THERAPEUTIC USE OF ENGINEERED POSTBIOTICS COMPRISING BACTERIOCINS AND/OR ENDOLYSINS

Abstract

The present invention concerns a postbiotic composition comprising at least one postbiotic and at least one bacteriocin and/or endolysin, preferably formulated, and a postbiotic composition comprising at least one postbiotic and at least one bacteriocin and/or endolysin for use as a medicament, wherein said postbiotic is preferably a microbial lysate, preferably obtained from microorganisms heterologously expressing said at least one bacteriocin and/or endolysin and wherein said at least one postbiotic and said at least one bacteriocin and/or endolysin have a synergistic effect in the therapeutic treatment.

Claims

1. 1-30. canceled

31. A formulation comprising an engineered postbiotic composition comprising a microbial lysate and lysostaphin, wherein said lysostaphin is obtained from bacteria heterologously expressing said lysostaphin.

32. The formulation according to claim 31 further comprising at least one pharmaceutically acceptable excipient and/or adjuvant selected from the group consisting of disintegrants, binders, bulking agents/fillers, lubricants, glidants, wetting agents, penetration/permeation enhancers, mucoadhesive agents, preservatives, anti-foaming agents, suspending agents, viscosity modifying agents, coloring agents, antioxidants, and combinations thereof.

33. The formulation according to claim 31 further comprising an additional therapeutically active agent.

34. The formulation according to claim 32 further comprising an additional therapeutically active agent.

35. The formulation according to claim 31, said formulation being in the form of aqueous, hydroalcoholic or oily solutions, of dispersions in the form of solutions or dispersions of the lotion or serum type, of emulsions in particular with liquid or semi-liquid consistency of the milk type, typically obtained by dispersion of a fatty phase in an aqueous phase (O/W) or conversely (W/O), or suspensions or emulsions of soft semi-solid or solid consistency of the cream type, of cream, of aqueous or anhydrous gel, of microemulsions, of nanoemulsions, of microcapsules, of microparticles, of ionic and/or nonionic type vesicular dispersions, of stick, of aerosol spray, of pump spray, or of foam.

36. The formulation according to claim 35, said formulation being in the form of an emulsion, of a microemulsion or of a nanoemulsion.

37. The formulation according to claim 31, wherein said bacteria heterologously expressing said lysostaphin are GRAS and/or probiotic bacteria.

38. The formulation according to claim 31, wherein said bacteria heterologously expressing said lysostaphin have been genetically modified to express said lysostaphin.

39. The formulation according to claim 31, wherein said bacteria heterologously expressing said lysostaphin are Lactobacillus or Escherichia bacteria.

40. The formulation according to claim 39, wherein said bacteria heterologously expressing said lysostaphin are selected from the group consisting of Lactobacillus rhamnosus, Lactobacillus plantarum and Escherichia coli bacteria.

41. The formulation according to claim 40, wherein said bacteria heterologously expressing said lysostaphin are Lactobacillus rhamnosus bacteria.

42. The formulation according to claim 31, wherein said bacteria heterologously expressing said lysostaphin do not comprise any antibiotic resistance marker.

43. The formulation according to claim 31, wherein said postbiotic composition further comprises at least two different bacteriocins and/or endolysins.

44. The formulation according to claim 43, wherein said at least two different bacteriocins and/or endolysins target the same bacterial species.

45. The formulation according to claim 43, wherein said at least two different bacteriocins and/or endolysins target different bacterial species.

46. The formulation according to claim 31, wherein said postbiotic composition is in the form of a pharmaceutical formulation for topical application.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0276] FIG. 1: Lysostaphin expression plasmid. p1016 plasmid map with His tagged Lysostaphin expressed from PorfX IP-673 inducible promoter. Plasmid backbone contains pWV01 origin of replication and an erythromycin resistance marker (ermB).

[0277] FIG. 2: Lysolysate killing activity. Measurement of S. aureus turbidity reduction with time when mixing S. aureus Newman strain with L. rhamnosus lysolysate produced from lysed culture of L. rhamnosus GG LrOs11721+p1016 expressing lysostaphin and concentrated 40× in 20 mM acetate buffer pH 5. L. rhamnosus GG LrOs11721 lysate was used as control and L. rhamnosus GG LrOs11721 lysates spiked with lysostaphin (1 and 4 μg/mL final concentration) as references.

