 76-79, wherein the microbial infection is caused by a dermatophyte, and wherein the dermatophyte is optionally selected from the group consisting of Trichophyton rubrum, T. interdigitale, T. violaceum, T. tonsurans, T. soudanense, T. mentagrophytes, M. furfur, E. floccosum, and M. gypseum.

[0237] 81. The method of embodiments 76-80, wherein the microbial infection is caused by a microbe selected from the group consisting of Aspergillus, Cryptococcus, Penicillium, Rhizopus, Lecythophora, Mucor, Physcomitrella, Rhizomucor, Syncephalastrum, Cokeromyces, Mortierella, Pythium, Fusarium, Histoplasma, and Blastomyces species.

[0238] 82. The method of embodiments 76-81, wherein the microbial infection is caused by a microbe selected from the group consisting of Candida species, wherein the Candida species is selected from the group consisting of C. albicans, C. auris, C. glabrata, C. parapsilosis, C. tropicalis, and C. krusei.

[0239] 83. The composition of any one of embodiments la or b to 62, for use in a method of treating, preventing, or inhibiting a microbial infection in a subject in need thereof.

[0240] 84. The composition of embodiment 83, wherein the subject is selected from the group consisting of a human, a livestock, a poultry, a fish, and a companion animal.

[0241] 85. A plant part at least partially coated with the composition of any one of embodiments 1-62.

[0242] 86. The plant part of embodiment 85, wherein the plant part is selected from the group consisting of a seed and a pre-harvest or post-harvest grain.

[0243] 87. The plant part of embodiments 85-86, wherein the plant part is selected from the group consisting of a pre-harvest or post-harvest fruit, a pre-harvest or post-harvest vegetable, and a pre-harvest or post-harvest flower.

[0244] 88. The plant part of embodiments 85-87, wherein the plant part is selected from the group consisting of a cereal crop, a legume crop, a root or tuber crop, an oil crop, a fruit crop, a vegetable crop, a nut crop, a forage or turf grass crop, a forage legume, a medicinal crop, a spice or flavor crop, a fiber crop, and a biofuel crop plant part.

[0245] 89. The plant portion according to Examples 85 to 88, wherein the plant portion is selected from the group consisting of: wheat, rice, corn, barley, oats, sorghum, rye, millet, peanut, chickpea, pigeon pea, lentil, kidney bean, soybean, lima bean, potato, sweet potato, cassava, rapeseed, peanut, palm, coconut, safflower, cotton, sunflower, flax, olive, sugarcane, beet, banana, orange, apple, pear, breadfruit, pineapple, strawberry, grape, cherry, coffee, cocoa, tomato, lettuce, carrot, melon, asparagus, Brassica species, cashew, walnut, pistachio, almond, alfalfa, clover, castor bean, flaxseed, iris, switchgrass, miscanthus and jatropha plant portions.

[0246] * * * * *

[0247] The term “and / or” as used herein should be understood to explicitly disclose each of two specified features or components, with or without the other. Therefore, the term “and / or” as used in phrases such as “A and / or B” is intended to include “A and B”, “A or B”, “A” (alone), and “B” (alone). Similarly, the term “and / or” as used in phrases such as “A, B, and / or C” is intended to cover each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0248] Where terms are provided in the singular form, other embodiments described by the plural form of the term are also provided. As used herein, the terms “include,” “includes,” and “including” should be interpreted as having at least the features they refer to, without excluding any additional unspecified features. Those skilled in the art will understand that, unless otherwise indicated, terms are intended to be “open-ended” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “including but not limited to,” etc.). Phrases such as “at least one” and “one or more” and terms such as “a” or “an” include both singular and plural forms.

[0249] It should also be understood that when a feature or aspect of this disclosure is described in the form of a Markush group, this disclosure is also intended to be described in the form of any single member of the Markush group or a subgroup of members. Similarly, all scopes disclosed herein also cover all possible subscopes and combinations of subscopes, and terms such as “between,” “up to,” “at least,” “greater than,” and “less than” include the numbers listed within the scope and include each individual member.

[0250] All references cited herein, whether prior or later, including but not limited to patents, patent applications, and patent publications (whether US patents, PCT patents, or foreign foreign patents), and all technical and / or scientific publications, are incorporated by reference in their entirety.

[0251] Examples

[0252] While various embodiments have been disclosed herein, other embodiments will be apparent to those skilled in the art. The various embodiments disclosed herein are for purposes of illustration and are not intended to limit the true scope and spirit of the disclosure, which is indicated by the appended claims. The disclosure will be further described with reference to the following examples, which are provided to illustrate certain embodiments and are not intended to limit the scope or spirit of the disclosure or the claimed subject matter.

[0253] Example 1: Heterologous expression and purification of DEF PFV1 and DEF PFV2

[0254] Synthetic defensin genes encoding defensin peptides (see Tables 1 and 2) were cloned into Xhol and Xbal restriction sites in a SacI linearized pPICZ alpha-A integration vector (Invitrogen, Carlsbad, CA) in frame with the alpha mating factor secretion signal sequence (containing the KEX2 cleavage site but not the Glu-Ala repeat sequence) and expressed in Pichia pastoris X33.

