Bacterium and extracts of said bacterium and the use of same in dermatology

Abstract

The present invention relates to a novel bacterial strain isolated from groundwater. The invention also relates to bacterial extracts and to the use of same in the context of the treatment of inflammations. More particularly, the present invention relates to novel compositions of interest in the treatment and the prevention of inflammatory disorders, notably dermatological pathologies.

Claims

1. A cosmetic or dermatological composition comprising effective amounts of a fractionated bacterial extract and a preservative, wherein the fractionated bacterial extract is obtained froma Gram-negative non-filamentous bacterium belonging to the class of Betaproteobacteria, subfamily of Neisseriaceae which nucleotide sequence of the 16S rRNA gene comrprises SEQ ID NO: 1, and wherein the fractionated bacterial extract is prepared by a method comprising incubating and reacting said bacterium in a basic medium of pH 9 to 11 for a suitable time, and removing intracellular components of the bacterium therefrom.

2. The composition according to claim 1, wherein said bacterium comprises at least one plasmid comprising SEQ ID NO: 2, or any sequence with at least 80% identity with said SEQ ID NO: 2.

3. The composition according to claim 1 wherein said bacterium has been deposited under deposit number 1-4290 in the Collection Nationale de Cultures de Microorganisms (CNCM).

4. The composition according to claim 1, wherein said intracellular components comprise nucleic acids.

5. The composition according to claim 1 wherein said fractionated extract comprises secreted peptides, secreted proteins and secondary metabolites.

6. The composition according to claim 1 wherein said fractionated extract comprises membrane proteins, periplasmic proteins, proteins arising from the flagellum, secreted peptides, secreted proteins and secondary metabolites.

7. The composition according to claim 1, wherein said fractionated extract comprises a protein profile, obtained by SDS-PAGE, which includes three principal bands corresponding to molecular weights ranging between 30 kDa and 36 kDa, 41 kDa and 45 kDa, and 47 kDa and 51 kDa, respectively.

8. The composition according to claim 1 further comprising and one or more typical dermatologically-compatible excipients.

9. The composition according to claim 1 wherein said fractionated extract comprises membrane proteins, periplasmic proteins, and proteins arising from the flagellum.

10. The composition according to claim 9, wherein said membrane proteins are selected from porins, OmpA, lipopolysaccharides and lipoproteins.

Description

DESCRIPTION OF FIGURES

(1) FIG. 1 illustrates the phylogenetic position of the sequence coding for the 16S rRNA of strain LMB64. The sequences appearing on this tree are sequences from the GenBank database closest to the sequence of LMB64.

(2) FIGS. 2A and 2B present images of bacterium LMB64 under the transmission electron microscope (A) and the scanning electron microscope (B).

(3) FIG. 3 presents growth optima determined as a function of the temperature, pH and salinity of the R3 culture medium.

(4) FIG. 4 illustrates induction of cytokines IL-10 and IL-12 by extract E0 (dose-dependent effect).

(5) FIG. 5 illustrates induction of surface molecules CD80, CD86, CD83 and CD54 by extract E0 (dose-dependent effect).

(6) FIG. 6 illustrates inhibition of IgE receptors by extract E0.

(7) FIG. 7 illustrates activation of TLR2 by extract ES0.

(8) FIG. 8 illustrates activation of TLR4 by extract ES0.

(9) FIG. 9 illustrates activation of TLR5 by extract ES0.

(10) FIG. 10 illustrates specific PAR2 antagonist activity by extract ES0.

(11) FIG. 11 illustrates induction of antimicrobial peptides and proteins by extract ES0.

(12) FIG. 12 consists of an SDS-PAGE gel of extract ES0.

EXAMPLE 1

Selection and Characterization of Bacterium LMB64

(13) Bacterium AV13 was isolated from groundwater.

(14) The taxonomic position of novel bacterium LMB64 is proposed in FIG. 1.

(15) More particularly, bacterium LMB64 is rod-shaped with a length of roughly 2.3 m (0.3) and a width of roughly 1.0 m (0.1). A distinctive characteristic of this bacterium is the presence of a polar flagellum (FIGS. 2A and 2B). As can also be seen in these images, bacterium LMB64 is a nonfilamentous bacterium.

(16) As mentioned above, bacterium LMB64 has a circular plasmid of roughly 11 kpb. This plasmid was completely sequenced (SEQ ID No. 2).

(17) The gene coding for 16S rRNA was also sequenced (SEQ ID No. 1). The bacterium was cultured in a fermentor in a synthetic medium. The growth rate is higher when the medium has a low concentration of carbon substrates.

