Staphylococcus hominis daptide antibiotics, derivatives thereof, and methods of treatment

Abstract

Embodiments of the present disclosure generally relate to a new class of daptide antibiotics and compositions that include the daptide antibiotics. Embodiments described herein also generally relate to methods of treating various diseases or medical conditions in a patient utilizing the new class of daptide antibiotics and compositions thereof. In an embodiment, a composition for treating a skin condition in a patient is provided. The composition includes a daptide comprising: a dimethylated 7V-terminus; a Dmp-modified C- terminus; a dehydrobutyrine residue positioned between the dimethylated 7V-terminus and the Dmp-modified C-terminus; at least one alanine (A) residue or at least one dehydroalanine (Dha) residue positioned between the dimethylated 7V-terminus and the Dmp-modified C- terminus; and at least one amino acid residue positioned between the dimethylated 7V-terminus and the Dmp-modified C-terminus. In another embodiment is provided a daptide bacteriocin composition that is antimicrobial against a staphylococcal skin bacteria in a patient.

Description

PCT Application Attorney Docket No.: CUBR / 0006PC Title: Staphylococcus Hominis Daptide Antibiotics, Derivatives Thereof, and Methods of Treatment Inventors: Alexander Horswill; Amber Nguyen; Nadja Cech; Zoie Bunch; Jonathan Chekan CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of and priority to U.S. Provisional Patent Application No.63 / 647,820, filed on May 15, 2024, which is incorporated herein by reference in its entirety. REFERENCE TO SEQUENCE LISTING

[0002] This application contains references to amino acid and nucleic acid sequences which have been submitted as the sequence listing text file entitled “SEQ ID NOS 1-20”, file size 71.1 Kilobytes (KB), created April 19, 2025, which is hereby incorporated by reference into this application in its entirety. FIELD

[0003] Embodiments of the present disclosure generally relate to a new class of daptide antibiotics and compositions that include the daptide antibiotics. Embodiments described herein also generally relate to methods of treating various diseases or medical conditions in a patient utilizing the new class of daptide antibiotics and compositions thereof. BACKGROUND

[0004] The human skin is home to microorganisms, such as coagulase-negative staphylococci (CoNS), which coexist homeostatically with the host and play a vital role in the maintenance and health of the skin. One such CoNS is Staphylococcus hominis (S. hominis). S. hominis serves in a protective role during colonization but the mechanism of S. hominis protective function in colonization resistance remains poorly understood. Such mediators of colonization resistance that CoNS produce include bacteriocins. Bacteriocins are typically ribosomally-synthesized and post-translationally-modified peptides (RiPPs). CoNS harbor myriad bacteriocins, among which the most well-characterized are lantibiotics. Lantibiotics are known to exhibit antimicrobial activity against Gram-positive pathogens, like methicillin-resistant Staphylococcus aureus (MRSA). Lack of bacteriocin-producing CoNS has been associated with increased colonization by S. aureus on the skin of atopic dermatitisPCT Application Attorney Docket No.: CUBR / 0006PC patients. Furthermore, bacteriocins have been proposed as alternative therapies to traditional antibiotics with proven efficacy and potency in clinical trials and small animal models of skin infection. Given the decline of effective antibiotics coupled with the emergence of multi- drug-resistant bacteria, it is imperative to broaden the discovery of non-lantibiotic RiPPs and investigate their mechanism of action.

[0005] Daptides are a more recently discovered class of RiPPs characterized by two amino-modified termini: a dimethylated N-terminus and a N2,N2-dimethyl-1,2- propanediamine (Dmp)-modified C-terminus. Despite these chemical features, the biological significance of daptides as antimicrobial bacteriocins remains unclear, and the mechanism of resistance has not been investigated.

[0006] There is a need for new antibiotics and compositions thereof. There is also a need for new methods for treatment of, for example, various diseases or medical conditions in a patient. SUMMARY

[0007] Embodiments of the present disclosure generally relate to a new class of daptide antibiotics and compositions that include the daptide antibiotics. The new class of antibiotics include daptides. As described herein, the inventors found a novel daptide with antimicrobial properties made by a skin Staphylococcus hominis strain AH5011. The daptide may be hominicin or a derivative thereof. The daptide has broad activity against Staphylococcal skin bacteria, including Staphylococcus aureus. The daptide also shows activity against Streptococcus pyogenes, also known as Group A Strep. A mechanism of action of the daptide may be pore formation in the cytoplasmic membrane of bacteria. Derivatives of the daptide are also described herein. Embodiments described herein also generally relate to methods of treating various diseases or medical conditions in a patient utilizing the new class of daptide antibiotics and compositions thereof. For example, embodiments of the present disclosure may be utilized for, e.g., treating a skin condition in a patient, such as a skin disease, a skin pathogen, a skin bacteria, or combinations thereof.

[0008] In an embodiment, a composition for treating a skin condition in a patient is provided. The composition includes a daptide, the daptide comprising: a dimethylated N- terminus; a N2,N2-dimethyl-1,2-propanediamine (Dmp)-modified C-terminus; a dehydrobutyrine residue positioned between the dimethylated N-terminus and the Dmp-PCT Application Attorney Docket No.: CUBR / 0006PC modified C-terminus; at least one alanine (A) residue or at least one dehydroalanine (Dha) residue positioned between the dimethylated N-terminus and the Dmp-modified C-terminus; and at least one amino acid residue positioned between the dimethylated N-terminus and the Dmp-modified C-terminus.

[0009] In another embodiment, a daptide bacteriocin composition that is antimicrobial against a staphylococcal skin bacteria in a patient is provided. The daptide bacteriocin composition includes an amino acid sequence having at least 75% sequence identity to one or more of the amino acid sequences set forth in SEQ ID NOs: 1-20.

[0010] In another embodiment, a method for treating a skin condition in a patient. The method includes administering to the patient a composition that includes a daptide, the daptide comprising: a dimethylated N-terminus; a Dmp-modified C-terminus; a dehydrobutyrine residue positioned between the dimethylated N-terminus and the Dmp-modified C-terminus; at least one alanine residue or at least one dehydroalanine (Dha) residue positioned between the dimethylated N-terminus and the Dmp-modified C-terminus; and at least one amino acid residue positioned between the dimethylated N-terminus and the Dmp-modified C-terminus. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] So that the manner in which the above recited features of the present disclosure may be understood in detail, a more particular description of the disclosure, briefly summarized above, may be had by reference to embodiments, some of which are illustrated in the appended drawings. It is to be noted, however, that the appended drawings illustrate only exemplary embodiments and are therefore not to be considered limiting of its scope, and may admit to other equally effective embodiments.

[0012] FIGS.1A-1G show data indicating that S. hominis AH5011 inhibits with species- and strain-specificity in a contact-independent and concentration-dependent manner. S. aureus strain AH6350 (FIG.1A), S. hominis strains AH4553 (FIG.1D), and AH5011 (FIG. 1F) were incubated with 40% v / v CM from AH5011 or tryptic soy broth (TSB) for 8 h and monitored by cell density (OD600) and colony-forming units (CFUs) (n=3). FIG. 1B: Antimicrobial susceptibility of Staphylococcus sp., Micrococcus luteus, Streptococcus pyogenes, Streptococcus agalactiae, and Enterococcus faecalis (n=2). “N” values denote the number of bacterial strains tested. FIG. 1C: Representative images of spot-on-lawn bioactivity of AH5011. S. hominis strains AH4553 (FIG. 1E) and AH5011 (FIG. 1G) werePCT Application Attorney Docket No.: CUBR / 0006PC incubated in 40%, 20%, 10% v / v conditioned medium (CM) from AH5011 for 8 h (n=3). Means ± SEM (error bars) are plotted. *, p <0.05; **, p <0.01; ***, p <0.001; ****, p <0.0001; ns, not significant by repeated measures two-way ANOVA (FIGS.1A, 1D, 1F) and one-way ANOVA with Dunnett’s multiple comparisons (FIGS.1E, 1G).

[0013] FIGS.2A-2D shows data indicating that S. hominis AH5011 encodes a plasmid- borne daptide bacteriocin. FIG. 2A: Representative images of spot-on-lawn bioactivity of wild-type AH5011 and plasmid-cured mutants against a lawn of AH4553, with measurements of diameter of inhibition (n=5). The dashed line indicates the limit of detection. Means ± SEM (error bars) are plotted. ****, p <0.0001; ns, not significant by one-way ANOVA with Dunnett’s multiple comparisons. FIG.2B: Gene arrangement and annotation of the hom BGC that spans 9,596 base pairs (bp) in length. FIG.2C: HomA encodes a 65-amino acid precursor peptide, including an N-terminal leader and a C-terminal core sequence. FIG.2D: The post- translational modifications of the core peptide, with the modified sites shown and the modifying enzymes indicated.

[0014] FIGS. 3A-3D show detection and structural characterization of the AH5011 daptide hominicin. FIG.3A: Base peak chromatograms of conditioned media from AH5011, Δp1Δp2, or TSB blank control. A peak matching the predicted m / z of the [M+2H]2+of the daptide bacteriocin (1020.1103) within 2 ppm of the measured mass (1020.1114) elutes at a retention time of 5.32 minutes, as indicated by the trace for AH5011. FIG. 3B: Base peak chromatograms of conditioned media from S. aureus strains expressing homABCDIJM1M2PlanMD and empty vector. FIG. 3C: MS-MS spectrum showing major fragments for the precursor ion calculated at m / z 1020.1103 identified as the bacteriocin [M+2H]2+ion in conditioned media from AH5011. FIG. 3D: Molecular structure and annotated MS / MS fragments of the AH5011 hominicin in conditioned media with precursor ion m / z 1020.110.

[0015] FIGS. 4A-4C show data for the heterologous expression and product characterization of hom BGC. FIG. 4A: Gene organization of the minimal hom BGC (no flanking genes included), with the constructed gene omissions depicted. FIG. 4B: Representative images of spot-on-lawn bioactivity of S. aureus wild-type and expression mutants against a bacterial lawn of S. hominis AH4553, with measurements of diameter of inhibition (n=5). The dashed line indicates the limit of detection. FIG.4C: S. hominis AH4553PCT Application Attorney Docket No.: CUBR / 0006PC was cultured in 40% v / v CM from engineered S. aureus strains for 8 h (n=3). Means ± SEM (error bars) are plotted. *, p <0.05; ****, p <0.0001; ns, not significant by one-way ANOVA with Dunnett’s multiple comparisons (FIGS.5B and 5C).

[0016] FIGS. 5A-5E show data indicating that AH5011 hominicin is a membrane- disruptive, pore-forming antimicrobial. FIG. 5A: AH6350 cells were stained with SYTOX Green and treated with AH5011 or Δp1Δp2 extract prepared by n-butanol extraction, 35% ethanol, or left untreated. Fluorescence intensity was measured 3 h post-treatment (n=3). The concentrations of hominicin in the AH5011 extract were 4.1 µg (1x), 8.2 µg (2x), 16.3 (4x) µg. FIG.5B: AH6350 cells were stained with DiSC3(5) and treated with AH5011 or Δp1Δp2 extract, 1% Triton X-100, or left untreated. Fluorescence intensity was measured every 5 min (n=3). FIG. 5C: Current / voltage (I / V) relationship of hominicin, unmodified core peptide, alpha-toxin, and vehicle under symmetrical conditions of 1 M KCl +10 mM HEPES, pH 7.2 (n=5). FIGS.5D and 5E: Representative current traces showing single channel openings and closings recorded at +100 mV (FIG. 5D) and -100 mV (FIG. 5E) for hominicin (left), unmodified core peptide (middle), and vehicle control (right). Means ± SEM (error bars) are plotted. *, p <0.05; **, p <0.01; ****, p <0.0001; ns, not significant by two-tailed unpaired t-test (FIG.5A) and one-way ANOVA with Dunnett’s multiple comparisons (FIG.5B).

[0017] FIGS. 6A-6F show data indicating that HomI confers resistance to AH5011 hominicin. FIGS. 6A and 6B: Bacterial growth curves of S. aureus strains Newman (WT), homI mutant, and empty vector grown in TSB (FIG.6A) or 40% v / v CM from AH5011 (FIG. 6B) (n=3). FIG.6C: Images of spot-on-lawn bioactivity of AH5011 and RN4220 expressing hom BGC against lawns of S. aureus, with measurements of diameter of inhibition (n=5). The dashed line indicates the limit of detection. FIGS. 6D, 6E, 6F: Extrusion of DiSC3(5) from wild-type (FIG. 6D), homI mutant (FIG. 6E), and empty vector (FIG. 6F) (n=3). The 8x AH5011 extract contains about 32.7 µg of hominicin. Means ± SEM (error bars) are plotted. *, p <0.05; ***, p <0.001; ****, p<0.0001; ns, not significant by one-way ANOVA with Dunnett’s multiple comparisons (FIGS.6A-6F).

[0018] FIGS.7A-7E show data indicating that AH5011 hominicin is protective against S. aureus skin infection. FIG. 7A: Representative image of hemolysis from AH6350 on 5% sheep blood agar. FIG.7B: Representative images of skin lesions for indicated groups. FIG. 7C: Total bacterial burden in epicutaneous lesions at 72 h post-infection (hpi) (n=10 perPCT Application Attorney Docket No.: CUBR / 0006PC group). FIG.7D: Skin barrier integrity was assessed by measuring transepidermal water loss (TEWL) prior to infection and at 72 hpi (n=5 per group). FIG. 7E: The severity of skin inflammation was quantified using a total disease score, which is the sum of the individual grades for erythema, edema, erosion, and scaling. Means ± SEM (error bars) are plotted. ND, no detection; *, p <0.05; **, p <0.01; ****, p <0.0001; ns, not significant by two-tailed Mann-Whitney U-test (FIG.7C) and one-way ANOVA with Dunnett’s multiple comparisons (FIGS.7D and 7E).

[0019] FIG.8 shows an NMR spectrum of S. hominis AH5011 hominicin in methanol-d3 (700 MHz).

[0020] FIG. 9 shows minimum inhibitory concentration (MIC) curves for AH5011 hominicin and vancomycin against all tested strains. The starting concentration was 8 µg / mL, serially diluted down to 0.002 µg / mL. Error bars indicate the standard error calculated from three replicates.

[0021] FIGS. 10A-10E show data indicating single-channel properties of membrane channels formed by hominicin. FIG. 10A: Schematic of planar lipid bilayer setup and the electrophysiological methods used in this study. FIG.10B: Bioactivity disk diffusion assay with the purified hominicin, synthetic unmodified core peptide, and methanol (vehicle) against S. aureus strain AH6350. FIGS.10C-10E: Representative current traces of hominicin, unmodified core, and vehicle at the indicated holding potential of +50 mV (FIG. 10C), -50 mV (FIG.10D), and -100 mV (FIG.10E).