[0255] According to Sagaram, PLoS One 6(4)Purification of defensin peptides was performed as described in e18550 (2011) with slight modifications using CM-Sephadex C-25 cation exchange chromatography and C18 reverse phase HPLC. After induction, cells were harvested by centrifugation at 6,000 rpm for 20 min at 4°C and the pH of the supernatant was adjusted to 6.0. Cation exchange resin (CM-Sephadex C-25, Sigma, Cat# C25120) equilibrated with binding buffer (25 mM sodium acetate, pH 6.0) was added to the supernatant and incubated at 4°C with 110 rpm overnight. After the resin was collected and washed with binding buffer, bound proteins were eluted using elution buffer (1 M NaCl, 50 mM Tris, pH 7.6) using an AKTA FPLC. FPLC fractions containing DEF peptides were concentrated using Amicon Ultra-15 centrifugal filter units. Concentrated fractions were dialyzed against 10 mM Tris, pH 7.6 and further purified by reverse phase C18-HPLC. Peaks were observed and fractions containing DEF_PFV1 (peak 1) and DEF_PFV2 (peak 2) were collected using methods provided in the Agilent HPLC System Manual (Agilent Technologies Part Number: G1380-90000 (1999)). HPLC fractions containing DEF_PFV1 (peak 1) and DEF_PFV2 (peak 2) peptides were lyophilized and resuspended in nuclease-free water. Concentrations of DEF_PFV1 and DEF_PFV2 were determined using a BCA assay performed according to the manufacturer’s protocol (Thermo-Fisher Scientific, Inc.® “Protein Assay Technical Handbook”). Purity and size of DEF_PFV1 and DEF_PFV2 were verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). NMR analysis was performed on DEF_PFV1 and DEF_PFV2 peptides to determine the disulfide bonds present in each peptide.

[0256] Example 2: Fungal cultures and spore suspensions

[0257] Botrytis cinerea T-4 fungal strain was grown in 20% V8 growth medium. Alternaria alternata was grown on potato dextrose agar. Fungal spores were harvested by flooding the fungal growth plate with sterile water. The spore suspension was filtered through two layers of Miracle Cloth, centrifuged at 13,600 rpm for 1 min, washed and resuspended in low salt synthetic fungal medium (SFM) (see U.S. Patent No. 6,916,970). The spore suspension was adjusted to an equivalent spore density using a hemocytometer.

[0258] S. sclerotiorum and C. herbarum conidia were harvested from fully grown plates by gently scraping the conidia off with an L-shaped spreader in the presence of 5 ml sterile water. To remove mycelial debris, the spore suspension was filtered through three layers of miracloth and washed twice with sterile water at 13,000 rpm for 2 minutes each time. The spore concentration was adjusted to 1 x 10 5 spores / ml using a hemocytometer.

[0259] S. sclerotiorum 555 fungal culture (BD Diagnostics, Sparks, MD “Difco TM & BBL™ Manual, Second Edition”) was grown on potato dextrose agar (PDA) medium at room temperature.

[0260] Example 3: Determination of minimum inhibitory concentration (MIC) of DEF_PFV1 and DEF_PFV2 in the absence and presence of cations

[0261] The antifungal activity of DEF_PFV1 and DEF_PFV2 was evaluated at different concentrations using 2-fold serial dilutions of each peptide. The antifungal activity of each peptide was determined by spectrophotometry using a 96-well plate assay (Sagaram, PLoS One 6 (4) :e18550 (2011) and Sagaram, PLoS One 8(12) :e82485 (2013)). About 45 µL of each peptide at different concentrations was added to each well of a microtiter plate containing 45 µL of ~10 5 spores / ml spore suspension. After 48 h, the quantitative fungal growth inhibition was determined by measuring the absorbance at 595 nm using a microplate reader (Tecan Infinite® M200 Pro, Tecan Systems Inc., San Jose, CA). The fungal cell viability was determined by a resazurin cell viability assay (see, Li, MPMI 32 :1649-1664 (2019) and Velivelli, PNAS 117(27) :16043 (2020)).

[0262] After incubation of the pathogen / peptide mixture for 48 h, 10 μΐ of a 0.1% solution of resazurin was added to each well. After incubation of the mixture overnight, the color of the resazurin dye changed from blue to pink or colorless, indicating the presence of living fungal cells. The MIC of each peptide was the lowest concentration of each peptide at which no change in blue color occurred. Using this protocol, the MIC values of DEF_PFV1 and DEF_PFV2 were determined in the presence of 100 mM NaCl and 2 mM CaCl2.

