Molecular bacterial therapy to control skin enzyme activity

Abstract

The present invention provides a purified polypeptide that inhibits (i) the production and / or activity of proteases in keratinocytes, (ii) the production and / or activity of IL-6 in keratinocytes, (iii) the production of phenol-soluble modulin alpha 3 from Staphylococcus aureus, and / or (iv) the production and / or activity of agr by S. aureus. A topical formulation comprising the polypeptide is further provided. A recombinant microorganism comprising a vector encoding the polypeptide or a polynucleotide is provided. A probiotic composition comprising the recombinant microorganism is further provided. The present invention also provides kits and products comprising the polypeptide and / or recombinant microorganism.

Description

Technology Field

[0001] Statement regarding federally sponsored research

[0002] The present invention was made with government support under license numbers AI117673, AR067547, AR062496, and AR064781 granted by the National Institutes of Health. The government holds specific rights in the present invention.

[0003] Cross-reference regarding related applications

[0004] This application claims priority under 35 USC §119 to Provisional Application No. 62 / 553,025 filed on August 31, 2017, the disclosure of which is incorporated herein by reference.

[0005] Technology field

[0006] The present disclosure relates to a composition and method for treating dermatological diseases and disorders, and a composition for adjusting skin barrier permeability.

[0007] Microorganism deposit

[0008] An exemplary microorganism of the present disclosure (Staphylococcus epidermidis ( Staphylococcus epidermidis ) A11, Staphylococcus hominis( Staphylococcus hominis ) C5, Staphylococcus hominis A9 and Staphylococcus warneri ( Staphylococcus warneri ) G2) is ATCC number PTA-125202 (strain name S. epidermidis ( S. epidermidis ) A11 81618, deposited on August 28, 2018), ATCC No. PTA-125204 (Strain name S. hominis( S. hominis ) C5 81618, deposited August 28, 2018), ATCC No. PTA-125203 (Strain name S. hominis As A9 81618, deposited on August 28, 2018) and ATCC No. PTA-125205 (strain name S. warneri ( S. warneri ) G2 81618, deposited on August 28, 2018) was deposited on August 28, 2018, at the American Type Culture Collection, 10801 Manassas University Boulevard, Virginia 20110-2209, USA. This deposit will be maintained at an accredited depositary for a period of at least 5 years from the most recent request for disclosure of samples received by the depositary, a period of at least 30 years from the date of deposit, or the duration of the relevant patent's enforceability, whichever is longest, and will be replaced in the event of mutation, inviolability, or destruction. All restrictions on the public availability of these cell lines will be finally withdrawn upon patent grant from this application. Background Technology

[0009] The epidermis is the first line of immune defense and protects and regulates interactions between microorganisms and the host organism. Controlling these interactions is important because bacteria not only reside on the surface affecting superficial keratinocytes, but some bacterial species also penetrate into the dermis and beneath the stratum corneum, influencing immune function. For example, Staphylococcus epidermidis (S. epidermidis) S. epidermidis )) interacts with epidermal keratinocytes to prevent Toll-like receptor 3-mediated inflammation, mobilizes mast cells and T cells, and increases tight junctions and the production of antimicrobial peptides. In contrast to common skin commensal bacteria, S. epidermidis, Staphylococcus aureus ( Staphylococcus aureus )(S. Aureus( S. aureusIt is often pathogenic and has a negative effect on skin function. This is particularly evident in skin diseases such as atopic dermatitis (AD), where S. aureus promotes the disease.

[0010] The microbiome inhabiting the skin of subjects with AD has been found to exhibit a decrease in overall microbial diversity and an increase in the abundance of S. aureus. Increased S. aureus colony formation is associated with increased disease severity in patients with AD. Mechanistically, it is uncertain how S. aureus exacerbates the disease. Several products from S. aureus have been shown to impair the skin barrier and / or induce inflammation. These products include α-toxin, superantigen, 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 colonization in the absence of clear clinical signs of infection is crucial for understanding the etiology of AD and developing future treatments.

[0011] The present disclosure provides a purified polypeptide comprising a sequence that is at least 98% identical to SEQ ID NO: 4, 11, 12, 13, 14, 15, 16, or 17, which inhibits (i) the production and / or activity of proteases in keratinocytes, (ii) the production and / or activity of IL-6 in keratinocytes, (iii) the production of phenol-soluble modulin alpha 3 from Staphylococcus aureus (S. aureus), and / or (iv) the production and / or activity of agr 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 another embodiment, the polypeptide consists of SEQ ID NOs: 4, 11, 12, 13, 14, 15, 16, or 17. In another or additional embodiment of any of the foregoing, the polypeptide comprises one or more D-amino acids. In another or additional embodiment, the polypeptide comprises a compound of Formula I, IA, or IB (see below).

[0012] The present disclosure also provides a topical formulation comprising a polypeptide of the present disclosure or a compound of formula I, IA or IB.

[0013] The present disclosure also provides an isolated polynucleotide encoding the polypeptide of the present disclosure. In one embodiment, the polynucleotide includes a sequence that hybridizes to a polynucleotide comprising SEQ ID NO: 1 or 3 under strict conditions and codes for a polypeptide comprising SEQ ID NO: 4. In another embodiment, the polynucleotide comprises SEQ ID NO: 1 or 3.

[0014] The present disclosure also provides a vector comprising the polynucleotide of the present disclosure. The vector may be any vector suitable for expression in a cell or microbial host.

[0015] 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 the polypeptide of the present disclosure and is engineered by recombinant engineering to express the polynucleotide of the present disclosure. In another embodiment, the microorganism is attenuated in such a way that it is non-pathogenic or has reduced pathogenicity compared to a wild-type organism of the same species. In another embodiment, the recombinant microorganism is a microorganism (e.g., a commensal) that is normally found on the skin of mammals (e.g., humans).

[0016] The present disclosure also provides a probiotic composition comprising the recombinant microorganism of the present disclosure.

[0017] The present disclosure also provides a probiotic composition comprising a microorganism expressing the polypeptide of the present disclosure (e.g., SEQ ID NOs: 4, 11, 12, 13, 14, 15, 16, and / or 17). In one embodiment, the microorganism is S. hominis, S. epidermidis, S. warneri, or any combination thereof. In a further embodiment, the microorganism is S. hominis C5, S. hominis A9, S. epidermidis A11, and / or S. warneri G2. In another or additional embodiment, the composition comprises a microorganism selected from the group of microorganisms having ATCC No. PTA-125202 (strain name S. epidermidis A11 81618, deposited August 28, 2018), ATCC No. PTA-125204 (strain name S. hominis C5 81618, deposited August 28, 2018), ATCC No. PTA-125203 (strain name S. hominis A9 81618, deposited August 28, 2018), ATCC No. PTA-125205 (strain name 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-natural (e.g., does not include the entire range of microorganisms found on the skin, includes an amount of microorganisms per unit volume not found on the skin, the microorganisms are genetically modified, or the composition contains components or compounds not normally found on the skin).

[0018] The present disclosure describes an effective amount of coagulase-negative Staphylococcus species sufficient to inhibit protease activity on the skin. Staphylococcus sp.The present invention also provides a method for treating a dermatological disorder, comprising the step of administering )(CoNS), or an effective amount of a fermented extract of CoNS, wherein the CoNS comprises a sequence at least 98% identical to SEQ ID NO: 4, 11, 12, 13, 14, 15, 16, or 17 and produces a polypeptide that inhibits protease production. In one embodiment, the dermatological disorder is selected from the group consisting of Netherton syndrome, atopic dermatitis, contact dermatitis, eczema, psoriasis, acne, epidermal hyperkeratosis, acanthosis, epidermal inflammation, dermal inflammation, and pruritus. In another embodiment, the step of administration is by topical application. In another or additional embodiment, the CoNS is Staphylococcus epidermidis, Staphylococcus capitis ( Staphylococcus capitis ), Staphylococcus caprae( Staphylococcus caprae ), Staphylococcus saccharoliticus( Staphylococcus saccharolyticus ), Staphylococcus warneri, Staphylococcus pasteuri( Staphylococcus pasteuri ), Staphylococcus hemolitiscus( Staphylococcus haemolyticus ), Staphylococcus debriesse ( Staphylococcus devriesei ), Staphylococcus hominis, Staphylococcus Zetensis ( Staphylococcus jettensis ), Staphylococcus petrasii( Staphylococcus petrasii ), and Staphylococcus rugdunensis ( Staphylococcus lugdunensisIt is selected from the group consisting of ). In another or additional embodiment of any of the foregoing, the fermented extract of CoNS comprises the polypeptide sequence of SEQ ID NO: 4 and / or a compound of Formula I, IA, or IB. In another embodiment, 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.

[0019] The present disclosure also provides a method for treating a skin disease or disorder, comprising the steps of: measuring the protease activity of a culture from the skin of a subject or of the skin from a subject; comparing the protease activity with a normal control; administering a symbiotic skin bacteria composition and / or fermentation extract from coagulase-negative Staphylococcus, wherein the symbiotic skin bacteria composition or fermentation extract comprises a polypeptide that is at least 98% identical to SEQ ID NO: 4, 11, 12, 13, 14, 15, 16, or 17, and / or comprises a compound of Formula I, IA, or IB, wherein the composition is formulated into a cream, ointment, or pharmaceutical composition that maintains the growth and replication ability of the symbiotic skin bacteria. In one embodiment, the coagulase-negative Staphylococcus is selected from the group consisting of Staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus saccharoliticus, Staphylococcus warneri, Staphylococcus pasteuri, Staphylococcus hemoliticus, Staphylococcus debriesei, Staphylococcus hominis, Staphylococcus zetensis, Staphylococcus petrasii, and Staphylococcus rugdunensis.

[0020] The present disclosure also provides a method for treating a skin disease or disorder, comprising the step of administering a probiotic composition comprising bacteria that produce a polypeptide that is at least 98% identical to the purified polypeptide of the present disclosure or SEQ ID NO: 4, 11, 12, 13, 14, 15, 16, or 17 and inhibits kallikrein production or activity.

[0021] The present disclosure also provides a method for treating a skin disease or disorder, comprising the step of administering a composition that inhibits phenol-soluble modulin expression, wherein the composition comprises the purified polypeptide of the present disclosure or a compound of formula I, IA, or IB. In one embodiment, the step of administration is topical. In another embodiment, the composition is a fermented extract of a coagulase-negative Staphylococcus.

[0022] The present disclosure also provides a topical probiotic composition comprising probiotic symbiotic skin bacteria 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 one embodiment, the composition is formulated as a lotion, shake lotion, cream, ointment, gel, foam, powder, solid, paste, or tincture.

[0023] The present disclosure also provides a drug composition comprising a S. aureus fermented extract or an S. aureus-probiotic comprising a drug and soluble phenolmodulin alpha 3. The present disclosure also provides a use of the composition for delivering a drug through the skin of a subject.

[0024] The present disclosure provides symbiotic / health-beneficial bacteria and / or their products to prevent increased protease activity in the skin. This is important in many disease conditions, including atopic dermatitis, Netterton syndrome, and other skin pathologies suffering from abnormally high protease activity and barrier disruption.

[0025] The present disclosure also provides factors and compositions that induce protease activity and thus aid in the proteolytic remodeling of the skin in the treatment of disorders related to wound repair, aging, sun damage, pigment abnormalities, and scarring.

[0026] The present disclosure provides a method for treating a dermatological disorder, comprising the step of administering an effective amount of a coagulase-negative Staphylococcus species (CoNS) or an effective amount of a fermented extract of CoNS, sufficient to inhibit protease activity on the skin. In one embodiment, the dermatological disorder is selected from the group consisting of Netterton syndrome, atopic dermatitis, contact dermatitis, eczema, psoriasis, acne, epidermal hyperkeratosis, acanthosis, epidermal inflammation, dermal inflammation, and pruritus. In another embodiment, the step of administration is by topical application. In another embodiment, CoNS is selected from the group consisting of Staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus saccharoticus, Staphylococcus warneri, Staphylococcus pasteeuri, Staphylococcus hemolticus, Staphylococcus debriesei, Staphylococcus hominis, Staphylococcus zetensis, Staphylococcus petracyi, and Staphylococcus rugdunensis. In a specific embodiment, CoNS is S. epidermidis.

[0027] The present disclosure also provides a method for treating a skin disease or disorder, comprising the steps of: measuring the protease activity of a culture from the skin of a subject or of the skin of a subject; comparing the protease activity with a normal control; and administering a symbiotic skin bacteria composition and / or fermented extract from coagulase-negative Staphylococcus, wherein the symbiotic skin bacteria composition comprises at least one symbiotic bacterium that reduces the serine protease activity of the culture or skin, and wherein the at least one symbiotic bacterium is formulated into a cream, ointment, or pharmaceutical composition that maintains the growth and replication ability of the symbiotic skin bacteria. In one embodiment, the coagulase-negative Staphylococcus is selected from the group consisting of Staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus saccharoliticus, Staphylococcus warneri, Staphylococcus pasteuri, Staphylococcus hemoliticus, Staphylococcus debriesei, Staphylococcus hominis, Staphylococcus zetensis, Staphylococcus petrasii, and Staphylococcus rugdunensis.

[0028] The present disclosure also provides a method for treating a skin disease or disorder, comprising the step of administering an agent that inhibits kallikrein expression. The present disclosure also provides a method for treating a skin disease or disorder, comprising the step of administering an agent that inhibits phenol-soluble modulin expression. In one embodiment of either of the foregoing, the step of administration is topical. In another embodiment, the agent is a fermented extract of a coagulase-negative Staphylococcus. In another embodiment, the coagulase-negative Staphylococcus is selected from the group consisting of Staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus saccharoliticus, Staphylococcus warneri, Staphylococcus pasteuri, Staphylococcus hemoliticus, Staphylococcus debriesei, Staphylococcus hominis, Staphylococcus zetensis, Staphylococcus petrasii, and Staphylococcus rugdunensis.

[0029] The present disclosure also provides a topical composition comprising a plurality of skin bacteria. In one embodiment, the probiotic commensal skin bacteria are coagulase-negative Staphylococcus species. In another distinct embodiment, the probiotic commensal skin bacteria comprise Staphylococcus aureus. In one embodiment of either of the foregoing embodiments, the bacteria are formulated into a cream, lotion, tincture, gel, or other topical preparation, wherein the bacteria remain viable.

[0030] The present disclosure provides a topical probiotic composition comprising a probiotic symbiotic skin bacteria fermentation extract, wherein the probiotic symbiotic skin bacteria fermentation extract is obtained from a coagulase-negative Staphylococcus (CoNS) species. In one embodiment, the CoNS is selected from the group consisting of Staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus saccharoliticus, Staphylococcus warneri, Staphylococcus pasteuri, Staphylococcus hemoliticus, Staphylococcus debriesei, Staphylococcus hominis, Staphylococcus zetensis, Staphylococcus petracii, and Staphylococcus rugdunensis.

[0031] In any one of the described embodiments, the topical probiotic composition is formulated as a lotion, shake lotion, cream, ointment, gel, foam, powder, solid, paste, or tincture.

[0032] The present disclosure provides a drug composition comprising a drug and a S. aureus fermented extract or an S. aureus-biotic composition.

[0033] The present disclosure provides a method for delivering a drug through the skin, comprising the step of contacting the skin with a composition comprising a drug and a S. aureus fermentation extract or an S. aureus-biotic composition. In one embodiment, the drug is a topical drug that is absorbed or adsorbed through the skin.

[0034] The present disclosure also provides a method for delivering a topical drug, comprising the step of contacting the skin of a subject with a composition comprising S. aureus or a fermented extract of S. aureus for a certain period of time and under certain dose and conditions to increase the penetration of the skin, and then contacting the skin with the drug to be delivered.

[0035] The present disclosure provides a composition comprising a fermented extract from S. aureus or a lotion, shake lotion, cream, ointment, gel, foam, powder, solid, paste, or tincture containing viable S. aureus.