(18) The culture media tested are R3, MS-glucose and LB media whose compositions are described below in tables 1a, 1b and 1c, respectively.

(19) TABLE-US-00001 TABLE 1a COMPOSITION OF R3 MEDIUM Yeast extract 1 g/l Difco proteose peptone 1 g/l Casamino acids 1 g/l Glucose 1 g/l Soluble starch 1 g/l Sodium pyruvate 0.5 g/l.sup. K.sub.2HPO.sub.4 0.6 g/l.sup. MgSO.sub.4, 7H.sub.2O 0.1 g/l.sup.

(20) TABLE-US-00002 TABLE 1b COMPOSITION OF MS-GLUCOSE MEDIUM Glucose 6.0 g/l Citric acid 0.84 g/l MgSO.sub.4, 7H.sub.2O 0.25 g/l NH.sub.4Cl 1.06 g/l Anhydrous K.sub.2HPO.sub.4 8.75 g/l Pyruvic acid sodium salt 0.5 g/l Zinc sulfate, 7H.sub.2O 4 mg/l Cobalt chloride, 6H.sub.2O 3.5 mg/l Sodium molybdate, 2H.sub.2O 3.5 mg/l Manganese sulfate, 1H.sub.2O 5 mg/l Boric acid 2 mg/l Concentrated hydrochloric acid 50 mg/l Copper sulfate, 5H.sub.2O 4 mg/l Iron chloride, 6H.sub.2O 27 mg/l

(21) TABLE-US-00003 TABLE 1c COMPOSITION OF LB MEDIA Tryptone 10 g/l Yeast extract 5 g/l NaCl 5 g/l

(22) The growth rates of bacterium LMB64 as a function of culture medium are presented in table 2 below.

(23) TABLE-US-00004 TABLE 2 Growth rate (/h) LB 0.25 (0.05) LB ( dilution) 0.46 (0.11) LB ( dilution) 0.60 (0.14) LB ( 1/10 dilution) 0.69 (0.15) MS-glucose 0.13 (0.04) R3 0.62 (0.14)

(24) The growth optima were determined as a function of the temperature, pH and salinity of the R3 culture medium (FIG. 3).

(25) The sources of carbon assimilable by the bacterium were characterized using an API 50CH gallery (incubation temperature: 25 C.). The results are summarized in table 3 below.

(26) TABLE-US-00005 TABLE 3 Incubation time 4 days 5 days 1. Glycerol 2. Erythritol 3. D-arabinose 4. L-arabinose 5. D-ribose 6. D-xylose 7. L-xylose 8. D-adonitol 9. Methyl--D-xylopyranoside 10. D-galactose 11. D-glucose + + 12. D-fructose + + 13. D-mannose 14. L-sorbose 15. L-rhamnose 16. Dulcitol 17. Inositol l + 18. D-mannitol 19. D-sorbitol 20. Methyl--D-mannopyranoside 21. Methyl--D-glucopyranoside 22. N-acetylglucosamine 23. Amygdaline 24. Arbutin 25. Esculin/iron citrate 26. Salicin 27. D-cellobiose 28. D-maltose l + 29. D-lactose (bovine origin) 30. D-melibiose 31. D-sucrose + + 32. D-trehalose l + 33. Inulin 34. D-melezitose 35. D-raffinose 36. Starch 37. Glycogen 38. Xylitol 39. Gentiobiose 40. D-turanose l + 41. D-lyxose 42. D-tagatose 43. D-fucose 44. L-fucose 45. D-arabitol 46. L-arabitol 47. Potassium gluconate 48. Potassium 2-ketogluconate 49. Potassium 5-ketogluconate +: usable substrate, l: low use

(27) The enzymatic activities demonstrated on the API ZYM gallery are: alkaline phosphatase, esterase (C4), esterase/lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, and -glucosidase.

(28) Bacterium LMB64 is sensitive to all the antibiotics tested as seen in table 4 below.