[0022] FIG.11 shows an illustrative, but non-limiting, working model for the hominicin daptide. The hom BGC from S. hominis strain AH5011 encodes the precursor peptide, immunity factor, and post-translationally modifying enzymes (bottom). Hominicin displays bactericidal activity against the genus Staphylococcus. To exert its mechanism of action, hominicin partitions into the hydrophobic core of the bacterial membrane and then self- assembles into an ion-conducting transmembrane pore (top left). The formation of peptidic channels consequently impairs cell membrane integrity by dissipating the bacterial transmembrane potential. HomI functions as the cognate resistance factor that protects from daptide-induced membrane damage (top right). FIG.11 was created using BioRender.

[0023] FIGS.12A and 12B show data indicating the effects of heat and protease treatment on AH5011 activity. FIG.12A: S. hominis strains were incubated with Proteinase K-treated,PCT Application Attorney Docket No.: CUBR / 0006PC or untreated 20% v / v CM from AH5011 for 8 h (n=3). FIG. 12B: S. hominis strains were incubated with 100oC treated, or untreated 20% v / v CM for 8 h (n=3). Data represent relative growth compared to the untreated control at 8 h post-incubation. Means ± SEM (error bars) are plotted. ****, p <0.0001; ns, not significant by two-tailed unpaired t-test (FIG.12A) and one-way ANOVA with Dunnett’s multiple comparisons (FIG.12B).

[0024] FIGS.13A-13D show data on the isolation of plasmid-cured mutants. FIG.13A: S. hominis AH4553 was incubated with 40% v / v CM from AH5011, or plasmid-cured mutants for 8 h (n=3). FIGS.13B-13D: Isogenic Δp1Δp2 (FIG.13B), p2 Δp1 (FIG.13C), and p1 Δp2 (FIG. 13D) mutants were treated with 40% v / v CM from wild-type AH5011, or untreated (TSB) for 24 h (n=3). Means ± SEM (error bars) are plotted. *, p <0.05; **, p <0.01; ns, not significant by one-way ANOVA with Dunnett’s multiple comparisons (FIG. 13A) and repeated measures two-way ANOVA (FIGS.13B-13D).

[0025] To facilitate understanding, identical reference numerals have been used, where possible, to designate identical elements that are common to the figures. It is contemplated that elements and features of one embodiment may be beneficially incorporated in other embodiments without further recitation. DETAILED DESCRIPTION

[0026] Embodiments of the present disclosure generally relate to a new class of daptide antibiotics and compositions that include the daptide antibiotics. As used herein, a “composition” may include component(s) of the composition, reaction product(s) of two or more components of the composition, a remainder balance of remaining starting component(s), or combinations thereof. Embodiments described herein also generally relate to methods of treating various diseases or medical conditions in a patient utilizing the new class of daptide antibiotics and compositions thereof. The term “patient”, “subject”, or “individual” are used interchangeably herein and refer to a vertebrate, such as a mammal. Mammals include, but are not limited to, humans.

[0027] Human skin serves as both a physical and an immunologic barrier between the body and the environment. Yet, the skin microbiota stably colonizes in this niche, preventing pathogen colonization, fortifying the epithelial barrier, and priming immune responses. Coagulase-negative staphylococci (CoNS) are prominent members of the skin microbiome, comprising a heterogenous group of thirty-eight (38) known species. CoNS actively maintainPCT Application Attorney Docket No.: CUBR / 0006PC skin homeostasis by secreting natural products that antagonize microbial competitors and pathogens. While studies of CoNS’s role in the skin microbiome have primarily focused on Staphylococcus epidermidis, emerging evidence underscores the beneficial roles of Staphylococcus hominis, a common CoNS on human skin, in defending against bacterial pathogens. S. hominis secretes autoinducing peptides that inhibit the accessory gene regulator (agr) system of the skin pathogen Staphylococcus aureus, thereby preventing S. aureus- mediated inflammation and cutaneous damage. Furthermore, S. hominis produces an arsenal of bacteriocins, including lantibiotics and other peptidic antibiotics, which inhibit S. aureus in vitro and in vivo.

[0028] Bacteriocins are often ribosomally synthesized and post-translationally modified peptides (RiPPs) with targeted antibacterial activity against specific genera and species. These RiPPs are generally encoded by complex and variable biosynthetic gene clusters (BGCs) that include the precursor bacteriocin, post-translationally modifying enzymes, and accessory proteins involved in export and producer immunity. Bacteriocin production is traditionally regarded as a probiotic trait, as it facilitates the producer strain in colonizing a niche, eliminating competing strains or pathogens, and potentially modulating the immune responses. Contextually, the loss of bacteriocin-producing CoNS is associated with increased burden of S. aureus on the skin of atopic dermatitis, underscoring the importance of commensal bacteria in maintaining and fortifying the skin’s antimicrobial barrier. In a post- antibiotic era, bacteriocins present a promising source of potent antimicrobials that have the potential to inhibit pathogens. Human clinical trials have demonstrated the safe delivery and effectiveness of lantibiotic-producing S. hominis as a bacteriotherapy treatment for S. aureus- colonized atopic dermatitis (AD) skin, highlighting the effect of a bacteriocin producer in reducing S. aureus colonization and disease severity.

[0029] Over 40 classes of RiPPs have been reported, categorized by class-defining chemical features and / or modifications, with heterogeneity in peptide structures, biosynthetic machinery, modes of action, immunity mechanisms, and target receptors. Daptides have emerged as a new class of RiPPs, characterized by an unusual (S)-N2,N2-dimethyl-1,2- propanediamine (Dmp)-modified C-terminus and defined in Microbacterium paraoxydans. Previous investigations on M. paraoxydans (Mpa) daptides reported varying degrees of activity but have not conclusively demonstrated target specificity, mode of action, orPCT Application Attorney Docket No.: CUBR / 0006PC mechanisms of resistance. Such knowledge is critical to the field of drug discovery for developing novel and rational interventions that reduce morbidity and mortality from bacterial infections.

[0030] Herein, the inventors identified a daptide bacteriocin produced by a human skin isolate of S. hominis. The expression of the reconstituted biosynthesis genes sufficiently conferred antimicrobial activity, with the Dmp modification being critical for this activity. The inventors isolated the daptide from culture supernatant and solved its structure using mass spectrometry and nuclear magnetic resonance (NMR). Mechanistically, the S. hominis daptide may induce membrane damage by dissipating the transmembrane potential of susceptible bacteria through the formation of peptidic channels. Additional genetic studies identified a novel protein, HomI, as the determinant for the cognate resistance against daptide- induced membrane damage. Also, the inventors demonstrated that topical treatment with the purified daptide reduces S. aureus colonization and cutaneous injury in a murine model of epicutaneous infection. The study presented herein elucidates the mechanisms of action, biosynthesis, and resistance for an understudied class of natural products called daptides.

[0031] Various issues in the art of daptides exist. For example, there is a lack of genetic and biosynthesis information for S. hominis MBBL 2-9 strain and it is unclear whether MBBL 2-9 hominicin is plasmid-encoded. There are no relevant animal studies using the pure hominicin peptide or the hominicin-producing strain. There is no reported resistance mechanism and the art lacks a proposed mechanism of action. There is also limited strain susceptibility testing. Here, the state-of-the-art has not tested for Streptococcus pyogenes and CoNS killing. Instead, studies exist on killing activity against only two S. aureus strains (MRSA ATCC 11435 and vancomycin-intermediate CCARM 3501). There have been no Marfey’s analyses performed to confirm the stereochemistry of the peptide hominicin and the presence of D-alanines has not been reported. In addition, hominicin has been classified as a class I peptide without the thioether bridge typical of lantibiotics. Further, prior studies have not suggested whether hominicin represents a new class of RiPP. These questions are answered herein.

[0032] Embodiments of the present disclosure generally relate to a new class of daptide antibiotics and compositions that include the daptide antibiotics. In general, daptides are polypeptides that include two amino-modified termini: a dimethylated N-terminus and a C-PCT Application Attorney Docket No.: CUBR / 0006PC terminus that is modified with N2,N2-dimethyl-1,2-propanediamine (Dmp). N2,N2-dimethyl- 1,2-propanediamine (Dmp) has the following chemical structure:

[0033] Daptides and derivatives thereof described herein are made up of amino acids. When in the form of a daptide or a derivative thereof, the amino acids may be interchangeably referred to as amino acid residues.

[0034] Daptides and derivatives thereof described herein may further include one or more dehydrobutyrine residues positioned between the dimethylated N-terminus and the modified Dmp-modified C-terminus. Dehydrobutyrine (Dhb) is a non-proteinogenic amino acid. Dhb is an unsaturated amino acid having a carbon-carbon double bond (C=C bond) in its molecular structure. Dhb has the following chemical structure:

[0035] Additionally, or alternatively, daptides and derivatives thereof of the present disclosure may further include one or more alanine residues, one or more dehydroalanine (Dha) residues, or both positioned between the dimethylated N-terminus and the Dmp- modified C-terminus of the daptide or derivative thereof. Dha is a non-proteinogenic amino acid. Dha is an unsaturated amino acid having a carbon-carbon double bond (C=C bond) in its molecular structure. Dha has the following chemical structure:

[0036] Daptides and derivatives thereof described herein may further include at least one amino acid residue positioned between the dimethylated N-terminus and the Dmp-modified C-terminus of the daptide or derivative thereof, wherein the at least one amino acid residue is not Dhb, Dha, or alanine. Amino acid residues positioned between the two terminal amino acid residues—including Dhb, Dha, and alanine, among others—are referred to herein as internal amino acid residues.PCT Application Attorney Docket No.: CUBR / 0006PC

[0037] The dimethylated N-terminus of daptides and derivatives thereof may include a dimethylated isoleucine residue where the nitrogen atom of the isoleucine residue is dimethylated. The Dmp-modified C-terminus may include an alanine residue modified with the Dmp. Here, the carboxylic acid group of the terminal alanine residue is modified with the Dmp to form an amide adduct.

[0038] Daptides and derivatives thereof described herein may have any suitable number of amino acid residues. For example, the daptide or derivative thereof may include from 15 to 25 amino acid residues, such as from 17 to 24 amino acid residues, such as from 18 to 23 amino acid residues, such as from 19 to 22 amino acid residues, such as from 20 to 21 amino acid residues, such as 20 amino acid residues, excluding the Dmp moiety.

[0039] Daptides and derivatives thereof of the present disclosure may comprise, consist essentially of, or consist of an amino acid sequence set forth in SEQ ID NO: 1 (IXPAXPFXPXITXIXAXVIA). In the amino acid sequence set forth in SEQ ID NO: 1, the N-terminus isoleucine (located at residue 1) is dimethylated (that is, the nitrogen atom has two methyl (CH3) groups); each X at residues 2, 5, 8, and 15 may be Dhb; each X at residues 10 and 17 may be, independently, alanine or Dha; X at residue 13 may be E (glutamic acid), F (phenylalanine), L (leucine), I (isoleucine), Y (tyrosine), W (tryptophan), V (valine), M (methionine), P (proline), A (alanine), Q (glutamine), or K (lysine); and the C-terminus alanine (located at residue 20) is modified with Dmp.

[0040] Daptides and derivatives thereof described herein may comprise, consist essentially of, or consist of an amino acid sequence set forth in SEQ ID NO: 2 (IXPAXPFXPXITXIXAXVIA). In the amino acid sequence set forth in SEQ ID NO: 2, the N-terminus isoleucine (located at residue 1) is dimethylated; each X at residues 2, 5, 8, and 15 is Dhb; each X at residues 10 and 17 is Dha; X at residue 13 is E, F, L, I, Y, W, V, M, P, A, Q, or K; and the C-terminus alanine (located at residue 20) is modified with Dmp.

[0041] Daptides and derivatives thereof described herein may comprise, consist essentially of, or consist of an amino acid sequence set forth in SEQ ID NO: 3 (IXPAXPFXPXITXIXAAVIA). In the amino acid sequence set forth in SEQ ID NO: 3, the N-terminus isoleucine (located at residue 1) is dimethylated; each X at residues 2, 5, 8, and 15 is Dhb; X at residue 10 is Dha; X at residue 13 is E, F, L, I, Y, W, V, M, P, A, Q, or K; and the C-terminus alanine (located at residue 20) is modified with Dmp.PCT Application Attorney Docket No.: CUBR / 0006PC

[0042] Daptides and derivatives thereof described herein may comprise, consist essentially of, or consist of an amino acid sequence set forth in SEQ ID NO: 4 (IXPAXPFXPAITXIXAXVIA). In the amino acid sequence set forth in SEQ ID NO: 4, the N-terminus isoleucine (located at residue 1) is dimethylated; each X at residues 2, 5, 8, and 15 is Dhb; X at residue 17 is Dha; X at residue 13 is E, F, L, I, Y, W, V, M, P, A, Q, or K; and the C-terminus alanine (located at residue 20) is modified with Dmp.

[0043] Daptides and derivatives thereof described herein may comprise, consist essentially of, or consist of an amino acid sequence set forth in SEQ ID NO: 5 (IXPAXPFXPAITXIXAAVIA). In the amino acid sequence set forth in SEQ ID NO: 5, the N-terminus isoleucine (located at residue 1) is dimethylated; each X at residues 2, 5, 8, and 15 is Dhb; X at residue 13 is E, F, L, I, Y, W, V, M, P, A, Q, or K; and the C-terminus alanine (located at residue 20) is modified with Dmp.

[0044] Daptides and derivatives thereof described herein may comprise, consist essentially of, or consist of an amino acid sequence set forth in SEQ ID NO: 6 (IXPAXPFXPAITEIXAAVIA). In the amino acid sequence set forth in SEQ ID NO: 6, the N-terminus isoleucine (located at residue 1) is dimethylated; each X at residues 2, 5, 8, and 15 is Dhb; and the C-terminus alanine (located at residue 20) is modified with Dmp.

[0045] SEQ ID NO: 6 is the AH5011 daptide hominicin (also referred to herein as “hominicin”) and is represented by the chemical structure shown as formula (I):PCT Application Attorney Docket No.: CUBR / 0006PC

[0046] Hominicin includes 20 amino acid residues, including 4 Dhb residues. Both the N-terminus and the C-terminus of hominicin are modified. As shown in formula (I), the nitrogen atom of the isoleucine residue located at the N-terminus is modified with two methyl (CH3) groups. The C-terminus residue of hominicin is alanine and the carboxylic acid group of the alanine is also modified (CH3CH(N(CH3)2)CH2NH−) to an amide group as the adduct of the carboxylic acid and the Dmp.