[0036] Details of one or more embodiments of the present invention are set forth in the following accompanying drawings and description. Other features, objects, and advantages of the present invention will be apparent from the description and drawings and from the claims. Brief explanation of the drawing

[0037] FIGS. 1a to d Is ( a to c) This indicates that NHEK was treated for 24 hours with sterile filtered supernatants of S. aureus (SA; Newman, USA300, 113; Sanger, 252) and S. epidermidis (ATCC12228, ATCC1457), and that the NHEK-conditioned medium was analyzed for specific trypsin-like, elastase-like, and MMP protease substrates. d The effect of proteases secreted by S. aureus (Newman) on trypsin activity was analyzed. Data are presented as mean ± SEM (n = 4) and represent at least three independent experiments. One-way ANOVA (aec) and two-way ANOVA (d) were used, and significance was indicated as *P < 0.05, ***P < 0.001, and ****P < 0.0001. ANOVA, Analysis of Variance; MMP, Matrix Metalloproteinase; NHEK, Normal Human Epidermal Keratinocytes. FIGS. 2a to c Is ( a Total protease activity (5 μg ml BODIPY FL casein) was measured in NHEK-conditioned medium after treatment with S. aureus (SA, Newman) supernatant for 0–48 hours, and ( bMeanwhile, this indicates that the serine protease inhibitor aprotinin (800 μg ml) was applied to the conditioned medium after 24 hours of treatment. c The effects of ) S. aureus (USA300 LAC) WT and protease-null strains on trypsin activity (Boc-Val-Pro-Arg-AMC, 200 mM) in NHEK-conditioned medium were compared. Two-way analysis of variance ( a, b ) and one-way ANOVA( c Both were used, and significance was indicated as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ANOVA, Analysis of Variance; NHEK, Normal Human Epidermal Keratinocytes; WT, Wild Type. FIGS. 3a to f This indicates that S. aureus increases KLK expression in human keratinocytes. a The relative abundance of KLK mRNA expression in NHEK after 24-hour treatment with S. aureus (SA, Newman) supernatant was analyzed by qPCR. b to e ) We analyzed the fold change of KLK5, 6, 13, and 14 in NHEK treated with S. aureus supernatant for 0–48 hours. All mRNA expression levels were normalized to the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). fChanges in protein expression of KLK5, 6, 13, and 14 were analyzed by immunoblotting after 24 hours of treatment with SA (Newman) supernatant using NHEK-conditioned medium and cell lysates, with both revealed and predicted molecular weights. The housekeeping gene, α-tubulin, was used as a loading control for the cell lysates. Data are presented as mean ± SEM (n = 3) and represent at least three independent experiments. Two-way ANOVA (BEE) was used, and significance was indicated as **P < 0.01, ***P < 0.001, ****P < 0.0001. ANOVA; KLK, kallikrein; NHEK, normal human epidermal keratinocytes; qPCR, quantitative real-time PCR; SEM, standard error of mean. FIGS. 4a to d This indicates that multiple KLKs are the cause of S. aureus-induced serine protease activity in human keratinocytes. NHEK was treated with KLK6, 13, or 14 siRNA (15 nM) prior to CaCl2 differentiation and the addition of S. aureus (Newman) supernatant. siRNA scrambled (-) controls 1 and 2 were used at 15 nM and 45 nM, respectively. a The conditioned medium was analyzed for changes in trypsin activity (Boc-Val-Pro-Arg-AMC, 200 μM). b to dTranscriptional levels of KLK6, KLK13, and KLK14 were evaluated by qPCR, and siRNA knockdown efficiency was confirmed by normalizing to the housekeeping gene, GAPDH. Data are presented as mean ± SEM (n = 4) and represent at least three independent experiments. One-way ANOVA (a) was used, and significance was indicated as *P < 0.05, **P < 0.01, ***P < 0.001. ANOVA, Analysis of Variance; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; KLK, Kallikrein; NHEK, Normal human epidermal keratinocytes; qPCR, Quantitative Real-Time PCR; siRNA, Small Interfering RNA; SEM, Standard Error of Mean. FIGS. 5a to c This indicates that multiple KLKs regulate S. aureus-induced DSG-1 and FLG cleavage in human keratinocytes. NHEKs treated with S. aureus (Newman) supernatant for 24 hours were subjected to siRNA knockdown of KLK6, 13, and 14 (15 nM) by immunoblotting ( a ) Desmoglein-1 (DSG-1) and ( b Changes in profilaggrin (Pro-FLG) cleavage were evaluated. The housekeeping gene, α-tubulin, was used as a loading control. DSG-1 (full length) and Pro-FLG are indicated by black arrows. c Densitometry analysis of both DSG-1 (whole length) and Pro-FLG, expressed as the mean pixel count (n = 1) normalized to α-tubulin. Immunoblots represent at least three independent experiments. KLK, kallikrein; NHEK, normal human epidermal keratinocytes; siRNA, small interfering RNA. Fig. 6 This describes the method for preparing the fermented extract and the assay for its activity. Fig. 7This indicates that under the control of a quorum sensing system, S. aureus phenol-soluble modulin (PSM) causes increased keratinocyte serine protease activity. Fig. 8 This indicates that S. aureus PSM increases mouse serine protease activity and skin barrier damage. Fig. 9 This indicates that S. aureus isolates from skin with atopic dermatitis (AD) lesions can induce serine protease activity in keratinocytes in an agr-type-dependent manner. Fig. 10 This indicates that the coagulase-negative Staphylococcus (CoNS) strain ATCC14490 (S. epidermidis) can turn off S. aureus agr activity by producing an auto-inducing peptide (AIP). Fig. 11 This shows the effects of S. aureus and symbiotic bacteria on serine protease activity in atopic dermatitis. Fig. 12 This represents the effect of S. hominis C5 on S. aureus agr activity. Fig. 13 This shows the effects of various CoNS strains on S. aureus agr activity. FIGS. 14a to jThis indicates that S. aureus PSMα causes disruption of epithelial barrier homeostasis. Human keratinocytes (NHEK) were stimulated for 24 hours with sterile-filtered supernatant of S. aureus (SA) from wild-type (WT), PSMα (ΔPSMα), or PSMβ (ΔPSMβ) knockout strains, and (a) trypsin activity and (b) KLK6 mRNA were analyzed compared to the housekeeping gene GAPDH (n=4). (c) changes in trypsin activity were analyzed by adding PSM synthetic peptides to NHEK for up to 24 hours. (d, e) gene ontology (GO) analysis was performed after evaluating transcripts by RNA-Seq of genes that changed ≥2-fold after PSMα3 treatment. 8-week-old male C57BL / 6 mice (n=6) were fed SA WT, SA ΔPSMα, or SA 10 secreted protease knockout strain (Δprotease) for 72 hours (1e 7 (f, g) Representative photographs of murine skin (dashed lines indicate the treatment area) and changes in epidermal thickness after treatment (scale = 200 μm). (h to k) Changes in the back skin of murine by WT or mutant SA strains in transepidermal water loss (TEWL) and SA CFU / cm2 were also evaluated. All error bars are expressed as the standard error of the mean (SEM), and statistical significance was determined using one-way ANOVA, indicated as p<0.05 *, p<0.01 **, p<0.001 ***, p<0.0001 ****. FIGS. 15a to g It indicates the characterization and deficiency of Staphylococcus epidermidis agr type I auto-induced peptide in AD skin. a, b Inhibition of S. epidermidis agr type I-III supernatant of S. aureus (SA) USA300 LAC agr type I activity after 24 hours (n=4) and indication of a known structure of an S. epidermidis agr type I self-inducing peptide (AIP). cEffects of Staphylococcus epidermidis (S. epi) agr type I strain RP62A wild-type (WT) or auto-induced peptide knockout (ΔAIP) on SA agr activity after 24 hours. d NHEK trypsin activity was measured after applying SA sterile-filtered supernatant growths with or without S. epi WT or ΔAIP supernatants to NHEK for an additional 24 hours (n=4). e ) Common (Consensus) of S. epidermidis agr type I-III genomes found on AD skin. ( f, g Ratio of S. epidermidis agr type I to the relative presence of SA in the redness zones of 8 individual AD subjects based on 'least severe' to 'most severe' AD scores from overall combined data of all subjects based on AD severity and objective SCORAD. All error bars are expressed as the standard error of the mean (SEM) and one-way ANOVA( a, c, d ) and (nonparametric) unpaired Mann-Whitney test( f Statistical significance was determined using ) as indicated by p<0.05 *, p<0.01 **, p<0.001 ***, and p<0.0001 ****. FIGS. 16a to f This indicates that multiple clinically isolated coagulase-negative Staphylococcus species inhibit S. aureus agr activity. a The sterile-filtered supernatant of clinically isolated coagulase-negative Staphylococcus (CoNS) was added to the S. aureus (SA) USA300 LAC agr type I P3-YFP reporter strain for 24 hours, and SA agr activity was analyzed (n=3). b, cThe genome of the S. hominis C5 strain was further sequenced, and the agrD gene was analyzed for auto-induced peptide (AIP) sequences. Biochemical analysis of the S. hominis C5 supernatant was performed using <3 kDa size exclusion centrifugation filtration, 80% ammonium sulfate precipitate, and supernatant treated at pH 11 for 1 hour to perform SA agr activity. d to f SA grown in the presence of S. hominis C5 supernatant for 24 hours was sterile filtered and added to human keratinocytes (NHEK) for 24 hours, after which trypsin activity, KLK6 mRNA expression compared to the housekeeping gene GAPDH, and IL-6 protein levels were analyzed. All error bars are expressed as the standard error of the mean (SEM), and statistical significance was determined using one-way ANOVA, indicated as p<0.05 *, p<0.01 **, p<0.001 ***, and p<0.0001 ****. FIGS. 17a to h This indicates that the AD clinical CoNS isolate inhibits SA-induced murine skin barrier damage. Live S. hominis C5(1e 8 S. aureus (SA) USA300 LAC agr type I pAmi P3-Lux reporter strain (1e) with or without CFU) 7 CFU) was administered to 8-week-old female C57BL / 6 mice (n=5) for 48 hours. a, b ) SA agr activity was evaluated on the dorsal skin of murine by changes in luminescence. c Representative image of murine skin after 48 hours of SA treatment (dashed box indicates treatment area). d to h ) SA CFU / cm 2Murine skin barrier damage and inflammation were evaluated by determining and analyzing changes in Il6 mRNA expression, transepidermal water loss (TEWL), trypsin activity, and Klk6 mRNA expression normalized to the housekeeping gene Gapdh. All error bars are expressed as the standard error of the mean (SEM), and statistical significance was determined using one-way ANOVA, indicated as p<0.05*, p<0.01**, p<0.001**, and p<0.0001****. FIGS. 18a to h This indicates that S. aureus PSMα alters the expression of essential barrier genes and cytokines in human keratinocytes. a to d Human keratinocytes treated with synthetic PSMα3 were evaluated for changes in trypsin activity and KLK6 transcript expression normalized to the housekeeping gene GAPDH using both dose- and time-dependent methods. E ) GO-term analysis of genes ≥2-fold downregulated from the control in human keratinocytes treated with PSMα3 for 24 hours. f to h Changes in human keratinocyte cytokine protein expression of IL-6, TNF-α, or IL-1α treated with SA WT, SA Δpsmα, or SA Δpsmβ supernatant for 24 hours. All error bars are expressed as the standard error of the mean (SEM), and statistical significance was determined using one-way ANOVA, indicated as p<0.05 *, p<0.01 **, p<0.001 ***, p<0.0001 ****. FIGS. 19a to h This indicates that S. aureus PSMα and protease cause barrier damage and inflammation induction on murine skin. S. aureus (SA)(1e 7 CFU) wild-type (WT), PSMα knockout (Δpsmα), and protease-deficient (Δprotease) strains were applied to the dorsal skin of male murines for 72 hours (n=6) ( a, e ) Trypsin activity, ( b, f ) Klk6, ( c, g ) Il6, and ( d, h Changes in IL17a / f mRNA expression (normalized by the housekeeping gene Gapdh) were measured. All error bars are expressed as the standard error of the mean (SEM), and statistical significance was determined using one-way ANOVA, indicated as p<0.05 *, p<0.01 **, p<0.001 ***, and p<0.0001 ****. FIGS. 20a to c indicates that the CoNS strain does not perform SA growth. The coagulase-negative Staphylococcus (CoNS) supernatant is ( a ) CoNS clinical isolates, ( b ) S. epidermidis (S. epi) agr types I-III, and ( c ) Effects on SA agr type I P3-YFP reporter strain growth as evaluated by OD600 nm (n=3-4) including S. epidermidis (S. epi) wild-type (WT) or auto-induced peptide knockout (ΔAIP) supernatant added to SA agr type I reporter strains for 24 hours. All error bars are expressed as the standard error of the mean (SEM). FIGS. 21a to b This indicates that S. hominis C5 inhibits SA agr types I-III but not type IV. The supernatant of S. hominis C5 was added to SA agr type I-IV P3-YFP reporter strains for 24 hours (n=3). a ) SA reporter strain agr type I-IV activity and ( b Measurement of growth by OD600nm when cultured in the presence of S. hominis C5 supernatant. All error bars are indicated as the standard error of the mean (SEM), and statistical significance was determined using one-way ANOVA, indicated as p<0.05 *, p<0.01 **, p<0.001 ***, p<0.0001 ****. FIGS. 22a to fThis indicates that the S. hominis C5 supernatant inhibits SA-induced skin barrier damage. S. aureus (SA)(1e) with or without 10x concentrated <3 kDa S. hominis C5 supernatant 7 CFU) was applied to the dorsal skin of female murines for 48 hours (n=3). a to b Representative image of the back of a murine after SA treatment (dashed line indicates the treated area) and SA CFU / cm² recovered from the murine skin 2 . ( c to f SA induced skin barrier damage markers including Il6, transepidermal water loss (TEWL), trypsin activity, and Klk6 mRNA expression (compared to the housekeeping gene Gapdh). All error bars are expressed as the standard error of the mean (SEM), and statistical significance was determined using one-way ANOVA, indicated as p<0.05 *, p<0.01 **, p<0.001 ***, and p<0.0001 ****. Specific details for implementing the invention

[0038] details

[0039] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include multiple referents unless the context clearly indicates otherwise. Accordingly, for example, a reference to “agent” includes multiple such agents, and a reference to “microorganism” includes one or more microorganisms and their equivalents known to a person skilled in the art.

[0040] Additionally, the use of "or" implies "and / or" unless otherwise noted. Similarly, "include," "include," "including," "includes," and "included" are interchangeable and are not intended to be restrictive.

[0041] Where the description of various embodiments uses the term "comprising," a person skilled in the art should additionally understand that in some specific cases, one embodiment may alternatively be described using the expressions "essentially composed of" or "composed of."

[0042] Unless otherwise defined, all technical terms used herein have the same meaning as generally understood by a person skilled in the art to which this disclosure pertains. Any method and reagent similar or equivalent to that described herein may be used in the practice of the disclosed method and composition.

[0043] Atopic dermatitis (AD) is one of the most common immune disorders, posing a significant financial burden on patients' quality of life and a serious risk of comorbidities. Defects in skin barrier function are a key characteristic of AD. Eczematous skin lesions in patients with AD are associated with elevated levels of Th2 cytokines, such as IL4 and IL13. Th2 cytokines promote a decline in skin barrier function by inhibiting the expression of filaggrin. These cytokines also inhibit the expression of human antimicrobial peptides, such as cathelicidin and β-defensin-2, which are defects in AD that can lead to dysbiosis of the skin bacterial community and enhanced colonization by S. aureus. Therapies targeting IL4 receptor alpha result in significant improvement of the disease. The strong association between Th2 cytokine activity, barrier function, antimicrobial activity, and disease outcomes supports efforts to define the causal relationships between these intrinsic epidermal functions.

[0044] The skin barrier of patients with AD can be impaired by increased proteolytic activity because they have been found to exhibit increased kallikrein (KLK) expression. KLK is a family of 15 serine proteases, some of which are primarily found in the upper granular and stratum corneum of the epidermis. In Netterton syndrome, increased serine protease activity is observed due to reduced activity of the serine protease inhibitor Kazal-type 5. The resulting increase in enzyme activity leads to increased epidermal desquamation, altered processing of antimicrobial peptides and filaggrin (FLG), and activation of protease-activated receptor 2, as well as inflammation. Increased protease activity may also play a crucial role in the communication of the microbiome with the skin immune system and has recently been shown to directly influence epidermal cytokine production and inflammation by enhancing bacterial penetration through the epidermis.

[0045] Gut bacterial imbalance in the skin microbiome and skin colonization by Staphylococcus aureus are associated with the exacerbation of atopic dermatitis (AD). The present disclosure demonstrates that S. aureus has the ability to induce specific KLK expression from keratinocytes and increase overall proteolytic activity in the skin. This describes a signaling system between bacteria on the skin and the host and suggests a previously unknown but likely important mechanism regarding how S. aureus colonization can increase disease severity in patients with AD.

[0046] S. aureus can secrete numerous proteases onto the skin that alter skin barrier integrity. Serine protease V8 and serine-like protease exfoliating toxins have been shown to cause increased epidermal shedding by cleaving corneodesmosome adhesion proteins, including DSG-1. The MMP aureolicin is known to cleave and inactivate LL-37, an important antimicrobial peptide for the skin. However, these direct proteolytic actions of S. aureus products require high levels of enzymes and bacteria and are more consistent with events occurring during infection of this organism.

[0047] Increased digestion of barrier proteins was observed after keratinocytes were activated by S. aureus. FLG is known to be cleaved from the larger Pro-FLG (400 kDa) into a monomeric form (37 kDa) that plays a crucial role in forming the physical barrier of the stratum corneum with keratin. Accelerated Pro-FLG cleavage has been shown to be associated with increased epidermal shedding of the skin (Hewett). et al. (2005). Interestingly, increased cleavage of Pro-FLG was observed in human keratinocytes treated with S. aureus supernatant. Pro-FLG cleavage was partially blocked when KLK6 or KLK13 was silenced, indicating that S. aureus can reduce skin barrier integrity in a KLK-dependent manner through the cleavage of Pro-FLG.

[0048] DSG-1 is a critical keratinodesmotic adhesion protein that causes increased epidermal exfoliation upon cleavage. In keratinocytes, full-length DSG-1 (160 kDa) is readily cleaved by KLK activity stimulated by S. aureus. It has been reported that KLK5, 6, 7, and 14 can cleave DSG-1, whereas KLK13 is not. This indicated that upregulated KLK6 and KLK14 can cause enhanced cleavage of full-length DSG-1, while providing evidence contrary to the notion that KLK13 is not involved in DSG-1 cleavage. Therefore, while S. aureus can cause KLK to alter FLG cleavage, it can also cause increased DSG-1 cleavage as another method of reducing epidermal skin barrier integrity. Specific siRNA knockdown suggested that increased expression of KLK is, at least in part, the cause of increased serine protease activity stimulated by S. aureus. Fig. 2C This demonstrates that proteases secreted by S. aureus are a cause of the induction of increased trypsin activity in keratinocytes. Because bacteria including S. aureus can penetrate the skin surface and elicit a strong skin immune response (Nakatsuji et al. , 2013, 2016; Zhang et al. (, 2015), these bacteria can also affect the protease activity of skin cells. These observations are also associated with rosacea or Netterton syndrome.

[0049] The present disclosure demonstrates that soluble factor(s) produced by S. aureus possess a potent and previously unexpected ability to alter endogenous protease activity produced by keratinocytes. This occurred in diluted solutions of S. aureus products in which bacterial protease activity was undetectable. Thus, S. aureus can significantly alter the balance of total epidermal protease activity by promoting the epidermis to increase the expression of endogenous protease activity.

[0050] Different strains of S. aureus (Newman, USA300, 113, and Sanger 252) and S. epidermidis (ATCC12228 and ATCC1457) had different effects on human keratinocyte protease activity. S. aureus strains, including Newman and USA300, increased trypsin activity, whereas other strains of S. aureus and S. epidermidis increased elastase or MMP activity. Therefore, bacteria may alter epidermal protease activity depending on both the bacterial species and the strain. It is possible that other bacterial species and strains of S. aureus may additionally and uniquely affect the enzyme balance of human skin. Interestingly, preliminary data revealed that purified Toll-like receptor ligands did not induce trypsin activity or KLK expression in keratinocytes.

