Molecular bacteriotherapy to control skin enzyme activity

Abstract

To provide compositions and methods for treating dermatological diseases and disorders, and compositions that modulate cutaneous permeability.SOLUTION: The invention provides a purified polypeptide which comprises a specific amino acid sequence and which inhibits (i) protease production and / or activity of keratinocytes, (ii) IL-6 production and / or activity of keratinocytes, (iii) production of phenol-soluble modulin α3 from S. aureus, and / or (iv) agr production and / or activity by S. aureus.SELECTED DRAWING: None

Description

Statement Regarding Government-Sponsored Research

[0001] This invention was made with government support under award numbers AI117673, AR067547, AR062496 and AR064781 awarded by the National Institutes of Health. The government has certain rights in this invention. Cross-Reference to Related Applications

[0002] This application claims priority under 35 U.S.C. § 119 to U.S. Provisional Patent Application No. 62 / 553,025, filed August 31, 2017, the disclosure of which is incorporated herein by reference. [Technical Field]

[0003] The present disclosure relates to compositions and methods for treating skin diseases and disorders, as well as compositions that modulate the permeability of the skin barrier.

[0004] Exemplary microorganisms of the present disclosure (Staphylococcus epidermidis A11, Staphylococcus hominis C5, Staphylococcus hominis A9, and Staphylococcus warneri G2) were deposited under the Budapest Treaty on August 28, 2018, with the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209, under ATCC numbers 111111 and 111121. PTA-125202 (strain designation: S. epidermidis A11 81618, deposited on August 28, 2018), ATCC no. PTA-125204 (Stock designation: S. hominis C5 81618, deposited on August 28, 2018), ATCC number PTA-125203 (Stock designation: S. hominis A9 81618, deposited on August 28, 2018) and ATCC number PTA-125205(Strain designation: S. warneri G2 81618, deposited August 28, 2018). The deposit will be maintained at the depository institution, but will be replaced in the event of authorized mutation, non-viability, or disruption for at least 5 years from the depositor's most recent request for sample release, at least 30 years from the date of deposit, or the life of any relevant patent, whichever is longer. If a patent is granted upon application, all restrictions on the availability of these cell lines to the public will be irrevocably lifted. [Background technology]

[0005] The epidermis is the first line of immune defense, protecting and regulating interactions between microorganisms and the host organism. Controlling this interaction is crucial because bacteria not only reside on the surface, affecting superficial keratinocytes, but also penetrate beneath the stratum corneum and into the dermis, where several bacterial species have been shown to affect immune function. For example, Staphylococcus epidermidis (S. epidermidis) interacts with epidermal keratinocytes, preventing inflammation via Toll-like receptor 3, recruiting mast cells and T cells, and increasing tight junction and antimicrobial peptide production. In contrast to common skin commensal bacteria, S. epidermidis and S. aureus are often pathogenic and negatively impact skin function. This is particularly evident in skin diseases such as atopic dermatitis (AD), which is promoted by S. aureus.

[0006] The skin microbiome of AD subjects has been shown to have decreased overall microbial diversity and increased abundance of Staphylococcus aureus. Increased S. aureus microbiota is associated with increased disease severity in AD patients. The mechanism by which S. aureus exacerbates disease is unclear. Several S. aureus products have been shown to damage the skin barrier or cause inflammation. These products include α-toxin, superantigens, toxic shock syndrome toxin-1, enterotoxin, protein A, Panton-Valentine leukocidin, exfoliative toxin, and V8 serine protease. Due to the potential pathogenic effects of these molecules, understanding the skin's response to S. aureus microbiota in the absence of clear clinical signs of infection is important for understanding AD pathogenesis and developing future treatments. Summary of the Invention

[0007] The present disclosure provides a purified polypeptide comprising a sequence at least 98% identical to SEQ ID NO:4, 11, 12, 13, 14, 15, 16, or 17, and which (i) inhibits protease production and / or keratinocyte activity, (ii) inhibits keratinocyte IL-6 production and / or keratinocyte activity, (iii) inhibits phenol-soluble modulin α3 production from S. aureus, and / or (iv) inhibits agr production and / or activity by S. aureus. In one embodiment, the polypeptide is at least 98% identical to SEQ ID NO:2. In another embodiment, the polypeptide comprises SEQ ID NO:4, 11, 12, 13, 14, 15, 16, or 17. In yet another embodiment, the polypeptide consists of SEQ ID NO:4, 11, 12, 13, 14, 15, 16, or 17. In another or further embodiment of any of the foregoing, the polypeptide comprises one or more D-amino acids. In yet another further embodiment, the polypeptide comprises a compound of Formula I, IA, or IB (see below).

[0008] The present disclosure also provides topical formulations comprising a polypeptide of the present disclosure or a compound of formula I, IA, or IB.

[0009] The present disclosure also provides isolated polynucleotides encoding polypeptides of the present disclosure. In certain embodiments, the polynucleotides comprise a sequence that hybridizes under stringent conditions to a polynucleotide consisting of SEQ ID NO: 1 or 3 and encodes a polypeptide comprising SEQ ID NO: 4. In other embodiments, the polynucleotide comprises SEQ ID NO: 1 or 3.

[0010] The present disclosure also provides a vector comprising a polynucleotide of the present disclosure, which may be any vector suitable for expression in a cellular or microbial host.

[0011] The present disclosure also provides a recombinant microorganism comprising a vector or polynucleotide of the present disclosure. In some embodiments, the microorganism does not naturally express a polypeptide of the present disclosure, but is engineered to express a polypeptide of the present disclosure by recombinant engineering. In another embodiment, the microorganism is attenuated, i.e., made non-pathogenic or has reduced pathogenicity compared to the wild-type organism of the same species. In another embodiment, the recombinant microorganism is a microorganism that is normally found (e.g., commensal) on the skin of a mammal (e.g., human).

[0012] The present disclosure also provides live bacterial compositions comprising the recombinant microorganisms of the present disclosure.

[0013] The present disclosure also provides live bacterial compositions comprising a microorganism expressing a polypeptide of the disclosure (e.g., SEQ ID NOs: 4, 11, 12, 13, 14, 15, 16, and / or 17). In certain embodiments, the microorganism is S. hominis, S. epidermidis, and / or S. warneri, or any combination thereof. In further embodiments, the microorganism is S. hominis C5, S. hominis A9, S. epidermidis All, and / or S. warneri G2. In yet further embodiments, the composition comprises a strain of S. hominis designated as ... (strain designation: S. epidermidis A11 81618, deposited on August 28, 2018), ATCC no. (Stock designation: S. hominis C5 81618, deposited on August 28, 2018), ATCC number (Stock designation: S. hominis A9 81618, deposited on August 28, 2018) and ATCC number (strain designation: S. warneri G2 81618, deposited August 28, 2018) and any combination of the aforementioned strains. In another embodiment, the probiotic composition of the present disclosure is non-naturally occurring (e.g., does not contain the full spectrum of microorganisms found on skin, or contains a quantity of microorganisms per unit volume not found on skin, or the microorganisms are genetically modified, or the composition contains an ingredient or compound not normally found on skin).

[0014] The present disclosure also provides a method for treating a skin disorder, comprising administering to the skin an effective amount of coagulase-negative Staphylococcus species (CoNS) or a fermentation extract of CoNS sufficient to inhibit protease activity, wherein the CoNS produces a polypeptide comprising a sequence at least 98% identical to SEQ ID NOs: 4, 11, 12, 13, 14, 15, 16, and 17, thereby inhibiting protease production. In certain embodiments, the skin disorder is selected from the group consisting of Netherton syndrome, atopic dermatitis, contact dermatitis, eczema, psoriasis, acne, epidermal hyperkeratosis, acanthosis nigricans, epidermal inflammation, dermal inflammation, and pruritus. In another embodiment, the administration is topical. In yet other further embodiments, the CoNS is selected from the group consisting of Staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus saccharolyticus, Staphylococcus warneri, Staphylococcus pasteuri, Staphylococcus haemolyticus, Staphylococcus devriesei, Staphylococcus hominis, Staphylococcus jettensis, Staphylococcus petrasii, and Staphylococcus lugdunensis. In other or further embodiments of any of the foregoing, a fermentation extract of the CoNS comprises the polypeptide sequence of SEQ ID NO:4 and / or a compound of Formula I, IA, or IB. In another embodiment, the CoNS is selected from the group consisting of S. epidermidis A11, S. hominis C4, S. hominis C5, S. hominis A9, S. warneri G2, and any combination thereof.

[0015] The present disclosure also provides a method for treating a skin disease or disorder, comprising measuring protease activity in a culture from the skin of a subject or the skin of the subject, comparing the protease activity to a normal control, and administering a composition and / or fermentation extract of a probiotic skin bacterium from coagulase-negative Staphylococcus aureus, wherein the composition or fermentation extract of the probiotic skin bacterium comprises a polypeptide at least 98% identical to SEQ ID NO: 4, 11, 12, 13, 14, 15, 16, or 17, or comprises a compound of Formula I, IA, or IB, and the composition is formulated into a cream, ointment, or pharmaceutical composition to maintain the growth and replication of the probiotic skin bacterium. In certain embodiments, the coagulase-negative staphylococcus is selected from the group consisting of Staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus saccharolyticus, Staphylococcus warneri, Staphylococcus pasteuri, Staphylococcus haemolyticus, Staphylococcus devriesei, Staphylococcus hominis, Staphylococcus jettensis, Staphylococcus petrasii, and Staphylococcus lugdunensis.

[0016] The present disclosure also provides a method of treating a skin disease or disorder comprising administering a live bacterial composition containing a bacterium that produces a purified polypeptide of the present disclosure or a polypeptide that is at least 98% identical to SEQ ID NO:4, 11, 12, 13, 14, 15, 16, or 17 and that inhibits the production or activity of kallikrein.

[0017] The present disclosure also provides a method for treating a skin disease or disorder, comprising administering a composition that inhibits expression of a phenol-soluble modulin, wherein the composition comprises a purified polypeptide of the present disclosure or a compound of Formula I, IA, or IB. In some embodiments, the administration is topical. In another embodiment, the composition is a fermentation extract of coagulase-negative Staphylococcus aureus.

[0018] The present disclosure also provides a probiotic composition for topical use comprising a probiotic probiotic skin bacterium selected from the group consisting of S. epidermidis A11, S. hominis C4, S. hominis C5, S. hominis A9, S. warneri G2, and any combination thereof. In some embodiments, the composition is formulated as a lotion, shake lotion, cream, ointment, gel, foam, powder, solid, paste, or tincture.

[0019] The present disclosure also provides a pharmaceutical composition comprising a drug and a fermentation extract of Staphylococcus aureus or live Staphylococcus aureus cells having phenol-soluble modulin α3. The present disclosure also provides a use of the composition for delivering a drug through the skin of a subject.

[0020] The present disclosure provides probiotic / beneficial bacteria and / or their products to prevent increased protease activity in the skin, which is important in many disease states including atopic dermatitis, Netherton syndrome, and other skin conditions with elevated protease activity and barrier disruption.

[0021] The present disclosure also provides factors and compositions that induce proteolytic enzyme activity and therefore aid in the proteolytic remodeling of skin in the treatment of disorders related to wound repair, aging, sun damage, pigmentation disorders, and scarring.

[0022] The present disclosure provides a method for treating a skin disorder, comprising administering an effective amount of coagulase-negative Staphylococcus species (CoNS) or an effective amount of a fermentation extract of CoNS sufficient to inhibit the activity of proteases in the skin. In some embodiments, the skin disorder is selected from the group consisting of Netherton syndrome, atopic dermatitis, contact dermatitis, eczema, psoriasis, acne, epidermal hyperkeratosis, acanthosis nigricans, epidermal inflammation, dermal inflammation, and pruritus. In other embodiments, the administration is topical. In certain embodiments, the CoNS is selected from the group consisting of Staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus saccharolyticus, Staphylococcus warneri, Staphylococcus pasteuri, Staphylococcus haemolyticus, Staphylococcus devriesei, Staphylococcus hominis, Staphylococcus jettensis, Staphylococcus petrasii, and Staphylococcus lugdunensis. In certain embodiments, the CoNS is Staphylococcus epidermidis.

[0023] The present disclosure also provides a method for treating a skin disease or disorder, comprising measuring protease activity in a culture from the skin of a subject or in the skin of the subject, comparing the protease activity to a normal control, and administering a composition of probiotic skin bacteria and / or a fermentation extract from coagulase-negative Staphylococcus aureus, wherein the composition of probiotic skin bacteria comprises at least one probiotic bacterium that reduces the activity of a serine protease in the culture or skin, and the at least one probiotic bacterium is formulated into a cream, ointment, or pharmaceutical composition to maintain the growth and replication capacity of the probiotic skin bacteria. In certain embodiments, the coagulase-negative staphylococcus is selected from the group consisting of Staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus saccharolyticus, Staphylococcus warneri, Staphylococcus pasteuri, Staphylococcus haemolyticus, Staphylococcus devriesei, Staphylococcus hominis, Staphylococcus jettensis, Staphylococcus petrasii, and Staphylococcus lugdunensis.

[0024] The present disclosure also provides a method of treating a skin disease or disorder, comprising administering an agent that inhibits the expression of kallikrein. The present disclosure also provides a method of treating a skin disease or disorder, comprising administering an agent that inhibits the expression of phenol-soluble modulin. In any of the foregoing embodiments, the administration is topical. In another embodiment, the agent is a fermentation extract of coagulase-negative staphylococcus. In another embodiment, the coagulase-negative staphylococcus is selected from the group consisting of Staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus saccharolyticus, Staphylococcus warneri, Staphylococcus pasteuri, Staphylococcus haemolyticus, Staphylococcus devriesei, Staphylococcus hominis, Staphylococcus jettensis, Staphylococcus petrasii, and Staphylococcus lugdunensis.

[0025] The present disclosure also provides a topical composition comprising a plurality of skin bacteria. In certain embodiments, the live, probiotic skin bacteria is a coagulase-negative Staphylococcus species. In another specific embodiment, the live, probiotic skin bacteria comprises Staphylococcus aureus. In one embodiment of any of the foregoing embodiments, the bacteria are formulated into a cream, lotion, tincture, gel, or other topical formulation in which the bacteria remain viable.

[0026] The present disclosure also provides a probiotic composition for topical use, comprising a fermentation extract of probiotic skin bacteria. The fermentation extract of probiotic skin bacteria is obtained from a coagulase-negative staphylococcus (CoNS) species. In some embodiments, the CoNS is selected from the group consisting of Staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus saccharolyticus, Staphylococcus warneri, Staphylococcus pasteuri, Staphylococcus haemolyticus, Staphylococcus devriesei, Staphylococcus hominis, Staphylococcus jettensis, Staphylococcus petrasii, and Staphylococcus lugdunensis.

[0027] In any of the described embodiments, the topical probiotic composition is formulated into a lotion, shake lotion, cream, ointment, gel, foam, powder, solid, paste, or tincture.

[0028] The present disclosure provides a pharmaceutical composition comprising a drug and a fermentation extract of Staphylococcus aureus or a biological composition of Staphylococcus aureus.

[0029] The present disclosure provides a method of drug delivery through the skin, comprising contacting the skin with a composition comprising a drug and a fermentation extract of Staphylococcus aureus or a biological composition of Staphylococcus aureus, in some embodiments, the drug is a topical drug that is absorbed or adsorbed through the skin.

[0030] The present disclosure also provides a method for topical drug delivery, comprising contacting the skin of a subject with a composition comprising Staphylococcus aureus or a fermentation extract of Staphylococcus aureus for a period of time at a dosage and under conditions that increase the permeability of the skin, and then contacting the skin with the drug to be delivered.

[0031] The present disclosure provides compositions comprising a fermentation extract from Staphylococcus aureus, or lotions, shakes, creams, ointments, gels, foams, powders, solids, pastes, or tinctures containing live Staphylococcus aureus.