[0047] Daptides and derivatives thereof described herein may include at least one additional amino acid residue positioned between the two terminal amino acid residues of the daptide represented by formula (I). Daptide derivatives are interchangeably referred to herein as daptides. The at least one additional amino acid residue may include a hydrophobic amino acid residue. A hydrophobic amino acid residue is an amino acid residue that includes a hydrophobic side chain. Illustrative, but non-limiting, examples of hydrophobic amino acid residues may include a phenylalanine (F) residue, a leucine (L) residue, an isoleucine (I) residue, a tyrosine (Y) residue, a tryptophan (W) residue, a valine (V) residue, a methionine (M) residue, a proline (P) residue, or combinations thereof.

[0048] Daptides and derivatives thereof described herein may include at least one hydrophobic amino acid residue that replaces the glutamic acid residue (residue 13) of the daptide represented by formula (I). For example, a daptide or derivative thereof of the present disclosure may comprise, consist essentially of, or consist of one or more amino acid sequences set forth in SEQ ID NOs: 7-14:PCT Application Attorney Docket No.: CUBR / 0006PC

[0049] The amino acid sequences set forth in SEQ ID NOs: 7-14 are the same as that set forth in SEQ ID NO: 6, except that the glutamic acid (E) residue (residue 13) is replaced with F, L, I, Y, W, V, M, or P residue, respectively.

[0050] Alternatively, daptides and derivatives thereof described herein may include an alanine (A) residue that replaces the glutamic acid residue (residue 13) of the daptide represented by formula (I). Here, a daptide or derivative thereof may comprise, consist essentially of, or consist of an amino acid sequence set forth in SEQ ID NO: 15 (IXPAXPFXPAITAIXAAVIA). The amino acid sequence set forth in SEQ ID NO: 15 is the same as that set forth in SEQ ID NO: 6, except that the glutamic acid (E) residue (residue 13) is replaced with an alanine (A) residue.

[0051] Alternatively, daptides and derivatives thereof described herein may include a glutamine (Q) residue that replaces the glutamic acid residue (residue 13) of the daptide represented by formula (I). Here, a daptide or derivative thereof may comprise, consist essentially of, or consist of an amino acid sequence set forth in SEQ ID NO: 16 (IXPAXPFXPAITQIXAAVIA). The amino acid sequence set forth in SEQ ID NO: 16 is the same as that set forth in SEQ ID NO: 6, except that the glutamic acid (E) residue (residue 13) is replaced with a glutamine (Q) residue.

[0052] Alternatively, daptides and derivatives thereof described herein may include an lysine (K) residue that replaces the glutamic acid residue of the daptide represented by formula (I). Here, a daptide or derivative thereof may comprise, consist essentially of, or consist of an amino acid sequence set forth in SEQ ID NO: 17 (IXPAXPFXPAITKIXAAVIA). The amino acid sequence set forth in SEQ ID NO: 17 is the same as that set forth in SEQ ID NO: 6, except that the glutamic acid (E) residue (residue 13) is replaced with a lysine (K) residue.

[0053] While not wishing to be bound by any theory, it is believed that the daptide represented by formula (I) (SEQ ID NO: 6) may serve as an ion channel. The ion channel may be largely driven by the glutamic acid residue (at residue 13) and therefore be a cation channel. Replacement of this glutamic acid residue may alter such a mode of action. For example, replacement of the glutamic acid residue with an alanine residue may remove ion selectivity (SEQ ID NO: 15). As another example, replacement of the glutamic acid residue with a glutamine residue may neutralize the charge (SEQ ID NO: 16). As another example,PCT Application Attorney Docket No.: CUBR / 0006PC replacement of the glutamic acid residue with a lysine residue may change the daptide represented by formula (I) into an anion channel (SEQ ID NO: 17).

[0054] Daptides and derivatives thereof described herein may further include at least one additional proline residue positioned between any two amino acid residues two terminal amino acid residues of the daptide represented by Formula (I) (or the amino acid sequence set forth in SEQ ID NO: 6).

[0055] Daptides and derivatives thereof described herein may include a branched amino acid residue that replaces at least one of the isoleucine (I) residues of the daptide represented by formula (I). A branched amino acid residue includes an amino acid residue having a branched side chain. Illustrative, but non-limiting, examples of branched amino acid residues may include a valine (V) residue, a leucine (L) residue, a threonine (T) residue, or combinations thereof. Replacement of at least one of the I residues with a V, L, or T residue may change the hydrophobicity of the daptide relative to the daptide represented by formula (I). In some embodiments, which may be combined with other embodiments, the at least one I residue replaced with a V, L, or T residue is an internal isoleucine residue, for example, one or more of the isoleucine residues located at residues 11, 14, and / or 19 of daptides described herein. Such a daptide may comprise, consist essentially of, or consist of an amino acid sequence set forth in SEQ ID NO: 18 (IXPAXPFXPAXTEXXAAVXA). In the amino acid sequence set forth in SEQ ID NO: 18, the N-terminus isoleucine (located at residue 1) is dimethylated; each X at residues 2, 5, 8, and 15 is Dhb; the C-terminus alanine (located at residue 20) is modified with Dmp; and each X at residues 11, 14, and 19 may be, independently, an isoleucine residue, a valine residue, a leucine residue, or a threonine residue, wherein at least one X at residues 11, 14, or 19 is not isoleucine.

[0056] Additionally, or alternatively, daptides and derivatives thereof described herein may include an aromatic amino acid residue that replaces at least one of the isoleucine (I) residues of the daptide represented by formula (I). An aromatic amino acid residue is an amino acid residue having a side chain with an aromatic ring. Illustrative, but non-limiting, examples of aromatic amino acid residues may include a phenylalanine (P) residue, a tyrosine (Y) residue, a tryptophan (W) residue, or combinations thereof. Replacement of at least one of the I residues with a V, L, or T residue may change the hydrophobicity and / or the sterics of the daptide relative to the daptide represented by formula (I). In some embodiments, whichPCT Application Attorney Docket No.: CUBR / 0006PC may be combined with other embodiments, the at least one I residue replaced with a P, Y, or T residue is an internal isoleucine residue, for example, one or more of the isoleucine residues located at residues 11, 14, and / or 19 of daptides described herein. Such a daptide may comprise, consist essentially of, or consist of an amino acid sequence set forth in SEQ ID NO: 19 (IXPAXPFXPAXTEXXAAVXA). In the amino acid sequence set forth in SEQ ID NO: 19, the N-terminus isoleucine (located at residue 1) is dimethylated; each X at residues 2, 5, 8, and 15 is Dhb; the C-terminus alanine (located at residue 20) is modified with Dmp; and each X at residues 11, 14, and 19 may be, independently, an isoleucine residue, a phenylalanine residue, a tyrosine residue, or a tryptophan residue, wherein at least one X at residues 11, 14, and / or 19 is not isoleucine.

[0057] Additionally, or alternatively, daptides and derivatives thereof described herein may include a serine (S) residue, a glycine (G) residue, a threonine (T) residue, or combinations thereof that replaces at least one of the alanine (A) residue of the daptide represented by formula (I). In some embodiments, which may be combined with other embodiments, the at least one A residue replaced with a S, G, or T residue is an internal alanine residue, for example, one or more of the alanine residues located at residues 4, 10, 16, and / or 17 of daptides described herein. Such a daptide may comprise, consist essentially of, or consist of an amino acid sequence set forth in SEQ ID NO: 20 (IXPXXPFXPXITEIXXXVIA). In the amino acid sequence set forth in SEQ ID NO: 20, the N-terminus isoleucine (located at residue 1) is dimethylated; each X at residues 2, 5, 8, and 15 is Dhb; the C-terminus alanine (located at residue 20) is modified with Dmp; and each X at residues 4, 10, 16, and 17 may be, independently, an alanine residue, a serine residue, a glycine residue, or a threonine residue, wherein at least one X at residues 4, 10, 16, and / or 17 is not alanine.

[0058] While not wishing to be bound by any theory, replacement of at least one of the alanine residues with a threonine residue may result in more dehydrobutyrine residues in the daptide relative to the daptide represented by SEQ ID NO: 6 (formula (I)). The biosynthetic machinery encodes a lanthipeptide dehydratase (LanMD), which converts serines and threonines into dehydroalanines and dehydrobutryines, respectively. Replacing alanines with threonines may lead to the incorporation of dehydrobutryines by the LanMDenzyme, potentially impacting the peptide backbone structure. This modification may enhancePCT Application Attorney Docket No.: CUBR / 0006PC structural stability, improve antimicrobial activity, and increase resistance to proteolytic degradation.

[0059] Additionally, or alternatively, daptides and derivatives thereof described herein may include at least one amino acid residue removed from the amino acid residues positioned between the two terminal amino acid residues of the daptide represented by formula (I). For example, at least one internal amino acid residue may be removed from a daptide having an amino acid sequence set forth in SEQ ID NO: 6 (formula (I)).

[0060] Daptides or derivatives thereof described herein may have an amino acid sequence having at least 75% sequence identity, such as at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 91% sequence identity, such as at least 92% sequence identity, such as at least 93% sequence identity, such as at least 94% sequence identity, such as at least 95% sequence identity, such as at least 96% sequence identity, such as at least 97% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity, such as 100% sequence identity to the amino acid sequences set forth in SEQ ID NOs: 1-20.

[0061] In some embodiments, which may be combined with other embodiments, one or both of residues 11 and 19 of any one of SEQ ID NOs: 1-20 may be modified with a cysteine residue. For example, one or both isoleucine residues at residue 11 and residue 19 may have a side chain that is modified with a cysteine residue. When isoleucine is not present at residues 11 and 19 (for example, SEQ ID NOs: 18 or 19), the side chain of residue 11 and / or residue 19 may be modified with a cysteine residue. This allows for chemical labeling and microscopy tracking of hominicin or a hominicin derivative described herein.

[0062] As used herein, the term “sequence identity” refers to the extent to which two sequences (amino acid) have the same residue at the same positions in an alignment. For example, “an amino acid sequence is X% identical to SEQ ID NO: Y” refers to % identity of the amino acid sequence to SEQ ID NO: Y and is elaborated as X% of residues in the amino acid sequence are identical to the residues of sequence disclosed in SEQ ID NO: Y. The degree of sequence identity between two or more nucleotide sequences may be calculated using a known computer algorithm for sequence alignment such as NCBI BLAST, using standard settings. In determining the degree of sequence identity between two amino acid sequences, a skilled artisan can consider “conservative” amino acid substitutions, which canPCT Application Attorney Docket No.: CUBR / 0006PC be described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue of similar chemical structure and which has little or essentially no influence on the function, activity, or other biological properties of the daptide.

[0063] Such conservative substitutions may be substitutions in which one amino acid within the following groups (a) - (e) is substituted by another amino acid residue within the same group: (a) small aliphatic, nonpolar or slightly polar residues: alanine (Ala), serine (Ser), threonine (Thr), proline (Pro), and glycine (Gly); (b) polar, negatively charged residues and their (uncharged) amides: aspartic acid (Asp), asparagine (Asn), glutamic acid (Glu), and glutamine (Gln); (c) polar, positively charged residues: histidine (His), arginine (Arg), and lycine (Lys); (d) large aliphatic, nonpolar residues: methionine (Met), leucine (Leu), isoleucine (Ile), valine (Val), and cysteine (Cys); and (e) aromatic residues: phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp). Particularly suitable conservative substitutions are as follows: Ala into Gly or into Ser; Arg into Lys; Asn into Gln or into His; Asp into Glu; Cys into Ser; Gln into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gln; Ile into Leu or into Val; Leu into Ile or into Val; Lys into Arg, into Gln, or into Glu; Met into Leu, into Tyr, or into Ile; Phe into Met, into Leu, or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and / or Phe into Val, into Ile, or into Leu.

[0064] Any suitable amino acid substitutions applied to daptides described herein may also be based on the analysis of the frequencies of amino acid variations between homologous proteins of G.E. Schulz and R.H. Schirmer, Principles of Protein Structure, Springer, New York, 1978, on the analyses of structure forming potentials developed by P.Y. Chou and G.D. Fasman, Biochemistry, 1974, 13, 211, and Adv. Enzymol., A. Meister ed., J. Wiley and Sons, 1978, 47, 45–149 and on the analysis of hydrophobicity patterns in proteins developed by D. Eisenberg et al., Proc. Nat. Acad. Sci. USA, 1984, 81, 140–144; J. Kyte and R.F. Doolittle, J. Mol. Biol., 1982, 157, 105–132, and D.M. Engelman et al., Annu. Rev. Biophys. Biophys. Chem., 1986, 15, 321–53, all of which are incorporated herein in their entirety by reference.

[0065] Daptides and derivatives thereof described herein may form at least part of a composition. Such compositions may be utilized for treating any suitable skin condition in a patient. Skin conditions may include skin diseases, skin pathogens, skin bacteria, or combinations thereof. Illustrative, but non-limiting, examples of skin diseases may include atopic dermatitis, eczema, psoriasis, acne, rosacea, or combinations thereof. Skin pathogensPCT Application Attorney Docket No.: CUBR / 0006PC may include bacteria such as, for example, Staphylococcus, Streptococcus (for example, Group A Streptococcus), coryneform, or combinations thereof.

[0066] Besides the daptide or derivative thereof described herein, compositions of the present disclosure may further include any suitable carrier. Carriers may include a cream, a paste, a fluid, a coating, a paint, a spray, a detergent, an emulsion, or combinations thereof. Carriers may include an antimicrobial cream, an antimicrobial paste, an antimicrobial fluid, an antimicrobial coating, an antimicrobial paint, an antimicrobial spray, an antimicrobial detergent, an antimicrobial soap, a mouthwash, a skin wash, a skin cream, a nasal wash, a toothpaste, a tooth wash, a dish detergent, a laundry detergent, a dishwasher detergent, a nasal spray, a mouth spray, a throat spray, a skin spray, a douche fluid, an enema fluid, a wound cleanser, a wound covering, an eyewash, a shampoo, a facial wash, a facial cream, a facial soap, or combinations thereof.

[0067] Compositions described herein may be antimicrobial compositions or antibacterial compositions useful to treat any suitable microbial condition in a patient. In at least one embodiment, which may be combined with other embodiments, the microbial condition includes a bacterial condition. The bacterial condition may be a disease resulting from a Staphylococcus infection. Additionally, or alternatively, compositions described herein may be utilized for treating staphylococcal skin bacteria in a patient. The staphylococcal skin bacteria may include Staphylococcus aureus.

[0068] Daptides and derivatives thereof described herein may form at least a portion of a daptide bacteriocin composition. The daptide bacteriocin composition may further include one or more carriers such as those carriers described herein. The daptide bacteriocin composition may be antimicrobial against staphylococci by compromising a target cell membrane through pore formation in the cytoplasmic membrane of the bacteria.

