JP2020519560A - Compositions and methods for inhibiting biofilm deposition and formation - Google Patents

Abstract

The present invention provides a method of combating a biofilm comprising contacting the biofilm with an antimicrobial agent or antimicrobial essential oil in combination with a composition comprising an effective amount of one or more biofilm degrading enzymes. provide. The biofilm may be on a biological or non-living surface, providing both medical and non-medical uses and methods. In a preferred embodiment, the present invention provides compositions and methods for use in treating or preventing biofilm infections in a subject, especially the oral cavity. [Selection diagram] None

Description

This application claims priority to PCT/US Patent Application Publication No. 2017/032437 filed May 12, 2017, as if its entire disclosure is incorporated herein by this reference. Incorporated herein.

This invention was made with government support under grant numbers: R01 HL107904 and R01 HL109442 awarded by the National Institutes of Health. The government has certain rights in this invention.

The present invention relates to the field of biofilm deposition and disease treatment. More specifically, the present invention provides compositions and methods useful in the treatment of caries and other oral disorders. The present invention also provides a method of coating a biomedical device to prevent unwanted biofilm deposition.

Several publications and patent documents are cited throughout the specification to describe the state of the art to which the invention pertains. Each of these citations is hereby incorporated by reference as if fully set forth.

Biopharmaceuticals produced by current systems are prohibitively expensive and not a reasonable price for the majority of the world population. The cost of protein drugs ($140 billion in 2013) exceeds 75% of GDP in the world [Walsh 2014], making it an unreasonable price. One-third of the world's population earns less than $2 a day and cannot afford to buy protein medicines (including the disadvantaged elderly and younger socioeconomic groups of the United States). Such high costs have been associated with protein production, purification, refrigerated shipping/storage, reduced shelf life, and aseptic delivery methods in exorbitantly expensive fermentors [Daniell et al 2015, 2016].

Biofilms are formed by a complex group of microbial cells that adhere to the exopolysaccharide matrix present on the surface of medical devices. Biofilm-related infections associated with the implantation of medical devices pose serious problems and adversely affect device function. Medical implants used in oral and orthopedic surgery are manufactured using alloys such as stainless steel and titanium. Bacterial adherence is associated with damage to surrounding tissues and frequently causes implant dysfunction, thus improving the surface properties, promoting biointegration, and controlling bacterial adherence by various physical and chemical agents. Surface treatment of medical implants has been attempted.

Many infectious diseases in humans have been caused by biofilms, including those that occur in the mouth [Hall-Stodley et al. , 2004; Marsh, et al 2011]. For example, caries (or tooth decay), the single most common biofilm-related oral disease, irritates most disadvantaged children and adults in the United States and around the world, spending more than $81 billion annually. [Beiker and Flemmig, 2011; Dye et al. , 2015; Kassebaum et al, 2015]. A caries-inducing (cariogenic) biofilm develops when bacteria accumulate on the tooth surface and firmly attaches to the extracellular matrix composed of high molecular substances such as exopolysaccharide (EPS), resulting in entangled bacteria. Form tissue clusters of cells [Bowen and Koo, 2011]. Current topical antibacterial methods for controlling cariogenic biofilms are limited. Chlorhexidine (CHX) is considered the “gold standard” for oral antibacterial therapy, but it has side effects such as tooth stains and calculus formation and is not recommended for daily use [Jones, 1997; Audio]. -Gold, 2008]. Alternatively, several antimicrobial peptides (AMPs) with potential anti-biofilm effects against caries-causing oral pathogens such as Streptococcus mutans have appeared [da Silva et al. , 2012; Guo et al. , 2015].

Antimicrobial peptides (AMPs) are evolutionarily conserved components of the innate immune response and are naturally found in various organisms, including humans. When compared to conventional antibiotics, AMP is less likely to develop resistance. They are strongly active against bacteria, fungi, viruses and can be tailored to target specific pathogens by fusion with surface antigens (Lee et al 2011; DeGray et al 2001; Gupta et al. 2015). Linear AMPs have poor stability or antibacterial activity when compared to AMPs with complex secondary structure. For example, retrocyclins or protegrins show high antibacterial activity or stability when cyclized (Wang et al 2003) or when forming a hairpin structure by disulfide bond formation (Chen et al 2000). RC 101 is extremely stable at pH 3, 4, 7 at temperatures of 25°C to 37°C and in human vaginal fluid for 48 hours (Sassi et al 2011a), but its antibacterial activity was maintained. It was up to 6 months (Sassi et al 2011b). Similarly, protegrin is very stable in salt or human body fluids (Lai et al 2002; Ma et al 2015) but loses potency upon linearization. These interesting features of antimicrobial peptides with complex secondary structures may facilitate the development of new therapeutic agents. However, the high cost of producing sufficient quantities of antimicrobial peptides is a major barrier to their clinical development and commercialization.

According to the present invention there is provided a multi-component composition comprising at least one antimicrobial peptide (AMP) which acts synergistically to degrade biofilm structure and inhibit biofilm deposition and at least one biofilm degrading enzyme. To be done. In certain embodiments, the AMP is selected from Protegrin 1, RC-101, and the AMPs listed in Table 1. Biofilm degrading enzymes include, for example, mutanases, dextranases, glucoamylases, deoxyribonucleases I, DNAases, dispersins B, glycoside hydrolases, and the enzymes provided in Table 2. In certain embodiments, the coding sequences for these enzymes are codon optimized for expression in plant chloroplasts. In a particularly preferred embodiment, at least one AMP and at least one biofilm degrading enzyme are recombinantly produced. In a particularly preferred embodiment, AMP and biofilm degrading enzyme are expressed as a fusion protein. In some embodiments, the enzyme is chemically synthesized, obtained from a microorganism, or obtained from a commercial supplier. When the composition is for the treatment of oral disorders, the composition may optionally further comprise antibiotics, fluorides, CHX, essential oils, or all of the above. The composition may be contained within a chewing gum, hard candy, or mouth rinse. Preferred fusion proteins of the invention include PG-1-Mut, PG-1-Dex, PG-1-Mut-Dex, RC-101-Mut, RC-, used alone or in combination for biofilm degradation. 101-Dex, RC-101-Mut-Dex are included, but are not limited thereto. In particular, any of the AMPs listed in Table 1 can replace either PG-1 or RC-101 of the fusion protein described above to alter or improve the bactericidal action of the fusion protein. To alter the degradative activity of the fusion protein, the enzymes listed above and below may replace Mut, Dex or both of the fusion proteins of the invention. In another embodiment, when two different EPS enzymes are used in the composition, such enzymes are used in different ratios, eg 1:2, 1:3, 1:4, 1:5, 1:6. , 1:7, 1:8, etc. When Mut and Dex are delivered together such as in a gum or buccal rinse, a preferred ratio is 5:1 Dex:Mut.

In another aspect, the invention provides for contacting a surface having a biofilm with the composition, wherein the composition has a bactericidal effect and reduces or eliminates the biofilm containing one or more unwanted microorganisms. Providing a method of disintegrating and/or removing a biofilm, the biofilm being present in or on an animal subject in need of said reduction or removal. In certain embodiments, the biofilm is in the mouth. In other embodiments, the biofilm is on an implanted medical device. This method involves the presence of a bio-form present on the inner or outer body surface selected from the group consisting of the surface of the urinary tract, middle ear, prostate, intima, heart valve, skin, scalp, nails, teeth, and inside wounds. It can also be used to remove film.

In yet another embodiment, the composition of the invention comprising said at least one AMP and said at least one biofilm-degrading enzyme is produced in a plant plastid. The plant may be a tobacco plant and the sequences encoding said AMP and enzyme are codon optimized for expression in plant plastids. In a preferred embodiment, AMP and biofilm degrading enzyme are expressed in lettuce plants as fusion proteins under the control of endogenous regulatory elements present in the lettuce plastid.

In another aspect of the invention, one or more essential oils and at least one biofilm that act synergistically to degrade biofilm structure and inhibit biofilm deposition in a biologically acceptable carrier for delivery. Compositions containing degrading enzymes are disclosed. In some embodiments, the essential oil comprises 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. In another embodiment, the essential oils are menthol, thymol, eucalyptol, and methyl salicylate and the biofilm-degrading enzymes are mutanases and dextranases. In another embodiment, the mutanase and dextranase are present in a 5:1 ratio. In certain embodiments, the enzyme is commercially available, purified from biological sources, or produced in plant plastids. In certain embodiments, the essential oil comprises mouthwash, mouthrinse, mouthspray, toothpaste, chewing gum, toothpaste gel, subgingival gel, mousse, foam, chewable tablet, dentifrice, lozenge, soluble strips and the like. Present on a carrier selected from the group. In certain embodiments, the carrier is mouthwash. Examples of commercially available mouthwashes containing essential oils such as menthol, thymol, eucalyptol, and/or methyl salicylate are marketed under the alcohol-free mouthwash, eg, Listerine® Zero™ brand. And disinfectant mouth rinses, such as those sold under the Listerine® brand.

The invention also provides an effective amount of one or more biofilm degrading enzymes selected from the group consisting of mutanases, dextranases, and glucoamylases, and menthol, thymol, eucalyptol, methyl salicylate, and two of these. A composition comprising one or more essential oils selected from the group consisting of the above in a carrier suitable for oral delivery, wherein the carrier is mouthwash, mouthrinse, mouth spray, toothpaste, chewing gum, teeth. There is provided a composition selected from the group consisting of a gel, a subgingival gel, a mousse, a foam, a chewable tablet, a dentifrice, a lozenge, and a dissolvable strip. In certain embodiments, the carrier is a liquid carrier such as mouthwash, mouthrinse, mouthspray. In certain embodiments, the carrier is a solid or semi-solid carrier such as chewing gum, chewable tablets, lozenges, or dissolvable strips. In certain embodiments, the carrier is a toothpaste.

In yet another aspect, a method of degrading and/or removing unwanted biofilm from the oral cavity is disclosed. One exemplary method is an effective amount of one or more biofilm degrading enzymes selected from the group consisting of mutanases, dextranases, glucoamylases, and combinations of two or more thereof, and menthol, thymol, eucalyptus. Administering to the surface of the oral cavity having the biofilm, one or more essential oils selected from the group consisting of putol, methyl salicylate, and combinations of two or more thereof. In certain embodiments, the essential oil comprises a combination of menthol, thymol, eucalyptol, and methyl salicylate. Another exemplary method comprises contacting the surface of the oral cavity with the biofilm with an effective amount of a pretreatment composition comprising a biofilm-degrading enzyme in a suitable carrier, followed by treatment with an antimicrobial oral rinse. Including, said contacting and treating exerts a bactericidal effect, thereby synergistically reducing or eliminating said unwanted biofilm. In a particular embodiment, the antimicrobial oral rinse comprises at least two essential oils wherein the antimicrobial oral rinse is selected from menthol, thymol, eucalyptol, and methyl salicylate. In another aspect, the essential oil is present in Listerine® mouthwash. In particular, the method described above is based on the symbiotic bacterium A. naeslundii and S. pathogenic S. oralis without adversely affecting Oralis. It is effective in selectively killing mutans.

The present invention also provides a surface of the oral cavity susceptible to biofilm deposition and one or more biofilms selected from the group consisting of mutanases, dextranases, glucoamylases, and combinations of two or more thereof. One or more essential oils selected from the group consisting of menthol, thymol, eucalyptol, methyl salicylate, and combinations of two or more thereof in contact with an effective amount of a degrading enzyme, Provided is a prophylactic method of inhibiting biofilm deposition on the surface of the oral cavity, including contacting the surface. In certain embodiments, the essential oil comprises a combination of menthol, thymol, eucalyptol, and methyl salicylate. Another exemplary method is to contact the surface of the oral cavity susceptible to biofilm deposition with an effective amount of a pretreatment composition comprising a biofilm-degrading enzyme in a suitable carrier, followed by treatment with an antimicrobial oral rinse. And the contacting and treating exert a bactericidal effect, thereby synergistically inhibiting unwanted biofilm deposition. In a particular embodiment, the antimicrobial oral rinse comprises at least two essential oils wherein the antimicrobial oral rinse is selected from menthol, thymol, eucalyptol, and methyl salicylate. In another aspect, the essential oil is in Listerine®. In particular, the method described above is based on the symbiotic bacterium A. naeslundii and S. pathogenic S. oralis without adversely affecting Oralis. It is effective in selectively killing mutans.

Purification of GFP-fused retrocyclin (RC101) and protegrin (PG1) expressed in tobacco chloroplasts-FIG. 1A. Western blot analysis of purified GFP-RC101 fusions using anti-GFP antibody. FIG. 1B. Native fluorescent gel of purified GFP-RC101 fusion. FIG. 1C. Western blot of purified GFP-PG1 fusions using anti-GFP antibody. FIG. 1D. Natural fluorescent gel of purified GFP-PG1. Note-All samples in Figures 1A-1D were loaded based on total protein values obtained from the Bradford method. Densitometry using Image J software was performed to determine expression levels of GFP concentrations, purity, and yield. Expression levels and yields were calculated from GFP concentration relative to total protein value. The yield was determined by multiplying the GFP concentration by the recovered amount after purification. Individual peptide yields were determined by dividing the GFP yield by the molar coefficient 14 (ratio of GFP MW to peptide MW). Double concentration was calculated by dividing the percent purity of the crude plant extract by the percentage expressed. Antimicrobial activity of AMPs (GFP-PG1 and GFP-RC101) against Streptococcus mutans and other oral microorganisms. Cell viability was determined by absorbance (A _600nm ) and colony forming unit (CFU) counts over time. (FIG. 2A) S. cerevisiae treated with different concentrations of GFP-PG1 and synthetic PG1. mutans time kill curve (A600 nm). (FIG. 2B) S. coli treated with GFP-PG1 and synthetic PG1 at each time point. Live cells of mutans (CFU/ml). (FIG. 2C) S. cerevisiae treated with different concentrations (A _{600 nm} ) of GFP-RC101. mutans time kill curve. (FIG. 2D) S. cerevisiae treated with GFP-RC101 at each time point. Live cells of mutans (CFU/ml). (FIG. 2E) S. cerevisiae treated with 10 μg/ml GFP-PG1 for 1 and 2 hours. gordonii, A.; naeslundii, and C.I. live cells of C. albicans (CFU/ml). Bacterial killing by GFP-PG1 determined via confocal fluorescence and SEM imaging (FIG. 3A) S. cerevisiae treated with 10 μg/ml GFP-PG1. Mutans time-lapse killing. The control group (FIG. 3B) was treated with S. It was composed of mutans cells. Propidium iodide (PI) (red) was used to determine bacterial viability over time at the single cell level using confocal microscopy. PI is cell impermeable and only penetrates into membrane-damaged cells. In dying cells and dead cells, bright red fluorescence is generated when PI binds to DNA. GFP-PG1 is shown in green. (FIG. 3C) S. cerevisiae exposed to GFP-PG1 at a concentration of 10 μg/ml for 1 hour using a scanning electron microscope. Morphological observation of mutans. Red arrows indicate extrusion of dimple membrane and intracellular components. Inhibition of biofilm formation by a single topical treatment of GFP-PG1. This figure shows the S. Figure 3 shows a representative image of a three-dimensional (3D) rendering of a mutans biofilm. Bacterial cells were stained with SYTO 9 (green) and EPS labeled with Alexa Fluor 647 (red). Disc surfaces of saliva coated hydroxyapatite (sHA) were treated with a single topical treatment of GFP-PG1 with a short 30 minute exposure (FIG. 4B). The control group (FIG. 4A) was treated with buffer only. The treated sHA disks were then treated with 1% (w/v) sucrose and actively growing S. The cells were transferred to a medium containing mutans cells (10 ⁵ cfu/ml) and incubated at 37°C, 5% CO ₂ for 19 hours. After growth of the biofilm, the biofilm was analyzed with a two-photon confocal microscope. (FIG. 4C) S. cerevisiae by quantitative PCR (qPCR) with and without propidium monoazide (PMA) treatment. Quantitative analysis of the ratio of live and dead cells of mutans (Klein et al., 2012). The combination of PMA and qPCR (PMA-qPCR) quantifies live cells with intact membranes. Prior to isolation of genomic DNA and quantification of qPCR, PMA is added to selectively crosslink dead cell DNA, thereby preventing PCR amplification (Klein et al., 2012). Asterisk indicates that the value of GFP-PG1 treatment was significantly different from the control (P<0.05). An EPS degrading enzyme that digests biofilm matrix. S. cerevisiae treated with a combination of dextranase and mutanase. Representative time-lapse image of EPS degradation of mutans biofilm. Bacterial cells were stained with SYTO 9 (green) and EPS labeled with Alexa Fluor 647 (red). White arrows indicate "open" spaces between bacterial cell clusters and "uncovered" cells after enzymatic degradation of EPS. Biofilm disruption by synthetic PG1 alone or in combination with EPS degrading enzyme. (FIG. 6A) Time-lapse quantification of EPS degradation in intact biofilms using COMSTAT. (FIG. 6B) Synthetic PG1 and EPS degrading enzymes (Dex/Mut) alone or in combination treated with ImageJ. Viability of mutans biofilm. (FIG. 6C) The anti-biofilm activity of synthetic PG1 was enhanced by EPS degrading enzyme (Dex/Mut). The asterisk indicates that the values of the different experimental groups are significantly different from each other (P<0.05). Different human periodontal cell lines by confocal microscopy: human periodontal ligament stem cells (HPDLS), maxillary mesenchymal stem cells (MMS), human head and neck squamous cell carcinoma cells (SCC), gingival-derived mesenchymal stromal cells ( GMSC), adult gingival keratinocytes (AGK) and osteoblasts (OBC) purified fusion proteins CTB-GFP, PTD-GFP, protegrin-1-GFP (PG1-GFP) and retrocyclin 101-GFP (RC101-). In vitro uptake of GFP). 2×10 ⁴ cells of the human cell lines HPDLS, MMS, SCC, GMSC, AGK and OBC were cultured overnight on an 8-well chamber slide (Nunc) at 37° C., then purified GFP fusion protein: CTB-GFP in 100 μl PBS. (8.8 μg), PTD-GFP (13 μg), PG1-GFP (1.2 μg), and RC101-GFP (17.3 μg) were each added with 1% FBS and incubated at 37° C. for 1 hour. After fixing with 2% paraformaldehyde for 10 minutes at RT and washing three times with PBS, the cells were stained with anti-fading mounting medium containing DAPI. For negative controls, cells were incubated with commercial GFP (2 μg) in PBS with 1% FBS and treated under the same conditions. All fixed cells were imaged using a confocal microscope. Green fluorescence indicates GFP expression. Blue fluorescence indicates DAPI labeled cell nuclei. Images were viewed under a 100× objective and at least 10-15 GFP positive cells or images were observed in each cell line. Scale bar represents 10 μm. All image studies have been analyzed in triplicate. Downstream processing of GFP fusions from transplastomic tobacco: Flow diagram showing the various steps involved in producing purified GFP fusions from greenhouse-grown transplastomic tobacco plants. Mutanase (FIG. 9A) and dextranase (FIG. 9B) vector and codon optimized sequences. Codon optimized mutanase: SEQ ID NO: 1. Codon optimized dextranase: SEQ ID NO:2. FIG. 10 is a schematic diagram of a chloroplast vector expressing a tandem repeat of AMP fused with GVGVP for use alone or expression of a fusion protein containing the EPS protein of FIG. 9. Novel purification strategy: Reverse temperature cycling purification process shows high yield. Expression of functional codon optimized mutanases in E. coli. FIG. 12 shows a Western blot showing mutanase expression in E. coli. FIG. 12B shows E. coli spread on 0.5% blue dextran plates. The transformed clones are usually S. It produces a recombinant dextranase made in mutans, which makes the blue halo around the colony visible. A gel diffusion assay comparing the degradation activity (total protein loading) of recombinant dextranase present in crude lysates from transformed E. coli against blue dextran compared to a commercially purified enzyme derived from penicillin. Represent FIG. 6 is a flow diagram of steps for manipulating lettuce plants for AMP/biofilm degrading enzyme production. 7 shows the preparation of chewing gum tablets. Although GFP is exemplified herein, chewing gum containing AMP-enzyme fusion proteins (eg, those provided in Figures 9 and 10) are also within the scope of the invention. The gum table was evaluated via fluorescence and by Western blot to confirm the concentration of GFP. (I) Western blotting (ii) Quantification of GFP release from chewing gum based on a fluorimeter (Fluoroskan Ascent™ Microplate Fluorometer-Thermo; λ _ex 485 nm; λ _em 538 nm). Commercially available GFP (Vector Laboratories, Catalog No. MB-0752) was used as a standard. The chewing gum was ground with protein extraction buffer. A chewing simulator using artificial saliva to assess the release kinetics of biofilm degraders from the gum tablets of the present invention is shown. Graph showing quantification of GFP released from chewing gum. Gum tablets containing increased concentrations of GFP expressed in lettuce leaves were evaluated in a chewing simulator in the presence of artificial saliva to determine GFP release kinetics. Graph showing that crude extracts containing enzymes expressed from chloroplast vectors are effective in inhibiting CFU formation when mixed with Listerine® mouthwash, similar to commercially available enzymes. S. cerevisiae in vitro using mutanase and dextranase from E. coli supplemented with Listerine (ratio 1:5). Enzymatic degradation of mutans biofilm. Commercially available mutanases (Bacillus sp. from Amano) and dextranases (Penicillium sp. from Sigma) were used as positive controls, while crude E. coli extracts were used as negative controls. CFU/ml is expressed as mean±standard deviation (n=2). ***, P<0.001 for E. coli extract. Glucanohydrolase is a S. Enhance the antibacterial and bactericidal effect of mutans biofilm. (FIG. 19A) Saliva coated hydroxyapatite (sHA) biofilm model used in this study. (FIG. 19B) Schematic of treatment regimen and preformed S. Hypothesis of EPS degradation/antibacterial (EDA) approach to mutans biofilm. (FIG. 19C) Effect of dextranase/mutanase treatment on antimicrobial killing efficacy. 19 hours preformed S. s. Mutans biofilms were topically treated with different combinations of glucanohydrolases for 120 minutes and then immediately exposed to antimicrobial agents (EOs) for 1 minute. The antibacterial bactericidal effect was judged based on the recovery rate of CFU. The most potent killing was consistently achieved using 8.75 U/mL dextranase and 1.75 U/mL mutanase prior to antimicrobial exposure (5:1 enzyme activity ratio, antimicrobial alone). Is more effective than about 3 logs). Data are shown as mean±standard deviation (n=4). NS, no significant difference; *, p<0.05; **, p<0.01***, p<0.001 (Student's t-test). (FIG. 19D) Screening for the combined effect of dextranase and mutanase on EPS degradation. Representative image of checkerboard microdilution assay. (FIG. 19E) Combined effect of dextranase and mutanase (decrease in biomass) determined by checkerboard microdilution assay. Red arrow, optimal combination selected for further experiments in this study (8.75 U/mL Dex and 1.75 U/mL Mut, 5:1 activity ratio). EPS degrading enzymes degrade the biofilm matrix in situ and promote antimicrobial targeting within the biofilm. (FIG. 20A) 19 hours S. cerevisiae after 120 minutes of matrix degradation with glucanohydrolase. Confocal microscope showing morphology of mutans biofilm. Green, bacterial cells stained with SYTO9. Red, exopolysaccharide (EPS) labeled with Alexa Fluor 647. White arrow, bacterial dispersal induced by double enzyme treatment. (FIG. 20B) Time-lapse killing assay performed using real-time bacterial live/dead staining and imaging. Preformed (19 hours) S. Mutans biofilms were treated with 8.75 U/mL dextranase + 1.75 U/mL mutanase or vehicle control for 120 minutes, both loaded with sterilization (EOs). Images of single microcolonies were acquired at 0, 1, and 5 minutes after antimicrobial loading. Green, live cells (SYTO 9); magenta, dead cells (propidium iodide); red, EPS (Alexa Fluor 647). The white arrows in the vehicle-treated group indicate that the antimicrobial agent readily killed the bacteria near the surface of the microcolonies while most of the cells inside were alive. (FIG. 20C) Total biomass per biofilm (dry weight) after 120 minutes of matrix degradation by glucanohydrolase. (FIG. 20D) S. after matrix degradation by glucanohydrolase for 120 minutes. Biochemical properties of exopolysaccharides of mutans biofilm. (FIG. 20E) Synergistic anti-biofilm effect of glucanohydrolase and antibacterial agent. D, dextranase; M, mutanase; D+M, dextranase+mutanase. Data are shown as mean±standard deviation (n=4). NS, no significant difference; ***, p<0.001 (Student's t-test). (FIG. 20F) Combination of the three enzymes with dextranase, mutanase, and glucoamylase further enhances antimicrobial killing efficacy. G: 20 U/mL glucoamylase; D+M: 8.75 U/mL dextranase+1.75 U/mL mutanase; D+M+G: dextranase+mutanase+glucoamylase. *, p<0.05; ***, p<0.001 (Student's t-test). Kinetics of enzymatic degradation of EPS matrix and associated refractory protective mechanisms. Preformed (19 hours) S. mutans biofilm (EPS labeled with Alexa Fluor 647) was imaged with a 0.1 M sodium acetate buffer solution containing 5 μM SYTO9 and 30 μM propidium iodide, with continuous labeling and alive and dead. Real-time visualization of bacterial cells was performed over time. Glucanohydrolase was added to the buffer to give a final concentration of 8.75 U/mL dextranase and 1.75 U/mL mutanase, while the same volume of buffer was used as a vehicle control. (FIGS. 21A and 21B) Matrix-decomposed four-dimensional time-lapse confocal imaging. (FIG. 21C) and (FIG. 21D) Final images of microcolonies obtained after 1 minute antimicrobial killing (EOs). Green, live cells (SYTO 9); magenta, dead cells (propidium iodide); red, EPS (Alexa Fluor 647). The black dotted circle is the core of the vehicle-treated microcolony, which is composed mainly of living bacteria after challenge with antibacterial agents. White arrows, EPS within microcolonies, were efficiently degraded by glucanohydrolase. Inserted dashed box, live bacteria in outer layer after antimicrobial exposure. (FIG. 21E) EPS degradation promotes antimicrobial killing of bacterial cells inside microcolony (time-lapse image of dead bacteria only). Magenta, dead cells (propidium iodide). The mechanical stability and integrity of biofilm scaffolds are damaged by EPS degradation. (FIG. 22A) S. cerevisiae with extensive cell dispersion caused by matrix degradation (8.75 U/mL dextranase and 1.75 U/mL mutanase) within 120 minutes. Time-lapse imaging showing "implosion-like" collapse of the physical structure of mutans biofilm. Green, bacterial cells stained with SYTO9. Red, EPS labeled with Alexa Fluor 647. (FIG. 22B) Time resolved EPS degradation (red squares) and microcolony spatial displacement (green circles) curves. The biological volume of EPS in the biofilm was algorithmically analyzed using COMSTAT2. The movement of each microcolony was quantified by calculation as cumulative displacement using TrackMate. Cumulative displacement data are shown as median ± quartile range. (FIG. 22C) Confocal image of glucan formation of sHA beads with and without glucanohydrolase pretreatment. Gray: hydroxyapatite surface; red: EPS glucan. EDA locally degrades EPS to enhance targeting of EPS-producing pathogens in mixed-species biofilms. (FIG. 23A) Schematic representation of the treatment plan and hypothesis of the EDA approach to preformed mixed species biofilms. (FIG. 23B) Ecological changes in the mixed-species biofilm model used in this study. The dashed box, EDA approach was tested at 43 hours (early stage) and 67 hours (late stage). (FIG. 23C) Effect of EDA on microbial ecology of early mixed-species biofilms (43 h). Above, CFU of various bacterial species recovered from mixed-species biofilm after EDA treatment. Below, the proportion of different species. (FIG. 23D) Effect of EDA on microbial ecology of late mixed-species biofilms (67 hours). Above, CFU of various bacterial species recovered from mixed-species biofilm after EDA treatment. Below, the proportion of different species. (FIG. 23E) EPS, S. cerevisiae in late mixed biofilm. mutans, and fluorescence in situ hybridization (FISH) showing the spatial distribution of symbionts (67 hours). a. Overview of mixed-species biofilm of sHA; b. Cross-sectional enlarged image (fusion); c. S. mutans only (green); d. EPS only (red); e. S. Oralis only (yellow); f. A. naeslundii only (cyan). (FIG. 23F) Dynamics of local degradation of EPS within mixed-species biofilm exposes embedded bacteria (yellow arrow). Gray, all bacteria stained with SYTO9; red, exopolysaccharide (EPS) labeled with Alexa Fluor 647. D, dextranase; M, mutanase; D+M, dextranase+mutanase. Data are shown as mean±standard deviation (n=4). NS, no significant difference; *, p<0.05**, p<0.01***, p<0.001 (Student's t-test) The EPS-degrading enzyme of the experimental salivary pellicle was S. cerevisiae in situ. Inhibits mutans biofilm formation. (FIG. 24A) S. cerevisiae by pretreatment with EPS degrading enzyme. Schematic of treatment plan and hypothesis for mutans biofilm prophylaxis. (FIG. 24B) Representative confocal images showing synergistic inhibition of biofilm formation by glucanohydrolase in experimental salivary pellicle. sHA discs were topically treated with EPS-degrading enzymes (8.75 U/mL dextranase and/or 1.75 U/mL mutanase) or vehicle control for 60 minutes prior to inoculation and 19 hours S. Incubated to allow formation of mutans biofilm. Green, bacterial cells stained with SYTO9; red, EPS labeled with Alexa Fluor 647; white arrow, biofilm formed on sHA pre-treated with a single enzyme, although the structure is altered, It showed microcolony-like formation. (FIG. 24C) Salivary pellicle glucanohydrolase reduces biomass (dry weight) accumulation. (FIG. 24D) CFU of biofilm formed on salivary pellicle pretreated with glucanohydrolase. (FIG. 24E) CFU recovered from biofilm of salivary pellicle pretreated with glucanohydrolase, followed by antibacterial killing at 19 hours. S. cerevisiae formed with sHA pretreated with EPS degrading enzyme or vehicle. The mutans biofilm was loaded with antimicrobial agents (EOs) for 1 minute at 19 hours, and the efficacy of the anti-biofilm was analyzed by measuring the CFU recovered from the biofilm. (FIG. 24F) Dose-dependent inhibitory effect of dextranase and/or mutanase on soluble and insoluble glucan synthesis by purified GtfB. Purified GtfB (10 U) was mixed with dextranase or/and mutanase and incubated with ([ ¹⁴ C]glucosyl)-sucrose substrate for 4 hours at 37° C. to synthesize glucan. Insoluble glucan was collected by centrifugation (13,400g, 4°C, 10 minutes). Soluble glucan was collected from the supernatant after precipitation with ethanol (final concentration: 70%) at −20° C. for 18 hours. The amount of radiolabeled insoluble and soluble glucan was quantified by scintillation counting. D, dextranase; M, mutanase; D+M, dextranase+mutanase. Data are shown as mean±standard deviation (n=4). NS, no significant difference; *, p<0.05**, p<0.01***, p<0.001 (Student's t-test) EDA is an S. cerevisiae in mixed-species biofilm. Selectively prevent early colonization of mutans. (FIG. 25A) S. cerevisiae by EPS degrading enzyme pretreatment. Schematic of treatment plan and hypothesis for mutans biofilm prophylaxis. S. cerevisiae by pretreatment with EPS degrading enzyme. Schematic representation of the treatment plan and hypothesis for mutans biofilm prophylaxis. (FIG. 25B) Microbial population of mixed-species biofilms after initial colonization with sHA. sHA discs were treated topically with EPS-degrading enzymes (8.75 U/mL dextranase and 1.75 U/mL mutanase) or vehicle control for 60 minutes prior to inoculation to allow formation of mixed-species biofilms. Incubated. (FIG. 25C) FISH image of a mixed-species community in the early stages of colonization. Green, S. mutans; yellow, S. oralis; cyan, A.; naeslundii; red, EPS. (FIG. 25D) Enzyme pretreatment enhances the overall bactericidal effect and the mixed seed biofilm S. Helps remove mutans. A 43-hour mixed-species biofilm formed with sHA pretreated with EPS-degrading enzyme or vehicle was loaded with antimicrobial agents (EOs) for 1 minute and by measuring the CFU of different bacterial species recovered from the biofilm. , The efficacy of anti-biofilm was analyzed. (FIG. 25E) Bacterial adhesion assayed by ³ H-thymidine radioisotope tracking spectroscopy. sHA beads were pretreated with vehicle or EPS degrading enzyme prior to GtfB immobilization and glucan was synthesized in situ in the presence of sucrose substrate. The beads were treated with 1.0×10 ⁹ cells/mL of radiolabeled S. mutans, S.M. oralis and A. Each was incubated in Naeslundii and washed to remove unbound bacteria. The number of attached bacteria was quantified by scintillation counting. D, dextranase; M, mutanase; D+M, dextranase+mutanase. NS, no significant difference; *, p<0.05; **, p<0.01***, p<0.001 (Student's t-test)

