WO2018209345A1 - Compositions and methods for inhibiting biofilm deposition and production - Google Patents
Compositions and methods for inhibiting biofilm deposition and production Download PDFInfo
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- WO2018209345A1 WO2018209345A1 PCT/US2018/032549 US2018032549W WO2018209345A1 WO 2018209345 A1 WO2018209345 A1 WO 2018209345A1 US 2018032549 W US2018032549 W US 2018032549W WO 2018209345 A1 WO2018209345 A1 WO 2018209345A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01084—Glucan 1,3-alpha-glucosidase (3.2.1.84), i.e. mutanase
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- A61K38/1729—Cationic antimicrobial peptides, e.g. defensins
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01011—Dextranase (3.2.1.11)
Definitions
- the present invention relates to the fields of biofilm deposition and the treatment of disease. More specifically, the invention provides compositions and methods useful for the treatment of dental caries and other oral diseases. The invention also provides methods for coating biomedical devices for inhibiting undesirable biofilm deposition thereon.
- Biopharmaceuticals produced in current systems are prohibitively expensive and are not affordable for large majority of the global population.
- the cost of protein drugs ($140 billion in 2013) exceeds GDP of >75% of countries around the globe [Walsh 2014], making them unaffordable.
- One third of the global population earns ⁇ $2 per day and can't afford any protein drug (including the underprivileged, elderly and lower socio-economic groups in the US).
- Such high costs are associated with protein production in prohibitively expensive fermenters, purification, cold transportation/ storage, short shelf life and sterile delivery methods [Daniell et al 2015, 2016].
- Biofilms are formed by a complex group of microbial cells that adhere to the
- Caries- causing (cariogenic) biofilms develop when bacteria accumulate on tooth-surfaces, forming organized clusters of bacterial cells that are firmly adherent and enmeshed in a extracellular matrix composed of polymeric substances such as exopolysaccharides (EPS) [Bowen and Koo, 2011].
- EPS exopolysaccharides
- Current topical antimicrobial modalities for controlling cariogenic biofilms are limited. Chlorhexidine (CHX) is considered the 'gold standard' for oral antimicrobial therapy, but has adverse side effects including tooth staining and calculus formation, and is not recommended for daily therapeutic use [Jones, 1997; Autio-Gold, 2008].
- AMPs antimicrobial peptides
- Antimicrobial peptides are an evolutionarily conserved component of the innate immune response and are naturally found in different organisms, including humans. When compared with conventional antibiotics, development of resistance is less likely with AMPs. They are potently active against bacteria, fungi and viruses and can be tailored to target specific pathogens by fusion with their surface antigens (Lee et al 2011; DeGray et al 2001; Gupta et al 2015). Linear AMPs have poor stability or antimicrobial activity when compared to AMPs with complex secondary structures.
- retrocyclin or protegrin have high antimicrobial activity or stability when cyclized (Wang et al 2003) or when forming a hairpin structure (Chen et al 2000) via disulfide bond formation.
- RC 101 is highly stable at pH 3, 4, 7 and temperature 25°C to 37°C as well as in human vaginal fluid for 48 hours (Sassi et al 201 la), while its antimicrobial activity was maintained for up to six months (Sassi et al 201 lb).
- protegrin is highly stable in salt or human fluids (Lai et al 2002; Ma et al 2015) but lost potency when linearized.
- a multi-component composition comprising at least one antimicrobial peptide (AMP) and at least one biofilm degrading enzyme which act synergistically to degrade biofilm structures and inhibit biofilm deposition
- the AMP is selected from protegrin 1, RC-10land the AMPs listed in Table 1.
- the biofilm degrading enzyme includes, for example, mutanase, dextranase, glucoamylase, deoxyribonuclease I, DNAase, dispersin B, glycoside hydrolases and the enzymes provided in Table 2.
- the coding sequences for these enzymes are codon optimized for expression in a plant chloroplast.
- the at least one AMP and at least one biofilm degrading enzyme are produced recombinantly.
- the AMP and biofilm degrading enzyme(s) are expressed as a fusion protein.
- the enzymes are chemically synthesized, obtained from a microorganism or obtained from a commercial supplier.
- the composition may optionally further comprise an antibiotic, fluoride, CHX, essential oils, or all of the above.
- the composition may be contained within chewing gum, hard candy, or within an an oral rinse.
- Preferred fusion proteins of the invention include, without limitation, PG-l-Mut, PG-l-Dex, PG-l-Mut-Dex, RC-101-Mut, RC-101-Dex, RC-101-Mut-Dex for use alone or in combination for the degradation of biofilms.
- any of the AMPs listed in Table 1 can replace either PG-1 or RC-101 in the aforementioned fusion proteins to alter or improve the bacteriocidal action of the fusion protein.
- the enzymes listed above and hereinbelow may replace Mut, Dex or both in the fusion proteins of the invention.
- such enzymes may be delivered at different ratios, e.g., 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8 etc.
- a preferred ratio is 5: 1 Dex:Mut.
- the invention provides a method of degrading and/or removing biofilm comprising contacting a surface harboring said biofilm with the compositions described above, the composition having a bactericidal effect, and reducing or eliminating said biofilm comprising one or more undesirable microorganisms, wherein when said biofilm is present in or on an animal subject in need of said reduction or elimination.
- the biofilm is present in the mouth. In other embodiments, the biofilm is present on an implanted medical device.
- the method may also be used to remove biofilms present in an internal or external body surface iselected from the group consisting of a surface in a urinary tract, a middle ear, a prostate, vascular intima, heart valves, skin, scalp, nails, teeth and an interior of a wound.
- composition of the invention comprising said at least one AMP and said at least one biofilm degrading enzyme are produced in a plant plastid.
- the plant may be a tobacco plant and the sequences encoding said AMP and enzyme is codon optimized for expression in a plant plastid.
- the AMP and biofilm degrading enzyme are expressed in a lettuce plant as a fusion protein under the control of endogenous regulatory elements present in lettuce plastids.
- a composition comprising one or more essential oils and at least one biofilm degrading enzyme which act synergistically to degrade biofilm structures and inhibit biofilm deposition, in a biologically acceptable carrier for delivery.
- the essential oils comprise one or more of menthol, thymol, eucalyptol and methyl salicylate and the at least one biofilm degrading enzyme is selected from mutanase, dextranase and glucoamylase.
- the essential oils are menthol, thymol, eucalyptol and methyl salicylate, and the biofilm degrading enzymes are mutanase and dextranase.
- the mutanase and dextranase are present in a 5: 1 ratio.
- the enzymes are obtained commercially, purified from biological sources or produced in a plant plastid.
- the essential oils are present in acarrier is selected from the group consisting of mouthwash, mouth rinse, mouth spray, toothpaste, chewing gum, tooth gel, sub-gingival gel, mousse, foam, chewable tablet, dentifrice, lozenge, and dissolvable strip.
- the carrier is a mouthwash. Examples of
- mouthwashes comprising essential oils such as menthol, thymol, eucalyptol and/or methyl salicylate
- alcohol-free mouthwashes e.g. those sold under the Listerine®ZeroTMbrand and antiseptic mouthrinses, e.g. those sold under the Listerine® brand.
- the invention also provides a composition
- a composition comprising an effective amount of one or more biofilm degrading enzymes selected from mutanase, dextranase and glucoamylase and one or more essential oils selected from the group consisting of group consisting of menthol, thymol, eucalyptol, methyl salicylate, and combinations of two or more thereof in a carrier suitable for oral delivery, wherein the carrier is selected from the group consisting of mouthwash, mouth rinse, mouth spray, toothpaste, chewing gum, tooth gel, sub-gingival gel, mousse, foam, chewable tablet, dentifrice, lozenge, and dissolvable strip.
- the carrier is a liquid carrier such as a mouthwash, mouth rinse, or mouth spray.
- the carrier is a solid or semi-solid carrier such as a chewing gum, chewable tablet, lozenge, or dissolvable strip.
- the carrier is a toothpaste.
- a method of degrading and/or removing unwanted biofilm from the oral cavity comprises administering an effective amount of one or more biofilm degrading enzymes selected from the group consisting of mutanase, dextranase, glucoamylase, and combinaitons of two or more thereof, and one or more essential oils selected from the group consisting of menthol, thymol, eucalyptol, methyl salicylate, and combinations of two or more thereof, to a surface of the oral cavity having said biofilm.
- said essential oils comprise a combination of menthol, thymol, eucalyptol and methyl salicylate.
- Another exemplary method comprises contacting a surface of the oral cavity having said biofilm with an effective amount ofa pretreatment composition comprising biofilm degrading enzymes in a suitable carrier, followed by treatment with an antimicrobial oral rinse, wherein said contacting and treating exert a bactericidal effect, thereby synergistically reducing or eliminating said unwanted biofilm.
- the antimicrobial oral rinse comprises at least two essential oils selected from menthol, thymol, eucalyptol and
- methylsalicylate In other aspects the essential oils are present in Listerine® mouthwash. Notably, the aforementioned methods are effective to selectively kill pathogenic S. mutans without adversely affecting commensal bacteria, A. naeslundii and S. oralis.
- the invention also provides a prophylactic method for inhibiting biofilm deposition on the surface in the oral cavity, comprising contacting a surface of the oral cavity susceptible to biofilm deposition with an effective amount of one or more biofilm degrading enzymes selected from the group consisting of mutanase, dextranase, glucoamylase, and combinaitons of two or more thereof, and one or more essential oils selected from the group consisting of menthol, thymol, eucalyptol, methyl salicylate, and combinations of two or more thereof, to a surface of the oral cavity having said biofilm.
- the essential oils comprise a combination of menthol, thymol, eucalyptol and methyl salicylate.
- Another exemplary method comprises contacting a surface of the oral cavity susceptible to biofilm deposition with an effective amount of a pretreatment composition comprising biofilm degrading enzymes in a suitable carrier, followed by treatment with an antimicrobial oral rinse, wherein said contacting and treating exert a bactericidal effect, thereby synergistically inhibiting deposition of the unwanted biofilm.
- the antimicrobial oral rinse comprises at least two essential oils selected from menthol, thymol, eucalyptol and methylsalicylate.
- the essential oils are present in Listerine®.
- the aforementioned methods are effective to selectively kill pathogenic S. mutans without adversely affecting commensal bacteria, A.
- FIG. 1A Figures 1A- ID - Purification of GFP fused Retrocyclin (RC101) and Protegrin (PG1) expressed in tobacco chloroplasts -
- Fig. 1 A Western blot analysis of purified GFP-RC101 fusion using Anti-GFP antibody.
- Fig. IB Native fluorescence gel of purified GFP -RC 101 fusion.
- Fig. lC Western blot of purified GFP-PG1 fusion using Anti-GFP antibody.
- Fig. ID Native fluorescence gel of purified GFP-PG1. Note - All the samples for Fig 1A -ID were loaded based on total protein values obtained from the Bradford method. Densitometry using Image J software was done to determine GFP concentration Expression level, purity and yield.