(29) TABLE-US-00006 TABLE 4 Zone of inhibition diameter (mm) Inhibitory Antibiotics tested R3 LB LB activity Ampicillin (10 g) 29 28 29 + Chloramphenicol (30 g) 29 26 24 + Ciprofloxacin (5 g) 38 34 34 + Kanamycin (30 g) 27 30 27 + Penicillin (6 g) 21 26 20 + Polymyxin B (50 g) 11 15 13 + Rifampicin (30 g) 20 19 15 + Tetracycline (30 g) 30 25 20 + Streptomycin (10 g) 25 25 24 + Vancomycin (30 g) 20 21 21 +

EXAMPLE 2

Method for Extracting Fractions E0, S0 and ES0

(30) Preculture: Strain AV13 is inoculated in an Erlenmyer flask containing 250 ml of MS glucose pyruvate medium (see table 5 below), followed by incubation under stirring for roughly 40 hours at 30 C. (pH 7) and 200 rpm until an OD.sub.6001.5 is obtained.

(31) TABLE-US-00007 TABLE 5 MS Glucose Pyruvate Citric acid 0.84 g MgSO.sub.4, 7H.sub.2O 0.25 g NH.sub.4Cl 1.06 g Anhydrous K.sub.2HPO.sub.4 8.75 g Pyruvic acid sodium salt 0.5 g Oligo mix 1 ml ddH.sub.2O qsp 1000 ml Verify pH 7 Autoclave 121 C. 30 min After autoclaving add: 20% glucose 30 ml OLIGO MIX Dissolve in 100 ml of distilled water: Zinc sulfate, 7H.sub.2O 4 g Cobalt chloride, 6H.sub.2O 3.5 g Sodium molybdate, 2H.sub.2O 3.5 g Manganese sulfate, 1H.sub.2O 5 g Boric acid 2 g Concentrated hydrochloric acid 50 g Copper sulfate, 5H.sub.2O 4 g Dissolve in 50 ml of distilled water: Iron chloride, 6H.sub.2O 27 g ddH.sub.2O qsp 1000 ml

(32) Culture: The preculture is then inoculated in a fermentor (Applikon) containing 3.71 of MS pyruvate medium+114 ml of 20% glucose solution. A temperature sensor regulates the temperature preferably near 30 C. An oxygen sensor (AppliSens) is used to maintain the concentration of dissolved oxygen in the medium at 18-25%. A pH sensor (AppliSens) is used to maintain the pH at 7 by the addition of 10% NH.sub.4OH via a fixed flow-rate pump. A Wedgewood Analytical sensor is used to monitor changes in optical density in real time. The culture is programmed in fed-batch mode; via a variable flow-rate pump the culture is supplied with 20% glucose solution. Fermentation is stopped when OD.sub.60022-26, in general after roughly 30 hours.

(33) Extraction S0: The supernatant is separated from the biomass by centrifugation for 1 hour at 4 C. and 4000 g.

(34) Extraction E0: The wet biomass is taken up in NaCl solution (1 M). After centrifugation for 15 minutes at 4 C. and 9000 g, the supernatant is discarded and the pellet is taken up in 1 M NaCl solution. The sample tube is then plunged into a cooled ultrasonic bath at a power setting of 50-60 W for several minutes. After centrifugation for 30 minutes at 4 C. and 6000 g, the pellet is discarded and the supernatant is recovered. Two volumes of cold ethanol are added and the suspension is left overnight at 4 C. After centrifugation for 30 minutes at 4 C. and 6000 g, the supernatant is discarded and the pellet is taken up in 25 mM Tris buffer, pH 8.8.

(35) Extraction ES0: The culture is brought to basic pH (pH 9-11) with a base buffer. The next step is incubation under stirring for 5 hours at a temperature of 4 C. After centrifugation, the supernatant is prefiltered to eliminate remaining biomass debris and then filtered on a 0.2 m filter. A clear yellow solution is obtained (ES0).

(36) Proteins are assayed according to the DC Protein Assay Kit II (Bio-Rad) protocol. Sugars are assayed in glucose equivalent according to the phenol/sulfuric acid method (Dubois, M. et al., 1956).

(37) As an example, table 6 below presents certain specific characteristics of extract ES0 as obtained under the conditions described above.

(38) TABLE-US-00008 TABLE 6 Test batch Preclinical batch 1 Organoleptic Homogeneous and translucent yellow-orange liquid characteristics Density near that of water pH 10.0 10.2 (in the presence of base buffer) Dry residue 5.9% 5.1% (thermobalance) Protein profile 12 detectable bands (including 3 principal bands (SDS-PAGE) roughly 34 kDa, 43 kDa and 49 kDa in size, respectively) Total protein assay 2.9 mg/ml 3.0 mg/ml (BCA)

(39) It is clearly understood that the data above are presented here only for illustrative purposes.

(40) More precisely, the data relate to a protein profile obtained by SDS-PAGE exhibiting three principal bands.