[0032] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. [Brief explanation of the drawings]

[0033] [Figure 1A] NHEKs were treated with sterile-filtered supernatants of Staphylococcus aureus (SA; Newman, USA300, 113, SANGER252) and Staphylococcus epidermidis (ATCC12228, ATCC1457) for 24 hours, and NHEK-conditioned medium was analyzed with specific trypsin-like substrates. [Figure 1B] NHEKs were treated with sterile-filtered supernatants of Staphylococcus aureus (SA; Newman, USA300, 113, SANGER252) and Staphylococcus epidermidis (ATCC12228, ATCC1457) for 24 h, and NHEK-conditioned medium was assayed with specific elastase-like substrates. [Figure 1C] NHEKs were treated with sterile-filtered supernatants of Staphylococcus aureus (SA; Newman, USA300, 113, SANGER252) and Staphylococcus epidermidis (ATCC12228, ATCC1457) for 24 h, and NHEK-conditioned media was assayed with substrates of specific MMP proteases. [Figure 1D] Proteolytic enzymes secreted by Staphylococcus aureus (Newman) were analyzed for their effect on trypsin activity. Data represent mean ± SEM (n = 4) and are representative of at least three independent experiments. One-way analysis of variance (aec) and two-way analysis of variance (d) were used, and significance was indicated by *P<0.05, ***P<0.001, and ****P<0.0001. ANOVA, analysis of variance; MMP, matrix metalloproteinase; NHEK, healthy human epidermal keratinocytes. [Figure 2A]Total protease activity (5 μg / ml BODIPY FL casein) was measured in NHEK-conditioned medium after treatment with supernatants of Staphylococcus aureus (SA, Newman) for 0 to 48 hours. [Figure 2B] The conditioned medium was added 24 hours after treatment with aprotinin (800 μg / ml), an inhibitor of serine proteases. [Figure 2C] The effect of NHEK-conditioned medium (Boc-Val-Pro-Arg-AMC, 200 mM) on trypsin activity was compared between wild-type and protease-deficient S. aureus (USA300 LAC) strains. Both two-way ANOVA (A, B) and one-way ANOVA (C) were used; significance is indicated by *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. ANOVA, analysis of variance; NHEK, healthy human epidermal keratinocytes; WT, wild-type. [Figure 3A-E] Staphylococcus aureus increases KLK expression in human keratinocytes. Figure 3A shows the relative amount of KLK mRNA expression in NHEKs treated with S. aureus (SA, Newman) supernatant for 24 hours, analyzed by qPCR. Figures 3B-E show that KLKs 5, 6, 13, and 14 were analyzed for fold change in mRNA expression in NHEKs treated with S. aureus supernatant for 0 to 48 hours. All mRNA expression levels were normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). [Figure 3F]NHEK-conditioned medium and cell lysates were treated with SA (Newman) supernatant for 24 hours using both published and predicted molecular weights, followed by immunoblotting for changes in protein expression of KLKs 5, 6, 13, and 14. The housekeeping gene α-tubulin was used as a loading control for cell lysates. Data represent mean ± SEM (n = 3) and are representative of at least three independent experiments. Two-way analysis of variance (BEE) was used, and significance was indicated by **P < 0.01, ***P < 0.001, and ****P < 0.0001. ANOVA, analysis of variance; KLK, kallikrein; NHEK, normal human epidermal keratinocytes; qPCR, quantitative real-time PCR; SEM, standard error of the mean. [Figure 4A-D] We demonstrate that multiple KLKs are responsible for S. aureus-induced serine protease activity in human keratinocytes. NHEKs were treated with KLK6, KLK13, or KLK14 siRNA (15 nM), differentiated with CaCl2, and cultured with S. aureus (Newman) supernatant. siRNA scramble (-) controls 1 and 2 were used at 15 nM and 45 nM, respectively. Conditioned media were analyzed for changes in trypsin activity (Boc-Val-Pro-Arg-AMC, 200 μM) (Figure 4A). Transcription levels of KLK6, KLK13, and KLK14 were assessed by qPCR and normalized to the housekeeping gene GAPDH to confirm siRNA knockdown efficiency (Figure 4B-D). Data represent the mean ± SEM (n = 4) and are representative of at least three independent experiments. One-way analysis of variance (a) was used, and significance was indicated by *P<0.05, **P<0.01, and ***P<0.001. ANOVA, analysis of variance; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; KLK, kallikrein; NHEK, healthy human epidermal keratinocytes; qPCR, quantitative real-time PCR; siRNA, small interfering RNA; SEM, standard error of the mean. [Figure 5A-C]We demonstrate that multiple KLKs regulate Staphylococcus aureus-induced DSG-1 and FLG cleavage in human keratinocytes. NHEKs were treated with S. aureus (Newman) supernatant for 24 hours, and changes in DSG-1 (Figure 5A) and profilaggrin (Pro-FLG) (Figure 5B) cleavage were assessed by immunoblotting after siRNA knockdown of KLKs 6, 13, and 14 (15 nM). The housekeeping gene α-tubulin was used as a loading control. DSG-1 (full-length) and Pro-FLG are indicated by black arrows. Densitometric analysis of both DSG-1 (full-length) and Pro-FLG is expressed as the average number of pixels normalized to α-tubulin (n = 1) (Figure 5C). Immunoblots are representative of at least three independent experiments. KLK, kallikrein; NHEK, normal human epidermal keratinocytes; siRNA, small interfering RNA. [Figure 6] The method for preparing the fermentation extract and assaying the activity are shown. [Figure 7] We show that Staphylococcus aureus phenol-soluble modulin (PSM) under the control of the agr quorum-sensing system is responsible for the increased activity of serine proteases in keratinocytes. [Figure 8] We show that S. aureus PSM increases serine protease activity and skin barrier damage in mice. [Figure 9] We show that Staphylococcus aureus isolates from atopic dermatitis (AD) lesional skin can induce serine protease activity in keratinocytes in an agr type-dependent manner. [Figure 10] We show that the coagulase-negative staphylococcus aureus (CoNS) strain ATCC14490 (S. epidermidis) produces an autoinducing peptide (AIP) that abolishes the agr activity of S. aureus. [Figure 11] 1 shows the effects of Staphylococcus aureus and commensal bacteria on serine protease activity in atopic dermatitis. [Figure 12] 1 shows the effect of S. hominis C5 on the agr activity of S. aureus. [Figure 13]shows the effect of various CoNS strains on agr activity in S. aureus. [Figures 14A-E] We demonstrate that S. aureus PSMα leads to disruption of epithelial barrier homeostasis. Human keratinocytes (NHEKs) were stimulated for 24 hours with sterile-filtered supernatants of S. aureus (SA) from wild-type (WT), PSMα (ΔPSMα), or PSMβ (ΔPSMβ) knockout strains, and trypsin activity (Figure 14A) and KLK6 mRNA (Figure 14B) were analyzed relative to the housekeeping gene GAPDH (n=4). PSM synthetic peptides were added to NHEKs for up to 24 hours, and changes in trypsin activity were analyzed (Figure 14C). Genes that changed more than two-fold after treatment with PSMα3 were evaluated by transcript analysis using RNA-Seq, and gene ontology (GO) analysis was performed. Eight-week-old male C57BL / 6 mice (n = 6) were treated with SA WT, SAΔPSMα, or a knockout strain of SA 10-secreted protease (Δprotease) (1e7 CFU) for 72 hours (Figure 14D-E). [Figure 14F-J] These results demonstrate that S. aureus PSMα leads to disruption of epithelial barrier homeostasis. Figures 14F-G show representative photographs of mouse skin (dashed lines indicate treatment sites) and the change in epidermal thickness after treatment (scale = 200 μm). Changes in the dorsal skin of mice treated with WT or mutant SA strains were also assessed for transepidermal water loss (TEWL) and SA CFU / cm2 (Figures 14H-K). All error bars represent the standard error of the mean (SEM). One-way analysis of variance was used to determine statistical significance, indicated by p<0.05*, p<0.01**, p<0.001***, and p<0.0001****. [Figures 15A-F]The characteristics of S. epidermidis agr type I autoinducer peptides and their absence in AD skin are shown. Figures 15A-B show the inhibition of S. epidermidis agr type I-III supernatants on the type I activity of Staphylococcus aureus (SA) USA300 LAC agr after 24 hours (n = 4) and the known structure of the S. epidermidis agr type I autoinducer peptide (AIP). Figure 15C shows the effect of the autoinducer peptide knockout (ΔAIP) on the agr activity of S. epidermidis agr type I strain RP62A wild type (WT) or SA after 24 hours. SA sterile-filtered supernatants with or without S. epidermidis WT or ΔAIP supernatants were added to NHEKs for an additional 24 hours, after which NHEK trypsin activity was measured (n = 4) (Figure 15D). Figure 15E shows the identity of S. epidermidis agr type I-III genomes found in AD skin. [Figure 15G] Figures 15F–G show the relative abundance ratios of S. epidermidis agr type I and SA in the flare areas of each of eight AD subjects, ranging from "mild" to "very severe." AD scores were based on the objective SCORAD score; the combined data for all subjects was based on AD severity. All error bars represent the standard error of the mean (SEM). Statistical significance was determined using one-way ANOVA (Figures 15A, C, D) and a (nonparametric) unpaired Mann-Whitney test (Figure 15F), with p<0.05*, p<0.01**, p<0.001***, and p<0.0001****. [Figures 16A-C]We demonstrate that multiple clinically isolated coagulase-negative staphylococci inhibit the agr activity of S. aureus. Sterile-filtered supernatants from clinically isolated coagulase-negative staphylococci (CoNS) were added to the S. aureus (SA) USA300 LAC agr type I P3-YFP reporter strain for 24 hours, and the agr activity of SA was analyzed (n = 3) (Figure 16A). The S. hominis C5 strain genome was further sequenced, and the sequence of the autoinducing peptide (AIP) was analyzed in the agrD gene. Biochemical analysis of S. hominis C5 supernatants included <3 kDa size-exclusion centrifugal filtration, 80% ammonium sulfate precipitation, and treatment at pH 11 for 1 hour, and the effect of the supernatant on SA agr activity was tested (Figure 16B-C). [Figure 16D-F] We demonstrate that clinically isolated, multiple coagulase-negative staphylococci inhibit the agr activity of S. aureus. SA grown for 24 hours in the presence of S. hominis C5 supernatant was sterile filtered and added to human keratinocytes (NHEK) for 24 hours. Trypsin activity, KLK6 mRNA expression relative to the housekeeping gene GAPDH, and IL-6 protein levels were analyzed (Figures 16D-F). All error bars represent the standard error of the mean (SEM). One-way analysis of variance was used to determine statistical significance, indicated by p<0.05*, p<0.01**, p<0.001***, and p<0.0001****. [Figures 17A-C] We demonstrate that a clinical CoNS isolate from AD inhibits SA-induced skin damage in mice. Eight-week-old 7BL / 6 mice were treated with the agr type I pAmi P3-Lux reporter strain (1e7 CFU) of Staphylococcus aureus (SA) USA300 LAC with or without live S. hominis C5 (1e8 CFU) for 48 hours (n=5). The agr activity of SA in the dorsal skin of mice was assessed by changes in luminescence (Figures 17A-B). Figure 17C shows representative images of mouse skin after 48 hours of SA treatment (the dashed box indicates the treatment area). [Figure 17D-H]We demonstrate that clinical CoNS isolates from AD inhibit SA-induced skin damage in mice. We measured SA CFU / cm2 and assessed mouse skin damage and inflammation by analyzing changes in Il6 mRNA expression, transepidermal water loss (TEWL), trypsin activity, and Klk6 mRNA expression normalized to the housekeeping gene Gapdh (Figures 17D-H). All error bars represent standard error of the mean (SEM). One-way analysis of variance was used to determine statistical significance, indicated by p<0.05*, p<0.01**, p<0.001***, and p<0.0001****. [Figures 18A-D] We demonstrate that S. aureus PSMα alters essential gene and cytokine expression in human keratinocytes. Human keratinocytes treated with synthetic PSMα3 were assessed for changes in trypsin activity and KLK6 transcript expression normalized to the housekeeping gene GAPDH in both a dose- and time-dependent manner (Figures 18A-D). [Figure 18E-H] These results show that S. aureus PSMα alters essential gene and cytokine expression in human keratinocytes. Figure 18E shows GO-term analysis of genes that were reduced by 2-fold or more compared to control in human keratinocytes treated with PSMα3 for 24 hours. Figures 18F-H show changes in cytokine protein expression of IL-6, TNF-α, or IL-1α in human keratinocytes treated with the supernatant of SA WT, SAΔpsmα, or SAΔpsmβ for 24 hours. All error bars represent the standard error of the mean (SEM). One-way analysis of variance was used to determine statistical significance, indicated by p<0.05*, p<0.01**, p<0.001***, and p<0.0001****. [Figure 19A-H-1]We demonstrate that S. aureus PSMα and protease are responsible for inducing damage and inflammation in mouse skin. S. aureus (SA) (1e7 CFU) wild-type (WT), PSMα knockout (Δpsmα), and protease-less (Δprotease) strains were inoculated onto the dorsal skin of male mice for 72 hours (n=6). Changes in trypsin activity (Figures 19A and 19E), Klk6 (Figures 19B and 19F), Il6 (Figures 19C and 19G), and IL17a / f mRNA expression normalized to the housekeeping gene Gapdh (Figures 19D and 19H) were measured. All error bars represent standard error of the mean (SEM). One-way analysis of variance was used to determine statistical significance, with p<0.05*, p<0.01**, p<0.001***, and p<0.0001****. [Figure 19A-H-2] We demonstrate that S. aureus PSMα and protease are responsible for inducing damage and inflammation in mouse skin. S. aureus (SA) (1e7 CFU) wild-type (WT), PSMα knockout (Δpsmα), and protease-less (Δprotease) strains were inoculated onto the dorsal skin of male mice for 72 hours (n=6). Changes in trypsin activity (Figures 19A and 19E), Klk6 (Figures 19B and 19F), Il6 (Figures 19C and 19G), and IL17a / f mRNA expression normalized to the housekeeping gene Gapdh (Figures 19D and 19H) were measured. All error bars represent standard error of the mean (SEM). One-way analysis of variance was used to determine statistical significance, with p<0.05*, p<0.01**, p<0.001***, and p<0.0001****. [Figure 20A-C]CoNS strains do not affect SA growth. Coagulase-negative staphylococci (CoNS) supernatants affect the growth of SA agr type I P3-YFP reporter strains, as assessed by OD (n=3-4). This includes CoNS clinical isolates (Figure 20A), S. epidermidis (S. epi) agr types I-III (Figure 20B), and S. epidermidis (S. epi) wild-type (WT) or autoinducing peptide knockout (ΔAIP) supernatants added to SA agr type I reporter strains for 24 hours (Figure 20C). All error bars represent standard error of the mean (SEM). [Figure 21A-B] Figure 21A shows the activity of SA agr types I-III, but not type IV. S. hominis C5 supernatant was added to the SA agr types I-IV P3-YFP reporter strain for 24 hours (n=3). Figure 21A shows the activity of SA agr types I-IV in the SA reporter strain. Figure 21B shows the OD600nm growth measurements when cultured in the presence of S. hominis C5 supernatant. All error bars represent the standard error of the mean (SEM). One-way analysis of variance was used to determine statistical significance, with p<0.05*, p<0.01**, p<0.001***, and p<0.0001****. [Figure 22A-F]We demonstrate that S. hominis C5 supernatant inhibits SA-induced skin barrier damage. Staphylococcus aureus (SA) (1e7 CFU) was applied to the dorsal skin of female mice for 48 hours (n=3) with or without 10x concentrated <3 kDa S. hominis C5 supernatant. Figures 22A-B show representative images of the mouse back (dashed lines indicate the treatment area) and the CFU / cm2 of SA recovered from mouse skin after SA treatment. SA induced skin damage markers, including Il6, transepidermal water loss (TEWL), trypsin activity, and Klk6 mRNA expression, relative to the housekeeping gene Gapdh (Figures 22C-F). All error bars represent the standard error of the mean (SEM). One-way analysis of variance was used to determine statistical significance, with p<0.05*, p<0.01**, p<0.001***, and p<0.0001****. Detailed Description of the Invention

[0034] As used in this specification and the appended claims, the singular forms "a," "the," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a reagent" includes a plurality of such reagents, reference to "the microorganism" includes one or more microorganisms and equivalents thereof known to those skilled in the art, and so forth.

[0035] Also, unless expressly stated otherwise, "or" means "and / or." Similarly, "include," "including," "including" and "comprising" are interchangeable and are not intended to be limiting.

[0036] It should be further understood that when the term "comprising" is used in describing various embodiments, those skilled in the art will understand that in some specific instances, the embodiments may instead be described using the words "consisting essentially of" or "consisting of."

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Any methods and reagents similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions.

[0038] Atopic dermatitis (AD) is one of the most common immune disorders, impairing patients' quality of life, causing significant economic burden, and increasing the risk of serious complications. Defects in skin function are a key feature of AD. Eczematous skin lesions from AD patients exhibit elevated levels of Th2 cytokines, such as IL-4 and IL-13. Th2 cytokines promote skin dysfunction by inhibiting filaggrin expression. These cytokines also suppress the expression of human antimicrobial peptides, such as cathelicidin and b-defensin-2. Defects in AD may lead to dysbiosis of skin bacterial communities and enhanced colonization by Staphylococcus aureus. Targeting IL-4 receptor alpha has been shown to significantly improve disease outcomes. The strong associations between Th2 cytokine activity, barrier function, antimicrobial activity, and disease prognosis are helping to define the causal relationships with these important epidermal functions.

[0039] The skin barrier of patients with AD has been shown to exhibit increased kallikrein (KLK) expression, potentially impairing its proteolytic activity. KLKs are a family of 15 serine proteases, some of which are found primarily in the upper granular and stratum corneum layers of the epidermis. In Netherton syndrome, increased serine protease activity is observed due to decreased activity of the serine protease inhibitor Kazal type 5. The resulting increased enzyme activity leads to increased desquamation, altered processing of antimicrobial peptides and filaggrin (FLG), activation of protease-activating receptor 2, and inflammation. Increased protease activity may also play an important role in communication between the microbiome and the skin's immune system, and has recently been shown to directly influence epidermal cytokine production and inflammation by facilitating bacterial penetration through the epidermis.