Many human infections are caused by pathogenic biofilms, including those that occur in the oral cavity (such as caries and periodontal disease). Caries (or tooth decay) continues to be the single and most costly and common biofilm-related oral disease in the United States and in the world. It is the main reason for urgent visits to the treatment room, which both children and adults are equally frustrated and absent from work or school. Unfortunately, the prevalence of caries remains high (>90% of the US adult population) and is the most common chronic illness that afflicts children and adolescents, especially in poor socioeconomic backgrounds. In addition, poor oral health often results in systemic effects that affect overall health. Importantly, the cost of treating the destruction of this disease (such as caries lesions and pulp infections) exceeds $40 billion annually in the United States alone. Fluoride is the mainstay of caries prevention. However, its widespread use provides incomplete protection against disease. Fluoride is effective in reducing and enhancing demineralization of early caries lesions, but has limited efficacy on biofilms. Conversely, current antimicrobial modalities for controlling caries-causing biofilms are largely ineffective.

There is an urgent need to develop effective treatments for controlling toxic oral biofilms. The present invention provides a low cost method for the production and delivery of therapeutically effective plant expressed biopharmaceuticals over current anti-biofilm/dental modalities. In certain embodiments, antibacterial agents and enzymes are obtained from commercial sources.

Definition:
As used herein, antimicrobial peptides are small peptides that have any bacterial activity. "RC-101" is an analog of the cyclic octadecapeptide, retrocyclin, which protects human CD4+ cells in vitro from infection with T- and M-tropic strains of HIV-1 in human cervical tissue. To prevent HIV-1 infection. RC-101's ability to prevent HIV-1 infection and retain full activity in the presence of vaginal fluid makes it well suited for other topical fungicide applications, especially in oral biofilms. The sequence of RC-101 is described in Plant Biotechnol J. et al., herein incorporated by reference. 2011 Jan;9(1):100-115.

"C16G2" is an S. It is a novel synthetic antimicrobial peptide with specificity for mutans.

"Protegrin-1 (PG)" is an 18-residue β-sheet peptide rich in cysteine. It has potent antibacterial activity against a wide range of microorganisms including bacteria, fungi, viruses, and especially clinically important antibiotic resistant bacteria. For example, the pathogenic microorganisms E. coli and the fungal opportunistic C. Albicans is effectively killed by PG in laboratory tests. The sequence of PG-1 is described in Plant Biotechnol J. et al., herein incorporated by reference. 2011 Jan;9(1):100-115.

Additional antimicrobial peptides include those shown in Table 1 below.

As used herein, "antimicrobial agent" includes CHX, triclosan/copolymer dentifrice, clindamycin, doxycycline gel, minocycline powder, macrolides, sulfonamides, penicillin and its derivatives, tetracycline, quinolone. , Levofloxacin, ciprofloxacin, and fluconazole.

The "essential oil" may include one or more selected from the group consisting of menthol, thymol, eucalyptol, methyl salicylate, and combinations of two or more thereof. In a particular embodiment, the essential oil comprises menthol. In a particular embodiment, the essential oil comprises thymol. In certain preferred embodiments, the essential oil comprises a combination of menthol, thymol, eucalyptol, and methyl salicylate.

A "biofilm" is a complex structure that attaches to surfaces that come into regular contact with water and colonies of bacteria and other such yeasts, fungi, protozoa, etc. that secrete a slimy protective coating that they normally enclose. Composed of microorganisms. Biofilms can be formed on solid or liquid surfaces, as well as on soft tissues of living organisms, and are resistant to conventional, conventional disinfection methods. Plaque, mucous coatings that foul pipes and tanks, and algae mats in water are examples of biofilms. Biofilms are generally pathogenic in the body and cause diseases such as caries, cystic fibrosis and otitis media.

“Biofilm-degrading enzyme” includes dextranase, mutanase, DNAse, endonuclease, deoxyribonuclease I, dispascin B, and glycoside hydrolase, such as 1→3)-α-D-glucan hydrolase. Although the use of chloroplast codon-optimized sequences encoding dextranase and mutanase, including but not limited to exopolysaccharide degrading enzymes, is preferred, one of skill in the art will recognize many different biofilm degrading enzymes in the art. I know well. Additional enzyme sequences for use in the fusion proteins of the invention are provided below. As mentioned above, suitable enzymes can be purified from the bacteria that produce them or can be obtained from commercial sources.

As used herein, the term "administering" or "administration" of an agent, drug, or peptide into a subject introduces or delivers to the subject a compound that performs its intended function. Including any route to Administration or administration can be performed by any suitable route, including oral, topical, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), or rectal. .. Administering or administration includes self-administration and administration by others. In one embodiment, the single dose involves oral delivery of a single formulation containing the enzyme and the antimicrobial agent. In other embodiments, administration can be continuous. In a preferred embodiment, the biofilm degrading enzyme is administered first, followed by delivery of the antimicrobial agent. Enzymatic treatment is the extracellular exposure of the gingiva and teeth to the enzyme in the oral cavity 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. It is performed for a suitable time to decompose the matrix. Preferably the teeth and gums are treated with the enzyme for 30-120 minutes. Most preferably, the enzymatic treatment is carried out for at least 30 minutes. After this period, an antimicrobial oral rinse is administered for 20, 30, 40, 50, 60, 90, or 120 seconds. In a preferred embodiment, at least two or more of methol, eucalyptol, thymol and methylsalicylic acid are present in the rinse. In a particularly preferred embodiment, the rinse is Listerine®. The enzyme-containing composition comprises a group consisting of mouthwash, mouthrinse, mouth spray, toothpaste, toothpaste gel, subgingival gel, mousse, foam, denture care product, chewable tablet, dentifrice, lozenge, soluble strip and the like. It may be a selected form.

As used herein, the term “disease”, “disorder”, or “complication” refers to any deviation from a subject's normal condition.

As used herein, terms such as "effective amount" and "amount effective" mean an amount that is effective over the dose and time period necessary to achieve the desired result. To do. In certain embodiments, the amount is effective to act prophylactically to inhibit biofilm formation. In another embodiment, the amount is effective to disperse an existing biofilm.

As used herein, the term “inhibit” or “prevent” does not exacerbate or develop the clinical symptoms of a disease state in a subject who is exposed to or susceptible to the disease state, For example, it means that the development of the disease is suppressed, but that the disease state has not yet undergone or has not yet exhibited the symptoms.

As used herein, the term "expression" in the context of a gene or polynucleotide involves transcription of the gene or polynucleotide into RNA. The term may also, but need not necessarily, include subsequent translation of RNA into polypeptide chains and their assembly into proteins.

Plant debris includes one or more molecules derived from a plant in which the protein of interest is expressed, including but not limited to proteins and fragments thereof, minerals, nucleotides and fragments, plant structural components, and the like. Can be Thus, whole plant material (such as whole or part of plant leaves, stems, fruits, etc.), or compositions related to crude plant extracts will have high levels of plant debris and no detectable plant debris. Certainly, compositions containing one or more purified proteins of interest are included. In certain embodiments, the plant debris is rubisco.

In another embodiment, the present invention can be administered to treat or prevent biofilm formation on biomedical devices useful in situ (eg, in the mouth) and surgical implants such as stents, artificial joints and the like. It relates to a composition. In this embodiment, the device is coated with the composition to prevent unwanted biofilm deposition of the device. The composition comprises a therapeutically effective amount of one or more antimicrobial peptides (AMPs), and one or more enzymes in combination having biofilm degrading activity, each of said AMPs and its enzymes depending on the plant and plant debris. It is expressed and acts synergistically to degrade the biofilm. In certain embodiments, AMP and enzyme are expressed from separate plastid transformation vectors. In another embodiment, a plastid transformation vector comprising a polycistronic coding sequence in which both AMP and enzyme are expressed from a single vector.

In other embodiments, the antimicrobial peptide is optional and one or more enzymes are used in combination with one or more antimicrobial agents and/or essential oils.

Proteins expressed in accordance with certain embodiments taught herein can be used in vivo by administering to a subject, human or animal in a variety of ways. The pharmaceutical composition may be administered orally, topically, subcutaneously, intramuscularly or intravenously, with oral topical administration being preferred.

The oral compositions produced by the embodiments of the present invention can be administered by consumption of food produced from transgenic plants that produce therapeutic proteins derived from plastids. The edible part of the plant or part thereof is used as a food ingredient. Therapeutic compositions can be formulated in a classical manner using solid or liquid vehicles, diluents and additives suitable for 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 such as starch, calcium carbonate, sucrose, lactose, gelatin and the like. The therapeutic protein of interest may optionally be purified from plant homogenates. The preparation may be emulsified. The active ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol and the like, and combinations thereof. In addition, if desired, the composition may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants. In a preferred embodiment, the edible plant, fruit juice, cereal, leaf, tuber, stem, seed, root, or other plant part of the pharmaceutical-producing transgenic plant is ingested by a human or animal, so that it is very useful in the treatment of disease. Provide an inexpensive means.

In certain embodiments, plant material (eg, lettuce material) comprising chloroplasts expressing AMPs, and biofilm-degrading enzymes, and combinations thereof are homogenized and encapsulated. In one particular embodiment, the extract of lettuce material is encapsulated. In another embodiment, the lettuce material is powdered prior to encapsulation. As mentioned above, biofilm degrading proteins can also be purified from plants after expression.

In another embodiment, for convenience of administration, the composition may be provided with the juice of a transgenic plant. For the purposes mentioned above, the transformed plants are preferably selected from the edible plants consisting, inter alia, of tomatoes, carrots and apples, which are usually consumed in the form of juices.

According to another embodiment, the present invention relates to a transformed chloroplast genome transformed with a vector containing a heterologous gene expressing a combination of the peptides disclosed herein.

Of particular interest are transformed chloroplast genomes transformed with a vector containing one or more AMP and biofilm degrading enzymes or combinations thereof, a heterologous gene expressing a polypeptide. In a related embodiment, the invention relates to a plant comprising at least one cell transformed to express a peptide disclosed herein.

Reference herein to a gene sequence refers to a single-stranded or double-stranded nucleic acid sequence and includes the coding sequence of the polypeptide of interest or the complement of the coding sequence. % Of the degenerate nucleic acid sequence encoding the polypeptide, as well as homologs that are at least about 50%, 55%, 60%, 65%, 60%, preferably about 75%, 90%, 96%, or 98% identical to the cDNA. Nucleotide sequences can be used with respect to the polynucleotides taught herein. The percent sequence identity between the sequences of two polynucleotides is determined using an affine gap search with a gap open penalty of -12 and a gap expansion penalty of -2, using a computer program such as ALIGN using the FASTA algorithm. Will be judged. Complementary DNA (cDNA) molecules, species homologues, and nucleic acid sequence variants that encode biologically active polypeptides are also useful polynucleotides.

Variants and homologues of the above nucleic acid sequences are also useful nucleic acid sequences. Typically, homologous polynucleotide sequences can be identified by hybridizing a candidate polynucleotide to a known polynucleotide under stringent conditions, as is known in the art. For example, use the following wash conditions: 2X SSC (0.3M NaCl, 0.03M sodium citrate, pH 7.0), 0.1% SDS, room temperature twice, 30 minutes each; then 2X. SSC, 0.1% SDS, once at 50° C., 30 minutes; then 2×SSC, twice at room temperature, 10 minutes each, each homologous sequence containing up to about 25-30% base pair mismatch can be identified. More preferably, homologous nucleic acid strands contain 15-25% base pair mismatches, and even more preferably 5-15% base pair mismatches.

Species homologues of the polynucleotides referred to herein can also be identified by making appropriate probes or primers and screening a cDNA expression library. It is well known that the Tm of double-stranded DNA decreases by 1 to 1.5°C for every 1% decrease in homology (Bonner et al., J. Mol. Biol. 81, 123 (1973). ). Nucleotide sequences that hybridize to the polynucleotide of interest, or their complements following stringent hybridization and/or wash conditions, are also useful polynucleotides Stringent wash conditions are known in the art. It is well known and understood, and is disclosed, for example, in Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed., 1989, at pages 9.50-9.51.

The following materials and methods are provided to facilitate the practice of Examples I and II.

Microorganisms and growth conditions In this study, Streptococcus mutans UA159 serotype c (ATCC 700610), Actinomyces naeslundii ATCC 12104, Streptococcus gordonii DL-1 and Candida albicans were used. These strains were selected by S. This is because mutans is a well-established virulent cariogenic bacterium [Ajdic D et al, 2002]. S. gordonii is a pioneering engraftment of dental biofilms, and A. naeslundii has also been detected in the early stages of dental biofilm formation and may be associated with the development of root caries [Dige I et al, 2009]. C. albicans is a fungal organism that colonizes the mucosal surface of humans and is also detected in the dental plaque of young children with caries in early childhood [Hajeshengalis E et al, 2015]. All strains were stored at -80°C in trypsin soy broth containing 20% glycerol. S. mutans, S.M. gordonii, A.; Blood agar plates were used for culturing naeslundii. C. Sabouraud agar plates were used for Albicans. All of these strains, with to the middle exponential phase _{(37 ° C, 5% CO} 2) 1% glucose prior to use, ultrafiltration (10 kDa molecular weight cutoff membrane; Prep / Scale, Millipore, MA) in Grown in buffered tryptone yeast extract broth (UFTYE; 2.5% tryptone and 1.5% yeast extract, pH 7.0).

Generation of Plastomic Lines Expressing Different Tagged GFP Fusion Proteins Transplastomic plants expressing GFP fused with CTB, PTD, retrocyclin and protegrin were generated as described in previous studies [Limaye. Kal et al 2013; Xiao et al 2016; Lee et al 2011]. A transplastomic strain expressing the GFP fusion protein was confirmed using the Southern blot assay as previously described [Verma et al 2008]. Expression of GFP-tagged protein was also confirmed by visualizing green fluorescence from the leaves of each construct under UV illumination.