(41) SDS-PAGE Protocol:

(42) Extract ES0 is taken up in buffer (20 mM Tris-HCl, pH 8.0; 1 mM EDTA; 2.5% SDS and 0.01% bromophenol blue) and 1 M DTT (1,4-dithiothreitol). The sample and the mixture of molecular weight markers (WesternC, Bio-Rad) were deposited respectively in wells of an 8-16% SDS-PAGE acrylamide gel (GeBaGel, Gene Bio-Application). The migration buffer contains 2.5 mM Tris, 19.2 mM glycine and 0.01% SDS (w/v). Migration is allowed to proceed under a constant voltage of 160 V for approximately 1 hour (GeBaGel system). The protein bands were then stained with Coomassie Blue (Instant Blue, Expedeon). Sizes were calculated in relation to known standards (STD).

(43) The gel obtained is presented in FIG. 12.

(44) According to one embodiment of the invention, these three bands have molecular weights of approximately 34 kDa, 43 kDa and 49 kDa, respectively.

EXAMPLE 3

Demonstration of the Pharmacological Activities of Fractions E0 and ES0

(45) Langerhans cells (LC) are generated in vitro from human monocytes isolated from Buffy-Coat pouches from the French National Blood Service (Etablissement Franais du Sang (EFS) Pyrnes Mditerrane): isolation on a Ficoll gradient (Lymphocyte Separation Medium, density 1.077 g/ml) and purification by magnetic immunoselection (Miltenyi Biotec); LC differentiation is carried out for 6 days in the presence of a cytokine cocktail (GM-CSF/IL-4/TGF). LC distributed on 24-well plates in RPMI-5% FCS culture medium are incubated for 24 hours with extract ES0.

(46) Surface molecules are analyzed by flow cytometry (FACSCalibur, BD Biosciences) with triple or quadruple staining: CD1a/CD54/CD80/CD83/CD86/FcRI; cytokines secreted in the culture supernatants are analyzed with the Cytometry Bead Array (cat. no. 550749, BD) in flow cytometry: IL-6, IL-8, TNF, IL-4, IL-10, IL-12.

(47) 3.1 Induction of Key Cytokines for Th1 Polarization

(48) Extract E0 induces according to a dose-dependent effect the expression of cytokines IL-10 and IL-12 by Langerhans cells (FIG. 4). These cytokines promote the induction of TH1 polarity of naive T lymphocytes.

(49) 3.2 Langerhans Cell Maturation and IgE Receptor (FcRI) Inhibition

(50) Extract E0 induces the maturation of Langerhans cells observed by dose-dependent induction of surface molecules CD80, CD86, CD83 and CD54 (FIG. 5). Similarly, extract E0 inhibits the expression of IgE receptors (FcRI) according to a dose-dependent effect (FIG. 6).

(51) 3.3 Activation of Toll-Like Receptors (TLRs)

(52) The TLR activity of ES0 was evaluated on TLR2, TLR4 and TLR5 using the model of HEK293 cells cotransfected by the gene for TLR2, TLR4 or TLR5 and by the reporter gene NFB-sAP (secreted alkaline phosphatase). The binding of a ligand to its TLR leads to the activation of the transcription factor NFB; the sAP gene is placed under the control of a promoter that can be induced by NFB. This reporter gene makes it possible to monitor cell signaling via TLRs: the release of sAP induced by ES0 and measured by colorimetric assay makes it possible to determine the activity of this active ingredient as a TLR2, TLR4 or TLR5 agonist.

(53) The study was carried out on the following human embryonic kidney (HEK293) cell lines: HEK-Blue-2 cells for TLR2, HEK-Blue-4 cells for TLR4, HEK-Blue-5 cells for TLR5,

(54) These cell lines were maintained in HEK-Blue Selection 10% FCS culture medium and then distributed in 96-well plates in HEK-Blue Detection medium in the presence of ES0 for 18 hours. The plates are read using calorimetry at 620 nm

(55) 3.3.1 Activation of TLR2

(56) Extract ES0 induces the activation of TLR2 according to a dose-dependent effect with a maximum activity at 100 ng/ml (FIG. 7).

(57) 3.3.2 Activation of TLR4

(58) Extract ES0 induces the activation of TLR4 with a maximum activity at 10 ng/ml (FIG. 8).

(59) 3.3.3 Activation of TLR5

(60) Extract ES0 induces the activation of TLR5 in a dose-dependent manner. This activity is inhibited in the presence of anti-TLR5 antibody, demonstrating the activation specificity of extract ES0 on TLR5 (FIG. 9).