[0051] Protease activity is highly upregulated in many skin conditions, leading to a compromised skin barrier. This is associated with exacerbated disease states in almost all cases. In one aspect, the present disclosure demonstrates that symbiotic microorganisms and their bacterial products are useful in preventing increased protease activity in the skin. In particular, the present disclosure demonstrates that coagulase-negative Staphylococcus can prevent serine protease activity induced by Staphylococcus aureus in the skin by inhibiting the agr quorum sensing system. Staphylococcus aureus, a pathogenic bacterial strain, can induce serine protease activity in the skin. Increased protease activity disrupts the skin barrier and causes exacerbated disease states, including Netterton syndrome and atopic dermatitis. The present disclosure demonstrates that such increased serine protease activity can be prevented through the use of symbiotic, or health-beneficial, skin bacteria and factors derived therefrom.

[0052] The present 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 reduce the integrity of the skin barrier in a KLK-dependent manner.

[0053] The present disclosure demonstrates that S. aureus not only secretes proteases but can also increase the expression of endogenous proteases by specifically activating keratinocytes. The present disclosure demonstrates that phenol-soluble modulin alpha (PSMα) is secreted by S. aureus and induces auto-digestion of the epidermis. For example, three members of the KLK family appear to play a significant role in this increased enzymatic activity.

[0054] The present disclosure also identifies symbiotic bacteria, genes, and polypeptides that inhibit the accessory gene regulator (agr) quorum sensing system of S. aureus and turn off PSMα to inhibit protease activity. Accordingly, the present disclosure provides targets for modulating atopic dermatitis as well as agents and probiotic preparations that modulate atopic dermatitis and protease activity on the skin.

[0055] The present disclosure demonstrates that coagulase-negative Staphylococcus (CoNS) species normally residing on the skin, such as S. epidermidis and S. hominis, protect against this biological activity of S. aureus by producing an auto-induced peptide (AIP) that inhibits the helper gene regulator (agr) quorum sensing system of S. aureus and turns off PSMα.

[0056] In fact, virtually all S. aureus toxins are under the control of virulence co-gene regulators (agrs). The agr system induces changes in gene expression at specific cell densities through a process called quorum sensing. In addition to toxins, agrs are known to upregulate various virulence determinants, such as exoenzymes (proteases, lipases, nucleases), and downregulate the expression of surface-binding proteins. These adaptations are thought to control the production of specific virulence determinants of infection when necessary (e.g., protein binding initially when cell density is low and adhesion to host tissue is important, and toxins and degradative exoenzymes when infection is established and nutrients need to be obtained from host tissue).

[0057] Multiple clinical isolates of different CoNS species inhibited protease activity and prevented epithelial damage in both in vitro and in vivo without altering the abundance of S. aureus (e.g., inhibited the biological activity of protease / agr activity without altering S. aureus density). Furthermore, the present disclosure indicates that patients with active AD exhibit a decrease in the relative abundance of these beneficial microorganisms (e.g., CoNS) compared to S. aureus, thereby overcoming the inhibition of quorum sensing and enabling barrier disruption by S. aureus. Taken together, the present disclosure demonstrates how members of the normal human skin microbiome maintain immune homeostasis by contributing as a community to the control of S. aureus toxin production.

[0058] The present disclosure also identified polynucleotide sequences, polypeptide sequences, and fragments thereof that provide a product inhibiting agr quorum sensing activity. These polynucleotides and polypeptides may be used to provide therapeutic agents and recombinant non-pathogenic or attenuated skin bacteria for use in topical formulations for treating S. aureus infection and / or atopic dermatitis.

[0059] For example, the present disclosure provides an auto-induced peptide (AIP) that downregulates agr activity. A polynucleotide encoding the AIP is also provided herein.

[0060] The present disclosure provides a correlation between increased S. aureus colony formation and increased serine protease activity in AD skin, and provides novel targets and therapies comprising, but not limited to, fermented extracts for upregulating protease activity (e.g., fermented extracts from S. aureus) or fermented extracts from symbiotic bacteria for downregulating protease activity in skin (e.g., containing one or more of the AIPs of the present disclosure). Furthermore, the present disclosure provides topical formulations comprising (i) such extracts or purified AIP peptides, and (ii) symbiotic probiotic bacteria (e.g., non-pathogenic or attenuated bacteria transformed with an AIP coding sequence, or a topical formulation of purified symbiotic bacteria). Additional therapeutic targets may be antibodies against KLK, and / or DSG-1 and / or FLG therapies (e.g., increased expression or delivery of these factors to AD subjects).

[0061] In one embodiment, the AIP polypeptide of the present disclosure is X1X2X3X4CX5X6X7X 8(It has a common sequence of sequence number: 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; X8 is F, L, or Y, where amino acids 5-9 of sequence number: 10 form a thiolactone ring. Exemplary peptide sequences belonging to the common sequence of SEQ NO: 10 include SYNVCGGYF (SEQ NO: 4), KYNPCSNYL (SEQ NO: 11), SYSPCATYF (SEQ NO: 12), SQTVCSGYF (SEQ NO: 13), GANPCALYY (SEQ NO: 14), TINTCGGYF (SEQ NO: 15), VQDMCNGYF (SEQ NO: 16), and GYSPCTNFF (SEQ NO: 17). In additional embodiments, the polypeptide produces a structure of formula I or IA. In another embodiment, the polypeptide may comprise a combination of D- or L-amino acids. In any one of the foregoing embodiments, the polypeptide inhibits S. aureus protease activity, agr activity, or keratinocyte protease activity.

[0062] The present disclosure provides a compound of Formula I.

[0063]

[0064] Chemical Formula I

[0065] Here, X1 is 1-6 amino acids; X2 is an amino acid selected from valine (V), proline (P), methionine (M) and threonine (T); where R 5 Is , and Selected from a group consisting of; where R 6 silver , and Selected from a group consisting of; where R 7 silver , and Selected from a group consisting of; where R 8 silver , and It is selected from a group consisting of.

[0066] In one embodiment, the present disclosure provides a compound of formula IA:

[0067]

[0068] Chemical formula IA

[0069] Here, X1 is 1-6 amino acids; X2 is an amino acid selected from valine (V), proline (P), methionine (M) and threonine (T); where R 1 silver , and Selected from a group consisting of; where R 2 Is , and Selected from a group consisting of; where R 3 silver , and Selected from a group consisting of; where R 5 Is , and Selected from a group consisting of; where R 6 silver , and Selected from a group consisting of; where R 7 silver , and Selected from a group consisting of; where R 8 silver , and It is selected from a group consisting of.

[0070] The present disclosure provides a purified polypeptide (e.g., AIP peptide) comprising a sequence at least 98% identical to SEQ ID NO: 4, which inhibits (i) protease production and / or protease activity of keratinocytes, (ii) IL-6 production and / or activity of keratinocytes, (iii) production of phenol-soluble modulin alpha 3 from Staphylococcus aureus (S. aureus), and / or (iv) agr production and / or activity by S. aureus. In another embodiment, the present disclosure provides a compound of formula IB:

[0071]

[0072] Chemical formula IB

[0073] In another embodiment, the present disclosure provides a purified polypeptide comprising or composed of SEQ ID NOs: 4, 11, 12, 13, 14, 15, 16, or 17. In a further embodiment, the polypeptide forms a structure of Formula I, IA, or IB.

[0074] In one embodiment, the AIP peptide of the present disclosure may comprise one or more D-amino acids.

[0075] The present disclosure provides a topical formulation comprising an AIP peptide having the common sequence of SEQ ID NO: 10, a peptide of SEQ ID NO: 4, 11, 12, 13, 14, 15, 16, or 17, or a compound of formula I, IA, or IB.

[0076] "Substantially identical" means that while the amino acid sequences are generally identical, they are not identical in whole, but retain the functional activity of the associated sequences. The percentage of identity regarding the sharing of polypeptide or polynucleotide sequences is based on sequence alignment. It is common in the relevant technical field to perform alignment and determine identity using various programs. Generally, two polypeptides or domains are "substantially identical" if their sequences are at least 85%, 90%, 95%, 98%, or 99% identical, or if there is a conservative variation in the sequences. Computer programs, such as the BLAST program (Altschul et al. Sequence identity can be compared using ( , 1990).

[0077] The present disclosure also provides a polynucleotide encoding the AIP polypeptide of the present disclosure (i.e., "AIP polynucleotide"). For example, the present disclosure provides a polynucleotide encoding SEQ ID NO: 2 or 4. In one embodiment, the polynucleotide hybridizes to a polynucleotide consisting of SEQ ID NO: 3 under strict conditions and codes for the polypeptide of SEQ ID NO: 4. The "strictness" of the hybridization reaction can be easily determined by a person skilled in the art and is generally an empirical calculation that depends on probe length, washing temperature, and salt concentration. Generally, longer probes require higher temperatures for proper annealing, while shorter probes require lower temperatures. Hybridization generally depends on the ability of denatured DNA to re-anneal when the complementary strand is present in an environment below its melting temperature. The higher the desired degree of homology between the probe and the hybridizable sequence, the higher the relative temperature that can be used. Consequently, the higher the relative temperature, the stricter the reaction conditions will be, while the lower the temperature, the less strict they tend to be. Further details and explanations regarding the rigor of hybridization reactions are in the literature [Ausubel et al.Refer to [Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995)]. “Strict conditions” or “high severity conditions” as defined herein typically include: (1) using low ionic strength and high temperature for washing, e.g., 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50°C; or (2) using a denaturant, e.g., formamide, e.g., 50% (v / v) formamide and 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer and 750 mM sodium chloride, 75 mM sodium citrate at pH 6.5 at 42°C during hybridization; (3) High-rigidity washing consisting of 50% formamide, 5xSSC (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 μg / ml), 0.1% SDS, and 10% dextran sulfate at 42°C, washing in 0.2xSSC (sodium chloride / sodium citrate) at 42°C, washing in 50% formamide at 55°C, and washing in 0.1xSSC containing EDTA at 55°C. The polynucleotide sequences encoding SEQ NOs: 11, 12, 13, 14, 15, 16 and 17 can be inferred using a codon chart.

[0078] AIP polynucleotides can be cloned into various vectors for use in the present disclosure. For example, AIP polynucleotides can be cloned into expression vectors or plasmids for use in transformation and / or expression in recombinant host cells. Vectors for use in bacterial transformation are known. The four main types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. Common to all engineered vectors are replication origins, multiple cloning sites, and selectable markers. Any of these are suitable for use in the present specification. AIP polynucleotides can be inserted into clones, vectors, shuttles, plasmids, or BACs, or incorporated into bacterial genomes. If plasmids are used, the copy number of the plasmid may be 5 to 500 copies per cell. Exemplary plasmids and expression vectors include, but are not limited to: 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, pC30il(Skaugen 1989), pCD034-1, pCD034-2, pCD256, pC12000, pC1305, pC1528, pCIS3, pCL2.1, pCT1138, pD125, pE194, pE194 / PLS1, pEGFP-C1, pEH, pF8801, pFG2, pFK-시리즈, pGK-시리즈, pGK12, pGK13, pIA, pIAV1,5,6,7,9, pIL.CatT, pIL252 / 3, pIL253, pIL7, pISA(E. 콜라이( E. coli )의 경우 낮음), 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 및 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; van 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), plul631, L. reuteri possessing the erythromycin-resistance gene ( L. reuteri 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, pC194 and pE194 are B. subtilis (B. subtilis Not functional in ) (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, System BetL.

[0079] In one embodiment, the present disclosure provides a topical composition comprising the AIP polypeptide or peptide of the present disclosure. For example, in one embodiment, the topical composition comprises a purified polypeptide (e.g., AIP peptide) comprising the common sequence of SEQ ID NO: 10, or a sequence that is at least 98% identical to SEQ ID NO: 4, 11, 12, 13, 14, 15, 16, or 17, which inhibits (i) protease production and / or protease activity of keratinocytes, (ii) IL-6 production and / or activity of keratinocytes, (iii) production of phenol-soluble modulin alpha 3 from Staphylococcus aureus (S. aureus), and / or (iv) 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).

[0080] In another embodiment, the topical composition may comprise a non-pathogenic microorganism (including an attenuated microorganism engineered to reduce or eliminate pathogenic activity), wherein the microorganism is engineered to express an AIP polypeptide. The microorganism may be engineered to contain a vector and / or an AIP polynucleotide. In one embodiment, the microorganism produces a compound of Formula I, IA and / or IB.

[0081] In one embodiment, the composition and method of this specification use non-pathogenic bacteria engineered to produce compounds of Formula I, IA and / or IB by transforming the bacteria with the AIP polynucleotide of this disclosure. In one embodiment, the bacterial population is a non-pathogenic and non-invasive microorganism, and in certain embodiments, may be a Gram-positive food-grade bacterial strain. In another embodiment, the bacterial population transformed is prepared from bacteria that occur naturally in the skin microbiome.

[0082] In certain embodiments, bacteria formed in the composition and transformed to express compounds of formulas I, IA and / or IB may be a collection of the same bacteria or a mixture of different bacteria at different phylogenetic levels. Bacteria residing on healthy human skin are Corynebacterium ( Corynebacterium ) genus and Propionibacterium ( Propionibacterium Actinobacteria, including bacteria of the genus Actinobacteria It includes bacterial species that typically reside on the human face, such as ). In another embodiment, bacteria residing on the skin of a healthy human subject are, for example, Bacteroidetes ( Bacteroidetes ) and Proteobacteria( Proteobacteria It includes bacterial species that typically reside on skin other than the face, including bacteria of the genus. Other bacteria in the skin microbiome include those listed in the present specification below.