- GFP concentrations were calculated from GFP concentrations relative to total protein values. Yield was determined by multiplying GFP concentration with recovered volume after purification. Individual peptide yield was determined by dividing GFP yield with molar factor 14 (ratio of GFP MW to peptide MW). The fold enrichment was calculated by dividing % purity with % expression in plant crude extracts.
- FIGS 2A -2E Antimicrobial activity of AMPs (GFP-PGl and GFP-RC101) against Streptococcus mutatis and other oral microbes. Cell viability was determined by absorbance (A 6 oonm) and counting colony forming units (CFU) over-time.
- FIG. 2A Time-killing curve of S. mutans treated with different concentrations of GFP-PGl and synthetic PG1 (A600 nm).
- FIG. 2B Viable cells (CFU/ml) of S. mutans treated with GFP-PGl and synthetic PG1 at each time point.
- FIG. 2C Time-killing curve of S. mutans treated with GFP-RC101 at different concentrations (A 6 oonm).
- FIG. 2D Viable cells (CFU/ml) of S. mutans treated with GFP -RC 101 at each time point.
- FIG. 2E Viable cells (CFU/ml) of S. gordonii, A. naeslundii and C. albicans treated with GFP-PGl at 10 ⁇ g/ml for 1 h and 2 h.
- FIGS 3A - 3C Bacterial killing by GFP-PGl as determined via confocal fluorescence and SEM imaging
- Fig. 3 A Time-lapse killing of S. mutans treated with GFP-PGl at 10 ⁇ g/ml.
- the control group (Fig. 3B) consisted of S. mutans cells treated with buffer only.
- PI Propidium iodide
- PI Propidium iodide
- Fig. 3C Morphological observations of S. mutans subjected to GFP-PGl at a concentration of 10 ⁇ g/ml for 1 h using scanning electron microscopy. Red arrows show dimpled membrane and extrusion of intracellular content.
- Figures 4A -4C Inhibition of biofilm formation by a single topical treatment of GFP-PGl.
- This figure displays representative images of three-dimensional (3D) rendering of S. mutans biofilm.
- Bacterial cells were stained with SYTO 9 (in green) and EPS were labeled with Alexa Fluor 647 (in red).
- Saliva-coated hydroxyapatite (sHA) disc surface was treated with a single topical treatment of GFP-PGl with a short-term 30 min exposure (Fig. 4B).
- the control group (Fig. 4A) was treated with buffer only. Then, the treated sHA disc was transferred to culture medium containing 1% (w/v) sucrose and actively growing S. mutans cells (10 5 cfu/ml) and incubated at 37°C, 5%C0 2 for 19h.
- FIG. 4C Quantitative analysis of proportion of live and dead S. mutans cells via quantitative PCR (qPCR) with or without propidium monoazide (PMA) treatment (Klein et al., 2012).
- PMA propidium monoazide
- the combination of PMA and qPCR (PMA-qPCR) quantify viable cells with intact membrane.
- PMA is added to selectively cross-link DNA of dead cells, and thereby prevent PCR amplification (Klein et al., 2012).
- Asterisks indicate that the values from GFP-PG1 treatment are significantly different from control (. ⁇ 0.05).
- FIG. 1 EPS-degrading enzymes digesting biofilm matrix. Representative time-lapsed images of EPS degradation in S. mutans biofilm treated with combination of dextranase and mutanase. Bacterial cells were stained with SYTO 9 (in green) and EPS were labeled with Alexa Fluor 647 (in red). The white arrows show Opening' of spaces between the bacterial cell clusters and 'uncovering' cells following enzymatic degradation of EPS.
- FIGS 6A -6C Biofilm disruption by synthetic PG1 alone or in combination with EPS- degrading enzymes.
- FIG. 6A Time-lapse quantification of EPS degradation within intact biofilms using COMSTAT.
- FIG. 6B The viability of S. mutans biofilm treated with synthetic PG1 and EPS-degrading enzymes (Dex/Mut) either alone or in combination by ImageJ.
- Fig. 6C Antibiofilm activity of synthetic PG1 was enhanced by EPS-degrading enzymes (Dex/Mut). Asterisks indicate that the values for different experimental groups are significantly different from each other ( ⁇ 0.05).
- Figure 7 In vitro uptake of purified fused protein CTB-GFP, PTD-GFP, Protegrin-l-GFP (PG1-GFP) and Retrocyclin10l-GFP (RC101- GFP) in different human periodontal cell lines: human periodontal ligament stem cells (HPDLS), maxilla mesenchymal stem cells (MMS), human head and neck squamous cell carcinoma cells (SCC), gingiva-derived mesenchymal stromal cells (GMSC), adult gingival keratinocytes (AGK) and osteoblast cell (OBC) with confocal microscopy.
- HPDLS human periodontal ligament stem cells
- MMS maxilla mesenchymal stem cells
- SCC human head and neck squamous cell carcinoma cells
- GMSC gingiva-derived mesenchymal stromal cells
- ATK adult gingival keratinocytes
- OBC osteoblast cell
- 2xl0 4 cells of human cell lines HPDLS, MMS, SCC, GMSC, AGK and OBC were cultured in 8-well chamber slides (Nunc) at 37°C for overnight, followed by incubation with purified GFP fusion proteins: CTB-GFP (8.8 PTD-GFP (13 PG1- GFP (1.2 ⁇ g), RC101-GFP (17.3 in 100 ⁇ PBS supplemented with 1% FBS, respectively, at 37°C for 1 hour. After fixing with 2% paraformaldehyde at RT for 10 min and washing with PBS for three times, the cells were stained with antifade mounting medium with DAPI.
- FIG. 1 Downstream processing of GFP fusions from transplastomic tobacco: Flow diagram illustrating the different steps involved in generation of purifed GFP fusions from transplastomic tobacco plants grown in greenhouse.
- Figures 9A - 9B Vectors and codon optimized sequences for mutanase (Fig. 9A) and dextranase (Fig. 9B). Codon optimized mutanase: SEQ ID NO: 1. Codon Optimized dextranase: SEQ ID NO: 2.
- Figure 10. A schematic diagram of a choloroplast vector expressing tandem repeats of AMPs fused with GVGVP for use alone or for expressing fusion protiens comprising the EPS proteins in Figure 9.
- Figures 12A - 12B Expression of functional codon optimized mutanase in E. coli.
- Fig. 12 shows western blots showing mutanase expression in E. coli.
- Fig. 12B shows E. coli spread on 0.5% blue dextran plates. Transformed clones are able to produce recombinant dextranase normally made in S. mutans and able to clear a blue halo around the colony.
- Fig. 12C represents a gel diffusion assay comparing the degradation activity of recombinant dextranase present in the crude lysate (Total Protein loading) from the transformed E. coli against blue dextran as compared to commercially purified enzyme from Penicillin.
- FIG 13. A flow diagram of the steps for engineering lettuce plants for AMP/biofilm degrading enzyme production.
- Figure 14. Chewing gum tablet preparation is shown. While GFP is exemplified herein, chewing gum comprising the AMP-enzyme fusion proteins (e.g., those provided in Figs. 9 and 10) is also within the scope of the invention.
- FIG. 16 A chewing simulator is shown which uses artificial saliva for assessing release kinetics of biofilm degrading agents from the gum tablets of the invention.
- Figure 17 A graph showing quantification of GFP released from chewing gum. Gum tablets comprising increasing concentrations of GFP expressed in lettuce leaves were assessed in a chewing simulator in the presence of artificial saliva to determine GFP release kinetics.
- FIGS 19A - 19E Glucanohydrolases enhance antimicrobial killing efficacy in S. mutans biofilm with optimum activity ratio.
- Fig. 19 A Saliva-coated hydroxy apatite (sHA) biofilm model used in this study.
- Fig. 19B Schematic of treatment regimen and hypothesis of EPS- degrading/ Antimicrobial (EDA) approach on preformed S. mutans biofilms.
- Fig. 19C Effect of dextranase/mutanase treatments on antimicrobial killing efficacy. 19-h preformed S. mutans biofilms on sHA were topically treated with different combinations of glucanohydrolases for 120min and then immediately exposed to antimicrobial (EOs) for lmin.
- EOs antimicrobial
- FIGS 20A -20F EPS-degrading enzymes dismantle biofilm matrix in situ and facilitate antimicrobial targeting within biofilms.
- FIG. 20 A Confocal microscopy showing morphology of 19-h S. mutans biofilm after matrix degradation by glucanohydrolase(s) for 120min. Green, bacteria cells stained by SYT09; Red, Exopolysaccharides (EPS) labelled by Alexa Fluor 647; White arrow, bacterial dispersion induced by dual -enzyme treatment.
- FIG. 20B Time-lapse killing assay performed using real-time bacterial live/dead staining and imaging. Preformed (19- h) S.
- mutans biofilms were treated with 8.75U/mL dextranase + 1.75U/mL mutanase or vehicle control for 120min and were both challenged with antimicrobial (EOs). Images of a single microcolony were acquired at Omin, lmin and 5min after antimicrobial challenge. Green, live cells (SYTO 9); magenta, dead cells (propidium iodide); red, EPS (Alexa Fluor 647). White arrows, in vehicle-treated group, the antimicrobial readily killed bacteria close to the surface of the microcolony while cells residing inside remained mostly vital. (Fig. 20C) Total biomass(dry weight) per biofilm after matrix degradation by glucanohydrolase(s) for 120min. (Fig.
- FIG. 20D Biochemical properties of exopolysaccharides of S. mutans biofilm after matrix degradation by glucanohydrolase(s) for 120min.
- FIG. 20E Synergistic antibiofilm effect of glucanohydrolases and antimicrobial.
- D Dextranase
- M Mutanase
- NS not significantly different; ***, p ⁇ 0.001 (Student t test).
- FIG. 20F Three-enzyme combination with dextranase, mutanase and glucoamylase further enhances antimicrobial killing efficacy.
- mutans biofilms (EPS labelled with Alexa Fluor 647) were imaged in 0.1M sodium acetate buffer solution containing 5 ⁇ SYT09 and 30 ⁇ propidium iodide for continuous labeling and real-time visualization of live and dead bacterial cells over time.
- Glucanohydrolases were added to the buffer solution to yield a final concentration of 8.75U/mL dextranase and 1.75U/mL mutanase while the same volume of buffer solution was used as vehicle control.
- Fig. 21 A and Fig. 2 IB 4-dimentional time-lapse confocal imaging of matrix degradation.
- Fig. 21C and (Fig. 2 ID) Final image of the microcolony acquired after the 1-min antimicrobial killing (EOs).
- FIGs 22A-22C Mechanical stability and integrity of the biofilm scaffold is damaged by EPS-degradation.
- FIG. 22A Time-lapse imaging showing "implosion-like" collapse of the physical structure of S. mutans biofilm accompanied with extensive cellular dispersion caused by matrix degradation (8.75U/mL dextranase and 1.75U/mL mutanase) within 120min. Green, bacteria cells stained by SYT09; Red, EPS labelled by Alexa Fluor 647.