Purification of tag-fused GFP protein Purification of GFP-fused protegrin-1 (PG1) and retrocyclin (RC101) from transplastomic tobacco was achieved by hydrophobic chromatography previously performed following organic extraction (Lee et. al, 2011). About 0.2-1 g of freeze-dried leaf material was taken and 10-20 ml of plant extraction buffer (0.2 M Tris HCl pH 8.0, 0.1 M NaCl, 10 mM EDTA, 0.4 M sucrose, 0. Reconstituted with 2% Triton X, 2% phenylmethylsulfonyl fluoride and one protease inhibitor cocktail). The resuspension was incubated in ice for 1 hour and vortex homogenized every 15 minutes. The homogenate was then spun down at 45000C and 75000g for 1 hour (Beckman LE-80K optima ultracentrifuge) to obtain a clarified lysate. The lysate was pretreated with 70% saturated ammonium sulfate and 1/4 volume of 100% ethanol, followed by vigorous shaking for 2 minutes (Yakhnin et al, 1998). The treated solution was spun down at 2100 g for 3 minutes. The upper ethanol phase was collected and the process was repeated using 1/16 volume of 100% ethanol. The pooled ethanolic phases were further treated with 1/3 volume of 5M NaCl and 1/4 volume of 1-butanol, vigorously homogenized for 2 minutes and centrifuged at 2100 g for 3 minutes. The bottom phase was collected, loaded onto a 7 kDa MWCO zeba spin desalting column (Thermo Scientific) and desalted according to the 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 onto a Toyopearl butyl-650S hydrophobic interaction column (Tosoh bioscience) run on an FPLC unit (Pharmacia LKB-FPLC system). The column was equilibrated with 2.3 column volumes of salt buffer (10 mM Tris-HCl, 10 mM EDTA and 50% saturated ammonium sulphate) to eventually saturate 20% salt and facilitate binding of GFP to the resin. did. This was followed by a column wash with 5.8 column volumes of salt and salt-free buffer mixture, followed by elution with salt-free buffer (10 mM Tris-HCl, 10 mM EDTA). The GFP fraction was identified and collected based on the peaks observed in the chromatogram. The collected fractions were subjected to a final polishing step by overnight dialysis. After dialysis, the purified protein was lyophilized (labconco lyophilizer) to concentrate the final product and stored at -20°C.

Quantification of purified GFP fusion Quantification of GFP-RC101 and GFP-PG1 was performed by both Western blotting and fluorescence. The lyophilized purified protein was resuspended in sterile 1X PBS and total protein determined by the Bradford method. Next, the purified protein was quantified by SDS-PAGE method by loading a sample of denatured protein on a 12% SDS gel with a commercial GFP standard (Vector labs), and diluted 1:3000 mouse Anti-GFP antibody (Millipore). ), then probed with a 1:4000 dilution of secondary HRP-conjugated goat anti-mouse antibody (Southern biotech).

Purified protein was also quantified using GFP fluorescence. 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. To obtain a standard curve, GFP concentration was determined in both Western and plain fluorescence methods by densitometry analysis using Image J software with a commercial GFP standard. Purity was determined based on GFP quantification of total protein values determined by Bradford method.

Uptake of purified tag-fused GFP protein by human periodontal cell line As described above (Xiao, et al 2016), human periodontal ligament stem cells (HPDLS), maxillary mesenchymal stem cells (MMS), human head and neck squamous cell carcinoma cells (SCC-1), gingival-derived mesenchymal stromal cells (GMSC), adult gingival keratinocytes (AGK) and osteoblasts (OBC), including four tags CTB, PTD, PG1 and in different human periodontal cell lines. To determine RC101 uptake, in brief, each human cell line cell (2×10 ⁴ ) was incubated overnight at 37° C. on an 8-well chamber slide (Nunc) followed by incubation with a purified GFP fusion tag. Were performed; CTB-GFP (8.8 μg), PTD-GFP (13 μg), GFP-PG1 (1.2 μg) and GFP-RC101 (17.3 μg) in 100 μl PBS supplemented with 1% FBS at 37° C. for 1 hour. ) I went there. After fixing with 2% paraformaldehyde for 10 minutes at RT and washing 3 times with PBS, all cells were stained with anti-fading mounting medium of DAPI (Vector Laboratories, Inc). For negative control, cells were incubated with commercial GFP (2 μg) in PBS with 1% FBS for 1 hour at 37°C. All fixed cells were imaged using a confocal microscope. Images were viewed under a 100× objective and three independent analyzes recorded at least 10-15 GFP-positive cells for each cell line.

Evaluation of antibacterial activity S. The killing kinetics of AMP (Gfp-PG1 and Gfp-RC101) against mutans was analyzed by a time-lapse killing assay. S. Mutans were grown to exponential phase and diluted to 10 ⁵ CFU/ml in growth medium. GFP-PG1 and GFP-RC101 were added to S. cerevisiae at concentrations of 0 to 10 μg/ml and 0 to 80 μg/ml, respectively. mutans suspension. Samples were taken at 0, 1, 2, 4, 8, and 24 hours, serially diluted with 0.89% NaCl, then spread on agar plates and colonies were counted after 48 hours. The absorbance at 600 nm was also checked at each time point. S. gordonii, A.; naeslundii, and C.I. albicans suspension was mixed with Gfp-PG1 at a concentration of 10 μg/ml, and aliquots were removed for CFU enumeration at 0, 1, and 2 hours.

Also, S. The effect of AMP on mutans cell viability was assessed by time-lapse measurement. S. Mutans were grown to exponential phase, harvested by centrifugation (5500 g, 10 min), the pellet washed once with sodium phosphate buffered saline (PBS) (pH 7.2) and resuspended in PBS, 1 The final concentration was adjusted to ×10 ⁵ CFU/ml. GFP-PG1 was transformed into S. The mutans suspension was added at a concentration of 10 μg/ml and 2.5 μM propidium iodide-PI (Molecular Probe Inc., Eugene, Oreg., USA) was added for dead cell labeling. For confocal imaging, 5 μl of the mixture was loaded on an agarose pad. Confocal images were acquired using a Leica SP5-FLIM inverted single photon laser scanning microscope equipped with a 100× (numerical aperture 1.4) oil immersion objective. Excitation wavelengths were 488 nm and 543 nm for GFP and PI, respectively. The GFP emission filter was a 495/540 OlyMPFC1 filter and the PI was a 598/628 OlyMPFC2 filter. In the time-lapse series, images of the same field of view were taken at 0, 10, 30, and 60 minutes and were created by ImageJ 1.44 (http://rsbweb.nih.gov/ij/download.html).

S. AMP treated with AMP. Morphological observations of mutans were also examined by scanning electron microscopy (SEM). S. Mutans were grown to exponential phase and diluted to 10 ⁵ CFU/ml in PBS. The bacterial suspension was mixed with GFP-PG1 (final concentration 10 μg/ml) for 1 hour at 37°C. After treatment, the bacteria were harvested by filtration using a 0.4 μm Millipore filter. The deposits were fixed with 2.5% glutaraldehyde and 2.0% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4) for 1 hour at room temperature and SEM (Quanta FEG 250, FEI, Hillsboro, Oregon). State) for observation. Only untreated or buffer treated bacteria served as controls.

Evaluation of anti-biofilm activity S. Biofilms were formed on saliva-coated hydroxyapatite disc surfaces using a well-characterized EPS matrix that produces mutans UA159. Briefly, hydroxyapatite discs (1.25 cm diameter, 2.7 ± 0.2 cm ² surface area, Clarkson, Chromatography Products, Inc., South Williamsport, PA) are filter sterilized for clear human saliva (sHA). ) Coated [Xiao J et alo. , 2012]. S. Mutans was grown to mid-exponential phase (37° C., 5% CO ₂ ) in UFTYE medium containing 1% (w/v) glucose. Each sHA disc contained 10 ⁵ CFU/ml of actively growing S. cerevisiae in UFTYE medium containing 1% (w/v) sucrose. mutans cells were inoculated and incubated at 37° C., 5% CO ₂ for 19 hours. Prior to inoculation, sHA discs were topically treated with GFP-PG1 solution (10 ug) for 30 minutes. The inhibitory effect of GFP-PG1 treatment on 3D biofilm architecture was observed via confocal imaging. Briefly, 2.5 μM Alexa Fluor 647 labeled dextran conjugate (10 kDa; 647/668 nm; Molecular Probes Inc.) was used to label EPS, while bacterial cells were labeled with 2.5 μM SYTO 9 (485/485/. 498 nm; Molecular Probes Inc.). Imaging was performed using a Leica SP5 microscope equipped with a 20× (numerical aperture, 1.00) water immersion objective. The excitation wavelength was 780 nm, the emission wavelength filter of SYTO 9 was a 495/540 OlyMPFEC1 filter, while the filter of Alexa Fluor 647 was a HQ655/40M-2P filter. The confocal image series was generated by optical sectioning at each selected position and the z-series scan step size was 2 μm. Amira 5.4.1 software (Visage Imaging, San Diego, CA) was used to create a 3D rendering of the biofilm architecture [Xiao J et al. 2012, Koo H et al. 2010].

19 hours S.S. on sHA disk. In order to investigate the effect of PG1 on the biofilm formed by mutans, 1) control, 2) EPS degrading enzyme only, 3) PG1 only, or 4) PG1 and EPS degrading enzyme were biofilm incubated for up to 60 minutes. Later, a time-lapse confocal microscope was used to investigate the 3D architecture of the EPS matrix, and in situ cell viability. The EPS degrading enzymes used here are dextranase and mutanase, and by hydrolyzing the α-1,6 glucoside bond and the α-1,3 glucoside bond, S. The EPS from mutans could be digested [Hayacibara et al. 2004]. Dextranase Produced from Penicillium sp. Commercially purchased from Sigma (St. Louis, Mo.) and produced from Trichoderma harzianum is a mutanase produced by Dr. William H. It was kindly provided by Bowen (Center of Oral Biology, University of Rochester Medical Center). Dextranase and mutanase were mixed in a ratio of 5:1 before application to biofilm [Mitsuue F. et al. 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 and dead cells. Biofilms were examined using confocal fluorescence imaging at 0, 10, 30, and 60 minutes 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 viable cells to total bacteria at each time point was calculated and the viability of viable cells (relative to viable cells at 0 minutes) was plotted. The initial number of viable cells at 0 minutes was considered 100%. Survival was determined by comparison to the 0 minute time point.

Microbiological Assay At selected time points (19 hours), biofilms were removed, homogenized by sonication, and microbiologically analyzed as detailed previously [Xiao J et al. 2012, Koo H et al. 2010]. Our sonication procedure provides optimal dispersion and maximum recoverable count without killing bacterial cells. Aliquots of the biofilm suspension were serially diluted and plated on blood agar plates using a spray vortex Jet Spiral Plater (IUL, SA, Barcelona, Spain). On the other hand, propidium monoazide (PMA) in combination with quantitative PCR (PMA-qPCR) was transformed into S. cerevisiae as described. It was used for analysis of viability of mutans cells. [Klein MI et al. 2012]. Since PMA and extracellular DNA cross-linked to the DNA of dead cells modify the DNA and inhibit PCR amplification of the extracted DNA, the combination of PMA and qPCR results in cells with intact membranes (ie, live cells). Only quantify. Briefly, biofilm pellets were resuspended in 500 μl TE (50 mM Tris, 10 mM EDTA, pH 8.0). The biofilm suspension was transferred to a 1.5 ml microcentrifuge tube using a pipette. Then mixed with PMA. 1.5 μl PMA (20 mM in 20% dimethylsulfoxide; Biotium, Hayward, CA) was added to the biofilm suspension. The tubes were incubated in the dark for 5 minutes at room temperature with occasional mixing. The sample was then exposed to light for 3 minutes (600W halogen light source). After photoinduced crosslinking, the biofilm suspension was centrifuged (13,000 g/10 min/4° C.) and the supernatant was discarded. The pellet was resuspended in 100 μl TE and then incubated with 10.9 μl lysozyme (100 mg/ml stock) and 5 μl mutanillidine (5 U/μl stock) (37°C/30 min). Genomic DNA was then isolated using the MasterPure DNA Purification Kit (Epicenter Technologies, Madison, WI). Ten pictograms of genomic DNA per sample and negative control (no DNA) were collected from iQ SYBR Green supermix (Bio-Rad Laboratories Inc., CA) and S. Amplified by MyiQ real-time PCR detection system using mutans specific primer (16S rRNA) [Klein MI et al 2010].

Statistical analysis Data are expressed as mean ± standard deviation (SD). All assays were performed in duplicate in at least 2 different experiments. Student's t-test was used to make pairwise comparisons of test and control. The level of significance selected for all statistical tests in this study was P<0.05.

The following examples are provided to illustrate particular embodiments of the invention. They are not intended to limit the invention in any way.

Example I
Generation and characterization of transplastomic strains All fusion tags (CTB, PTD, protegrin, retrocyclin) were fused to green fluorescent protein (smGFP) at the N-terminus to assess efficiency and specificity. Fusion constructs encoding these fusion proteins have been cloned into chloroplast transformation vectors, which are then described in US patent application Ser. No. 13/101,389, which is hereby incorporated by reference. Was used to transform the desired plant. To make plants expressing the GFP fusion protein, tobacco chloroplasts were transformed using a biolistic particle delivery system. As seen in FIG. 1B, each tag-fused GFP is driven by the same regulatory sequence-psbA promoter and a 5'UTR that is light regulated, and the transcribed mRNA is stabilized by the 3'psbA UTR. Since the psbA gene is the most highly expressed chloroplast gene, psbA regulatory sequences are used in our laboratory to express transgenes [7,34]. To facilitate integration of the expression cassette into the chloroplast genome, two flanking sequences, the isoleucyl-tRNA synthetase (trnI) and alanyl-tRNA synthetase (trnA) genes, flank the same expression cassette as the native chloroplast genome sequence. doing. Sprouts from the selection medium were investigated for specific integration of the transgene cassettes in the trnI and trnA spacer regions, followed by transformation of all chloroplast genomes in each plant cell (of the untransformed wild-type chloroplast genome). (Absence) was confirmed by Southern blot analysis. Therefore, stable integration of all GFP expression cassettes with chloroplast genome homoplasmy and transgenes was confirmed prior to extraction of the fusion protein. In addition, GFP expression was phenotypically confirmed by visualizing green fluorescence under UV light. Then, the confirmed homoplasmic line was transferred and cultured in an automatic greenhouse to increase the biomass.

To scale up the leaf material biomass of each GFP-tagged plant, each homocytoplasmic line was grown in a temperature and humidity controlled greenhouse. Full-grown mature leaves were harvested late in the evening to maximize accumulation of GFP fusion protein driven by light-regulated regulatory sequences. To further increase the content of fusion protein on a weight basis, frozen leaves were lyophilized under vacuum at -40°C. In addition to the protein concentration effect, freeze-drying extended the shelf life of the plant expressed therapeutic protein at room temperature for over a year [Daniell et al 2015; 2016]. Therefore, in this study, lyophilized and powdered plant cells expressing the GFP fusion tag protein were used for oral delivery to mice.

Expression and Purification of GFP Fusion Antibacterial Peptide from Transplastomic Tobacco Tobacco leaves expressing the GFP fusion antibacterial peptides RC101 and PG1 were harvested from the greenhouse and then lyophilized for protein extraction and purification. The average expression level of GFP-RC101 was found to be 8.8% of total protein in the crude extract, while GFP-PG1 expression was 3.8% of total protein based on densitometry. The differences in expression levels were similar to those previously reported (Lee et al 2011, Gupta et al, 2015).

Purification of GFP fused to different antimicrobial peptides (RC101 and PG1) was performed using plankton and biofilm-forming S. was performed to verify bactericidal activity against both mutans. Lyophilized tobacco material expressing different GFP fusions was used for extraction and subsequent downstream processing (see FIG. 8) to obtain the finished purified product, which was subsequently used to determine the concentration of GFP fusion peptide. Quantitatively. Quantification of purified GFP-RC101 and GFP-PG1 was performed by both Western blot and native GFP fluorescence. The purified GFP-RC101 showed an average purity of 94% with an average yield of 1624 μg GFP (116 μg RC101 peptide) per gram of lyophilized leaf material (FIGS. 1A and 1B). For GFP-PG1, both methods (FIGS. 1C and 1D) showed an average purity of 17% with an average yield of 58.8 μg GFP (4.2 μg, PG1 peptide) per gm of lyophilized leaf material. The differences in purity may be due to different types of tags fused to GFP, as seen in previous studies (Xiao et al 2015, Skosyrev et al 2003). The fold-concentration of purified GFP-RC101 and GFP-PG1 from plant extracts was 10.6 and 4.5, respectively. Western blots also show a 27 kDa GFP standard. This corresponds to a monomeric fragment with a 54 kDa GFP dimer with a load in the range of 6-8 ng GFP. In the GFP-RC101 Western blot, the 29 and 58 kDa fragments were clearly visible, corresponding to the monomeric and dimeric forms of the fusion (FIG. 1A). This may be due to the ability of GFP to form dimers (Ohashi et al, 2007). A Western blot of GFP-PG1 (FIG. 1D) shows that the 29 kDa monomer bound to a 40 kDa fragment along with other non-specific plant proteins that may have been co-purified as previously described. It is clearly shown that there is a possibility that the mobility may be shifted by (Morasuttia et al 2002). The native fluorescence of GFP-RC101 and GFP-PG1 (FIGS. 1B and 1D) shows that GFP is electrophoretic mobility at each GFP fragment described in previous studies because GFP is fused to a cationic peptide. It shows a multimeric band visible below the 27 kDa GFP standard size that may be causing the shift (Lee et al, 2011).

Antimicrobial activity of AMP As shown in Figure 2 (AE), we first examined the antimicrobial activity of GFP-PG1 using a dose response time-killing study. GFP-PG1 exhibits potent antibacterial activity against Streptococcus mutans, a proven biofilm-forming and dental caries-causing pathogen, and rapidly kills bacterial cells within 1 hour at low concentrations (FIG. 2A). GFP-PG1 also killed the early oral colonization bacteria Streptococcus gordonii and Actinomyces naeslundii, but showed limited antifungal activity against Candida albicans at the concentrations tested (FIG. 2E). Time-lapse confocal imaging is described by S. It shows that mutans viability is affected at 10 minutes as shown in FIG. 3A compared to untreated controls (FIG. 3B). By SEM imaging, S. Mutant membrane surface disruption was revealed, leading to intracellular content extrusion and irregular cell morphology, while untreated bacteria remained pristine and smooth without any visible cell lysis or debris. It showed a smooth surface (Fig. 3C). S. Having shown the antibacterial effect of GFP-PG1 on mutans, the possibility of preventing the biofilm formation of this antimicrobial peptide or the destruction of the preformed biofilm was investigated.

Inhibition of Biofilm Initiation by AMP It is difficult to prevent the formation of pathogenic oral biofilms because the drug must exert a therapeutic effect after topical application. To determine whether GFP-PG1 could interfere with the initiation of biofilms, saliva coated apatite (sHA) surfaces (tooth surrogates) were treated with a single topical treatment of GFP-PG1 for 30 minutes and then actively. Growing S. Incubated with mutans cells under cariogenic (sucrose-rich) conditions. S. cerevisiae with minimal accumulation of EPS matrix on GFP-PG1 treated sHA surface. Substantial impairment of biofilm formation by mutans was observed (FIGS. 4B and 4C). A small number of adherent cell clusters were almost non-viable compared to controls (FIG. 4A), indicating a strong effect of GFP-PG1 on the initiation of biofilm despite local short-term exposure.

Disruption of preformed biofilm with AMP with or without EPS-degrading enzyme Disruption of biofilm formed on the surface is difficult. Destruction of cariogenic biofilms is particularly important because the drug is often unable to reach clusters of pathogenic bacteria (such as S. mutans) because it incorporates and protects the surrounding exopolysaccharide (EPS)-rich matrix. Difficult [Bowen and Koo, 2011]. EPS-degrading enzymes such as dextranase and mutanase have no antibacterial effect but help digest the matrix of cariogenic biofilms. First, dextranase and/or mutanase required for EPS matrix disruption were optimized without affecting cell viability (data not shown). As shown in Figure 5, the combination of dextranase and mutanase digests the "open space" (see arrow) between EPS (red) and bacterial cell clusters (green) and "uncovered" cells (see arrow). it can. Therefore, the combination of GFP-PG1 and EPS degrading enzyme synergistically enhances the overall anti-biofilm effect.

To explore this concept, Streptococcus mutans biofilms were pre-formed on the sHA surface and treated locally with GFP-PG1 and EPS degrading enzyme (Dex/Mut) alone or in combination. Time-lapse confocal imaging and quantitative computational analysis were performed to analyze EPS matrix degradation and live/dead cells within the biofilm (FIG. 6A). The combination of enzyme and peptide degraded the EPS matrix by more than 60%, but increased bacterial killing when compared to GFP-PG or Dex/Mut alone. These findings were further validated via standard culture assays by determining colony forming units. S. cerevisiae combined with Dex/Mut. The antibacterial activity of PG against mutans biofilm was significantly enhanced over either alone. Local exposure of Dex/Mut alone had no effect on the viability of biofilm cells, whereas GFP-PG-1 alone showed limited killing activity (Fig. 6B). Together, the data demonstrate the potential of this combinatorial approach to synergistically enhance the antimicrobial effect of GFP-PG-1 on established biofilms (FIG. 6C).

Uptake of GFP fused with different tags by human periodontal cells.

The purified GFP fusion protein when incubated with human cultured cells including HPDLS, MMS, SCC-1, GMSC, AGK and osteoblasts (OBC) revealed interesting results. Although only one representative image for each cell line is presented, uptake studies were performed in triplicate and at least 10-15 images were recorded under a confocal microscope. Without the fusion tag, GFP did not enter the human cell lines tested. CTB-GFP and PTD-GFP effectively penetrated all cell types tested, but differed in their localization patterns. Upon incubation with CTB-GFP, GFP signals were mainly localized around HPDLSCs and MMSCs, uniform small cytoplasmic spots of SSC-1, large cytoplasmic foci of AGKs, OBCs and GMSCs. PTD-GFP was observed as small cytoplasmic foci of MMSC, HPDLSC, GMSC, AGK, OBC, and cytoplasmic spots of varying size around the cytoplasm and SCC-1 cells. PG1-GFP is the most efficient tag for entry into all human cells tested, as GFP can localize at a 10-fold lower concentration than any other fusion protein. PG1-GFP showed cytoplasmic localization only in HPDLSC, SCC-1, GMSC and AGK cells, and was localized both in the periphery and cytosol in MMSC, but only in the periphery of OBC. RC101-GFP was localized to SCC-1, GMSC, AGK and OBC, but its localization in HPDLSC was negligible and could not be detected in MMSC cells.

Discussion and Conclusions The assembly of cariogenic oral biofilms is a representative example of how pathogenic bacteria accumulate on the surface (tooth) as the extracellular EPS matrix develops. Prevention of cariogenic biofilm formation requires destruction of bacterial accumulation on the tooth surface by topical treatment. Chlorhexidine (CHX) is regarded as the "gold standard" for topical antibacterial therapy (Flemmig and Beikler 2011; Marsh et al 2011; Caufield et al 2001). CHX effectively suppresses mutans streptococcal levels in saliva, but has adverse side effects such as tooth staining and stone formation and is not recommended for daily prophylactic or therapeutic use (Auto-Gold 2008) .. Alternatively, several antimicrobial peptides (AMPs) have been developed and tested against oral bacteria, showing potential effects on biofilms (effects are diminished on plankton cells). (Review by Silva et al., 2012). Unfortunately, these studies tested the efficacy of anti-biofilms using continuous and prolonged biofilm exposure to AMPs (several hours), rather than a clinically used topical treatment regimen. .. Moreover, synthetic AMPs are expensive to manufacture and are not suitable for dental applications. Here we show plant-made AMP. This demonstrates the potent effect of controlling biofilm formation with a single short-term topical treatment of the surrogate surface of the tooth.