[0083] In one embodiment, the bacterium is Propiobacterium acidifaciens ( Propionibacterium acidifaciens ), Propiobacterium acidipropionis ( Propionibacterium acidipropionici ), Propiobacterium acidipropioni( Propionibacterium acidipropionici ) Strain 4900, Propiobacterium acnes( Propionibacterium acnes ), Propiobacterium australiens( Propionibacterium australiense ), Propiobacterium abidum ( Propionibacterium avidum ), Propiobacterium cyclohexanicum ( Propionibacterium cyclohexanicum ), Propiobacterium preudenreikii subspecies preudenreikii( Propionibacterium freudenreichii subsp. . Freudenreichii ), P. preudenreikii subspecies preudenreikii( P. freudenreichii ssp. freudenreichii ) Strain 20271, Propiobacterium preudenreikii subspecies shermanii( Propionibacterium freudenreichii subsp. of Sherman ), P. preudenreikii subspecies shemanii( P. freudenreichii ssp. shermanii ) Strain 4902, P. preudenreikii subspecies shermanii( P. freudenreichii ssp. shermanii) Strain 4902, Propiobacterium granulosum( Propionibacterium granulosum ), Propiobacterium inocium( Propionibacterium innocuous ), P. Jensenii( P. jensenii ) Strain 20278, Propiobacterium lymphophyllum( Propionibacterium lymphophilus ), Propiobacterium microarrowphyllum ( Propionibacterium microaerophilum ), Propiobacterium propionicum ( Propionibacterium propionicum ), Propiobacterium toenii( Propionibacterium thoenii ), and P. Toenii( P. thoenii It originates from the genus Propiobacterium, including but not limited to strain 20277. In one embodiment, the bacteria is not Propiobacterium acnes. In one embodiment, the bacteria is C. accholens ( C. accolens ), C. Afermentan( C. afermentan ), C. amicolatum( C. amycolatum ), Corynebacterium argentoratens( C. argentoratense ), Corynebacterium aquaticum( C. aquaticum ), Corynebacterium auris( C. auris ), Corynebacterium bovis ( C. ox ), Corynebacterium diphtheria( C. diphtheria ), C. Equi( C. equine )(currently Rhodococcus equi( Rhodococcus equi )), C. flavescens( C. yellow ), C. glucuronolaiticum( C. glucuronolyticum ), C. glutamicum ( C. glutamicum ), C. granulosum( C. granulosum ), C. Haemollaiticum( C. haemolyticum ), C. Halopitica( C. halophytica ), C. J.K.ium( C. jeikeium )(Military JK), C. McGinley( C. macginleyi ), C. Matruchoti ( C. matruchotii ), C. minutisimum( C. minutissimum ), C. Parbum( C. small )(Propionibacterium acnes), C. propincum( C. close relative ), C. Pseudodiphtericum( C. pseudodiphtheria )(C. Hoffmann-Nye( C. hofmannii )), C. pseudotuberculousis( C. pseudotuberculosis )(C. Obis( C. sheep )), C. Phyogenes( C. pyogenes ), C. urealyticum( C. urealyticum )(Group D2), C. Renale( C. renal ), C. Spec( C. spec. ), C. Striatum( C. striatum ), C. Tenuis( C. tenuis ), C. Ulcerance( C. ulcerans ), C. urealyticum( C. urealyticum ) and C. xerosis( C. xerosis It originates from the genus Corynebacterium, including but not limited to ). Bacteria having lipophilic and non-lipophilic groups are considered, and non-lipophilic bacteria may include fermenting Corynebacteria and non-fermenting Corynebacteria. In one embodiment, the bacteria are not C. diphtheria, C. amicolatum, C. striatum, C. zecheum, C. urealyticum, C. xerosis, pseudotuberculousis, C. tenuis, C. striatum, or C. minutismum, because they may be pathogenic. In one embodiment, the bacteria are the GRAS bacterial species Artrobacter arylaitensis ( Arthrobacter arilaitensis ), Artrobacter bergerei ( Arthrobacter bergerei ), Artrobacter globiformis( Arthrobacter globiformis ), Artrobacter Nicotianae ( Arthrobacter nicotianae ), Cochlea rhizophila( Kocuria rhizophila ), Cochlea Variants( Kocuria varians ), Micrococcus luteus( Micrococcus luteus ), Micrococcus Lilae ( Micrococcus lylae ), Microbacterium gubeenens ( Microbacterium gubbeenense ), Brevibacterium aurantiacum ( Brevibacterium aurantiacum ), Brevibacterium casei( Brevibacterium casei ), Brevibacterium linens( Brevibacterium linens ), Brachibacterium allimentarium( Brachybacterium alimentarium ) and Brachibacterium tyrofermentans( Brachybacterium thyrofermentansIn microcosinea including but not limited to ) Micrococcinea It originates from the ) suborder. In another embodiment, the bacteria are Staphylococcus agnetis ( Staphylococcus aureus ), S. Arletae( S. arlettae ), S. Auricularis( S. auricularis ), S. Capitis( St. of the head ), S. Capra ( S. caprae ), S. Carnossus( S. carnosus ), Staphilococcus caseeoliticus( Staphylococcus caseolyticus ), S. Chromogenes S. chromogenes ), S. Konii( S. cohnii ), S. Condiment( Holy condiment ), S. Delphini( S. delphini ), S. Debrie Essay (S. devriesei ) , S. Equorum Horse health ), S. Felice( S. felis ), S. Pleureti ( S. fleurettii ), S. Gallinarum( S. hens ), S. Hemolitisus( S. haemolyticus ), S. Hominis( Holy man ), S. Hikers S. hyicus ), S. Intermedius( S. intermedius ), S. cloosyi( S. kloosii ), S. Reay( S. leei ), S. Lentus( S. lentus ), S. lugdunensis( S. lugdunensis ), S. Lutrae( S. otter ), S. masiliensis( S. massiliensis ), S. Microti( S. microti ), S. Muscaé( S. flies ), S. nephilensis( S. nepalensis ), S. Pasteuri( St. pastor ), S. Pettenkoferry ( S. pettenkoferi ), S. piscipermentans( S. piscifermentans ), S. Shudintermedius( S. pseudintermedius ), S. pseudorugdunensis( S. pseudolugdunensis ), S. Pulverelli( S. pulvereri ), S. Rostra( S. rostra ), S. Sacharoticus( S. saccharolyticus ), S. Sapropaticus( S. saprophyticus ), S. Schleifery ( S. schleiferi ), S. Sushi Woori( S. squirrels ), S. Simiae( S. simiae ), S. Simulans( S. simulans ), S. Stefanovici ( S. stepanovicii ), S. succinus( S. amber ), S. Bitulinus( S. vitulinus ), S. Warneri( S. warneri ) and S. xylosus( S. xylosus It originates from the genus Staphylococcus, including but not limited to ). In one embodiment, the bacteria is not S. aureus or S. epidermidis. In another embodiment, the bacteria is Streptococcus acidomimus ( Streptococcus acidominimus ), Streptococcus adjacens( Streptococcus adjacens ), Streptococcus agalactiae( Streptococcus agalactiae ), Streptococcus alactolyticus( Streptococcus alactolyticus ), Streptococcus anginosus ( Streptococcus anginosus ), Streptococcus australis( Streptococcus australis ), Streptococcus bovis( Streptococcus bovis ), Streptococcus cavalli( Streptococcus cavalli ), Streptococcus canis( Streptococcus canis ), Streptococcus caprinus( Streptococcus caprineus ), Streptococcus castoreus( Streptococcus castoreum ), Streptococcus secorum( Streptococcus cecum ), Streptococcus constellatus( Streptococcus constellatus ), Streptococcus constellatus subspecies constellatus( Constellated ), Streptococcus constellatus subspecies paringis( Pharynx ), Streptococcus cremoris( Streptococcus cremoris ), Streptococcus chrysetti( Hamster Streptococcus ), Streptococcus cristatus( Streptococcus cristatus ), Streptococcus Danieliae( Streptococcus danielii ), Streptococcus defectives( Streptococcus defectives ), Streptococcus dentapri( Streptococcus dentaris ), Streptococcus dentiroseti( Streptococcus dentirousetti ), Streptococcus didelphis( Streptococcus didelphis ), Streptococcus difficilis( Streptococcus difficile ), Streptococcus durans( Streptococcus duranus ), Streptococcus disgalactiae( Streptococcus dysgalactiae ), Streptococcus disgalactiae subspecies disgalactiae( Streptococcus dysgalactiae subsp. Dysgalactiae ), Streptococcus disgalactiae subspecies equisimilis( Streptococcus equi subsp. Equivalent ), Streptococcus entericus( Enteric Streptococcus ), Streptococcus equi( Streptococcus equi ), Streptococcus equity subspecies equity ( Streptococcus equi subsp. Equi ), Streptococcus equi subspecies luminatorum( Ruminants ), Streptococcus equi subspecies Zuephidemicus ( Zooepidemic ), Streptococcus equines( Streptococcus equine ), Streptococcus faecalis( Streptococcus faecalis ), Streptococcus faecium( Streptococcus faecium ), Streptococcus ferus( Streptococcus ferus ), Streptococcus galinaceus( Streptococcus gallinaceous ), Streptococcus galoliticus( Streptococcus gallolyticus ), Streptococcus galoliticus subspecies galoliticus( Streptococcus gallolyticus subsp. Gallolytic ), Streptococcus galoliticus subspecies macedonicus( Macedonian ), Streptococcus galoliticus subspecies Pasteurianus( Pasteurian ), Streptococcus garbieae ( Streptococcus garvieae ), Streptococcus gordoniae ( Streptococcus gordonii ), Streptococcus halicoeri ( Streptococcus halichoeri ), Streptococcus hansenii( Streptococcus hansenii ), Streptococcus henri ( Streptococcus henryi ), Streptococcus hyointestinalis( Streptococcus hyointestinalis ), Streptococcus hyobaginalis(S Streptococcus hyovaginalis ), Streptococcus ichthalluri ( Streptococcus ictaluri ), Streptococcus infantarius( Streptococcus infantis ), Streptococcus infantarius subspecies coli ( Streptococcus infantarius subsp. Cultivate ), Streptococcus infantarius subspecies infantarius( Infantryman ), Streptococcus infantis( Streptococcus infantis ), Streptococcus ainiae( Streptococcus pneumoniae ), Streptococcus intermedius( Streptococcus intermedius ), Streptococcus intestinalis( Streptococcus intestinalis ), Streptococcus lactarius( Lactococcus lactis ), Streptococcus lactis( Streptococcus lactis ), Streptococcus lactis subspecies cremoris( Streptococcus lactis subsp. Creamy ), Streptococcus lactis subspecies diacetyllactis ( Diacetylactide ), Streptococcus lactis subspecies lactis ( Milk ), Streptococcus lutetiensis( Streptococcus lutetensis ), Streptococcus macacae( Streptococcus macaque ), Streptococcus macedonicus( Streptococcus macedonicus ), Streptococcus marimammalium( Streptococcus marimammalius m), Streptococcus masiliensis ( Streptococcus massiliensis ), Streptococcus mellionis( Streptococcus merionis ), Streptococcus minor( Streptococcus minor ), Streptococcus mitis( Streptococcus milius ), Streptococcus morbilorum( Streptococcus measles ), Streptococcus mutans( Streptococcus mutans ), Streptococcus oligofermentans( Streptococcus oligofermentans ), Streptococcus oralis( Oral Streptococcus ), Streptococcus odoris ( Streptococcus orisrattus ), Streptococcus ovis( Streptococcus ovis ), Streptococcus parasanguinis( Streptococcus parasanguinis ), Streptococcus parauberis( Streptococcus parauberis ), Streptococcus parbulus( Streptococcus minor ), Streptococcus Pasteurianus( Streptococcus pasteurianus ), Streptococcus ferroris( Streptococcus pneumoniae ), Streptococcus pocae( Streptococcus phocae ), Streptococcus plantarum( Streptococcus plantarum ), Streptococcus pleomorphus( Streptococcus pleomorphus ), Streptococcus pluranimalium( Streptococcus pluranimum ), Streptococcus fluorextrorum( Streptococcus pluronicum ), Streptococcus pneumoniae( Streptococcus pneumoniae ), Streptococcus forsi( Streptococcus porcine ), Streptococcus forcinus ( Streptococcus porcine ), Streptococcus porcorum( Streptococcus porcine ), Streptococcus pseudopneumoniae ( Streptococcus pseudopneumoniae ), Streptococcus pseudoporcinus ( Streptococcus pseudoporcine ), Streptococcus pyogenes( Streptococcus pyogenes ), Streptococcus raffinolactis( Streptococcus raffinolactis ), Streptococcus lati( Streptococcus rattus ), Streptococcus rupicaprae( Streptococcus rupicaprae ), Streptococcus saccharoliticus( Saccharolytic Streptococcus ), Streptococcus salivarius( Streptococcus salivarius ), Streptococcus salivarius subspecies salivarius( Streptococcus salivarius subsp. Saliva ), Streptococcus salivarius subspecies thermophilus( Thermophilus ), Streptococcus sanguinis( Streptococcus sanguis ), Streptococcus siloi( Streptococcus shiloi ), Streptococcus sinensis ( Streptococcus sinensis ), Streptococcus sobrinus( Streptococcus sobrinus ), Streptococcus suis( Streptococcus suis ), Streptococcus thermophilus( Streptococcus thermophilus ), Streptococcus toraltensis( Streptococcus thoraltensis ), Streptococcus tigrinus( Streptococcus tigurinus ), Streptococcus trogloditae( Streptococcus troglodytes ), Streptococcus trogloditedis( Streptococcus troglodytidis ), Streptococcus uberis( Streptococcus uberis), Streptococcus urinalis( Streptococcus urinary ), Streptococcus vestibularis( Vestibular Streptococcus ) and Streptococcus wyius( Streptococcus vaius It originates from the genus Streptococcus, including but not limited to ). In another embodiment, the bacteria are Lactococcus garbiae ( Lactococcus garvieae ), Lactococcus lactis( Lactococcus lactis ), Lactococcus lactis subspecies cremoris ( Lactococcus lactis subsp. cremoris ), Lactococcus lactis subspecies in Hordnia ( hordes ), Lactococcus lactis, Lactococcus lactis subspecies lactis( Milk ), Lactococcus pistisium( Lactococcus piscifolia ), Lactococcus plantarum( Lactococcus plants ), Lactococcus raffinolactis( Lactococcus raffinolactis ), Lactobacillus acetotolerans ( Lactobacillus acetotolerans ), Lactobacillus acidophilus( Lactobacillus acidophilus ), Lactobacillus agilis( Lactobacillus agilis ), Lactobacillus algidus( Lactobacillus algidus ), Lactobacillus alimentarius( Lactobacillus alimentarius ), Lactobacillus amyloricus( Lactobacillus amylolyticus ), Lactobacillus amylopillus( Lactobacillus amylophilus ), Lactobacillus amylovorus( Lactobacillus amylovorus ), Lactobacillus animalis( Lactobacillus animalis ), Lactobacillus aviarius( Lactobacillus aviarius ), Lactobacillus aviarius subspecies arapinosus ( Lactobacillus aviarius subsp. araffinate ), Lactobacillus aviarius subspecies aviarius( aviary ), Lactobacillus barbaricus( Lactobacillus bavaria ), Lactobacillus bifermentans( Lactobacillus bifermentans ), Lactobacillus brevis ( Lactobacillus brevis ), Lactobacillus buccneri( Lactobacillus buchneri ), Lactobacillus bulgaricus( Lactobacillus bulgaricus ), Lactobacillus carnis( Lactobacillus carnivorus), Lactobacillus casei ( Lactobacillus casei ), Lactobacillus casei subspecies allactosus ( Lactobacillus casei subsp. milky ), Lactobacillus casei subspecies casei( cheese ), Lactobacillus casei subspecies Pseudoplantarum( pseudoplants ), Lactobacillus casei subspecies rhamnosus (rhamnosus) ), Lactobacillus casei subspecies tolerance ( tolerant ), Lactobacillus catenaformis( Lactobacillus catenaformis ), Lactobacillus cellobiosus ( Lactobacillus cellobius ), Lactobacillus colinoides( Lactobacillus collinoides ), Lactobacillus confusus ( Lactobacillus confuscus ), Lactobacillus coriniformis( Lactobacillus coryniformis ), Lactobacillus corniformis subspecies corniformis( Lactobacillus coryniformis subsp. coryniform ), Lactobacillus coriniformis subspecies torquens( twisting ), Lactobacillus crispatus( Lactobacillus crispatus ), Lactobacillus curvatus( Lactobacillus curvatus ), Lactobacillus curvatus subspecies curvatus ( Lactobacillus curvatus subsp. curvatus ), Lactobacillus curbatus subspecies melibiosus ( honey-scented ), Lactobacillus delbrueckii ( Lactobacillus delbrueckii ), Lactobacillus delbrueckii subspecies bulgaricus( Lactobacillus delbrueckii subsp . Bulgarian ), Lactobacillus delbrueckii subspecies delbrueckii ( delbrueckii ), Lactobacillus delbrueckii subspecies lactis ( milk ), Lactobacillus dibergens( Lactobacillus divergens ), Lactobacillus parciminis ( Lactobacillus farciminis ), Lactobacillus fermentum( Lactobacillus fermentum ), Lactobacillus formalicalis ( Lactobacillus formicus ), Lactobacillus fructivorans( Lactobacillus fructivorans ), Lactobacillus fructosus ( Lactobacillus fructosus ), Lactobacillus galinarum( Lactobacillus gallinum ), Lactobacillus gasseri( Lactobacillus gasseri ), Lactobacillus graminis( Lactobacillus graminis ), Lactobacillus halotolerans( Lactobacillus halotolerans ), Lactobacillus hamsteri( Lactobacillus hamsteri ), Lactobacillus helveticus( Lactobacillus helveticus ), Lactobacillus heterothiocchii( Lactobacillus heterochii ), Lactobacillus hyalgardi ( Lactobacillus hilgardii ), Lactobacillus homohiocii ( Lactobacillus homohiochii ), Lactobacillus iners ( Lactobacillus inert ), Lactobacillus intestinalis( Lactobacillus intestinalis ), Lactobacillus gensenii( Lactobacillus jensenii ), Lactobacillus johnsonii ( Lactobacillus johnsonii ), Lactobacillus candleteri( Lactobacillus kandleri ), Lactobacillus kefiri( Lactobacillus kefiri ), Lactobacillus cepuranofaciens( Lactobacillus kefuranofaciens ), Lactobacillus kefirgranum( Lactobacillus kefir grain ), Lactobacillus cuncea ( Lactobacillus kunkeei ), Lactobacillus lactis( Lactobacillus lactis ), Lactobacillus reikmannii ( Lactobacillus leichmannii ), Lactobacillus lindenneri ( Lactobacillus lindneri ), Lactobacillus malefermentans( Lactobacillus malfermentans ), Lactobacillus mali ( Lactobacillus mali ), Lactobacillus maltaromicus( Lactobacillus maltaromicus ), Lactobacillus mannihotivorans( Lactobacillus manihotivorans ), Lactobacillus minor( Lactobacillus minor ), Lactobacillus minutus ( Lactobacillus minutus ), Lactobacillus mucosae( Lactobacillus mucosae ), Lactobacillus murinus( Lactobacillus murineus ), Lactobacillus nagelii( Lactobacillus nagelii ), Lactobacillus oris ( Lactobacillus oris ), Lactobacillus panis( Lactobacillus bread ), Lactobacillus parabukneri( Lactobacillus parabuchneri ), Lactobacillus paracasei( Lactobacillus paracasei ), Lactobacillus paracasei subspecies paracasei ( Lactobacillus paracasei subsp. cheese ), Lactobacillus paracasei subspecies tolerance ( tolerant ), Lactobacillus parakephiri ( Lactobacillus parakefiri ), Lactobacillus paralimentarius( Lactobacillus paraalimentarius ), Lactobacillus paraplantarum( Lactobacillus paraplantarum ), Lactobacillus pentosus ( Lactobacillus pentosus ), Lactobacillus ferulens( Lactobacillus perolens ), Lactobacillus piscicolas ( Lactobacillus piscicola ), Lactobacillus plantarum( Lactobacillus plantarum ), Lactobacillus pontis( Lactobacillus pontis ), Lactobacillus reuteri( Lactobacillus reuteri ), Lactobacillus rhamnosus ( Lactobacillus rhamnosus ), Lactobacillus rhamnosus strain 5 / E5a, Lactobacillus limae( Lactobacillus cracks ), Lactobacillus logosa ( Lactobacillus rogosae ), Lactobacillus luminis( Lactobacillus rumen ), Lactobacillus sakei( Lactobacillus sakei ), Lactobacillus sakei subspecies camosus ( Lactobacillus sakei subsp. grey ), Lactobacillus sakei subspecies sakei( sake ), Lactobacillus salivarius( Lactobacillus salivarius ), Lactobacillus salivarius subspecies salicinius ( Lactobacillus salivarius subsp. willow tree ), Lactobacillus salivarius subspecies salivarius( salivary ), Lactobacillus sanfrancisensis( Lactobacillus sanfranciscensis ), Lactobacillus sativae ( Lactobacillus sharpeae ), Lactobacillus suevicus ( Lactobacillus suebicus ), Lactobacillus trichodes( Lactobacillus trichodes ), Lactobacillus woolly ( Lactobacillus uli ), Lactobacillus vaccinostercus ( Lactobacillus vaccinostercus ), Lactobacillus vaginalis ( Lactobacillus vaginalis ), Lactobacillus viridescens( Lactobacillus viridescens ), Lactobacillus bitulinus( Lactobacillus vitulinus ), Lactobacillus xylostosus( Lactobacillus xylosus ), Lactobacillus yamanashiensis( Lactobacillus yamanashiensis ), Lactobacillus yamanaxiensis subspecies Mali( Lactobacillus yamanashiensis subsp. mali ), Lactobacillus yamanashiensis subspecies yamanashiensis( Yamanashiensis ) and Lactobacillus zea( Lactobacillus zeae It originates from the genus Lactobacillus, including but not limited to ). In another embodiment, the bacteria are Lactococcus schleifer ( Lactococcus Schleifer ), Lactococcus chungangensis( Lactococcus chungangensis ), Lactococcus fuziensis( Lactococcus fujiensis ), Lactococcus garviae, Lactococcus lactis, Lactococcus lactis subspecies cremoris, Lactococcus lactis subspecies hordniae, Lactococcus lactis subspecies lactis, Lactococcus lactis subspecies tructae( Tructae It is derived from the genus Lactococcus, including but not limited to ), Lactococcus plantarum, and Lactococcus raffinolacti.