- FIG. 22B Time- resolved EPS degradation (red squares) and microcolony spatial displacement (green circle) curves. The biovolume of EPS in the biofilm was algorithmically analyzed using COMSTAT2.
- FIG. 22C Confocal image of glucan formation on sHA beads with/without glucanohydrolase pretreatment. Grey: hydroxyapatite surface; Red: EPS glucan.
- FIGS 23A - 23F EDA locally degrade EPS for enhanced targeting of EPS-producing pathogen in mixed-species biofilms.
- FIG. 23 A Schematic of treatment regimen and hypothesis of EDA approach on preformed mixed-species biofilms.
- FIG. 23B Ecological shift in the mixed-species biofilm model used in this study. Dashed boxes, EDA approach was tested at 43h (the early stage) and 67h (the late stage).
- Fig. 23C Impact of EDA on microbial ecology in early mixed-species biofilm (43h). Top, CFU of different bacterial species recovered from the mixed-species biofilm after EDA treatment. Bottom, proportion of different species.
- Fig. 23 A Schematic of treatment regimen and hypothesis of EDA approach on preformed mixed-species biofilms.
- FIG. 23B Ecological shift in the mixed-species biofilm model used in this study. Dashed boxes, EDA approach was tested at 43h (the early stage) and 67h (the late stage).
- FIG. 23C Impact
- FIG. 23D Impact of EDA on microbial ecology in late mixed-species biofilm (67h). Top, CFU of different bacterial species recovered from the mixed-species biofilm after EDA treatment. Bottom, proportion of different species.
- Fig. 23E Fluorescence in situ hybridization (FISH) showing spatial distribution of EPS, S. mutans and commensals in late mixed-species biofilms (67h). a. overview of mixed-species biofilm on sHA; b. cross-sectional magnified image(merged); c. S. mutans only (green); d. EPS only (red); e. S. oralis only (yellow); f. A. naeslundii only (cyan).
- FIG. 23F Dynamics of localized degradation of EPS inside the mixed-species biofilm exposing the embedded bacteria(yellow arrows). Grey, all bacteria stained by SYT09; Red,
- EPS Exopolysaccharides labelled by Alexa Fluor 647.
- D Dextranase
- M Mutanase
- D+M Dextranase+Mutanase.
- NS not significantly different; *, p ⁇ 0.05 **, p ⁇ 0.01 ***, pO.001 (Student t test)
- Figures 24A- 24F EPS-degrading enzymes in experimental salivary pellicle inhibit S.
- FIG. 24A Schematic of treatment regimen and hypothesis for S. mutans biofilm prevention by EPS-degrading enzyme pretreatment.
- FIG. 24B Schematic of treatment regimen and hypothesis for S. mutans biofilm prevention by EPS-degrading enzyme pretreatment.
- sHA discs were topically treated with EPS- degrading enzymes (8.75U/mL dextranase and/or 1.75U/mL mutanase) or vehicle control for 60 min before inoculum and were incubated to allow S. mutans biofilm formation for 19h before imaging.
- Green bacteria cells stained by SYT09; Red, EPS labelled by Alexa Fluor 647; White arrow, biofilms formed on single enzyme pretreated sHA showed formation of microcolony-like, albeit altered structures.
- Glucanohydrolases in salivary pellicle reduce biomass(dry weight) accumulation.
- Fig. 24D CFU of the biofilm formed on glucanohydrolase-pretreated salivary pellicle.
- Fig. 24E CFU recovered from the biofilm on glucanohydrolase-pretreated salivary pellicle followed by antimicrobial killing at 19h.
- S. mutans biofilms formed on sHA pretreated with EPS-degrading enzymes or vehicle were challenged with antimicrobial (EOs) for lmin at 19h and antibiofilm efficacy was assayed by determining the CFU recovered from the biofilm.
- EOs antimicrobial
- FIGs 25A - 25E EDA selectively prevents early colonization of S. mutatis in mixed- species biofilms.
- FIG. 25A Schematic of treatment regimen and hypothesis for S. mutans biofilm prevention by EPS-degrading enzyme pretreatment.
- FIG. 25B Microbial population of mixed-species biofilms after early colonization on sHA. sHA discs were topically treated with EPS-degrading enzymes(8.75U/mL dextranase and 1.75U/mL mutanase) or vehicle control for 60 min before inoculum and were incubated to allow mixed-species biofilm formation.
- FIG. 25C FISH image of early-colonizing mixed-species community. Green, S.
- Fig. 25D Enzyme pretreatment potentiates overall killing efficacy and helps eliminate S. mutans in mixed-species biofilm. 43h Mixed-species biofilms formed on sHA pretreated with EPS-degrading enzymes or vehicle were challenged with antimicrobial (EOs) for lmin and antibiofilm efficacy was assayed by determining the CFU of different bacterial species recovered from the biofilm.
- Fig. 25E Bacterial adhesion assayed by 3 H-thymidine radioisotope tracing spectroscopy.
- sHA beads were pretreated with either vehicle or EPS-degrading enzymes before GtfB immobilization and glucans were synthesized in situ in the presence of sucrose substrate.
- the beads were incubated with 1.0x 10 9 cells/mL radiolabeled S. mutans, S. oralis and A. naeslundii, respectively and were washed to remove unbound bacteria. The number of adhered bacterial was quantified by scintillation counting.
- D Dextranase
- M Mutanase
- D+M Dextranase+Mutanase.
- NS not significantly different; *, p ⁇ 0.05; **, p ⁇ 0.01 ***, p ⁇ 0.001 (Student t test)
- Fluoride is effective in reducing demineralization and enhancing demineralization of early carious lesions, but has limited effects against biofilms. Conversely, current antimicrobial modalities for controlling caries-causing biofilms are largely ineffective.
- the antimicrobials and enzymes are obtained from commercial sources.
- antimicrobial peptides are small peptides having any bacterial activity.
- "RC-101” is an analogue of retrocyclin, a cyclic octadecapeptide, which can protect human CD4+ cells from infection by T- and M-tropic strains of HIV-1 in vitro and prevent HIV-1 infection in human cervicovaginal tissue.
- the ability of RC-101 to prevent HIV-1 infection and retain full activity in the presence of vaginal fluid makes it a good candidate for other topical microbicide applications, especially in oral biofilms.
- the sequence of RC-101 is provided in Plant Biotechnol J. 2011 Jan; 9(1): 100-115 which is incorporated herein by reference.
- C16G2 is a novel synthetic antimicrobial peptide with specificity for S. mutatis,
- Protegrin-1 is a cysteine-rich, 18-residue ⁇ -sheet peptide. It has potent antimicrobial activity against a broad range of microorganisms, including bacteria, fungus, virus, and especially some clinically relevant, antibiotic-resistant bacteria. For example, bacterial pathogens E. coli and fungal opportunist C. albicans are effectively killed by PG in laboratory testing.
- the sequence of PG-1 is provided in Plant Biotechnol J. 2011 Jan; 9(1): 100-115 which is incorporated herein by reference. Additonal antimicrobial peptides include those set forth below in Table 1 below.
- an "antimicrobial” as used herein includes without limitation, CHX, triclosan/copolymer dentrifices, clindamycin, doxycycline gels, minocycline powder, macrolides, sulfonamides, penicillin and derivatives thereof, tetracycline, quinolones, levofloxacin, ciprofloxacin and fluconazole.
- “Essential oils” may include one or more selected from the group consisting of menthol, thymol, eucalyptol, methyl salicylate and combinations of two or more thereof.
- the essential oils comprises menthol.
- the essential oils comprises thymol.
- the essential oils comprise a combination of menthol, thymol, eucalyptol, and methyl salicylate.
- a "biofilm” is a complex structure adhering to surfaces that are regularly in contact with water, consisting of colonies of bacteria and usually other microorganisms such as yeasts, fungi, and protozoa that secrete a mucilaginous protective coating in which they are encased.
- Biofilms can form on solid or liquid surfaces as well as on soft tissue in living organisms, and are typically resistant to conventional methods of disinfection.
- Dental plaque the slimy coating that fouls pipes and tanks, and algal mats on bodies of water are examples of biofilms.
- Biofilms are generally pathogenic in the body, causing such diseases as dental caries, cystic fibrosis and otitis media.
- Biofilm degrading enzymes include, without limitation, exo-polysaccharide degrading enzymes such as dextranase, mutanase, DNAse, endonuclease, deoxyribonuclease I, dispersin B, and glycoside hydrolases, such as 1 ⁇ 3) -a-D-glucan hydrolase, although use of chloroplast codon optimized sequences encoding dextranase and mutanase are preferred, the skilled person is well aware of many different biofilm degrading enzymes in the art. Additional enzyme sequences for use in the fusion proteins of the invention are provided below. As noted above, suitable enzymes can also be purified from the bacteria which produce them or obtained from commercial sources.
- administering or “administration” of an agent, drug, or peptide to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function.
- the administering or administration can be carried out by any suitable route, including orally, topically, intranasally, parenterally (intravenously,
- administration includes self-administration and the administration by another.
- a single admintration entail oral delivery of a single formulation comprising enzymes and antimicrobials.
- the administration can be sequential.
- biofilm degrading enzymes are first administered, followed by delivery of antimicrobials. Enzyme treatment is performed for a suitable time to degraded the
- gums and teeth are exposed to enzymes for at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 45 minutes, at least 60 minutes.
- the teeth and gums are treated with enzyme for between 30 and 120 minutes. Most preferably the enzyme treatment is performed for at least 30 minutes.
- an antimicrobial oral rinse is administered for 20, 30, 40, 50, 60, 90, or 120 seconds.
- at least two or more of methol, eucalyptol, thymol and methylsalicylate are present in the rinse.
- the rinse is Listerine®.
- the enzyme containing composition may be in a form selected from the group consisting of a mouthwash, mouth rinse, mouth spray, toothpaste, tooth gel, sub-gingival gel, mousse, foam, denture care product, chewable tablet, dentifrice, lozenge, dissolvable strip, and the like.
- disease As used herein, the terms “disease,” “disorder,” or “complication” refers to any deviation from a normal state in a subject.
- an amount effective at dosages and for periods of time necessary to achieve the desired result it is meant an amount effective at dosages and for periods of time necessary to achieve the desired result. In certain embodiments, the amount is effective to act prophylactically to inhibit formation of biofilm. In other embodiments, the amounts are effective to disperse existing biofilms.
- the term “inhibiting” or “preventing” means causing the clinical symptoms of the disease state not to worsen or develop, e.g., inhibiting the onset of disease, in a subject that may be exposed to or predisposed to the disease state, but does not yet experience or display symptoms of the disease state.
- the term "expression" in the context of a gene or polynucleotide involves the transcription of the gene or polynucleotide into RNA.