The cariogenic biofilm developed is characterized by bacteria embedded in an EPS matrix, making biofilm processing and removal very difficult (Paes Leme et al 2006; Koo et al 2013). The EPS-rich matrix promotes microbial attachment, aggregation, protection and prevents diffusion (Koo et al 2013; Flemming and Wingender 2010). EPS matrix creates spatial and microenvironmental heterogeneity in the biofilm, locally regulates pathogen growth and protection against antimicrobial agents, and firmly adheres the biofilm to the tooth surface Scaffolding (Flemming and Wingender 2010; Peterson et al. 2015). CHX is much less effective on cariogenic biofilms formed (Hope and Wilson, 2004; Van Strydonck et al 2012; Xiao et al., 2012). EPS is mainly composed of a mixture of insoluble (high content of α1,3-bond glucose) and soluble (mainly α1,6-bond glucose) glucan (Bowen and Koo 2011). Thus, the possibility of using EPS matrix-degrading dextranase or mutanase (of fungal origin) to disrupt biofilms and prevent caries was investigated and commercially available mouthwashes (eg, Biotene) were investigated. PBF). However, topical application of the enzyme alone produced a clinically moderate anti-biofilm/anti-caries effect, probably due to lack of antibacterial activity and reduced enzymatic activity in the mouth (Hull 1980) (Balakrishnan et al 2000). ). Interestingly, recent in vitro studies have shown that a chimeric glucanase composed of a fusion of dextranase and mutanase is more effective at disrupting plaque biofilms than either enzyme alone. (Jiao et al 2014). However, the approach of combining antimicrobial agents and both EPS matrix-degrading enzymes into a single therapeutic system has not yet been developed, probably due to cost and formulation-related difficulties. This study demonstrates that PG1 and matrix-degrading enzymes act synergistically and effectively to destroy cariogenic biofilms. This feasible and effective topical anti-biofilm approach can simultaneously degrade the scaffold of the biofilm matrix and use antimicrobial peptides in combination with EPS digestive enzymes to kill the embedded bacteria.

Retention of high levels of antibacterial activity by the fusion of protegrin and GFP opens the door to many clinical applications to improve oral health beyond biofilm disruption. In addition to biofilm disruption, enhancing wound healing of gingival tissue is an important clinical need. We have recently reported that both protegrin and retrocyclin can enter human mast cells and induce degranulation, an important step in the wound healing process (Gupta et al 2015). Therefore, the antimicrobial peptides protegrin and retrocyclin play an important role in killing biofilm bacteria and initiate wound healing through mast cell degranulation. In addition, it is important to effectively deliver growth hormone or other proteins to enhance cell adhesion and stimulate bone formation, angiogenesis, bone regeneration, osteoblast or endothelial cell differentiation. The cell-penetrating peptides previously identified have some limitations. CTB enters all cell types via the ubiquitous GM1 receptor, which requires the pentameric form of CTB. On the other hand, PTD does not invade immune cells (Xiao et al 2016).

In this study, the ability of PG1-GFP or RC101-GFP to enter periodontal and gingival cells was tested. PG1-GFP is the most efficient tag for periodontal or gingival human cell entry because it can detect GFP signals at concentrations 10 times lower than other fusion proteins. PG1-GFP effectively entered HPDLSC, SCC-1, GMSC, AGK, MMSC, OBC, although there was some variation in intracellular localization. In contrast, RC101-GFP entered SCC-1, GMSC, AGK and OBC, but its localization in HPDLSC and MMSC cells was poor or undetectable. Therefore, this study identified a new role for protegrin in delivering drugs to osteoblasts, periodontal ligament cells, gingival epithelial cells, or fibroblasts to improve oral health. It is possible to release the protein drug synthesized in plant cells by mechanical milling, and the protein drug bioencapsulated in freeze-dried plant cells embedded in chewing gum is for long and slow sustained release. To provide an ideal drug delivery mode of This overcomes the major limitation of current mouthwashes, the brief contact of the antibacterial agent on the gum/tooth surface.

Beyond topical application, protein drugs fused with plant cell-expressed protegrin can be delivered orally to the deeper layers of gingival tissue in a non-invasive manner, improving patient compliance. Plant-bioencapsulated protein drugs can be stored for many years at room temperature without loss of potency (Su et al 2015; Daniell et al 2016). The high cost of current protein pharmaceuticals is due to exorbitantly expensive fermentor production, purification, refrigerated shipping/storage, short shelf life, and aseptic delivery methods. All of these challenges can improve oral health using this new concept of drug delivery. The recent FDA approval of plant cells for the production of protein pharmaceuticals (Walsh 2014) is on track for the clinical advancement of this new concept.

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Example II
Generation of AMP-expressing chloroplast vectors, biofilm-degrading enzymes and their fusion proteins Effective treatment of infectious diseases associated with biofilms reaches clusters of microorganisms where antimicrobial agents reside within the surrounding extracellular matrix This is often a problem because you can't do that and they are captured and protected. Furthermore, the development of new treatments and maintenance of oral hygiene for biofilm-related oral diseases need to be cost-effective and easily accessible.

To ensure a continuous supply of reagents, dextranase/mutanase and protegrin/retrocyclin are independently expressed as chloroplast fusion proteins in other plants such as tobacco and lettuce. Proteins are produced and used in low cost purification strategies. Since tobacco plants produce 1 million seeds, production can be easily expanded. Each acre of tobacco produces up to 40 metric tons of biomass, facilitating low-cost, large-scale production of AMP, enzyme, and the fusion construct encoding it. In another approach, proteins are produced in edible plants such as lettuce.

Several dextranases (Dex) and mutanases (Muts) have been isolated from fungi and bacteria and characterized for their enzymatic activity. Optimal dextranase and mutanase enzymes should have enzymatic properties suitable for the human oral environment. Based on short-term oral treatment, strong binding/retention properties to plaque biofilms and catalytic activity to both types of EPS (dextran and mutans) are highly desirable. Enzymes added to commercially available dextranase-containing mouthwashes (such as bioten) are mainly derived from fungi (Penicillium spp. and Chetium elaticum). However, fungal dextranase exhibits optimum values at higher temperatures (50-60°C) than bacterial dextranase (35-40°C). In addition, bacterial dextranase is more stable and effective at oral temperature (about 37°C) and is suitable for caries prevention. Recently, the dextranase of Arthrobacter sp strain Arth410 has been found to have optimum temperature (35-45°C) and pH values (pH 5-7) found in oral and cariogenic biofilms as compared to fungal dextranase. Showed excellent dextran degradation characteristics. Moreover, topical application of the bacterial dextran enzyme is more effective in reducing caries in vivo than the fungal dextran. Similarly, Paenibacillus sp. The bacterial mutanases of strain RM1 show that the biofilms were effectively degraded by the 6 hour incubation even after removal of the mutanases, which was preceded by a 3 minute initial incubation with the biofilms. Also, RM1 mutanases show improved biofilm degradation properties when compared to other microbial species. In particular, fungal enzymes require glycosylation, which prevents their expression in chloroplasts. Furthermore, the immunogenicity of glycoproteins in the human system may further raise regulatory concerns. Thus, the invention includes the use of bacterial dextranases and mutanases for chloroplast expression.

Both sequences have been codon-optimized for chloroplast expression in order to increase the production of Arth410 dextranase and RM1 mutanase proteins in chloroplasts. See Figures 9A and 9B.

To maximize retrocyclin and protegrin synthesis and reduce toxicity of AMPs, ten tandem repeats of PG1 or RC101 separated by a protease cleavage site are used, as shown in FIG. For each copy of the expressed gene, 10 functional copies of PG1 or RC 101 are made. For this purpose, Tobacco Etch Virus (TEV) protease with high specificity and a short cleavage site of 7 amino acids was selected. Alternatively, a furin cleavage site can be used. The vector can also be designed to include a nucleic acid encoding a biofilm degrading enzyme. The coding region can be expressed under the promoter utilized to express AMP or can be linked to a vector operably linked to a second promoter region. The biofilm degrading enzyme coding sequence may include a TEV protease cleavage site to facilitate release of the enzyme. This approach offers a safer and cleaner option than the chemical cleavage method. Most importantly, the individual PG1 peptides in the fusion protein do not form secondary structures prior to cleavage, thereby avoiding accumulation of functional peptides that can be fatal to the host production system. .. The cleaved PG1/RC101 antibacterial activity, biofilm degrading enzymes or fusion proteins thereof can be used to degrade biofilms using the methods disclosed in Example I.

As mentioned above, the sequence encoding the AMP/biofilm degrading enzyme is optionally codon optimized prior to insertion into a chloroplast transformation vector such as pLD. Transformation of chloroplasts is dependent on a double homologous recombination event. Therefore, the chloroplast vector contains a region of homology with the chloroplast genome that flanks the expression cassette encoding the heterologous protein of interest, which does not disrupt the functional gene and thus the intergenic spacer region of the chloroplast genome. Facilitate the insertion of the transgene cassette into. Although any intergenic spacer region can be used for transgene insertion, the most commonly used transgene integration site is the trnI-trnA gene (within the rrn operon) located within the IR region of the chloroplast genome. It is a transcriptionally active intergenic region between (FIG. 10). Since Escherichia coli and chloroplast have similar protein synthesis mechanisms, codon optimization efficiency can also be evaluated in E. coli, and thus plants can be created. Both systems can be used to purify and evaluate the expression of AMPs, biofilm degrading enzymes, or fusion proteins thereof, and AMPs or enzyme activity.

Purification strategy A hydrophobic interaction column (HIC; TOSOH Butyl Toyopearl 650m) can be used to purify PG1 fused with Green Florescent Scientine (GFP). GFP selectively binds to HIC, making Rc101/PG1 purity >90%. Despite the use of expensive HIC chromatography methods, the recovery is very low (<20%). To address this issue and increase yield, 10 tandem repeats of elastin-like biopolymer (GVGVP (SEQ ID NO: 11), Figure 10) and PG1 have been incorporated into the vector. This biopolymer has the unique thermal property of precipitating out of solution when raised above the reverse transition temperature (Tt). GVGVP remains in the soluble monomer state below Tt and forms insoluble aggregates above Tt. This phase transition from the dissolved state to the insoluble state is reversible by changing the temperature of the solution, which facilitates protein purification. The fusion protein is then resolubilized by cooling below Tt to remove any co-precipitated insoluble contaminants, as shown in FIG. The process of heating (37°C) and cooling (4°C) is known as inverse transition cycling (ITC), and 3-5 rounds of ITC can be performed to obtain highly pure protein (purity >98%, (Fig. 11).

In another approach, the signal peptide is fused to dextranase or mutanase for expression in E. coli, where the signal peptide results in secretion of the enzyme into the extracellular medium. Furthermore, secreted proteins have to cross the two membrane system of E. coli, passing through the periplasmic environment in which the disulfide isomerase, folderase and chaperone reside. Thus, the correct folding of secreted proteins and the formation of disulfide bonds are promoted by enzymes, which enhances the biological activity of proteins (optimal for AMPs). Another advantage of this production strategy is the low level of proteolytic activity in the medium that serves to enhance the stability of the recombinant protein. The signal sequence of the secreted protein is cleaved during the export process, creating a genuine N-terminus to the native protein. There are several molecules that help transfer proteins to the extracellular medium, such as the TAT, SRP, or SecB dependent pathways. However, rather than functioning independently, different pathways interact closely with each other. Pathways that depend on both SRP and SecB can work together in targeting a single protein. Also, under Sec deficiency conditions, translocation of Sec pathway substrates is rescued by the TAT system.

Out of a large number of signal sequences, outer membrane protein A (OmpA) and Seq X (derived from lac Z) signal peptides showed excellent export function, and the fusion protein was added to extracellular medium at a maximum of 4 g/L and 1 g/L, respectively. Can be exported. Therefore, these signal sequences are used to efficiently export Arth 410 Dex and RM1 Mut to the extracellular environment. Accumulation of dextranase and mutanase exported to the medium is determined by protein quantification and enzymatic assays.

Successful expression of these proteins in E. coli was achieved. See the Western blot results shown in FIG. Chloroplast vectors carrying these sequences collide with tobacco or lettuce leaves to produce plants capable of large-scale production of extranase/mutanase/AMP proteins. After harvesting large scale biomass, the leaves are lyophilized and stored at room temperature. In another approach, clinically proven caries-preventing compounds such as (250 ppm fluoride) and the broad spectrum fungicide chlorhexidine 0.12% can be evaluated to determine if these agents enhance efficacy.

The AMP-enzyme combination effectively disrupts the formation of cariogenic biofilms and the onset of cavitation in vivo. Moreover, the AMP-enzyme fusion protein appears to be superior to current chemotherapy for antimicrobial therapy and prevention of dental caries.

As described above, lettuce chloroplast-effective AMP enzymes (independently or in combination) can be expressed under the control of endogenous lettuce regulatory elements for large-scale GLP production and stability evaluation. it can. The main advantage is the reduction of production costs by eliminating exorbitantly expensive refining processes. Freeze-dried leaf material expressing AMP/enzyme can be stored at ambient temperature for months or years, while maintaining its integrity and functionality. See FIG. 13. In addition to long-term storage, increased protein drug concentration and reduced microbial contamination are other advantages. Lettuce leaves showed a 20-25 fold increase in protein drug concentration after lyophilization compared to fresh leaves, thus reducing the amount of material used for oral or topical delivery. After lyophilization, the plant material can be incorporated into chewing gum to deliver the biofilm degrading composition contained therein.

The steps for producing the AMP/enzyme or its fusion are shown in FIG. Lettuce chloroplast vectors useful for expressing the proteins of the invention have been previously described in US patent application Ser. No. 12/059,376, which is incorporated herein by this reference. For therapeutic proteins such as lettuce chloroplast proinsulin, up to 70% expression levels of total protein can be achieved using this system.

The AMP enzyme expressed in edible plants is preferably delivered orally (topically) when used for the treatment of oral diseases and the prevention and inhibition of caries formation. To enhance lysis of plant cells in the oral cavity, AMP/enzyme expressing plant cells express the cell wall degrading enzyme described in US patent application Ser. No. 12/396,382, which is incorporated herein by reference. Optionally mixed with plant cells.

The preparation of chewing gum tablets is shown in FIG. Using GFP as an example of the protein of interest, this data shows the amount of GFP that can be incorporated into chewing gum tablets. GFP levels were assessed by both fluorescence and Western blot. The results are shown in Fig. 15. The present inventors evaluated GFP release kinetics from gum tablets containing GFP using the chewing simulator and artificial saliva shown in FIG. 16. FIG. 17 shows a graph showing the release kinetics over time from gum tablets containing different amounts of GFP present in recombinant lettuce.

From these data it is clear that gum tablets containing the AMP enzyme fusion protein of the invention deliver the active agent for a suitable period of time to achieve bacterial killing and plaque or biofilm degradation. However, oral rinses such as Listerine® can also be used to deliver the AMP-enzyme fusion protein or combinations of the invention. FIG. 18 demonstrates that a crude extract containing the enzyme of the present invention mixed with LISTERINE mouthwash is as effective as a commercially produced and purified enzyme that is very expensive to prepare. To do. The data show that various combinations (both different ratios and amounts) of dual enzyme S. It shows that the biomass of mutans biofilm was significantly reduced in a dose-dependent manner. Of the various combinations, 25U Dex and 5U Mut (5:1, Dex:Mut ratio) were the most effective and provided over 80% of the total biomass degradation within 120 minutes. Further experiments confirmed that a Dex/Mut activity ratio of 5:1 showed the highest effect on both EPS degradation and bacterial killing by LISTERINE mouthwash. Excitingly, dual enzyme pretreatment dramatically enhanced the effect of LISTERINE mouthwash-mediated bacterial killing (3 log reduction relative to vehicle pretreatment and LISTERINE mouthwash). Inclusion of the third enzyme further improved overall anti-biofilm activity. Furthermore, the results of the mixed-species model show that the combination of dual enzymes not only enhances the overall antibacterial activity, but also when using the listerine mouthrinse after enzyme pretreatment (proportion of symbiotic/probiotic S. oralis S.). It was shown that a targeted reduction of mutans dominance could be induced. Therefore, the enzyme + LISTERINE mouthwash strategy is based on S. mutans, while selectively targeting the pathogen of mutans, There is a need to increase the proportion of oralis and thereby prevent microecological imbalances within mixed-species biofilms.

In addition to its antibacterial activity, AMPS has the ability to stimulate innate immunity and wound healing. In addition to its antibacterial properties, exploiting this new mast cell host defense function of AMPs will extend its clinical application. Biofilm-related caries is the most challenging model for the development of topical therapeutics. Once developed, such topical drug delivery can be readily adapted to other biofilms because matrix formation hinders efficacy in many other biofilm-related diseases. Applications are beyond oral biofilms because the matrix is unique to all biofilms. The biofilm inhibiting compositions described herein can also be used for coating stents, artificial joints, implants, valves, and other medical devices that are inserted into the human body for the treatment of disease.

As mentioned above, the AMP/enzyme, or leaves expressing it, can be incorporated into chewing gum for effective topical application to the chewing gum for the treatment of oral diseases. The composition can also be incorporated into an oral rinse such as a LISTERINE mouthwash. As mentioned above, other anti-dental agents such as fluoride and CHX may be included in such gums or oral rinses.

The references in Table 2 below describe many different mutanases from various biological sources. Each of these references is incorporated herein by this reference.

Additional biofilm degrading enzymes that encode sequences useful in the practice of the invention include, but are not limited to:

I) Paenibacillus humicus NA1123
See also http://www.ncbi.nlm.nih.gov/nuccore/AB489092 Genbank AB489092
Length: 1,146
Mass (Da): 119,007
Reference: Otsuka R, et al. Microbiol Immunol. 2015 Jan;59(1):28-36.
2. Paenibacillus humicus NA1123 Mutanase Protein Sequence
>gi|257153265|dbj|BAI23187.1| putative mutanase [Paenibacillus humicus]
MRIRTKYMNWMLVLVLIAAGFFQAAGPIAPATAAGGANLTLGKTVTASGQSQTYSPDNVKDSNQGTYWESTNNAFPQWIQVDLGASTSIDQIVLKLPSGWETRTQTLSIQGSANGSTFTNIVGSAGYTFNPSVAGNSVTINFSAASARYVRLNFTANTGWPAGQLSELEIYGATAPTPTPTPTPTPTPTPTPTPTPTVTPAPSATPTPTPPAGSNIAVGKSITASSSTQTYVAANANDNNTSTYWEGGSNPSTLTLDFGSNQSITSVVLKLNPASEWGTRTQTIQVLGADQNAGSFSNLVSAQSYTFNPATGNTVTIPVSATVKRLQLNITANSGAPAGQIAEFQVFGTPAPNPDLTITGMSWTPSSPVESGDITLNAVVKNIGTAAAGATTVNFYLNNELAGTAPVGALAAGASANVSINAGAKAAATYAVSAKVDESNAVIEQNEGNNSYSNPTNLVVAPVSSSDLVAVTSWSPGTPSQGAAVAFTVALKNQGTLASAGGAHPVTVVLKNAAGATLQTFTGTYTGSLAAGASANISVGSWTAASGTYTVSTTVAADGNEIPAKQSNNTSSASLTVYSARGASMPYSRYDTEDAVLGGGAVLRTAPTFDQSLIASEASGQKYAALPSNGSSLQWTVRQGQGGAGVTMRFTMPDTSDGMGQNGSLDVYVNGTKAKTVSLTSYYSWQYFSGDMPADAPGGGRPLFRFDEVHFKLDTALKPGDTIRVQKGGDSLEYGVDFIEIEPIPAAVARPANSVSVTEYGAVANDGKDDLAAFKAAVTAAVAAGKSLYIPEGTFHLSSMWEIGSATSMIDNFTVTGAGIWYTNIQFTNPNASGGGISLRIKGKLDFSNIYMNSNLRSRYGQNAVYKGFMDNFGTNSIIHDVWVEHFECGMWVGDYAHTPAIYASGLVVENSRIRNNLADGINFSQGTSNSTVRNSSIRNNGDDGLAVWTSNTNGAPAGVNNTFSYNTIENNWRAAAIAFFGGSGHKADHNYIIDCVGGS GIRMNTVFPGYHFQNNTGITFSDTTIINSGTSQDLYNGERGAIDLEASNDAIKNVTFTNIDIINAQRDGVQIGYGGGFENIVFNNITIDGTGRDGISTSRFSGPHLGAAIYTYTGNGSATFNNLVTRNIAYAGGNYIQSGFNLTIK (SEQ ID NO: 12)
3. Sequence of mRNA from Paenibacillus genus NA1123
>gi|257153264|dbj|AB489092.1|Paenibacillus humicus mut gene of putative mutanase, complete cd
1 aaaggaggat cgccaaccaa tcatcccagc aaagaaggtg atggcagccc aagaattgaa
61 agcgctttga atttggaata tacggatttg gccgacctgc tgattcagtc gtattcaagc
121 gattatgccg cgaaccaatc gaacccgagg aggactataa tgcgtatccg cactaaatat
181 atgaactgga tgttggtgct cgtcctgatc gccgccggct tcttccaggc tgccggcccc
241 atcgctcccg ccaccgctgc aggaggcgcg aatctgacgc tcggcaaaac cgtcaccgcc
301 agcggccagt cgcagacgta cagccccgac aatgtcaagg acagcaatca gggaacttac
361 tgggaaagca cgaacaacgc cttcccgcag tggatccaag tcgaccttgg cgccagcacg
421 agcatcgacc agatcgtgct caagcttccg tccggatggg agactcgtac gcaaacgctc
481 tcgatacagg gcagcgcgaa cggctcgacg ttcacgaaca tcgtcggatc ggccgggtat
541 acattcaatc catccgtcgc cggcaacagc gtcacgatca acttcagcgc tgccagcgcc
601 cgctacgtcc gcctgaattt cacggccaat acgggctggc cagcaggcca gctgtcggag
661 cttgagatct acggagcgac ggcgccaacg cctactccca cgcctactcc aacaccaacg
721 ccaacgccaa caccaacgcc aacccctaca gtaacccctg cgccttcggc cacgccgact
781 ccgactcctc cggcaggcag caacatcgcc gtagggaaat cgattacagc ctcttccagc
841 acgcagacct acgtagctgc aaatgcaaat gacaacaata catccaccta ttgggaggga
901 ggaagcaacc cgagcacgct gactctcgat ttcggttcca accagagcat cacttccgtc
961 gtcctcaagc tgaatccggc ttcggaatgg gggactcgca cgcaaacgat ccaagttctt
1021 ggagcggatc agaacgccgg ctccttcagc aatctcgtct ctgcccagtc ctatacgttc
1081 aatcccgcaa ccggcaatac ggtgacgatt ccggtctccg cgacggtcaa gcgcctccag
1141 ctgaacatta cggcgaactc cggcgcccct gccggccaga ttgccgagtt ccaagtgttc
1201 ggcacgccag cgcctaatcc ggacttgacc attaccggca tgtcctggac tccgtcttct
1261 ccggtcgaga gcggcgacat tacgctgaac gccgtcgtca agaacatcgg aactgcagct
1321 gcaggcgcca cgacggtcaa tttctacctg aacaacgaac tcgccggcac cgctccggta
1381 ggcgcgcttg cggcaggagc ttctgcaaat gtatcgatca atgcaggcgc caaagcagcc
1441 gcaacgtatg cggtaagcgc caaagtcgac gagagcaacg ccgtcatcga gcagaatgaa
1501 ggcaacaaca gctactcgaa cccgactaac ctcgtcgtag cgccggtgtc cagctccgac
1561 ctcgtcgccg tgacgtcatg gtcgccgggc acgccgtcgc agggagcggc ggtcgcattt
1621 accgtcgcgc ttaaaaatca gggtacgctg gcttccgccg gcggagccca tcccgtaacc
1681 gtcgttctga aaaacgctgc cggagcgacg ctgcaaacct tcacgggcac ctacacaggt
1741 tccctggcag caggcgcatc cgcgaatatc agcgtgggca gctggacggc agcgagcggc
1801 acctataccg tctcgacgac ggtagccgct gacggcaatg aaattccggc caagcaaagc
1861 aacaatacga gcagcgcgag cctcacggtc tactcggcgc gcggcgccag catgccgtac
1921 agccgttacg acacggagga tgcggtgctc ggcggcggag ctgtcctgag aacggcgccg
1981 acgttcgatc agtcgctcat cgcttccgaa gcatcgggac agaaatacgc cgcacttccg
2041 tccaacggct ccagcctgca gtggaccgtc cgtcaaggcc agggcggtgc aggcgtcacg
2101 atgcgcttca cgatgcccga cacgagcgac ggcatgggcc agaacggctc gctcgacgtc
2161 tatgtcaacg gaaccaaagc caaaacggtg tcgctgacct cttattacag ctggcagtat
2221 ttctccggcg acatgccggc tgacgctccg ggcggcggca ggccgctctt ccgcttcgac
2281 gaagtccact tcaagctgga tacggcgttg aagccgggag acacgatccg cgtccagaag
2341 ggcggtgaca gcctggagta cggcgtcgac ttcatcgaga tcgagccgat tccggcagcg
2401 gttgcccgtc cggccaactc ggtgtccgtc accgaatacg gcgctgtcgc caatgacggc
2461 aaggatgatc tcgccgcctt caaggctgcc gtgaccgcag cggtagcggc cggaaaatcc
2521 ctctacatcc cggaaggcac cttccacctg agcagcatgt gggagatcgg ctcggccacc
2581 agcatgatcg acaacttcac ggtcacgggt gccggcatct ggtatacgaa catccagttc
2641 acgaatccca atgcatcggg cggcggcatc tccctgagaa tcaaaggaaa gcttgatttc
2701 agcaacatct acatgaactc caacctgcgt tcccgttacg ggcagaacgc cgtctacaaa
2761 ggctttatgg acaatttcgg cactaattcg atcatccatg acgtctgggt cgagcatttc
2821 gaatgcggca tgtgggtcgg cgactacgcc catactcctg cgatctatgc gagcgggctc
2881 gtcgtggaaa acagccgcat ccgcaacaat cttgccgacg gcatcaactt ctcgcaggga
2941 acgagcaact cgaccgtccg caacagcagc atccgcaaca acggcgatga cggcctcgcc
3001 gtctggacga gcaacacgaa cggcgctccg gccggcgtga acaacacctt ctcctacaac
3061 acgatcgaga acaactggcg cgcggcggcc atcgccttct tcggcggcag cggccacaag
3121 gctgaccaca actacatcat cgactgtgtc ggcggctccg gcatccggat gaatacggtg
3181 ttcccaggct accacttcca gaacaacacc ggcatcacct tctcggatac gacgatcatc
3241 aacagcggca ccagccagga tctgtacaac ggcgagcgcg gagcgattga tctggaagct
3301 tccaacgacg cgatcaaaaa cgtcaccttc accaacatcg acatcatcaa tgcccagcgc
3361 gacggcgttc agatcggcta tggcggcggc ttcgagaaca tcgtgttcaa caacatcacg
3421 atcgacggca ccggccgcga cgggatatcg acatcccgct tctcgggacc tcatcttggc
3481 gcagccatct atacgtacac gggcaacggc tcggcgacgt tcaacaacct ggtgacccgg
3541 aacatcgcct atgcaggcgg caactacatc cagagcgggt tcaacctgac gatcaaatag
3601 gctgcaaaaa aaaggaagct cctcggagct tccttttttt (SEQ ID NO: 13)