[0263] The MIC determination of DEF_PFV1 and DEF_PFV2 against Botrytis cinerea was performed in 2X SFM to determine the antifungal activity. It was hypothesized that antifungal peptides would significantly lose their antifungal activity in the presence of cations, as the electrostatic interaction between positively charged peptides and negatively charged fungal membranes is significantly weakened in the presence of cations. Therefore, the antifungal activity of DEF_PFV1 and DEF_PFV2 could be determined in SFM supplemented with 100 mM NaCl or 2 mM CaCl2. The loss of antifungal activity in the presence of NaCl or CaCl2indicated that the antifungal activity was salt-sensitive.

[0264] Example 4: Semi-plant antifungal activity of DEF_PFV against Sclerotinia homoeocarpa 555

[0265] For semi-plant antifungal assays, 1 mm plugs were taken from the edge of freshly grown 2-day-old fungal colonies from PDA medium and placed on about 3- to 4-week-old detached soybean leaves and pods (6 weeks old). Subsequently, 40 μΐ of different concentrations of DEF_PFV solution or water were immediately applied to the plugs, and evaluation was performed after 2-3 days of incubation under high humidity. The severity of disease damage on each leaf was evaluated using the CropReporter system described by Li et al. Mol Plant Microbe Interact. 2019 Dec;32(12): 1649-1664. doi: 10.1094 / MPMI-08-19-0224-R. Epub 2019 Oct 28. PMID: 31425003. High-resolution fluorescent images could be taken using CropReporter (PhenoVation, Wageningen, The Netherlands).

[0266] Example 5: Antifungal activity of DEF_PFV1 and DEF_PFV2 against Botrytis cinerea infection in tomato fruits

[0267] Tomato fruits commercially available were subjected to antifungal activity assays. Tomato fruits were washed with 0.01% bleach and rinsed three times with sterile distilled water. Silicon carbide powder (Fisher Chemical, USA) was gently rubbed onto the surface of the fruits to cause micro-wounding. Twenty μΐ of Botrytis cinerea conidia (1 X 10 5 conidia ml"1) were inoculated onto the fruits by drop inoculation. Conidia of B. cinerea were prepared as described above. After drop inoculation, the fruits were incubated in a sealed, weatherproof box containing a wet paper towel to maintain high humidity. Twelve hours after inoculation, 20 μΐ of peptide solution (0.75 μΜ DEF_PFV1 or DEF_PFV2) was added to the drop inoculated conidial suspension and incubated for 5 days. Fungal growth on the fruits was observed daily.

Claims

1. A composition comprising a defensin peptide fold variant 1 (DEF PFV1), wherein the defensin peptide is a cationic defensin peptide comprising, in sequential amino acid sequence from its N-terminus: (1) a first cysteine pair motif comprising C1 and C2, (2) a first intervening sequence (IS1), (3) a second cysteine pair motif comprising C3 and C4, (4) a second intervening sequence (IS2), (5) a third cysteine pair motif comprising C5 and C6, (6) a third intervening sequence (IS3), and (7) a fourth cysteine pair motif comprising C7 and C8, wherein the cysteine residues C1, C2, C3, C4, C5, C6, C7, and C8 of the DEF PFV1 form a first set of disulfide bonds, and thereby DEF PFV1 adopts a first conformation that exhibits greater antimicrobial activity, increased protease resistance, or increased protease sensitivity compared to a defensin peptide fold variant 2 (DEF PFV2) having the same amino acid sequence as DEF PFV1 but forming a different second set of disulfide bonds between the cysteine residues C1, C2, C3, C4, C5, C6, C7, and C8 and adopting a different second conformation, and wherein the composition is substantially free of DEF PFV2.

2. The composition of claim 1, wherein the defensin peptide is a native defensin peptide.

3. The composition of claim 1, wherein the defensin peptide is a synthetic variant of a native defensin peptide comprising one or more amino acid insertions, deletions, and / or substitutions in the native defensin peptide.

4. The composition of claim 1, wherein the substitutions in the native defensin peptide comprise at least one of: substitution of a hydrophobic amino acid with a hydrophobic amino acid, substitution of a cationic amino acid with a cationic amino acid, or substitution of an anionic amino acid with a cationic or anionic amino acid.

5. The composition of claim 1, wherein the first set of disulfide bonds comprises a C1-C8 disulfide bond, a C2-C5 disulfide bond, a C3-C6 disulfide bond, and a C4-C7 disulfide bond.

6. The composition of claim 1, wherein the second set of disulfide bonds comprises a disulfide bond other than a C1-C8 disulfide bond, a C2-C5 disulfide bond, a C3-C6 disulfide bond, and a C4-C7 disulfide bond.

7. The composition of claim 1, wherein the first cysteine pair motif is a C1-10AA-C2 motif flanked by an N-terminal C1 and a C-terminal C2, comprising ten amino acids (A1, A2, A3, A4, A5, A6, A7, A8, A9, and A10) in sequential order from its N-terminus, wherein A1 is C1, A2 is C2, A3 is C1, A4 is C2, A5 is C1, A6 is C2, A7 is C1, A8 is C2, A9 is C1, and A10 is C2. 10 ).