[0278] FIG. 3: Lysolysate specific and efficient killing of S. aureus. Log reduction (-log10 (Final CFU/Initial CFU)) in S. aureus and other staphylococcal strains after 1 hour incubation with lysolysate.

[0279] FIGS. 4 and 5: Synergistic effect of lysostaphin and bacterial lysates. FIG. 4: Measurement of S. aureus turbidity reduction with time for L. rhamnosus GG lysate (concentrated 40×) and 20 mM acetate buffer pH 5 with or without lysostaphin (1 μg/mL). Time to reduce by half initial absorbance (IC50) is lower for lysostaphin in lysate compared to lysostaphin in acetate buffer. Lysate and acetate buffer in absence of lysostaphin have identical IC50. FIG. 5: Measurement of S. aureus CFU reduction with time for L. rhamnosus GG lysate (concentrated 40×) and 20 mM acetate buffer pH 5 with or without lysostaphin (1 μg/mL)

[0280] FIG. 6: Effect of lysate concentration on synergy. Measurement of S. aureus turbidity reduction with time for different concentrations of L. rhamnosus GG lysate (4×, 0.4×, 0.04×, 0.004×, 0.0004×) supplemented with Lysostaphin (1 μg/mL final concentration). Below a given concentration of lysate (between 0.004× and 0.0004×) the Lysostaphin killing activity is equivalent or lower than the same Lysostaphin concentration in acetate buffer.

[0281] FIG. 7: Synergy effect on different bacterial strains. Measurements of S. aureus killing efficiency (1/IC50) for different bacterial lysates spiked with different concentrations of lysostaphin. A higher killing efficiency in bacterial lysate than in acetate buffer adjusted at equivalent pH is observed.

[0282] FIG. 8: Effect of molecular crowding on lysostaphin activity in acetate buffer. Measurements of S. aureus turbidity reduction in acetate buffer pH 5 with lysostaphin 4 μg/mL, supplemented with different concentrations of Bovine Serum Albumin (BSA) to increase molecular crowding, show a reduction in lysostaphin activity with higher molecular crowding.

[0283] FIG. 9: Stimulation of S. epidermidis growth by L. rhamnosus lysate. Growth curve of S. epidermidis in diluted TSB (12.5% v/v) supplemented with 20 mM acetate buffer pH 5 or L. rhamnosus lysate.

[0284] FIG. 10: Anti-inflammatory effect of L. rhamnosus lysate. IL-10 ELISA on supernatant of LPS stimulated RAW blue Macrophages after treatment with L. rhamnosus GG lysate. IL-10 concentration in supernatants of RAW-BLUE cells stimulated with LPS and treated with L. rhamnosus lysate is higher than when treated with 20 mM acetate buffer pH 5.

[0285] FIGS. 11 and 12: Wound Healing effect of L. rhamnosus lysate. FIG. 11: Microscopy picture of scratch assay. Scratch assay on a Normal Human Epidermal Keratinocytes layer (NHEK) was performed and closure of the wound was monitored at T=0, 6, 12, 24 hours. Treatment with L. rhamnosus lysate allows a faster closure of the wound compared to treatment with IFN-γ or no treatment (control). FIG. 12: Quantification of wound-healing. Percentages of wound surface area decrease faster in presence of lysolysate compared to control or IFN-γ.

EXAMPLES

[0286] In the present examples, the inventors developed engineered postbiotics suitable to tackle diseases due to the growth of S. aureus, and demonstrated: [0287] a S. aureus specific killing activity based on a lysate containing lysostaphin, [0288] a synergistic increase in lysostaphin killing activity in presence of bacterial lysate, [0289] an anti-inflammatory effect to dampen the inflammatory response, [0290] a wound healing activity to repair barrier function, and [0291] a beneficial effect on host commensal microbiota.

[0292] As a proof of principle, probiotic strain L. rhamnosus GG (LrOs11721) was engineered to express lysostaphin, a bacteriocin with high specificity for S. aureus. Lysostaphin was cloned on a plasmid (FIG. 1) under the control of the sakacin inducible promoter PorfX (Sørvig et al. (2005) Microbiology (Reading, England) 151:2439-2449) and transformed into L. rhamnosus. Transformants were grown, lysostaphin expression was induced at mid-log phase and cells were harvested at high density (OD˜1). Bacterial cells were concentrated 40× in 25 mM sodium acetate buffer pH 5 by centrifugation before being lysed mechanically and filtered sterilized leading to a lysate (herein called lysolysate) containing both L. rhamnosus cell components and lysostaphin.