- the term can also, but not necessarily, involves the subsequent translation of the RNA into polypeptide chains and their assembly into proteins.
- a plant remnant may include one or more molecules (such as, but not limited to, proteins and fragments thereof, minerals, nucleotides and fragments thereof, plant structural components, etc.) derived from the plant in which the protein of interest was expressed. Accordingly, a composition pertaining to whole plant material (e.g., whole or portions of plant leafs, stems, fruit, etc.) or crude plant extract would certainly contain a high concentration of plant remnants, as well as a composition comprising purified protein of interest that has one or more detectable plant remnants. In a specific embodiment, the plant remnant is rubisco.
- the invention pertains to an administrable composition for treating or preventing biofilm formation in situ (e.g., in the mouth) and on biomedical devices useful for surgical implantation such as stents, artificial joints, and the like.
- the devices are coated with the composition to inhibit unwanted biofilm deposition on the device.
- the composition comprises a therapeutically-effective amount of one or more antimicrobial peptides (AMP) and one or more enzymes having biofilm degrading activity in combination, each of said AMP and enzyme thereof having been expressed by a plant and a plant remnant and acting synergisticall to degrade said biofilm.
- AMP(s) and enzymes(s) are expressed from separate plastid transformation vectors.
- the plastid transformation vectors comprising polycistronic coding sequences where both the AMP and the enzymes are expressed from a single vector.
- the antimicrobial peptides are optional and one or more enzymes are employed in combination with one or more antimicrobials, and/or essential oils.
- Proteins expressed in accord with certain embodiments taught herein may be used in vivo by administration to a subject, human or animal in a variety of ways.
- the pharmaceutical compositions may be administered orally, topically, subcutaneously, intramuscularly or intravenously, though oral topical administration is preferred.
- the therapeutic compositions can be formulated in a classical manner using solid or liquid vehicles, diluents and additives appropriate to the desired mode of administration. Orally, the composition can be administered in the form of tablets, capsules, granules, powders, gums, and the like with at least one vehicle, e.g., starch, calcium carbonate, sucrose, lactose, gelatin, etc.
- the therapeutic protein(s) of interest may optionally be purified from a plant homogenate. The preparation may also be emulsified.
- the active ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient.
- excipients are, e.g., water, saline, dextrose, glycerol, ethanol or the like and combination thereof.
- the compositions may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants.
- the edible plant, juice, grain, leaves, tubers, stems, seeds, roots or other plant parts of the pharmaceuticalproducing transgenic plant is ingested by a human or an animal thus providing a very inexpensive means of treatment of disease.
- plant material e.g.
- lettuce material comprising chloroplasts expressing AMPs and biofilm degrading enzymes and combinations thereof, is homogenized and encapsulated.
- an extract of the lettuce material is encapsulated.
- the lettuce material is powderized before encapsulation.
- the biofilm degrading proteins may also be purified from the plant following expression.
- compositions may be provided with the juice of the transgenic plants for the convenience of administration.
- the plants to be transformed are preferably selected from the edible plants consisting of tomato, carrot and apple, among others, which are consumed usually in the form of juice.
- the subject invention pertains to a transformed chloroplast genome that has been transformed with a vector comprising a heterologous gene that expresses a combination of peptides as disclosed herein.
- the subject invention pertains to a plant comprising at least one cell transformed to express a peptide as disclosed herein.
- Reference to genetic sequences herein refers to single- or double-stranded nucleic acid sequences and comprises a coding sequence or the complement of a coding sequence for polypeptide of interest.
- Degenerate nucleic acid sequences encoding polypeptides, as well as homologous nucleotide sequences which are at least about 50, 55, 60, 65, 60, preferably about 75, 90, 96, or 98% identical to the cDNA may be used in accordance with the teachings herein polynucleotides.
- Percent sequence identity between the sequences of two polynucleotides is determined using computer programs such as ALIGN which employ the FASTA algorithm, using an affine gap search with a gap open penalty of -12 and a gap extension penalty of -2.
- cDNA Complementary DNA
- homologous polynucleotide sequences can be identified by hybridization of candidate polynucleotides to known polynucleotides under stringent conditions, as is known in the art. For example, using the following wash conditions: 2 X SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0), 0.1% SDS, room temperature twice, 30 minutes each; then 2X SSC, 0.1% SDS, 50° C. once, 30 minutes; then 2X SSC, room temperature twice, 10 minutes each homologous sequences can be identified which contain at most about 25-30% basepair mismatches. More preferably, homologous nucleic acid strands contain 15-25% basepair mismatches, even more preferably 5-15% basepair mismatches.
- Species homologs of polynucleotides referred to herein also can be identified by making suitable probes or primers and screening cDNA expression libraries. It is well known that the Tm of a double-stranded DNA decreases by 1-1.5 ° C with every 1% decrease in homology (Bonner et al., J. Mol. Biol. 81, 123 (1973). Nucleotide sequences which hybridize to polynucleotides of interest, or their complements following stringent hybridization and/or wash conditions also are also useful polynucleotides. Stringent wash conditions are well known and understood in the art and are disclosed, for example, in Sambrook et al., MOLECULAR CLONING: A
- Streptococcus mutans UA159 serotype c ATCC 700610
- Actinomyces naeslundii ATCC 12104
- Streptococcus gordonii DL-1 Streptococcus gordonii DL-1
- Candida albicans SC5314 were used in present study. These strains were selected because S. mutans is a well-established virulent cariogenic bacteria [Ajdic D et al, 2002].
- S. gordonii is a pioneer colonizer of dental biofilm, and naeslundii is also detected during the early stages of dental biofilm formation and may be associated with development of root caries [Dige I et al, 2009].
- albicans is a fungal organism that colonizes human mucosal surfaces, and it is also detected in dental plaque from toddlers with early childhood caries [Hajeshengallis E et al, 2015]. All strains were stored at -80°C in tryptic soy broth containing 20% glycerol. Blood agar plates were used for cultivating S.mutans, S. gordonii and A.naeslundii. Sabouraud agar plates were used for C. albicans.
- transplastomic plants expressing GFP fused with CTB, PTD, retrocyclin and protegrin were created as described in previous studies [Limaye et al 2006; Kwon et al 2013; Xiao et al 2016; Lee et al 2011].
- Transplastomic lines expressing GFP fusion proteins were confirmed using Southern blot assay as described previously [Verma et al 2008]. Also, expression of GFP tagged proteins were confirmed by visualizing green fluorescence from the leaves of each construct under UV illumination.
- transplastomic tobacco was accomplished by organic extraction followed by hydrophobic chromatography done previously (Lee et al, 2011). About 0.2-1 gm of lyophilized leaf material was taken and reconstituted in 10-20 ml of plant extraction buffer (0.2M Tris HCl pH 8.0, 0.1M NaCl, 10mM EDTA, 0.4M sucrose, 0.2 % Triton X supplemented with 2%
- Phenylmethylsulfonylfluoride and 1 protease inhibitor cocktail The resuspension was incubated in ice for 1 hour with vortex homogenization every 15 min. The homogenate was then spun down at 75000g at 4°C for 1 hour (Beckman LE-80K optima ultracentrifuge) to obtain the clarified lysate. The lysate was subjected to pretreatment with 70% saturated ammonium sulfate and 1/4* 11 volume of 100% ethanol, followed by vigorous shaking for 2 min (Yakhnin et al, 1998). The treated solution was spun down at 2100 g for 3 min. The upper ethanol phase was collected and the process was repeated with ⁇ / ⁇ " 1 volume of 100% ethanol.
- the pooled ethanol phases were further treated with l/3 rd volume of 5M NaCl and 1/4* 11 volume of 1-butanol, homogenized vigorously for 2 min and spun down at 2100 g for 3 min.
- the lowermost phase was collected and loaded onto a 7kDa MWCO zeba spin desalting column (Thermo scientific) and desalted as per manufacturer's recommendations.
- the desalted extract was then subjected to hydrophobic interaction chromatography during the capture phase for further purification.
- the desalted extract was injected into a
- Toyopearl butyl - 650S hydrophobic interaction column (Tosoh bioscience) which was run on a FPLC unit (Pharmacia LKB-FPLC system).
- the column was equilibriated with 2.3 column volumes of salted buffer (10mM Tris-HCl, 10mM EDTA and 50% saturated ammonium sulfate) to a final 20% salt saturation to facilitate binding of GFP onto the resin. This was followed by a column wash with 5.8 column volumes of salted and unsalted buffer mix and then eluted with unsalted buffer (10mM Tris-HCl, 10mM EDTA).
- the GFP fraction was identified based on the peaks observed in the chromatogram and collected. The collected fractions were subjected to a final polishing step by overnight dialysis. After dialysis the purified proteins were lyophilized (labconco lyophilizer) in order to concentrate the finished product and then stored in -20°C.
- GFP-RC101 and GFP-PGl Quantification of GFP-RC101 and GFP-PGl was done by both western blot and fluorescence based methods.
- the lyophilized purified proteins were resuspended in sterile IX PBS and the total protein was determined by Bradford method.
- the purified protein was then quantified by SDS-PAGE method by loading denatured protein samples along with commercial GFP standards (Vector labs) onto a 12 % SDS gel and then western blotting was done using 1 :3000 dilution of mouse Anti-GFP antibody (Millipore) followed by probing with 1 :4000 dilution of secondary HRP conjugated Goat-Anti Mouse antibody (Southern biotech).
- the purified proteins were also quantified using GFP fluorescence.
- the protein samples were run on a 12 % SDS gel under native conditions. After the run, the gel was placed under a UV lamp and then photographed.
- fluorescence methods was determined by densitometric analysis using Image J software with commercial GFP standards in order to obtain the standard curve. Purity was determined based on GFP quantitation with respect to total protein values determined in Bradford method.
- each human cell line cells (2xl0 4 ) were cultured in 8 well chamber slides (Nunc) at 37°C overnight, followed by incubation with purified GFP-fused tags: CTB-GFP (8.8 ⁇ g), PTD-GFP (13 ⁇ g), GFP-PGl (1.2 ⁇ g) and GFP-RC101 (17.3 ⁇ g) in 100 ⁇ PBS supplemented 1% FBS at 37°C for 1 hour.
- FIPDLS human periodontal ligament stem cells
- MMS maxilla mesenchymal stem cells
- SCC-1 human head and neck squamous cell carcinoma cells
- GMSC gingiva-derived mesenchymal stromal cells
- ATK adult gingival keratinocytes
- OBC osteoblast cells
- the killing kinetics of AMPs (Gfp-PGl and Gfp-RC10l) against S.mutans were analyzed by time-lapse killing assay.
- S.mutans were grown to log phase and diluted to 10 5 CFU/ml in growth medium.
- GFP-PG1 and GFP-RC101 were added to S.mutans suspensions at
- S. mutans were grown to log phase and harvested by centrifugation (5500 g, 10min) and the pellet was washed once with sodium phosphate-buffered saline (PBS) (pH 7.2), re-suspended in PBS and adjusted to a final concentration of 1 x 10 5 CFU/ml.