[0069] Daptides and derivatives thereof described herein may form at least a portion of a probiotic composition. The probiotic composition may further include one or more carriers such as those carriers described herein. The probiotic composition may be utilized for treating, for example, bacteria on a skin in a patient.

[0070] Daptides and derivatives thereof described herein may form at least a portion of an anti-infective composition. The anti-infective composition may further include one orPCT Application Attorney Docket No.: CUBR / 0006PC more carriers such as those carriers described herein. The anti-infective composition may be utilized for treating, for example, a staphylococcal skin infection in a patient.

[0071] Daptides, derivatives thereof, and compositions described herein may act in any suitable manner. While not wishing to be bound by any theory, it is believed that daptides, derivatives thereof, or composition thereof described herein may act by: (a) disrupting cell membrane of a bacteria, a pathogen, a microbe, or combinations thereof; (b) compromising a cell membrane by, for example, forming pores in a cytoplasmic membrane of a bacteria, a pathogen, a microbe, or combinations thereof; (c) inducing permeabilization, depolarization, or both of a cytoplasmic membrane of a bacteria, a pathogen, a microbe, or combinations thereof; or (d) combinations thereof. (See, for example, FIG.5 and FIGS.10A-10E).

[0072] Daptides, derivatives thereof, and / or compositions thereof described herein may have broad activity against Staphylococcal skin bacteria, including Staphylococcus aureus. Daptides, derivatives thereof, and / or compositions thereof described herein may show activity against Group A Strep.

[0073] Additionally, or alternatively, daptides, derivatives thereof, and / or compositions thereof described herein may be used to reduce bacteria, microbes, pathogens, or combinations thereof on and / or in proximity to the area of treatment. The area of treatment is the area where the daptide, derivative thereof, and / or composition thereof is applied to the patient.

[0074] Embodiments described herein also generally relate to various methods that include use of daptides, derivatives thereof, or compositions thereof described herein. Such methods may include methods of treating a skin condition in a patient (for example, a skin disease, a skin pathogen, a skin bacteria, or combinations thereof in a patient).

[0075] Methods may include treating any suitable skin condition in a patient such as staphylococcal skin bacteria, a microbial condition, a skin disease, atopic dermatitis, eczema, psoriasis, acne, among other treatments using daptides, derivatives thereof, or compositions thereof described herein.

[0076] Methods described herein may include administering to a patient an amount, such as a therapeutically effective amount, of daptides, derivatives thereof, or compositions thereof described herein.PCT Application Attorney Docket No.: CUBR / 0006PC

[0077] The term “therapeutically effective amount” refers to a non-toxic, but sufficient amount of the daptide or derivative thereof described herein (or a composition containing the daptide or derivative thereof described herein) to provide the desired level in the bloodstream or at the site of action (for example, intracellularly) in the patient to be treated, and / or to provide a desired physiological, biophysical, biochemical, pharmacological or therapeutic response, such as amelioration of the manifestations of a skin condition. The exact amount will vary from patient to patient, and will depend on numerous factors, such as the daptide or derivative thereof, the activity of the composition, the delivery device employed, the physical characteristics of the composition, intended patient use (for example, the number of doses administered per day), as well as patient considerations, such as species, age, and general condition of the patient, the severity of the condition being treated, additional drugs being taken by the patient, mode of administration, and the like. These factors and considerations can be determined by one skilled in the art.

[0078] Daptides, derivatives thereof, or compositions thereof described herein may be administered to a patient by any suitable technique including, but not limited to topical, oral, aerosol, parenteral (for example, intravenously), ophthalmic, transdermal, or extracorporeal administration, among other routes of administration. A patient may take or may be administered the composition in any suitable dosage form, such as topical, oral, intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (for example, by injection or infusion).

[0079] In methods described herein, the administration of daptides, derivatives thereof, or compositions thereof described herein may induce permeabilization and / or depolarization of a cytoplasmic membrane of a bacteria, microbe, or pathogen. For example, administration of daptides, derivatives thereof, or compositions thereof described herein may induce permeabilization and / or depolarization of a cytoplasmic membrane of a staphylococcal skin bacteria. Additionally, or alternatively, the administration of daptides, derivatives thereof, or compositions thereof described herein may induce pore formation in a cytoplasmic membrane of a bacteria, microbe, or pathogen. For example, administration of daptides, derivatives thereof, or compositions thereof described herein may induce pore formation in a cytoplasmic membrane of staphylococcal skin bacteria.PCT Application Attorney Docket No.: CUBR / 0006PC

[0080] Daptides, derivatives thereof, or compositions thereof described herein may be used therapeutically in combination with a pharmaceutically acceptable carrier, diluent, agent, or other material. “Pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non-toxic, for example, the material may be administered to an individual without causing an undesirable biological effect or interacting in a deleterious manner with components of the composition in which it is contained.

[0081] Daptides, derivatives thereof, or compositions thereof described herein may be useful for topical formulation. Besides the daptides, derivatives thereof, or compositions thereof described herein, formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, powders, or combinations thereof. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be used.

[0082] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use aspects of the present disclosure, and are not intended to limit the scope of aspects of the present disclosure. Efforts have been made to ensure accuracy with respect to numbers used (for example, amounts, dimensions, etc.) but some experimental errors and deviations should be accounted for. EXAMPLES A. Introduction

[0083] The inventors found a plasmid-borne daptide bacteriocin (hominicin) from a human skin isolate of Staphylococcus hominis, which features an N2,N2-dimethyl-1,2- propanediamine C-terminus. Heterologous expression of the reconstituted biosynthetic loci yielded a daptide product of the same molecular mass, with antimicrobial activity against the skin pathogen Staphylococcus aureus. Membrane permeability and voltage-clamp lipid bilayer experiments supported a mechanism by which the daptide rapidly dissipates the transmembrane potential by forming peptidic channels. Additionally, the inventors found a novel immunity gene, homI, that confers resistance against membrane damage. The inventors also found that the purified daptide effectively protects mouse skin from S. aureus-induced epicutaneous injury in vivo, as evidenced by reduced bacterial burden, inflammation, andPCT Application Attorney Docket No.: CUBR / 0006PC transepithelial water loss. Collectively, the study presented herein identifies the molecular target and mechanism of daptide activity, showcasing the therapeutic application of daptides for treating bacterial skin infections. B. Methods B.1. Ethics statement

[0084] All vertebrate animal experiments were approved and conducted in accordance with the Institutional Animal Care and Use Committee of the University of Colorado Anschutz Medical Campus under protocol number 00941. Conventionally raised C57BL6 / J male and female mice, aged 8-10 weeks old were purchased from the Jackson Laboratory and housed in specific pathogen-free facilities at the University of Colorado Anschutz Medical Center Animal Facility. At experimental endpoints, mice were euthanized via CO2inhalation followed by cervical dislocation. B.2. Collection of bacteria from human subjects

[0085] Collection of bacterial isolates from human skin was carried according to protocols approved by Colorado Multiple Institutional Review Board (protocol no.19-2218) and University of California San Diego Institutional Review Board (project no. 071032). Informed consent was obtained from all subjects. B.3. Growth conditions and reagents

[0086] The bacterial strains and plasmids used in this study are listed in Table 1. Table 1PCT Application Attorney Docket No.: CUBR / 0006PC

[0087] S. hominis isolates were confirmed to be S. hominis by matrix-assisted laser desorption / ionization (MALDI)-time of flight (TOF) mass spectrometry prior to experimentation. All staphylococcal strains and Micrococcus luteus were grown in tryptic soy broth (TSB) at 37ºC with shaking at 250 revolutions per minute (rpm). Escherichia coli was grown in Luria broth (LB) at 37ºC with shaking. Enterococcus faecalis was grown in Brain Heart Infusion at 37°C with shaking. Streptococcus agalactiae was grown statically in Todd Hewitt broth at 37°C. Streptococcus pyogenes was grown statically in Todd Hewitt broth supplemented with yeast extract at 37°C + 5% CO2. For strains with pCM28, chloramphenicol was added to a final concentration of 10 µg / mL. The unmodified core peptide was custom synthesized by AnaSpec, Inc. B.4. Bacteriocin antimicrobial activity assaysPCT Application Attorney Docket No.: CUBR / 0006PC

[0088] For conditioned medium (CM) assays, overnight bacterial cultures of S. hominis or S. aureus strains were pelleted, and the spent media was filtered through a 0.22-µm cellulose acetate Spin-X filter (Costar).100 µL of 80% (vol / vol) CM was added to a 96-well culture plate unless otherwise indicated. The indicator strains were prepared by diluting overnight cultures 1:50 in fresh TSB.100 µL of the indicator strain was added to the culture plate to achieve a final volume of 200 µL per well. Cultures were grown in a Stuart humidified incubator at 37°C with shaking at 800 rpm. Cell density (OD600) was measured on a Tecan Group Ltd. Infinite Pro plate reader. At specified time-points, 20 µL of culture was removed, serially diluted, and plated on TSA for colony counting.

[0089] For the spot-on-lawn assays, S. hominis AH4553 was used as the sensitive strain for all experiments, unless otherwise stated.100 µL of the 1:10 diluted suspension of AH4553 was spread on TSA using sterile glass beads and air dried.10 µL of the daptide-producing or nonproducing strains were inoculated onto the bacterial lawn. The plates were incubated at 37°C for 16-20 h, and images of the zones of inhibition were collected and analyzed using ImageJ software. B.5. Isolation of plasmid-cured mutants

[0090] Overnight culture of AH5011 was subcultured 1:100 in fresh TSB and allowed to reach an OD600 of 0.50. Acriflavine was added to the bacterial culture at a concentration of 25 µg / mL and incubated for 1 h. The culture was diluted and plated on TSA to obtain single colonies, which were then picked and patched on fresh TSA. Bacterial strains cured of p1 failed to produce inhibition zones when spotted on a lawn of AH4553. Strains cured of p2 failed to grow on agar containing 1 µg / mL mupirocin. To confirm the loss of plasmids, polymerase chain reaction (PCR) was performed using primers for the p1- and p2- specific genes, homA and mupA, respectively. B.6. Cloning of the hom biosynthetic gene cluster

[0091] All primers used in this study are listed in Table 2. All species of genes were S. hominis. Table 2PCT Application Attorney Docket No.: CUBR / 0006PC

[0092] The hom gene cluster was cloned into pCM28 by a Gibson assembly strategy. The pCM28 vector was linearized by restriction digestion using BamHI-HF and EcoRI-HF (NEB). The hom gene cluster was amplified in four parts from S. hominis AH5011 genomic DNA using primers homBGC_p1_Frw / _Rev, homBGC_p2_Frw / _Rev, homBGC_p3_Frw / _Rev, and homBGC_p4_Frw / _Rev. The resulting 15-kb construct, pAN1, was introduced into chemically competent E. coli DH5-alpha cells and selected on LB agar with 100 µg / mL ampicillin. The plasmid was verified by DNA sequencing. The pAN1 andPCT Application Attorney Docket No.: CUBR / 0006PC empty vector pCM28 were electroporated into competent S. aureus RN4220, as previously described. Transformants were selected on TSA with 10 µg / mL chloramphenicol.

[0093] For gene omission studies, expression constructs were generated as mentioned above. To construct pCM28-homABCDJM1M2PlanMD, the gene cluster was amplified in three parts using primers homBGC_p3_Fwd / _Rev, homBGC_p4_Fwd / _Rev, and homBGC_p7_Fwd / _Rev. The resulting 14-kb construct was designated as pAN2. To construct pCM28-homACIM1PlanMD, the gene cluster was amplified in three parts using primers homBGC_p4_Fwd / _Rev, homBGC_p5_Fwd / _Rev, and homBGC_p6_Fwd / _Rev. The resulting 11-kb construct was designated as pAN3.

[0094] Cloning of the immunity gene, homI, was performed as follows: The pCM28 vector was linearized by restriction digestion using BamHI-HF and PstI-HF (NEB). The insert was PCR-amplified using primers homI_Frw / _Rev. Purified vector and insert were then ligated using T4 ligase. The resulting 6.6-kb construct, designated as pAN4, was introduced into DH5-alpha cells and verified by DNA sequencing. pAN4 was passaged through RN4220 and subsequently introduced into the appropriate strains by electroporation. B.7. Mass spectrometric identification of the purified daptide and in conditioned media

[0095] Samples were analyzed on a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific, Waltham, MA) with a heated electrospray ionization source coupled to an Acquity ultrahigh-performance liquid chromatography (UPLC) system (Waters Corp., Milford, MA). Each sample was injected with a 3-7 µL volume and eluted from an Acquity UPLC BEH C18 1.7 µm 2.1 × 50 mm column (Waters Corporation) at a 0.3 mL / min flow rate using a binary solvent system that includes 0.1% formic acid (A) and acetonitrile (CH3CN) with 0.1% formic acid (B). Mass spectra were collected using positive ion mode electrospray ionization with two scan events, a full-scan event over a mass range of 300 to 2,000 at a resolving power of 35,000, and a data-dependent tandem mass spectrometry (MS / MS) scan event selecting the calculated m / z or a data-independent scan event using all ion fragmentation. The solvent gradient began with a 1.5 min isocratic hold at 20% B which was diverted to waste. Subsequently, the flow was diverted to the mass spectrometer and a linear increase to 60% B was performed over 5 min. The gradient was held isocratic from 6.5 min to 7.0 min and then increased to 100% B at 8.0 min. The column was washed at 100% B for 1 min and then returned to the starting conditions to allow equilibration for 1.0 min prior to the next injection.PCT Application Attorney Docket No.: CUBR / 0006PC B.8. Butanol extraction of AH5011 hominicin

[0096] The bacteriocin was partially purified from culture media by n-butanol extraction. n-Butanol (100 mL) was added to 300 mL of media and thoroughly mixed before setting aside for several minutes until the phases settled. After centrifugation at 2000 x g for 5 min, the upper butanol phase was collected and evaporated in a rotatory vacuum evaporator (Buchi Rotavapor R-3000) at 45°C. The dried extract was resuspended and concentrated in nuclease- free water or PBS, which was stored at -80°C until use. B.9. Isolation of the AH5011 hominicin