II) Paenibacillus genus curdlanolyticus MP-1
1. General Information of Paenibacillus curdlanolyticus MP-1 Mutanase
http://www.ncbi.nlm.nih.gov/nuccore/HQ640944
Genbank HQ640944; Length: 1,261; Mass (Da): 131,631
Reference: Pleszczynska M, et al. Protein Expr Purif. 2012 Nov;86(1):68-74.
2. Paenibacillus curdlanolyticus MP-1 mutanase protein sequence
>gi|315201261|gb|ADT910363.1|alpha-1,3-glucanase [Paenibacillus curdlanolyticus]
MRNKYVTWTLALTMLFSSFFLAVGPNKVVHAAGGTNLALGKNVTASGQSQTYSPNNVKDSNQSTYWESTNNAFPQWIQVDLGATTSIDQIVLKLPAGWGTRTQTLAVQGSTDGSSFTNIVGSAGYVFNPAVANNAVTINFSAASTRYVRLNVTANTAWPAAQLSEFEIYGAGGTTTPPTTPAGTYEAESAALSGGAKVNTDHTGYTGTGFVDGYWTQGATTTFTANVSAAGNYDVTLKYANASGSAKTLSVYVNGTKIRQTTLASLANWDTWGTKVETLSLNAGNNTIAYKYEASDSGNVNIDSIAVAPSTSTPVDPEPPITPPTGSNIAIGKAISASSNTQAFVAANANDNDTNTYWEGGAASSTLTLDLGANQNVTSIVLKLNPSSAWSTRTQTIQVLGHNQSTTTFSNLVSSQSYTFNPATGNSVTIPVTATVKRLQLSITANSGSGAGQIAEFQVYGTPAPNPDLTITGMSWTPASPIETDAVTLNATVKNSGNADAPATTVNFYLNNELVGSSPVGALAAGASSTVSLNVGTKTAATYAVSAKVDESNSIIEQNDANNSYTNASSLVVAPVASSDLVGATTWTPSTPVAGNAIGFMVNLKNQGTIASASGAHGITVVVKNAAGAALQSFSGTYSGAIAAGASVNVTLPGTWTAVNGSYTVTTTVAVDANELTNKQGNNVSTSNLVVYAQRGASMPYSRYDTEDATRGGGATLQTAPTFNQAQIASEASGQSYIALPSNGSSAQWTVRQGQGGAGVTMRFTMPDSTDGMGLNGSLDVYVNGVKVKTVSLTSYYSWQYFSGDMPGDAPSAGRPLFRFDEVHWKLDTPLQPGDTIKIQKGNGDSLEYGIDFLEIEPVPTAIAKPANSLSVTEYGAVANDGQDDLAAFKATVTAAVAAGKSVYIPAGTFNLSSMWEIGSANNMINNITITGAGYWHTNIQFTNPNAAGGGISLRISGQLDFSNVYMNSNLRSRYGQNAIYKGFMDNFGTNSKIHDVWVE HFECGMWVGDYAHTPAIYATGLVVENSRIRNNLADGINYSQGTSNSIVRNSSIRNNGDDGLAVWTSNTNGAPAGVNNTFSYNTIENNWRAGGIAFFGGGGHKADHNLIVDTVGGSGIRMNTVFPGYHFQNNTGITFSDNTLINTGTSQDLYNGERGAIDLEASNDAIKNVTFTNIDIINTQRDAIQFGYGGGFENIVFNNININGTGLDGVTTSRFAGPHKGAAIYTYTGNGSATFNNLTTSNVAYPGLNFIQQGFNLVIQ (SEQ ID NO: 14)
3. Sequence of mRNA from Paenibacillus curdlanolyticus MP-1
1 atgcgcaaca agtatgtcac atggacgctc gccctgacga tgctattttc gagcttcttc
61 cttgcagtag gtcccaacaa ggtcgttcac gcagcaggcg gaacgaattt agcgctcggc
121 aaaaacgtta cggcaagcgg ccaatcgcaa acgtatagtc ccaacaatgt aaaagacagc
181 aatcaatcga cgtactggga aagcacgaac aatgcattcc cgcaatggat tcaagtagac
241 ttaggcgcaa cgacgagcat tgaccaaatc gtactgaagc tgcccgctgg atggggtacg
301 cgtacgcaaa cgttagctgt tcaaggaagc acggacggtt cctcgttcac gaatatcgtg
361 ggctccgcag gctatgtatt taatcctgct gttgccaata acgccgttac gattaacttc
421 tctgctgcaa gcacgcgtta tgttcgtctg aacgtaacag cgaacacggc ttggccagca
481 gcgcagctgt ccgaattcga gatttatggc gctggcggca cgacgacgcc tccaacaacg
541 ccagcaggca catatgaagc tgaatccgca gcattgtccg gcggtgcgaa agtgaacacg
601 gatcataccg gctacacggg tacgggcttt gttgacggct actggacaca aggcgcgaca
661 acgacgttca cggctaacgt gtccgcagct ggcaactatg acgttacatt gaaatatgcc
721 aacgcaagcg gcagtgccaa gacgctaagc gtttacgtca acggcacgaa gattcgccag
781 acgacgctgg caagcctggc aaactgggac acttggggca cgaaggttga gacgctgagc
841 ttgaatgccg gcaataatac gattgcatac aagtatgagg ctagcgactc gggcaacgtg
901 aatatcgact ccattgccgt ggcgccatcg acttcgacac cggtagatcc agaaccgccg
961 atcacgccgc caacgggcag caatatcgca atcggcaaag cgatcagcgc atcttcgaat
1021 acgcaagcat tcgtagctgc caacgcgaac gataacgata cgaacacgta ctgggaaggc
1081 ggagctgcat cgagcacgct gacgctggat cttggcgcga accaaaatgt aacctcgatc
1141 gtgctgaagc tgaatccttc ttcggcatgg agcacgcgta cgcaaacgat ccaagtgctt
1201 ggccacaacc aaagcacgac gacgttcagc aatctggtat cttcgcaatc gtatacgttc
1261 aatcctgcaa cgggcaactc cgtgacgatt ccggttacgg caacagttaa gcgcttgcag
1321 ctgagcatta cggcgaactc gggttccggc gctggtcaaa ttgcggaatt ccaagtgtat
1381 ggaacgccgg caccaaaccc agacctgacg atcacaggca tgtcctggac gcctgcttcg
1441 ccaattgaaa cggatgcagt tacgctgaat gcaacggtta aaaacagcgg aaatgcagac
1501 gctcctgcaa cgacggtaaa cttctacctg aacaatgagc tcgtaggctc ctcgccagtt
1561 ggcgcacttg ctgcaggcgc ttcctcgacg gtttcgctga atgttggtac gaaaacggct
1621 gcaacttatg cagttagcgc gaaagtcgat gagagcaatt cgattatcga gcaaaatgat
1681 gcgaacaaca gttatacgaa cgcatcctcg ctcgtcgtcg ctcctgtcgc aagctctgac
1741 ttggttggcg cgacgacgtg gacgcctagc acgccggttg ccggcaatgc aattggcttc
1801 atggtaaatc ttaaaaacca aggaacgatt gcatctgcaa gcggcgcgca tggcattaca
1861 gttgtcgtga aaaatgccgc aggcgctgcg ctccaatcgt tcagcggcac ctacagcgga
1921 gcaatcgcag ctggcgcatc cgttaacgta accctgccag gtacgtggac ggctgtgaat
1981 ggcagctaca cggtaacgac aacggttgct gtcgatgcta acgagctgac gaacaaacaa
2041 gggaacaacg taagcacttc gaacctcgtt gtttatgcac aacgtggcgc aagcatgcct
2101 tacagccgtt atgacacgga agacgctaca cgtggcggcg gtgcaacgct gcaaaccgca
2161 ccaaccttca accaagcgca aatcgcttcg gaagcatccg gacaaagcta tatcgcgctg
2221 ccttcgaacg gctcctccgc acaatggacg gtccgtcaag gacaaggcgg agctggcgtt
2281 acgatgcgct tcacgatgcc ggattcgact gacggtatgg gtttgaacgg ttcgctcgac
2341 gtttatgtca acggcgttaa agtaaaaacg gtatcgctca cgtcctacta cagctggcag
2401 tatttctcgg gcgatatgcc tggcgatgcg ccgtccgctg gccgtccgtt gttccgcttt
2461 gacgaagtac actggaagct tgacacgcct cttcaaccag gcgacacgat caaaatccaa
2521 aaaggcaacg gagatagcct ggaatacggc attgacttcc tcgaaatcga gccggttcca
2581 acagcaatcg ctaaacctgc caactcgctt tccgttacgg agtatggcgc tgtagcaaac
2641 gatggccaag acgaccttgc cgcattcaaa gcaacggtta cggctgcagt tgctgctggc
2701 aaatccgttt acattcctgc tggcacgttc aatctgagca gcatgtggga aatcggatcg
2761 gctaacaaca tgatcaacaa cattacgatt acaggcgcag gctactggca tacgaacatt
2821 caattcacga atccgaatgc agcaggcggc ggcatttcgc tccggatttc cggacagctt
2881 gatttcagca atgtttacat gaactccaac ctgcgttcgc gttatggtca aaatgcgatt
2941 tacaaaggct tcatggacaa cttcggcaca aactccaaaa tccatgacgt atgggttgag
3001 cacttcgagt gcggcatgtg ggtaggcgat tacgcgcata cgccagcgat ctatgcaacg
3061 ggtcttgtcg ttgaaaacag ccggattcgc aacaaccttg cagacggcat caactactcg
3121 caaggcacga gcaattcgat cgtacgcaac agcagtatcc gcaataacgg tgatgacggt
3181 ctggcggttt ggacgagtaa cacgaatggc gcgccagcag gcgtgaacaa cacgttctcg
3241 tacaacacga tcgaaaacaa ctggcgtgca ggcggtatcg cattcttcgg cggcggcggc
3301 cacaaggctg accacaacct gatcgttgat acggttggcg gctccggcat ccggatgaac
3361 acggtattcc caggctacca cttccaaaac aacacgggta ttacgttctc cgacaacacg
3421 ctgatcaaca caggcacaag ccaagatttg tacaacggcg agcgcggtgc gatcgatctc
3481 gaagcatcga acgatgcaat caagaacgtc acgttcacga acatcgacat catcaacacc
3541 cagcgcgatg cgatacaatt cggctacggc ggcggattcg agaacatcgt atttaacaac
3601 attaacatta acggtacggg gcttgacggc gttacaacct cacggtttgc tggaccgcat
3661 aaaggtgctg caatctacac gtacacgggc aatggctctg caacgttcaa taacctgacg
3721 acgagcaacg tggcatatcc aggcttgaat ttcattcagc aaggctttaa tctggtgatc
3781 cagtag (SEQ ID NO: 15)

III) Paenibacillus sp. RM1 strain.

1. General Information on Mutanase Genbank E16590; Length: 1,291; Mass (Da): 135 kD
Reference: Shimotsuura I, et al. Appl Environ Microbiol. 2008 May;74(9):2759-65.
2. Paenibacillus sp. Protein sequence of the mutanase of strain RM1.

Deduced amino acid sequence of mutanase RM1. The signal peptide region is underlined and the linker region is boxed. The arrow indicates the cleavage site of the N-terminal domain of the protein. The DNA sequence was registered under GenBank Accession No. E16590. (Sequence ID number: 16)
3. Paenibacillus sp. Sequence of mRNA from RM1 strain
1 cccgggtacc agacctatcg ggaaaaacgc gagcggccct tcgcgcctta tgcgctacgg
61 acggtgctgg cgggcggttt gtttttcatc atcattcccc tgatgatcta cacggcatcg
121 tatatcccgt ttttgctcgt gccgggtccc ggacacgggt tgaaagacgt cgtctccgcc
181 cagaagttca tgttcaatta tcatagccgg cttaacgcca cccacccatt ctcgtcgctg
241 tggtgggagt ggcctctcat ccgcaagccg atctggtatt acggagccgc ggaattggcg
301 ccgggaaaaa tggcgagcat cgtgggcatg ggcaatccgg cggtgtggtg gacgggaacg
361 attgcggtaa tcgcggccct tcgctcggcc tggaagaagc gggaccggag catgaccgtc
421 gtcttcgttg gaatcgcctc gtcttatctt ccgtgggttt tcgtatccag actcaccttt
481 atttatcact ttttcgcttg cgttccgttt ctcgttcttt gcatcgttta ttggattcga
541 aaaatggaat agcgtaagcc gggatatcgg attgcgacgc tcctttacgc aggcgcggtt
601 ctggtgctgt tcattttgtt ttacccgatt ttgtcgggga ccgaaataga cgtttcttac
661 gcggaccgcg ttctgaagtg gttcggcggg tggatttttc acgggtaagc gagcgttgga
721 agcaaggaag ggaaggaaga cgagcgtctc cttcccgaaa tccatccaat atcttgaaat
781 tgcatacatt tttcgtaaga ttgcttctta tctgtctccc tcccctgttc ttataatggg
841 ggtatcccaa cgaaaggagg gtttgtaagc gctgtcagcs tgtttgccga aagttctcgc
901 atttgctgac ctacactttg aggaggagga atttaatgcg ctgcaaattt gtcgcatggt
961 cgcttgttac agccatgctg atggccagtt tgctgacggc tgtaggaccg ttcggccccg
1021 cttccgccgc gggaggaccg aatctgacgc cgggcaaacc cattacggcg agcggccaat
1081 cccaaaccta cagccctcag aacgtaaaag acggcaatca aaatacgtat tgggaaagca
1141 cgaacaacgc gttcccgcaa tggattcaag tggatttggg cgcaagcacg ggcatcgacc
1201 aaattgtgct gaagctgccc gcaagctggg aagcgcgcac gcaaacgctg gccgttcaag
1261 gcagcttgaa cggttcgacg ttcacggaca ttgtcggctc cgccaattat gtattcagtc
1321 cgtctgtcgg gaacaacacg gttacgatca actttaccgc gaccagcacg cgctacgtgc
1381 gcttgtatgt aacggccaac acgggctggc cggcggcgca gctgtccgaa ttcgaaattt
1441 acggctccgg cgaccagacg ccggcgcctg atacgtatca agccgaatcc gcggctctgt
1501 ccggcggcgc gaaagtcaac acggaccatg ccggatatat cggcacgggc tttgttgacg
1561 gttactggac gcaaggcgcg acgacgacct tttcggtcaa cgcgccgacg gcgggcaact
1621 acgatgtaac gctgaggtac ggcaacgcaa ccggcagcaa caaaacggta agcctctacg
1681 tcaatggagc gaagattcgc cagaccacgc tgcccagcct gcctaactgg gattcatgga
1741 gcagcaagac ggagacgctt aacctgaatg caggcagcaa caccattgcg tacaaatacg
1801 acccgggcga ttccggcaac gtcaatcttg accaaatcac ggtcgaagcg tcgacttcaa
1861 cgcctactcc tactccatcc cctactccta cacctacgcc aacgccgacg cctacgccta
1921 cgcctacacc cacacctact ccgaccccga cgcctacgcc tacacctaca cctacaccta
1981 cgccgacgcc tcctccgggc ggcaacatcg ccatcggcaa atcgatttcc gcatcctccc
2041 acacgcagac gtacgttgcg gagaacgcga acgataacga tgtcaacacg tactgggaag
2101 gcggcggcaa tccgagcacg ctgacgctcg atctcggagc gaactacaat attacgtcca
2161 tcgtgctgaa gctgaacccg tcctcgatat gggctgcgcg tacgcaaacg attcaagtgc
2221 tcggacacga tcagaacacg acgaccttca gcaatctggt ctcggcgaaa tcgtactcgt
2281 tcgatccggc ctccggcaat actgtgacca ttccggttac ggcgacggtg aaacgtttgc
2341 agttgaacat tacgtcgaac tccggcgccc cggccggaca agtcgccgag ttccaggtgt
2401 tcggcacgcc tgcgccgaat ccggacctga cgattaccgg catgtcctgg tcgccttctt
2461 ctccggttga gaccgacgcc attacgctaa acgcaacggt gaagaacaac gggaatgcca
2521 gcgccgcggc gaccaccgtc aatttctacc tgaacaacga gctggcgggt tccgcgccgg
2581 tagccgcgct ggcggcaggc gcttcggcaa cggtgccgct gaatgtcggc gcgaaaaccg
2641 ccgcgacata cgcggtcggc gccaaagtag acgagagcaa cgcggtcatc gagctgaacg
2701 agtcgaacaa cagctacacg aatccggctt cactcgttgt ggcccccgtt tccagctcgg
2761 atctggtggg cacggtttcg tggacgccga gcactccgat tgccaacaat gccgtttctt
2821 ttaacgtaaa tcttaaaaat caaggaacga ttgcttccgc cggcgggtct cacggcgtga
2881 cggtcgtgct taaaaatgct tccggttcga ccgttcaaac gttcagcggt tcctataccg
2941 gcagcctggc tccgggagcg tccgtcaaca tcacccttcc ggggacctgg acggcggcag
3001 ccggcagcta cacggtaacg gccaccgttg cggcagacgc caacgaactt ccgatcaagc
3061 aagccaacaa cgcgaacacc gcaagcctga ccgtatattc cgcccgcggc gcgagcatgc
3121 cgtacagccg gtatgacacc gaggacgcca ccctcggcgg cggcgccacg ctgaagtccg
3181 cgccgacatt cgatcaggcg cttacggcat cggaagccac cggccaactc tatgcggcgc
3241 tgccctcgaa cggctcctat cttcaatgga ccgtcagaca gggtcagggc ggcgcaggcg
3301 tgacgatgag atttacgatg cccgactcgg cggacggcat gggattaaac ggttcgctag
3361 acgtttacgt caacggcacc aaagtcaaaa ccgtatcgct gacctcctac tacagctggc
3421 agtatttctc gggcgatatg cccggagacg ctcccagcgc gggccgtccg ctcttccgct
3481 ttgacgaagt gcactggaag ctggatactc cgctcaaacc cggagacacg attcgcatcc
3541 agaagaacaa cggcgacagc ctggaatacg gtgtcgactt tattgaaatc gaaccggttc
3601 cggctgcgat ctcccgtccg gccaactcgg tttccgtaac ggattacggc gctgtgccga
3661 acgacggaca ggacgatctc accgccttta aagccgccgt aaacgcggcg gtcgcatccg
3721 acaagatctt gtacattccg gaaggaacgt tccacctcgg caacatgtgg gagatcggtt
3781 ccgtcagcaa catgatcgat cacattacga ttacgggagc cggtatctgg tatacgaaca
3841 tccagtttac caacgccaat ccggcgtccg gcggcatctc gctccggatt acgggcaagc
3901 ttgatttcag caacgtgtac ctcaactcca atttgcggtc gcggtatggt caaaatgcgg
3961 tttacaaagg ctttatggac aacttcggga ccaattccgt catccgcgac gtctgggtcg
4021 agcacttcga atgcggcttc tgggtcgggg actacgggca tacgccggcg atccgcgcga
4081 gcgggctgct gattgaaaac agccgaatcc gcaacaacct ggccgatggc gtcaacttcg
4141 cccaagggac cagcaattcg accgtacgca acagcagcct gcgcaacaac ggcgacgacg
4201 cccttgccgt atggacgagt aatacgaacg gcgcgcccga aggcgtaaac aataccttct
4261 cgtacaacac catcgaaaac aactggcgcg cgggaggcat cgccttcttc ggaggaagcg
4321 gacacaaggc cgaccacaac tacatcgtcg actgcgtcgg cggttccggc atccggatga
4381 acaccgtgtt ccccggatac cacttccaga acaataccgg cattgtgtgttc tcggacacga
4441 ccatcgtcaa ctgcggcacg agcaaagacc tatacaacgg cgaacgcggc gccatcgatc
4501 tggaagcttc gaacgacgcc atccggaacg tgacgtttac caacatcgat attatcaact
4561 ctcagcgcga tgcgatccag ttcggttacg gcggcggctt caccaacatc gtgttcaaca
4621 acatcaacat taacggaacc ggtcttgacg gcgtaaccac ctcgcggttc tcgggaccgc
4681 atctgggcgc ggcgatcttc acctataccg gcaacggctc cgccacgttc aacaatctga
4741 ggaccagcaa tatcgcttac cccaatctgt attacatcca gagcgggttc aatctgatca
4801 tcaataatta gatatctggg cccgtctgcg ggggaggaac tcttcggagc tcgaattcgt
4861 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
4921 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
4981 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa ctgtcgtgcc agctgcatta
5041 atgaatcggc caacgcgcgg ggagaggcsg tttkcgtatt gggcgccctt (SEQ ID NO: 17)