- PBS sodium phosphate-buffered saline
- GFP-PG1 was added to S.mutans suspensions at concentrations of 10 ⁇ g/ml and 2.5 ⁇ propidium iodide-PI (Molecular Probe Inc., Eugene, OR, USA) was added for labeling dead cells. 5 ⁇ of mixtures were loaded on an agarose pad for confocal imaging.
- mutans were grown to log phase and diluted to 10 5 CFU/ml in PBS.
- Bacteria suspension was mixed with GFP-PG1 (final concentration of 10 ⁇ g/ml) for 1 h at 37°C.
- the bacteria were collected by filtration using 0.4 ⁇ m Millipore filters.
- the deposits were fixed in 2.5% glutaraldehyde and 2.0% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4) for 1 hour at room temperature and processed for SEM (Quanta FEG 250, FEI, Hillsboro, OR) observation. Untreated or bacteria treated with buffer only served as controls.
- EPS was labeled using 2.5 ⁇ Alexa Fluor 647-labeled dextran conjugate (10 kDa; 647/668 nm; Molecular Probes Inc.), while the bacteria cells were stained with 2.5 ⁇ SYT09 (485/498 nm; Molecular Probes Inc.).
- the imaging was performed using Leica SP5 microscope with 20X (numerical aperture, 1.00) water immersion objective.
- the excitation wavelength was 780 nm
- the emission wavelength filter for SYTO 9 was a 495/540 OlyMPFEC 1 filter
- the filter for Alexa Fluor 647 was a HQ655/40M-2P filter.
- the confocal image series were generated by optical sectioning at each selected positions and the step size of z-series scanning was 2 ⁇ m.
- Amira 5.4.1 software (Visage Imaging, San Diego, CA, USA) was used to create 3D renderings of biofilm architecture [Xiao J et al. 2012, Koo H et al. 2010].
- mutans by hydrolyzing a-1,6 glucosidic linkages and a-1,3 glucosidic linkages [Hayacibara et al. 2004].
- Dextranase produced from Penicillium sp. was commercially purchased from Sigma (St. Louis, MO) and mutanase produced from Trichoderma harzianum was kindly provided by Dr. William H. Bowen (Center for Oral Biology, University of Rochester Medical Center). Dextranase and mutanase were mixed at ratio of 5: 1 before applying to biofilms [Mitsue F. Hayacibara et al. 2004].
- Alexa Fluor 647-labeled dextran conjugate was used to label the EPS-matrix, while SYTO 9 and PI were used to label live cells and dead cells.
- Biofilms were examined using confocal fluorescence imaging at 0, 10, 30 and 60 min, and subjected to AMIRA/COMSTAT/ImageJ analysis.
- the total biomass of EPS matrix, live and dead cells in each series of confocal images was quantified using COMSTAT and ImageJ.
- the ratio of live to the total bacteria at each time point was calculated, and the survival rate of live cells (relative to live cells at 0 min) was plotted. The initial number of viable cells at time point 0 min was considered to be 100%.
- the percent-survival rate was determined by comparing to time point 0 min.
- Microbiological assays At selected time point (19 h), biofilms were removed, homogenized via sonication and subject to microbiological analyses as detailed previously [Xiao J et al. 2012, Koo H et al. 2010]; our sonication procedure does not kill bacteria cells while providing optimum dispersal and maximum recoverable counts. Aliquots of biofilm suspensions were serially diluted and plated on blood agar plates using an antomated Eddy Jet Spiral Plater (IUL, SA, Barcelona, Spain). Meanwhile, propidium monoazide (PMA) combined with quantitative PCR (PMA-qPCR) was used for analysis of S.mutans cell viability as described. [Klein MI et al. 2012].
- PMA propidium monoazide
- PMA-qPCR quantitative PCR
- biofilm pellets were resuspended with 500 ⁇ TE (50 mM Tris, 10 mM EDTA, pH 8.0). Using a pipette, the biofilm suspensions were transferred to 1.5 ml microcentrifuge tubes; then mixed with PMA. 1.5 ⁇ PMA (20 mM in 20% dimethyl sulfoxide; Biotium, Hayward, CA) was added to the biofilm suspensions. The tubes were incubated in the dark for 5 min, at room temperature, with occasional mixing.
- fusion tags (CTB, PTD, protegrin, retrocyclin) were fused to the green fluorescent protein (smGFP) at N-terminus to evaluate their efficiency and specificity. Fusion constructs encoding these fusion proteins were cloned into chloroplast transformation vectors which were then used to transform plants of interest as described in US Patent application no. 13/101,389 which is incorporated herein by reference. To create plants expressing GFP fusion proteins, tobacco chloroplasts were transformed using biolistic particle delivery system. As seen in the Fig. IB, each tag-fused GFP is driven by identical regulatory sequences - the psbA promoter and 5' UTR regulated by light and the transcribed mRNA is stabilized by 3 ' psbA UTR.
- the psbA gene is the most highly expressed chloroplast gene and therefore psbA regulatory sequences are used for transgene expression in our lab [7, 34].
- two flanking sequences isoleucyl-tRNA synthetase (trnl) and alanyl-tRNA synthetase (trnA) genes, flank the expression cassette, which are identical to the native chloroplast genome sequence.
- the emerging shoots from selection medium were investigated for specific integration of the transgene cassette at the trnl and trnA spacer region and then transformation of all chloroplast genomes in each plant cell (absence of untransformed wild type chloroplast genomes) was confirmed by Southern blot analysis.
- stable integration of all GFP expression cassettes and homoplasmy of chloroplast genome with transgenes were confirmed before extracting fusion proteins.
- by visualizing the green fluorescence under UV light GFP expression of was phenotypically confirmed.
- each homoplasmic line was grown in a temperature- and humidity-controlled greenhouse. Fully grown mature leaves were harvested in late evenings to maximize the accumulation of GFP fusion proteins driven by light-regulated regulatory sequences. To further increase the content of the fusion proteins on a weight basis, frozen leaves were freeze-dried at -40°C under vacuum. In addition to the concentration effect of proteins, lyophilization increased shelf life of therapeutic proteins expressed in plants more than one year at room temperature [Daniell et al 2015; 2016].
- GFP fused antimicrobial peptides RC101 and PG1 were harvested from greenhouse and subsequently lyophilized for protein extraction and purification.
- the average expression level of GFP-RC101 was found to be 8.8% of total protein in crude extracts while expression of GFP-PG1 was that of 3.8% of total protein based on densitometry. The difference in expression levels was similar to what was reported previously (Lee et al 2011, Gupta et al, 2015).
- GFP fused to different antimicrobial peptides was done in order to test the microbicidal activity against both planktonic and biofilm forming S.mutans. Lyophilized tobacco material expressing different GFP fusions was used for extractions and subsequent downstream processing (See Figure 8) to obtain the finished purified product which was subsequently quantified to determine concentration of GFP fused peptides.
- GFP-RC10l and GFP-PGl Quantitation of purified GFP-RC10l and GFP-PGl was done by both western blot and Native GFP fluorescence method where purified GFP -RC 101 show 94% average purity with an average yield of 1624 ⁇ g of GFP (116 ⁇ g of RC101 peptide) per gm of lyophilized leaf material (Fig 1 A and IB). In GFP- PGl both methods (Fig 1C and ID) show 17% average purity with an average yield of 58.8 ⁇ g of GFP (4 ⁇ g PG1 peptide) per gm of lyophilized leaf material.
- the difference in purity can be attributed to difference in the type of tags fused to GFP as seen in previous studies (Xiao et al 2015, Skosyrev et al 2003).
- the fold enrichment of purified GFP-RC10l and GFP-PGl from plant extracts was 10.6 and 4.5 respectively.
- the western blots also show GFP standards at 27 kDa which corresponds to the monomer fragment along with a 54kDa GFP dimer with loadings ranging from 6-8 ng of GFP.
- 29 kDa and 58 kDa fragments are clearly visible which correspond to the monomer and dimer forms of the fusion (Fig. 1 A).
- GFP-RC10l and GFP-PGl show multimeric bands with some of them visible below the 27 kDa GFP standard size which could be because of GFP being fused to cationic peptides causing a electrophoretic mobility shift with each GFP fragment as described in previous studies (Lee et al, 2011).
- Streptococcus mutans a proven biofilm-forming and caries-causing pathogen, rapidly killing the bacterial cells within lh at low concentrations (Fig. 2A).
- GFP-PGl also killed the early oral colonizers Streptococcus gordonii and Actinomyces naeslundii, but showed limited antifungal activity against Candida albicans at the concentrations tested (Fig 2E).
- Time-lapse confocal imaging shows that S. mutans viability is affected as early as 10 minutes as shown in Fig 3 A relative to the untreated controls (Fig. 3B).
- SEM imaging revealed disruption of S.
- EPS-degrading enzymes such as dextranase and mutanase could help digest the matrix of cariogenic biofilms, although they are devoid of antibacterial effects.
- Streptococcus mutans biofilms were pre-formed on sHA surface, and treated topically with GFP-PGl and EPS-degrading enzymes (Dex/Mut) either alone or in combination.
- GFP-PGl and EPS-degrading enzymes Dex/Mut
- Time-lapsed confocal imaging and quantitative computational analyses were conducted to analyze EPS-matrix degradation and live/dead bacterial cells within biofilms (Fig 6A).
- the enzymes-peptide combination resulted in more than 60% degradation of the EPS- matrix, while increasing the bacterial killing when compared to either GFP-PG or Dex/Mut alone.
- GFP fusion proteins when incubated with human cultured cells, including HPDLS, MMS, SCC-1, GMSC, AGK and osteoblast cells (OBC) revealed interesting results. Although only one representive image of each cell line is presented, uptake studies were performed in triplicate and at least 10-15 images were recorded under confocal microscopy. Without a fusion tag, GFP did not enter any tested human cell line. Both CTB-GFP and PTD- GFP effectively penetrated all tested cell types, although their localization patterns differed.
- PG1-GFP is the most efficient tag in entering all tested human cells because GFP could be localized at tenfold lower concentrations than any other fusion proteins.
- PG1-GFP showed exclusively cytoplasmic localization in FIPDLSC, SCC-1, GMSC and AGK cells and localized to both the periphery and cytosol in MMSC, but it is only localized to the periphery of OBC.
- RC101-GFP was localized in SCC-1, GMSC, AGK and OBC, but its localization in FIPDLSC was negligible and was undetectable in MMSC cells. Discussion and Conclusions
- Chlorhexidine is considered 'gold standard' for topical antimicrobial therapy (Flemmig and Beikler 2011; Marsh et al 2011; Caufield et al 2001). CHX effectively suppresses mutans streptococci levels in saliva, but it has adverse side effects including tooth staining and calculus formation, and is not recommended for daily preventive or therapeutic use (Autio-Gold 2008).