[0097] The bacteriocin was concentrated by liquid-liquid partitioning of culture media with chloroform (1:1, v / v). The chloroform was removed by evaporation under nitrogen gas and the material was subjected to reversed phase flash chromatography using an automated CombiFlash RF system (Teledyne-Isco). A 34.7-minute method was implemented with a gradient of water (A) and methanol (B). The gradient was performed at a flow rate of 75 mL / min on a 130 g C18 RediSep column beginning at 50 % B and linearly increasing to 100% B, followed by an isocratic hold at 100% B for approximately 8.5 minutes. Subsequently, preparative scale reversed phase high performance liquid chromatography (HPLC) was performed for further purification on a Varian HPLC system (Agilent, Santa Clara, CA) equipped with ProStar 210 pumps, a ProStar 710 fraction collector, a ProStar 335 photodiode array detector with and Galaxie Chromatography Workstation software (version 1.9.3.2). A 40-minute method was implemented with a gradient of water (A) and acetonitrile (B) on a Phenomenex Gemini – NX C18 column (5 µm; 250 × 21.20 mm). The gradient separation was performed at a flow rate of 21.2 mL / min and began at 30 % B and linearly increased to 50% B. B.10. NMR data collection

[0098] 1D and 2D NMR data were collected on an Agilent 700 MHz NMR spectrometer (Agilent Technologies, Inc.) equipped with a cryoprobe, operating at a frequency of 700 MHz for1H and 175 MHz for13C. Samples were dissolved in methanol-d3 and residual solvent signals (δH = 3.31 ppm and δC = 49.0 ppm) were used as internal reference peaks. B.11. Determination of minimum inhibitory concentration (MIC)

[0099] The MICs of the purified hominicin were determined for a panel of skin microbes using a broth microdilution assay modified from the Clinical Laboratory Standards InstitutePCT Application Attorney Docket No.: CUBR / 0006PC (CLSI) guidelines. Serial dilutions of hominicin were prepared in 96-well microtiter plates containing bacterial cultures adjusted to 5 x 105CFU / mL in either TSB media (staphylococci) or in cation adjusted Mueller Hinton Broth with laked horse blood (streptococci). As a control, the MICs of vancomycin were also obtained for the same panel of skin microbes. Plates were incubated overnight at 37°C at 250 rpm, and bacterial growth was assessed by measuring optical density at 600 nm using a BioTek Synergy H1 microplate reader. The MIC was defined as the lowest concentration of hominicin that completely inhibited visible bacterial growth. B.12. SYTOX Green influx to detect membrane permeabilization

[0100] SYTOX Green assay was performed as outlined in the literature but modified to use nuclease-free water instead of TSB. Mid-log S. aureus cells were washed and then suspended in a final concentration of 2 µM SYTOX Green (Invitrogen).100 µL of SYTOX- bacterial suspension was dispensed into a 96-well black culture plate (Corning). Plates were incubated for 15 min in the dark. Then, 100 µL of extract from AH5011 or Δp1Δp2, 70% ethanol, or water were added to achieve a final volume of 200 µL per well. Fluorescence intensity was measured with a Tecan plate reader at an excitation / emission wavelength of 480 nm / 522 nm and normalized to “no bacteria” controls. B.13. DiSC3(5) efflux to detect membrane depolarization

[0101] To measure DiSC3(5) efflux, mid-log S. aureus cells were washed and suspended in a final concentration of 4 µM DiSC3(5) (Invitrogen) in buffer (5 mM HEPES, 20 mM glucose, pH 7.2). The labeled bacteria were dispensed into a 96-well black culture plate and allowed to reach equilibrium state. Next, 100 µL of extract from AH5011 or Δp1Δp2, 1% Triton X-100, or HEPES / glucose buffer were added to achieve a final volume of 200 µL per well. Fluorescence measurements were taken every 5 min at an excitation / emission wavelength of 620 nm / 670 nm and normalized to “no bacteria” controls. B.14. Voltage-clamp planar lipid bilayer

[0102] Electrophysiology experiments were performed using a 4-channel micro- electrode cavity array (100 µm MECA, Ionera Technologies) on an Orbit Mini (Nanion Technologies) system. Recordings were collected using the Elements Data Reader software, with parameters set to 200 pA gain and 1.25 kHz sampling rate and subsequently analyzed using the Elements Data Analyzer (v. 1.4.6). For preparation of the horizontal lipid bilayerPCT Application Attorney Docket No.: CUBR / 0006PC system, 150 µL of the bathing solution (1 M KCl, 10 mM HEPES, pH 7.2) was added to the measurement chamber.1,2-diphytanoyl-sn-glycerol-3-phosphocholine (DphpC) from Avanti Polar Lipids was dissolved in n-octane to a concentration of 10 mg / mL (Sigma, electronics grade). Membranes were painted over the microcavities using the air bubble technique56 until the capacitance was between 15-30 pF as recommended by the manufacturer. A transmembrane voltage of +50 mV was applied, unless otherwise stated. Approximately 40 ng of hominicin, 40 ng of synthetic unmodified core peptide, or 8 ng of alpha-toxin was added per aperture, and each membrane was monitored for fusion spikes to indicate insertion of peptides into the lipid bilayer. Current traces were monitored until single-channel conductance events were observed at the indicated voltage. For current-voltage analysis, the current was recorded for 10 seconds at 20 mV increments with a return to 0 mV between each step, starting from -100 mV to +100 mV. Single-channel conductance was determined from the current divided by the transmembrane voltage. B.15. Murine epicutaneous infection with S. aureus

[0103] A detailed description of the mouse model for epicutaneous S. aureus exposure has been described elsewhere. 24 h prior to bacterial inoculation, the dorsal skin of anesthetized C57BL6 / J mice (2% isoflurane) was shaved and depilated with Nair (Church & Dwight Co., Inc.). S. aureus strain AH6350 was subcultured 1:50 in TSB and allowed to reach an OD600 of 1. Cells were pelleted, washed, and then resuspended in PBS to achieve an inoculum of 1 x 108CFU in 100 µL volume. S. aureus was inoculated on a sterile 2-cm2 gauze pad and affixed to the back skin with Tegaderm dressing and secured with adhesive bandages (BAND-AID, Johnson and Johnson) for 72 h. For mice receiving treatment, hominicin (100 µg) or vehicle (methanol) were combined with S. aureus immediately prior to application on the gauze pad. Inoculum concentration was verified by serial dilution, plating, and colony counting. Transepithelial water loss was measured using a Tewameter TM300 device (Courage & Khazaka Electronic GmbH) before infection and at 72 h post- infection. Two sites per lesion were analyzed to minimize error in measurements. The disease score as a measure of skin inflammation severity was evaluated by a blinded observer from digital photographs and quantified as a sum of three individual grades for erythema, edema (each graded: 0, 1, 2, 3) and scaling / erosion (scaling graded: 0, 1, 2, 3 while erosion graded as 4, 5, 6, 7). To enumerate bacterial burden, the full-thickness 2-cm2atopic lesion werePCT Application Attorney Docket No.: CUBR / 0006PC excised, suspended in 0.50 mL PBS with 1-mm zirconia-silica homogenization beads (Biospec), and subsequently homogenized for three 1-minute intervals. The tissue homogenates were serially diluted and plated on nonselective (TSA) and selective (mannitol salt agar [MSA]) media. Plates were incubated overnight before counting colonies. B.16. Whole-genome assembly and annotation

[0104] Genomic DNA from S. hominis AH5011 and S. aureus AH6350 were isolated by phenol-chloroform extraction for whole-genome sequencing, genome assembly, and annotation at the SeqCenter facility (Pittsburgh, USA). SeqCenter-prepared DNA libraries were sequenced on the NextSeq 2000 Illumina and the ONT MinION sequencer. Quality control and adapter trimming were performed with blc2fastq (version 2.20.0.445) and porechop (version 0.2.3_seqan2.1.1) for Illumina and Oxford Nanopore sequencing, respectively. Unicycler (version 0.5.0) was used to assemble Illumina and ONT reads. Bandage (version 0.8.1) and BUSCO (version 5.2.2) were used to assess assembly completeness. Genomes were annotated using Prokka (version 1.14.5). BAGEL4 was used to detect biosynthetic gene clusters. Multi-locus sequence typing of S. aureus genomes was performed using PubMLST database. Unless otherwise stated, default parameters were used for all software. The hom BGC and its homologs were identified using BLASTp. Genomes of various staphylococci containing the hom BGC were downloaded from NCBI and are listed in Table 3. Table 3PCT Application Attorney Docket No.: CUBR / 0006PC

[0105] Comparative gene cluster analysis and visualization were performed using CAGECAT, a web-based platform that integrates Cblaster and Clinker modules. Multi- sequence alignment was performed using Clustal Omega and visualized using Geneious Prime (version 2024.0.7). B.17. Quantification and statistical analysis

[0106] Statistical details of experiments, including the methods used and defined significance values, can be found in the figure legends. Data represent mean ± standard error of the mean. All statistical analyses were performed with GraphPad Prism software. Two- tailed Student’s t-tests were used to assess the statistical significance of differences between two groups. A one-way analysis of variance (ANOVA) with Dunnett’s multiple-comparison test was chosen for multiple comparisons involving more than two groups. A repeated measures two-way ANOVA analysis was selected for bacterial growth curve experiments. C. Non-limiting Results C.1. Antimicrobial activity of a skin commensal S. hominis strain AH5011

[0107] The cell-free, conditioned media (CM) from a human skin isolate collection of S. hominis for antimicrobial activity was screened. It was determined that the strain AH5011 (D11), an isolate from non-lesional skin of a subject with atopic dermatitis, suppressed the growth of S. aureus strain AH6350 as shown in FIG.1A.

[0108] To determine the antimicrobial specificity of AH5011, the inventors tested the susceptibility of skin commensals and pathogenic strains of Staphylococcus, including S. aureus, S. hominis, S. epidermidis, S. capitis, S. haemolyticus, S. lugdunensis, S. simulans, S.PCT Application Attorney Docket No.: CUBR / 0006PC warneri, and S. pseudintermedius, as well as Micrococcus luteus and Streptococcus pyogenes (FIG. 1B). Streptococcus agalactiae and Enterococcus faecalis were included as non-skin- associated bacteria. Antimicrobial susceptibility was assessed by calculating the ratio of optical density of the test condition (40% v / v CM) to untreated cells grown in tryptic soy broth (TSB) for 8 hours. Inhibition is defined as a ratio of < 0.5.

[0109] This analysis revealed that AH5011 CM was inhibitory towards staphylococci, M. luteus (Micrococcus luteus), and S. pyogenes (Streptococcus pyogenes), but not S. agalactiae or E. faecalis. However, the inhibitory activity was determined to be strain dependent, as evident by a bimodal distribution of susceptibility for S. aureus (Staphylococcus aureus), S. hominis (Staphylococcus hominis), S. epidermidis (Staphylococcus epidermidis), S. lugdunensis (Staphylococcus lugdunensis), and S. warneri (Staphylococcus warneri). This bimodal distribution was not observed for S. agalactiae (Streptococcus agalactiae) and E. faecalis (Enterococcus faecalis). Therefore, it was hypothesized that S. hominis AH5011 may target specific members of the skin community, largely the Staphylococcus genus. The inventors found that the antimicrobial function was strain-dependent (FIG. 1C) and growth suppressive to the S. hominis strain AH4553 (FIG.1D) in a concentration-dependent manner (FIG. 1E). Furthermore, it was determined that the inhibitory function of the CM may be inactivated by the serine protease Proteinase K but remains thermally stable at 100°C (FIGS. 12A and 12B). This confirmed the secreted product as a proteinaceous and heat-stable molecule. Additionally, it was found that AH5011 was resistant to the inhibitory effect from its own CM (FIG. 1F), regardless of the concentration (FIG. 1G), which suggested a self- protection mechanism. C.2. The S. hominis AH5011 bacteriocin is a member of the daptide family of RiPPs

[0110] The whole-genome sequence of AH5011 revealed a 2.1-Mb genome with two plasmids, p1 and p2, that are 19,752 bp and 22,074 bp, respectively. Mb refers to million base pairs (2.1 Mb is 2,100,000 base pairs). Bacteriocin biosynthesis machineries are often found on genetically mobile plasmids. To assess whether the inhibitory function was plasmid- encoded, plasmid-cured mutants of AH5011 were generated upon exposure to acriflavine and tested for loss of activity. As illustrated in FIG.2A, the absence of p1, but not p2, resulted in a complete loss of activity, as shown by the lack of an inhibitory zone from the Δp1 and Δp1Δp2 mutants spotted against control strain AH4553. Furthermore, the CM from the Δp2PCT Application Attorney Docket No.: CUBR / 0006PC mutant, but not from Δp1 and Δp1Δp2, was inhibitory (FIG.13A). When treated with wild- type CM, the growth of the Δp1Δp2 (FIG. 13B) and Δp1 (FIG. 13C) mutants were significantly attenuated, but not that of the Δp2 mutant (FIG. 13D). These results indicated that the p1 plasmid from S. hominis AH5011 was the main contributor to the antimicrobial activity and producer immunity.

[0111] The AH5011 plasmids were analyzed using the BAGEL4 database, and a gene cluster containing a lanthipeptide dehydratase and serine peptidase in the p1 plasmid was identified. The 9.6-kb gene cluster included ten genes encoding the precursor peptide, seven biosynthesis enzymes, a peptidase, and a hypothetical protein (FIG. 2B). Table 4 shows a listing of locus tags for the S. hominis AH5011 BGCs. Table 4

[0112] Based on sequence similarity with proteins of known functions and / or domains, homB encodes a domain of unknown function (DUF)-fused RiPP recognition element (RRE) protein; homC encodes an enoyl-(acyl carrier protein) reductase family member; homD encodes a class-III pyridoxal phosphate-dependent aminotransferase; homJ encodes a NAD(P)H-dependent oxidoreductase, belonging to the flavoprotein-like superfamily; homM1 and homM2 both encode S-adenosyl-L-methionine-dependent methyltransferases; homP encodes a serine peptidase, belonging to the subtilisin family; and homMDencodes a DUF4135 domain-containing protein that represents only the dehydratase domain of class IIPCT Application Attorney Docket No.: CUBR / 0006PC lanthipeptide synthetases (LanM). Therefore, the inventors proposed to designate this single domain enzyme as LanMD to indicate its proposed dehydration activity.

[0113] RiPP precursor peptides include an N-terminal leader sequence, which serves as the binding element for modifying enzymes, and a C-terminal core sequence, which receives the post-translational modifications. The homA gene encodes a 65-residue precursor bacteriocin (FIG. 2C), with the core peptide directly mapping to the previously reported hominicin, a peptidic natural product that features the unusual Dmp-modified C-terminus. While the genome of hominicin-producing strain MBBL 2-9 is not publicly available, it was inferred that the AH5011 bacteriocin is structurally related to hominicin, with key differences at the 10th and 17th residues between the precursor AH5011 bacteriocin and the final structure of hominicin.