[0293] Example 1: S. aureus Specific Killing Using a L. rhamnosus Lysolysate

[0294] In order to check the staphylolytic activity of the lysolysate, a turbidity reduction experiment was performed using S. aureus Newman strain mixed with lysolysate (FIG. 2). As shown in FIG. 2, a rapid decrease of the S. aureus population, as measured by absorbance at 600 nm, can be observed in the presence of the lysolysate. No decrease in absorbance was observed when S. aureus cells were put in the presence of L. rhamnosus lysate, indicating that the expressed Lysostaphin is responsible for the turbidity reduction and so the staphylolytic activity. Inventors decided to test the lysolysate killing specificity towards S. aureus.

[0295] Specificity of endolysin is generally at the genus level meaning that for example staphylococcal endolysins are able to kill both non-commensal species such as S. aureus but also commensal species such as S. epidermidis. Unlike endolysins, Lysostaphin has been shown to be specifically targeting S. aureus and show a much lower activity against other Staphylococcal species such as S. epidermidis. To test if the Lysostaphin-containing L. rhamnosus lysate has also a high S. aureus specificity, a killing assay was performed (FIG. 3) using lysolysate in presence of both Coagulase positive strains (CoPS) including 75 S. aureus strains and Coagulase negative strains (CoNS) including 35 S. epidermidis strains. An average of 4.58 log reduction was obtained for S. aureus strains against a 1.54 log reduction obtained against the 35 S. epidermidis strains. Thus lysolysate shows a high specificity towards S. aureus species.

[0296] To quantify the effect of the bacterial lysate on Lysostaphin activity, a turbidity reduction experiment and a CFU experiment were performed (FIG. 4-5). The IC50, time to decrease initial OD by half, was measured for (i) L. rhamnosus lysate alone, (ii) 20 μg/ml of purified Lysostaphin (Sigma reference L9043) resuspended in 20 mM acetate buffer pH 5 (NaOAc) buffer and (iii) 20 μg/ml of purified Lysostaphin resuspended in L. rhamnosus lysate. Surprisingly, the inventors observed a higher turbidity reduction (lower IC50) and a faster decrease in CFU for the Lysostaphin in lysate compared to Lysostaphin in acetate buffer. No difference in turbidity reduction or CFU counts were measured between L. rhamnosus lysate and acetate buffer indicating that there is no activity of the L. rhamnosus lysate alone and the improvement of Lysostaphin activity in lysate is not the result of an additive effect of Lysostaphin activity and L. rhamnosus lysate activity but rather a synergistic effect of the lysate on the Lysostaphin activity. Such synergistic effect of Lysostaphin and bacterial lysate has not been documented and offers an advantageous and non-obvious effect for the killing of S. aureus with lysolysate compare to Lysostaphin alone.

[0297] This synergistic effect between Lysostaphin and the L. rhamnosus lysate was observed for different concentrations of lysate (FIG. 6) and for different concentrations of Lysostaphin (FIG. 7).

[0298] To test if this effect was specific of L. rhamnosus lysate, S. aureus killing activity of Lysostaphin mixed with lysates from different bacteria (Lactobacillus plantarum and Escherichia coli) was measured. A synergistic effect was observed for both Lactobacillus plantarum and E. coli even if at higher Lysostaphin concentration (6 μg/ml) E. coli lysate killing activity was similar to the activity in buffer (FIG. 7).

[0299] The inventors also tested if the pH of the lysate could explain this synergy as it is known that acidic pH is not optimum for Lysostaphin activity. Lysate from L. rhamnosus, L. plantarum and E.coli were respectively pH 6.8, 6.2 and 6.2. S. aureus bactericidal activity was measured for different concentrations of Lysostaphin in acetate buffer with equivalent pH (6.2 and 6.8). A higher killing was still observed for the lysate compared to buffer at same pH (FIG. 7) indicating that the pH cannot explain the synergy observed.