- EPS-rich matrix promotes microbial adhesion, cohesion and protection as well as hindering diffusion (Koo et al 2013; Flemming and Wingender 2010.
- EPS matrix creates spatial and microenvironmental heterogeneity in biofilms, modulating the growth and protection of pathogens against antimicrobials locally as well as a highly adhesive scaffold that ensures firm attachment of biofilms on tooth surfaces (Flemming and Wingender 2010; Peterson et al. 2015).
- CHX is far less effective against formed cariogenic biofilms (Hope and Wilson, 2004; Van Strydonck et al 2012; Xiao et al., 2012).
- the EPS are comprised primarily of a mixture of insoluble (with high content of al,3 linked glucose) and soluble (mostly al,6 linked glucose) glucans (Bowen and Koo 2011).
- EPS-matrix degrading dextranase or mutanase (from fungi) to disrupt biofilm and prevent dental caries has been explored and included in commercially available over-the-counter mouthwashes (e.g. Biotene PBF).
- antimicrobial peptides protegrin and retrocyclin play an important role in killing bacteria in biofilms and initiate wound healing through degranulation of mast cells.
- it is important to effectively deliver growth hormones or other proteins to enhance cell adhesion, stimulate osteogenesis, angiogenesis, bone regeneration, differentiation of osteoblasts or endothelial cells.
- Previously identified cell penetrating peptides have several limitations. CTB enters all cell types via the ubiquitous GM1 receptor and this requires pentameric form of CTB. PTD on the other hand does not enter immune cells (Xiao et al 2016).
- PG1-GFP is the most efficient tag in entering periodontal or gingival human cells because GFP signal could be detected even at ten-fold lower concentrations than any other fusion proteins.
- PG1-GFP effectively entered HPDLSC, SCC-1, GMSC, AGK, MMSC and OBC.
- RC101-GFP entered SCC-1, GMSC, AGK and OBC but its localization in HPDLSC and MMSC cells were poor or undetectable.
- protein drugs fused with protegrin expressed in plant cells can be orally delivered to deeper layers of gum tissues in a non-invasive manner and increase patient compliance.
- Protein drugs bioencapsulated in plants can be stored for many years at room temperature without losing their efficacy (Su et al 2015; Daniell et al 2016).
- the high cost of current protein drugs is due to their production in prohibitively expensive ferm enters, purification, cold transportation/ storage, short shelf life and sterile delivery methods. All these challenges could be eliminated using this novel drug delivery concept to enhance oral health.
- Recent FDA approval of plant cells for production of protein drugs (Walsh 2014) augurs well for clinical advancement of this novel concept.
- Chloroplast genomes diversity, evolution and applications in genetic engineering. Genome Biology, in press DeGray G, Rajasekaran K, Smith F, Sanford J, Daniell H. 2001. Expression of an antimicrobial peptide via the chloroplast genome to control phytopathogenic bacteria and fungi. Plant Physiol. 127(3):852-62.
- Hayacibara MF Koo H, Vacca-Smith AM, Kopec LK, Scott-Anne K, Cury JA, Bowen WH. 2004.
- Kwon KC Verma D, Singh ND, Herzog R, Daniell H. 2013. Oral delivery of human biopharmaceuticals, autoantigens and vaccine antigens bioencapsulated in plant cells. Adv Drug Deliv Rev. 65(6):782-99.
- Oral delivery of bioencapsulated exendin- 4 expressed in chloroplasts lowers blood glucose level in mice and stimulates insulin secretion in beta-TC6 cells. Plant Biotechnol J. 11 :77-86.
- Trotti A Garden A, Warde P, Symonds P, Langer C, Redman R, Pajak TF, Fleming TR, Henke M, Bourhis J, Rosenthal DI, Junor E, Cmelak A, Sheehan F, Pulliam J, Devitt-Risse P, Fuchs H, Chambers M, O'Sullivan B, Ang KK. 2004.
- dextranase /mutanase and protegrin/retrocyclin are expressed independently and as fusion proteins in tobacco and other plant chloroplasts, such as lettuce. Proteins will be produced and used in low cost purification strategies. Tobacco plants produce a million seeds, and thus, it is feasible to scale up production easily. Each acre of tobacco will produce up to 40 metric tons of biomass, facilitating low cost large scale production of AMP, enzymes and fusion constructs encoding the same. In another approach, the proteins are produced in an edible plant such as lettuce.
- dextranases Dex and mutanases (Mut) have been isolated from fungi and bacteria and characterized for their enzymatic activity.
- Optimal dextranase and mutanase enzymes should have enzymatic properties suitable for human oral environment. Based on short duration of oral treatments, strong binding/retention property to plaque-biofilms and catalytic activity to both types of EPS (dextrans and mutans) are highly desirable.
- the enzymes added in commercial dextranase-containing mouthwashes e.g. Biotene
- fungi Piericillium sp. and Chaetomium erraticum
- fungal dextranases show higher temperature optima (50-60°C) than bacterial dextranases (35-40°C). Furthermore, bacterial dextranases are more stable and effective at oral temperature ( ⁇ 37°C) and are suitable for dental caries-prevention. Recently, a dextranase from Arthrobacter sp strain Arth410 showed superior dextran degradation properties at optimal temperatures (35-45°C) and pH values (pH 5-7) found in mouth and in cariogenic biofilms when compared to fungal dextranases. In addition, topical applications of bacterial dextranase are more effective in reducing dental caries in vivo than fungal dextranse.
- a bacterial mutanase from Paenibacillus sp. strain RM1 shows that biofilm was effectively degraded by 6 hr incubation even after removal of the mutanase, preceded by first incubation with the biofilms for 3 min.
- RM1 mutanase shows enhanced biofilm-degrading property.
- fungal enzymes require glycosylation, which precludes their expression in chloroplasts.
- the present invention involves use of bacterial dextranase and mutanase for expression in chloroplasts.
- PG1 or RC101 In order to maximize synthesis and reduce toxicity of AMPs, ten tandem repeats of PG1 or RC101, separated by protease cleavage sites as shown in Figure 10 are employed. For each copy of expressed gene, ten functional copies of PG1 or RC 101 will be made. For this purpose we have chosen the Tobacco Etch Virus (TEV) protease, which has high specificity and a short cleavage site of seven amino acids. Alternatively, furin cleavage sites can also be employed.
- TSV Tobacco Etch Virus
- This vector can also be engineered to include a nucleic acid encoding a biofilm degrading enzyme.
- the coding region can be expressed under the promoter utilized to express the AMP or can be ligated into the vector operably linked to a second promoter region.
- the biofilm degrading enzyme coding sequence may also contain TEV protease cleavage sites to facilitate release of the enzyme. This approach provides a safer and cleaner option than chemical cleavage methods. Most importantly, individual PG1 peptides in the fusion protein will not form secondary structures before cleavage, thereby avoiding accumulation of functional peptides which can be lethal to the host production systems. Antimicrobial activity of the cleaved
- biofilm degrading enzymes or fusion proteins thereof can be used to degrade biofilms using the methods disclosed in Example I.
- sequences encoding the AMP/biofilm degrading enzymes are optionally codon-optimized prior to insertion into chloroplast transformation vectors, such as pLD. Chloroplast transformation relies upon a double homologous recombination event.
- chloroplast vectors comprise homologous regions to the chloroplast genome which flank the expression cassette encoding the heterologous proteins of interest, which facilitate insertion of the transgene cassettes into the intergenic spacer region of the chloroplast genome, without disrupting any functional genes.
- any intergenic spacer region could be used to insert transgenes, the most commonly used site of transgene integration is the transcriptionally active intergenic region between the trnl-trnh genes (in the rrn operon), located within the IR regions of the chloroplast genome ( Figure 10). Because of similar protein synthetic machinery between E. coli and chloroplasts, efficiency of codon-optimization can also be assessed in E.coli and then plants can be created. Both systems could be used for expression of AMPs, biofilm degrading enzymes or fusion proteins thereof, as well as for purification and evaluation of AMPs or enzymatic activities. Purification strategies
- a hydrophobic interaction column (HIC; TOSOH Butyl Toyopearl 650m) can be used to purify PG1 fused with Green Florescent Protein (GFP).
- GFP Green Florescent Protein
- the GFP selectively binds to the HIC and facilitates Rc10l/PGl to >90% purity.
- recovery is very poor ( ⁇ 20%).
- 10 tandem repeats of PG1 with an elastin like biopolymer (GVGVP (SEQ ID NO: 11); Fig.10) are engineered into the vector.
- This biopolymer has a unique thermal property of precipitating out of solution upon increasing temperature above its inverse transition temperature (Tt).
- GVGVP remains in soluble monomelic state below Tt and form insoluble aggregates above it. This phase transition from soluble to insoluble state is reversible by changing the temperature of the solution and this facilitates protein purification. Subsequently fused protein is re-solubilized by cooling below Tt and to remove any insoluble contaminants that have co-precipitated as shown in Figure 11.
- the process of heating (37°C ) and cooling (4°C) is known as Inverse Transition Cycling (ITC) and performing 3-5 rounds of ITC results in highly purified proteins (>98% purity, Figure 11).
- a signal peptide is fused with dextranase or mutanase for expression in E. coli, where the signal peptide will result in secretion of the enzymes into the extracellular media.
- secretory proteins should pass through two membrane systems of E.coli, during which they pass through the periplasmic environment where disulfide isomerases, foldases and chaperones are present. Therefore, correct folding and disulfide bond formation of secretory proteins are facilitated by the enzymes, resulting in enhancement of biological activity of proteins (ideal for AMPs).
- Another merit of this production strategy is the low level of proteolytic activity in the culture medium which serves to enhance the stability of the recombinant protein.
- the signal sequence of the secreted protein is cleaved during the export process, creating an authentic N-terminus to the native protein.
- outer membrane protein A (OmpA) and Seq X (derived from lac Z) signal peptide demonstrate superior export functions and are capable of exporting fused protein into extracellular medium at up to 4g /L and lg/L, respectively.
- extranase/mutanase/AMP proteins After harvesting large scale biomass, leaves will be lyophilized and stored at room temperature.
- clinically-proven anti -caries compounds such as (fluoride 250ppm) and a broad-spectrum bactericidal, chlorhexidine 0.12% can be included to assess whether these agents increase efficacy.
- the AMP-enzyme combination effectively disrupts cariogenic biofilm formation and the onset of cavitation in vivo.
- AMP-enzyme fusion protein appears to be superior to current chemical modalities for antimicrobial therapy and caries prevention.
- AMP-enzyme (independently or in combination) can be expressed in lettuce chloroplasts under the control of endogenous lettuce regulatory elements, for large scale GLP production and stability assessment.
- a key advantage is the lower production cost by elimination of prohibitively expensive purification processes.
- Freeze-dried leaf material expressing AMP/enzymes can be stored at ambient temperatures for several months or years while maintaining their integrity and functionality. See Figure 13.