IV) Trichoderma harzianum (CCM F-470)
1. General Information on Trichoderma harzianum Mutanase
See also http://www.uniprot.org/uniprot/Q8WZM7Length:635 Mass (Da): 67,726
Last modified date: March 1, 2002-v1
Checksum: iBB0D864E2F432C58
2. Protein sequence of mutanase from Trichoderma harzianum
(http://www.uniprot.org/uniprot/Q8WZM7.fasta)
>tr|Q8WZM7|Q8WZM7_TRIHA Alpha-1,3-glucanase OS=Trichoderma harzianum GN=p3 PE=2 SV=1
MLGVFRRLRLGALAAAALSSLGSAAPANVAIRSLEERASSADRLVFCHFMIGIVGDRGSSADYDDDMQRAKAAGIDAFALNIGVDGYTDQQLGYAYDSADRNGMKVFISFDFNWWSPGNAVGVGQKIAQYANRPAQLYVDNRPFASSFAGDGLDVNALRSAAGSNVYFVPNFHPGQSSPSNIDGALNWMAWDNDGNNKAPKPGQTVTVADGDNAYKNWLGGKPYLAPVSTWVFNHFGPEVSYSKNWVFPSGPLIYNRWQQVLQQGFPRVEIVTWNDYGESHYVGPLKSKQFHDGNSKWVNDMPHDGFLDLSKPFIAAYKNRDTDISKYVQNEQLVYWYRRNLKALDCDATDTTSNRPANNGSGNYFEGRPDGWQTMDDTVYVAALLKTAGSVTVTSGGTTQTFQANAGANLFQIPASIGQQKFALTRNGQTVFSGTSLMDITNVCSCGIYNFNPYVGTIPAGFDDPLQADGLFSLTIGLHVTTCQAKPSLGTNPPVTSGPVSSLPASSTTRASSPPPVSSTRVSSPPVSSPPVSRTSSAPPPPGNSTPPSGQVCVAGTVADGESGNYIGLCQFSCNYGYCPPGPCKCTAFGAPISPPASNGRNGCPLPGEGDGYLGLCSFSCNHNYCPPTACQYC (SEQ ID NO: 18)
3. Sequence of mRNA (Trichoderma harzianum
(http://www.ebi.ac.uk/ena/data/view/AJ243799&display=fasta)
>ENA|AJ243799|AJ2437999.1 Trichoderma harzianum mRNA for alpha-1,3-glucanase (p3 gene)
ATGTTGGGCGTTTTCCGCCGCCTCAGGCTCGGCGCCCTTGCCGCCGCAGCTCTGTCTTCTCTCGGCAGTGCCGCTCCCGCCAATGTTGCTATTCGGTCTCTCGAGGAACGTGCTTCTTCTGCTGACCGTCTCGTATTCTGTCATTTCATGATTGGGATCGTGGGTGACCGTGGCAGCTCGGCAGATTATGATGACGATATGCAACGTGCCAAAGCCGCTGGCATTGACGCCTTCGCCCTGAACATCGGCGTTGACGGCTATACCGACCAGCAGCTCGGCTATGCCTATGACTCTGCCGATCGTAATGGCATGAAAGTCTTCATTTCATTTGATTTCAACTGGTGGAGCCCCGGCAATGCAGTTGGTGTTGGCCAGAAGATTGCGCAGTATGCCAACCGCCCTGCCCAGCTGTATGTCGACAACCGGCCATTCGCCTCTTCCTTCGCCGGTGACGGTCTGGATGTAAATGCGTTGCGCTCTGCTGCAGGCTCCAACGTTTACTTTGTGCCCAACTTCCACCCTGGTCAATCTTCCCCCTCCAACATTGATGGCGCCCTTAACTGGATGGCCTGGGATAATGATGGAAACAACAAGGCACCCAAGCCGGGCCAGACTGTCACAGTGGCAGACGGTGACAACGCTTATAAGAATTGGTTGGGTGGCAAGCCTTACCTGGCGCCTGTCTCAACTTGGGTTTTCAACCATTTCGGGCCCGAAGTTTCATATTCCAAGAACTGGGTTTTCCCAAGTGGGCCTCTGATCTATAACCGGTGGCAACAAGTCTTGCAGCAAGGGTTCCCAAGGGTTGAGATCGTTACCTGGAATGACTACGGGGAATCTCACTACGTCGGTCCCCTGAAGTCTAAGCAATTTCATGATGGGAACTCCAAATGGGTCAATGATATGCCCCACGATGGATTCCTGGATCTTTCGAAGCCGTTCATAGCCGCATATAAAAACAGGGATACCGACATCTCCAAGTATGTTCAAAATGAGCAGC TTGTTTACTGGTACCGCCGCAACTTAAAGGCACTGGACTGTGACGCCACCGACACAACCTCTAACCGCCCGGCTAACAATGGAAGCGGCAATTACTTTGAGGGACGCCCCGATGGTTGGCAAACTATGGATGATACGGTTTACGTGGCGGCACTTCTCAAGACTGCCGGTAGCGTCACGGTCACGTCTGGTGGCACCACTCAAACGTTCCAGGCCAACGCCGGAGCCAATCTCTTCCAAATCCCGGCCAGCATCGGCCAGCAAAAGTTTGCTCTGACTCGTAACGGTCAGACCGTCTTTAGCGGAACCTCATTGATGGATATCACCAACGTTTGCTCTTGCGGTATCTACAACTTCAACCCATATGTTGGCACCATTCCTGCCGGCTTTGACGACCCTCTTCAGGCTGACGGTCTTTTCTCTTTGACCATCGGATTGCACGTCACAACTTGTCAGGCCAAGCCATCTCTTGGAACTAACCCTCCTGTCACTTCCGGCCCTGTGTCCTCGCTTCCAGCTTCCTCCACCACCCGCGCATCCTCGCCGCCTCCTGTTTCTTCAACTCGTGTCTCTTCTCCCCCTGTCTCTTCCCCTCCAGTTTCTCGCACCTCTTCTGCCCCTCCCCCTCCGGGCAACAGCACGCCGCCATCGGGTCAGGTTTGCGTTGCCGGCACCGTTGCCGACGGCGAGTCTGGCAACTACATCGGCCTGTGCCAATTCAGCTGCAACTACGGTTACTGCCCACCAGGACCGTGTAAGTGCACCGCCTTTGGTGCTCCCATCTCGCCACCGGCATCCAACGGCCGCAACGGCTGCCCTCTGCCGGGAGAAGGCGATGGTTATCTGGGCCTGTGCAGTTTCAGTTGTAACCATAATTACTGCCCGCCAACGGCATGTCAATACTGCTAGGAGGGATCAATCTCAGTATGAGTATATGGAGGCTGCTGAAGGACCAATTAGCTGTTCTTATCGGCAGACGAAACCCATAGAGTAAGAAGTTA AATAAAATGCAATTAATGTGTTTTCAAAAAAAAAAAAAAAA (SEQ ID NO: 19)
(Since Trichoderma harzianum is a fungus, it has a poly A tail)

V) Trichoderma harzianum
1. General Information on Trichoderma harzianum Mutanase
See also http://www.uniprot.org/uniprot/Q8WZM7.

2. Protein sequence of mutanase from Trichoderma harzianum (see http://www.uniprot.org/uniprot/Q8WZM7.fasta)
>tr|Q8WZM7|Q8WZM7_TRIHA Alpha-1,3-glucanase OS=Trichoderma harzianum GN=p3 PE=2 SV=1
MLGVFRRLRLGALAAAALSSLGSAAPANVAIRSLEERASSADRLVFCHFMIGIVGDRGSSADYDDDMQRAKAAGIDAFALNIGVDGYTDQQLGYAYDSADRNGMKVFISFDFNWWSPGNAVGVGQKIAQYANRPAQLYVDNRPFASSFAGDGLDVNALRSAAGSNVYFVPNFHPGQSSPSNIDGALNWMAWDNDGNNKAPKPGQTVTVADGDNAYKNWLGGKPYLAPVSTWVFNHFGPEVSYSKNWVFPSGPLIYNRWQQVLQQGFPRVEIVTWNDYGESHYVGPLKSKQFHDGNSKWVNDMPHDGFLDLSKPFIAAYKNRDTDISKYVQNEQLVYWYRRNLKALDCDATDTTSNRPANNGSGNYFEGRPDGWQTMDDTVYVAALLKTAGSVTVTSGGTTQTFQANAGANLFQIPASIGQQKFALTRNGQTVFSGTSLMDITNVCSCGIYNFNPYVGTIPAGFDDPLQADGLFSLTIGLHVTTCQAKPSLGTNPPVTSGPVSSLPASSTTRASSPPPVSSTRVSSPPVSSPPVSRTSSAPPPPGNSTPPSGQVCVAGTVADGESGNYIGLCQFSCNYGYCPPGPCKCTAFGAPISPPASNGRNGCPLPGEGDGYLGLCSFSCNHNYCPPTACQYC (SEQ ID NO: 20)
3. Sequence of mRNA (Trichoderma harzianum, further information can be found below.

(http://www.ebi.ac.uk/ena/data/view/AJ243799&display=fasta)
>ENA|AJ243799|AJ2437999.1 Trichoderma harzianum mRNA for alpha-1,3-glucanase (p3 gene)
ATGTTGGGCGTTTTCCGCCGCCTCAGGCTCGGCGCCCTTGCCGCCGCAGCTCTGTCTTCTCTCGGCAGTGCCGCTCCCGCCAATGTTGCTATTCGGTCTCTCGAGGAACGTGCTTCTTCTGCTGACCGTCTCGTATTCTGTCATTTCATGATTGGGATCGTGGGTGACCGTGGCAGCTCGGCAGATTATGATGACGATATGCAACGTGCCAAAGCCGCTGGCATTGACGCCTTCGCCCTGAACATCGGCGTTGACGGCTATACCGACCAGCAGCTCGGCTATGCCTATGACTCTGCCGATCGTAATGGCATGAAAGTCTTCATTTCATTTGATTTCAACTGGTGGAGCCCCGGCAATGCAGTTGGTGTTGGCCAGAAGATTGCGCAGTATGCCAACCGCCCTGCCCAGCTGTATGTCGACAACCGGCCATTCGCCTCTTCCTTCGCCGGTGACGGTCTGGATGTAAATGCGTTGCGCTCTGCTGCAGGCTCCAACGTTTACTTTGTGCCCAACTTCCACCCTGGTCAATCTTCCCCCTCCAACATTGATGGCGCCCTTAACTGGATGGCCTGGGATAATGATGGAAACAACAAGGCACCCAAGCCGGGCCAGACTGTCACAGTGGCAGACGGTGACAACGCTTATAAGAATTGGTTGGGTGGCAAGCCTTACCTGGCGCCTGTCTCAACTTGGGTTTTCAACCATTTCGGGCCCGAAGTTTCATATTCCAAGAACTGGGTTTTCCCAAGTGGGCCTCTGATCTATAACCGGTGGCAACAAGTCTTGCAGCAAGGGTTCCCAAGGGTTGAGATCGTTACCTGGAATGACTACGGGGAATCTCACTACGTCGGTCCCCTGAAGTCTAAGCAATTTCATGATGGGAACTCCAAATGGGTCAATGATATGCCCCACGATGGATTCCTGGATCTTTCGAAGCCGTTCATAGCCGCATATAAAAACAGGGATACCGACATCTCCAAGTATGTTCAAAATGAGCAGC TTGTTTACTGGTACCGCCGCAACTTAAAGGCACTGGACTGTGACGCCACCGACACAACCTCTAACCGCCCGGCTAACAATGGAAGCGGCAATTACTTTGAGGGACGCCCCGATGGTTGGCAAACTATGGATGATACGGTTTACGTGGCGGCACTTCTCAAGACTGCCGGTAGCGTCACGGTCACGTCTGGTGGCACCACTCAAACGTTCCAGGCCAACGCCGGAGCCAATCTCTTCCAAATCCCGGCCAGCATCGGCCAGCAAAAGTTTGCTCTGACTCGTAACGGTCAGACCGTCTTTAGCGGAACCTCATTGATGGATATCACCAACGTTTGCTCTTGCGGTATCTACAACTTCAACCCATATGTTGGCACCATTCCTGCCGGCTTTGACGACCCTCTTCAGGCTGACGGTCTTTTCTCTTTGACCATCGGATTGCACGTCACAACTTGTCAGGCCAAGCCATCTCTTGGAACTAACCCTCCTGTCACTTCCGGCCCTGTGTCCTCGCTTCCAGCTTCCTCCACCACCCGCGCATCCTCGCCGCCTCCTGTTTCTTCAACTCGTGTCTCTTCTCCCCCTGTCTCTTCCCCTCCAGTTTCTCGCACCTCTTCTGCCCCTCCCCCTCCGGGCAACAGCACGCCGCCATCGGGTCAGGTTTGCGTTGCCGGCACCGTTGCCGACGGCGAGTCTGGCAACTACATCGGCCTGTGCCAATTCAGCTGCAACTACGGTTACTGCCCACCAGGACCGTGTAAGTGCACCGCCTTTGGTGCTCCCATCTCGCCACCGGCATCCAACGGCCGCAACGGCTGCCCTCTGCCGGGAGAAGGCGATGGTTATCTGGGCCTGTGCAGTTTCAGTTGTAACCATAATTACTGCCCGCCAACGGCATGTCAATACTGCTAGGAGGGATCAATCTCAGTATGAGTATATGGAGGCTGCTGAAGGACCAATTAGCTGTTCTTATCGGCAGACGAAACCCATAGAGTAAGAAGTTA AATAAAATGCAATTAATGTGTTTTCAAAAAAAAAAAAAAAA (SEQ ID NO: 21)
(Since Trichoderma harzianum is a fungus, it has a poly A tail)
Dextranase (Dex) gene of Penicillium minioluteum GenBank: L41562.1
(Http://www.ncbi.nlm.nih.gov/nuccore/L41562.1)
The mature protein is 574 amino acids and has a molecular weight of 67KD. Optimal reaction conditions are pH 5.5 and 40°C. The pH range is 3-6.

Amino acid sequence
MATMLKLLALTLAISESAIGAVMHPPGNSHPGTHMGTTNNTHCGADFCTWWHDSGEINTQTPVQPGNVRQSHKYSVQVSLAGTNNFHDSFVYESIPRNGNGRIYAPTDPPNSNTLDSSVDDGISIEPSIGLNMAWSQFEYSHDVDVKILATDGSSLGSPSDVVIRPVSISYAISQSDDGGIVIRVPADANGRKFSVEFKTDLYTFLSDGNEYVTSGGSVVGVEPTNALVIFASPFLPSGMIPHMTPDNTQTMTPGPINNGDWGAKSILYFPPGVYWMNQDQSGNSGKLGSNHIRLNSNTYWVYLAPGAYVKGAIEYFTKQNFYATGHGILSGENYVYQANAGDNYIAVKSDSTSLRMWWHNNLGGGQTWYCVGPTINAPPFNTMDFNGNSGISSQISDYKQVGAFFFQTDGPEIYPNSVVHDVFWHVNDDAIKIYYSGASVSRATIWKCHNDPIIQMGWTSRDISGVTIDTLNVIHTRYIKSETVVPSAIIGASPFYASGMSPDSRKSISMTVSNVVCEGLCPSLFRITPLQNYKNFVVKNVAFPDGLQTNSIGTGESIIPAASGLTMGLNISNWTVGGQKVTMENFQANSLGQFNIDGSYWGEWQIS (SEQ ID NO: 22)
DNA sequence
1 ggcatagtaa tcccgacagc cgagtatgat ggagcttctt cggataatga tagcgccacc
61 agaccttgct tgagctggag agctaaaaca ttaaacgcca cacgaccaac actctcatta
121 gttgcgatag atgatgctcg gagctgttga aactcagaaa ttccttctat gcggggtctc
181 caagatcgat cctgggggat gtgaatacta cggtggacct aattgacgcc ttgacaggtg
241 atgttaagcg aaccaaggaa gaataatctg gggctagatg aagatgttga gctgtaaggt
301 acggtacgtt cctattggct ttatcggagc ttctccgggt tactcagtct ttccgggagc
361 atgatcattt ttgtattgtc caatagtaag cagaaactga gagccaccac aaactcaaaa
421 cctcggtagc gaagtttccc ggaaccagtc aggattctca gaaactgtgc tcgtgttgcg
481 gggaatccgc attctacgtc gtctggagca aggaaatgtt cgtgctggat tgaggaggat
541 aggtaggttg gagaatctct tcagctaacc aatctataag catgctccgg taacctttag
601 agtttcacat tcaacgtaat ttccaagata gccagagcgt ccttgaatta ctatgtagaa
661 atcctaaaat ttcccctgta aaatgcaagt caacgagatg cgtgccctca atgtctctcg
721 gcgctacccc ggaaatgatg cataaggcca agaatgtcac ccggtaactt tttcttcaga
781 atatcctaag atttccatca aacacagtcg aataggtcaa tgctcgcgag agactttctg
841 ccttcactct acgtcctact catagaagtt caacggctca attccggggt aatctagagt
901 ttggacctca agggagatgt tgcaacaaat tgtactagaa cgatgcgctt gctttccaat
961 acagtagttg acttcatata gcttccaaca aaagggatgg ggatgaaggc tctatagcga
1021 gaagtctata agaaagtgtc ctcatacctg tatctctcag tcgttcgaga acaatcccgg
1081 aaactatctt atcttgcgag aaagaagaca atatctcaaa cttatggcca caatgctaaa
1141 gctacttgcg ttgacccttg caattagcga gtccgccatt ggagcagtca tgcacccacc
1201 tggcaattct catcccggta cccatatggg cactacgaat aatacccatt gcggcgccga
1261 tttctgtacc tggtggcatg attcagggga gatcaatacg cagacacctg tccaaccagg
1321 gaacgtgcgc caatctcaca agtattccgt gcaagtgagc ctagctggta caaacaattt
1381 tcatgactcc tttgtatatg aatcgatccc ccggaacgga aatggtcgca tctatgctcc
1441 caccgatcca cccaacagca acacactaga ttcaagtgtg gatgatggaa tctcgattga
1501 gcctagtatc ggccttaata tggcatggtc ccaattcgag tacagccacg atgtagatgt
1561 aaagatcctg gccactgatg gctcatcgtt gggctcgcca agtgatgttg ttattcgccc
1621 cgtctcaatc tcctatgcga tttctcagtc tgacgatggt gggattgtca tccgggtccc
1681 agccgatgcg aacggccgca aattttcagt tgagttcaaa actgacctgt acacattcct
1741 ctctgatggc aacgagtacg tcacatcggg aggcagcgtc gtcggcgttg agcctaccaa
1801 cgcacttgtg atcttcgcaa gtccgtttct tccttctggc atgattcctc atatgacacc
1861 cgacaacacg cagaccatga cgccaggtcc tatcaataac ggcgactggg gcgccaagtc
1921 aattctttac ttcccaccag gtgtatactg gatgaaccaa gatcaatcgg gcaactcggg
1981 gaagttagga tctaatcata tacgtctaaa ctcgaacact tactgggtct accttgcccc
2041 cggtgcgtac gtgaagggtg ctatagagta ttttaccaag cagaacttct atgcaactgg
2101 tcatggtatc ctatcgggtg aaaactatgt ttaccaagcc aatgccggcg acaactacat
2161 tgcagtcaag agcgattcaa ccagcctccg gatgtggtgg cacaataacc ttgggggtgg
2221 tcaaacatgg tactgcgttg gcccgacgat caatgcgcca ccattcaata ctatggattt
2281 caatggaaat tctggcatct caagtcaaat tagcgactat aagcaggtgg gagccttctt
2341 cttccagacg gatggaccag aaatatatcc caatagtgtc gtgcacgacg tcttctggca
2401 cgtcaatgat gatgcaatca aaatctacta ttcgggagca tctgtatcgc gggcaacgat
2461 ctggaaatgt cacaatgacc caatcatcca gatgggatgg acgtctcggg atatcagtgg
2521 agtgacaatc gacacattaa atgttattca cacccgctac atcaaatcgg agacggtggt
2581 gccttcggct atcattgggg cctctccatt ctatgcaagt gggatgagtc ctgattcaag
2641 aaagtccata tccatgacgg tttcaaacgt tgtttgcgag ggtctttgcc cgtccctatt
2701 ccgcatcaca ccccttcaga actacaaaaa ttttgttgtc aaaaatgtgg ctttcccaga
2761 cgggctacag acgaatagta ttggcacagg agaaagcatt attccagccg catctggtct
2821 aacgatggga ctgaatatct ccaactggac tgttggtgga caaaaagtga ctatggagaa
2881 ctttcaagcc aatagcctgg ggcagttcaa tattgacggc agctattggg gggagtggca
2941 gattagctga attccagctc tcggagcgcg tgagtgcttc tacccgctcc tttacccttg
3001 tcgagagata aaggcataag ttagctcatg tgaaggcgat ttcagttcat tctctctttt
3061 tggagcttat ttcctgttcg accaattgtg acaccaactt gcctttcaaa agacgtggac
3121 gatatgtgta cggtaatcag tcaaatgaac gtcaacattc atttaataag gacatttcca
3181 ggtttcctta ctctgtcgat tatgcctaac tcgggttgat gtcttgtcag gatggaaaat
3241 ctcgttgtgt acttccagtg aaatgggcag ggctaagccc taaaccctaa cgcatacaat
3301 ttgtaggcac ctacccatgt aagttcacac ccagtcgact tataagtcta gatatttatg
3361 ctatgcaggc tctggaatga tttacattcc atgctataca tagttatttg caagaatttg
3421 cagacgagat aaaaatcaat ggacgaataa tcacgcatta ctccacaggc tcatgccacg
3481 gagcaagggt tcccccgaat ctaggccaga ccgggatgat attcaaccga ttctttttgc
3541 agtaactatc tccgtacgag ctgcacgagc taaacggatt atataaaggt gctaactgag
3601 cattggatcc gtcagttata tgaaatgca (SEQ ID NO: 23)
2. Dextranase (Dex) gene of Penicillium aculeatum (Talaromyces aculeatus strain z01) GenBank: ncbi.nlm.nih.gov/nuccore/KF999646.1 KF999946.1 on the World Wide Web
The optimum pH is about 5. The pH range is 3-6.