[0040] Commensalization of the skin microbiome and skin colonization by Staphylococcus aureus are associated with the exacerbation of atopic dermatitis (AD). This study demonstrates that S. aureus has the ability to induce the expression of specific KLKs from keratinocytes and increase overall proteolytic activity in the skin. This suggests a previously unknown bacterial communication system with the host and a potentially important mechanism by which S. aureus colonization exacerbates disease severity in AD patients.

[0041] Staphylococcus aureus can secrete multiple proteases into the skin that alter skin integrity. The serine protease V8 and serine-like protease exfoliative toxins have been shown to cleave keratodesmosomal adhesive proteins, including DSG-1, leading to increased desquamation. The MMP aureolysin is known to cleave and inactivate LL-37, an important antimicrobial peptide on the skin. However, these direct proteolytic actions of S. aureus products require high levels of enzymes and bacteria, more consistent with events occurring during infection with this organism.

[0042] Increased protein digestion was observed after keratinocytes were activated by S. aureus. FLG is known to be cleaved from the larger Pro-FLG (400 kDa) to a monomeric form (37 kDa) that plays an important role in forming the physical barrier between keratin and the stratum corneum. It has been shown that enhanced Pro-FLG cleavage may be associated with increased skin desquamation (Hewett et al., 2005). Interestingly, increased Pro-FLG cleavage was observed in human keratinocytes treated with S. aureus supernatant. When KLK6 or KLK13 were silenced, Pro-FLG cleavage was partially blocked, indicating that S. aureus reduces skin integrity through a KLK-dependent process of Pro-FLG cleavage.

[0043] DSG-1 is an important corneodesmosomal adhesion protein, and its cleavage increases desquamation. Full-length DSG-1 (160 kDa) in keratinocytes is readily cleaved by KLK activity stimulated by Staphylococcus aureus. It has been reported that KLKs 5, 6, 7, and 14 can cleave DSG-1, but KLK13 cannot. This indicates that increased expression of KLK6 and KLK14 promotes full-length DSG-1 cleavage, providing evidence against the notion that KLK13 is not involved in DSG-1 cleavage. Thus, while S. aureus can alter FLG cleavage by KLKs, it also increases DSG-1 cleavage as another way to reduce epidermal skin integrity. Specific siRNA knockdown studies suggested that increased KLK expression is at least partially responsible for the increased serine protease activity stimulated by S. aureus. Figure 2C shows that proteases secreted by S. aureus contribute to the induction of increased trypsin activity in keratinocytes. Because bacteria, including S. aureus, can penetrate the skin surface and induce strong cutaneous immune responses (Nakatsuji et al., 2013, 2016; Zhang et al., 2015), these bacteria may also affect protease activity in skin cells. These observations are also relevant to rosacea syndrome or Netherton syndrome.

[0044] This disclosure demonstrates that soluble factors produced by Staphylococcus aureus have a potent and previously unsuspected ability to alter the activity of endogenous proteases produced by keratinocytes. This occurred in dilutions of S. aureus products in which bacterial protease activity was undetectable. Thus, S. aureus can stimulate the epidermis to increase the expression of endogenous proteolytic activity, thereby drastically altering the balance of overall epidermal proteolytic activity.

[0045] Different strains of Staphylococcus aureus (Newman, USA300, 113, and SANGER252) and Staphylococcus epidermidis (ATCC12228 and ATCC1457) had different effects on proteolytic enzyme activity in human keratinocytes. While S. aureus strains, including Newman and USA300, increased trypsin activity, other strains of S. aureus and S. epidermidis increased elastase or MMP activity. Thus, bacteria may alter the activity of epidermal proteases depending on both the bacterial species and strain. Other bacterial species and strains of S. aureus may have additional unique effects on the enzyme balance in human skin. Interestingly, preliminary data showed that purified Toll-like receptor ligands did not induce trypsin activity or KLK expression in keratinocytes.

[0046] In several skin diseases that result in skin barrier damage, protease activity is significantly increased. This is almost always associated with worsening disease states. In one aspect, the present disclosure demonstrates that probiotic microorganisms and their bacterial products are useful for preventing increased protease activity in the skin. In particular, the present disclosure demonstrates that coagulase-negative staphylococci can suppress Staphylococcus aureus-induced serine protease activity in the skin by inhibiting the AGR quorum-sensing system. Pathogenic strains of Staphylococcus aureus can induce serine protease activity in the skin. Increased protease activity leads to skin breakdown and exacerbation of conditions such as Netherton syndrome and atopic dermatitis. The present disclosure demonstrates that this increased serine protease activity can be suppressed by the use of probiotic or beneficial skin bacteria and factors derived therefrom.

[0047] This disclosure presents an unexpected response of keratinocytes to S. aureus: due to increased DSG-1 and FLG cleavage, S. aureus produces one or more factors that are KLK-dependent and reduce skin integrity.

[0048] This disclosure demonstrates that Staphylococcus aureus not only secretes proteolytic enzymes but also specifically activates keratinocytes to increase the expression of endogenous proteases. This disclosure demonstrates that phenol-soluble modulin alpha (PSMα) is secreted by Staphylococcus aureus and causes epidermal autolysis. For example, three members of the KLK family appear to play an important role in increasing the activity of the enzyme.

[0049] The present disclosure also identifies probiotic bacteria, genes, and polypeptides that inhibit the accessory gene regulator (agr) quorum-sensing system of Staphylococcus aureus, blocking PSMα and inhibiting protease activity. Thus, the present disclosure provides targets for modulating atopic dermatitis, as well as pharmaceutical and probiotic formulations for modulating atopic dermatitis and protease activity on the skin.

[0050] This disclosure demonstrates that coagulase-negative staphylococci (CoNS) species, such as S. epidermidis and S. hominis, which normally inhabit the skin, protect against this biological activity of S. aureus by producing an autoinducing peptide (AIP), which inhibits the accessory gene regulation (agr) quorum-sensing system of S. aureus and blocks PSMα secretion.

[0051] Virtually all S. aureus toxins are under the control of the virulence accessory gene regulator (AGR). The AGR system triggers changes in gene expression at specific cell densities through a process called quorum sensing. In addition to toxins, AGR is known to increase the expression of a wide variety of virulence determinants, such as exoenzymes (proteolytic, lipolytic, and nucleolytic enzymes), and decrease the expression of surface-binding proteins. This adaptation is thought to control the production of certain virulence determinants when needed (e.g., initial binding proteins at low cell densities, which are important for adhesion to host tissues, toxins and degradative exoenzymes when infection is established and nutrients must be obtained from host tissues).

[0052] Multiple clinical isolates of different CoNS species inhibited protease activation and prevented epithelial damage both in vitro and in vivo without altering the abundance of S. aureus (e.g., inhibiting the biological activity of protease / agr activity without altering S. aureus density). Furthermore, the present disclosure demonstrates that patients with active AD exhibit a decreased relative abundance of these beneficial microorganisms (e.g., CoNS) compared to S. aureus, thereby overcoming the inhibition of quorum sensing and enabling S. aureus subversion. Collectively, the present disclosure demonstrates how members of the skin microbiome in healthy individuals collectively maintain immune homeostasis by contributing to the control of S. aureus toxin production.

[0053] The present disclosure also identifies polynucleotide and polypeptide sequences and fragments thereof that provide products that inhibit the activity of agr quorum sensing. These polynucleotides and polypeptides can be used to provide therapeutic agents and recombinant non-pathogenic or attenuated skin bacteria for topical formulations to treat Staphylococcus aureus infections and / or atopic dermatitis.

[0054] For example, the present disclosure provides an autoinducing peptide (AIP) that reduces the activity of agr. Also provided herein are polynucleotides encoding AIP.

[0055] The present disclosure provides a link between increased Staphylococcus aureus flora and increased serine protease activity in AD skin, and also provides new targets and treatments. Examples include, but are not limited to, fermented extracts (e.g., fermented extracts from Staphylococcus aureus) that increase protease activity, or fermented extracts from commensal bacteria (e.g., containing one or more AIPs of the present disclosure) that decrease protease activity in the skin. Furthermore, the present disclosure provides (i) topical formulations containing such extracts or purified AIP peptides, and (ii) topical formulations containing commensal bacteria (e.g., non-pathogenic or attenuated bacteria transformed with AIP coding sequences, or purified commensal bacterial preparations in topical formulations). Additional therapeutic targets may be antibodies to KLKs and / or DSG-1 and / or FLG therapy (e.g., increasing the expression or delivery of these factors to AD subjects).

[0056] In certain embodiments, an AIP polypeptide of the disclosure has the consensus sequence X1X2X3X4CX5X6X7X8 (SEQ ID NO: 10), where X1 is S, K, V, G, or T; X2 is Y, Q, A, or I; X3 is N, S, T, or D; X4 is V, P, M, or T; X5 is G, S, A, N, or T; X6 is G, N, T, or L; X7 is Y or F; and X8 is F, L, or Y, where amino acids 5-9 of SEQ ID NO: 10 form a thiolactone ring. Exemplary peptide sequences corresponding to the consensus sequence of SEQ ID NO:10 include SYNVCGGYF (SEQ ID NO:4), KYNPCSNYL (SEQ ID NO:11), SYSPCATYF (SEQ ID NO:12), SQTVCSGYF (SEQ ID NO:13), GANPCALYY (SEQ ID NO:14), TINTCGGYF (SEQ ID NO:15), VQDMCNGYF (SEQ ID NO:16), and GYSPCTNFF (SEQ ID NO:17). In further embodiments, the polypeptide produces a structure of Formula I or IA. In another embodiment, the polypeptide can include a combination of D- or L-amino acids. In any of the foregoing embodiments, the polypeptide inhibits the activity of Staphylococcus aureus protease, agr activity, or keratinocyte protease activity.

[0057] The present disclosure provides compounds of formula I: JPEG0007720639000001.jpg155138

[0058] In some embodiments, the disclosure provides a compound of formula IA: JPEG0007720639000002.jpg202137

[0059] The present disclosure provides a purified polypeptide (e.g., an AIP peptide) comprising a sequence at least 98% identical to SEQ ID NO:4, and which (i) inhibits keratinocyte protease production and / or protease activity, (ii) inhibits keratinocyte IL-6 production and / or activity, (iii) inhibits phenol-soluble modulin alpha 3 production from S. aureus, and / or (iv) inhibits agr production and / or activity by S. aureus. In another embodiment, the disclosure provides a compound of formula IB: JPEG0007720639000003.jpg103138

[0060] In still further embodiments, the disclosure provides a purified polypeptide comprising or consisting of SEQ ID NO: 4, 11, 12, 13, 14, 15, 16, or 17. In further embodiments, the polypeptide produces a structure of Formula I, IA, or IB.

[0061] In certain embodiments, the AIP peptides of the present disclosure can include one or more D-amino acids.

[0062] The present disclosure provides topical formulations comprising an AIP peptide having the consensus sequence of SEQ ID NO: 10, or a peptide of SEQ ID NO: 4, 11, 12, 13, 14, 15, 16, or 17, or a compound of formula I, IA, or IB.

[0063] "Substantially identical" means that the amino acid sequences are largely, but not completely, identical, but maintain the functional activity of the related sequence. The percentage of identity shared by polypeptide or polynucleotide sequences is based on alignment of the sequences. It is common in the art to use various programs to perform alignments and measure identity. In general, two polypeptide or domain sequences are "substantially identical" if they are at least 85%, 90%, 95%, 98%, or 99% identical, or if there are conservative variations in the sequences. Computer programs such as the BLAST program (Altschul et al., 1990) can be used to compare sequence identity.

[0064] The present disclosure also provides polynucleotides encoding the AIP polypeptides of the present disclosure (i.e., "AIP polynucleotides"). For example, the present disclosure provides polynucleotides encoding SEQ ID NO:2 or 4. In certain embodiments, the polynucleotide hybridizes to a polynucleotide consisting of SEQ ID NO:3 under stringent conditions and encodes the polypeptide of SEQ ID NO:4. The "stringency" of a hybridization reaction can be readily determined by one of skill in the art and is generally an empirical calculation dependent on the length of the probe, the temperature of washing, and the salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes require lower temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of homology between the desired probe and the hybridizable sequence, the higher the relative temperature that can be used. Consequently, higher relative temperatures result in more stringent reaction conditions, while lower temperatures result in less stringent conditions. For further details and explanations regarding stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995). As defined herein, "stringent conditions" or "high stringency conditions" generally refer to the following: (1) low ionic strength and high temperature washes;For example, 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50°C; (2) for hybridization, a denaturing agent such as formamide is used (e.g., 50% (v / v) formamide containing 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer at pH 6.5 containing 750 mM sodium chloride, 75 mM sodium citrate at 42°C); or (3) 50% formamide, 5x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution, sonicated salmon sperm DNA (50 The PCR reaction was performed using a 50% 5'-diaminobenzyl 1,1 ...

[0065] The AIP polynucleotide can be cloned into a variety of vectors for use in the present disclosure. For example, the AIP polynucleotide can be cloned into an expression vector or plasmid for use in transformation and / or expression in a recombinant host cell. Vectors for bacterial transformation are known. Four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. Common to all engineered vectors are an origin of replication, a multiple cloning site, and a selectable marker. Any of these are suitable for use herein. The AIP polynucleotide can be inserted into a clone, vector, shuttle, plasmid, BAC, or integrated into the bacterial genome. When a plasmid is used, the copy number of the plasmid can be between 5 and 500 copies per cell. Exemplary plasmids and expression vectors include p252, p256, p353-2 (Leer et al., 1992), p8014-2, pA1, pACYC, pAJ01, pAl-derived (Vujcic & Topisirovic 1993), pall, pAM-beta-1,2,3,5,8 (Simon and Chopin 1988), pAR1411, pBG10, pBK, pBM02, pBR322, pBR328, pBS-slpGFP, pC194 (McKenzie et al. 1986, 1987; Horinouchi & Weisblum 1982b), PC194 / PUB110, pC30il, and pC30il (Skaugen et al., 1992). 1989), pCD034-1, pCD034-2, pCD256, pC12000, pC1305, pC1528, pCIS3, pCL2.1, pCT1138, pD125, pE194, pE194 / PLS1, pEGFP-C 1, pEH, pF8801, pFG2, pFK-series, pGK-series, pGK12, pGK13, pIA, pIAV1,5,6,7,9, pIL.CatT, pIL252 / 3, pIL253, pIL7, pISA (low for E.coli), pJW563, pKRV3, pLAB1000 (Josson et al. 1990), pLB4 (Bates & Gilbert 1989, pLBS, pLE16, pLEB124, pLEB590, pLEB591, pLEB600, pLEB604, pLEP24Mcop, pLJ1 (Takiguchi et al. 1989), pLKS, pLTK2, pWCFS101 and pMD5057 (Bates & Gilbert, 1989; Skaugen, 1989; Leer et al., 1992; Vujcic & Topisirovic, 1993; Eguchi et al., 2000; Kaneko et al., 2000; Danielsen, 2002; Daming et al., 2003; de las Rivas et al., 2004; Kranenburg et al. 2005), pLP1 / 18 / 30, pLP18, pLP317, pLP317cop, pLP3537, pLP3537xyl, pLP402, pLP825, pLP825 and pLPE323, pLP82H, pLPC37, pLPE23M, pLPE323, pLPE350, pLPI (Bouia et al. 1989), pLS1, pLS1 and pE194 (Lacks et al. 1986; Horinouchi & Weisblum 1982a), plu631, and L. harboring the erythromycin resistance gene.pLUL631, pM3, pM4, pMD5057, pMG36e, pND324, pNZ-series, pPSC series, pSH71 (de vos, 1987), pSIP-series, pSK11L, pSL2, PSN2, pSN2 (Khan & Novick 1982), pT181 (Koepsel et al. 1987), (Khan & Novick 1983), pT181 in B. reuteri; pC194 and pE194 are not functional in B. subtilis (Gruss et al. 1987), pT181, pE194 / pLS1, pC194 / pUB110 and pSN2 (Khan, 2005), pTL, pTRK family, pTRT family, pTUAT35, pUBII0 and pC194. (McKenzie et al. 1986, 1987; Horinouchi & Weisblum 1982b), pUCL22, pULP8 / 9, pVS40, pWC1, pWCFS101, pWV02, pWV04, pWV05, RepA, and the BetL system.

[0066] In certain embodiments, the present disclosure provides topical compositions comprising the AIP polypeptide or peptide of the present disclosure. For example, in certain embodiments, the topical composition comprises a purified polypeptide (e.g., an AIP peptide) that comprises the consensus sequence of SEQ ID NO: 10 or a sequence at least 98% identical to any of SEQ ID NOs: 4, 11, 12, 13, 14, 15, 16, or 17, and that (i) inhibits keratinocyte protease production and / or protease activity, (ii) inhibits keratinocyte IL-6 production and / or activity, (iii) inhibits phenol-soluble modulin α3 production from S. aureus, and / or (iv) inhibits agr production and / or activity by S. aureus. In another embodiment, the topical composition comprises a compound of Formula I, IA, or IB (as defined above).

[0067] In another embodiment, the topical composition can include a non-pathogenic microorganism (including an attenuated microorganism engineered to reduce or eliminate pathogenic activity), wherein the microorganism has been engineered to express an AIP polypeptide. The microorganism may also be engineered to contain a vector and / or an AIP polynucleotide. In some embodiments, the microorganism produces a compound of Formula I, IA, and / or IB.