[0114] The Dmp-modified C-terminus is a class-defining feature of daptides. The Dmp moiety arises from enzymatic modifications of the invariant threonine residue, beginning with the oxidative decarboxylation of the hydroxyl group to a ketone, catalyzed by the DUF-RRE and alcohol dehydrogenase DapBC. This is followed by transamination of the ketone to yield a primary amine via transaminase DapD and a dimethylation step by the methyltransferase DapM to produce the Dmp. Indeed, the homBGC contains the necessary tailoring enzymes, HomBCDM2, to modify the C-terminal Thr-21 into Dmp (FIG. 2D). Additionally, LanMD likely functions as a dehydratase to convert the core threonines into dehydrobutyrines and serines into dehydroalanines. It was anticipated that the flavin-dependent oxidoreductase HomJ likely catalyzes the reduction of dehydroalanines to D-alanines. The modified core is proteolytically cleaved from the leader peptide by the peptidase HomP while the second methyltransferase HomM1is likely responsible for the dimethylation of the free N-terminal isoleucine, which results in a fully modified and bioactive daptide. C.3. Detection and structural characterization of AH5011 daptide

[0115] The CM from daptide-positive AH5011 and daptide-negative Δp1Δp2 strains were analyzed using ultraperformance liquid chromatography-mass spectrometry (UPLC-MS). A distinct chromatographic peak at retention time 5.32 min was detected. The chromatographic peak had a measured m / z value of 1020.1114 in the CM from AH5011 but absent from Δp1Δp2. The measured mass was within 2 ppm of the predicted m / z (1020.1103) for a doubly- charged protonated ion of the daptide (FIG.3A). The observed MS-MS fragmentation patternPCT Application Attorney Docket No.: CUBR / 0006PC of this m / z in combination with the genetic data led the inventors to propose the daptide bacteriocin sequence as: DmIle–Dhb–Pro–Ala–Dhb–Pro–Phe–Dhb–Pro–Ala–Ile–Thr–Glu–Ile–Dhb– Ala–Ala–Val–Ile–Ala–Dmp

[0116] See FIGS. 3C and 3D. The initial hominicin investigation lacked genetic information for S. hominis strain MBBL 2-9, so the precursor peptide sequence was not proposed. The determination of the accurate mass of hominicin from S. hominis AH5011 confirmed its molecular weight to be 2038.5110 Da. Interpretation of 1D and 2D NMR spectra was used as an orthogonal approach to confirm the proposed structure of AH5011 hominicin (FIG. 8). Table 5 shows1H (700 MHz) and13C (175 MHz) NMR spectroscopic data of AH5011 hominicin in methanol-d3. Table 5PCT Application Attorney Docket No.: CUBR / 0006PCPCT Application Attorney Docket No.: CUBR / 0006PCPCT Application Attorney Docket No.: CUBR / 0006PC

[0117] To determine whether the hom BGC from AH5011 is sufficient to produce the daptide, the inventors cloned the entire 9.6-kb BGC on the plasmid pCM28 and heterologously expressed the construct in the daptide-resistant S. aureus strain RN4220. From the CM of the BGC-expressing S. aureus, but not the empty vector, an ion was detected with the same retention time (5.33 mins), accurate mass (1020.1115 m / z), and MS-MS fragmentation pattern as the ion detected in the AH5011 strain (FIG. 3B). These results indicated that the expression of the hom BGC was sufficient for the biosynthesis and secretion of hominicin.

[0118] Given the antimicrobial potency from CM and optimal aerobic growth conditions, chloroform extraction was utilized to enrich the hominicin, and the resulting material was then subjected to flash chromatography, followed by preparative high-pressure liquid chromatography (HPLC) (data not shown). The peptide of >95% purity exhibited an [M+2H]2+m / z and fragmentation pattern within 5 ppm of the [M+2H]2+m / z determined for the compound from the strain AH5011 (data not shown). A simpler extraction by n-butanol also provided a partially purified and enriched antimicrobial fraction from conditioned media (data not shown). The AH5011 extract obtained via n-butanol extraction was then adopted for further experiments. C.4. Activity of AH5011 hominicin against skin commensals and pathogens

[0119] Based on the inhibitory effect of the CM against a wide range of Gram-positive bacteria, as shown in FIG. 1, the inventors next measured the minimum inhibitory concentration (MIC) of the purified daptide against a selection of skin commensal and pathogenic bacteria using a broth microdilution assay adapted from the Clinical Laboratory Standards Institute (CLSI) guidelines. These results are shown in Table 6. MIC values arePCT Application Attorney Docket No.: CUBR / 0006PC reported as the lowest concentration (in µg / mL) that completely inhibited visible bacterial growth. Table 6

[0120] As shown in Table 6, hominicin demonstrated antimicrobial potency comparable to that of the clinically relevant antibiotic vancomycin across all tested strains. Full MIC curves are shown in FIG.9. C.5. Contribution of N2,N2-dimethyl-1,2-propanediamine modification on AH5011 hominicin activity

[0121] There is a lack of available genetic data for hominicin, and the AH5011 hom BGC represents the first fully assembled gene cluster for a staphylococcal daptide. Accordingly, next investigated was the minimal genes required for antimicrobial activity. Plasmid constructs lacking the homBDJM2 genes and the homI gene of unknown function were generated (FIG. 4A). Each construct was heterologously expressed into RN4220 and spot plated against AH4553 (FIG. 4B). As expected, the expression of the full-length hom BGC conferred antimicrobial activity, as evidenced by an inhibitory zone with a mean diameter of 17.29 ± 0.62 mm, compared to the empty vector (9.99 ± 0.19 mm). The omission of homBDJM2 genes abolished antimicrobial activity (9.84 ± 0.19 mm). Interestingly, the omission of homI gene retained antimicrobial activity, suggesting that homI likely does not contribute to the biosynthesis and / or modifications of the daptide.

[0122] Next tested was whether the activity, or lack thereof, could be detected in the CM of each mutant (FIG. 4C). The CM from homACIM1PlanMD was not growth suppressive, whereas CM from homABCDIJM1M2PlanMDand homABCDJM1M2PlanMDattenuated the growth of AH4553. To corroborate the activity results, mass spectrometric analysis of thePCT Application Attorney Docket No.: CUBR / 0006PC CM from each mutant was performed to confirm the presence or absence of hominicin. For homABCDJM1M2PlanMD, a 21-residue product was detected with an m / z value for the [M+2H]2+ion of 1020.1095 [Δ = 1 ppm] (data not shown), corresponding to hominicin, with a C-terminal Dmp. For homACIM1PlanMD, the calculated daptide mass was not detected in the CM; instead, multiple peptide species of similar mass to hominicin were detected with the most abundant ion displayed an [M+2H]2+of 1026.5679 (data not shown). The MS / MS spectra displayed a fragmentation pattern that was distinct from hominicin (data not shown). This structure likely differed from hominicin in the lack of C-terminal modification and oxidoreduction of dehydroalanine residues, matching the predicted m / z of 1026.5659 [Δ = 2 ppm] (data not shown). C.6. AH5011 hominicin is a membrane-disruptive, pore-forming antimicrobial

[0123] The structural characteristics of daptides include a hydrophobic core and positively charged amino termini. These features are found in many membrane-active, pore- forming antimicrobial peptides. Whether daptides also form membrane pores have not been demonstrated.

[0124] The inventors next investigated whether hominicin could permeabilize the cytoplasmic membranes of S. aureus by measuring the uptake of the impermeant SYTOX Green fluorescent probe into bacterial cells (FIG. 5A). Upon cell entry, the fluorescence intensity of SYTOX Green was enhanced after binding to nucleic acids. S. aureus cells treated with 2x or 4x conditioned media from AH5011, prepared from n-butanol extraction, which contained 8.2 µg and 16.3 µg of hominicin respectively, were significantly permeabilized compared to Δp1Δp2-treated cells. Next examined were the effects of hominicin on the membrane potential by measuring the release of potentiometric, cationic 3,3’- dipropylthiadicarbocyanine iodide [DiSC3(5)] dye from bacterial cells (FIG. 5B). DiSC3(5) accumulates on polarized membranes and translocates into lipid bilayers, resulting in fluorescence self-quenching and reduced fluorescence signals in the culture medium. It was found that the S. aureus cells treated with AH5011 extract were depolarized in a dose- dependent manner, as indicated by the increased fluorescence intensity in the culture medium due to the extrusion of DiSC3(5).

[0125] To test whether hominicin exhibits ion channel activity, the pure peptide was reconstituted in artificial lipid bilayers bathed in a solution of 1 M KCl and 10 mM HEPES.PCT Application Attorney Docket No.: CUBR / 0006PC Then the ion flux across the membrane was measured using voltage-clamp electrophysiology (FIG. 10A). The current / voltage relationship of hominicin displayed an ohmic relationship with a calculated channel conductance of 692.4 ± 70.4 pS while the synthetic unmodified core peptide and buffer alone exhibited no channel activity (FIG.5C). Alpha-toxin, a known pore- forming protein formed by S. aureus, was used as a control and a channel conductance of 489.9 ± 82.8 pS was observed.

[0126] At both positive and negative holding potentials, hominicin exhibited ion- conducting activity, as evidenced by observable openings and closings of single channels (FIG. 5D, FIG. 5E). While hominicin exhibited antimicrobial activity, its unmodified form was inactive against S. aureus (FIG. 10B). This result suggested that the post-translational modifications of hominicin may be important for its membrane insertion and channel activity. The unmodified peptide did not alter the current trace from baseline, whereas hominicin caused stepwise increases in the current trace at +50 mV (FIG.10C) and decreases at -50 mV (FIG.10D) and -100 mV holding potentials (FIG.10E). The changes in current traces were indicative of ion flux across the bilayer through the opening of membrane pores. These results suggested the mode of action of AH5011 hominicin may involve impairing the membrane integrity by depolarizing the bacterial transmembrane potential through the formation of transmembrane pores. C.7. The genetic diversity and conservation of daptide BGC in Staphylococcus

[0127] A gene cluster comparison using the CAGECAT pipeline was performed to assess the diversity and conservation of the hom BGC in Staphylococcus. This gene cluster comparison enables detailed homology search, filtering, gene neighborhood estimation, and visualization of resulting variant BGCs. Fourteen assemblies were identified, carrying homologs of the precursor peptide along with at least one or two associated biosynthesis genes (Table 3).

[0128] These assemblies were sourced from various staphylococci, including S. aureus, S. pseudintermedius, S. felis, S. epidermidis, S. warneri, S. agnetis, and S. cohnii. Some of the BGCs exhibited variations in the composition of tailoring enzymes and the number of precursor peptides (data not shown). Notably, seven assemblies lack the genes encoding for the dehydratase LanMDand oxidoreductase HomJ, suggesting that the installation of dehydroamino acids and the conversion of serines to D-alanines may have evolvedPCT Application Attorney Docket No.: CUBR / 0006PC independently from the Dmp biosynthesis. Additionally, accessory genes likely involved in bacteriocin export and immunity were identified. These accessory genes were predicted to encode ATP-binding cassette (ABC) transporters, previously reported in the export of bacteriocins across the cytoplasmic membrane and / or expelling bacteriocins as a resistance mechanism. These were not found in S. hominis AH5011; however, the gene of unknown function, homI, was present in seven BGCs, including AH5011 and seems to correlate with hominicin-like molecules. It was also observed that these daptide BGCs are located on or near mobile genetic elements, such as plasmids, recombinases, and transposes. This result suggested the acquisition of daptides via horizontal gene transfer. Alignment of the precursor peptides revealed diversity in sequence and length while conservation of the C-terminal threonine was observed (data not shown). C.8. HomI is the immunity protein that provides resistance to AH5011 hominicin

[0129] Producer strains are often protected from the activity of their bacteriocin by self- immunity systems. The roles of these immunity proteins, whether anchored to or embedded in the membrane, involve several mechanisms, including bacteriocin sequestration, efflux, degradation, and / or interference with binding to target receptor. The mechanisms of producer immunity to daptides have never been investigated. The inventors ascertained the role of homI given that 6 out of the 14 daptide BGCs encode this gene (data not shown). It was hypothesized that HomI is the immunity protein that provides cognate resistance to hominicin. Using a genetically tractable and daptide-susceptible S. aureus strain Newman, homI was cloned on an expression plasmid and no growth defects between the wild-type and homI mutant in TSB were observed (FIG.6A). Under growth conditions with AH5011 CM, the homI mutant exhibited significant resistance while the growth of wild-type and empty vector were attenuated (FIG. 6B). HomI-mediated resistance was confirmed by the lack of inhibitory zones in the presence of daptide-producing AH5011 and S. aureus strain expressing homABCDIJM1M2PlanMD(FIG.6C).

[0130] HomI is predicted to be a 32.8 kDa membrane-associated protein with five transmembrane helices (data not shown). Protein sequence alignment of S. hominis HomI with other daptide BGCs encoding HomI showed sequence identity as high as 79% with S. aureus and as low as 55% with S. pseudintermedius and S. agnetis (data not shown). To identify structural homologs, the structure of S. hominis HomI was predicted using AlphaFoldPCT Application Attorney Docket No.: CUBR / 0006PC and used this structure as a query in Foldseek. This analysis revealed that HomI shares structural homology with the YidC family of membrane protein insertases (data not shown), suggesting that HomI may function at the bacterial membrane interface. Alignments of the HomI model with an AlphaFold-predicted structure of S. aureus YidC and a crystal structure of B. halodurans YidC (PDB 3WO7_A) yielded RMSD values of 1.41 Å and 1.76 Å, respectively.

[0131] Next examined was the potential function of HomI in protecting against daptide- induced membrane damage by measuring DiSC3(5) fluorescence in the culture medium. Under Δp1Δp2-treated and untreated conditions, S. aureus wild-type, homI mutant, and empty vector strains exhibited DiSC3(5) quenching as shown by a decrease in fluorescence intensity, which reflects dye uptake and the energized and / or polarized state of the cells. When exposed to AH5011 CM, the wild-type (FIG. 6D) and empty vector (FIG. 6E) strains displayed membrane depolarization as evident by a rise in fluorescence intensity in the culture medium. However, the homI mutant retained its membrane potential, comparable to the untreated control (FIG.6F). Altogether,identified as the genetic determinant that conferred protection against membrane damage caused by daptides. C.9. AH5011 hominicin restricts S. aureus skin infection in vivo

[0132] The translational potential of daptides to protect against invading pathogens has never been tested. Therefore, hominicin-mediated skin protection from S. aureus induced epicutaneous injury was investigated. S. aureus is the most common cause of skin and soft tissue infections in the United States and frequently associates with high morbidity, morality, and healthcare costs. As an opportunistic pathogen, S. aureus exacerbates diseases, such as atopic dermatitis (AD), an inflammatory skin disease with a complex pathogenesis marked by impaired skin barrier function and structure, immunological abnormalities, and dysbiosis. Nasal carriage is a known risk factor for invasive S. aureus infections.