8. The composition of claim 1, wherein the second cysteine pair motif is a C3-3AA-C4 motif flanked by an N-terminal C3 and a C-terminal C4, comprising three amino acids (A 11 , A 12 , and A 13 ) in sequential order from its N-terminus.

9. The composition of claim 1, wherein the third cysteine pair motif is a C5-5-6AA-C6 motif flanked by N-terminal C5 and C-terminal C6, comprising three amino acids (A 14 , A 15 , A 16 , A 17 , A 18 , and optionally A 19 in sequential order from its N-terminus.

10. The composition of claim 1, wherein the fourth cysteine pair motif is a C7-3AA-C8 motif flanked by an N-terminal C7 and a C-terminal C8, comprising three amino acids (A 20 , A 21 , and A 22 ) in sequential order from its N-terminus.

11. A composition comprising a defensin peptide fold variant 1 (DEF PFV1), wherein the defensin peptide is a synthetic variant of a native defensin peptide comprising, in sequential amino acid sequence from its N-terminus: (1) a first amino acid pair motif comprising X1 and C2, (2) a first intervening sequence (IS1), (3) a first cysteine pair motif comprising C3 and C4, (4) a second intervening sequence (IS2), (5) a second cysteine pair motif comprising C5 and C6, (6) a third intervening sequence (IS3), and (7) a second amino acid pair motif comprising C7 and Y1, wherein the cysteine residues C3, C4, C5, C6, C7, and C8 of the DEF PFV1 form a first set of disulfide bonds, and thereby DEF PFV1 adopts a first conformation that exhibits greater antimicrobial activity, increased protease resistance, or increased protease sensitivity compared to a defensin peptide fold variant 2 (DEF PFV2) having the same amino acid sequence as DEF PFV1 but forming a different second set of disulfide bonds between the cysteine residues C3, C4, C5, C6, C7, and C8 and adopting a different second conformation, and wherein the composition is substantially free of DEF PFV2. wherein the amino acids X1and Y1are each independently selected from the group of amino acids other than cysteine, and wherein the cysteines C2, C3, C4, C5, C6, and C7 of DEF_PFV1form a first set of disulfide bonds, and whereby DEF_PFV1adopts a first conformation that exhibits greater antimicrobial activity, increased protease resistance, or increased protease sensitivity compared to Defensin Peptide Fold Variant 2 (DEF_PFV2) having the same amino acid sequence as DEF_PFV1but forms a different second set of disulfide bonds between cysteines C2, C3, C4, C5, C6, and C7 and adopts a different second conformation, and wherein the composition is substantially free of DEF_PFV2.

12. The composition of claim 11, wherein the amino acids X1and Y1are each Ser (S) or Thr (T).

13. The composition of claim 11, wherein the first set of disulfide bonds includes a C2-C5 disulfide bond, a C3-C6 disulfide bond, and a C4-C7 disulfide bond.

14. The composition of claim 11, wherein the second set of disulfide bonds includes a disulfide bond other than a C2-C5 disulfide bond, a C3-C6 disulfide bond, and a C4-C7 disulfide bond.

15. The composition of claim 11, wherein the first amino acid pair motif is an X1-10AA-C2 motif flanked by an N-terminal X1 and a C-terminal C2, comprising ten amino acids (A1, A2, A3, A4, A5, A6, A7, A8, A9, and A10) in sequential order from its N-terminal end, wherein A1 is X1, A10 is C2, and A2-A9 are any amino acid. 10 ).

16. The composition of claim 11, wherein the first cysteine pair motif is a C3-3AA-C4 motif flanked by an N-terminal C3 and a C-terminal C4, comprising three amino acids (A 11 , A 12 , and A 13 ) in sequential order from its N-terminus.

17. The composition of claim 11, wherein the second cysteine pair motif is a C5-5-6AA-C6 motif flanked by an N-terminal C5 and a C-terminal C6, comprising three amino acids (A 14 , A 15 , A 16 , A 17 , A 18 , and optionally A 19 in sequential order from its N-terminus.

18. The composition of claim 11, wherein the second amino acid pair motif is a C7-3AA-Y1 motif flanked by an N-terminal C7 and a C-terminal Y1, comprising three amino acids (A 20 , A 21 , and A 22 ) in sequential order from its N-terminal end.

19. A composition comprising a Defensin Peptide Fold Variant 1 (DEF_PFV1), wherein the defensin peptide is a synthetic variant of a native defensin peptide comprising, in sequential amino acid sequence from its N-terminus: (1) a first amino acid pair motif including X1and Y1, (2) a first intervening sequence (IS1), (3) a first cysteine pair motif including C3and C4, (4) a second intervening sequence (IS2), (5) a second cysteine pair motif including C5and C6, (6) a third intervening sequence (IS3), and (7) a second amino acid pair motif including X2and Y2, wherein the amino acids X1, Y1, X2, and Y2are each independently selected from the group of amino acids other than cysteine, and wherein the cysteines C3, C4, C5, and C6 of DEF_PFV1form a first set of disulfide bonds, and whereby DEF_PFV1adopts a first conformation that exhibits greater antimicrobial activity, increased protease resistance, or increased protease sensitivity compared to Defensin Peptide Fold Variant 2 (DEF_PFV2) having the same amino acid sequence as DEF_PFV1but forms a different second set of disulfide bonds between cysteines C3, C4, C5, and C6 and adopts a different second conformation, and wherein the composition is substantially free of DEF_PFV2.