- increase of protein drug concentration and decrease of microbial contamination are other advantages. Lettuce leaves, after lyophilization showed 20-25 fold increase in protein drug concentration when compared to fresh leaves, thereby reducing the amount of materials used for oral or topical delivery. Following lyophilization, the plant material can be incorporated into a chewing gum to deliver the biofilm degrading compositions contained therein.
- lettuce chloroplast vectors useful for expressing the proteins of the invention have been previously described in US Patent Application No. 12/059,376, which is incorporated herein by reference. Expression levels of up to 70% of total protein in case of therapeutic proteins like proinsulin in lettuce chloroplasts can be achieved using this system.
- AMP-enzyme(s) expressed in the edible plants are preferably orally delivered (topically) when used for treatment of oral diseases and the prevention and inhibition of dental carie formation.
- AMP/enzyme expressing plant cells are optionally mixed with plant cells expressing cell wall degrading enzymes, described in US Patent Application, 12/396,382, also incorporated herein by reference.
- Chewing gum tablet preparation is shown in Figure 14.
- GFP as an example of the protein of interest, this data shows the amounts of GFP that can be incorporated in to a chewing gum tablet. GFP levels were assessed both via fluorescence and by western blot. The results are shown in Figure 15.
- the present inventors employed the chewing simulator shown in Figure 16 and artificial saliva to assess GFP release kinetics from the gum tablets comprising GFP.
- Figure 17 shows a graph illustrating the release kinetics over time from gum tablets comprising different amounts of GFP present in recombinant lettuce. It is clear from these data that gum tablets comprising the AMP-enzyme fusion proteins of the invention will deliver the active material for a suitable time period to achieve bacterial kill and plaque or biofilm degradation. However, oral rinses, such as Listerine®, can also be employed to deliver the AMP-enzyme fusion proteins or combinations of the invention.
- Figure 18 demonstrates that crude extracts comprising the enzymes of the invention mixed with
- LISTERINE mouthwash are as effective as commercially produced and purified enzymes that are quite costly to prepare.
- the data reveal that the dual-enzyme at various combinations (both different ratio and amounts) markedly reduced the biomass of S. mutans biofilm, in a dose- dependent manner.
- 25U Dex and 5U Mut (5: 1, Dex:Mut ratio) was the most effective, resulting in more than 80% of the total biomass degradation within 120 minutes.
- 5: 1 Dex/Mut activity ratio displayed the highest effectiveness for both EPS degradation and bacterial killing by LISTERINE mouthwash.
- LISTERINE mouthwash The inclusion of a third enzyme further enhanced the overall anti- biofilm activity. Furthermore, results from the mixed-species model indicated that the dual- enzyme combination was capable of not only enhancing the overall antibacterial activity, but also inducing targeted reduction of S. mutans dominance (while increasing the proportion of commensal/probiotic S. oralis) when LISTERINE mouthwash was used after enzymes pre- treatment. Accordingly, the enzyme+LISTERINE mouthwash strategy should selectively target the pathogen S. mutans, while increasing the proportion of commensal S. oralis, thereby preventing microecological imbalance within mixed-species biofilm.
- AMPS have the ability to stimulate innate immunity and wound healing, in addition to antimicrobial activity. Harnessing this novel mast cell host defense feature of AMPs in addition to their antimicrobial properties expands their clinical applications.
- Biofilm-associated caries is the most challenging model for development of topical therapeutics. When developed, such topical drug delivery can be easily adapted to other biofilms, as matrix formation hinders drug efficacy in many other biofilm-associated diseases. Matrix is inherent in all biofilms thus the application goes beyond the biofilm in the mouth.
- the biofilm inhibiting compositions described herein can also be employed in coating stents, artificial joints, implants, valves and other medical devices inserted into the human body for the treatment of disease.
- the AMP/enzymes, or leaves expressing the same can be incorporated into a chewing gum for effective topical application of the same for the treatment of oral disease.
- the compositions may also be incorporated into an oral rinse, such as LISTERINE mouthwash.
- other anti dental carrie agents such as fluoride or CHX may included in such gums or oral rinses.
- biofilm degrading enzyme encoding sequences useful in the practice of the invention, include without limitation,
- biofilms are difficult to treat using conventional antimicrobial monotherapy as they comprise structured microbial communities embedded in an extracellular matrix associated with bacterial adhesion-cohesion and drug tolerance.
- EPS exopolysaccharides
- Our data showed that dextranase synergized with mutanase to breakdown EPS glucan-matrix in pre-formed oral biofilms, while markedly potentiating bacterial killing by antimicrobials (3-log increase vs antimicrobial alone).
- S. mutans UA159 serotype c (ATCC 700610), a proven cariogenic dental pathogen and also the primary producer of water-insoluble EPS matrix (26) was used for single-species biofilm model.
- Actinomyces naeslundii (ATCC ® 12104TM), and Streptococcus oralis (ATCC ® 35037TM) were selected commensals to generate mixed-species biological biofilm with S. mutans, because these three species are all detected in high abundance in the supragingival plaque of human(37).
- S. oralis is one of the earliest pioneer colonizers on the saliva-coated tooth surface (37). They were known to produce soluble glucans from sucrose and to be acid-tolerant(38).
- naeslundii also colonizes on saliva pellicle during the early stages of plaque formation.
- Strains were stored at -80°C in tryptic soy broth (TSB) containing 25% glycerol. All strains were grown in ultrafiltered (10kDa molecular-weight cut-off membrane; Prep/Scale, Millipore, MA) buffered tryptone-yeast extract broth (UFTYE medium; 2.5% tryptone and 1.5% yeast extract, pH 7.0) supplemented with 1% (w/v) glucose at 37°C and 5% C0 2 to mid-exponential phase before use(25).
- TTB tryptic soy broth
- EPS-degrading enzymes are glucanohydrolases that can cleave the glucosidic linkages of polysaccharides.
- Dextranase[a-(1 ⁇ 6) glucanase; EC 3.2.1.11] which can catalyze the hydrolysis of glucoside bonds in (l ⁇ 6)-a-D-glucans of different origins was purchased from Sigma (St. Louis, MO).
- Mutanase[a-(1 ⁇ 3) glucanase; EC 3.2.1.59] that hydrolyzes glucoside bonds in (l ⁇ 3)-a-D-glucans was a kind gift from Johnson & Johnson (New Brunswick, NJ).
- One unit (U) of activity of dextranase was defined as the amount of enzyme which will literate ⁇ . ⁇ of reducing sugar (measured as maltose) per minute from a-(l ⁇ 6)-linked glucans at pH 5.5 at 37°C.
- One unit (U) of mutanase was defined as the quantity of enzyme which will liberate 1.0 ⁇ m ⁇ of reducing sugar (measured as glucose) per minute from a-(l ⁇ 3)-linked glucans at pH 5.5 at 37°C.
- An alcohol-free essential oils (EOs)-based solution comprising the antimicrobial combination of menthol, thymol, eucalyptol, and methyl salicylate was used for Example III. LISTERINE ZERO ® comprising these EOs, was kindly provided by Johnson & Johnson, was used as model antimicrobial agent.
- Single- and mixed-species biofilms were formed on vertically-suspended saliva-coated hydroxyapatite (sHA), a model to mimic the biological dental surface, as detailed previously(25). Briefly, Hydroxyapatite discs (1.25 cm in diameter, surface area of 2.7 ⁇ 0.2 cm2, Clarkson, Chromatography Products, Inc., South Williamsport, PA) were coated with filter-sterilized human whole saliva. For single-species biofilm, each sHA disk was inoculated with 10 5 CFU/mL actively growing S. mutans in UFTYE medium containing 1% (w/v) sucrose, and was grown for 19h (37°C and 5% C0 2 )(25).
- sHA saliva-coated hydroxyapatite
- the mixed-species biofilm model was designed to mimic the biofilm formation based on the "ecological plaque-biofilm" concept(41), as described by Xiao et al. (24).
- the bacterial cultures of S. mutans, S. oralis and A naeslundii were diluted to provide an inoculum with a defined microbial population of S. mutans (10 2 CFU/mL), S. oralis (10 7 CFU/mL), and A naeslundii (10 6 CFU/mL) in UFTYE medium with 0.1% (w/v) sucrose.
- the organisms were grown for the first 29h to form the initial biofilm community, with a medium change at 19 h.
- biofilms were transferred to UFTYE medium containing 1% (w/v) sucrose to induce environmental changes to simulate a cariogenic challenge.
- Biofilms were analyzed at 43h (early cariogenic biofilm) or 67h (mature cariogenic biofilm). The culture medium was replaced twice daily (8 am and 6 pm) with UFTYE medium with 1% (w/v) sucrose for experimental period longer than 43h.
- the final concentrations of dextranase and mutanase ranged from 0-17.50U/mL.
- each well was briefly washed with 0.89% sodium chloride to remove solubilized/detached biomass and residual biofilms were stained with 0.2% crystal violet for visualization and quantification. Relative reduction rate was calculated using that of the vehicle control group as control(0% reduction).
- Coefficient of Drug Interaction(CDI) was used to evaluate the synergism/antagonism between the two
- biofilms were exposed to EOs at full strength for lmin and analyzed upon harvest. Quantitative biofilm analysis
- Biofilms were subjected to biochemical and microbiological analysis, as detailed elsewhere (22, 25). Briefly, biofilms on HA surfaces were harvested, homogenized by sonication and plated on blood agar after serial dilution using an automated Eddy Jet Spiral Plater (IUL, SA, Barcelona, Spain) to determine the CFU on each hydroxyapatite disk (CFU per biofilm). For mixed-species biofilms, the three species were differentiated by observation of colony
- biofilm suspension was centrifuged (5,500g, 10min, 4°C), and the pellet was washed with water and dried in oven (105°C, 24h) before measuring water-insoluble dry weight.
- Water-soluble and water-insoluble polysaccharides in biofilm matrix were extracted and colormetrically quantified using phenol-sulfuric acid method as detailed previously (44, 45). At least 3 independent biofilm experiments were performed for each of the assays.
- EPS were dynamically labelled via incorporation of Alexa Fluor 647 dextran conjugate (Final concentration molecular weight, 10 kDa;
- the sHA disk was transferred to a Petri dish (diameter 35mm) containing 5 ⁇ ⁇ SYT09 (for staining live cells; Molecular Probes) and 30 ⁇ M propidium iodide (for staining dead, or membrane compromised cells; Molecular Probes) in 4mL of 0.1M sodium acetate buffer(pH5.5), which enabled continuous labeling and real-time visualization of live and dead bacterial cells over the entire experimental period.
- GtfB was obtained from Streptococcus milleri KSB8 and purified to near homogeneity by hydroxyapatite column chromatography as detailed by Venkitaraman et al. and Koo et al. (50, 51). Gtf activity was measured by the incorporation of 14 C-glucose from radiolabeled sucrose (PerkinElmer, MA, USA) into glucans (50). One unit(U) of GtfB activity is defined as the amount of enzyme that incorporates 1 ⁇ mol of glucose over a 4-h reaction. Glucan synthesis in the presence of EPS-degrading enzymes was performed as detailed by Hayacibara et al. (21) with some modifications.