[0133] Therefore, the strain AH6350, a high toxin-producing isolate from an acute lesion of pediatric AD, was selected as a model clinical organism to study in vivo (FIG. 7A). AH6350 is highly susceptible to hominicin, with a minimum inhibitory concentration of 1 µg / mL (Table 6). Genomic analysis revealed that strain AH6350 is closely related to strains from sequence type (ST) 15 lineage with an accessory gene regulator (agr) quorum-sensingPCT Application Attorney Docket No.: CUBR / 0006PC system of type II (data not shown). ST15 is commonly associated as a nasal carriage lineage and often express surface proteins with skin binding capacity.

[0134] Epicutaneous infection of C57BL6 / J mice with AH6350 alone resulted in severe skin scaling, erosion, and erythema (FIG. 7B). Infection with S. aureus was augmented by the topical addition of hominicin to the skin surface, as evidenced by a reduced bacterial burden compared to the untreated group (FIG. 7C). Additionally, the dorsal skin of mice treated with hominicin retained barrier function and integrity, as measured by transepithelial water loss (FIG. 7D). The severity of local inflammation on mouse skin at 72 hours post- infection was assessed using a skin disease score, which represents the sum of individual grades for erythema, edema, erosion, and scaling. Topical application of hominicin resulted in a significant reduction in skin inflammation and cutaneous injury (FIG.7E). D. Non-limiting Discussion

[0135] In a polymicrobial setting, skin microbes contend with neighboring competitors and have evolved mechanisms for interbacterial competition, including the production of antimicrobial natural products. Daptides are an unstudied class of RiPPs. Herein, the inventors describe a member of this class produced by S. hominis and explore its biosynthesis and mechanism of action using bioinformatic, microbiological, and biophysical techniques and propose a comprehensive model for hominicin (FIG.11). Bacteriocins inhibit similar or closely related bacteria that the producer strain encounters in its niche.

[0136] The study presented herein is the first investigation into the breadth of daptide activity against members of the skin community. While varying levels of growth inhibition against related skin staphylococci was observed, the lack of inhibition against S. agalactiae and E. faecalis aligns with this principle, as these species are not commonly found in the human skin microbiome. Moreover, inhibition against M. luteus and S. pyogenes—the former is a commensal species and the latter a known etiological agent of skin and soft infections— points to the microbiome-shaping role of daptides. The results described herein indicate that AH5011 hominicin may have evolved to eliminate specific members of the skin community (FIG.1), suggesting that it is improved for efficacy and tolerance at the skin surface.

[0137] Structural characterization of AH5011 hominicin revealed several post- translational modifications, which include a Dmp-modified C-terminus, dimethylated N- terminus, and dehydrobutyrines (FIG. 3). Various biosynthetic machinery coordinates thePCT Application Attorney Docket No.: CUBR / 0006PC three-step pathway to convert the C-terminal threonine to (S)-N2,N2-dimethyl-1,2- propanediamine. The hom BGC encodes the dehydratase LanMD that modifies Thr-2, Thr-5, Thr-8, Ser-10, Thr-15, and Ser-17 into dehydroamino acids. The inventors observed that the Thr-12 remains unmodified, which may be explained by possible structural constraints, enzyme specificity, or interference by other modifications that may influence its accessibility. Furthermore, the inventors identified a tertiary modification likely mediated by the HomJ oxidoreductase and are currently investigating its role in converting dehydroalanines, derived from Ser-10 and Ser-17, into D-alanines. Incorporation of D-amino acids into RiPPs imparts several advantageous properties, such as stabilization of peptide structures, reduced susceptibility to proteolysis, and enhancement of bioactivity. Lastly, amino-modified termini are common features present in many defense peptides. Notably, the recently discovered non- ribosomal aquimarins from a marine bacterial genus Aquimarina feature a C-terminal amine, which was shown to be indispensable for antibacterial activity. Other bodies of work corroborate the importance of amino-modified termini for peptide-membrane interaction and structural stabilization. The inventors observed that omission of the genes required for the Dmp modification resulted in a pronounced loss of activity, suggesting that the Dmp moiety plays a role in hominicin’s mode of action (FIG.4).

[0138] Collectively, the MS, genetic, and NMR data described herein indicated that AH5011 daptide is structurally identical to hominicin. Hominicin was originally classified as a lanthipeptide lacking the lanthionine thioether bridge, but it is more correctly assigned as being among the class of peptides called daptides. Based on gene sequence data and expression studies, the study presented herein provides insight into the biosynthesis and antimicrobial function of daptides from S. hominis. Here, the inventors have chosen to adopt the hominicin name for the peptide to be consistent with the daptide nomenclature. Based on the genetic information, the study provided herein is the first to propose that the precursor peptide contains serines at the 10th and 17th residues, which are likely post-translationally converted into D-Ala to form the hominicin peptide. This finding is significant, as it resolves the biosynthetic gap between the primary sequence of the precursor peptide and its mature form, a connection not identified in the original report.

[0139] In contrast to the results presented herein, other researchers have found that the Mpa daptides do not exhibit antibacterial activity against strains of Bacillota, including thePCT Application Attorney Docket No.: CUBR / 0006PC genus Staphylococcus. Worth highlighting is that the Mpa daptides do not harbor dehydroamino acids, which are known to play a role in conferring antibacterial properties, structural stability, and resistance to proteolysis. Mpa daptides display hemolytic activity against bovine erythrocytes, suggesting their capability to interact with cell membranes. Using in-vivo membrane potential measurements coupled with electrophysiology, the study presented herein provides evidence that hominicin permeabilizes the cytoplasmic membrane of S. aureus, likely driven by its capacity to penetrate and self-organize into ion-conducting channels in the membrane (FIG.5). The initial interaction at the membrane interface is likely facilitated by the positively charged amino termini via electrostatic interactions. With its hydrophobic moieties, hominicin efficiently partitions into the hydrophobic core of the membrane and spontaneously self-assembles individual monomers to form a transmembrane pore. The lipid bilayer experiment presented herein supports this working model given that hominicin associated with the phospholipid membrane without the mediation of other surface receptors or proteins. The results presented herein provide a molecular picture of the daptide as a membrane-depolarizing, pore-forming agent that consequently leads to the loss of membrane integrity and a lethal collapse of transmembrane potential in susceptible bacteria. It is worth noting that the observed channel activity may not be sufficient to fully encapsulate the hominicin’s structure within the membrane.

[0140] Additionally, the inventors identified the novel daptide resistance protein, HomI. To the inventors’ knowledge, there has been no previous report of an immunity mechanism for daptides. The inventors observed that HomI sufficiently protected against membrane depolarization, thereby conferring resistance against membrane damage (FIG. 6). HomI shares structural homology with the YidC family of membrane insertase proteins, with five transmembrane helices predicted to localize the protein to the membrane (data not shown). YidC is involved in facilitating membrane insertion of small proteins or assisting the protein- conducting channel SecYEG, which mediates the translocation of secretory proteins across the membrane. As such, the Enterocin P bacteriocin from Enterococcus faecium has been shown to require the sec-dependent pathway for its secretion, and the sec pathway can be alternatively used to secrete dehydrated lanthipeptides in the absence of their lantibiotic transporter, NisT. Interestingly, the inventors detected homologs of ABC transporters in aPCT Application Attorney Docket No.: CUBR / 0006PC number of daptide BGCs (data not shown), which may represent alternative resistance mechanisms.

[0141] In this study, the inventors demonstrated the effectiveness of treating S. aureus- infected skin with daptides in vivo (FIG. 7). The inventors’ discovery of a commensal bacterium producing a potent daptide is a strain-specific feature that is often overlooked in large genomic datasets. Nevertheless, the presence of small molecule biosynthetic gene clusters is prevalent in human-associated metagenomes, highlighting the commensal bacteria as an untapped reservoir of useful bioactive molecules. The results presented herein illustrate that S. hominis is a protective skin commensal, showing its beneficial role in the production of bacteriocins. The translatability of characterizing these natural products and evaluating their utility as antimicrobial therapies presents an avenue for therapeutic development. Utilizing hominicin or daptide-producing staphylococci could be an effective strategy for preventing S. aureus colonization and infection. The introduction of hom genes into an exclusively commensal species could be utilized in a precision-based bacteriotherapy to target specific colonizing pathogens or provide colonization resistance against future infections. Collectively, the inventors’ discoveries provide insights into the molecular mechanisms of daptides and highlight their antibacterial property as anti-staphylococcal agents. EMBODIMENTS LISTING

[0142] The present disclosure provides, among others, the following embodiments, each of which can be considered as optionally including any alternate embodiments:

[0143] Clause A1. A composition for treating a skin condition in a patient, the composition comprising a daptide, the daptide comprising: a dimethylated N-terminus; a N2,N2-dimethyl-1,2-propanediamine (Dmp)-modified C-terminus; a dehydrobutyrine residue positioned between the dimethylated N-terminus and the Dmp-modified C-terminus; at least one alanine (A) residue or at least one dehydroalanine (Dha) residue positioned between the dimethylated N-terminus and the Dmp-modified C-terminus; and at least one amino acid residue positioned between the dimethylated N-terminus and the Dmp-modified C-terminus.

[0144] Clause A2. The composition according to Clause A1, wherein the daptide comprises from 15 to 25 amino acid residues, such as from 17 to 24 amino acid residues, suchPCT Application Attorney Docket No.: CUBR / 0006PC as from 18 to 23 amino acid residues, such as from 19 to 22 amino acid residues, such as from 20 to 21 amino acid residues, such as 20 amino acid residues.

[0145] Clause A3. The composition according to any one of Clauses A1-A2, wherein: the dimethylated N-terminus of the daptide comprises a dimethylated isoleucine; and the Dmp- modified C-terminus of the daptide comprises an alanine residue modified with Dmp.

[0146] Clause A4. The composition according to any one of Clauses A1-A3, wherein the daptide comprises: an amino acid sequence having at least 75% sequence identity, such as at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 98% sequence identity, such as 100% sequence identity to one or more of the amino acid sequences set forth in SEQ ID NOs: 1-6. Clause A5. The composition according to any one of Clauses A1-A4, wherein the daptide comprises: an amino acid sequence having at least 75% sequence identity, such as at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 98% sequence identity, such as 100% sequence identity to one or more of the amino acid sequences set forth in SEQ ID NOs: 7-14.

[0147] Clause A6. The composition according to any one of Clauses A1-A5, wherein the daptide comprises: an amino acid sequence having at least 75% sequence identity, such as at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 98% sequence identity, such as 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15.

[0148] Clause A7. The composition according to any one of Clauses A1-A6, wherein the daptide comprises: an amino acid sequence having at least 75% sequence identity, such as at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 98% sequence identity, such as 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16.

[0149] Clause A8. The composition according to any one of Clauses A1-A7, wherein the daptide comprises: an amino acid sequence having at least 75% sequence identity, such as at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90%PCT Application Attorney Docket No.: CUBR / 0006PC sequence identity, such as at least 95% sequence identity, such as at least 98% sequence identity, such as 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17.

[0150] Clause A9. The composition according to any one of Clauses A1-A8, wherein the daptide comprises: an amino acid sequence having at least 75% sequence identity, such as at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 98% sequence identity, such as 100% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 18 or 19.

[0151] Clause A10. The composition according to any one of Clauses A1-A9, wherein the daptide comprises: an amino acid sequence having at least 75% sequence identity, such as at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 98% sequence identity, such as 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 20.

[0152] Clause A11. The composition according to any one of Clauses A1-A10, wherein the skin condition comprises: a skin bacteria, a skin disease, a skin pathogen, or combinations thereof.

[0153] Clause A12. The composition according to Clause A11, wherein the skin pathogen or the skin bacteria comprises: Staphylococcus, Streptococcus (for example, Group A Streptococcus), coryneform, or combinations thereof.

[0154] Clause A13. The composition according to any one of Clauses A11-A12, wherein the skin pathogen or the skin bacteria comprises: M. luteus, S. pyogenes, S. aureus, S. hominis, S. epidermidis, S. lugdunensis, S. warneri.

[0155] Clause A14. The composition according to any one of Clauses A11-A13, wherein the skin disease comprises: atopic dermatitis, eczema, psoriasis, acne, rosacea, or combinations thereof.

[0156] Clause A15. The composition according to any one of Clauses A1-A14, wherein the composition is configured to: (a) induce dissipation of a transmembrane potential through pore formation, thereby causing cell membrane damage in a bacteria, a pathogen, a microbe, or combinations thereof; (b) penetrate and self-organize into ion-conductingPCT Application Attorney Docket No.: CUBR / 0006PC channels in a cytoplasmic membrane of a bacteria, a pathogen, a microbe, or combinations thereof; (c) induce permeabilization and depolarization of a cytoplasmic membrane of a bacteria, a pathogen, a microbe, or combinations thereof; or (d) a combination thereof.

[0157] Clause A16. A daptide bacteriocin composition that is antimicrobial against a staphylococcal skin bacteria in a patient, the daptide bacteriocin composition comprising: the composition according to any one of Clauses A1-A15 (for example, an amino acid sequence having at least 75% sequence identity, such as at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 98% sequence identity, such as 100% sequence identity to one or more of the amino acid sequences set forth in SEQ ID NOs: 1-20).

[0158] Clause A17. The daptide bacteriocin composition according to claim 16, wherein the daptide bacteriocin composition is configured to: (a) induce dissipation of a transmembrane potential through pore formation, thereby causing cell membrane of the staphylococcal skin bacteria; (b) penetrate and self-organize into ion-conducting channels in a cytoplasmic membrane of the staphylococcal skin bacteria; (c) induce permeabilization and depolarization of a cytoplasmic membrane of the staphylococcal skin bacteria; or (d) a combination thereof.

[0159] Clause A18. A method for treating a skin condition in a patient, the method comprising: administering to the patient the composition according to any one of Clauses A1- A17.

[0160] Clause A19. The method according to Clause A18, wherein the daptide comprises: an amino acid sequence having at least 75% sequence identity to one or more of the amino acid sequences set forth in SEQ ID NOs: 1-20.

[0161] Clause A20. The method of according to any one of Clauses A18-A19, wherein the daptide comprises: an amino acid sequence having at least 90% sequence identity to one or more of the amino acid sequences set forth in SEQ ID NOs: 6-20. Clause B1. A composition for treating a skin bacteria, the composition comprising a daptide or a derivative thereof, the daptide or derivative thereof comprising: a dimethylated N-terminus; a N2,N2-dimethyl-1,2-propanediamine (Dmp)-modified C-terminus; a dehydrobutyrine residue positioned between the termini; a dehydroalanine residue positioned between the termini; and at least one amino acid residue positioned between the termini.PCT Application Attorney Docket No.: CUBR / 0006PC

[0162] Clause B2. A composition for treating staphylococcal skin bacteria in a patient, the composition comprising a daptide represented by formula (I), a derivative thereof, or a combination thereof.