20. The composition of claim 19, wherein the amino acids X1, Y1, X2, Y2are each Ser (S) or Thr (T).

21. The composition of claim 19, wherein the first amino acid pair motif is an X1-10AA-Y1 motif flanked by an N-terminal X1 and a C-terminal Y1, comprising ten amino acids (A1, A2, A3, A4, A5, A6, A7, A8, A9, and A10) in sequential order from its N-terminal end. 10 ).

22. The composition of claim 19, wherein the first cysteine pair motif is a C3-3AA-C4 motif flanked by an N-terminal C3 and a C-terminal C4, comprising three amino acids (A 11 , A 12 , and A 13 ) in sequential order from its N-terminus.

23. The composition of claim 19, wherein the second cysteine pair motif is a C5-5-6AA-C6 motif flanked by an N-terminal C5 and a C-terminal C6, comprising three amino acids (A 14 , A 15 , A 16 , A 17 , A 18 and optionally A 19 in sequential order from its N-terminus.

24. The composition of claim 19, wherein the second amino acid pair motif is an X2-3AA-Y2 motif flanked by an N-terminal X2 and a C-terminal Y1, comprising three amino acids (A 20 , A 21 , and A 22 ) in sequential order from its N-terminus.

25. The composition of any one of claims 19-24, wherein the defensin peptide comprises a defensin gamma core peptide sequence GXC5X3-9C6 (SEQ ID NO: 455) or a variant gamma core peptide sequence GXC5X3-10C6 (SEQ ID NO: 456).

26. The composition of any one of claims 19-24, wherein the A1 is an anionic amino acid selected from the group consisting of Asp (D) and Glu (E).

27. The composition of any one of claims 19-24, wherein the A4 is a polar neutral amino acid selected from the group consisting of Ser (S) and Thr (T).

28. The composition of any one of claims 19-24, wherein the A7 is an aromatic amino acid selected from the group consisting of Phe (F), Tyr (Y), and Trp (W).

29. The composition of any one of claims 19-24, wherein the A9 is an aliphatic amino acid selected from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), Ile (I), and Pro (P).

30. The composition of any one of claims 19-24, wherein the second cysteine pair motif is a C3-3AA-C4 motif flanked by an N-terminal C3 and a C-terminal C4, comprising three amino acids (A 11 , A 12 , and A 13 ) in sequential order from its N-terminus.

31. The composition according to any one of claims 19 to 24, wherein the A 11 is an aliphatic amino acid selected from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), lie (I), and Pro (P) or a cationic amino acid selected from the group consisting of His (H), Arg (R), and Lys (K).

32. The composition according to any one of claims 19 to 24, wherein the A 13 is an aliphatic amino acid selected from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), lie (I), and Pro (P) or a polar neutral amino acid selected from the group consisting of Ser (S) and Thr (T).

33. The composition according to any one of claims 19 to 24, wherein the third cysteine pair motif is a C5-5-6AA-C6 motif flanked by an N-terminal C5 and a C-terminal C6, comprising three amino acids (A 14 , A 15 , A 16 , A 17 , A 18 and optionally A 19 ) in sequential order from its N-terminus.

34. The composition according to any one of claims 19 to 24, wherein the A 14 is a cationic amino acid selected from the group consisting of His (H), Arg (R) and Lys (K), or an aliphatic amino acid selected from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), lie (I) and Pro (P), or a polar neutral amino acid selected from the group consisting of Ser (S) and Thr (T).

35. The composition according to any one of claims 19 to 24, wherein the A 16 is an aliphatic amino acid selected from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), lie (I), and Pro (P) or an aromatic amino acid selected from the group consisting of Phe (F), Tyr (Y), and Trp (W).

36. The composition of any one of claims 19-24, wherein the A 18 is a cationic amino acid selected from the group consisting of His (H), Arg (R), and Lys (K).

37. The composition according to any one of claims 19 to 24, wherein the A 20 is a polar neutral amino acid selected from the group consisting of Ser (S) and Thr (T).

38. The composition of any one of claims 19-24, wherein the A 21 is a cationic amino acid selected from the group consisting of His (H), Arg (R), and Lys (K).

39. The composition of any one of claims 19-24, wherein the A 22 is an aliphatic amino acid selected from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), lie (I), and Pro (P).