- GtfB (10U) was mixed with dextranase or mutanase (ranging from 0-50U), and incubated with ([ 14 C]glucosyl)-sucrose substrate (0.2 ⁇ Ci/mL; 200.0 mM sucrose, 40 ⁇ M dextran T-10, 0.02% sodium azide in Adsorption Buffer: 50mM KC1, 0.35mM K2HPO4, 0.65mM KH 2 P0 4 , lmM CaCb, 0.1 mM MgCl 2 - 6H 2 0, pH 6.5) for 4h at 37°C to allow glucan synthesis.
- Adsorption Buffer 50mM KC1, 0.35mM K2HPO4, 0.65mM KH 2 P0 4 , lmM CaCb, 0.1 mM MgCl 2 - 6H 2 0, pH 6.5
- mutanase For the combinatory effect of dextranase and mutanase, 1U of mutanase with various amount of dextranase(O-10U) was used in the assay. Insoluble glucans were collected after centrifugation (13,400g, 4°C, 10min) and washed three times with water. Soluble glucans were precipitated with ethanol(final concentration: 70%) for 18h at -20°C. The amount of radiolabeled insoluble and soluble glucans were quantified by scintillation counting (50).
- the cariogenic pathogen S. mutans produce glucan-binding proteins which greatly promote its colonization on tooth surface in the presence of Gtf-synthesized glucans (56) while other commensals rely on adhesin-receptor interaction that mediates preferential adhere to salivary pellicle(57).
- Gtf-synthesized glucans 56) while other commensals rely on adhesin-receptor interaction that mediates preferential adhere to salivary pellicle(57).
- EPS-degrading enzymes we assayed bacterial adherence using radioisotope tracing spectroscopy (21, 58). Briefly, S. mutans, S.
- Saliva-coated Hydroxyapatite beads (Bio-rad, CA, USA; 80 ⁇ m particle size) were pretreated with either vehicle or glucanohydrolases at the optimum combination before GtfB immobilization, and glucans were synthesized in situ in the presence of sucrose substrate(200.0 mM sucrose, 40 ⁇ M dextran T-10, 0.02% sodium azide in Adsorption Buffer, pH6.5) for 4h. The glucan-coated surfaces were then incubated with 1.0 X 10 9 cells/mL isotope-labeled bacteria in Adsorption Buffer for lh at 37°C. Hydroxyapatite beads with salivary pellicle only was used as the control.
- Unbound bacteria were removed by washing the beads with Adsorption Buffer 3 times. Affinity of bacterial adhesion was quantified by scintillation counting. The actual number of adherent cells on the sHA beads was calculated using calibration curves of measured radioactivity (counts per minutes, CPM) versus number of radiolabeled bacteria.
- EPS-degrading enzymes synergistically enhance antimicrobial killing at the optimum activity ratio.
- EPS produced by streptococcal Gtfs are key components in cariogenic biofilm matrix, and contain a mixture of glycosidic linkages, comprised predominantly of a-(l ⁇ 3)-, a-(l ⁇ 6)- and a-(l ⁇ 4)-linked glucans with high structural polymorphism(21). Therefore, targeting the EPS-rich matrix is challenging, and may require breakdown of more than one specific chemical bond.
- Glucanohydrolase such as dextranase[a-(l ⁇ 6) glucanohydrolase] and mutanase[a-(l ⁇ 3) glucanohydrolase] were shown to disrupt the glucan synthesis by Gtfs and digest preformed EPS matrix(16, 21, 22).
- EPS-degrading activity of various combinations of dextranase and mutanase using a high-throughput biofilm model ( Figure 19D). Neither dextranase nor mutanase alone could efficiently degrade the biofilm, even at the highest concentration tested (17.5U/mL, ⁇ 25% of reduction rate).
- EPS-matrix has been recognized as an important factor for biofilm drug recalcitrance (4).
- how the structural organization of the matrix and biofilm 3D architecture hinder antimicrobial efficacy remains poorly understood.
- Using high-resolution time-lapse confocal microscopy we observed the spatiotemporal morphological changes that biofilms undergo following EPS-degradation.
- the vehicle-treated biofilm display clusters of densely-packed bacterial (in green) cells, termed microcolonies (1-3) embedded by abundant amount of EPS matrix (in red), typically found in cariogenic biofilms.
- Time-lapse killing assay was performed using real-time bacterial live/dead staining and EPS imaging ( Figure 20 B and Figure 2 IE).
- the biofilm without dual-enzyme treatment contained many live cells (in green) after brief (1 min) and prolonged (5min) exposure to topical antimicrobial agent (EOs) ( Figure 20B, top).
- EOs topical antimicrobial agent
- exopolysaccharide matrix provides essential scaffold for mechanical stability and modulates the resistance of biofilms to mechanical clearance from the substratum (23), posing another challenge for the development of biofilm-disruptive strategies.
- disassembling the matrix-scaffold could impact the physical integrity and stability of the biofilm architecture.
- time-lapsed imaging we tracked the dynamic changes of the biofilm structure during the EPS degradation.
- EDA locally degrade EPS for enhanced targeting of EPS-producing pathogen in mixed- species biofilms.
- EPS-degrading enzymes prevent biofilm formation by inhibiting glucan synthesis in situ
- EPS formed on hydroxyapatite and microbial surfaces promote bacterial adhesion and cohesion.
- EPS glucans synthesized from dietary sucrose by streptococcal Gtfs are of central importance in adhesive interactions providing scaffolding material for biofilm initiation (26).
- Our data indicate that EPS-degrading enzymes could also prevent biofilm formation given their potent efficacy on pre-formed biofilms.
- EPS-degrading enzymes were topically applied to the experimental salivary pellicle surface (sHA) before biofilm formation (Figure 24A).
- EDA approach selectively prevents early colonization S. mutans in mixed-species biofilms.
- the surface-formed EPS were shown to provide binding sites for glucan-binding cariogenic pathogens (e.g. S. mutans) in the presence of sucrose and enhance their colonization, disrupting the commensal -rich community (22).
- glucan-binding cariogenic pathogens e.g. S. mutans
- sucrose glucan-binding cariogenic pathogens
- Biofilm drug recalcitrance imposes great challenges for existing antimicrobial monotherapies and indicates urgent need for multi -targeted or combinatorial approaches(2).
- the resistance to mechanical clearance represents another important mechanism by which the biofilms persistently attach to abiotic and biotic surfaces, making physical removal challenging (13, 32).
- Our data highlight the ability of EPS -degrading enzymes to weaken the mechanical stability and cause structural collapse of the biofilm architecture.
- the enzymes can also induce dispersal of the bacterial cells by degrading the adhesive polysaccharide that encases and stabilizes the bacterial cells. In the clinical setting, the dispersed cells from biofilms will return to an planktonic state and become more sensitive to killing by antimicrobial agents and host defenses(2).
- biofilm dispersal also contributes to the
- S.mutans greatly promote their adhesion by glucan-dependent mechanism such as surface-adsorbed Gtf and the glucan-binding proteins(Gbps) (36).
- glucanohydrolases selectively interfered with the colonization of the EPS-producing pathogens by disrupting glucan production in situ.
- glucan-coated sHA surface favored S. mutans cells adhesion and further accumulation.
- EPS- degradation of the glucan-coated surface reestablished colonization by commensals likely by exposing the pellicle receptors for adhesin-mediated binding by the commensal bacteria.
Abstract
Description
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US16/301,000 US20230241186A1 (en) | 2016-05-12 | 2018-05-14 | Compositions and methods for inhibiting biofilm deposition and production |
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CN113025447A (en) * | 2021-02-05 | 2021-06-25 | 广东省科学院生物工程研究所 | Composition for removing bacterial biofilm and application thereof |
WO2023110900A1 (en) * | 2021-12-16 | 2023-06-22 | Novozymes A/S | Oral care composition comprising enzymes |
WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5741487A (en) * | 1996-05-16 | 1998-04-21 | Lion Corporation | Mutanase-containing oral compositions |
US20040136924A1 (en) * | 2002-12-30 | 2004-07-15 | Boyd Thomas J. | Oral care compositions and methods |
US20120128599A1 (en) * | 2009-08-12 | 2012-05-24 | Colgate-Palmolive Company | Oral care composition |
US20120171128A1 (en) * | 2010-12-30 | 2012-07-05 | Jr Chem, Llc | Dental cleaning composition |
US20120315260A1 (en) * | 2011-06-13 | 2012-12-13 | Svetlana A. Ivanova | Compositions and Methods to Prevent and Treat Biofilms |
Family Cites Families (8)
Publication number | Priority date | Publication date | Assignee | Title |
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JPS60130509A (en) * | 1983-12-20 | 1985-07-12 | Lion Corp | Composition for oral cavity |
JPH0812541A (en) * | 1994-06-29 | 1996-01-16 | Lion Corp | Oral cavity composition |
JP2002020254A (en) * | 2000-07-07 | 2002-01-23 | Lion Corp | Dentifrice preparation composition |
US7354569B2 (en) * | 2003-07-11 | 2008-04-08 | Colgate-Palmolive Company | Chewable antiplaque confectionery dental composition |
US9271904B2 (en) * | 2003-11-21 | 2016-03-01 | Intercontinental Great Brands Llc | Controlled release oral delivery systems |
US20060263306A1 (en) * | 2005-05-19 | 2006-11-23 | Pauline Pan | Compositions having improved substantivity |
US20070140990A1 (en) * | 2005-12-21 | 2007-06-21 | Nataly Fetissova | Oral Compositions Comprising Propolis |
US9125841B2 (en) * | 2013-02-26 | 2015-09-08 | Johnson & Johnson Consumer Inc. | Oral care compositions |
-
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5741487A (en) * | 1996-05-16 | 1998-04-21 | Lion Corporation | Mutanase-containing oral compositions |
US20040136924A1 (en) * | 2002-12-30 | 2004-07-15 | Boyd Thomas J. | Oral care compositions and methods |
US20120128599A1 (en) * | 2009-08-12 | 2012-05-24 | Colgate-Palmolive Company | Oral care composition |
US20120171128A1 (en) * | 2010-12-30 | 2012-07-05 | Jr Chem, Llc | Dental cleaning composition |
US20120315260A1 (en) * | 2011-06-13 | 2012-12-13 | Svetlana A. Ivanova | Compositions and Methods to Prevent and Treat Biofilms |
Non-Patent Citations (1)
Title |
---|
See also references of EP3454877A4 * |
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CN113025447A (en) * | 2021-02-05 | 2021-06-25 | 广东省科学院生物工程研究所 | Composition for removing bacterial biofilm and application thereof |
WO2023110900A1 (en) * | 2021-12-16 | 2023-06-22 | Novozymes A/S | Oral care composition comprising enzymes |
WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
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