[0163] Clause B3. The composition according to Clause B2, wherein: the derivative comprises at least one additional amino acid residue positioned between the two terminal amino acid residues of the daptide represented by formula (I); and the at least one additional amino acid residue comprises a hydrophobic amino acid residue, the hydrophobic amino acid residue comprising an amino acid residue with a hydrophobic side chain.

[0164] Clause B4. The composition according to Clause B3, wherein the at least one additional amino acid residue comprising a hydrophobic side chain comprises a phenylalanine residue, a leucine residue, an isoleucine residue, a tyrosine residue, a tryptophan residue, a valine residue, a methionine residue, a proline residue, or combinations thereof.

[0165] Clause B5. The composition according to any one of Clauses B2-B4, wherein: the derivative comprises a hydrophobic amino acid residue that replaces the glutamic acid residue of the daptide represented by formula (I).

[0166] Clause B6. The composition according to any one of Clauses B2-B5, wherein: the derivative comprises an alanine residue that replaces the glutamic acid residue of the daptide represented by formula (I).

[0167] Clause B7. The composition according to any one of Clauses B2-B5, wherein: the derivative comprises a glutamine residue that replaces the glutamic acid residue of the daptide represented by formula (I).

[0168] Clause B8. The composition according to any one of Clauses B2-B5, wherein: the derivative comprises an lysine residue that replaces the glutamic acid residue of the daptide represented by formula (I).

[0169] Clause B9. The composition according to any one of Clauses B2-B8, wherein: the derivative comprises at least one additional proline residue positioned between the two terminal amino acid residues of the daptide represented by formula (I).

[0170] Clause B10. The composition according to any one of Clauses B2-9, wherein: the derivative comprises an additional proline residue positioned between any two amino acid residues of the daptide represented by formula (I).PCT Application Attorney Docket No.: CUBR / 0006PC

[0171] Clause B11. The composition according to any one of Clauses B2- B10, wherein: the derivative comprises a branched amino acid residue that replaces at least one of the isoleucine residues of the daptide represented by formula (I).

[0172] Clause B12. The composition according to Clause B11, wherein: the branched amino acid residue comprises a valine residue, a leucine residue, a threonine residue, or combinations thereof.

[0173] Clause B13. The composition according to any one of Clauses B1- B12, wherein: the derivative comprises an aromatic amino acid residue that replaces at least one of the isoleucine residues of the daptide represented by formula (I).

[0174] Clause B14. The composition according to any one of Clauses B1- B13, wherein: the aromatic amino acid residue comprises a phenylalanine residue, a tyrosine residue, a tryptophan residue, or combinations thereof.

[0175] Clause B15. The composition according to any one of Clauses 1-14, wherein: the derivative comprises a serine residue, a glycine residue, a threonine residue, or combinations thereof that replaces at least one of the alanine residues of the daptide represented by formula (I).

[0176] Clause B16. The composition according to any one of Clauses 2-15, wherein: the derivative comprises at least one amino acid residue removed from the amino acid residues positioned between the two terminal amino acid residues of the daptide represented by formula (I).

[0177] Clause B17. A daptide bacteriocin composition that is antimicrobial against staphylococci by compromising a target cell membrane through pore formation in a cytoplasmic membrane of bacteria, the daptide bacteriocin composition comprising the composition according to any one of Clauses B1-B16.

[0178] Clause B18. A probiotic composition for treating bacteria on the skin in a patient, comprising the composition according to any one of Clauses B1- B17.

[0179] Clause B19. A composition for treating a skin disease in a patient, comprising the composition according to any one of Clauses B1-B18.

[0180] Clause B20. A composition for treating a skin pathogen in a patient, comprising the composition according to any one of Clauses B1-1B9.PCT Application Attorney Docket No.: CUBR / 0006PC

[0181] Clause B21. The composition according to Clause B20, wherein the skin pathogen comprises Staphylococcus, Streptococcus, coryneform, or combinations thereof.

[0182] Clause B22. A composition that disrupts cell membrane integrity, comprising the composition according to any one of Clauses B1-B21.

[0183] Clause B23. A composition for treating atopic dermatitis in a patient, psoriasis in a patient, acne in a patient, or combinations thereof, comprising the composition according to any one of Clauses B1-B23.

[0184] Clause B24. An anti-infective composition for treating a staphylococcal skin infection in a patient, comprising the composition according to any one of Clauses B1-B23.

[0185] Clause B25. A composition for treating staphylococcal skin bacteria in a patient, comprising the composition according to any one of Clauses B1-B24.

[0186] Clause B26. A method for treating a skin pathogen in a patient, the method comprising: administering to the patient the composition according to any one of Clauses B1- B25.

[0187] Clause B27. The method according to Clause B26, wherein the skin pathogen comprises Staphylococcus, Streptococcus, coryneform, or combinations thereof.

[0188] Clause B28. A method for treating staphylococcal skin bacteria in a patient, the method comprising: administering to the patient the composition according to any one of Clauses B1-B25.

[0189] Clause B29. The method according to Clause B28, wherein the staphylococcal skin bacteria comprises staphylococcus aureus.

[0190] Clause B30. A method for treating a skin disease in a patient, the method comprising: administering to the patient the composition according to any one of Clauses B1- B25.

[0191] Clause B31. The method according to Clause B30, wherein the skin disease comprises atopic dermatitis, psoriasis, acne, or combinations thereof.

[0192] Clause B32. The method according to any one of Clauses B26-B31, wherein the method induces permeabilization and depolarization of a cytoplasmic membrane of staphylococcal skin bacteria.

[0193] Clause B33. The method according to any one of Clauses B26-B32, wherein the method induces pore formation in a cytoplasmic membrane of staphylococcal skin bacteria.PCT Application Attorney Docket No.: CUBR / 0006PC

[0194] All documents described herein are incorporated by reference herein, including any priority documents and / or testing procedures to the extent they are not inconsistent with this text. As is apparent from the foregoing general description and the specific embodiments, while forms of the embodiments have been illustrated and described, various modifications may be made without departing from the spirit and scope of the present disclosure. Accordingly, it is not intended that the present disclosure be limited thereby. Likewise, the term “comprising” is considered synonymous with the term “including.” Likewise whenever a composition, an element, a group of elements, or a method is preceded with the transitional phrase “comprising,” it is understood that we also contemplate the same composition, method, or group of elements with transitional phrases “consisting essentially of,” “consisting of,” “selected from the group of consisting of,” or “Is” preceding the recitation of the composition, element, elements, or method, and vice versa, such as the terms “comprising,” “consisting essentially of,” “consisting of” also include the product of the combinations of elements listed after the term.

[0195] In the foregoing, reference is made to embodiments of the disclosure. However, it should be understood that the disclosure is not limited to specific described embodiments. Instead, any combination of the following features and elements, whether related to different embodiments or not, is contemplated to implement and practice the disclosure. Furthermore, although embodiments of the disclosure may achieve advantages over other possible solutions and / or over the prior art, whether or not a particular advantage is achieved by a given embodiment is not limiting of the disclosure. Thus, the foregoing aspects, features, embodiments, and advantages are merely illustrative and are not considered elements or limitations of the appended claims except where explicitly recited in a claim(s). Likewise, reference to “the disclosure” shall not be construed as a generalization of any inventive subject matter disclosed herein and shall not be considered to be an element or limitation of the appended claims except where explicitly recited in a claim(s).

[0196] “Treatment” and “treating” includes the medical management of a subject with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease,PCT Application Attorney Docket No.: CUBR / 0006PC pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. It is understood that treatment, while intended to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder, need not actually result in the cure, ameliorization, stabilization or prevention. The effects of treatment can be measured or assessed as described herein and as known in the art as is suitable for the disease, pathological condition, or disorder involved. Such measurements and assessments can be made in qualitative and / or quantitative terms. Thus, for example, characteristics or features of a disease, pathological condition, or disorder and / or symptoms of a disease, pathological condition, or disorder can be reduced to any effect or to any amount.

[0197] For purposes of this present disclosure, and unless otherwise specified, all numerical values within the detailed description and the claims herein are modified by “about” or “approximately” the indicated value, and consider experimental error and variations that would be expected by a person having ordinary skill in the art. For the sake of brevity, only certain ranges are explicitly disclosed herein. However, ranges from any lower limit may be combined with any upper limit to recite a range not explicitly recited, as well as, ranges from any lower limit may be combined with any other lower limit to recite a range not explicitly recited, in the same way, ranges from any upper limit may be combined with any other upper limit to recite a range not explicitly recited. For example, the recitation of the numerical range 1 to 5 includes the subranges 1 to 4, 1.5 to 4.5, 1 to 2, among other subranges. As another example, the recitation of the numerical ranges 1 to 5, such as 2 to 4, includes the subranges 1 to 4 and 2 to 5, among other subranges. Additionally, within a range includes every point or individual value between its end points even though not explicitly recited. For example, the recitation of the numerical range 1 to 5 includes the numbers 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, among other numbers. Thus, every point or individual value may serve as its own lower or upper limit combined with any other point or individual value or any other lower or upper limit, to recite a range not explicitly recited.PCT Application Attorney Docket No.: CUBR / 0006PC

[0198] As used herein, the indefinite article “a” or “an” shall mean “at least one” unless specified to the contrary or the context clearly indicates otherwise. For example, embodiments comprising “an amino acid” include embodiments comprising one, two, or more amino acids, unless specified to the contrary or the context clearly indicates only one amino acid is included.

[0199] While the foregoing is directed to embodiments of the present disclosure, other and further embodiments of the disclosure may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow.

Claims

PCT Application Attorney Docket No.: CUBR / 0006PC CLAIMS What is claimed is:

1. A composition for treating a skin condition in a patient, the composition comprising a daptide, the daptide comprising: a dimethylated N-terminus; a N2,N2-dimethyl-1,2-propanediamine (Dmp)-modified C-terminus; a dehydrobutyrine residue positioned between the dimethylated N-terminus and the Dmp-modified C-terminus; at least one alanine (A) residue or at least one dehydroalanine (Dha) residue positioned between the dimethylated N-terminus and the Dmp-modified C-terminus; and at least one amino acid residue positioned between the dimethylated N-terminus and the Dmp-modified C-terminus.

2. The composition according to claim 1, wherein the daptide comprises from 15 to 25 amino acid residues.

3. The composition according to claim 1, wherein: the dimethylated N-terminus of the daptide comprises a dimethylated isoleucine; and the Dmp-modified C-terminus of the daptide comprises an alanine residue modified with Dmp.

4. The composition according to claim 1, wherein the daptide comprises an amino acid sequence having at least 75% sequence identity to one or more of the amino acid sequences set forth in SEQ ID NOs: 1-6.

5. The composition according to claim 1, wherein the daptide comprises an amino acid sequence having at least 75% sequence identity to one or more of the amino acid sequences set forth in SEQ ID NOs: 7-14.PCT Application Attorney Docket No.: CUBR / 0006PC 6. The composition according to claim 1, wherein the daptide comprises an amino acid sequence having at least 75% sequence identity to the amino acid sequence set forth in SEQ ID NO:

15.

7. The composition according to claim 1, wherein the daptide comprises an amino acid sequence having at least 75% sequence identity to the amino acid sequence set forth in SEQ ID NO:

16.

8. The composition according to claim 1, wherein the daptide comprises an amino acid sequence having at least 75% sequence identity to the amino acid sequence set forth in SEQ ID NO:

17.

9. The composition according to claim 1, wherein the daptide comprises an amino acid sequence having at least 75% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 18 or 19.

10. The composition according to claim 1, wherein the daptide comprises an amino acid sequence having at least 75% sequence identity to the amino acid sequence set forth in SEQ ID NO:

20.

11. The composition according to claim 1, wherein the skin condition comprises a skin bacteria, a skin disease, a skin pathogen, or combinations thereof.

12. The composition according to claim 11, wherein the skin pathogen or the skin bacteria comprises Staphylococcus, Streptococcus, coryneform, or combinations thereof.

13. The composition according to claim 11, wherein the skin pathogen or the skin bacteria comprises M. luteus, S. pyogenes, S. aureus, S. hominis, S. epidermidis, S. lugdunensis, S. warneri.PCT Application Attorney Docket No.: CUBR / 0006PC 14. The composition according to claim 11, wherein the skin disease comprises atopic dermatitis, eczema, psoriasis, acne, rosacea, or combinations thereof.

15. The composition according to claim 1, wherein the composition is configured to: (a) induce dissipation of a transmembrane potential through pore formation, thereby causing cell membrane damage in a bacteria, a pathogen, a microbe, or combinations thereof; (b) penetrate and self-organize into ion-conducting channels in a cytoplasmic membrane of a bacteria, a pathogen, a microbe, or combinations thereof; (c) induce permeabilization and depolarization of a cytoplasmic membrane of a bacteria, a pathogen, a microbe, or combinations thereof; or (d) a combination thereof.

16. A daptide bacteriocin composition that is antimicrobial against a staphylococcal skin bacteria in a patient, the daptide bacteriocin composition comprising an amino acid sequence having at least 75% sequence identity to one or more of the amino acid sequences set forth in SEQ ID NOs: 1-20.

17. The daptide bacteriocin composition according to claim 16, wherein the daptide bacteriocin composition is configured to: (a) induce dissipation of a transmembrane potential through pore formation, thereby causing cell membrane of the staphylococcal skin bacteria; (b) penetrate and self-organize into ion-conducting channels in a cytoplasmic membrane of the staphylococcal skin bacteria; (c) induce permeabilization and depolarization of a cytoplasmic membrane of the staphylococcal skin bacteria; or (d) a combination thereof.

18. A method for treating a skin condition in a patient, the method comprising: administering to the patient a composition comprising a daptide, the daptide comprising: a dimethylated N-terminus;PCT Application Attorney Docket No.: CUBR / 0006PC a N2,N2-dimethyl-1,2-propanediamine (Dmp)-modified C-terminus; a dehydrobutyrine residue positioned between the dimethylated N-terminus and the Dmp-modified C-terminus; at least one alanine residue or at least one dehydroalanine (Dha) residue positioned between the dimethylated N-terminus and the Dmp-modified C-terminus; and at least one amino acid residue positioned between the dimethylated N- terminus and the Dmp-modified C-terminus.

19. The method according to claim 18, wherein the daptide comprises an amino acid sequence having at least 75% sequence identity to one or more of the amino acid sequences set forth in SEQ ID NOs: 1-20.

20. The method according to claim 18, wherein the daptide comprises an amino acid sequence having at least 90% sequence identity to one or more of the amino acid sequences set forth in SEQ ID NOs: 6-20.

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