Amino acid sequence
MATMLKLLTLALAISESAIGAVLHPPGSSHPSTRTDTTNNTHCGADFCTWWHDSGEINTQTPVQPGNVRQSHKYSVQVSLAGANNFQDSFVYESIPRNGNGRIYAPTDPPNSNTLDSSVDDGISIEHSIGLNMAWSQFEYSQDVDIKILAADGSSLGSPSDVVIRPVSISYAISQSDDGGIVIRVPADANGRKFSVEFKNDPYTFLSDGNEYVTSGGSVVGVEPTNALVIFASPFLPSGMIPHMTPDNTQTMTPGPINNGDWGSKSILYFPPGVYWMNQDQSGNSGKLGSNHIRLNSNTYWVYFAPGAYVKGAIEYFTKQNFYATGHGVLSGENYVYQANAGENYVAVKSDSTSLRMWWHNNLGGGQTWYCVGPTINAPPFNTMDFNGNSGISSQISDYKQVGAFFFQTDGPEIYPNSVVHDVFWHVNDDAIKIYYSGASVSRATIWKCHNDPIIQMGWTSRDISGVTIDTLNVIHTRYIKSETVVPSAIIGASPFYASGMSPDSSKSISMTVSNVVCEGLCPSLFRITPLQNYKNFVVKNVAFPDGLQTNSIGTGESIIPAASGLTMGLDISNWSVGGQKVTMQNFQANSLGQFDIDGSYWGEWQIN (SEQ ID NO: 24)
DNA sequence
1 atggccacaa tgctaaagct acttacgttg gcccttgcaa ttagcgagtc tgccattgga
61 gcagtcctgc acccacctgg cagttctcat cccagtaccc gtacggacac tacgaataat
121 acccattgcg gtgccgactt ctgtacctgg tggcatgatt caggcgagat caacacacag
181 acacctgtcc aaccggggaa cgtgcgccaa tctcacaagt attccgtaca agtgagccta
241 gctggtgcga acaactttca ggactccttt gtatatgaat cgatccctcg gaacggaaat
301 ggtcgcatct atgctcccac cgatccaccc aacagcaaca cactagattc aagtgttgat
361 gatggaatct cgattgaaca tagtattggc ctcaatatgg catggtccca attcgagtac
421 agccaggatg tcgatataaa gatcctggcc gctgatggct catcgttggg ctcaccaagt
481 gatgttgtta ttcgccccgt ctcaatctcc tatgcaattt ctcaatccga cgatggcgga
541 attgtcattc gggtcccagc cgatgcgaac ggccgcaaat tttcagtcga gttcaaaaat
601 gacccgtaca cgttcctctc tgacggcaac gagtacgtca catcgggagg cagcgttgtc
661 ggcgttgagc ctaccaacgc acttgtgatc ttcgcaagcc cgtttcttcc gtcaggcatg
721 attcctcata tgacacccga caacacgcag accatgacac caggacctat caataacggc
781 gactggggct ccaagtcaat tctttatttc ccaccgggcg tatactggat gaaccaagat
841 caatcaggca actcggggaa attaggatct aatcatatac gcctgaactc gaacacctac
901 tgggtctact ttgccccagg tgcgtacgtg aagggtgcta tagagtattt caccaagcag
961 aacttctatg caactggtca tggtgtccta tcgggtgaaa actatgttta ccaagccaat
1021 gctggcgaaa actacgttgc ggtcaagagc gattcgacta gcctccggat gtggtggcac
1081 aataacctgg gaggtggaca aacatggtac tgcgttgggc ctacgatcaa tgcgccgcca
1141 tttaacacaa tggatttcaa tggaaattcc ggtatctcaa gtcaaattag cgactataag
1201 caggtgggag ctttcttctt tcagacggat ggaccagaaa tttatcccaa tagtgtcgtg
1261 cacgacgtct tctggcatgt caatgatgat gcaatcaaaa tctactattc cggagcatct
1321 gtctcgcggg caacgatctg gaaatgtcac aacgatccaa tcatccagat gggatggacg
1381 tctcgggata tcagtggagt gacaatcgac acattgaatg tcatccacac ccgctacatc
1441 aagtcggaga cggtggtgcc ttcggctatc attggggctt ctccattcta tgcaagtggg
1501 atgagtcctg attcaagcaa gtctatatcc atgacggttt caaacgttgt ctgcgaggga
1561 ctttgcccgt ctctgttccg aatcacacct ttacagaact acaagaattt tgttgtcaaa
1621 aatgtggctt tcccagatgg gctacagacg aatagtattg gcacgggaga aagcattatt
1681 ccagccgcat ctggtctaac gatgggactg gatatctcca actggtctgt tggtggtcag
1741 aaggtgacta tgcagaactt tcaagccaat agtctggggc aattcgacat tgacggcagc
1801 tattgggggg agtggcagat taactagctg aataatattg cagctttcag ggcgcatgag
1861 tgcttgtacc cgctccttta cccttgtc (SEQ ID NO: 25)
3. Dextranase Penicillium funiculosum dex gene GenBank: AJ272066.1 (http://www.ncbi.nlm.nih.gov/nuccore/7801166)
The optimum pH is about 5.5. The optimum temperature is 60°C. The pH range is 5 to 7.5 (http://www.sciencedirect.com/science/article/pii/S0032959298001277)
Amino acid sequence
MATMLKLLALTLAISESAIGAVMHPPGVSHPGTHTGTTNNTHCGADFCTWWHDSGEINTQTPVQPGNVRQSHKYSVQVSLAGTNNFHDSFVYESIPRNGNGRIYAPTDPSNSNTLDSSVDDGISIEPSIGLNMAWSQFEYSQDVDIKILATDGSSLGSPSDVVIRPVSISYAISQSNDGGIVIRVPADANGRKFSVEFKNDLYTFLSDGNEYVTSGGSVVGVEPTNALVIFASPFLPSGMIPHMKPHNTQTMTPGPINNGDWGAKSILYFPPGVYWMNQDQSGNSGKLGSNHIRLNSNTYWVYLAPGAYVKGAIEYFTKQNFYATGHGVLSGENYVYQANAGDNYVAVKSDSTSLRMWWHNNLGGGQTWYCVGPTINAPPFNTMDFNGNSGISQISDYKQVGAFFFQTDGPEIYPNSVVHDVFWHVNDDAIKIYYSGASVSRATIWKCHNDPIIQMGWTSRDISGVTIDTLNVIHTRYIKSETVVPSAIIGASPFYASGMSPDSSKSISMTVSNVVCEGLCPSLFRITPLQNYKNFVVKNVAFPDGLQTNSIGTGESIIPAASGLTMGLNISSWTVGGQKVTMENFQANSLGQFNIDGSYWGEWQISRISSSQSA (SEQ ID NO: 26)
DNA sequence
1 atggccacaa tgctaaagct acttgcgttg acccttgcaa ttagcgagtc cgccattgga
61 gcagtcatgc acccacctgg cgtttctcat cccggtaccc atacgggcac tacgaataat
121 acccattgcg gcgccgactt ctgtacctgg tggcatgatt caggggagat caacacgcag
181 acacctgtcc aaccagggaa cgtgcgccaa tctcacaagt attccgtgca agtgagtcta
241 gctggtacaa acaactttca tgactccttt gtatatgaat cgatcccccg gaacggaaat
301 ggtcgcatct atgctcccac cgatccatcc aacagcaaca cattagattc aagcgtggat
361 gatggaatct cgattgagcc tagtatcggc ctcaatatgg catggtccca attcgagtac
421 agccaggatg tcgatataaa gatcctggca actgatggct catcgttggg ctcaccaagt
481 gatgttgtta ttcgccccgt ctcaatctcc tatgcgattt ctcagtccaa cgatggcggg
541 attgtcatcc gggtcccagc cgatgcgaac ggccgcaaat tttcagtcga attcaaaaat
601 gacctgtaca ctttcctctc tgatggcaac gagtacgtca catcgggagg tagcgtcgtc
661 ggcgttgagc ctaccaacgc acttgtgatc ttcgcaagtc cgtttcttcc ttctggcatg
721 attcctcata tgaaacccca caacacgcag accatgacgc caggtcctat caataacggc
781 gactggggcg ccaagtcaat tctttacttc ccaccaggtg tatactggat gaaccaagat
841 caatcgggca actcgggtaa attaggatct aatcatatac gtctaaactc gaacacttac
901 tgggtctacc ttgcccccgg tgcgtacgtg aagggtgcta tagagtattt caccaagcaa
961 aacttctatg caactggtca tggtgtccta tcaggtgaaa actatgttta ccaagccaat
1021 gctggcgaca actatgttgc agtcaagagc gattcgacca gcctccggat gtggtggcac
1081 aataaccttg ggggtggtca aacatggtac tgcgttggcc cgacgatcaa tgcgccacca
1141 ttcaacacta tggatttcaa tggaaattct ggcatctcaa gtcaaattag cgactataag
1201 caggtgggag ccttcttctt ccagacggat ggaccagaaa tctatcccaa tagtgtcgtg
1261 cacgacgtct tctggcacgt caatgatgat gcaatcaaaa tctactattc gggagcatct
1321 gtatcgcggg caacgatctg gaaatgtcac aatgacccaa tcatccagat gggatggaca
1381 tctcgggata tcagtggagt gacaatcgac acattaaatg ttattcacac ccgctacatc
1441 aaatcggaga cggtggtgcc ttcggctatc attggggcct ctccattcta tgcaagtggg
1501 atgagtcccg attcaagcaa gtccatatcc atgacggttt caaacgttgt ttgcgagggt
1561 ctttgcccgt ccctgttccg catcacaccc ctacagaact acaaaaattt tgttgtcaaa
1621 aatgtggctt tcccagatgg gctacagaca aatagtattg gcacaggaga aagcattatt
1681 ccagccgcat ctggtctaac gatgggacta aatatctcca gctggactgt tggtggacaa
1741 aaagtgacaa tggagaactt tcaagccaat agcctggggc agttcaatat tgacggcagc
1801 tattgggggg agtggcagat tagtcgaatt tccagctctc agagcgcgtg agtgcttcta
1861 cccgctcctt tacccttgtc gaaggatcaa ggcataagtt agctcatgtg aaggcgattt
1921 cagttcattc tctctttttt ggagctcatt tccttttcga ccaattgtga caccaaattg
1981 ccatgtgtac tgtaattggt caaatgaacg ttaaccttcg atttaatatg gacatttcca
2041 ggtttcctta ctctgtcgat tatgcctaac tcgggttgat gtcttgtcag gatgaaaatc
2101 tcgttgtcat gtacttcgag tgaaatgggc agggctaacc cctaagccct aacgcccaat
2161 cgacttataa gtctagatgt ttatgctatg caggctctgg aatgatttac attccatgct
2221 ataca (SEQ ID NO: 27)

Example III
Dual-Targeting Antibiofilm Approach for EPS Matrix Degradation and Enhanced Bacterial Killing As mentioned above, biofilms are structured microbial communities embedded in an extracellular matrix associated with bacterial adhesion-aggregation and drug resistance. Is difficult to treat using conventional antimicrobial monotherapy. In the present example, a multi-targeted approach to enhance anti-biofilm efficacy was further investigated by combining exopolysaccharide (EPS) matrix-degrading glucanohydrolase with clinically used essential oil-based antimicrobial agents. Our data show that dextranase synergizes with mutanase to degrade the EPS glucan matrix in preformed oral biofilms while significantly enhancing bacterial killing by antimicrobial agents. (3 log increase vs antibacterial agent alone). Further analysis shows that the EPS degradation/antibacterial dual approach (EDA) disrupts matrix scaffolds, causes “physical disruption” of bacterial clusters, induces cell dispersal, and exposes bacterial cells to rapid antibacterial killing. Became clear. Interestingly, a single pretreatment of the apatite surface using the described EDA approach prevented biofilm accumulation by inhibiting EPS synthesis and bacterial binding in situ. It was also unexpectedly discovered that the EDA approach could selectively target the EPS-producing oral pathogen Streptococcus mutans, destroying colonization and overgrowth in a mixed-species ecological biofilm model. An interesting mechanism was observed that localized killing of EPS and exposure of embedded pathogens enhanced killing (compared to other species) and promoted the predominance of symbiotic bacteria. Together, these results demonstrate an approach that can enhance the efficacy and precision of anti-biofilms by disassembling the EPS matrix and its protective microenvironment, thereby enhancing killing of internal bacterial cells. There is.

The following materials and methods are provided to facilitate implementation of Example III.

Bacterial strain and growth conditions mutans UA159 serotype c (ATCC 700610), a proven cariogenic dental pathogen, and a major producer of water-insoluble EPS matrix (26) was used in the monospecies biofilm model. Actinomyces naeslundii (ATCC® 12104®) and Streptococcus oralis (ATCC® 35037®) are S. is a symbiont selected to produce mixed-species biological biofilms with mutans. All three are abundant in human supragingival plaques (37). S. oralis is one of the earliest pioneering engraftments on saliva-covered tooth surfaces (37). They produced soluble glucan from sucrose and were known to be acid resistant (38). A. naeslundii also colonizes salivary pellicles in the early stages of plaque formation. Strains were stored in trypsin soy broth (TSB) containing 25% glycerol at -80°C. All strains were ultrafiltered (10 kDa molecular weight cutoff membrane; Prep/Scale, Millipore, supplemented with 1% (w/v) glucose at 37° C. and 5% CO ₂ before use until mid-exponential phase. , Massachusetts) buffered tryptone yeast extract broth (UFTYE medium, 2.5% tryptone and 1.5% yeast extract, pH 7.0) (25).

EPS Degrading Enzyme and Antibacterial Agent EPS degrading enzyme is a glucanohydrolase capable of cleaving the glucosidic bond of a polysaccharide. Dextranase [α-(1→6) glucanase; EC 3.2.1.11], which can catalyze the hydrolysis of the glucosidic bond of (1→6)-α-D-glucan of different origin, is described by Sigma (St. Louis). , Missouri). Mutanase [α-(1→3) glucanase; EC 3.2.1.59], which hydrolyzes the glucosidic bond of (1→3)-α-D-glucan, is from Johnson & Johnson (New Brunswick, NJ). It was a kind gift. Glucanohydrolase activity has been reported by Kopec et al. It was determined as described in (39) with some modifications. Briefly, purified S. mutans GtfD glucan [α-(1→6)-linked glucan] or GtfB glucan [α-(1→3)-linked glucan] in dextranase of 0.1 M sodium acetate buffer (pH=5.5) or Incubated with mutanase for 1 hour at 37°C. The amount of reducing sugars released from the glucan by each glucanohydrolase was colorimetrically quantified using the Somogyi-Nelson method (40). One unit (U) of dextranase activity is the amount of enzyme read as 1.0 μmol reducing sugar (measured as maltose) per minute from α-(1→6)-linked glucan at pH 5.5 at 37°C. Defined One unit of mutanase (U) was defined as the amount of enzyme that liberates 1.0 μmol reducing sugar (measured as glucose) per minute from α-(1→3) linked glucan at pH 5.5 at 37°C. An alcohol-free Essential Oil (EO) based solution containing a combination of menthol, thymol, eucalyptol, and methyl salicylate antimicrobial agents was used in Example III. LISTERINE ZERO® containing these EOs was kindly provided by Johnson & Johnson and used as a model antimicrobial agent.

Single- and mixed-species biofilm models Single- and mixed-species biofilms were formed into vertically suspended saliva-coated hydroxyapatite (sHA), a model that mimics the surface of biological teeth, as described above. (25). Briefly, hydroxyapatite discs (1.25 cm diameter, 2.7 ± 0.2 cm ² surface area, Clarkson, Chromatography Products, Inc., South Williamsport, PA) were coated with filter-sterilized whole human saliva. For single species biofilms, 10 ⁵ CFU/mL actively growing S. cerevisiae in UFTYE medium containing 1% (w/v) sucrose on each sHA disc. mutans were inoculated and grown for 19 hours (37° C. and 5% CO ₂ ) (25). A mixed-species biofilm model is described by Xiao et al. It was designed to mimic biofilm formation based on the concept of "ecological plaque-biofilm" (41), as described by (24). S. mutans, S.M. oralis, and A. Naeslundii bacterial cultures were diluted to give S. cerevisiae in UFTYE medium containing 0.1% (w/v) sucrose. mutans (10 ² CFU/mL), S. oralis (10 ⁷ CFU/mL), and A. Provide a defined microbial population of naeslundii (10 ⁶ CFU/mL) for inoculation. The organism grew for the first 29 hours to form the first biofilm community, and at 19 hours the medium changed. At 29, biofilms were transferred to UFTYE medium containing 1% (w/v) sucrose to induce environmental changes that simulate a cariogenic task. Biofilms were analyzed at 43 hours (early cariogenic biofilm) or 67 hours (mature cariogenic biofilm). The culture medium was changed twice a day (8 am and 6 pm) with UFTYE medium containing 1% (w/v) sucrose for an experimental period of more than 43 hours.

Screening of the combined effect of dextranase and mutanase on EPS degradation For screening the combined effect on the degradation of EPS by dextranase and mutanase at different activity ratios, 96-well plates were coated with filter sterilized human whole saliva. In UFTYE medium containing 1% (w/v) sucrose, 10 ⁵ CFU/mL of actively growing S. mutans was inoculated. S. Prior to the combined effect of dextranase and mutanase for destroying mutans biofilm, the biofilm was allowed to grow for 19 hours (37°C, 5% CO ₂ ), followed by a slight modification as described above. Determined by the Board microdilution assay (42). Briefly, the biofilm in each well was incubated with 0.1M sodium acetate buffer (pH=5.5) in combination with serially diluted test glucanohydrolase for 120 minutes at 37°C. Final concentrations of dextranase and mutanase ranged from 0 to 17.50 U/mL. After incubation, each well was briefly washed with 0.89% sodium chloride to remove solubilized/separated biomass and residual biofilm was stained with 0.2% crystal violet for visualization and quantification. .. Relative reduction rates were calculated using that of the vehicle control group as a control (0% reduction). Using the drug interaction coefficient (CDI), the synergism/antagonism between two glucanohydrolases was calculated by the following formulas (R _Dex and R _Mut , relative reduction with dextranase or mutanase alone; R _{Dex + Mut} , (Relative reduction of Dex/Mut combination).

CDI<0.7 shows a significant synergistic effect. CDI=1 indicates an additive effect. CDI>1 shows antagonism (43).

Therapeutic Regimen of EPS Degradation/Antibacterial (EDA) Approach The anti-biofilm effect of the EDA approach depends on the preformed biofilm (efficacy of biofilm disruption) and the process of biofilm formation (efficacy of biofilm prevention). Tested using both. For biofilm disruption, preformed biofilms of sHA (19 hours for single species, 43 hours/67 hours for mixed species biofilms) were treated with: 1) Vehicle control ( 0.1 M sodium acetate buffer, pH=5.5); 2) 120 minutes optimal concentration of EPS degrading enzyme, then biofilm for 1 minute with full strength antimicrobial agents (EOs) and harvested for further analysis. did. For the prevention of biofilm, sHA discs were treated topically with EPS-degrading enzyme (optimum concentration) or vehicle control (0.1 M sodium acetate buffer, pH=5.5) for 60 minutes before inoculation, single species or mixed The seeds were incubated to allow colonization and biofilm formation. At the end of the experimental period (19 hours for single-species biofilm, 43 hours for mixed-species biofilm), biofilms were exposed to EO at maximum intensity for 1 minute and analyzed at harvest.

Quantitative biofilm analysis Biofilms have been the subject of biochemical and microbiological analysis, as described elsewhere in detail (22,25). Briefly, HA surface biofilms were harvested, homogenized by sonication, serially diluted using an automated Eddy Jet Spiral Plater (IUL, SA, Barcelona, Spain) and plated on blood agar. The CFU (CFU per biofilm) of each hydroxyapatite disc was determined. In mixed-species biofilms, three were distinguished by observing colony morphology on blood agar and the percentage of various species was calculated (total CFU per biofilm was considered 100%). An aliquot of the biofilm suspension was centrifuged (5,500 g, 10 min, 4°C), the pellet washed with water, oven dried and the water insoluble dry weight was determined (105°C, 24 hours). Water-soluble and water-insoluble polysaccharides in the biofilm matrix were extracted and colorimetrically determined using the phenol-sulfuric acid method as previously detailed (44,45). At least 3 independent biofilm experiments were performed for each assay.

The effect of the EDA approach on biofilm 3D architecture was analyzed by super-resolution fluorescence confocal microscopy at various time points using a well-established protocol (24). Briefly, EPS acts as a primer for the synthesis of glucan during biofilm formation, Alexa Fluor 647 dextran, while bacterial cells were stained with 2.5 μM SYTO 9 green fluorescent nucleic acid stain (485/498 nm, Molecular Probes). Dynamically labeled by incorporation of conjugate (final concentration, 1 μM, molecular weight, 10 kDa, 647/668 nm, maximum absorbance/fluorescence emission of Molecular Probes, Eugene, Oreg.). Imaging was performed using a single photon laser scanning microscope (LSM800, Zeiss, Germany) equipped with a 20× (1.0 numerical aperture) water immersion objective. Each biofilm was scanned in five randomly selected areas and a confocal image series was generated by optical sectioning at each of these locations. Computational analysis of confocal images using Comstat2 (www.comstat.dk) was performed to determine bacterial and EPS biovolumes to complement biochemical and microbiological analysis (46). Amir 5.4.1 software (Visage Imaging, San Diego, Calif., USA) was used to create a 3D rendering of the biofilm architecture.

Four-Dimensional Time-Lapse Confocal Live Imaging and Analysis The dynamic impact of EDA on biofilm EPS structure, mechanical stability, and killing effect is described by Xiao et al. EPS evaluated using 4D (x, y, z, t) time-lapse confocal live imaging modified by (24) was performed using 1 mM Alexa Fluor 647 dextran conjugate supplemented to the medium. Labeled At the end of biofilm formation (19 hours for single-species biofilm, 67 hours for mixed-species biofilm), sHA discs were treated with 5 μM SYTO9 (for live cell staining; Molecular Probes) and 4 mL of 0.1 M sodium acetate buffer ( Transferred to petri dishes (35 mm diameter) containing 30 μM propidium iodide (pH 5.5) for staining dead or membrane-damaged cells, Molecular Probes. This allowed continuous labeling and real-time visualization of live and dead cells throughout the experimental period. After initial staining at 37° C. for 30 minutes, multi-channel images were acquired using a LSM800 single photon high resolution confocal microscope (Zeiss) equipped with a 20× water immersion objective. Immediately after completion of the first scan, EPS degrading enzyme or vehicle buffer was carefully added to the solution and mixed well. In the 4D time-lapse series, images of the same field of view were acquired every 10 minutes after adding EPS degrading enzyme. At 120 minutes, antimicrobial agents (EOs) were added to the same buffer and mixed until the final concentration was 25% of maximum strength. This concentration was optimized in preliminary experiments to obtain the highest killing effect with minimal impact on imaging. Imaging of the same field was initiated 1 minute after the antimicrobial challenge. The total EPS biomass in each series of confocal images was algorithmically analyzed using COMSTAT2 (46). Computational analysis of the dynamics of biofilm structural stability (single particle tracking) was performed using the open source platform TrackMate (47, 48). The movement of each microcolony is quantified as a cumulative displacement and is a measure of how much it has moved during the observation period. Fiji (Distributed by ImageJ) (49) and Amira (Visage Imaging) were used to create the image processing and time-lapse rendering of the biofilm architecture.