40. The composition of any one of claims 19-24, wherein the IS1 comprises five amino acids.

41. The composition of any one of claims 19-24, wherein the IS1 consists of five amino acids.

42. The composition of any one of claims 19-24, wherein the IS2 comprises nine to eleven amino acids.

43. The composition of any one of claims 19-24, wherein the IS2 consists of nine amino acids.

44. The composition of any one of claims 19-24, wherein the IS3 comprises one amino acid that is an aliphatic amino acid selected from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), Ile (I), and Pro (P) or an aromatic amino acid selected from the group consisting of Phe (F), Tyr (Y), and Trp (W).

45. The composition of any one of claims 19-24, wherein the IS3 consists of one amino acid.

46. A composition comprising a defensin peptide fold variant 1 (DEF PFV1), wherein the defensin is a cationic defensin peptide comprising a defensin gamma core peptide sequence GXC5X3-9C6 (SEQ ID NO: 455) or a variant gamma core peptide sequence GXC5X3-10C6 (SEQ ID NO: 456), GXC5X3-9(F / W / Y) (SEQ ID NO: 457), or GXC5X3-10(F / W / Y) (SEQ ID NO: 458), and at least two or three additional cysteine residues selected from the group consisting of cysteine residues corresponding to Cl, C2, C3, C4, C7, and C8 of a reference defensin peptide, wherein the cysteine residues of DEF_PFV1 form a first set of disulfide bonds, and whereby DEF_PFV1 adopts a first conformation that exhibits greater antimicrobial activity, increased protease resistance, or increased protease sensitivity compared to a defensin peptide fold variant 2 (DEF_PFV2) having the same amino acid sequence as DEF_PFV1 but forming a different second set of disulfide bonds between the cysteine residues and adopting a different second conformation, and wherein the composition is substantially free of DEF_PFV2.

47. The composition of claim 46, wherein the cationic defensin peptide comprises at least four additional cysteine residues selected from the group consisting of cysteines corresponding to Cl, C2, C3, C4, C5, C6, C7, and C8 of a reference defensin peptide.

48. The composition of claim 46, wherein one to four of the cysteine residues corresponding to Cl, C2, C3, C4, C7, and C8 of a reference defensin peptide are substituted with a different amino acid or are deleted, optionally wherein the cysteine is substituted with a Ser (S) or Thr (T) residue.

49. The composition of claim 46, wherein at least one of the cysteine residues corresponding to Cl, C2, C3, C4, C7, and C8 of the reference defensin peptide is substituted with a different amino acid, at least one of the cysteine residues corresponding to Cl, C2, C3, C4, C5, C6, C7, and C8 of the reference defensin peptide is deleted, and wherein at least two of the cysteine residues corresponding to Cl, C2, C3, C4, C7, and C8 of the reference defensin peptide are retained.

50. The composition of claim 46, wherein the defensin peptide comprises cysteine residues corresponding to C5, C6, C7, and C8 of a reference defensin peptide.

51. The composition of claim 46, wherein the defensin peptide is a native defensin peptide.

52. The composition of claim 46, wherein the defensin peptide is a synthetic variant of a native defensin peptide comprising one or more amino acid insertions, deletions, and / or substitutions in a native defensin peptide.

53. The composition of claim 46, wherein the substitution in a native defensin peptide comprises at least one of: a substitution of a hydrophobic amino acid with a hydrophobic amino acid, a substitution of a cationic amino acid with a cationic amino acid, or a substitution of an anionic amino acid with a cationic or anionic amino acid.

54. The composition of claim 46, wherein the defensin peptide comprises a C5-5-6 AA-C6 motif flanked by N-terminal C5 and C-terminal C6, comprising three amino acids (A 14 , A 15 , A 16 , A 17 , A 18 and optionally A 19 ) in sequential order from its N-terminus.

55. The composition of claim 46, wherein: (i) Said A 14 The amino acids selected are cationic amino acids from the group consisting of His (H), Arg (R), and Lys (K), or aliphatic amino acids from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), Ile (I), and Pro (P), or polar neutral amino acids from the group consisting of Ser (S) and Thr (T); (ii) the A 16 The amino acid is an aliphatic amino acid selected from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), Ile (I), and Pro (P) or an aromatic amino acid selected from the group consisting of Phe (F), Tyr (Y), and Trp (W); and / or (iii) wherein the A is... 18 These are cationic amino acids selected from the group consisting of His (H), Arg (R), and Lys (K).

56. The composition of claim 46, wherein the defensin peptide comprises a C7-3AA-C8 motif flanked by a N-terminal C7 and a C-terminal C8, comprising three amino acids (A 20 , A 21 , and A 22 ) in sequential order from its N-terminus.

57. The composition of claim 46, wherein: (i) said A is a polar neutral amino acid selected from the group consisting of Ser (S) and Thr (T); (ii) said A 20 is a cationic amino acid selected from the group consisting of His (H), Arg (R) and Lys (K); and / or (iii) said A 21 is an aliphatic amino acid selected from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), lie (I) and Pro (P). 22 is an aliphatic amino acid selected from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), lie (I) and Pro (P).