[0300] One difference between Lysostaphin in acetate buffer and Lysostaphin in lysate is the presence of a large amount of proteins and other cellular molecules that might provide a high molecular crowding environment for Lysostaphin to act. In order to test if molecular crowding could explain the increased activity of Lysostaphin in bacterial lysate the inventors performed a turbidity reduction experiment in presence of increasing concentrations of Bovine Serum Albumin (BSA) (FIG. 8). All concentrations of BSA led to a slower decrease in turbidity indicating a lower Lysostaphin activity. Thus molecular crowding does not seem to explain the synergy effect observed between lysate and Lysostaphin.

[0301] The inventors have shown that a lysolysate, produced from the lysis of L. rhamnosus bacterial cells heterologously expressing cytoplasmic Lysostaphin, allows highly efficient and specific killing of S. aureus strains. Surprisingly inventors demonstrated a synergistic effect between Lysostaphin and L. rhamnosus lysate increasing Lysotaphin killing activity. This synergistic effect is not specific to L. rhamnosus lysate, and depends on the lysate concentration.

[0302] Materials and Methods:

[0303] Bacterial Strains:

[0304] L. plantarum s15998 was isolated from fermented cabbage. Lysolysate was produced from strain s18195 (L. rhamnosus +p1016).

[0305] Production of Bacterial Lysates:

[0306] Overnight cultures of L. plantarum s15998 was inoculated from cryostock in 50 mL of MRS (NutriSelect Merck) and incubated in anaerobic conditions at 37° C. Overnight culture of L. rhamnosus was inoculated from cryostock in 50 mL of SPY2 (Heenan, C. N., et al.(2002). Lwt-Food Sci Technology 35, 171-176) and incubated in anaerobic conditions at 37° C. Overnight culture of E. coli K-12 MG1655 liquid culture was grown in 50 mL LB (Difco) and incubated overnight in aerobic conditions at 37° C.

[0307] Overnight cultures were diluted 1/10 in 500 mL of the appropriate media pre-reduced in anaerobic conditions and incubated at 37° C. in anaerobic conditions except for E. coli that was incubated at 37° C. in aerobic conditions. At OD.sub.600nm≈[1-2], bacterial cultures were put on ice, and the following steps were performed at 4° C. First cells were washed twice in deionized water using centrifugation and finally resuspended in 12.5 mL of 20 mM acetate buffer pH 5 (40× concentration of the initial cell culture). The concentrated culture was then lysed using bead beating at 30 Hz for 2 cycles of 20 minutes. Bacterial lysate was centrifuged for 10 min at 10 000 g and supernatant was then filtered (0.4 μm) and stored at 4° C. CFU counting was performed before and after bead beating treatment to measure lysis efficiency.

[0308] Production of Lysolysate:

[0309] Overnight culture of L. rhamnosus p1016 was inoculated from cryostock in 50 mL of SPY2 medium (Heenan, C. N., et al, (2002). Lwt-Food Sci Technology 35, 171-176) with erythromycin at a final concentration of 5 μg/mL and incubated in anaerobic conditions at 37° C. Overnight culture was diluted 1/10 in 500 mL of SPY2 medium pre-reduced in anaerobic conditions and incubated at 37° C. in anaerobic conditions. At an OD.sub.600nm of 0.3 the culture was induced with 200 ng/mL of inducing peptide IP-673 (Novopro Cat.#: 300935) and incubated at 37° C. until OD.sub.600nm=1.0. Bacterial culture was put on ice, and the following steps were performed at 4° C. First cells were washed twice in deionized water using centrifugation and finally resuspended in 12.5 mL of 20 mM acetate buffer pH 5 (40× concentration of the initial cell culture). The concentrated culture was then lysed using bead beating at 30 Hz for 2 cycles of 20 minutes, placing the sample on ice for 2 minutes in between cycles. Bacterial lysate was centrifuged for 10 min at 10 000 g and supernatant was then filtered (0.4 μm) and stored at 4° C. CFU quantification was performed before and after bead beating treatment to measure lysis efficiency.