(61) 3.4 Inhibition of Protease-activated Receptor 2 (PAR2)

(62) The inhibition of protease-activated receptors by extract ES0 is evaluated on human keratinocytes from a cell line (HaCaT) by measuring the intracellular calcium influx induced after specific stimulation of PAR2 with stratum corneum tryptic enzyme (SCTE). The fluorescent probe Fluo-4/AM is used: its esterified form facilitates its penetration by passive diffusion in the cell; only the deesterified form bound to calcium ions is excitable under 485 nm fluorescence and emits at 535 nm.

(63) The fluorescent probe is incorporated for 30 minutes in cells inoculated in 96-well plates and then extract ES0 is incubated for 30 minutes. Calcium flow is measured well by well in real time according to kinetics before and after injection of SCTE. The plates are read using a Mithras LB940 reader (Berthold Technologies).

(64) Extract ES0 inhibits in a dose-dependent manner activation of PAR2 induced by human SCTE (FIG. 10).

(65) 3.5 Modulation of Targets of Atopic Dermatitis on Keratinocytes

(66) The study was carried out on normal human epidermal keratinocytes (NHEK, K-SFM culture medium) in the context of the induction of an atopic dermatitis phenotype. The activity of ES0 was studied on keratinocytes exhibiting an atopic dermatitis phenotype after stimulation for 24 hours with Poly I:C+IL-4+IL-13+TNF- and analyzed by PCR array on the expression of a panel of 32 selected genes.

(67) On keratinocytes, extract ES0 inhibited according to a dose-dependent effect 15 targets among the mediators involved in atopic dermatitis pathology, as can be seen clearly in table 7 below (the results indicating for each target gene the percentage of inhibition obtained).

(68) TABLE-US-00009 TABLE 7 ES0 Dexamethasone 10 g/ml 30 g/ml 60 g/ml 2 M Cyto- TSLP 56% 75% 92% 91% kines IL-1 35% 46% 59% 54% IL-18 27% 44% 65% 44% IFN-1 66% 82% 90% 49% Chemo- IL-8 37% 55% 88% 75% kines MlP-1 10% 43% 75% 76% RANTES 15% 44% 65% 12% MCP-3 43% 63% 88% Pro 20% TARC 58% 64% 39% Pro 20% MIP-3 41% 61% 80% 40% MDC 16% 44% 58% 45% Skinkine 28% 32% 39% 59% Recep- IL-4-R 30% 45% 69% 75% tors RARRES3 30% 47% 63% 28% TLR3 22% 50% 60% pro 29%

(69) 3.6 Induction of Antimicrobial Peptides

(70) The activity of extract ES0 on the expression of antimicrobial peptides and proteins is studied on the HaCaT keratinocyte cell line: after 3 hours of treatment in the presence of ES0, the cells are recovered for an analysis of the expression of antimicrobial targets by quantitative RT-PCR; total RNA are extracted and assayed; after reverse transcription of mRNA into cDNA, the quantitative PCR amplification step is carried out in 96-well plates on an iCycler quantitative PCR system (Bio-Rad). The results obtained are expressed as the relative quantity (RQ) of mRNA after treatment by ES0 in relation to the control without the active ingredient. IL-1 is used in parallel as a reference positive inducer of antimicrobial peptide expression. Expression of the gene of interest is considered regulated when RQ2 (induction) or RQ0.5 (inhibition).

(71) Extract ES0 induces the expression of antimicrobial peptides and proteins hBD2, hBD3, S1007A, LL37, P13, RNase 7 and NOD2 (FIG. 11).

EXAMPLE 4

Formulation of a Body and Face Cream Comprising Bacterial Extract ES0

(72) Extract ES0: 0.1-5%

(73) Evening primrose oil: 1-3%

(74) Glycine: 0.1-0.4%

(75) Ceramides: 0.1-0.3%

(76) Humectants: 5-20%

(77) Emulsifier: 2-7%

(78) Capric/caprylic triglycerides: 1-10%

(79) Preservatives

(80) Water qsp 100%

EXAMPLE 5

Formulation of a Body and Face Cleansing Gel Comprising Bacterial Extract ES0

(81) Extract ES0: 0.1-5%

(82) Evening primrose oil: 0.5-2%

(83) Glycine: 0.1-0.4%

(84) Ceramides: 0.1-0.4%

(85) Surfactants: 10-20% in active matter

(86) Humectants: 5-15%

(87) Preservatives

(88) Water qsp 100%