[0068] In certain embodiments, the compositions and methods herein employ non-pathogenic bacteria engineered to produce compounds of Formula I, IA, and / or IB by transforming the bacteria with an AIP polynucleotide of the present disclosure. In certain embodiments, the bacterial population is a non-pathogenic, non-invasive microorganism, which in certain embodiments may be a Gram-positive, food-grade bacterial strain. In another embodiment, the transformed bacterial population is prepared from bacteria naturally present in the skin microbiome.

[0069] In some embodiments, the bacteria forming the bacterial population in the composition and transformed to express the compounds of Formula I, IA, and / or IB can be a mixture of the same bacteria or different bacteria at different phylogenetic levels. Bacteria native to the skin of healthy subjects typically include bacterial species native to the human face, such as Actinomycetes (including bacteria of the genera Corynebacterium and Propionibacterium). In another embodiment, bacteria native to the skin of healthy subjects typically include bacterial species native to skin other than the face, including, for example, bacteria of the genera Bacteroides and Proteobacteria. Other bacteria in the skin microbiome include those listed herein below.

[0070] In certain embodiments, the bacterium is from the genus Propionibacterium. Examples include Propionibacterium acidifaciens, Propionibacterium acidipropionici, Propionibacterium acidipropionici strain 4900, Propionibacterium acnes, Propionibacterium australiense, Propionibacterium avidum, Propionibacterium cyclohexanicum, Propionibacterium freudenreichii subsp, Freudenreichii, P. freudenreichii ssp. freudenreichii strain 20271, Propionibacterium freudenreichii subsp. Shermanii, P. freudenreichii ssp. shermanii strain 4902, P. freudenreichii ssp. innocuum, P. Examples of suitable bacteria include, but are not limited to, Propionibacterium jensenii 20278, Propionibacterium lymphophilum, Propionibacterium microaerophilum, Propionibacterium propionicum, Propionibacterium thoenii, and P. thoenii strain 20277. In some embodiments, the bacterium is not Propionibacterium acnes. In some embodiments, the bacterium is from the genus Corynebacterium, including, but not limited to, C. accolens, C. afermentan, C. amycolatum, C. argentoratense, C. aquaticum, C. auris, C. bovis, C. diphtheria, C. equi (now Rhodococcus equi), C. flavescens, C. glucuronolyticum, C. glutamicum, C. granulosum, C.Examples of suitable bacteria include, but are not limited to, C. haemolyticum, C. halofytica, C. jeikeium (group JK), C. macginleyi, C. matruchotii, C. minutissimum, C. parvum (Propionibacterium acnes), C. propinquum, C. pseudodiphtheriticum (C. hofmannii), C. pseudotuberculosis, (C. ovis), C. pyogenes, C. urealyticum (group D2), C. renale, C. spec, C. striatum, C. tenuis, C. ulcerans, C. urealyticum, and C. xerosis. Bacteria with lipophilic and non-lipophilic groups are contemplated, and non-lipophilic bacteria may include fermentative and non-fermentative corynebacteria. In some embodiments, the bacterium is not C. diphtheria, C. amicolatum, C. striatum, C. jeikeium, C. urealyticum, C. xerosis, C. pseudotuberculosis, C. tenuis, C. striatum, or C. minutissimum, although these may be pathogenic. In some embodiments, the bacterium is from the suborder Micrococcineae, including Arthrobacter arilaitensis, Arthrobacter bergerei, Arthrobacter globiformis, Arthrobacter nicotianae, Kocuria rhizophila, Kocuria varians, Micrococcus luteus, Micrococcus lylae, Microbacterium gubbeenense, Brevibacterium aurantiacum, Brevibacterium casei, Brevibacterium linens, Brachybacterium alimentarium, and BrachybacteriumIn another embodiment, the bacterium is from the genus Staphylococcus. Examples include Staphylococcus agnetis, S. arlettae, S. auricularis, S. capitis, S. caprae, S. carnosus, Staphylococcus caseolyticus, S. chromogenes, S. cohnii, S. condiment, S. delphini, S. devriesei, S. equorum, S. felis, S. fleurettii, S. gallinarum, S. haemolyticus, S. hominis, S. hyicus, S. intermedius, S. kloosii, S. leei, S. lentus, S. lugdunensis, S. lutrae, S. massiliensis, S. microti, S. muscae, S. nepalensis, S. pasteuri, S. pettenkofer, S. piscifermentans, S. pseudointermedius, S. Examples of suitable bacterium include, but are not limited to, S. pseudolugdunensis, S. pulvereri, S. rostra, S. saccharolyticus, S. saprophyticus, S. schleiferi, S. sciuri, S. simiae, S. simulans, S. stepanovicii, S. succinus, S. vitulinus, S. warneri, and S. xylosus. In some embodiments, the bacterium is not Staphylococcus aureus or Staphylococcus epidermidis. In other embodiments, the bacterium is from the genus Streptococcus. Examples include Streptococcus acidominimus, Streptococcus adjacens, Streptococcus agalactiae, Streptococcus alactolyticus, Streptococcus anginosus, Streptococcus australis, Streptococcus bovis, and Streptococcuscaballi, Streptococcus canis, Streptococcus caprinus, Streptococcus castoreus, Streptococcus cecorum, Streptococcus constellatus, Streptococcus constellatus subsp. Constellatus, Streptococcus constellatus subsp. Pharyngis, Streptococcus cremoris, Streptococcus criceti, Streptococcus cristatus, Streptococcus danieliae, Streptococcus defectives, Streptococcus dentapri, Streptococcus dentirousetti, Streptococcus didelphis, Streptococcus difficilis, Streptococcus durans, Streptococcus dysgalactiae, Streptococcus dysgalactiae subsp. Dysgalactiae, Streptococcus dysgalactiae subsp. Equisimilis, Streptococcus entericus, Streptococcus equi, Streptococcus equi subsp. Equi, Streptococcus equi subsp. Ruminatorum, Streptococcus equi subsp. Zooepidemicus, Streptococcus equines, Streptococcus faecalis, Streptococcus faecium, Streptococcus ferus, Streptococcus gallinaceus, Streptococcus gallolyticus, Streptococcus gallolyticus subsp. Gallolyticus, Streptococcus gallolyticus subsp. Macedonicus, Streptococcus gallolyticus subsp.Pasteurianus, Streptococcus garvieae, Streptococcus gordonii, Streptococcus halichoeri, Streptococcus hansenii, Streptococcus henryi, Streptococcus hyointestinalis, Streptococcus hyovaginalis, Streptococcus ictaluri, Streptococcus infantarius, Streptococcus infantarius subsp. Coli, Streptococcus infantarius subsp. Infantarius, Streptococcus infantis, Streptococcus iniae, Streptococcus intermedius, Streptococcus intestinalis, Streptococcus lactarius, Streptococcus lactis, Streptococcus lactis subsp. Cremoris, Streptococcus lactis subsp. Diacetilactis, Streptococcus lactis subsp. Lactis, Streptococcus lutetiensis, Streptococcus macacae, Streptococcus macedonicus, Streptococcus marimammalium, Streptococcus massiliensis, Streptococcus merionis, Streptococcus minor, Streptococcus mitis, Streptococcus morbillorum, Streptococcus mutans, Streptococcus oligofermentans, Streptococcus oralis, Streptococcus orisratti, Streptococcus ovis, Streptococcus parasanguinis, Streptococcus parauberis, Streptococcusparvulus, Streptococcus pasteurianus, Streptococcus peroris, Streptococcus phocae, Streptococcus plantarum, Streptococcus pleomorphus, Streptococcus pluranimalium, Streptococcus plurextorum, Streptococcus pneumonia, Streptococcus porci, Streptococcus porcinus, Streptococcus porcorum, Streptococcus pseudopneumoniae, Streptococcus pseudoporcinus, Streptococcus pyogenes, Streptococcus raffinolactis, Streptococcus ratti, Streptococcus rupicaprae, Streptococcus saccharolyticus, Streptococcus salivarius, Streptococcus salivarius subsp. Salivarius, Streptococcus salivarius subsp. Thermophilus, Streptococcus sanguinis, Streptococcus shiloi, Streptococcus sinensis, Streptococcus sobrinus, Streptococcus suis, Streptococcus thermophilus, Streptococcus thoraltensis, Streptococcus tigurinus, Streptococcus troglodytae, Streptococcus troglodytidis, Streptococcus uberis, Streptococcus urinalis, Streptococcus vestibularis, and Streptococcus waius. In another embodiment, the bacteria are from the genus Lactobacillus. For example, Lactococcusgarvieae, Lactococcus lactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. hordniae, Lactococcus lactis, Lactococcus lactis subsp. lactis, Lactococcus piscium, Lactococcus plantarum, Lactococcus raffinolactis, Lactobacillus acetotolerans, Lactobacillus acidophilus, Lactobacillus agilis, Lactobacillus algidus, Lactobacillus alimentarius, Lactobacillus amylolyticus, Lactobacillus amylophilus, Lactobacillus amylovorus, Lactobacillus animalis, Lactobacillus aviarius, Lactobacillus aviarius subsp. arafinfosus, Lactobacillus aviarius subsp. aviarius, Lactobacillus bavaricus, Lactobacillus bifermentans, Lactobacillus brevis, Lactobacillus buchneri, actobacillus bulgaricus, Lactobacillus carnis, Lactobacillus casei, Lactobacillus casei subsp. alactosus, Lactobacillus casei subsp. casei, Lactobacillus casei subsp. pseudoplantarum, Lactobacillus casei subsp. rhamnosus、Lactobacillus casei subsp. tolerans、Lactobacillus catenaformis、Lactobacillus cellobiosus、Lactobacillus collinoides、Lactobacillusconfusus, Lactobacillus coryniformis, Lactobacillus coryniformis subsp. coryniformis, Lactobacillus coryniformis subsp. torquens, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus curvatus subsp. curvatus, Lactobacillus curvatus subsp. melibiosus, Lactobacillus delbrueckii, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. delbrueckii, Lactobacillus delbrueckii subsp. lactis, Lactobacillus divergens, Lactobacillus farciminis, Lactobacillus fermentum, Lactobacillus formicalis, Lactobacillus fructivorans, Lactobacillus fructosus, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus graminis, Lactobacillus halotolerans, Lactobacillus hamsteri, Lactobacillus helveticus, Lactobacillus heterohiochii, Lactobacillus hilgardii, Lactobacillus homohiochii, Lactobacillus iners, Lactobacillus intestinalis, Lactobacillus jensenii, Lactobacillus johnsonii, Lactobacillus kandleri, Lactobacillus kefiri, Lactobacillus kefuranofaciens, Lactobacillus kefirgranum, Lactobacilluskunkeei, Lactobacillus lactis, Lactobacillus leichmannii, Lactobacillus lindneri, Lactobacillus malefermentans, Lactobacillus mali, Lactobacillus maltaromicus, Lactobacillus manihotivorans, Lactobacillus minor, Lactobacillus minutus, Lactobacillus mucosae, Lactobacillus murinus, Lactobacillus nagelii, Lactobacillus oris, Lactobacillus panis, Lactobacillus parabuchneri, Lactobacillus paracasei, Lactobacillus paracasei subsp. paracasei, Lactobacillus paracasei subsp. tolerans, Lactobacillus parakefiri, Lactobacillus paraalimentarius, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus perolens, Lactobacillus piscicola, Lactobacillus plantarum, Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus rhamnosus strain 5 / E5a, Lactobacillus rimae, Lactobacillus rogosae, Lactobacillus ruminis, Lactobacillus sakei, Lactobacillus sakei subsp. camosus, Lactobacillus sakei subsp. sakei, Lactobacillus salivarius, Lactobacillus salivarius subsp. salicinius, Lactobacillus salivarius subsp.In another embodiment, the bacterium is from the genus Lactococcus. Examples of suitable Lactococcus species include, but are not limited to, Lactobacillus salivarius, Lactobacillus sanfranciscensis, Lactobacillus sharpeae, Lactobacillus suebicus, Lactobacillus trichodes, Lactobacillus uli, Lactobacillus vaccinostercus, Lactobacillus vaginalis, Lactobacillus viridescens, Lactobacillus vitulinus, Lactobacillus xylosus, Lactobacillus yamanashiensis, Lactobacillus yamanashiensis subsp. mali, Lactobacillus yamanashiensis subsp. Yamanashiensis, and Lactobacillus zeae. In another embodiment, the bacterium is from the genus Lactococcus. Examples of suitable Lactococcus species include, but are not limited to, Lactococcus salivarius, Lactobacillus sanfranciscensis, Lactobacillus sharpeae, Lactobacillus suebicus, Lactobacillus trichodes, Lactobacillus uli, Lactobacillus vaccinostercus, Lactobacillus vaginalis, Lactobacillus viridescens, Lactobacillus vitulinus, Lactobacillus xylosus, Lactobacillus yamanashiensis, Lactobacillus yamanashiensis subsp. mali, Lactobacillus yamanashiensis subsp. Yamanashiensis, and Lactobacillus zeae. cus Schleifer, Lactococcus chungangensis, Lactococcus fujiensis, Lactococcus garvieae, Lactococcus lactis, Lactococcus lactis subsp. Cremoris, Lactococcus lactis subsp. Hordniae, Lactococcus lactis subsp. Lactis, Lactococcus lactis subsp. Tructae, Lactococcus piscium, Lactococcus plantarum and Lactococcus raffinolacti.

[0071] In yet another embodiment, the present disclosure provides a probiotic composition for topical delivery comprising the CoNS probiotic skin bacteria of the present disclosure. In some embodiments, the CoNS bacteria comprise bacteria that produce an AIP polypeptide and / or a compound of Formula I. In further embodiments, the topical composition contains only a single species of microorganism that produces an AIP polypeptide or a compound of Formula I. In yet another embodiment, the probiotic skin bacteria of the present disclosure comprise a microorganism selected from the group consisting of S. epidermidis A11, S. hominis A9, S. hominis C4, S. hominis C5, and S. warneri G2. In yet another embodiment, the topical probiotic composition of the present disclosure can comprise or consist of a probiotic skin bacteria selected from the group consisting of S. epidermidis A11, S. hominis A9, S. hominis C4, S. hominis C5, S. warneri G2, and any combination thereof.

[0072] The commensal bacteria of the present disclosure can be isolated from human skin and identified using the methods described herein. For example, the present disclosure provides methods for collecting, identifying, and culturing the commensal bacteria described herein by swabbing a human skin surface with a foam-tip swab or similar. The swabs were placed in tryptic soy broth. The broth was diluted onto mannitol salt agar (MSA) plates supplemented with 3% egg yolk. Pink colonies representing halo-free coagulase-negative Staphylococcus aureus (CoNS) strains were picked and grown in tryptic soy broth (TSB), and 25% of the volume of sterile-filtered supernatant was added to a S. aureus agrI-type YFP reporter strain grown in fresh TSB. (For measuring inhibition of S. aureus agr activity, a 24-hour incubation was performed.) The Agr activity of the S. aureus reporter strain was measured using a fluorometer. Strains that strongly inhibited S. aureus agr activity were further characterized by gDNA isolation and sequencing. Isolate gDNA using any commercially available kit (e.g., DNeasy UltraClean Microbial Kit, Qiagen). Sequence gDNA using various sequencing platforms (e.g., MiSeq; Illumin Inc., San Diego, CA) for two cycles, generating two 250-bp paired-end reads. Remove adapters using cutadapt (see, for example, the worldwide web at cutadapt.readthedocs.io / en / stable / ). Remove low-quality sequences using Trim Galore with default parameters (see, for example, www / bioinformatics.babraham.ac.uk / projects / trim_galore / ). Remove sequences mapping to the human genome from the quality-trimmed dataset using the Bowtie2 program (Ver. 2.28) (I) with the following parameters: -D 20 -R 3 -N 1 -L 20 -very-sensitive-local, and the human reference genome hg19. De novo assemble the filtered reads using SP-des (Ver. 3.8.0) with k-mer lengths ranging from 33 to 127.Annotate the genome using the subsystem technology (RASY) of the initial setting parameters. Align the amino acid sequences of the annotated CDS (coding DNA sequences) with the agr proteins of bacteria obtained from the Uniprot database. Identify the agr genes from the assembled genome based on three criteria: (i) sequence identity > 60%, (ii) e-value < e100, and (iii) agr locus organization, an operon of four genes, agrBDCA. Microorganisms having a sequence that is at least 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 1 or 3 are useful in the methods and compositions of the present disclosure.

[0073] As used herein, the terms "live bacteria composition", "topical live bacteria composition", or "live bacteria skin composition" include live commensal skin bacteria, fermentation extracts of live commensal skin bacteria, attenuated or modified microorganisms expressing AIP polypeptide and (i) reagents that inhibit the activity of proteolytic enzymes, or (ii) reagents that promote the activity of proteolytic enzymes, and a pharmaceutical carrier composition that maintains the survival of the commensal skin bacteria.

[0074] As used herein, the term "topical" can include topical administration to the skin and shallow injections (e.g., intradermal and intralesional) such that the topical live bacteria composition comes into direct contact with the skin.