[0310] Turbidity Reduction Experiment:

[0311] Overnight culture of S. aureus strain Newman was inoculated from an isolated colony in 15 mL of TSB (Tryptic Soy Broth, Difco) and incubated at 37° C., aerobically. Overnight culture was diluted 1/100 in a final volume of 1.5 L of TSB and incubated aerobically at 37° C. At OD600 nm=1 culture was washed twice with deionised water at 4° C., centrifuged at 4° C., at 4000 g for 10 minutes, resuspended in 7.5 mL of 1× PBS (Phosphate Buffered Saline, Fisher BioReagents, pH 7.4) and finally frozen as 500 μL aliquots at −20° C.

[0312] Bacterial suspension for the turbidity reduction assay was prepared from an aliquot of the frozen stock of S. aureus strain Newman. Lysostaphin solution and bacterial suspension were mixed in a ratio 1:10 in duplicates in a 96-well plate (Microlon 200, transparent, flat bottom) in a final volume of 200 μL and absorbance at 600 nm (Tecan Infinite 200 pro) was measured every 1.3 minutes, at 37° C. without agitation for 1 hour.

[0313] Example 2: Beneficial Effect of Lysate on Skin Microbiota

[0314] The approach of the inventors aims at killing specifically S. aureus, as shown in example 1, but also helping the skin commensal bacteria to restore homeostasis by helping them grow and occupy the niche left empty from S. aureus decolonization.

[0315] To test such an effect, the inventors investigated the effect of the lysate on the growth of S. epidermidis (FIG. 9). S. epidermidis (ATCC® 12228TM) was grown in poor nutrient conditions supplemented or not with L. rhamnosus lysate and cell density was followed by absorbance using OD.sub.600nm measurements. S. epidermidis shows a higher growth rate and final density in presence of L. rhamnosus lysate compared to buffer indicating a beneficial effect of the lysate on S. epidermidis.

[0316] Materials and Methods: Production of L. Ramnosus Lysate:

[0317] Overnight culture of L. rhamnosus was inoculated from cryostock in 50 mL of SPY2 (Heenan, C. N., et al.(2002). Lwt-Food Sci Technology 35, 171-176) and incubated in anaerobic conditions at 37° C. Overnight culture was diluted 1/10 in 500 mL of the appropriate media pre-reduced in anaerobic conditions and incubated at 37° C. in anaerobic conditions. At OD600 nm≈1, bacterial culture was put on ice, and the following steps were performed at 4° C. First cells were washed twice in deionized water using centrifugation and finally resuspended in 12.5 mL of 20 mM acetate buffer pH 5 (40× concentration of the initial cell culture). The concentrated culture was then lysed using bead beating at 30 Hz for 2 cycles of 20 minutes. Bacterial lysate was centrifuged for 10 min at 10 000 g and supernatant was then filtered (0.4 μm) and stored at 4° C. CFU was performed before and after bead beating treatment to measure lysis efficiency.

[0318] Growth Curve Experiment:

[0319] A preculture of S. epidermidis (ATCC® 12228TM) was inoculated from cryostock into 10 mL TSB and incubated at 37° C. overnight. Overnight culture was washed twice in 12.5% (v/v) TSB, normalized to OD.sub.600nm=1 and diluted 1/100 in 12.5% (v/v) TSB. In a 96 well plate, 180 μL of normalized bacterial culture was supplemented with 20 μL of L. rhamnosus lysate or 20 μL of 20 mM acetate buffer pH 5. Absorbance at 600 nm (Tecan Infinite 200 pro) was measured every 10 minutes, at 37° C. with agitation for a total of 10 hours.

[0320] Example 3: Anti-Inflammatory Effect

[0321] Another aspect of the engineered postbiotic of the present invention is its anti-inflammatory property. L. rhamnosus has already been shown to have anti-inflammatory properties, as a live probiotic (Sultana et al. (2013) Applied and Environmental Microbiology 79:4887-4894; Garcia et al. (2015) Applied and Environmental Microbiology 81:2050-2062), as a lysate or as killed probiotic (Mohammedsaeed et al. (2015) Scientific Reports 5:16147; Li et al. (2009) Pediatr Res 66:203-207). In order to test the anti-inflammatory properties of the lysate, the inventors used a LPS inflammation model on macrophage RAW-BLUE cells.

[0322] Briefly, growth culture of RAW-BLUE cells was supplemented with LPS or PBS and the cells were then treated with L. rhamnosus lysate or the buffer the lysate was prepared in.