[0084] In another embodiment, the present disclosure provides a probiotic composition for topical delivery comprising the CoNS symbiotic skin bacteria of the present disclosure. In one embodiment, the CoNS bacteria comprise bacteria that produce an AIP polypeptide and / or a compound of Formula I. In a further embodiment, the topical composition contains only a single species of microorganism that produces the AIP polypeptide or a compound of Formula I. In another embodiment, the symbiotic skin bacteria of the present disclosure comprise microorganisms selected from the group consisting of S. epidermidis A11, S. hominis A9, S. hominis C4, S. hominis C5, and S. warneri G2. In another embodiment, the topical probiotic composition of the present disclosure may comprise or consist of symbiotic 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.

[0085] The symbiotic bacteria of the present disclosure can be isolated from human skin and identified using the methods described herein. For example, the present disclosure provides a method for obtaining, identifying, and culturing the symbiotic bacteria described herein by swabbing the surface of human skin, for example, using a foam tip swab. The swab is placed in tryptic soy broth. The broth is diluted onto a mannitol salt agar plate (MSA) supplemented with 3% egg yolk. Halo-free pink colonies representing coagulase-negative Staphylococcus (CoNS) strains are collected and grown in tryptic soy broth (TSB) before adding 25 volume% of sterile-filtered supernatant to a S. aureus agr type I YFP reporter strain grown in fresh TSB (for measurement of S. aureus agr activity inhibition after 24 hours of incubation). The Agr activity of S. aureus reporter strains is measured using a fluorescence analyzer. S. aureus strains exhibiting strong inhibition of agr activity are further characterized by gDNA isolation and sequencing. gDNA is isolated using any number of commercially available kits (e.g., DNeasy UltraClean Microbial Kit, Qiagen). gDNA can be sequenced for two cycles using various sequencing platforms (e.g., MiSeq; Illumina Inc., San Diego, California), which can generate two 250 bp paired-end reads. Adapters are removed using cutadapt (e.g., refer to the World Wide Web at cutadapt.readthedocs.io / en / stable / ). Trim Galore with default parameters (e.g., bioinformatics.babraham.ac.Low-quality sequences can be removed using the World Wide Web at uk / projects / trim_galore / . Sequence mapping to the human genome was performed using the Bowtie 2 program (ver. 2.28) with the human reference genome hg19 and parameters (-D20-R3-N1-L20-highly-sensitive-local). I It is removed from the quality-trimmed dataset using ). Filtered reads are reassembled using SPAdes (version 3.8.0) with k-mer lengths ranging from 33 to 127. The genome is annotated with rapid annotation of microbial genomes using subsystems technology (RASY) with default parameters. Amino acid sequences (coding DNA sequences) from the annotated CDS are aligned to bacterial agr proteins obtained from the Uniprot database. From the assembled genome Agr Genes are identified according to the following three criteria: (i) sequence identity >60%, (ii) e-value <e100; 및 (iii) agr 유전자자리 조직화(locus organization), 4개의 유전자의 오페론, 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.

[0086] As used herein, the terms “probiotic composition,” “topical probiotic composition,” or “probiotic skin composition” comprise a composition comprising probiotic symbiont skin bacteria, a fermented extract of probiotic symbiont skin bacteria, an attenuated or engineered microorganism expressing an AIP polypeptide, an agent that (i) inhibits protease activity or (ii) promotes protease activity, and a pharmaceutical carrier that maintains the viability of the symbiont skin bacteria.

[0087] As used herein, the term “local” may include administration outside the skin as well as shallow injection (e.g., intradermal and lesion) so that the topical probiotic composition comes into direct contact with the skin.

[0088] As used herein, the term “fermented extract” refers to a product produced by fermenting probiotic symbiotic skin bacteria in a culture and under appropriate fermentation conditions. For example, a culture of S. aureus can produce PSMα3, which is useful for increasing skin barrier permeability. Extracts from S. aureus contain PSMα3, which can be applied to the skin to improve permeability, induce skin remodeling, or promote skin barrier permeability for drug delivery. Similarly, fermented extracts of CoNS bacteria producing AIP of this disclosure may be cultured, and extracts from such cultures are used to inhibit S. aureus-associated pathologies (e.g., protease activity, dermatitis, etc.).

[0089] As used herein, the term “probiotic symbiont skin bacteria” includes microorganisms of the skin microbiome. Probiotic symbiont skin bacteria may include a composition of bacteria that promote protease activity (“protease-promoting probiotic symbiont skin bacteria”). Protease-promoting probiotic symbiont skin bacteria are typically bacteria of the skin that produce phenol-soluble module alpha 3 (PSMα3). A composition of protease-promoting probiotic symbiont skin bacteria (or its fermentation extract) is useful, for example, for promoting skin remodeling, wound healing, aging, sun damage, pigmentation abnormalities, and scar formation. In one embodiment, protease-promoting probiotic symbiont skin bacteria comprises one or more bacteria that have serine protease activity and / or induce serine protease activity in the skin. For example, protease-promoting probiotic symbiont skin bacteria may include a strain of S. aureus that produces phenol-soluble module alpha 3 (PSMα3).

[0090] In another embodiment, the probiotic symbiont skin bacteria may comprise a composition of bacteria that inhibit protease activity ("protease inhibiting probiotic symbiont skin bacteria"). The protease-inhibiting probiotic symbiont skin bacteria composition is useful for treating diseases such as rosacea, atopic dermatitis, and Netterton syndrome. In one embodiment, the probiotic-inhibiting probiotic symbiont skin bacteria comprises one or more bacteria that inhibit the serine protease activity of other bacteria on the skin and / or inhibit the serine protease activity of the skin. For example, the protease-inhibiting probiotic symbiont skin bacteria may comprise a coagulase-negative Staphylococcus species. In one embodiment, the coagulase-negative strain is selected from the group consisting of Staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus saccharolicus, Staphylococcus warneri, Staphylococcus pasteuri, Staphylococcus hemoliticus, Staphylococcus debriesei, Staphylococcus hominis, Staphylococcus zetensis, Staphylococcus petracii, and Staphylococcus rugdunensis. In one embodiment, the protease-inhibiting commensal skin bacteria are selected from the group consisting of S. epidermidis strains, S. hominis strains, S. warneri strains, and any combination thereof. In a specific embodiment, 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 another specific embodiment, the S. warneri strain is S. warneri G2. In one embodiment, the CoNS bacteria include bacteria that produce the AIP polypeptide and / or a compound of Formula I. In a further embodiment, the topical composition contains only a single species of microorganism that produces the AIP polypeptide or a compound of Formula I.In another embodiment, the symbiotic skin bacteria of the present disclosure comprise microorganisms selected from the group consisting of S. epidermidis A11, S. hominis A9, S. hominis C4, S. hominis C5, and S. warneri G2. In another embodiment, the topical probiotic composition of the present disclosure may comprise or be composed of symbiotic 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.

[0091] The term "contacting" refers to exposing the skin to a topical probiotic composition so that the probiotic skin composition can modulate protease activity (e.g., serine protease activity) on the skin.

[0092] The terms "inhibiting" or "inhibiting effective amount" refer to an amount of a probiotic skin composition consisting of one or more probiotic microorganisms and / or fermentation media or extracts and / or fermentation byproducts and / or synthetic molecules sufficient to cause inhibition of protease activity (e.g., serine protease activity) on the skin or in a skin culture, for example. The term "inhibiting" also includes preventing or improving signs or symptoms of a disorder (e.g., rash, sore, etc.).

[0093] For the treatment of a subject suffering from a disease or disorder, the term “therapeutic effective dose” as used herein means an amount of a probiotic skin composition or its extract sufficient to improve the signs or symptoms of the disease or disorder. For example, the therapeutic effective dose may be measured as an amount sufficient to reduce the subject’s symptoms of dermatitis or rash by measuring the frequency of the severity of skin erosion. Typically, the 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 dose will depend on factors such as the disorder and the subject’s body weight, the type of bacteria, the subject’s gender, and the severity of the symptoms. Nevertheless, a suitable dose can be easily determined by a person skilled in the art.

[0094] The terms “purified” and “substantially purified” as used herein refer to cultures or co-cultures of microorganisms or biological agents that are substantially free of other cells or components found in the natural environment to which the agent is naturally associated, or co-cultures (e.g., fermentation media and extracts, fractional fermentation media, fermentation byproducts, AIP peptides, polypeptides, genes, polynucleotides, compounds of Formula I, etc.). In some embodiments, the co-culture probiotic may comprise a plurality of symbiotic skin bacteria.

[0095] The present disclosure provides whole-cell preparations comprising substantially homogeneous preparations of S. epidermidis, S. hominis, and / or S. warneri. Such preparations may be used in the preparation of compositions for the treatment of inflammation and microbial infections. The whole-cell preparations are S. epidermidis ,It may include S. hominis and / or S. warneri, or may include a non-pathogenic (e.g., attenuated microorganism) vector as described below. The present disclosure also provides a fraction derived from these whole cells comprising an agent that reduces protease activity in the skin derived from S. aureus activity.

[0096] The ability of the first bacterial composition to inhibit the protease activity of the second bacterial composition can be determined by contact by measuring the protease activity of the second bacterial composition before and after contacting the second composition with the first composition. Contact between the topical probiotic composition of the present disclosure and an organism may occur in vitro, for example, by adding the topical probiotic composition to a bacterial culture medium to test the protease inhibitory activity of the bacteria. Alternatively, contact may occur in vivo, for example, by contacting the topical probiotic composition with a subject suffering from a skin disease or disorder.

[0097] Probiotic symbiotic skin bacteria preparations may be manufactured in any number of ways. Topical probiotic compositions may be administered to a subject using any of the various methods known in the relevant art. For example, the probiotic skin compositions, extracts, or synthetic preparations of the present disclosure may be formulated for topical administration (e.g., lotions, creams, sprays, gels, or ointments). Such topical preparations are useful for treating or inhibiting the presence of microorganisms, fungi, or viruses, or infections or inflammation on the skin. Examples of preparations include topical lotions, creams, soaps, wipes, etc.

[0098] In another embodiment, a topical probiotic composition comprising a plurality of probiotic symbiotic skin bacteria is provided. When used for the treatment of dermatitis or other skin diseases or disorders associated with increased protease (e.g., serine protease) activity, the composition comprises one or more bacteria that inhibit protease activity on the skin. In this case, the probiotic symbiotic skin bacteria are coagulase-negative Staphylococcus species. In one embodiment, the probiotic symbiotic skin bacteria are selected from the group consisting of S. epidermidis strains, S. hominis strains, S. warneri strains, and any combination thereof. Where increased protease activity is required (e.g., for wound healing, skin remodeling, etc.), the probiotic symbiotic bacteria composition contains bacteria that increase protease activity or stimulate skin protease activity (e.g., serine protease activity). In this embodiment, the exemplary symbiotic bacterial composition will comprise S. aureus bacteria or virulence-attenuated S. aureus that produces PSMα3.

[0099] In another embodiment, the topical probiotic composition comprises a probiotic symbiotic skin bacteria fermentation extract that promotes protease activity on the skin. In various aspects, the bacteria from which the extract is produced include Staphylococcus aureus.

[0100] In another embodiment, a topical probiotic composition is provided that is essentially composed of S. aureus fermented extract alone or in combination with S. aureus. According to a further aspect, the topical probiotic composition may be formulated as a lotion, shake lotion, cream, ointment, gel, foam, powder, solid, paste, or tincture.

[0101] In another embodiment, the topical probiotic composition comprises a probiotic symbiotic skin bacteria fermentation extract. In various aspects, the bacteria from which the extract is produced comprise coagulase-negative Staphylococcus species. In one embodiment, the Staphylococcus species is selected from the group consisting of S. epidermidis strains, S. hominis strains, S. warneri strains, and any combination thereof that produce an AIP that inhibits the agr quorum sensing system and / or protease production in the skin or skin microbiome. In one embodiment, the AIP comprises a common sequence of SEQ ID NO: 10 or a sequence that is at least 98% identical to SEQ ID NO: 4, 11, 12, 13, 14, 15, 16, or 17 having agr quorum modulating activity, and / or a compound of Formula I, IA, or IB.

[0102] In another embodiment, a topical probiotic composition is provided that is essentially composed of a coagulase-negative Staphylococcus species fermentation extract or S. epidermidis fermentation extract alone or in combination with a coagulase-negative Staphylococcus species or S. epidermidis. In another embodiment, the composition comprises one or more deposited microbial strains described herein (e.g., S. epidermidis A11, S. hominis A9, S. hominis C5 and / or S. warneri G2).

[0103] According to additional embodiments, the topical probiotic composition may be formulated as a lotion, shake lotion, cream, ointment, gel, foam, powder, solid, paste, or tincture.

[0104] In another embodiment, a fermented extract is provided that can be obtained by fermenting bacteria selected from the group consisting of S. epidermidis strains, S. hominis strains, S. warneri strains, and any combination thereof under fermentation conditions. In various aspects, such fermented extracts can be used to inhibit serine protease activity 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 may be formulated as a lotion, shake lotion, cream, ointment, gel, foam, powder, solid, paste, or tincture.

[0105] In another embodiment, a bandage or dressing is provided comprising the topical probiotic composition described above, the probiotic symbiotic skin bacteria fermentation extract described above, the probiotic symbiotic skin bacteria described above, and any combination thereof. In various aspects, a bandage or dressing is provided comprising a matrix and probiotic symbiotic skin bacteria whose main components inhibit protease activity on the skin. In various aspects, a bandage or dressing is provided comprising a matrix and probiotic symbiotic skin bacteria whose main components inhibit protease activity on the skin.

[0106] In another embodiment, a bandage or dressing is provided comprising the topical probiotic composition described above, the probiotic symbiotic skin bacteria fermentation extract described above, the probiotic symbiotic skin bacteria described above, and any combination thereof. In various aspects, a bandage or dressing is provided comprising a matrix and probiotic symbiotic skin bacteria whose main components promote protease activity on the skin. In various aspects, a bandage or dressing is provided comprising a matrix and probiotic symbiotic skin bacteria whose main components promote protease activity on the skin.

[0107] The present disclosure also provides a method for treating a skin disease or disorder associated with protease (e.g., serine protease activity). Examples of such disease or disorder include Netterton syndrome, atopic dermatitis, contact dermatitis, eczema, psoriasis, acne, epidermal hyperkeratosis, acanthosis, epidermal inflammation, dermal inflammation, and pruritus. In one embodiment, the presence of the disease or disorder is first determined by measuring the protease activity of a sample (e.g., skin or a culture of bacteria from the skin) from a subject suspected of having the disease or disorder. If the sample is found to be higher than normal protease activity (e.g., serine protease activity), the subject's skin is treated with a protease-inhibiting commensal bacterial preparation by contacting the preparation. In another embodiment, a culture from a subject containing bacteria having high protease activity is contacted with an in vitro preparation to determine the susceptibility of the culture to the preparation and its effect on protease inhibition.

[0108] Protease-inhibiting symbiotic bacterial preparations or fermentation extracts may be combined with one or more known serine protease inhibitors. There are a number of commercially and clinically relevant serine protease inhibitors that may be used in the methods and compositions of the present disclosure. For example, serine protease inhibitors disclosed, for example, in U.S. Patent No. 5,786,328, U.S. Patent No. 5,770,568, or U.S. Patent No. 5,464,820 (the contents of which are incorporated herein by reference). Exemplary serine protease inhibitors include antibodies that bind to and inhibit serine protease polypeptides or their functional fragments, enzymes that degrade serine protease polypeptides into inactive peptides, substrate analogs, etc. Serine protease expression inhibitors include, for example, antisense molecules, ribozymes, and small molecule agonists (e.g., vitamin D antagonists) that reduce 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 comprise peptides having amino acid sequences corresponding to positions 388 through 390 of tissue kallikrein. The peptides may be produced synthetically or genetically by recombinant engineering techniques, such as the cloning and expression of nucleic acid sequences, or may be purified from natural sources, such as bacterial, fungal, or cellular extracts. The structural, chemical, physicochemical, nomenclature, and analytical aspects of the amino acids are described in the literature [Chemistry of the Amino Acids (J.P. Greenstein and M. Winitz editors, John Wiley & Sons, New York, NY, 1961, reprinted 1984)], which is specifically incorporated herein by reference.Peptides consist of modified and / or unmodified amino acids, including naturally occurring amino acids, non-naturally occurring (non-coding) amino acids, synthetically produced amino acids, and combinations thereof. Naturally occurring amino acids include glycine (Gly), amino acids with alkyl side chains, such as glycine (Gly), amino acids with alkyl side chains, such as alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), and proline (Pro), aromatic amino acids phenylalanine (Phe), tyrosine (Tyr) and tryptophan (Trp), amino acids alcohols serine (Ser) and threonine (Thr), acidic amino acids aspartic acid (Asp) and glutamic acid (Glu), amides of Asp and Glu, asparagine (Asn) and glutamine (Gln), sulfur-containing amino acids cysteine ​​(Cys) and methionine (Met), and basic amino acids histidine (His), lysine (Lys), and arginine (Arg). Non-naturally occurring amino acids include, for example, ornithine (Orn), norleucine (Nle), citralin (Cit), homo-citralin (hCit), desmosine (Des), and isodesmosine (Ide). Modified amino acids include derivatives and analogs of naturally occurring, non-naturally occurring, and synthetically produced amino acids. These forms of amino acids have been chemically modified by, for example, halogenation of one or more active sites with chlorine (Cl), bromine (Br), fluorine (F), or iodine (I); alkylation with carbon-containing groups, for example, methyl (Me), ethyl (Et), butyl (Bu), amino (NH2 or NH3), amidino (Am), acetomidomethyl (Acm), or phenyl (Ph) groups; or addition of phosphorus (P), nitrogen (N), oxygen (O), or sulfur (S)-containing groups. Modification may also be achieved, for example, by hydration, oxidation, hydrogenation, esterification, or cyclization of another amino acid or peptide, or precursor chemical. Examples include the amino acids hydroxylase and decarboxylase, monocytic amino acids, polyamino acids, and amino acid derivatives.Specific examples include gamma-aminobutyric acid (GABA), hydroxyproline (Hyp), aminoadipic acid (Aad) that can be modified at the 2 or 3 position, o-aminobutyric acid (Aab or Abu), selenocysteine ​​(SeCys2), tert-butylglycine (Bug or tert-BuGly), N-carbamyl amino acid, amino acid methyl ester, amino-propionic acid (or β-alanine; 13-Ala), adamantylglycine (Adg), aminocaproic acid (Acp), N-ethylasparagine (Et-Asn), allo-hydroxylysine (aHyl), allo-isoleucine (aIle), phenylglycine (Phg), pyridylalanine (Pal), thienylalanine (Thi), α-Δ-aminobutyric acid (Kbu), and α-β-diaminopropionic acid (Kpr). It includes 1- or 2-naphthylalanine (1Nal or 2Nal), orthofluorophenylalanine (Phe(oF)), N-methylglycine (MeGly), N-methyl-isoleucine (Melle), N-methyl-valine (MeVal), 2-amino-heptanoic acid (Ahe), 2- or 3-amino-isobutyric acid (Aib), 2-amino-pimelic acid (Dbu), 2-2'-diaminopimelic acid (Dpm), 2,3-diaminopropionic acid (Dpr), and N-ethylglycine (EtGly). Chemically produced non-coding amino acids include, for example, phenylglycine (Ph-Gly), cyclohexylalanine (Cha), cyclohexylglycine (Chg), and 4-aminophenylalanine (Phe(4NH2) or Aph). Modified amino acids may also be chemical structures that are not amino acids at all, but are actually classified into other chemical forms such as alkylamines, sugars, nucleic acids, lipids, fatty acids, or other acids. Any of the modified or non-modified amino acids, including peptides, may be in a D- or L-stereotype or may contain one or more tautomers or resonance forms.