[0075] As used herein, the term "fermentation extract" means the product when live commensal skin bacteria are fermented in a medium under appropriate fermentation conditions. For example, when Staphylococcus aureus is cultured, PSMα3, which helps to enhance skin permeability, is produced. The extract from Staphylococcus aureus contains PSMα3 that can be applied to the skin to improve permeability, induce skin remodeling, or promote skin permeability for drug delivery. Similarly, the fermentation extract of CoNS bacteria that produces the AIP of the present disclosure can be cultured, and the extract from the culture can be used to inhibit Staphylococcus aureus-related conditions (e.g., proteolytic enzyme activity, dermatitis, etc.).

[0076] As used herein, the term "probiotic skin bacteria" includes microorganisms of the skin microbiome. The probiotic skin bacteria can include a composition of bacteria that promotes protease activity ("probiotic skin bacteria-promoting protease"). The probiotic skin bacteria-promoting protease is typically a skin bacterium that produces phenol-soluble module α3 (PSMα3). The protease-promoting probiotic skin bacteria composition (or a fermentation extract thereof) is useful, for example, for skin remodeling, promoting wound repair, aging, sun damage, pigmentation disorders, and scarring. In some embodiments, the probiotic skin bacteria-promoting protease includes one or more bacteria that have serine protease activity and / or induce skin serine protease activity. For example, the probiotic skin bacteria-promoting protease can include a Staphylococcus aureus strain that produces phenol-soluble module α3 (PSMα3).

[0077] In another embodiment, the protease-inhibiting probiotic skin bacteria can comprise a composition of bacteria that inhibits the activity of proteases ("protease-inhibiting probiotic skin bacteria"). Protease-inhibiting probiotic skin bacteria compositions are useful for treating conditions such as rosacea, atopic dermatitis, and Netherton syndrome. In certain embodiments, the protease-inhibiting probiotic skin bacteria can comprise one or more bacteria that inhibit the serine protease activity of other skin bacteria and / or that inhibit the serine protease activity of the skin. For example, the protease-inhibiting probiotic skin bacteria can comprise a coagulase-negative Staphylococcus species. In some embodiments, the coagulase-negative strain is selected from the group consisting of Staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus saccharolyticus, Staphylococcus warneri, Staphylococcus pasteuri, Staphylococcus haemolyticus, Staphylococcus devriesei, Staphylococcus hominis, Staphylococcus jettensis, Staphylococcus petrasii, and Staphylococcus lugdunensis. In some embodiments, the protease-inhibiting probiotic skin bacteria is selected from the group consisting of S. epidermidis, S. hominis, S. warneri, and any combination thereof. In certain embodiments, the S. epidermidis strain is S. epidermidis 14990 and / or S. epidermidis A11. In another embodiment, the S. hominis strain is S. hominis C4, S. hominis C5, and / or S. hominis A9. In yet another embodiment, the S. warneri strain is S. warneri G2. In some embodiments, the CoNS bacteria comprise bacteria that produce an AIP polypeptide and / or a compound of Formula I.In further embodiments, the topical composition comprises only a single species of microorganism that produces an AIP polypeptide or a compound of Formula I. In yet another embodiment, the probiotic skin bacteria of the present disclosure comprise a microorganism selected from the group consisting of S. epidermidis A11, S. hominis A9, S. hominis C4, S. hominis C5, and S. warneri G2. In yet another embodiment, the topical probiotic composition of the present disclosure can comprise or consist of a probiotic skin bacteria selected from the group consisting of S. epidermidis A11, S. hominis A9, S. hominis C4, S. hominis C5, S. warneri G2, and any combination thereof.

[0078] The term "contacting" refers to exposing the skin to a topical probiotic composition so that the probiotic composition for skin can modulate the activity of proteases (e.g., serine proteases) on the skin.

[0079] The term "inhibition" or "effective inhibiting amount" refers to the amount of a probiotic skin composition comprising one or more probiotic microorganisms, fermentation media or extracts, fermentation by-products, and / or synthetic molecules sufficient to cause inhibition of protease activity (e.g., serine protease activity) on the skin or in culture on the skin. The term "inhibition" also includes preventing or ameliorating signs or symptoms of a disorder (e.g., rash, pain, etc.).

[0080] The term "effective therapeutic amount" used herein for treating a disease or disorder in a subject refers to an amount of a probiotic composition for skin or an extract thereof sufficient to improve the signs or symptoms of the disease or disorder. For example, an effective therapeutic amount can be measured as an amount sufficient to reduce the symptoms of dermatitis or a rash in a subject, as measured by the frequency and severity of skin sores. Typically, a subject is treated with an amount that reduces the symptoms of the disease or disorder by at least 50%, 90%, or 100%. Generally, the optimal dosage depends on factors such as the disorder and the subject's weight, the type of bacteria, the subject's gender, and the severity of the symptoms. Nevertheless, an appropriate dose may be easily determined by one skilled in the art.

[0081] As used herein, the terms "purified" and "substantially purified" refer to a culture or co-culture of microorganisms or biological agents (e.g., fermentation media and extracts, fractionated fermentation media, fermentation by-products, AIP peptides, polypeptides, genes, polynucleotides, compounds of Formula I, etc.) that is substantially free of other cells or components present in the natural environment in which the biological agent is naturally associated when produced in vivo. In some embodiments, the co-culture can include a plurality of probiotic skin bacteria.

[0082] The present disclosure provides whole cell preparations containing substantially homogeneous preparations of S. epidermidis, S. hominis, and / or S. warneri. Such preparations can be used to prepare compositions for treating inflammation and microbial infections. The whole cell preparations can contain S. epidermidis, S. hominis, and / or S. warneri, or can contain non-pathogenic (e.g., attenuated microbial) vectors described below. The present disclosure also provides fractions derived from the whole cells containing pathogens that reduce proteolytic enzyme activity in the skin due to Staphylococcus aureus activity.

[0083] The ability of a first bacterial composition to inhibit the protease activity of a second bacterial composition can be measured by measuring the protease activity of the second bacterial composition before and after contacting the second composition with the first composition. Contacting an organism with a topical live bacterial composition of the present disclosure can occur in vitro, for example, by adding the topical live bacterial composition to a bacterial culture and measuring the bacterial protease inhibitory activity. Contacting can also occur in vivo, for example, by contacting a topical live bacterial composition with a subject suffering from a skin disease or disorder.

[0084] Formulations of probiotic skin bacteria can be prepared in numerous ways. Topical probiotic compositions can be administered to a subject using any of a variety of methods known in the art. For example, the probiotic skin compositions or extracts or synthetic formulations of the present disclosure may be formulated for topical administration (e.g., as lotions, creams, sprays, gels, or ointments). Such topical formulations are useful for treating or suppressing microbial, fungal, or viral presence, infection, or skin inflammation. Exemplary formulations include topical lotions, creams, soaps, wipes, and the like.

[0085] In yet another embodiment, a topical probiotic composition containing a plurality of probiotic probiotic skin bacteria is provided. When used to treat dermatitis or other skin diseases or disorders associated with increased protease (e.g., serine protease) activity, the composition contains one or more bacteria that inhibit protease activity on the skin. In such cases, the probiotic probiotic skin bacteria are coagulase-negative Staphylococcus species. In some embodiments, the probiotic probiotic skin bacteria are selected from the group consisting of S. epidermidis, S. hominis, S. warneri, and any combination thereof. When increased protease activity is desired (e.g., wound healing, skin reconstruction, etc.), the probiotic probiotic bacterial composition contains bacteria that have increased protease activity or that stimulate skin protease activity (e.g., serine protease activity). In this embodiment, an exemplary probiotic bacterial composition contains Staphylococcus aureus or a virulence-attenuated Staphylococcus aureus that produces PSMα3.

[0086] In another embodiment, the topical probiotic composition comprises a fermentation extract of probiotic skin bacteria that promotes the activity of proteolytic enzymes on the skin. In various aspects, the bacteria from which the extract is produced comprises Staphylococcus aureus.

[0087] In yet another embodiment, there is provided a probiotic composition for topical use consisting essentially of a fermentation extract of Staphylococcus aureus, or in combination with Staphylococcus aureus. According to a further aspect, the probiotic composition for topical use can be formulated into a lotion, shake, cream, ointment, gel, foam, powder, solid, paste, or tincture.

[0088] In another embodiment, the topical probiotic composition comprises a fermentation extract of probiotic, commensal skin bacteria. In various aspects, the bacteria from which the extract is produced comprise a coagulase-negative Staphylococcus species. In some embodiments, the Staphylococcus species is selected from the group consisting of S. epidermidis, S. hominis, S. warneri, and any combination thereof, which produce an AIP that inhibits the agr quorum sensing system and / or the production of proteolytic enzymes in the skin or the skin microbiome. In some embodiments, the AIP comprises the consensus sequence of SEQ ID NO: 10, or a sequence at least 98% identical to SEQ ID NOs: 4, 11, 12, 13, 14, 15, 16, or 17, which has agr quorum sensing-modulating activity, and / or a compound of Formula I, IA, or IB.

[0089] In yet another embodiment, there is provided a probiotic composition for topical use consisting essentially of an extract or fermentation extract of coagulase-negative Staphylococcus species, or an extract of S. epidermidis fermentation alone or in combination with coagulase-negative Staphylococcus species, or S. epidermidis. In another embodiment, the composition comprises one or more of the deposited microbial strains described herein (e.g., S. epidermidis A11, S. hominis A9, S. hominis C5, and / or S. warneri G2).

[0090] According to further embodiments, the topical probiotic composition can be formulated into a lotion, shake lotion, cream, ointment, gel, foam, powder, solid, paste, or tincture.

[0091] In another embodiment, a fermented extract is provided by fermenting a bacterium selected from the group consisting of S. epidermidis, S. hominis, S. warneri, and any combination thereof under fermentation conditions. In various aspects, such a fermented extract can be used to inhibit the activity of serine proteases on the skin. In another embodiment, the fermented extract is obtained from any one or more of the deposited microbial strains described herein (e.g., S. epidermidis A11, S. hominis A9, S. hominis C5, and / or S. warneri G2). According to further embodiments, the fermented extract can be formulated into a lotion, shake, cream, ointment, gel, foam, powder, solid, paste, or tincture.

[0092] In another embodiment, a bandage or dressing is provided that comprises the topical probiotic composition described above, a fermentation extract of the probiotic probiotic skin bacteria described above, the probiotic probiotic skin bacteria described above, and any combination thereof. In various aspects, a bandage or dressing is provided whose main component comprises a matrix and a probiotic probiotic skin bacteria that inhibits the activity of protease enzymes on the skin. In various aspects, a bandage or dressing is provided whose main component comprises a matrix and a fermentation extract of the probiotic probiotic skin bacteria that inhibits the activity of protease enzymes on the skin.

[0093] In another embodiment, a bandage or dressing is provided that includes the topical probiotic composition described above, a fermentation extract of the probiotic probiotic skin bacteria described above, the probiotic probiotic skin bacteria described above, or any combination thereof. In various aspects, a bandage or dressing is provided whose main components include a matrix and a fermentation extract of the probiotic probiotic skin bacteria that promotes the activity of proteolytic enzymes on the skin. In various aspects, a bandage or dressing is provided whose main components include a matrix and a fermentation extract of the probiotic probiotic skin bacteria that promotes the activity of proteolytic enzymes on the skin.

[0094] The present disclosure also provides methods for treating skin diseases or disorders associated with protease activity (e.g., serine protease activity). Examples of such diseases or disorders include Netherton syndrome, atopic dermatitis, contact dermatitis, eczema, psoriasis, acne, epidermal hyperkeratosis, epidermitis, epidermal inflammation, dermal inflammation, and pruritus. In one embodiment, the presence of a disease or disorder is first determined by measuring protease activity in a sample (e.g., skin or a bacterial culture from the skin) from a subject suspected of having the disease or disorder. If the sample exhibits higher than normal protease activity (e.g., serine protease activity), the subject is treated with a protease-inhibiting probiotic bacterial preparation by contacting the skin with the preparation. In another embodiment, a culture from a subject harboring bacteria with elevated protease activity is contacted in vitro with the preparation, and the susceptibility of the culture to the preparation and its inhibitory effect on the protease are measured.

[0095] Protease-inhibiting probiotic bacterial preparations or fermentation extracts can be combined with one or more known serine protease inhibitors. Many commercially available and clinical serine protease inhibitors are available for use in the methods and compositions of the present disclosure. For example, serine protease inhibitors are disclosed in U.S. Patent Nos. 5,786,328, 5,770,568, or 5,464,820, the disclosures of which are incorporated herein by reference. Exemplary serine protease inhibitors include antibodies that bind to and inhibit serine protease polypeptides or functional fragments thereof, enzymes that degrade serine protease polypeptides into inactive peptides, and substrate analogs. Serine protease expression inhibitors include, for example, antisense molecules, ribozymes, and small molecule drugs (e.g., vitamin D antagonists) that decrease the transcription or translation of serine protease polynucleotides (e.g., DNA or RNA). One embodiment of the present disclosure relates to substrate analogs of tissue kallikrein. These substrate analogs include peptides having amino acid sequences corresponding to positions 388 to 390 of tissue kallikrein. The peptides may be synthesized by recombinant engineering techniques, such as cloning and expression of nucleic acid sequences, or may be genetically produced, or purified from natural sources, such as bacterial, fungal, or cell extracts. The structural, chemical, physicochemical, nomenclature, and analytical aspects of amino acids are described in "Amino Acid Chemistry" (J.P. Greenstein and M. Winitz, editors, John Wiley & Sons, New York, NY, 1961, reprinted 1984), specifically incorporated herein by reference. The peptides may contain modified and / or unmodified amino acids, including naturally occurring amino acids, non-naturally occurring (non-coding) amino acids, synthetic amino acids, and combinations thereof.Such naturally occurring amino acids include glycine (Gly); amino acids with alkyl side chains such as alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), and proline (Pro); aromatic amino acids such as phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp); amino acid alcohols such as serine (Ser) and threonine (Thr); acidic amino acids such as aspartic acid (Asp) and glutamic acid (Glu); sulfur-containing amino acids such as asparagine (Asn), which is an amide of Asp and Glu, glutamine (Gln), cysteine (Cys), and methionine (Met); and basic amino acids such as histidine (His), lysine (Lys), and arginine (Arg). Unnatural amino acids include, for example, ornithine (Orn), norleucine (Nle), citraline (Cit), homocitraline (hCit), desmosine (Des), and isodesmosine (Ide). Modified amino acids include derivatives and analogs of natural and unnatural amino acids, as well as synthetic amino acids. Such amino acid forms are chemically modified, for example, by halogenation of one or more active sites with chlorine (Cl), bromine (Br), fluorine (F), or iodine (I), or by alkylation with carbon-containing groups [e.g., methyl (Me), ethyl (Et), butyl (Bu), amino (NH or NH), amidino (Am), acetomidomethyl (Acm), or phenyl (Ph) groups, etc.], or by the addition of phosphorus (P), nitrogen (N), oxygen (O), or sulfur (S)-containing groups. For example, modifications may be made to another amino acid or peptide, or precursor chemicals, by hydration, oxidation, hydrogenation, esterification, or cyclization. Examples include amino acid hydroxamates and decarboxylases, dansyl amino acids, polyamino acids and amino acid derivatives.Specific examples include gamma-aminobutyric acid (GABA), hydroxyproline (Hyp), aminoadipic acid modified at position 2 or 3 (Aad), o-aminobutyric acid (Aab or Abu), selenocysteine (SeCys2), tert-butylglycine (Bug or tert-BuGly), N-carbamyl amino acids, amino acid methyl esters, aminopropionic acid (or β-alanine; 13-Ala), adamentylglycine (Adg), aminocaproic acid (Acp), N-ethylasparagine (Et-asn), allohydroxylysine (aHyl), alloisoleucine (aIle), phenylglycine (Phg), pyridylalanine (Pal), thienylalanine (Thi), α-Δ-aminobutyric acid (Kbu), α-β-diaminopropionic acid (Kpr), and ), 1- or 2-naptitylalanine (1Nal or 2Nal), orthofluorophenylalanine (Phe(oF)), N-methylglycine (MeGly), N-methyl-isoleucine (Melle), N-methyl-valine (MeVal), 2-aminoheptanoic acid (Ahe), 2- or 3-aminoisobutyric acid (Aib), 2-aminopimelic acid (Dbu), 2-2'-diaminopimelic acid (Dpm), 2,3-diaminopropionic acid (Dpr), and N-ethylglycine (EtGly). Chemically generated non-encoded amino acids include, for example, phenylglycine (Ph-Gly), cyclohexylalanine (Cha), cyclohexylglycine (Chg), and 4-aminophenylalanine (Phe(4NH2) or Aph). Modified amino acids may be chemical structures that are not amino acids at all, but are actually classified as other chemical forms such as alkylamines, sugars, nucleic acids, lipids, fatty acids, or other acids. Any modified or unmodified amino acid, including peptides, may be in the D- or L-conformation and may contain one, two, or more tautomers or resonance forms.