58. The composition of any one of claims 46-57, wherein the cationic defensin peptide has an isoelectric point between 8.0 and 12.

0.

59. The composition of any one of claims 46-57, wherein the DEF_PFV1 exhibits greater protease resistance than the DEF_PFV2.

60. The composition of any one of claims 46-57, wherein the DEF_PFV1 exhibits lower protease resistance than the DEF_PFV2.

61. The composition of any one of claims 46-57, wherein the defensin peptide comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% sequence identity to any one of SEQ ID NO: 1-454 or SEQ ID NO: 459-SEQ ID NO:

472.

62. The composition of any one of claims 46-57, wherein the composition further comprises an agriculturally, pharmaceutically, or veterinarily feasible carrier, diluent, or excipient.

63. A method for making the composition of any one of claims 46-57, the method comprising: (a) separating a fraction comprising DEF_PFV1 from one or more fractions comprising DEF_PFV2 or DEF_PFV2 peptide fragments thereof from a mixture comprising a defensin peptide fold variant 1 (DEF_PFV1) and a defensin peptide fold variant 2 (DEF_PFV2) or DEF_PFV2 peptide fragments thereof; and (b) combining the fraction comprising DEF_PFV1 or a preparation further purified from the DEF_PFV1 fraction with an agriculturally, pharmaceutically, or veterinarily feasible carrier, diluent, or excipient, thereby making the composition.

64. The method of claim 63, further comprising obtaining the mixture comprising DEF_PFV1 and DEF_PFV2 or DEF_PFV2 peptide fragments thereof by (a) culturing a microorganism expressing a recombinant polynucleotide comprising a transcriptional promoter operably linked to a polynucleotide encoding a signal peptide that is in-frame with and upstream of a polynucleotide encoding DEF_PFV1 or a variant thereof in a fermentation broth, and (b) separating the microorganism from the fermentation broth comprising the mixture, 65. The method of claim 64, wherein the microorganism is a bacterium or a fungus. wherein the microorganism is optionally selected from the group consisting of Candida, Kluyveromyces, Hansenula, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia, Myceliopthora, and Escherichia.

65. The method of claim 63, further comprising obtaining the mixture comprising DEF PFV1 and DEF PFV2 or a DEF PFV2 peptide fragment thereof by (a) culturing a microorganism expressing a recombinant polynucleotide comprising a transcriptional promoter operably linked to a polynucleotide encoding DEF PFV1 or a variant thereof in a fermentation broth, and (b) isolating the microorganism comprising the mixture from the fermentation broth, (c) disrupting the microorganism to obtain a cell lysate, and (d) separating insoluble debris in the cell lysate from an aqueous fraction comprising the mixture, wherein the microorganism is optionally selected from the group consisting of Candida, Kluyveromyces, Hansenula, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia, Myceliopthora, and Escherichia.

66. The method of claim 63, further comprising treating the mixture comprising DEF PFV1 and DEF PFV2 with a protease under non-denaturing conditions sufficient to produce a DEF PFV2 peptide fragment but insufficient to produce a DEF PFV1 peptide fragment.

67. The method of claim 63, wherein the protease is a trypsin family serine protease that cleaves DEF PFV2 at the carboxy-terminal end of DEF PFV2 Arg (R) or Lys (K).

68. The method of claim 63, wherein the trypsin family serine protease is recombinant bovine, porcine, human, or microbial trypsin, and optionally, wherein the microbial trypsin is Streptomyces trypsin or a variant thereof.

69. The method of claim 63, further comprising separating the DEF PFV1 from the DEF PFV2 peptide fragment by size exclusion chromatography.

70. A method for preventing or reducing crop damage or post-harvest loss caused by a plant pathogenic microorganism, the method comprising contacting a plant, a plant seed, a pre-harvest or post-harvest grain, a pre-harvest or post-harvest fruit, or a pre-harvest or post-harvest vegetable with an effective amount of the composition of any one of claims 1 to 24 or 46 to 57 and under conditions suitable for preventing or reducing crop damage or post-harvest loss.

71. The method of claim 70, wherein the plant pathogenic microorganism is selected from the group consisting of a Fusarium species, an Alternaria species, a Verticillium species, a Phytophthora species, a Colletotrichum species, a Erysiphe species, a Cercospora species, a Phakopsora species, a Rhizoctonia species, a Sclerotinia species, a Pythium species, a Phoma species, an Aspergillus species, an Elsinoe species, a Puccinia species, a Septoria species, a Penicillium species, a Chaetomium species, a Phomopsis species, a Leveillula species, a Melampsora species, a Mycosphaerella species, a Catenaspora species, a Botrytis species, a Helminthosporium species, a Diplodia species, a Corynespora species, or a Passalora species.