[0109] Pharmaceutical compositions comprising the probiotic skin compositions disclosed herein, comprising symbiotic 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 genetically modified), or attenuated microorganisms comprising an AIP peptide-coding sequence may be formulated into any dosage form suitable for topical administration for local or systemic effects, including emulsions, solutions, suspensions, creams, gels, hydrogels, ointments, sprays, dressings, elixirs, lotions, suspensions, tinctures, pastes, foams, films, aerosols, irrigations, sprays, suppositories, bandages, and skin patches. Topical formulations comprising the probiotics disclosed herein may also comprise liposomes, micelles, microspheres, nanosystems, and mixtures thereof.

[0110] In one embodiment, a bandage or dressing is provided comprising a probiotic skin composition disclosed herein comprising symbiotic bacteria (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 genetically modified), or an attenuated microorganism comprising an AIP peptide-coding sequence described herein. In various aspects, a bandage or dressing is provided comprising a probiotic skin composition and a matrix comprising, wherein the principal components thereof comprise symbiotic bacteria (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 genetically modified), or an attenuated microorganism comprising an AIP peptide-coding sequence described above. In various embodiments, a bandage or dressing is provided in which its primary components include probiotic symbiotic skin bacteria or extracts and a matrix. In various aspects, a bandage or dressing is provided in which its primary components include probiotic symbiotic skin bacteria fermentation extracts and a matrix. In various aspects, a bandage or dressing is provided in which its primary components include glycerol and a matrix. In one embodiment, the bandage or dressing is applied to a site of skin damage or injury. In another embodiment, the bandage or dressing is applied to a site of infection.

[0111] "Pharmaceutical acceptable carriers" are intended to include solvents, dispersion media, coating agents, antibacterial and antifungal agents (where necessary, provided they are not harmful to probiotic symbiotic bacteria), isotonic agents, absorption retardants, etc. The use of such media and agents for pharmaceutical active substances is widely known in the relevant art. Where any conventional media or formulation is incompatible with the pharmaceutical composition, its use in therapeutic compositions and therapeutic methods is considered. Supplemental active compounds may also be incorporated into the composition.

[0112] Pharmaceutically acceptable carriers and excipients suitable for use in topical formulations disclosed herein include, but are not limited to, aqueous vehicles, water-miscible vehicles, non-aqueous vehicles, stabilizers, solubility enhancers, isotonic agents, buffers, antioxidants, local anesthetics, suspending agents and dispersants, wetting agents or emulsifiers, complexing agents, metal ion chelating agents or chelating agents, penetration enhancers, cryoprotectants, lyoprotectants, thickeners, and inert gases.

[0113] Pharmaceutical compositions containing probiotics may be formulated in the form of ointments, creams, sprays, and gels. Suitable ointment vehicles include oily or hydrocarbon vehicles, such as lard, benzoinated lard, olive oil, cottonseed oil, and other oils, and white petroleum jelly; emulsifying or absorbent vehicles, such as hydrophilic petroleum jelly, hydroxystearin sulfate, glycerol, and anhydrous lanolin; water-removable vehicles, such as hydrophilic ointments; water-soluble ointment vehicles, such as polyethylene glycol of various molecular weights; and emulsion vehicles, water-in-oil (W / O) emulsions, or oil-in-water (O / W) emulsions, such as cetyl alcohol, glyceryl monostearate, lanolin, and stearic acid (see Remington: The Science and Practice of Pharmacy). These vehicles require the addition of emollients or generally antioxidants and preservatives.

[0114] Suitable cream bases may be oil-in-water or oil-in-water. The cream vehicle may be washable with water and contains an oil phase, an emulsifier, and an aqueous phase. The oil phase is also referred to as the "internal" phase and generally consists of petroleum jelly and fatty alcohols such as cetyl or stearyl alcohol. The aqueous phase typically exceeds the oil phase by volume, though not strictly necessary, and generally contains a humectant. The emulsifier in the cream formulation may be a nonionic, anionic, cationic, or amphoteric surfactant.

[0115] A gel is a semi-solid, suspension-type system. A single-phase gel contains a substantially homogeneous material throughout the liquid carrier. Suitable gelling agents are cross-linked acrylic acid polymers, such as carbomers, carboxypolyalkylenes, and Carbopol. RTMHydrophilic polymers such as polyethylene oxide, polyoxyethylene-polyoxypropylene copolymer, and polyvinyl alcohol; cellulose-based polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and methylcellulose; gums such as tragacanth and xanthan gum; and sodium alginate are included. To produce a homogeneous gel, a dispersant such as alcohol or glycerin may be added, or the gelling agent may be dispersed by trituration, mechanical mixing, and / or stirring.

[0116] In another embodiment, a pharmaceutical composition comprising a compound of Formula I disclosed herein and / or a symbiotic probiotic, its derivative or analog may be formulated alone or in combination with one or more additional therapeutic agents, including but not limited to chemotherapy agents, antibiotics (as long as they do not impair the probiotic benefit), antifungal agents, antipruritic agents, analgesics, protease inhibitors and / or antiviral agents.

[0117] Topical administration, as used herein, includes administration to the skin (intra-), conjunctiva, cornea, intraocular, eye, ear, transdermal, nasal, vaginal, urethral, ​​respiratory, and rectal. Such topical formulations are useful for treating or inhibiting cancer of the eyes, skin, and mucous membranes (e.g., oral cavity, vagina, rectum). Examples of formulations on the market include topical lotions, creams, soaps, wipes, etc.

[0118] A solution or suspension for use in a pressurized vessel, pump, spray, atomizer, or nebulizer may be formulated to contain ethanol, aqueous ethanol, or a propellant as a suitable alternative solvent for dispersion, dissolution, or extended release of the active ingredient disclosed herein; and / or a surfactant, such as sorbitan trioleate, oleic acid, or oligolactic acid.

[0119] Substances useful for forming an erosive matrix include chitin, chitosan, dextran, and pullulan; gum agar, gum arabic, gum karaya, locust bean gum, gum tragacanth, carrageenan, gum ghatti, guar gum, xanthan gum, and scleroglucan; starch, e.g., dextrin and maltodextrin; hydrophilic colloids, e.g., pectin; phosphatides, e.g., lecithin; alginates; propylene glycol alginate; gelatin; collagen; and cellulose compounds, e.g., ethyl cellulose (EC), methyl ethyl cellulose (MEC), carboxymethyl cellulose (CMC), CMEC, hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), cellulose acetate (CA), cellulose propionate (CP), cellulose butyrate (CB), cellulose acetate butyrate (CAB), CAP, CAT, hydroxypropyl methyl cellulose (HPMC), HPMCP, HPMCAS, hydroxypropyl methyl cellulose acetate trimellitate (HPMCAT), and ethyl hydroxyethyl cellulose (EHEC); polyvinyl pyrrolidone; 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; and other acrylic acid derivatives, such as butyl methacrylate, methyl methacrylate, ethyl methacrylate, ethyl acrylate, (2-dimethylaminoethyl)methacrylate, and (trimethylaminoethyl)methacrylate chloride homopolymers and copolymers, but are not limited thereto.

[0120] In another embodiment, the composition provided herein (e.g., a probiotic composition or a composition comprising a peptide or compound of Formula I) may be combined with one or more steroid drugs known in the relevant art, including but not limited to aldosterone, beclomethasone, betamethasone, deoxycorticosterone acetate, fludrocortisone acetate, hydrocortisone (cortisol), prednisolone, prednisone, methylprenisolone, dexamethasone, and triamcinolone.

[0121] In another embodiment, the composition provided herein (e.g., a probiotic composition or a composition comprising a peptide or compound of Formula I) may be combined with one or more antifungal agents including, but not limited to, amorolfin, amphotericin B, anidulafungin, bifonazole, butenafin, butoconazole, caspofungin, ciclopirox, clotrimazole, iconazole, penticonazole, fin, fluconazole, isoconazole, itraconazole, ketoconazole, micapungin, miconazole, naftifine, natamycin, nystatin, oxyconazole, labuconazole, posaconazole, limosidine, sertaconazole, sulfonazole, terbinafine, terconazole, thioconazole, and voriconazole.

[0122] Kits and manufactured articles for use in the therapeutic applications described herein are also described herein. Such kits may comprise a carrier, package, or container compartmentalized to accommodate one or more containers, such as vials, tubes, etc., and each of the container(s) comprises one of the separate elements to be used in the described method. Suitable containers include, for example, bottles, vials, syringes, and test tubes. Containers may be formed from various materials, such as glass or plastic.

[0123] For example, the container(s) may contain one or more compositions provided herein (e.g., a probiotic composition or a composition comprising a peptide or compound of Formula I) in combination with another agent as disclosed herein. Such kits may optionally contain compositions disclosed herein, along with identification descriptions, labels, or instructions regarding their use in the methods described herein.

[0124] The following examples are provided to further explain the invention, but are not limited thereto.

[0125] Examples

[0126] Example 1

[0127] Culture of primary human keratinocytes Neonatal NHEKs (ThermoFisher Scientific, Waltham, Massachusetts) were cultured at 37°C and 5% CO2 in EpiLife medium (ThermoFisher Scientific) supplemented with 1x EpiLife defined growth supplement (ThermoFisher Scientific), 60 μM CaCl2, and 1x antibiotic-antifungal agent (PSA; 100 U / ml penicillin, 100 U / ml streptomycin, 250 ng / ml amphotericin B; ThermoFisher Scientific). For the experiment, NHEKs were grown to a 70% full growth rate and then differentiated in high-calcium EpiLife medium (2 mM CaCl2) for 48 hours before treatment with the bacterial sterile filtered supernatant. The use of these commercially available human-derived cell products does not require informed consent. For bacterial supernatant treatment, differentiated NHEK was treated with 5 volume% sterile filtered bacterial supernatant relative to Epilife medium. NHEK was used only in experiments of subculture 3 to 5.

[0128] Bacteria cultureAll bacteria were cultured in 3% trypsin soy broth (TSB; Sigma, St. Louis, Missouri) at 37°C with shaking at 300 rpm. S. aureus strains Newman, USA300, 113, Sanger 252 and S. epidermidis strains ATCC12228 and ATCC1457 were grown in a stationary phase for 24 hours, then centrifuged (4,000 rpm, room temperature [RT], 10 min) and the supernatant was sterile filtered (0.22 μm) and added to NHEK. Briefly, protease-deficient strains were cultured in 3% TSB containing 25 μg / ml lincomycin and 5 μg / ml erythromycin for 24 hours, and then subcultured in 3% TSB for an additional 24 hours. In the case of the murine-producing S. aureus colony formation assay, 2 x 10 of the bacteria 6 The colony-forming unit was applied to an 8-mm TSB agar disc and dried at room temperature for 30 minutes before being added to the skin of the back of a murine.

[0129] Murine bacteria disk model. Female C57BJ / 6L mice (8 weeks old) were used in a murine model of bacterial skin colonization. Briefly, to remove dorsal hair, the mice were shaved, nair was applied for 2 e3 minutes, and then the hair was removed with an alcohol wipe. After 24 hours of recovery, 3 x 8 mm TSB agar discs were treated for 12 hours with either TSB only (vehicle control) or 2 e of S. aureus (USA300) per disc. 6 It was applied to the dorsal skin of murine as a colony-forming unit. Tegaderm was applied to the top of the agar disc and fixed in place. After euthanizing the mice, 8-mm whole-skin punch biopsies were collected for analysis.

[0130] in situ zymographyMurine skin sections (10 μm thick) were rinsed 1x with 1% Tween-20 in water for 5 minutes. To measure total protease activity, sections were treated with 2 μg / ml of BODIPY FL casein total protease active substrate (Thermo-Fisher Scientific) in a humidified chamber at 37°C for 4 hours. The serine protease inhibitor AEBSF (50 mM; Sigma) was applied to the sections 30 minutes prior to the addition of BODIPY FL casein. After rinsing the slides 1x with phosphate-buffered saline, DAPI (ThermoFisher Scientific) and ProLong gold antifade mounting medium without cover slides were applied. Fluorescence signals were measured using an Olympus BX51 (Tokyo, Japan) fluorescence microscope.

[0131] Protease activity testAfter adding 50 ml of NHEK conditioned medium to a 96-well black bottom plate (Corning, New York), 150 ml of 5 μg ml BODIPY FL casein substrate, 2 μg ml elastin (elastase-like substrate; Thermo Fisher Scientific), or 4 μg ml gelatin (MMP substrate; Thermo Fisher Scientific) was added according to the manufacturer's instructions. In addition, 200 μM of peptide Boc-Val-Pro-Arg-AMC (trypsin-like substrate; Bachem, Bubendorf, Switzerland) was added to the NHEK conditioned medium at a volume of 150 μl in 1x digestion buffer (Thermo Fisher Scientific). Relative fluorescence intensity was analyzed by reading every 2 hours for 24 hours at room temperature using a SpectraMAX Gemini EM fluorescence meter (Thermo Fisher Scientific). BODIPY FL casein plates were read at ex: 485 nm and em: 530 nm. Elastin-like and MMP substrate plates were read at ex: 485 nm and em: 515 nm. Trypsin-like substrate plates were read at ex: 354 nm and em: 435 nm.

[0132] Quantitative Real-time PCR RNA was isolated from NHEK using a Purelink RNA isolation column (Thermo Fisher Scientific) according to the manufacturer's instructions. RNA was quantified using a Nanodrop spectrophotometer (Thermo Fisher Scientific), and 500 ng of RNA was reverse transcribed using an iScript cDNA synthesis kit (Bio-Rad, Irvine, California). Quantitative real-time PCR reactions were performed on a CFX96 real-time detection system (Bio-Rad) using gene-specific primers and TaqMan probes (Thermo Fisher Scientific).

[0133] Immunoblotting.For cell lysis, cold 1x radioimmunoprecipitation assay (RIPA) buffer (Sigma) containing a 1x protease inhibitor cocktail (Cell Signaling Technology, Danvers, Massachusetts) was applied to NHEK and scraped. The cell lysates were incubated on ice for 30 minutes and centrifuged (13,000 rpm, 15 min, 4°C) to remove cell debris. After measuring protein concentration using the bicinchonic acid (BCA) assay (Pierce, Rockford, Illinois), 40 mg of the sample was added to 4x Laemmli sample buffer (Bio-Rad) containing 1% β-mercaptoethanol and heated at 95°C for 7 minutes to prepare the sample. Samples were processed on a 4-20% tris-glycine precast TGX gel (Bio-Rad), transferred to a 0.22-μm polyvinylidene difluoride (PVDF) membrane (Bio-Rad) using a trans-blot turbo delivery system (Bio-Rad), blocked in a 1x Odyssey blocking solution containing 0.1% Tween-20 (LI-COR, Lincoln, Nebraska) at room temperature for 1 hour, and stained with the primary antibody overnight at 4°C. The Odyssey (LI-COR) fluorescent secondary antibody was applied to the membrane on an orbital shaker at room temperature for 1 hour after washing with 3x PBST (phosphate-buffered saline with 0.1% Tween-20). An additional 3x PBST wash was applied before analysis on an infrared imaging device (LI-COR). Primary antibodies from Santa Cruz Biotechnologies (Santa Cruz, California), KLK5 (H-55), KLK6 (H-60), DSG-1 (H-290), FLG (H-300), and α-tubulin (TU-02) 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.

[0134] KLK gene silencing NHEK was treated for 24 hours with 15 nM or 45 nM of specific KLK silencer-selected siRNA or siRNA scramble (-) controls (Thermo Fisher Scientific) using RNAiMAX (Thermo Fisher Scientific) and Opti MEM medium (Thermo Fisher Scientific). After differentiating NHEK in high-calcium medium (2 mM CaCl2) for 48 hours, it was treated for 24 hours with sterile filtered S. aureus (Newman) supernatant prior to analysis of NHEK lysates and conditioned medium.

[0135] Statistical analysis. Both one-way and two-way ANOVA were used for statistical analysis where a P value < 0.05 was significant. GraphPad Prism version 6.0 (GraphPad, La Jolla, California) was used for the statistical analysis of the results.