[0096] Pharmaceutical compositions, including the live bacterial skin compositions disclosed herein, contain probiotic bacteria (e.g., S. epidermidis A11, S. hominis A9, S. hominis C4, S. hominis C5, and / or S. warneri G2), engineered forms thereof (e.g., attenuated or recombinant), or attenuated microorganisms containing an AIP peptide coding sequence can be formulated into any dosage form suitable for topical administration for local or systemic effect, including emulsions, solutions, suspensions, creams, gels, hydrogels, ointments, powders, dressings, elixirs, lotions, suspensions, tinctures, pastes, foams, films, aerosols, washes, sprays, suppositories, bandages, and skin patches. Topical formulations containing the live bacteria disclosed herein may also include liposomes, micelles, microspheres, nanosystems, and mixtures thereof.

[0097] In certain embodiments, a bandage or dressing is provided that includes a probiotic skin composition as disclosed herein having a probiotic bacterium (e.g., S. epidermidis A11, S. hominis A9, S. hominis C4, S. hominis C5, and / or S. warneri G2), an engineered form thereof (e.g., attenuated or recombinant), or an attenuated microorganism comprising an AIP peptide coding sequence as described herein. In various aspects, a bandage or dressing is provided that includes a probiotic skin composition comprising a matrix and a probiotic bacterium (e.g., S. epidermidis A11, S. hominis A9, S. hominis C4, S. hominis C5, and / or S. warneri G2), an engineered form thereof (e.g., attenuated or recombinant), or an attenuated microorganism comprising an AIP peptide coding sequence as described herein. In various embodiments, a bandage or dressing is provided whose primary components comprise a matrix and a fermented extract of probiotic skin bacteria. In various aspects, a bandage or dressing is provided whose primary components comprise a matrix and a fermented extract of probiotic skin bacteria. In various aspects, a bandage or dressing is provided whose primary components comprise a matrix and glycerol. In some embodiments, the bandage or dressing is applied to a site of skin injury or wound. In other embodiments, the bandage or dressing is applied to a site of infection.

[0098] "Pharmaceutically acceptable carrier" is intended to include solvents, dispersion media, coatings, antibacterial and antifungal agents (if necessary, provided they are not harmful to probiotic bacteria), isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the pharmaceutical composition, its use in therapeutic compositions and methods is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0099] Pharmaceutically acceptable carriers and excipients suitable for use in the topical formulations disclosed herein include, but are not limited to, water-soluble solvents, water-miscible solvents, non-water-soluble solvents, stabilizers, solubility enhancers, isotonicity agents, buffers, antioxidants, topical anesthetics, suspending and dispersing agents, wetting or emulsifying agents, complexing agents, sequestering or chelating agents, penetration enhancers, cryoprotectants, cryoprotectants, thickening agents, and inert gases.

[0100] Pharmaceutical compositions containing live bacteria may be formulated into ointments, creams, sprays, and gels. Suitable ointment solvents include oily or hydrocarbon solvents (including lard, benzoated lard, olive oil, cottonseed oil, other oils, white petrolatum, etc.); emulsifying or absorbing solvents (hydrophilic petrolatum, hydroxystearin sulfate, glycerol, anhydrous lanolin, etc.); water-removable solvents (e.g., hydrophilic ointments); water-soluble ointment solvents (including polyethylene glycols of various molecular weights); emulsion solvents, water-in-oil (W / O) emulsions, or oil-in-water (O / W) emulsions (including cetyl alcohol, glyceryl monostearate, lanolin, and stearic acid) (see Remington: "The Science and Practice of Pharmacy"). These solvents are emollients, but generally require the addition of antioxidants and preservatives.

[0101] Suitable cream bases may be oil-in-water or water-in-oil. Cream vehicles may be water-washable and may contain an oil phase, an emulsifier, and an aqueous phase. The oil phase, also known as the "internal" phase, is generally composed of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol. The aqueous phase is usually, but not necessarily, larger in volume than the oil phase and generally contains a moisturizer. The emulsifier in a cream formulation may be a nonionic, anionic, cationic, or amphoteric surfactant.

[0102] Gels are suspension-type systems of semisolids. Single-phase gels contain a substantially uniform material throughout the liquid carrier. Suitable gelling agents include cross-linked acrylic polymers such as carbomer, carboxypolyalkylene, Carbopol™, etc.; hydrophilic polymers such as polyethylene oxide, polyoxyethylene-polyoxypropylene copolymers, and polyvinyl alcohol; cellulosic polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and methylcellulose; gums such as tragacanth gum and xanthan gum; sodium alginate; and gelatin. To prepare a uniform gel, a dispersing agent such as alcohol or glycerin may be added, and the gelling agent may be dispersed by trituration, mechanical mixing, and / or stirring.

[0103] In another embodiment, pharmaceutical compositions comprising the compounds of Formula I and / or probiotic bacteria, derivatives or analogs thereof disclosed herein can be formulated alone or in combination with one or more other therapeutic agents (including, but not limited to, chemotherapeutic agents, antibiotics, antifungals, antipruritics, analgesics, protease inhibitors and / or antivirals) so long as the benefits of the probiotics are not destroyed.

[0104] As used herein, topical administration includes (intradermal), conjunctival, intracorneal, intraocular, ocular, otic, transdermal, intranasal, vaginal, urethral, respiratory, and rectal administration. Such topical formulations are useful for treating or inhibiting cancer in the eyes, skin, and mucous membranes (e.g., mouth, vagina, rectum). Examples of commercially available formulations include topical lotions, creams, soaps, wipes, etc.

[0105] Solutions or suspensions for use in pressurized containers, pumps, sprays, nebulizers, or nebulizer inhalers can be formulated to contain ethanol, aqueous ethanol, or a suitable alternative agent for dispersing, dissolving, or extending the release of the active ingredients disclosed herein, a propellant as a solvent; and / or a surfactant (e.g., sorbitan trioleate, oleic acid, or oligolactic acid).

[0106] Materials useful for forming erodible matrices include chitin, chitosan, dextran, pullulan; agar, gum arabic, karaya gum, locust bean gum, tragacanth gum, carrageenan, gum ghatti, guar gum, xanthan gum, scleroglucan; starches such as dextrin and maltodextrin; hydrophilic colloids such as pectin; phospholipids such as lecithin; alginates; propylene glycol alginate; gelatin; collagen; celluloses such as ethyl cellulose (EC), methylethyl cellulose (MEC), carboxymethyl cellulose (CMC), CMEC, hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), cellulose acetate (CA), cellulose propionate (CP), and cellulose butyrate. Celluloses such as cellulose acetate butyrate (CB), cellulose acetate butyrate (CAB), cellulose acetate butyrate (CAP), cellulose acetate butyrate (CAT), hydroxypropyl methylcellulose (HPMC), HPMCP, HPMCAS, hydroxypropyl methylcellulose acetate trimellitate (HPMCAT), and ethyl hydroxyethyl cellulose (EHEC); polyvinylpyrrolidone; polyvinyl alcohol; polyvinyl acetate; glycerol fatty acid esters; polyacrylamide; polyacrylic acid; copolymers of ethacrylic acid or methacrylic acid (EUDRAGIT, Rohm America, Inc., Piscataway, NJ); poly(2-hydroxyethyl methacrylate); polylactide; copolymers of L-glutamic acid and ethyl-L-glutamate; degradable lactic acid-glycolic acid copolymers; poly-D-(-)-3-hydroxybutyric acid; Other acrylic acid derivatives such as homopolymers; copolymers of butyl methacrylate, methyl methacrylate, ethyl methacrylate, ethyl acrylate, (2-dimethylaminoethyl) methacrylate, and (trimethylaminoethyl) methacrylate chloride, are included, but are not limited to.

[0107] In still further embodiments, the compositions provided herein (e.g., live bacterial compositions or compositions comprising a peptide or compound of Formula I) can be combined with one or more steroid drugs known in the art, including, but not limited to, aldosterone, beclomethasone, betamethasone, deoxycorticosterone acetate, fludrocortisone acetate, hydrocortisone (cortisol), prednisolone, prednisone, methylprenisolone, dexamethasone, and triamcinolone.

[0108] In still further embodiments, the compositions provided herein (e.g., live bacterial compositions or compositions comprising a peptide or compound of Formula I) can be combined with one or more antifungal agents, including, but not limited to, amorolfine, amphotericin B, anidulafungin, bifonazole, butenafine, butoconazole, caspofungin, ciclopirox, clotrimazole, econazole, fenconazole, filipin, fluconazole, isoconazole, itraconazole, ketoconazole, micafungin, miconazole, naftifine, nastamiconazole, sertaconazole, sulconazole, terbinafine, terconazole, tioconazole, and voriconazole.

[0109] Kits and articles of manufacture are also described herein for use in the therapeutic applications described herein. Such kits can include a carrier, package, or container compartmentalized to house one or more containers, such as vials, tubes, etc., each containing one of the distinct elements used in the methods described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials, such as glass or plastic.

[0110] For example, the container can contain one or more compositions provided herein (e.g., a live bacterial composition, or a composition comprising a peptide or a compound of Formula I), optionally in combination with another agent disclosed herein. Such kits optionally include a composition disclosed herein along with an identifying description, label, or instructions for use in accordance with the methods described herein.

[0111] The following examples are provided to further illustrate, but not limit, the present invention. [Example]

[0112] Example 1 Culture of Primary Human Keratinocytes. Neonatal NHEKs (ThermoFisher Scientific, Waltham, MA) were cultured in EpiLife medium (ThermoFisher Scientific) supplemented with 1x EpiLife defined growth supplement (ThermoFisher Scientific), 60 μM CaCl2, and 1x antibiotic-antimycotic (PSA; 100 U / ml penicillin, 100 U / ml streptomycin, 250 ng / ml amphotericin B; ThermoFisher Scientific) at 37°C and 5% CO2. For experiments, NHEKs were grown to 70% confluency, differentiated in high-calcium EpiLife medium (2 mM CaCl2) for 48 hours, and then treated with sterile-filtered bacterial supernatant. The use of these commercially available human-derived cell products does not require informed consent. For bacterial supernatant treatment, differentiated NHEKs were treated with sterile-filtered bacterial supernatant at a concentration of 5% by volume of EpiLife medium. NHEKs were used for experiments between passages 3 and 5 only.

[0113] Bacterial Culture. All bacteria were grown in 3% tryptic soy broth (TSB; Sigma, St. Louis, MO) at 37°C with shaking at 300 RPM. S. aureus strains Newman, USA 300, 113, and SANGER 252 and S. epidermidis strains ATCC 12228 and ATCC 1457 were grown to stationary phase for 24 h, then centrifuged (4,000 RPM, room temperature, 10 min), and the supernatant was sterile filtered (0.22 μm) and added to NHEK. Briefly, protease-free strains were grown for 24 h in 3% TSB containing 25 μg / ml lincomycin and 5 μg / ml erythromycin, then subcultured in 3% TSB for an additional 24 h. For the mouse live S. aureus microbiota assay, 2 × 106 bacterial microbiota-generating units were plated onto 8-mm TSB agar disks, allowed to dry at room temperature for 30 min, and then added to the dorsal skin of mice.

[0114] Murine Bacterial Disc Model: Female C57BJ / 6L mice (8 weeks old) were used for the murine model of bacterial skin microbiota. Briefly, mice were shaved to remove hair from the dorsal skin, and Nair was applied for 2–3 minutes, followed by removal of hair with an alcohol wipe. After 24 hours of recovery, 3 x 8 mm TSB agar discs containing either TSB alone (solvent control) or 2e6 microbiota-generating units of Staphylococcus aureus (USA300) per disc were applied to the dorsal skin of mice for 12 hours. Tegaderm was applied over the agar discs for fixation. After euthanasia, 8-mm whole skin punch biopsies were taken for analysis.

[0115] On-section zymography. Mouse skin sections (10 μm thick) were rinsed once for 5 minutes in 1% Tween-20 solution. To measure total protease activity, the sections were treated with 2 μg / ml BODIPY FL casein total protease active substrate (Thermo-Fisher Scientific) for 4 hours in a humidified chamber at 37°C. Thirty minutes before the addition of BODIPY FL casein, the serine protease inhibitor AEBSF (50 mM; Sigma) was added to the sections. The sections were rinsed once with phosphate-buffered saline, followed by mounting with DAPI-free ProLong Gold Antifade Mounting Medium (ThermoFisher Scientific) and a cover slide. Fluorescence signals were measured using an Olympus BX51 (Tokyo, Japan) fluorescence microscope.

[0116] Protease activity measurements: 50 ml of NHEK-conditioned medium was added to a 96-well black-bottom plate (Corning, Corning, NY) and 150 μl of 5 μg / ml BODIPY FL casein substrate, 2 μg / ml elastin (elastase-like substrate; ThermoFisher Scientific), or 4 μg / ml gelatin (MMP substrate; ThermoFisher Scientific) was added according to the manufacturer's instructions. 200 μM of the peptide Boc-Val-Pro-Arg-AMC (trypsin-like substrate; BACHEM, Bubendorf, Switzerland) was added to 150 μl of NHEK-conditioned medium in 1x digestion buffer (ThermoFisher Scientific). Relative fluorescence intensity was analyzed every 2 hours for 24 hours at room temperature using a SpectraMAX Gemini EM fluorometer (ThermoFisher Scientific). The BODIPY FL casein plate was read at ex: 485 nm and em: 530 nm. The elastin-like and MMP substrate plate was read at ex: 485 nm and em: 515 nm. The trypsin-like substrate plate was read at ex: 354 nm and em: 435 nm.

[0117] Quantitative real-time PCR. RNA was isolated from NHEKs using Purelink RNA isolation columns (ThermoFisher Scientific) according to the manufacturer's instructions. RNA was quantified using a Nanodrop spectrophotometer (ThermoFisher Scientific), and 500 ng of RNA was reverse transcribed using an iScript cDNA synthesis kit (Bio-Rad, Irvine, CA). Quantitative real-time PCR reactions were performed on a CFX96 Real-Time Detection System (Bio-Rad) using gene-specific primers and TaqMan probes (ThermoFisher Scientific).

[0118] Immunoblotting: For cell lysis, NHEKs were added to cold 1x radioimmunoprecipitation assay (RIPA) buffer (Sigma) containing 1x protease inhibitor cocktail (Cell Signaling Technology, Danvers, MA) and then scraped. The cell lysate was incubated on ice for 30 minutes and centrifuged (13,000 RPM, 15 minutes, 4°C) to remove cellular debris. Protein concentration was measured using a bicinchoninic acid (BCA) assay (Pierce, Rockford, IL). Subsequently, 40 mg of the sample was added to 4x Laemmli sample buffer (Bio-Rad) containing 1% b-mercaptoethanol and heated at 95°C for 7 minutes. Samples were run on a 4-20% Tris-glycine precast TGX gel (Bio-Rad) and transferred to a 0.22-µm polyvinylidene fluoride (PVDF) membrane (Bio-Rad) using a Trans-Blot Turbo Transfer System (Bio-Rad). After blocking for 1 hour at room temperature in 1x Odyssey blocking solution containing 0.1% Tween-20 (LI-COR, Lincoln, NE), primary antibodies were stained overnight at 4°C. After three washes with PBST (phosphate-buffered saline containing 0.1% Tween-20), Odyssey (LI-COR) fluorescent secondary antibodies were applied to the membranes for 1 hour at room temperature on an orbital shaker. After three further washes with PBST, the membranes were analyzed using an infrared imager (LI-COR). Primary antibodies KLK5 (H-55), KLK6 (H-60), DSG-1 (H-290), FLG (H-300), and a-tubulin (TU-02) from Santa Cruz Biotechnologies (Santa Cruz, CA) were used at a 1:100 dilution. KLK13 (ab28569) and KLK14 (ab128957) antibodies from Abcam (Cambridge, UK) were used at a 1:1,000 dilution.

[0119] Silencing of KLK gene expression: NHEKs were treated with 15 nM or 45 nM of specific KLK silencer-selective siRNA or siRNA scramble (-) control (ThermoFisher Scientific) for 24 hours using RNAiMAX (ThermoFisher Scientific) and OptiMEM medium (ThermoFisher Scientific). NHEKs were differentiated in high-calcium medium (2 mM CaCl2) for 48 hours and then treated with sterile-filtered Staphylococcus aureus (Newman) supernatant for 24 hours. NHEK lysates and conditioned medium were analyzed.

[0120] Statistical analysis: Both one-way and two-way analysis of variance were used for statistical analysis, with a P value of <0.05 considered significant. GraphPad Prism version 6.0 (GraphPad, La Jolla, CA) was used for statistical analysis of the results.

[0121] Staphylococcus aureus affects the activity of proteolytic enzymes in human keratinocytes. To evaluate whether various bacterial strains found in human skin can induce keratinocyte protease activity, primary cultures of healthy human epidermal keratinocytes (NHEK) were treated with sterile-filtered culture supernatants from four different laboratory isolates of Staphylococcus aureus, including two methicillin-resistant S. aureus strains (USA300 and SANGER252) and two methicillin-susceptible S. aureus strains. Two S. epidermidis isolates (ATCC12228 and ATCC1457) were also tested. Twenty-four hours after exposure to the sterile bacterial culture supernatants, the keratinocyte culture medium was analyzed for protease activity using substrates selective for trypsin-like, elastase-like, or matrix meroproteinase (MMP) activity. NHEK-conditioned medium contained significantly more trypsin activity after treatment with S. aureus strains Newman and USA300 (Figure 1a). Both MMP and elastase activities were increased by S. epidermidis ATCC 12228, whereas S. aureus USA300 and SANGER 252 and S. epidermidis ATCC 1457 increased elastase activity to a lesser extent in NHEK-conditioned medium (Fig. 1b and c). To confirm that the increased proteolytic enzyme activity observed in NHEK-conditioned medium was due to NHEK and not produced by the bacteria themselves, trypsin activity was analyzed after adding S. aureus (Newman) supernatant to culture wells with and without NHEK. When the same concentration of diluted S. aureus supernatant was added to NHEK medium alone, no enzyme activity was detected in the absence of NHEK (Fig. 1d).