72. The method of claim 70, wherein the plant, plant seed, pre- or post-harvest grain, pre- or post-harvest fruit, or pre- or post-harvest vegetable is selected from the group consisting of a cereal crop, a legume crop, a root or tuber crop, an oil crop, a fruit crop, a vegetable crop, a nut crop, a forage or turf grass crop, a forage legume, a medicinal crop, a spice or flavor crop, a fiber crop, and a biofuel crop, a plant seed, a pre- or post-harvest grain, a pre- or post-harvest fruit, or a pre- or post-harvest vegetable.

73. The method of claim 70, wherein the plant, plant seed, pre- or post-harvest grain, pre- or post-harvest fruit, or pre- or post-harvest vegetable is selected from the group consisting of wheat, rice, maize, barley, oats, sorghum, rye, millet, peanut, chickpea, pigeon pea, lentil, bean, soybean, lima bean, potato, sweet potato, cassava, oilseed rape, peanut, palm, coconut, safflower, cotton, sunflower, flax, olive, sugarcane, sugar beet, banana, orange, apple, pear, breadfruit, pineapple, strawberry, grape, cherry, coffee, cacao, tomato, lettuce, carrot, melon, asparagus, Brassica species, cashew, walnut, pistachio, almond, alfalfa, clover, castor, camelina, borage, switchgrass, miscanthus, and jatropha plant, a plant seed, a pre- or post-harvest grain, a pre- or post-harvest fruit, or a pre- or post-harvest vegetable.

74. A medical device having antimicrobial properties, the medical device comprising a medical device in operable combination with the composition of any one of claims 1 to 24 or 46 to 57, wherein the medical device comprises at least one surface that is topically coated or impregnated with the composition.

75. The medical device of claim 74, wherein the medical device is selected from the group consisting of a stent, a catheter, a contact lens, a condom, a patch, and a diaphragm.

76. A method for treating, preventing, or inhibiting a microbial infection in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the composition of any one of claims 1 to 24 or 46 to 57 under conditions and for a duration suitable for treating, preventing, or inhibiting the microbial infection.

77. The method of claim 76, wherein the administering comprises introducing the composition locally, enterally, parenterally, or intravenously to the subject.

78. The method of claim 76, wherein the subject is selected from the group consisting of a human, a livestock, a poultry, a fish, and a companion animal.

79. The method of claim 76, wherein the microbial infection is a microbial infection of a mucosa, an eye, a skin, or a nail, and the composition is applied to the mucosa, the eye, the skin, or the nail.

80. The method of claim 76, wherein the microbial infection is caused by a dermatophyte, and wherein the dermatophyte is optionally selected from the group consisting of Trichophyton rubrum, T. interdigitale, T. violaceum, T. tonsurans, T. soudanense, T. mentagrophytes, Microsporum canis, E. floccosum, and M. gypseum.

81. The method of claim 76, wherein the microbial infection is caused by a microbe selected from the group consisting of Aspergillus, Cryptococcus, Penicillium, Rhizopus, Lecythophora, Mucor, Physarum, Rhizomucor, Syncephalastrum, Cokeromyces, Mortierella, Pythium, Fusarium, Histoplasma, and Blastomyces species.

82. The method of claim 76, wherein the microbial infection is caused by a microbe selected from the group consisting of Candida species, wherein the Candida species is selected from the group consisting of C. albicans, C. auris, C. glabrata, C. parapsilosis, C. tropicalis, and C. krusei.

83. The composition of any one of claims 1-24 or 46-57 for use in a method of treating, preventing, or inhibiting a microbial infection in a subject in need thereof.

84. The composition of claim 83, wherein the subject is selected from the group consisting of a human, a livestock, a poultry, a fish, and a companion animal.

85. A plant part at least partially coated with the composition of any one of claims 1-24 or 46-57.

86. The plant part of claim 85, wherein the plant part is selected from the group consisting of a seed and a pre-harvest or post-harvest grain.

87. The plant part of claim 85, wherein the plant part is selected from the group consisting of a pre-harvest or post-harvest fruit, a pre-harvest or post-harvest vegetable, and a pre-harvest or post-harvest flower.

88. The plant part of claim 85, wherein the plant part is selected from the group consisting of a cereal crop, a legume crop, a root or tuber crop, an oil crop, a fruit crop, a vegetable crop, a nut crop, a forage or turf grass crop, a forage legume, a medicinal crop, a spice or flavor crop, a fiber crop, and a biofuel crop plant part.

89. The plant part of claim 85, wherein the plant part is selected from the group consisting of wheat, rice, maize, barley, oat, sorghum, rye, millet, peanut, chickpea, pigeon pea, lentil, bean, soybean, lima bean, potato, sweet potato, cassava, oilseed rape, peanut, palm, coconut, safflower, cotton, sunflower, flax, olive, sugar cane, sugar beet, banana, orange, apple, pear, breadfruit, pineapple, strawberry, grape, cherry, coffee, cocoa, tomato, lettuce, carrot, melon, asparagus, Brassica species, cashew, walnut, almond, pistachio, alfalfa, clover, castor, camelina, pennycress, switchgrass, miscanthus, and jatropha plant parts.

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