[0136] Staphylococcus affects the protease activity of human keratinocytes.To evaluate whether different strains of bacteria found on human skin can induce protease activity in keratinocytes, primary cultures of normal human epidermal keratinocytes (NHEK) were treated with sterile filtered culture supernatants from four different laboratory isolates of S. aureus, including two methicillin-resistant S. aureus strains (USA300 and Sanger 252) and two methicillin-sensitive S. aureus strains. Two S. epidermidis isolates (ATCC12228 and ATCC1457) were also tested. 24 hours after exposure to sterile bacterial culture supernatants, the keratinocyte culture medium was analyzed for protease activity against substrates selective for trypsin-like, elastase-like, or matrix metalloproteinase (MMP) activity. NHEK-conditioned medium contained significantly more trypsin activity after treatment with S. aureus strains Newman and USA300 (Fig. 1a). Both MMP and elastase activities were increased by S. epidermidis strain ATCC12228, while S. aureus strains USA300 and Sanger 252 and S. epidermidis strain ATCC1457 increased elastase activity in NHEK-conditioned medium to a lesser extent (Figs. 1b and c). To confirm that the increased protease activity observed in NHEK-conditioned medium originated from NHEK and was not produced by the bacteria themselves, trypsin activity was analyzed after adding S. aureus (Newman) supernatant to culture wells in the presence and absence of NHEK. When the same concentration of diluted supernatant from S. aureus was added to NHEK medium alone, no enzyme activity was detected in the absence of NHEK (Fig. 1d).

[0137] S. aureus increases epidermal serine protease activity.Due to the significant increase in trypsin activity induced by specific S. aureus strains (Newman and USA300) and the potential role this activity may play in diseases mediated by S. aureus, experiments on these organisms were focused to better understand how bacteria induce protease activity in NHEK. To evaluate 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, after which NHEK conditioned medium was collected for protease analysis. Measurements of total protease activity in NHEK conditioned medium revealed a time-dependent increase in total proteolytic activity following exposure to S. aureus supernatant (Fig. 2a). The addition of the serine protease inhibitor aprotinin confirmed that this activity was attributed to serine protease (Fig. 2b), which was consistent with the observation of increased trypsin-like activity shown in Fig. 1a. A comparison of the wild-type and protease-deficient strains of S. aureus USA300 LAC showed that both the wild-type and protease-deficient strains increased trypsin activity in NHEK-conditioned medium, but the protease-deficient strains significantly reduced the ability to induce trypsin activity compared to the wild-type strains (Fig. 2c). Taken together, these data confirm that S. aureus can increase endogenous NHEK serine protease activity and that S. aureus protease and other S. aureus products are a cause of this bacterium's ability to activate keratinocytes.

[0138] To further demonstrate the action of S. aureus on epidermal protease activity, raw S. aureus (USA300) was applied to the dorsal skin of mice. Subsequently, the skin at the application site was biopsied and sectioned for the analysis of total proteolytic activity by in vitro geomography in the presence or absence of the serine protease inhibitor 4-benzenesulfonyl fluoride (AEBSF). Compared to skin treated with agar discs alone, total epidermal protease activity in the epidermis was qualitatively increased after treatment with S. aureus, and the increased activity detected by increased fluorescence was primarily eliminated by the inhibition of serine protease activity by AEBSF. Background autofluorescence in hair follicles was observed in all sections, including those without any substrate control. These observations further demonstrate that the presence of S. aureus can increase protease activity in the epidermis.

[0139] S. aureus increases KLK expression in keratinocytes. KLK is a family of serine proteases abundant in the epidermis with trypsin-like or chymotrypsin-like activity. To determine whether S. aureus can alter KLK mRNA expression in keratinocytes, NHEK was treated with S. aureus (Newman) supernatant for 24 hours, and the expression of KLK1-15 was measured by quantitative real-time PCR. KLK5 had the highest relative mRNA abundance, while KLK6, 13, and 14 consistently showed the largest fold increase after exposure to S. aureus (Fig. 3a-e). All other analyzed KLKs showed subtle increases in mRNA expression after exposure to S. aureus, with the exception of KLK1, which showed decreased expression. mRNA for KLK2, 3, and 15 was not detected.

[0140] Next, changes in KLK protein expression after treatment with S. aureus (Newman) supernatant were analyzed for both cell lysates and NHEK-conditioned media. Immunoblotting for KLK6 and 14 showed increased expression of these KLK proteins after treatment with S. aureus supernatant in both cell lysates and conditioned media, whereas KLK13 increased only in the conditioned medium. KLK5 showed no change in expression after treatment with S. aureus supernatant (Fig. 3f).

[0141] KLK6, 13, and 14 are a cause of increased keratinocyte serine protease activity. Since KLK6, 13, and 14 showed the greatest increase in expression in NHEK after S. aureus exposure, experiments were performed to investigate whether these KLKs were the cause of the observed increased serine protease activity. Their expression was selectively silenced using small interfering RNA (siRNA). siRNA for KLK6 and KLK13 significantly reduced S. aureus-induced trypsin activity, while KLK14 reduced trypsin activity to a lesser extent. Triple knockdown of KLK6, 13, and 14 also showed a significant reduction in trypsin activity from control siRNA, although no additive effect was observed (Fig. 4a). Interestingly, triple knockdown of KLK6, 13, and 14 resulted in reduced knockdown efficiency for KLK13 and KLK14, which may explain the lack of an additive effect on trypsin activity (Figs. 4b-d).

[0142] S. aureus promotes the degradation of desmoglein-1 and FLG by induction of KLK.Desmoglein-1 (DSG-1) and FLG are both important for regulating epidermal skin barrier integrity. Immunoblotting indicated that exposure of NHEK to S. aureus (Newman) supernatant promotes the cleavage of full-length DSG-1 (160 kDa), and that DSG-1 cleavage is blocked by siRNA silencing of KLK6, 13, or 14 (Fig. 5a). S. aureus-mediated cleavage of propyllagrin (Pro-FLG) in NHEK, indicated by a >250 kDa band in the immunoblot, was also partially blocked by siRNA silencing of KLK6 and KLK13 (Fig. 5b). Densitometric analysis further illustrates the knockdown ability of KLK6, 13, and 14 to prevent DSG-1 or Pro-FLG cleavage (Fig. 5c). Overall, these observations demonstrate that the ability of S. aureus to increase keratinocyte proteolytic activity by induction of KLK6, 13, and 14 can cause the digestion of molecules essential for maintaining a normal epidermal barrier.

[0143] Example 2

[0144] Bacteria manufacturing.All bacteria used in this study are listed in Table A. All Staphylococcus strains (S. aureus, S. epidermidis, S. hominis, S. warneri, S. capitis, and S. rugdunensis) were grown as a stationary phase in 3% trypsin soy broth (TSB) at 250 RPM for 24 hours in a 37°C incubator at a volume of 4 mL or 400 μL according to the assay. Specific strains were grown with the antibiotic selections listed in Table S1 at the following concentrations: 5 μg / mL Erm, 25 μg / mL Lcm, and 10 μg / mL Cm. For the treatment of bacterial supernatants on human keratinocytes or murine skin, bacteria cultured for 24 hours were pelleted (15 min, 4,000 RPM, RT), and the supernatant (0.22 μm) was filter-sterilized. For murine and human keratinocyte experiments using S. hominis C5 and S. epidermidis RP62A strains, the bacterial sterile-filtered supernatant was filtered through a 3 kDa exclusion column (Amicon Ultra-15 centrifuge filter, Millipore) to collect the <3 kDa fraction, freeze-dried, further concentrated by 10x, and resuspended in molecular-grade H2O prior to treatment. The S. hominis C5 supernatant was further biochemically tested using several techniques. Small peptides were isolated by ammonium sulfate precipitation (80%) for 1 hour at room temperature, followed by centrifugation (30 min, 4,000 RPM, RT) and resuspension of the precipitate (pellet) in H2O. Furthermore, the pH of the S. hominis C5 supernatant was raised to pH 11 with 2M NaOH for 1 hour, and then the pH of the supernatant was returned to a starting pH of approximately 6.5 using a pH 1-14 strip with 2M HCl before being added to the S. aureus agr reporter strain.

[0145] Bacteria and Plasmids bacteria strain name S. epidermidis RP62A WT (agr type I) S. epidermidis RP62A ΔAIP(#47) S. epidermidis ATCC1457 (agr type II) S. epidermidis 8247 (agr type III) S. Epidermidis A9 S. Epidermidis A11 S. Aureus USA300 LAC WT S. Aureus USA300 Δpsmα S. Aureus USA300 Δpsmβ S. Aureus USA300 LAC WT(AH1263) S. aureus USA300 LAC Δprotease (AH1919), Erm R , Lcm R S. Aureus USA300 LAC agr Type I pAmi P3-Lux(AH2759), Cm R S. Aureus USA300 LAC agr Type I P3-YFP(AH1677), Cm R S. aureus 502a agr Type II P3-YFP(AH430), Cm R S. Aureus MW2 agr Type III P3-YFP(AH1747), Cm R S. aureus MN TG agr type IV P3-YFP(AH1872), Cm R S. Hominis C4 S. Hominis C5 S. Hominis A9 S. Warneri G25 S. Capitis H8 S. rugdunensis E7 DC10B-CC10 Plasmid strain name pMAD(E. Amp in coli R , Erm in Staphilococcus R ) pMAD:: ΔAIP

[0146] Normal human keratinocyte cultureNormal neonatal human epidermal keratinocytes (NHEK; Thermo Fisher Scientific) were 60 μM CaCl2 supplemented with 1x Epilife Defined Growth Supplement (EDGS; Thermo Fisher Scientific) and 1x antibiotic-antifungal agent (PSA; 100 U / mL penicillin, 100 U / mL streptomycin, 250 ng / mL amphotericin B; Thermo Fisher Scientific). 2( It was cultured in Epilife medium containing Thermo Fisher Scientific at 37°C and 5% CO2. NHEK was used only in experiments between passages 3 and 5. For the experiment, NHEK was grown to a 70% full growth rate and then differentiated in high-calcium Epilife medium (2 mM CaCl2) for 48 hours to simulate the upper layer of the epidermis. For bacterial supernatant treatment, differentiated NHEK was treated with 5 vol% sterile-filtered bacterial supernatant relative to Epilife medium for 24 hours. Similar to synthetic PSM treatment, 5–50 μg-mL of peptide was added to NHEK in DMSO for 24 hours.

[0147] S. aureus epicutaneous mouse model. Sex- and age-matched male or female C57BL / 6 (Jackson) mice, 8 weeks of age, were used in all experiments (n=3-6) as specified in the drawing legend. All animal experiments were approved by the Institutional Animal Care and Use Committee. Mice were shaved, and Nair was applied for 2 to 3 minutes and immediately removed with an alcohol wipe. The skin barrier was allowed to recover for 48 hours from shaving prior to the application of the bacteria. S. aureus (1e7 CFU) in 3% TSB was applied at 1.5 cm 2A volume of 100 μL was applied to murine skin on a piece of sterile gauze for 48–72 hours. Tegaderm was applied over the gauze to secure it in place for maintenance during the treatment period. For the S. aureus agr inhibition experiment, raw S. hominis C5 (10:1) or a 10x concentrated <3 kDa sterile-filtered symbiotic bacterial supernatant (1:1) was combined with S. aureus in 3% TSB immediately before application to the gauze.

[0148] Preparation of synthetic phenol-soluble modulin. All synthetic phenol-soluble modulins (PSMs) were prepared by LifeTein (Hillsboro, New Jersey). The peptides were prepared to 95% purity by N-terminal formylation (f). The PSM sequences were as follows:

[0149] PSMα1: f-MGIIAGIIKVIKSLIEQFTGK(Sequence No.: 5),

[0150] PSMα2: f-MGIIAGIIKFIKGLIEKFTGK(Sequence No.: 6),

[0151] PSMα3: f-MEFVAKLFKFFKDLLGKFLGNN(Sequence No.: 7),

[0152] PSMα4: f-MAIVGTIIKIIKAIIDIFAK(SEQ number: 8),

[0153] PSMβ2: f-MTGLAEAIANTVQAAQQHDSVKLGTSIVDIVANGVGLLGKLFGF(Sequence No.: 9).

[0154] The peptide was resuspended in DMSO and concentrated by speedvac with 500 mg of powdered stock stored at -80℃ before being reconstituted in DMSO for the experiment.

[0155] RNA isolation and quantitative real-time PCR.All RNAs were 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 tissues, 0.5 cm 2 The entire thickness of the skin was bead-beated (2 x 30 seconds, 2.0 mm zirconia beads) in 750 μL of RNA lysis buffer on ice for less than 5 minutes. Subsequently, the tissue was centrifuged (10 min, 13,000 RPM, 4℃), 350 μL of clear lysate was added to 70% EtOH, and RNA was isolated based on the column. For S. aureus RNA isolation, 1 x 10⁹ CFU bacteria were incubated with a 2:1 ratio of RNAProtect (Qiagen) for 10 minutes, followed by centrifugation (10 min, 13,000 RPM, RT). The samples were then resuspended in 750 μL of RNA lysis buffer and subjected to beading (2 x 1 min, 6.5 speed) using lysis matrix tube B and Fastprep-24 (MP Biomedicals). Subsequently, the samples were centrifuged again, and 350 μL of the clear lysate was added to 70% EtOH as described above. After RNA isolation, the samples were quantified using 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 Takman probes (Thermo Fisher Scientific) were used with GAPDH as the housekeeping gene.

[0156] Generation and transformation of RP62A competent cells.Electro-competent RP62A cells were prepared. Briefly, overnight cultures of S. epidermidis RP62A were prepared in preheated Brain Heart Infusion (BHI) broth at an OD of 0.5. 600 The cells were diluted to nm, transferred to a centrifuge tube, incubated in a 37°C incubator for an additional 30 minutes while shaking, and then chilled on ice for 10 minutes. Cells were harvested by centrifugation (10 min, 4000 RPM, 4°C), washed sequentially with 1 volume, 1 / 10 volume, and then 1 / 25 volume of cold autoclaved water, and re-pelleted 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 tubes in 50 μL for storage at -80°C. Transfection with S. epidermidis RP62A was performed. Briefly, frozen eligible cells were thawed on ice for 5 minutes and then thawed at room temperature for 5 minutes. The thawed cells were centrifuged briefly (1 min, 5000 g, RT), and the pellet was 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 micropulser (Bio-Rad) at 2.1 kV with a time constant of 1.1 msec. Immediately after electroporation, the cells were resuspended in 1 mL of BHI broth supplemented with 500 mM sucrose, shaken at 30°C for 1 hour, and then plated on BHI agar with 10 μg / mL chloramphenicol (Cm) at 30°C.

[0157] Allele replacement of S. 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 1000 bp upstream and downstream fragments of the AIP sequence of RP62A were amplified by PCR and ligated by gene splicing via overlap extension or 'SOEing'. The sewn fragments and pMAD vector were digested with BamHI and SalI, ligated together by T4 DNA ligase (New England Biolabs), and then used to chemically transform the S. epidermidis clonal complex 10 plasmid-modified E. coli strain DC10B-CC10. The transformants were plated on LB at 37°C at 100 μg / mL Amp and 30 μg / mL Cm. Correct transformants were verified by restriction digest and sequencing. Identified constructs were annotated with pMAD::ΔAIP. Subsequently, pre-qualified RP62A were transformed with ~5 μg of pMAD::ΔAIP derived from DC10B-CC10 and then plated on BHI agar at 30°C with 10 μg / mL Cm and 50 μL of 40 mg / mL 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal). A single blue colony was selected and grown overnight in BHI at 30°C with 10 μg / mL Cm. The overnight culture was then diluted 1:100 without antibiotics in fresh, preheated BHI (for a final volume of 100 mL) and incubated at 43°C for 24 hours. A single crossover event was promoted by repeating dilution and growth at 43°C for an additional time and selecting pale blue colonies grown on BHI agar supplemented with 10 μg / mL Cm and 50 μL of 40 mg / mL X-Gal at 43°C.Pale blue colonies were selected and incubated overnight at 30°C in BHI without antibiotics to promote double crossover events. These overnight dilutions were plated onto BHI agar supplemented with 50 μL of 40 mg / mL X-Gal and incubated overnight at 37°C. White colonies were selected 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 selected and screened for deletions in the AIP coding sequence by sequencing. The identified mutant strains were annotated as S. epidermides RP62A ΔAIP.

[0158] RNA sequencing analysis. RNA was submitted to the University of California, San Diego (UCSD) Genome Core Facility for library preparation and sequencing. After using the TruSeq mRNA Library Prep Kit (Illumina) for library preparation, high-throughput sequencing was performed on a HiSeq 2500 sequencer (Illumina). Data were analyzed using Partek Flow and Partek Genomics Suite software, and gene ontology analysis was performed using the PANTHER classification system (http: / / ]pantherdb.org).

[0159] histology. Total thickness of murine skin (0.5 cm) 2The tissue was collected, fixed in paraformaldehyde (4%), washed with PBS, incubated overnight in 30% and 10% sucrose, and then frozen on OCT mounting medium with dry ice. Cryostat-cut sections (10 mm) were mounted on Superfrost Plus glass slides (Fisher Scientific) and stained with hematoxylin and eosin (H&E). The sections were incubated in a 75% to 100% EtOH gradient at 5-minute intervals, followed by xylene incubation and mounting on Paramount glass slides. Photographs were taken at 200x magnification using an Olympus BX51 (Tokyo, Japan) fluorescence microscope.

[0160] Determination of cytokine levels. Protein concentrations of various cytokines were quantified using conditioned medium (25 μL) from NHEK. A magnetic bead-based milliplex assay kit (Millipore) for three human cytokines (IL-6, IL-8, TNFα) was used according to the manufacturer's instructions for the Magpix 200 (Luminex) system. Human IL-1α and IL-36α were quantified by ELISA (R&D Systems).

[0161] Quantification of bacterial CFU 10 S. aureus colony-forming units (CFU) were incubated on Baird-Parker agar (BD) plates containing a 3% egg yolk emulsion with tellurite for 24 hours in a 37°C incubator. -1 to 10 -5 Serial dilutions (10 μL) were plated, quantified, and CFCs were counted. Bacterial CFU for all Staphylococcus strains were also approximated using a spectrophotometer and by measuring the OD600 nm of cells diluted 1:20 in PBS.

[0162] Transepidermal water loss measurement To determine damage to the epidermal skin barrier, transepidermal water loss (TEWL) of murine skin treated with S. aureus for 48-72 hours was measured using a TEWAMETER TM300 (C & K).