[0122] Staphylococcus aureus increases the activity of epidermal serine protease. Due to the significant increase in trypsin activity induced by certain S. aureus strains (Newman and USA300) and the potential role this activity may play in S. aureus-mediated disease, we conducted experiments focused on this organism to better understand how the bacteria induce protease activity in NHEKs. To assess the kinetics of the protease response to S. aureus, keratinocytes were treated with sterile-filtered culture supernatant from S. aureus (Newman) for 0, 8, 24, and 48 hours, and NHEK-conditioned medium was collected for protease analysis. Measurement of total protease activity in NHEK-conditioned medium showed a time-dependent increase in total proteolytic activity after exposure to S. aureus supernatant (Figure 2a). Addition of aprotinin, an inhibitor of serine proteases, confirmed that this activity was due to serine proteases (Figure 2b). This was consistent with the observed increase in trypsin-like activity, as shown in Figure 1a. Comparison of wild-type and protease-deficient S. aureus USA300 LAC strains showed that both wild-type and protease-deficient strains increased trypsin activity in NHEK-conditioned medium, but the protease-deficient strain had a significantly reduced ability to induce trypsin activity compared to the wild-type strain (Figure 2c). Collectively, these data confirm that S. aureus increases the activity of endogenous NHEK serine proteases and that S. aureus proteases and other S. aureus products contribute to the ability of this bacterium to activate keratinocytes.

[0123] To further examine the effect of S. aureus on epidermal protease activity, live S. aureus (USA300) was applied to the dorsal skin of mice. The skin at the application site was then biopsied and sectioned, and total protease activity was analyzed by zymography on the sections in the presence or absence of the serine protease inhibitor 4-benzenesulfonyl fluoride (AEBSF). Total epidermal protease activity was qualitatively increased in the epidermis after S. aureus treatment compared with skin treated with agar discs alone. The increase in activity, detected by increased fluorescence, was largely eliminated by the inhibition of serine protease activity with AEBSF. Background autofluorescence in hair follicles was observed in all sections, including the no-substrate control. These observations further demonstrated that the presence of S. aureus can increase epidermal protease activity.

[0124] Staphylococcus aureus increases KLK expression in keratinocytes. KLKs are a family of serine proteases abundant in the epidermis with trypsin- or chymotrypsin-like activity. To determine whether S. aureus can alter KLK mRNA expression in keratinocytes, NHEKs were treated with S. aureus (Newman) supernatant for 24 hours, and the expression of KLK1-15 was measured by quantitative real-time PCR. KLK5 showed the highest relative mRNA abundance, while KLK6, 13, and 14 consistently showed the greatest fold increase after exposure to S. aureus (Figure 3a-e). All other KLKs analyzed showed a slight increase in mRNA expression after exposure to S. aureus, except for KLK1, which showed decreased expression. KLK2, 3, and 15 mRNA were not detected.

[0125] Next, both cell lysates and NHEK-conditioned medium were analyzed for changes in KLK protein expression after treatment with S. aureus (Newman) supernatant. Immunoblotting of KLK6 and 14 showed increased expression of these KLK proteins after treatment with S. aureus supernatant in both cell lysates and conditioned medium, whereas KLK13 increased only in conditioned medium. There was no change in KLK5 expression after treatment with S. aureus supernatant (Fig. 3f).

[0126] KLK6, 13, and 14 contribute to increased keratinocyte serine protease activity. Because KLK6, 13, and 14 showed the greatest increase in expression in NHEKs after S. aureus exposure, we performed experiments to determine whether these KLKs were responsible for the observed increase in serine protease activity. We selectively silenced their expression using small interfering RNA (siRNA). KLK6 and KLK13 siRNA significantly reduced S. aureus-induced trypsin activity, whereas KLK14 did not significantly reduce trypsin activity. Although no additive effect was observed, triple knockdown of KLK6, 13, and 14 also significantly reduced trypsin activity compared to control siRNA (Figure 4a). Interestingly, triple knockdown of KLK6, 13, and 14 reduced the knockdown efficiency of KLK13 and KLK14. This may explain the lack of additive effect on trypsin activity (Fig. 4b–d).

[0127] Staphylococcus aureus promotes the degradation of desmoglein-1 and FLG through induction of KLKs. Both desmoglein-1 (DSG-1) and FLG are important in regulating epidermal skin integrity. Immunoblotting showed that exposure of NHEKs to S. aureus (Newman) supernatant promoted the cleavage of full-length DSG-1 (160 kDa), and DSG-1 cleavage was blocked by siRNA silencing of KLK6, 13, or 14 (Figure 5a). S. aureus-induced cleavage of profilaggrin (Pro-FLG) in NHEKs, indicated by a band of >250 kDa on the immunoblot, was also partially blocked by siRNA silencing of KLK6 and KLK13 (Figure 5b). Densitometric analysis further demonstrated the ability of KLK6, 13, and 14 knockdown to prevent DSG-1 or Pro-FLG cleavage (Figure 5c). Collectively, these observations indicate that the ability of S. aureus to increase the proteolytic activity of keratinocytes by inducing KLK6, 13, and 14 leads to the digestion of molecules essential for the maintenance of normal epidermis. Example 2

[0128] Bacterial Preparation. A list of all bacteria used in this study is provided in Table A. All staphylococcal strains (S. aureus, S. epidermidis, S. hominis, S. warneri, S. capitis, and S. lugdunensis) were grown to stationary phase in 3% tryptic soy broth (TSB) at 250 RPM in a 37°C incubator in a volume of 4 mL or 400 μL, depending on the assay, for 24 h. Antibiotics were selected as indicated in Table A, and specific strains were grown at concentrations of 5 μg / mL Erm, 25 μg / mL Lcm, and 10 μg / mL Cm. To process bacterial supernatants on human keratinocytes or mouse skin, the 24-h cultured bacteria were sedimented (15 min, 4,000 RPM, room temperature), followed by filter sterilization of the supernatant (0.22 μm). For mouse and human keratinocyte experiments using S. hominis C5 and S. epidermidis RP62A strains, sterile-filtered bacterial supernatants were filtered through a 3 kDa size-exclusion column (Amicon Ultra-15 centrifugal filter, Millipore). The fraction below 3 kDa was collected, further concentrated 10-fold using a freeze dryer, and resuspended in molecular-grade HO before processing. Further biochemical testing was performed on S. hominis C5 supernatants using several techniques. Ammonium sulfate precipitation (80%) was performed at room temperature for 1 hour, followed by centrifugation (30 min, 4,000 RPM, room temperature) to separate small peptides, and the pellet was resuspended in HO. Additionally, the supernatant of S. hominis C5 was raised to pH 11 with 2 M NaOH for 1 h, and then the pH of the supernatant was returned to an initial pH of approximately 6.5 using strips of pH 1–14 with 2 M HCl and added to the agr reporter strain of S. aureus .

[0129] [Table A]

[0130] Cultivation of keratinocytes from healthy individuals Normal neonatal human epidermal keratinocytes (NHEKs; Thermo Fisher Scientific) were cultured in EpiLife medium (Thermo Fisher Scientific) supplemented with 60 μM CaCl (Thermo Fisher Scientific), 1x EpiLife defined growth supplement (EDGS; Thermo Fisher Scientific), and 1x antibiotic-antimycotic (PSA; 100 U / ml penicillin, 100 U / ml streptomycin, 250 ng / ml amphotericin B; Thermo Fisher Scientific) at 37°C and 5% CO. NHEKs were used only for experiments between passages 3 and 5. For experiments, NHEKs were grown to 70% confluency and then differentiated in high-calcium EpiLife medium (2 mM CaCl) for 48 hours to stimulate the upper epidermal layer. For bacterial supernatant treatment, differentiated NHEKs were treated with 5% sterile-filtered bacterial supernatant in Epilife medium for 24 hours. For synthetic PSM treatment, NHEKs were treated with 5–50 μg / mL peptides in DMSO for 24 hours.

[0131] Staphylococcus aureus epidermidis mouse model Eight-week-old male or female C57BL / 6 (Jackson) mice, matched for sex and age as specified in the figure legend, were used for all experiments (n = 3–6). All animal experiments were approved by the Institutional Animal Care and Use Committee. Mice were shaved, and NAIR was applied for 2–3 minutes, then quickly removed with an alcohol wipe. The skin was allowed to recover from hair removal for 48 hours before bacterial application. Staphylococcus aureus (1e7 CFU) in 3% TSB was applied to mouse skin at a volume of 100 μL / 1.5 cm of sterile plaque for 48–72 hours. Tegaderm was applied over the plaque to maintain the treatment. For S. aureus agr inhibition experiments, live S. aureus C5 (10:1) or 10-fold concentrated <3 kDa sterile-filtered commensal bacterial supernatant (1:1) was mixed with S. aureus in 3% TSB immediately before application to the guaze.

[0132] Preparation of synthetic phenol-soluble modulin All synthetic phenol-soluble modulins (PSMs) were produced by LifeTein (Hillsborough, NJ). Peptides were produced with 95% purity by N-terminal formylation (f). The PSM sequences are as follows: PSMα1: f-MGIIAGIIKVIKSLIEQFTGK (SEQ ID NO: 5), PSMα2: f-MGIIAGIIKFIKGLIEKFTGK (SEQ ID NO: 6), PSMα3: f-MEFVAKLFKFFKDLLGKFLGNN (SEQ ID NO: 7), PSMα4: f-MAIVGTIIKIIKAIIDIFAK (SEQ ID NO: 8), PSMβ2: f-MTGLAEAIANTVQAAQQHDSVKLGTSIVDIVANGVGLLGKLFGF (SEQ ID NO: 9). Peptides were resuspended in DMSO, concentrated in a speedvac to a 500 mg powder stock, and stored at -80°C until reconstitution with DMSO for the experiment.

[0133] RNA isolation and quantitative real-time PCR Total RNA was isolated using the Purelink RNA Isolation Kit according to the manufacturer's instructions (Thermo Fisher Scientific). For NHEK, 350 μL of RNA lysis buffer (containing 1% β-mercaptoethanol) was added directly to the cells. For mouse tissue, 0.5 cm2 of full-thickness skin was beaded (2x 30 s, 2.0 mm zirconia beads) in 750 μL of RNA lysis buffer for 5 minutes on ice. The tissue was then centrifuged (10 minutes, 13,000 RPM, 4°C), and 350 μL of the cleared lysate was added to 70% EtOH, followed by column-based RNA isolation. For S. aureus RNA isolation, 1x109 CFU bacteria were incubated with a 2:1 ratio of RNAprotect (Qiagen) for 10 minutes, then centrifuged (10 minutes, 13,000 RPM, room temperature), resuspended in 750 μL of RNA lysis buffer, and subjected to beading (2x 1 min 6.5 speed) using Lysis Matrix B tubes and Fastprep-24 (MP Biomedicals). The sample was then centrifuged again, and 350 μL of the cleared lysate was added to 70% EtOH as described above. After RNA isolation, samples were quantified using a Nanodrop (Thermo Fisher Scientific), and 500 ng of RNA was reverse transcribed using the iScript cDNA Synthesis Kit (Bio-Rad). qPCR reactions were performed on a CFX96 Real-Time Detection System (Bio-Rad). For mammalian cells, gene-specific primers and TaqMan probes (Thermo Fisher Scientific) were used with GAPDH as the housekeeping gene.

[0134] Generation and transformation of RP62A competent cells Electrocompetent RP62A cells were prepared. Briefly, an overnight culture of S. epidermidis RP62A was diluted to an OD of 0.5 with prewarmed Brain Heart Infusion (BHI) broth, incubated with shaking at 37°C for an additional 30 minutes, transferred to a centrifuge tube, and chilled on ice for 10 minutes. Cells were harvested by centrifugation (10 minutes, 4000 RPM, 4°C) and washed successively with 1 volume, 1 / 10 volume, and then 1 / 25 volume of cold autoclaved water, resuspending at 4°C after each wash. After the final wash, cells were resuspended in 1 / 200 volume of cold 10% sterile glycerol and aliquoted into 50 μL tubes for storage at -80°C. Transformation of S. epidermidis RP62A was performed. Briefly, frozen competent cells were thawed on ice for 5 minutes and then at room temperature for 5 minutes. Thawed cells were briefly centrifuged (1 min, 5000 g, room temperature) and the pellet resuspended in 50 μL of 10% glycerol supplemented with 500 mM sucrose. After adding DNA, the cells were transferred to a 1 mm cuvette and pulsed on a Bio-Rad Micropulser (Bio-Rad) at 2.1 kV with a time constant of 1.1 ms. Immediately after electroporation, the cells were resuspended in 1 mL of BHI broth containing 500 mM sucrose, shaken at 30°C for 1 h, and then plated onto BHI agar containing 10 μg / mL chloramphenicol (Cm) at 30°C.

[0135] Allelic substitutions in Staphylococcus epidermidis RP62A AIP The allele replacement plasmid pMAD (50) was used to selectively generate an in-frame deletion of the AIP coding sequence of agrD in S. epidermidis RP62A. Briefly, approximately 1,000 bp fragments upstream and downstream of the AIP sequence in RP62A were amplified by PCR and joined by gene splicing via overlap extension, or "SOEing." The stitched fragment and the pMAD vector were digested with BamHI and SalI, ligated with T4 DNA ligase (New England Biolabs), and then used to chemically transform the S. epidermidis clonal complex 10 plasmid-engineered E. coli strain DC10B-CC10. Transformants were plated on LB medium containing 100 μg / mL Amp and 30 μg / mL Cm at 37°C. Transformants were verified for correctness by restriction digestion and sequencing. The verified construct was annotated pMAD:ΔAIP. Next, electrocompetent RP62A was transformed with ~5 μg of pMAD:ΔAIP from DC10B-CC10 and plated on BHI agar containing 10 μg / mL Cm and 50 μL of 40 mg / mL 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) at 30°C. Single blue colonies were picked and grown overnight at 30°C in BHI containing 10 μg / mL Cm. The overnight culture was then diluted 1:100 (final volume 100 mL) into fresh, prewarmed BHI without antibiotics and incubated at 43°C for 24 h. Single cross-over events were promoted by further dilution and growth at 43°C, followed by picking light blue colonies grown on BHI agar supplemented with 10 μg / mL Cm and 50 μL of 40 mg / mL X-Gal at 43°C. Light blue colonies were picked and incubated overnight at 30°C in antibiotic-free BHI to promote double-crossover. This overnight dilution was plated onto 50 μL of BHI agar supplemented with 40 mg / mL X-Gal and incubated overnight at 37°C. White colonies were picked and patched onto BHI agar supplemented with 10 μg / mL Cm or 50 μL of 40 mg / mL X-Gal.Colonies that failed to grow in the presence of Cm and remained white in the presence of X-Gal were picked and screened for the deletion of the AIP coding sequence by sequencing. The verified mutant was annotated as S. epidermidis RP62AΔAIP.

[0136] RNA-seq analysis RNA was submitted to the University of California, San Diego (UCSD) Genomics Core Facility for library preparation and sequencing. Libraries were prepared using the TruSeq mRNA Library Prep kit (Illumina) and high-throughput sequencing was performed on a HiSeq 2500 sequencer (Illumina). Data were analyzed using Partek Flow and Partek Genomics Suite software, and systematic analysis of gene concepts was performed using the PANTHER classification system (http: / / pantherdb.org).

[0137] Histological examination Whole mouse skin (0.5 cm2) was harvested, fixed in paraformaldehyde (4%), washed with PBS, incubated overnight in 30% and 10% sucrose, and frozen in OCT mounting medium containing dry ice. Cryostat sections (10 mm) were mounted on Superfrost Plus glass slides (Fisher Scientific) and stained with hematoxylin and eosin (H&E). Sections were incubated in a 75% to 100% EtOH gradient for 5 min intervals, then incubated in xylene and mounted with paramount and glass slides. Photographs were taken at 200x magnification using an Olympus BX51 (Tokyo, Japan) fluorescence microscope.

[0138] Measurement of cytokine levels Protein concentrations of various cytokines were measured using 25 μL of conditioned medium from NHEKs. Magnetic bead-based Milliplex assay kits (Millipore) for three human cytokines (IL-6, IL-8, and TNFα) were used according to the manufacturer's instructions on the Magpix 200 (Luminex) system. Human IL-1α and IL-36α were quantified by ELISA (R&D Systems).

[0139] Bacterial CFU quantification Serial dilutions (10 μL) from 10 to 10 were plated on Baird-parker agar (BD) plates containing 3% egg yolk emulsion with telluric acid for 24 hours in a 37°C incubator, and the CFU strains were counted to quantify the bacterial flora units (CFU) of S. aureus. The OD600nm of cells diluted 1:20 in PBS was also measured using a spectrophotometer to estimate the bacterial CFU of all staphylococcus strains.