[0323] Seventeen hours after treatment the anti-inflammatory cytokine IL-10 was measured by ELISA in RAW-BLUE cells supernatant (FIG. 10). IL-10 levels were higher in the inflammation model after treatment with lysate compared to LPS treatment alone indicating a potential anti-inflammatory effect of the L. rhamnosus lysate.

[0324] Materials and Methods:

[0325] Production of L. Rhamnosus Lysate:

[0326] Overnight culture of L. rhamnosus was inoculated from cryostock in 50 mL of SPY2 (Heenan, C. N., et al. (2002). Lwt-Food Sci Technology 35, 171-176) and incubated in anaerobic conditions at 37° C. Overnight culture was diluted 1/10 in 500 mL of the appropriate media pre-reduced in anaerobic conditions and incubated at 37° C. in anaerobic conditions. At OD600 nm≈1, bacterial culture was put on ice, and the following steps were performed at 4° C. First cells were washed twice in deionized water using centrifugation and finally resuspended in 12.5 mL of 20 mM acetate buffer pH 5 (40× concentration of the initial cell culture). The concentrated culture was then lysed using bead beating at 30 Hz for 2 cycles of 20 minutes. Bacterial lysate was centrifuged for 10 min at 10 000 g and supernatant was then filtered (0.4 μm) and stored at 4° C. CFU enumeration was performed before and after bead beating treatment to measure lysis efficiency.

[0327] RAW-BLUE Cells Experiment:

[0328] RAW-BLUE cells macrophage were grown in DMEM-P/S-FBS cell culture media (DMEM, 4.5 g/l glucose, 10% heat-inactivated FBS, 100 U/mL of penicillin/streptomycin and 1 mM sodium pyruvate) with 200 μg/ml Zeocin and used once a confluency of 70-80% was reached. Cells were collected and diluted in DMEM-P/S-FBS (no zeocin) to 5.88e5 cells/mL and 170 μL was added to each well (˜100,000 cells/well). After 7 hours cells were stimulated with 10 μL of 20 μg/mL LPS (1 μg/mL final concentration in well) or 10 μL PBS as a control, followed by treatment with 20 μL of lysate or 20 mM acetate buffer pH 5. Approximately 17 hours after, supernatant was collected and used for ELISA.

[0329] IL-10 ELISA:

[0330] Using culture supernatant harvested from the Raw Blue cells inflammation model exp. An IL-10 elisa was performed as per manufacturer's instructions for the ELISA kit (DuoSet® ELISA DEVELOPMENT SYSTEM ref:DY417-05). The standard curve was performed using the following concentrations of IL-10: 0, 7.8125, 15.625, 31.25, 62.5, 125, 250, 500 pg/mL.

[0331] Example 4: Wound Healing Activity to Repair Barrier Function

[0332] In addition to S. aureus bactericidal effects and stimulation of commensal bacteria, the inventors also explored the wound healing activity of the lysolysate. Indeed, L. rhamnosus lysate has been previously shown to promote wound healing (Mohammedsaeed et al. (2015) Scientific reports 5:16147) and stimulate barrier function (Sultana et al. (2013) Applied and Environmental Microbiology 79:4887-4894).

[0333] For that purpose, a scratch assay with keratinocytes in the presence of different concentrations of lysolysate was performed (FIGS. 11-12). A faster decrease in the surface of the wound was observed when lysolysate was added compared to control buffer or interferon gamma.

[0334] In conclusion the engineered postbiotics of the present invention show multiple activities that once combined on human skin should help resolve dysbiosis-induced disorders or diseases and reach homeostasis faster by: [0335] killing specifically the most frequent aetiological agent that is S. aureus, without negatively affecting the commensal skin population, [0336] stimulating growth of commensal skin population such as S. epidermidis, [0337] reducing inflammation and thus allowing de-escalation of associated symptoms (redness, itching), and [0338] promoting wound-healing thereby promoting barrier function of the skin.

[0339] Some of these activities should act synergistically to treat dysbiosis-induced disorders or diseases. For example by reducing inflammation and the associated production of human antimicrobial peptide, the engineered postbiotics could decrease impact on local skin microbiota whose growth will also be stimulated by the engineered postbiotics. In return, preserving the diversity of skin microbiota will help prevent dysbiosis resurgence and reappearance of diseases or disorders associated with such dysbiosis.