[0163] Trypsin activity analysis 50 μL of NHEK-conditioned medium was added to 96-well black-bottom plates (Corning), followed by the addition of 150 μL of the peptide Boc-Val-Pro-Arg-AMC (trypsin-like substrate; Bakem) to a final concentration of 200 μM in 1x digestion buffer (10 mM Tris-HCl pH 7.8), and incubation was performed at 37°C for 24 hours. Relative fluorescence intensity (ex: 354 nm, em: 435 nm) was analyzed using a Spectramax Gemini EM fluorescence analyzer (Thermo Fisher Scientific). For the analysis of murine cutaneous trypsin activity, 0.5 cm 2 The entire thickness of the skin was bead-beated in 1 mL of 1 M acetic acid (2.0 mm zirconia beads, 2 x 30 seconds with 5 minutes each) and then spun overnight at 4°C. The sample was centrifuged (10 min, 13,000 RPM, 4°C), added to a new microcentrifuge tube, and the protein was concentrated by removing all residual acetic acid using a speed bag. The protein was resuspended in molecular-grade water (500 μL), spun overnight at 4°C, and then centrifuged again. The clear protein lysate was added to a new tube, and a BCA (Bio-Rad) assay was used to determine the protein concentration. Finally, 10 μg of total protein was added to a 96-well plate and analyzed using the trypsin substrate as described above.

[0164] S. aureus agr activityS. aureus agr activity was detected using S. aureus USA300 LAC agr type I P3-YFP (AH1677) or S. aureus USA300 LAC agr type I pAmi P3-Lux (AH2759) reporter strains. For in vitro experiments, 1 e6 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 symbiotic supernatant (25 vol%) and shaken at 37°C for 24 hours at 250 RPM. Subsequently, the bacteria were diluted 1:20 in PBS (final 200 μL), and YFP (e.g., 495 nm, e.g., 530 nm) was detected using the fluorescence meter described above, and OD was measured on a spectrophotometer. 600 Bacterial density was determined using nm readings. For the murine experiment, the activity of S. aureus USA300 LAC agr type I pAmi P3-Lux was determined using an IVIS instrument, and luminescence intensity after 2 minutes of exposure was evaluated by measuring emitted photons (p / sec / cm2 / sr) using LiveImaging software (PerkinElmer).

[0165] Genome sequencing and assemblyGenomic DNA of S. hominis C5 was isolated using the DNA UltraClean Microvial Kit (Qiagen). The library was sequenced for 2 cycles using the MiSeq platform (Illumina Inc., San Diego, California) to generate 2x 250 bp paired-end reads. Adapters were removed using cutadapt (version 1.9.1) (http[: / / ]cutadapt.readthedocs.io / en / stable / ). Low-quality sequences (quality score <30) can be removed using trim_galore (version 1.9.1) (https[: / / ][www.]bioinformatics.babraham.ac.uk / projects / trim_galore / ) with default parameters. Sequence mapping for the human genome was removed from the quality-trimmed dataset using the human reference genome hg19 (UCSC Genome Browser) and the Voti2 program (version 2.2.8) (51) with the following parameters (-D 20 -R 3 -N 1 -L 20 --highly sensitive-local). Filtered reads were reassembled using SPAdes (version 3.8.0) (52) with k-mer lengths ranging from 33 to 127. The genome was annotated with the rapid annotation of the microbial genome using the subsystem technique (RAST) with default parameters. The amino acid sequences (coding DNA sequences) from the annotated CDS were aligned to bacterial agr proteins obtained from the Uniprot database (downloaded in October 2017). The Agr gene from the assembled genome was identified according to the following three criteria: (i) sequence identity > 60%, (ii) e-value < e100; and (iii) agr locus organization, a four-gene operon, agrBDCA.

[0166] Microbiome Data and Genome Comparative AnalysisPublicly available shotgun metagenomic data on atopic dermatitis skin were analyzed. The relative abundance of S. aureus and S. epidermidis strains was obtained directly from the published supplementary data ([www.]sciencetranslationalmedicine.org / cgi / content / full / 9 / 397 / eaal4651 / DC1). agrD characterization was limited to eight patients (AD01, AD02, AD03, AD04, AD05, AD08, AD09, and AD11) with information on seven distinct body sites on erythematous AD skin and differences in AD severity based on objective SCORAD. The 61 evaluated S. epidermidis strains were classified as agr type I, II, or III by comparing amino acid sequences with known agr type I-III sequences within the agrD gene region.

[0167] Quantification and statistical analysis. The nonparametric Mann-Whitney test was used to analyze the statistical significance of metagenome data from patients with AD. One-way or two-way ANOVA was used for statistical analysis as indicated in the various figure descriptions. All statistical analyses were performed using GraphPad PRISM version 6.0 (GraphPad, La Jolla, California). All data are presented as ± standard error of the mean (SEM), and a P-value ≤ 0.05 is considered significant.

[0168] PSMα and proteases produced by S. aureus induce epidermal barrier damage.The primary function of human skin is to establish a physical barrier against the external environment. Certain toxins produced by S. aureus, such as phenol-soluble modulin (PSM), can promote epithelial inflammation and have been suggested to be a key factor in causing disease in AD (19-22). Therefore, to understand how S. aureus on the skin surface can influence inflammatory activity across the epidermal barrier, normal human epidermal keratinocytes (NHEK) were evaluated for their ability to express proteolytic activity upon exposure to S. aureus USA300 LAC strains with targeted deletions in the PSMα or PSMβ operons. PSMα production was required for the induction of trypsin-like serine protease activity and increased mRNA levels of kallikrein 6 (KLK6) (Fig. 14A-B). In S. aureus, the PSMα and PSMβ operons contain distinct peptides, including PSMα1-4 and PSMβ1-2. Therefore, synthetic PSMα1-4 and PSMβ2 peptides were tested on NHEK (Fig. 14C), and it was found that all PSMα peptides could stimulate trypsin activity, whereas PSMβ2 would not. The strongest PSMα trypsin activity inducer in NHEK was selected, further indicating that it could stimulate trypsin activity and KLK6 mRNA expression in NHEK in both dose- and time-dependent ways (Fig. 18). Furthermore, transcriptional profiling by RNA-Seq of NHEK exposed to PSMα3 indicated that this toxin exerts extensive effects on the expression of skin barrier-related genes, including multiple proteases (KLK, MMP), components of the physical barrier (filagrin, desmoglein-1, loricrin, keratin), antimicrobial peptides, and cytokines (Figs. 14D-E, Fig. 18).

[0169] To verify the role of the PSMα operon on the in vivo epidermal barrier, mice were colonized on the skin surface for 72 hours with equal numbers of wild-type or PSMα mutant strains of S. aureus USA300 LAC. Wild-type S. aureus induced erythema, scaling, and epidermal thickening, whereas no change in bacterial prevalence was observed in the absence of PSMα (Fig. 14F). Despite the increased epidermal thickness, an increase in transepidermal water loss (TEWL), a widely established method for assessing skin barrier damage, was observed after exposure to wild-type S. aureus but not in the absence of PSMα (Fig. 14G). However, skin barrier disruption in fully differentiated epidermis in vivo was also dependent on S. aureus protease expression. Using S. aureus USA300 LAC mutant strains lacking 10 major secreted proteases, including aureolisin, V8, stapopine A / B, and SplA-F, visible evidence of damage and increased TEWL were reduced in the manner of S. aureus protease deficiency, despite completely damage-free PSMα expression (Fig. 14F, H). Consistent with total and histological changes observed to be associated with the expression of PSMα or bacterial proteases, increases in keratinocyte trypsin activity, Klk6 transcript expression, 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 (Fig. 19). Furthermore, despite changes in the skin barrier and the inflammatory environment of the skin, the presence of S. aureus on the skin surface remained unchanged under these conditions (Fig. 14I-J). Taken together, these data suggest that the generation of PSMα and protease activity from S. aureus results in damage to the epidermal barrier, and that this barrier damage is required for S. aureus to promote inflammation (Figs. 14I-J).

[0170] S. epidermidis auto-induced peptides inhibit S. aureus agr activity.Interestingly, both S. aureus PSMα peptides and secreted proteases are regulated by the agr quorum sensing system. Furthermore, clinical isolates of S. aureus possess four distinct agr types, with agr type I being found to be the most prominent in AD subjects. Although S. aureus skin colonization increases in AD, other bacterial species, such as coagulase-negative Staphylococcus (CoNS) strains including the abundant human skin commensal S. epidermidis, are also present, making it essential to understand how these bacteria signal. Lab isolates of S. epidermidis agr type I have been shown to produce self-inducing peptides (AIPs) that inhibit the S. aureus agr type I-III system via a crosstalk mechanism but not type IV, whereas 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 could affect the S. aureus agr system, conditioned culture supernatants from S. epidermidis strains with 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 I was the only potent inhibitor of S. aureus agr activity, while S. epidermidis agr types II and III had little effect (Fig. 15A). Targeted deletion of S. epidermidis agr type I AIP within the agrD gene region abolished S. epidermidis' ability to inhibit S. aureus agr activity (Figs. 15B-C). Since S. aureus PSMα-induced NHEK trypsin activity is a component of epidermal barrier damage, S.Wild-type or AIP knockout strains of Epidermidis agr type I were tested to determine if they could influence this result. When S. aureus was cultured in the presence of wild-type S. epidermidis agr type I supernatant but not cultured with S. epidermidis lacking this AIP, S. aureus-induced NHEK trypsin activity was observed to be inhibited (Fig. 15D). Overall, these experiments established that the ability of S. aureus to induce NHEK barrier damage can be affected by S. epidermidis agr type I AIP expression.

[0171] Deficiency of the relative abundance of S. epidermidis agr type I in the AD skin. Since the potential for laboratory strains of S. epidermidis to influence the effects of S. aureus on human keratinocyte function was established, experiments were conducted to determine the abundance of these bacteria in a clinical setting. Available metagenome data (based on objective SCORAD) from the skin microbiomes of eight subjects with varying severity of AD collected from seven body sites were analyzed for the relative abundance of S. epidermidis based on agr type. Sequence alignment identified the S. epidermidis genome based on agr type IIII in AD patients and determined that the most frequent S. epidermidis agr type on AD skin was agr type I (Fig. 15E). A comparison between S. epidermidis agr type I and S. aureus indicated that S. epidermidis agr type I was relatively less abundant in AD subjects with increased disease severity (Figs. 15F-G). These observations confirmed the presence of S. epidermidis agr type I in the AD skin microbiome and suggest a possible association with clinical disease.

[0172] Various Staphylococcus aureus 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, different AD clinical isolates of CoNS were tested for the ability of their culture supernatant to inhibit the quorum sensing activity of S. aureus USA300 LAC agr type I. Various species, including S. epidermidis, S. hominis, S. warneri, and S. capitis, exhibited potent inhibitory activity against the agr activity of S. aureus (Fig. 16A). Similar to the laboratory isolate of S. epidermidis, the CoNS strains inhibited S. aureus agr activity without inhibiting growth rate (Fig. 316S). In addition, genomic sequencing of the agrD AIP coding region of S. hominis strain C5 revealed a novel AIP sequence in the AIP coding region with a predicted octomer AIP sequence for S. hominis C5 (Fig. 16B; SEQ ID: 4) that is similar to the sequence of the agr type I coding region of S. epidermidis. Biochemical analysis of the active S. hominis C5 supernatant indicated that the inhibition of S. aureus agr activity depends on a <3 kDa (small size) and pH 11 sensitivity (thi-lactone ring) factor that can be precipitated with 80% ammonium sulfate (peptide) (Fig. 16C).

[0173] Next, S. aureus was cultured in the presence of sterile-filtered supernatant from S. hominis C5, and the subsequent culture supernatant was applied to NHEK as shown in Fig. 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 NHEK (Figs. 16D-F). Furthermore, S. hominis C5 would be able to inhibit multiple S. aureus agr systems, with the exception of the most common clinical isolates of agr type I, which include agr types II and III but not IV (Fig. 17S). This finding is consistent with what was observed in the S. epidermidis agr type I system. Overall, these observations suggest that clinical isolates of CoNS species other than S. epidermidis can inhibit S. aureus damage to keratinocytes using quorum sensing.

[0174] Clinical CoNS isolates inhibit the activity of S. aureus agr, which promotes AD. To establish the physiological relevance of quorum sensing interactions between CoNS and S. aureus in vivo, S. Aureus S. aureus agr activity was evaluated by IVIS using the USA300 LAC agr type I P3-Lux promoter (luminescent) strain. On the dorsal skin, S. aureus exhibited abundant agr activity, but the S. Hominis In the presence of C5, S. aureus agr activity was inhibited (Fig. 17A-B). Furthermore, S. hominis C5 also protected against S. aureus-induced skin erythema and scaling without altering S. aureus abundance (Fig. 17D) (Fig. 17C). This phenotype was associated with improved evidence regarding inflammation, barrier disruption, epidermal protease activity, and Klk6 expression (Fig. 17E-H). Moreover, when S. aureus <3 kDa concentrated S. hominis C5 supernatant was applied to the dorsal skin of murine, a similar reduction in barrier damage and inflammation was observed without changes in S. aureus abundance (Fig. 22). These data suggest that the skin CoNS microbial community may contain novel AIPs that promote epithelial barrier homeostasis through interspecies quorum sensing activity.

[0175] Many embodiments of the present invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the present disclosure. Accordingly, other embodiments are within the scope of the following claims.

[0177] Depository Name: American Type Culture Collection (ATCC)

[0178] Trustee Number: PTA125202

[0179] Date of Trust: 2018828

[0180] Depository Name: American Type Culture Collection (ATCC)

[0181] Trustee Number: PTA125203

[0182] Date of Trust: 2018828

[0183] Depository Name: American Type Culture Collection (ATCC)

[0184] Trustee Number: PTA125204

[0185] Date of Trust: 2018828

[0186] Depository Name: American Type Culture Collection (ATCC)

[0187] Trustee Number: PTA125205

[0188] Date of Trust: 2018828

Claims

Claim 1 A purified polypeptide comprising the sequence of SEQ ID NO: 4, wherein (i) inhibits protease production and / or activity of keratinocytes, (ii) inhibits IL-6 production and / or activity of keratinocytes, (iii) Staphylococcus aureus ( S. Inhibition of the production of phenol-soluble modulin alpha 3 from (aureus), and / or (iv) S. A purified polypeptide that inhibits the production and / or activity of an accessory gene regulator (agr) by Aureus. Claim 2 In claim 1, the purified polypeptide comprising the sequence of SEQ ID NO:

2. Claim 3 A purified polypeptide according to claim 1, wherein the polypeptide consists of SEQ ID NO:

4. Claim 4 A purified polypeptide according to any one of claims 1 to 3, wherein the polypeptide comprises one or more D-amino acids. Claim 5 In any one of claims 1 to 3, the purified polypeptide comprising a compound of formula IB: formula IB ,where X1 is 0 to 6 amino acids. Claim 6 delete Claim 7 An isolated polynucleotide encoding the polypeptide of any one of paragraphs 1 to 3. Claim 8 In claim 7, the isolated polynucleotide comprising SEQ ID NO: 1 or 3. Claim 9 A vector containing the polynucleotide of claim 7. Claim 10 A vector containing the polynucleotide of claim 8. Claim 11 A recombinant microorganism containing the polynucleotide of claim 7. Claim 12 A recombinant microorganism containing the polynucleotide of claim 8. Claim 13 In paragraph 11, a recombinant microorganism in which the microorganism is a weakened microorganism or a symbiotic microorganism. Claim 14 A topical probiotic composition comprising the recombinant microorganism of claim 13 for use in treating dermatological disorders selected from the group consisting of atopic dermatitis, Netterton syndrome, contact dermatitis, eczema, psoriasis, acne, epidermal hyperkeratosis, acanthosis, epidermal inflammation, dermal inflammation, and pruritus. Claim 15 A topical probiotic composition for use in treating a dermatological disorder selected from the group consisting of atopic dermatitis, Netterton syndrome, contact dermatitis, eczema, psoriasis, acne, epidermal hyperkeratosis, acanthosis, epidermal inflammation, dermal inflammation, and pruritus, comprising a microorganism expressing the polypeptide of claim 1 or a fermented extract of a microorganism expressing the polypeptide of claim 1, wherein the fermented extract comprises the polypeptide. Claim 16 In claim 15, a topical probiotic composition comprising a polypeptide containing a compound of formula IB: formula IB ,where X1 is 0 to 6 amino acids. Claim 17 In paragraph 15, the microorganism Staphylococcus hominis ( S. hominis ), Staphylococcus epidermidis ( S. epidermidis ), Staphylococcus warneri( S. warneri A topical probiotic composition, which is ), or any combination thereof. Claim 18 In Paragraph 17, microorganisms S. Hominis C5, S. Hominis A9, S. Epidermidis A11, and / or S. Warneri G2, a topical probiotic composition. Claim 19 In item 15, the composition is ATCC number PTA-125202 (strain name S. Epidermidis A11 81618, deposited August 28, 2018), ATCC number PTA-125204 (strain name S. Hominis C5 81618, deposited August 28, 2018), ATCC number PTA-125203 (strain name S. Hominis A9 81618, deposited August 28, 2018), ATCC number PTA-125205 (strain name S. Warneri A topical probiotic composition comprising a microorganism selected from the group of microorganisms having any combination of the strains described above (G2 81618, deposited on August 28, 2018), and any combination of the strains described above. Claim 20 In claim 15, a topical probiotic composition in which the topical probiotic composition is formulated as a lotion, shake lotion, cream, ointment, gel, foam, powder, solid, paste, or tincture. Claim 21 A pharmaceutical composition for treating a dermatological disorder selected from the group consisting of Netterton syndrome, atopic dermatitis, contact dermatitis, eczema, psoriasis, acne, epidermal hyperkeratosis, acanthosis, epidermal inflammation, dermal inflammation, and pruritus, comprising a purified polypeptide of any one of claims 1 to 3 or a topical probiotic composition of any one of claims 15 to 20. Claim 22 delete Claim 23 delete Claim 24 delete Claim 25 delete Claim 26 delete Claim 27 delete Claim 28 delete Claim 29 delete Claim 30 delete Claim 31 delete Claim 32 delete Claim 33 delete Claim 34 delete Claim 35 delete Claim 36 delete Claim 37 delete

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