[0140] Measurement of transepidermal water loss To measure damage to the epidermal skin, transepidermal water loss (TEWL) was measured using a TEWAMETER TM300 (C&K) in mouse skin treated with Staphylococcus aureus for 48 to 72 hours.

[0141] Assay of trypsin activity Fifty microliters of NHEK-conditioned medium was added to a black 96-well black-bottom plate (Corning), followed by 150 μL of the peptide Boc-Val-Pro-Arg-AMC (trypsin-like substrate; BACHEM) in 1x digestion buffer (10 mM Tris-HCl pH 7.8) at a final concentration of 200 μM and incubated at 37°C for 24 hours. Relative fluorescence intensity (ex: 354 nm, em: 435 nm) was analyzed using a SpectraMAX Gemini EM fluorometer (Thermo Fisher Scientific). To analyze mouse skin trypsin activity, 0.5 cm2 of full-thickness skin in 1 cm2 of 1 M acetic acid was bead-beaten (2.0 mm zirconia beads, 2x 30 s, 5 min each) and rotated overnight at 4°C. The samples were centrifuged (10 min, 13,000 RPM, 4°C) into new microfuge tubes, and the protein was concentrated using a speedvac to remove any residual acetic acid. The protein was resuspended in molecular-grade water (500 μL), rotated overnight at 4°C, and then centrifuged again. The cleared protein lysate was placed into a new tube, and the protein concentration was determined by BCA (Bio-Rad) assay. Finally, 10 μg of total protein was added to a 96-well plate and analyzed with trypsin substrate as described above.

[0142] AGR activity in Staphylococcus aureus Agr activity in S. aureus was detected using either the S. aureus USA300 LAC agr type I P3-YFP (AH1677) or the S. aureus USA300 LAC agr type I pAmi P3-Lux (AH2759) reporter strains. For in vitro experiments, 1e6 CFU of S. aureus USA300 LAC agr type I P3-YFP was added to 300 μL of 3% TSB along with 100 μL of sterile-filtered syncytial supernatant (25% by volume) and shaken (250 RPM) at 37°C for 24 hours. The bacteria were then diluted 20-fold with PBS (200 μL final volume), and YFP (ex: 495 nm, em: 530 nm) was detected using a fluorometer as described above. Bacterial density was measured using a spectrophotometer reading at OD600 nm. In mouse experiments, the activity of S. aureus USA300 LAC agr type I pAmi P3-Lux was measured using an IVIS machine to assess the luminescence intensity after 2 minutes of exposure by measuring the emitted photons (p / sec / cm2 / sr) using LiveImaging software (PerkinElmer).

[0143] Genome sequencing and assembly S. hominis C5 genomic DNA was isolated using the DNeasy UltraClean Microbial Kit (Qiagen). For the library, sequences were measured using the MiSeq platform (Illumina Inc., San Diego, CA) for 2 cycles to generate 2x250 bp paired-end reads. Adapters were removed using cutadapt (Ver.1.9.1) (http: / / cutadapt.readthedocs.io / en / stable / ). Low-quality sequences (quality score <30) were removed using Trim Galore (Ver.1.9.1) with the default parameters (https: / / www.bioinformatics.babraham.ac.uk / projects / trim_galore / ). Sequence mapping to the human genome was removed from the quality-trimmed dataset using Bowtie2 (Ver.2.2.8) (51) with the following parameters (-D 20 -R 3 -N 1 -L 20 --very-sensitive-local) and the human reference genome hg19 (UCSC Genome Browser). Reads processed with the SPAdes (Ver. 3.8.0) (52) filter with k-mer lengths in the range of 33 - 127 were newly assembled. For the genome, rapid annotation of the microbial genome was performed using the subsystems technology (RAST) with the default parameters. Amino acid sequences (coding DNA sequences) from the annotated CDS were aligned to the agr proteins of bacteria obtained from the Uniprot database (downloaded in October 2017). Agr genes from the assembled genome were identified according to the following three criteria: i) sequence identity > 60%; ii) e-value < e100 and iii) agr locus organization, an operon of four genes, agrBDCA.

[0144] Comparative analysis of microbiome data and genomes We analyzed publicly available shotgun metagenomic data on atopic dermatitis skin. S. aureus and S. epidermidis strains were obtained directly from the public supplementary material ([www.]sciencetranslationalmedicine.org / cgi / content / full / 9 / 397 / eaal4651 / DC1). Characterization of Agr D was limited to information on seven different body sites of erythematous AD skin and eight patients (AD01, AD02, AD03, AD04, AD05, AD08, AD09, and AD11) with varying AD severity levels based on the objective SCORAD. Sixty-one S. epidermidis strains were classified as agr type I, II, or III by comparison of their amino acid sequences with known agr type I-III sequences within the agrD gene region.

[0145] Quantitative and statistical analysis The nonparametric Mann-Whitney test was used to analyze the statistical significance of metagenomic data from AD patients. Either one-way or two-way analysis of variance was used for statistical analysis, as indicated in various figure legends. All statistical analyses were performed using GraphPad Prism Ver. 6.0 (GraphPad, La Jolla, CA). All data are presented as mean ± standard error of the mean (SEM), and a P value of 0.05 or less was considered significant.

[0146] PSMα and proteolytic enzymes produced by Staphylococcus aureus induce epidermal damage. The primary function of human skin is to establish a physical barrier against the external environment. Specific toxins produced by S. aureus, such as phenol-soluble modulins (PSMs), can promote epidermal inflammation and have been proposed to be key drivers of disease progression in AD (19-22). Therefore, to understand how surface S. aureus influences inflammatory activity in the epidermis, we assessed the ability of healthy human epidermal keratinocytes (NHEKs) to express proteolytic activity upon exposure to S. aureus USA300 LAC strain containing targeted deletions in either its PSMα or PSMb operon. PSMα production was required for the induction of trypsin-like serine protease activity and increased kallikrein 6 (KLK6) mRNA levels (Figure 14A-B). The PSMα and PSMβ operons in S. aureus contain distinct peptides, including PSMα1-4 and PSMβ1-2. Accordingly, we tested synthetic PSMα1-4 and PSMβ2 peptides in NHEKs and found that all PSMα peptides could stimulate trypsin activity, whereas PSMβ2 could not (Fig. 14C). We selected PSMα3, the most potent inducer of PSMα trypsin activity in NHEKs, and further demonstrated that it could stimulate trypsin activity and KLK6 mRNA expression in NHEKs in both a dose- and time-dependent manner (Fig. 18). Furthermore, transcriptional profiling by RNA-Seq of NHEKs exposed to PSMα3 showed that this toxin had a widespread effect on the expression of genes related to the skin, including multiple proteases (KLKs, MMPs), physiologic components (filaggrin, desmoglein-1, loricrin, involucrin, keratin), antimicrobial peptides, and cytokines (Figures 14D-E and 18).

[0147] To examine the role of the PSMα operon on epidermal function in vivo, mice were colonized on their skin surfaces with equal numbers of S. aureus USA300 LAC wild-type or PSMα mutant strains for 72 hours. Wild-type S. aureus induced erythema, scaling, and acanthosis, whereas no change in bacterial burden was observed in the absence of PSMα (Figure 14F). Despite the increased epidermal thickness, an increase in transepidermal water loss (TEWL), an established method for assessing skin damage, was observed after exposure to wild-type S. aureus but not in the absence of PSMα (Figure 14G). However, the breakdown of fully differentiated epidermal skin in vivo also depended on the expression of S. aureus proteases. Using a S. aureus USA300 LAC mutant lacking 10 major secreted proteases, including aureolysin, V8, staphopains A / B, and SplA-F, clear evidence of damage and increased TEWL were reduced upon S. aureus protease deficiency, despite fully intact PSMα expression (Figure 14F, H). Concurrent with macroscopically and histologically observed changes associated with PSMα or bacterial proteases, decreased keratinocyte trypsin activity, expression of Klk6 transcripts, and cytokines Il6, Il17a, and Il17f were measured only in mice exposed to wild-type S. aureus but not in PSMα- or protease-deficient strains (Figure 14I–J). Furthermore, despite alterations in the cutaneous and cutaneous inflammatory environment, the abundance of S. aureus did not change on the skin surface under these conditions (Figure 14I–J). Taken together, these data suggest that the active production of PSMα and protease activity from S. aureus leads to epidermal damage, and that this damage is required for S. aureus to promote inflammation.

[0148] S. epidermidis autoinducing peptides inhibit S. aureus AGR activity. Interestingly, both S. aureus PSMα peptides and secreted proteolytic enzymes are under the regulation of the AGR quorum-sensing system. Furthermore, clinical isolates of S. aureus have been found to possess four distinct AGR types, with AGR type I being most prominent in AD subjects. Although S. aureus skin microbiota increases in AD, other bacterial species, such as coagulase-negative staphylococci (CoNS) strains, including S. epidermidis, an abundant human skin commensal, are also present. This makes it essential to understand how these bacteria communicate. S. epidermidis AGR type I laboratory isolates have been shown to produce an autoinducing peptide (AIP) that inhibits S. aureus AGR type I-III systems but not type IV through an AGR crosstalk mechanism. However, little is known about the effects of other S. epidermidis AGR types II and III on S. aureus AGR activity. To investigate whether S. epidermidis agr activity affects the S. aureus agr system, conditioned culture supernatants from S. epidermidis strains expressing agr types I, II, or III were added to the S. aureus USA300 LAC agr type I reporter strain. This experiment confirmed that S. epidermidis agr type I was the only potent inhibitor of S. aureus agr activity, while S. epidermidis agr types II and III were largely ineffective (Figure 15A). Targeted deletion of the S. epidermidis agr type I AIP within the agrD gene region abolished S. epidermidis's inhibitory ability to S. aureus agr activity (Figure 15B-C). Because S. aureus PSMα-induced NHEK trypsin activity is a component of epidermal damage, we tested S. epidermidis agr type I wild-type or AIP knockout strains to determine whether it affected this outcome. When S. aureus was cultured in the presence of wild-type S. epidermidis agr type I supernatant, inhibition of S. aureus-induced NHEK trypsin activity was observed, but not in S. epidermidis lacking the AIP (Figure 15D).Overall, these experiments established that the ability of S. aureus to induce NHEK damage can be influenced by agr type I AIP expression in S. epidermidis.

[0149] Relative Abundance of S. epidermidis Agr Type I in AD Skin After establishing the potential for laboratory strains of S. epidermidis to influence the effects of S. aureus on human keratinocyte function, we conducted experiments to measure the abundance of these bacteria in a clinical setting. Metagenomic data collected from seven body sites from the skin microbiomes of eight AD subjects with varying severity (based on the objective SCORAD) were analyzed for the relative abundance of S. epidermidis based on agr type. Sequence alignment identified S. epidermidis genomes based on agr type III in AD patients, and the most frequent S. epidermidis Agr type in AD skin was found to be agr type I (Figure 15E). Comparison of S. epidermidis Agr type I with S. aureus showed that S. epidermidis Agr type I became relatively less abundant in AD subjects as the disease became more severe (Figure 15F-G). These observations confirm the presence of S. epidermidis agr type I in the AD skin microbiome and suggest a possible association with clinical disease.

[0150] Diverse Staphylococcus species and strains inhibit S. aureus agr activity. To further establish the physiological significance of quorum-sensing interactions between S. aureus and other members of the skin microbiome, we tested the ability of CoNS culture supernatants from different AD clinical isolates to inhibit the quorum-sensing activity of S. aureus USA300 LAC agr type I. Diverse species, including S. epidermidis, S. hominis, S. warneri, and S. capitis, exhibited potent inhibitory activity against S. aureus agr activity (Figure 16A). Similar to laboratory isolates of S. epidermidis, CoNS strains inhibited S. aureus agr activity without inhibiting growth rate (Figure 16S). Furthermore, genomic sequence analysis of the agrD AIP coding region of S. hominis strain C5 revealed a novel AIP sequence in the AIP coding region that is similar to the sequence of the S. epidermidis agr type I coding region and has the predicted octomeric AIP sequence of S. hominis C5. (Figure 16B; SEQ ID NO:4). Biochemical techniques on the supernatant of active S. hominis C5 showed that inhibition of S. aureus agr activity was dependent on a <3 kDa (small size), pH 11-sensitive (thiolactone ring) factor that was precipitable with 80% ammonium sulfate (peptide) (Figure 16C).

[0151] Next, S. aureus was cultured in the presence of sterile-filtered supernatant of S. hominis C5, and the culture supernatant was then applied to NHEKs as shown in Figure 14. Similar to S. epidermidis agr type I, S. hominis C5 inhibited S. aureus-induced trypsin activity, KLK6 transcript production, and IL-6 protein expression in NHEKs (Figures 16D-F). Furthermore, S. hominis C5 was able to inhibit multiple S. aureus agr systems, apart from the most common clinical isolates of agr type I, which lack agr type IV but contain agr types II and III (Figure 21). This finding is consistent with that observed with the S. epidermidis agr type I system. Overall, these observations suggest that, in addition to S. epidermidis, clinical isolates of CoNS species may utilize quorum sensing to suppress S. aureus damage to keratinocytes.

[0152] Clinical CoNS isolates inhibit the AD-promoting agr activity of S. aureus. To establish the physiological relevance of the quorum-sensing interaction between CoNS and S. aureus in vivo, we assessed S. aureus agr activity by IVIS using the S. aureus USA300 LAC agr type I P3-Lux promoter (luminescence) strain. S. aureus on dorsal skin exhibited abundant agr activity, but in the presence of live S. hominis C5, S. aureus agr activity was inhibited (Figure 17A-B). Furthermore, S. hominis C5 protected against S. aureus-induced skin erythema and scaling (Figure 17C) without altering S. aureus abundance (Figure 17D). This phenotype was associated with improved evidence of inflammation, destruction, epidermal protease activity, and Klk6 expression (Figure 17E-H). Furthermore, when S. aureus was applied to the dorsal skin of mice in the presence of <3 kDa concentrated S. hominis C5 supernatant, we observed a similar reduction in damage and inflammation without any change in S. aureus abundance (Figure 22). These data indicate that the microbial community of skin CoNS likely contains novel AIPs that promote epithelial homeostasis through interspecies quorum sensing.

[0153] While a number of embodiments of the present disclosure have been described, it will be understood that various modifications can be made without departing from the spirit and scope of the disclosure. Accordingly, other embodiments are within the scope of the following claims.

Claims

1. A composition comprising a microorganism expressing a polypeptide comprising the sequence of SEQ ID NO: 4, wherein the polypeptide comprises a thiolactone ring.

2. A composition comprising a fermentation extract of a microorganism expressing a polypeptide comprising the sequence of SEQ ID NO: 4, wherein the polypeptide comprises a thiolactone ring.

3. The composition of claim 2 , wherein the fermentation extract comprises the polypeptide.

4. The composition of any one of claims 1 to 3, wherein the microorganism is a recombinant microorganism comprising a polynucleotide comprising SEQ ID NO: 1 or 3.

5. The composition of claim 4, wherein the recombinant microorganism comprises a vector, and the vector comprises a polynucleotide comprising SEQ ID NO: 1 or 3.

6. The composition of any one of claims 1 to 5, wherein the microorganism is S. hominis, S. epidermidis, S. warneri, or any combination thereof.

7. 7. The composition of claim 6, wherein the microorganism is S. hominis C5, S. hominis A9, S. epidermidis A11 and / or S. warneri G2.

8. 7. The composition of claim 6, wherein the microorganism is selected from the group of microorganisms having ATCC No. PTA-125202 (strain designation: S. epidermidis A11 81618, deposited on August 28, 2018), ATCC No. PTA-125204 (strain designation: S. hominis C5 81618, deposited on August 28, 2018), ATCC No. PTA-125203 (strain designation: S. hominis A9 81618, deposited on August 28, 2018), ATCC No. PTA-125205 (strain designation: S. warneri G2 81618, deposited on August 28, 2018), and any combination of the aforementioned strains.

9. The composition of any one of claims 1 to 8, wherein the polypeptide (i) inhibits the production and / or activity of proteases in keratinocytes, (ii) inhibits the production and / or activity of IL-6 in keratinocytes, (iii) inhibits the production of phenol-soluble modulin α3 from Staphylococcus aureus, and / or (iv) inhibits the production and / or activity of agr by Staphylococcus aureus. The composition described above.

10. The composition of any one of claims 1 to 9, wherein the polypeptide comprises a compound of formula I, IA, or IB: In the formula, X １ is 1 to 6 amino acids.

11. The composition of any one of claims 1 to 10 formulated as a lotion, shake lotion, cream, ointment, gel, foam, powder, solid, paste or tincture.

12. Use of a composition according to any one of claims 1 to 11 in the manufacture of a medicament for treating a skin disorder.

13. 13. The use according to claim 12, wherein the skin disorder is selected from the group consisting of Netherton syndrome, atopic dermatitis, contact dermatitis, eczema, psoriasis, acne, epidermal hypersensitivity, acanthosis nigricans, epidermal inflammation, dermal inflammation and pruritus.

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