Effect of EPS Degrading Enzyme on Glucan Synthesis by Purified GtfB GtfB was obtained from Streptococcus milleri KSB8 and described in Venkitaraman et al. And Koo et al. Purified to near homogeneity by hydroxyapatite column chromatography as described in more detail in (50, 51). Gtf activity was measured by incorporation of ¹⁴ C-glucose into glucan from radiolabeled sucrose (PerkinElmer, Mass., USA) (50). One unit (U) of GtfB activity is defined as the amount of enzyme that takes up 1 μmol glucose in a 4-hour reaction. Glucan synthesis in the presence of EPS-degrading enzymes is described by Hayacibara et al. It was carried out with some modifications as described in detail by (21). Briefly, GtfB (10 U) was mixed with dextranase or mutanase (range 0-50 U) and added to ([ ¹⁴ C]glucosyl)-sucrose substrate (0.2 μCi/mL; 200.0 mM sucrose, 40 μM dextran). T-10,0.02% sodium azide adsorption buffer: 50 mM of KCl, 0.35 mM of _{K 2 HPO 4, KH 2 PO} 4, 1mM of CaCl ₂ of 0.65mM, MgCl ₂ · 6H of 0.1mM Glucan is synthesized by incubating for 4 hours at 37° C. in ₂ O, pH 6.5). To combine the effects of dextranase and mutanase, 1 U of mutanase with varying amounts of dextranase (0-10 U) was used in the assay. Insoluble glucan was collected after centrifugation (13,400 g, 4° C., 10 minutes) and washed with water three times. Soluble glucan was precipitated with ethanol (final concentration: 70%) at -20°C for 18 hours. The amount of radiolabeled insoluble and soluble glucan was quantified by scintillation counting (50).

Fluorescence in situ hybridization (FISH) of mixed-species biofilms
FISH was used to understand the mechanism of EDA-induced precision and improved efficacy of anti-biofilms, as described elsewhere with modifications, using morphogenesis of mixed-species biofilms. And the spatial distribution of multimicrobial populations in an ecological biofilm model was evaluated (52-54). Briefly, mixed seed biofilms formed of sHA were gently washed twice with phosphate buffered saline (PBS) and 4% paraformaldehyde (PBS at pH 7.4) at 4°C. I fixed the time. After washing with PBS, the samples were transferred to 50% ethanol (in PBS, pH 7.4) and stored at -20°C. For hybridization, bacterial cells were first permeabilized by treatment with lysozyme (Sigma, 400,000 mM Tris-HCl pH 7.5, 400,000 U/mL in 5 mM EDTA) for 14 minutes at 37°C. A hybridization solution (25% formamide, 0.9 M NaCl, 0.01% SDS, 20 mM Tris-HCl, pH 7 containing a FISH oligonucleotide probe (SMU587, 5'-ACTCCAGACTTTCCTGAC-3' (SEQ ID NO: 28)). .5), S. mutans; MIT588, 5'-ACAGCCTTTAACTTCAGACTTATCTAA-3' (SEQ ID NO: 29), Alexa Fluor 488; Of Cy3, ACT476 for A., and Alexa Fluor 594 for A. naeslundii, each probe at a final concentration of 1 μM, biofilms were incubated for 4 hours at 46° C. and washed (0.2 M NaCl, 20 mM Tris-HCl). , PH 7.5, 5 mM EDTA, 0.01% SDS) at 37°C for 15 minutes. High resolution images were acquired using a LSM800 single photon confocal microscope (Zeiss) with a 20× water immersion objective. Each field was imaged using continuous excitation with 640 nm, 561 nm, and 488 nm laser lines, and the entire emitted fluorescence spectrum was detected as a lambda stack (55). The Zeiss ZEN software applied the linear mixing algorithm to the lambda stack using the reference spectra of all fluorophores in the specimen. The unblended images were combined and false colored using Fiji (49).

Evaluation of initial bacterial attachments The cariogenic pathogen S. mutans produce a glucan-binding protein that greatly promotes tooth surface colonization in the presence of Gtf synthetic glucan (56), while other commensals adhesin- mediating preferential attachment to salivary pellicle. It depends on receptor interactions (57). In order to investigate the effect of EPS-degrading enzymes adsorbed on the surface on the binding affinity of multi-microorganisms and to mechanically understand the effect on the initial microbial colonization, radioisotope tracing spectroscopy was used to analyze bacterial Adhesion was analyzed (21,58). Briefly, S. mutans, S.M. oralis, and A. naeslundii was grown into radiolabeled bacteria at 37° C., 5% CO ₂ until exponential metaphase in UFTYE medium supplemented with 10 μCi ³ H-thymidine (PerkinElmer) and 1% (w/v) glucose to obtain cell density. (Cells/mL) was measured using a Petroff-Hausser count chamber. Hydroxyapatite beads coated with saliva (Bio-rad, CA, USA, 80 μm particle size) were pretreated with vehicle or glucanohydrolase in the optimal combination prior to GtfB immobilization, and the glucan was sucrose substrate (200 μm). 0.0 mM sucrose, 40 μM dextran T-10, 0.02% sodium azide (in adsorption buffer pH 6.5) were synthesized in situ for 4 hours. The glucan-coated surface was then incubated with 1.0×10 ⁹ cell/mL isotope-labeled bacteria in adsorption buffer at 37° C. for 1 hour. Hydroxyapatite beads with saliva pellicle alone were used as a control. Unbound bacteria were removed by washing the beads 3 times with adsorption buffer. The affinity of bacterial attachment was quantified by scintillation counting. The actual number of adherent cells of the sHA beads was calculated using a standard curve of the measured radioactivity (counts per minute, CPM) and the number of radiolabeled bacteria.

Results EPS degrading enzymes synergistically enhance antibacterial and killing effects at optimal activity ratios.

EPS produced by Streptococcus Gtfs is an important component of the cariogenic biofilm matrix and contains mainly α-(1→3)-, α-(1→6)-, and high structural polymorphisms. It contains a mixture of glycosidic bonds composed of α-(1→4)-linked glucans (21). Therefore, targeting EPS-rich matrices may be difficult and may require the breaking of multiple specific chemical bonds. Glucanohydrolases such as dextranase [α-(1→6)glucanohydrolase] and mutanase [α-(1→3)glucanohydrolase] disrupt glucan synthesis by Gtfs and form preformed EPS. It has been shown to digest the matrix (16, 21, 22). To obtain the optimal effect, a high throughput biofilm model was first used to screen the EPS degrading activity of various combinations of dextranase and mutanase (FIG. 19D). Even at the highest concentration tested (17.5 U/mL, <25% of reduction), neither dextranase nor mutanase was able to efficiently degrade biofilms. However, the combination of dextranase and mutanase showed a synergistic effect on EPS degradation, with the highest reduction rate (activity ratio 5:1) at 8.75 U/mL dextranase and 1.75 U/mL mutanase. >60%) and the highest synergistic effect (drug interaction coefficient, CDI=0.635, showing a marked synergistic effect) was achieved.

To test our concept of multi-targeting, we developed a combination therapy combining glucanohydrolase with a clinically used Essential Oils (EOs) based formulation as a model antimicrobial agent. The established saliva coated hydroxyapatite (sHA) oral biofilm model was used to assess anti-biofilm effects (FIG. 19A). Biofilms were topically treated with EPS-degrading enzymes and immediately thereafter exposed to antibacterial agents (FIG. 19B). Consistent with the optimization of glucanohydrolase, the most potent killing activity was observed using 8.75 U/mL dextranase and 1.75 U/mL mutanase (1000-fold effect over antimicrobial agent alone). Target) (FIG. 19C). Since the glucan matrix also possesses a significant proportion of α-(1→4) glycosidic bonds, it was also investigated whether inclusion of glucoamylase [α-(1→4)glucanase] could further improve efficacy. .. Only a modest increase in killing effect (about 0.5 log CFU) was observed when compared to the optimized dual enzyme combination (FIG. 20F). We show that the dual enzyme approach provides sufficient high EPS degrading activity to validate the concept proposed in this study. Overall, the data suggest that dextranase and mutanase can synergistically degrade the protective EPS matrix and greatly enhance the killing of biofilm cells.

EPS degrading enzymes degrade the biofilm matrix in situ and promote antimicrobial targeting within the biofilm.

The EPS matrix has been recognized as an important factor in drug resistance of biofilms (4). However, it remains unclear how the matrix and structural organization of the biofilm 3D architecture interferes with antimicrobial efficacy. A high-resolution time-lapse confocal microscope was used to observe the spatiotemporal morphological changes of the biofilm following EPS degradation. As shown in FIG. 20A, vehicle-treated biofilms are densely packed with bacteria (green) called microcolonies (1-3) embedded with the abundant amount of EPS matrix (red) normally found in cariogenic biofilms. ) Shows clusters of cells. In sharp contrast, biofilms treated with the combination of dextranase and mutanase, which were essentially lacking EPS, showed bacterial cell dispersal (white arrows; Figures 20A-d). Neither dextranase nor mutanase was able to completely degrade the preformed biofilm matrix. Consistent with the confocal image data, the dual enzyme treatment significantly reduced biomass (FIG. 20C) with minimal exopolysaccharide content (FIG. 20D), compared to glucanohydrolase alone, and a single Targeting glycosidic bonds was shown to be insufficient to completely degrade the biofilm matrix.

Next, we investigated how EPS degradation modulates the antimicrobial kill profile at the single microcolony level. The time-lapse killing assay was performed using real-time bacterial live/dead staining and EPS imaging (FIGS. 20B and 21E). The biofilm without dual enzyme treatment (vehicle treatment) contains many viable cells (green) after short-term (1 minute) and long-term (5 minutes) exposure to topical antibacterial agents (EOs). (FIG. 20B, top). It was found that bacterial cells near the surface (white arrows) are mostly dead (magenta) upon exposure to the antibacterial agent, whereas cells present within the microcolonies are mostly viable (green). However, following EPS degradation by glucanohydrolase, the antimicrobial effectively killed bacteria both inside and outside the disrupted microcolonies (FIG. 20B, bottom). These findings were further validated by microbial analysis and showed that glucanohydrolase in combination synergistically enhanced the antimicrobial killing of antimicrobial agents (about 3 logs more effective killing versus EOs alone, p<0. On the other hand, the enzyme alone had no antibacterial activity (p>0.05 vs. vehicle treated control) (FIG. 20E). Thus, glucanohydrolase appears to be the "disorder for the physical barriers" provided by the EPS matrix, thus exposing bacterial cells to enhance antibacterial activity.

To further understand the dynamics of enzymatic disassembly of EPS matrix and its associated rebellious protective mechanisms, time-resolved super-resolution confocal imaging was used to study the spatiotemporal decomposition of EPS in situ (Figure 21A and 21B). Visualizing the ultrastructural features of the matrix within a single microcolony reveals a complex network of cross-linked EPS (Figure 21A, red). Interestingly, the combination of dual enzymes efficiently degraded EPS, both outside and inside the microcolony (Fig. 21B, white arrow), and was more effective and uniform after 1 minute exposure to EOS. Bacteria were killed (Figure 21D, live and dead green and magenta, respectively). In contrast, vehicle-treated microcolonies showed a bacterial central nucleus (Fig. 21C, dashed circle) predominantly composed of live cells (green), while the outer layer was viable after antimicrobial exposure. A mixture of cells and dead cells was retained (live bacteria are shown in the dashed box inserted). The data show that the combination of dextranase and mutanase, in addition to degrading the surrounding EPS, can reach the interior of bacterial microcolonies and digest EPS scaffolds that enhance antibacterial effects.

The mechanical stability and integrity of biofilm scaffolds is compromised by EPS degradation.

The presence of the exopolysaccharide matrix provides an essential scaffold for mechanical stability, regulates biofilm resistance to mechanical clearance from the substratum (23), and poses another challenge for developing biofilm disruption strategies. To do. Here, we investigated whether disassembling the matrix scaffold could affect the physical integrity and stability of the biofilm architecture. Time-lapse imaging was used to follow dynamic changes in biofilm structure during EPS degradation. A "implosion-like" collapse of the physical structure was observed as a result of the removal of EPS. There, the entire microcolony essentially shattered to the bottom of the sHA surface with cell dispersal (FIG. 22A). To further measure this kinetic process, computational analyzes were performed to generate time-resolved EPS degradation (red squares) and microcolony spatial displacement (green circles) curves. As shown in FIG. 22B, EPS was removed over time with a higher failure rate during the first 30 minutes (a reduction of approximately 70%), at which point the decomposition of EPS slowed down over time. In particular, "physical disintegration" was detected late in EPS degradation (after 70 minutes) and appeared as a detectable displacement of microcolonies. This indicates that full disassembly of the EPS scaffold and 3D integrity may be required that completely compromises the mechanical stability of the biofilm. Overall, the data show that the EPS degradation/antimicrobial dual approach (EDA) produced EPS and cariogenic S. cerevisiae. It has been shown that biofilms mediated by mutans can be strongly destroyed.

EDA locally degrades EPS to enhance targeting of EPS-producing pathogens in mixed-species biofilms.

Production of EPS in the presence of sucrose regulates the transition from symbiont-rich communities to pathological biofilms. Promotes establishment of mutans (22). Here, we hypothesized that the EDA approach could target EPS-producing pathogens in a mixed species model that mimics the ecological development of cariogenic biofilms (22, 24). In the ecological biofilm model, S. mutans is S. oralis and A. Co-cultured with naeslundii, forming an early microbial community in sHA (FIGS. 23A and 23B). Next, sucrose was introduced to characterize the EPS-rich matrix and acidity from a symbiont-rich community (high in S. oralis, low in A. naeslundii cells, and low in S. mutans). Micro environment (22, 25) S. Induces ecological changes to biofilms that promote mutans growth (FIGS. 23A and 23B). Therefore, we investigated whether the EDA approach could disrupt the establishment of pathogen-dominant biofilms under cariogenic conditions (treatment plan shown in Figure 23A). To demonstrate this concept, the EDA approach was tested at two different time points representing different ecological stages of mixed-species biofilms (FIG. 23B). S. Similar killing by antimicrobials (EOs) was observed with or without EPS degradation at 43 hours (early stage), when mutans were not predominant in low EPS production (FIG. 23C). The accumulated EPS is particularly abundant and S. At 67 hours (late stage) when mutans became dominant, EDA had a higher overall killing effect compared to antimicrobial agents (EOs) alone (FIG. 23D, top). Interestingly, the inclusion of EPS degrading enzyme resulted in a more selective killing of the bacteria (FIG. 23D, top) and S. S. than oralis (about 0.5 log reduction). Very high potency against mutans (about 2.0 log reduction vs antibacterial), leading to a symbiotic advantage in biofilms (FIG. 23D, bottom).

Given the efficiency of disrupting the pathogenic predominance, we further characterized the spatial organization of the EPS matrix, pathogens and symbionts of mature mixed-species biofilms by fluorescence in situ hybridization (FISH). Representative confocal images showed that the bacterial components were organized into highly structured and characteristic spatial organization (FIG. 23E). The biofilm structure has a S. EPS-embedded S. It was characterized by the presence of mutans cell clusters (FIG. 23E b, cross section overlay). Using the phrase "core-corona", the dense S. elegans entangled in EPS (shown in red). S. mutans that form a halo-like structure surrounding a microcolony of M. mutans (shown in green). The consortium structure generated from the oralis community (shown in yellow) is described (Fig. 23Eb-f). Such spatial and protected tissue can be transformed into S. cerevisiae within established mixed biofilms using only antimicrobial agents. It seems to be difficult to target mutans. Conversely, time-lapse confocal microscopy revealed that the combination of dual enzymes allowed for the local degradation of EPS, exposing the embedded bacteria and enhancing the bactericidal activity of antimicrobial agents (Fig. 23F). Taken together, the EDA approach can target mixed-species EPS-producing pathogens by eliminating structural and toxic properties, thereby increasing the accuracy and efficacy of anti-biofilms.

EPS Degrading Enzyme Prevents Biofilm Formation by Inhibiting In Situ Glucan Synthesis Previous studies have shown that hydroxyapatite and EPS formed on the surface of microorganisms promote bacterial attachment and aggregation. There is. Specifically, EPS glucans synthesized from dietary sucrose by Streptococcus Gtfs have a central importance in adhesive interactions that provide a scaffolding material for initiation of biofilms (26). Our data indicate that EPS-degrading enzymes can prevent the formation of biofilms given their strong effect on preformed biofilms. To address this issue, EPS-degrading enzymes were topically applied to the experimental salivary pellicle surface (sHA) prior to biofilm formation (FIG. 24A). Glucanohydrolase activity on the sHA surface pretreated with dextranase/mutanase was confirmed (Table 3) and the surface-formed EPS could be degraded (Fig. 22C). S. cerevisiae formed on sHA. A representative 3D rendering of the mutans biofilm is shown in Figure 24B. Although biofilm formation of dual enzyme pretreated sHA was very defective and could not form EPS matrix or microcolonies, biofilm formed with single enzyme pretreated sHA showed changes with detectable EPS. It showed (albeit reduced) the formation of microcolony structures (white arrows, Figure 24B). Microbiological data showed that the combination of dual enzymes was more effective in suppressing biomass accumulation (Figure 24C) and reducing total biofilm CFU (Figure 24D) than dextranase or mutanase alone. .. Also, the biofilm formed on the pretreated sHA surface is exposed to an antibacterial agent to determine if it is more susceptible to killing. A 100-fold effective killing (total >4 log CFU or more reduction) was observed with enzyme pretreated sHA surface biofilms (relative to vehicle pretreated surface) (FIG. 23E). ).

To further understand EPS degradation in situ, a glucan synthesis assay was performed in the presence of dextranase and/or mutanase to determine if the biosynthesis and composition of Gtf-derived glucan was affected. In general, both glucanohydrolases interfered with the amount and proportion of soluble/insoluble glucan synthesized (Figure 24F). The presence of dextranase affected the ability of GtfB to synthesize insoluble glucan, but reached a plateau of inhibitory activity (up to 60% reduction in insoluble glucan relative to vehicle control) (FIG. 24F, left), previously. (21). Mutanase completely inhibited insoluble glucan synthesis at low concentrations, but enhanced production of soluble glucan (Fig. 24F, middle). However, the combination of the dual enzymes completely abolished the synthesis of insoluble and soluble glucans, confirming the synergistic effect of dextranase and mutanase (Fig. 24F, right). Taken together, these data indicate that EPS degrading enzymes can inhibit glucan biosynthesis by bacterial Gtf and thus prevent biofilm formation in situ via a glucan-dependent mechanism.

The EDA approach is based on the S. Selectively prevent early colonization of mutans.

Surface-formed EPS provides a binding site for glucan-binding cariogenic pathogens (such as S. mutans) in the presence of sucrose, promoting their colonization and destroying symbiotic-rich communities. Was shown (22). Using a mixed-species ecological model (supra), the effect of the EDA approach on the dynamics of microbial population change was first investigated using a therapeutic regimen, as shown in Figure 25A. At 43 hours (early cariogenic biofilm, 14 hours after sucrose introduction), dramatic changes in microbial population were observed. Early mixed biofilms formed with sHA pretreated with EPS-degrading enzyme showed S. cerevisiae compared to vehicle control. Although it showed a 2-log reduction in the mutans population (CFU), S. oralis is also A. The naeslundii population was also unaffected (FIG. 25B), suggesting a selective inhibitory effect on bacterial colonization. FISH using three species-specific probes (representative image shown in FIG. 25C) shows A. naeslundii (shown in cyan) and S. mutans (shown in green) cells associated with a large number of S. Oralis (displayed in yellow) was shown (FIG. 24C left). In contrast, symbiotic animals, especially S. oralis occupied the biofilm formed by enzyme-treated sHA (Fig. 25B right graph and 25C right graph). Notably, topical treatment with antibacterial agents was performed in the S. Although the mutans were completely removed, a large number of S. Mutans cells were detected (Fig. 25D).

S. Since mutans can express a glucan-binding protein that promotes heat adhesion to EPS(27) formed on the surface, it was considered that glucanohydrolase can selectively prevent colonization of EPS-producing pathogens. S. To further understand the selective destruction of mutans, bacterial adherence to glucan-coated sHA surfaces was assessed by ³ H-thymidine radioisotope tracing spectroscopy. Symbiotic bacteria (S. oralis and A. naeslundii) bind to salivary pellicle more effectively than pathogens (S. mutans), while glucan produced in pellicle significantly suppresses symbiotic binding, S. It was found to enhance the binding of mutans (Fig. 25E). Interestingly, glucanohydrolase pretreatment of pellicle prior to glucan formation has various effects on bacterial binding affinity, S. It was advantageous for symbiotic attachment with the harmful effects of mutans. This finding indicated that selective inhibition of bacterial colonization was associated with pathogen adhesion disruption by a glucan-dependent mechanism.

Discussion Biofilm drug resistance poses significant challenges to existing antimicrobial monotherapy, demonstrating the urgent need for a multitarget or combination approach (2). Here, a readily available EPS-degrading enzyme in combination with a clinically used antibacterial agent targets the protective matrix and synergizes anti-biofilm effects by amplifying local killing in local therapeutic regimens. Demonstrate that it can be enhanced. Importantly, this strategy can spatially target EPS-producing pathogens within mixed-species biofilms by interrupting colonization and exposing in situ to enhance antimicrobial clearance. Our studies show that matrix disruption with antibacterial action produces optimal biofilm control, while targeting EPS-producing pathogens in the context of mixed-species communities, thereby providing a powerful yet selective anti-biofilm. It shows that it provides an approach.

The importance of extracellular matrix in the antimicrobial resistance of biofilms is increasingly recognized (28). However, the mechanism by which the matrix enhances bacterial resistance to antimicrobial agents is not well understood. Explaining in detail the effects of the EPS matrix on spatiotemporal degradation and enhanced killing of bacteria and on the occurrence of degradation of biofilms, further on the role of the EPS matrix in creating a "protective physical scaffold" for existing bacteria. Gain insight. Using time-resolved biofilm imaging with a highly specific EPS-degrading enzyme, we have discovered how matrix disassembly optimizes biofilm prevention and destruction, and overcomes antimicrobial resistance. First, the enhancement of antibacterial effect by glucanohydrolase was closely associated with the effectiveness of EPS degradation. Our data indicate that simultaneous degradation of α-(1→3) and α-(1→6) glycosyl bonds in the glucan matrix is required to effectively digest the matrix and disrupt the microcolony structure. It was revealed. A significant enhancement of the antibacterial effect was observed by facilitating access and killing of bacterial cells inside the EPS-degraded microcolonies, concomitant with structural disassembly. Moreover, glucanohydrolase pretreated sHA surfaces can reduce EPS production in situ, prevent the establishment of EPS-embedded bacterial clusters, and promote killing of the resulting defective biofilms. Thus, despite the lack of intrinsic antibacterial activity, glucanohydrolases can be potent adjuvants for various types of antibacterial agents. While the combination of dextranase and mutanase was effective for EPS matrix degradation, additional glucosyl linkages such as α-(1→4) and other biopolymers were also found in the cariogenic biofilm matrix ( 29-31). For example, the presence of extracellular DNA and lipoteichoic acid within the dental biofilm matrix is also important for biofilm structure, and topical treatment with DNase has been shown to help disperse the initial biofilm. It was also found that the inclusion of a glucoamylase targeting α-(1→4) bond can improve the overall digestion of the EPS matrix and further enhance the antibacterial effect. Therefore, it is expected that matrix degradation performance can be maximized by targeting various extracellular polymeric substances. This may further enhance the effect of eliminating matrix-induced antimicrobial resistance.

Resistance to mechanical clearance represents another important mechanism by which biofilms adhere persistently to abiotic and biological surfaces, making physical removal difficult (13, 32). Our data highlight the ability of EPS-degrading enzymes to compromise mechanical stability and cause structural disruption of biofilm architecture. The observed "physical disintegration" strongly indicates that glucanohydrolase can degrade the biofilm core scaffold structure. In particular, the enzyme can also induce dispersal of the bacterial cells by degrading the adhesive polysaccharide that encapsulates and stabilizes the bacterial cells. In the clinical setting, dispersed cells from biofilms revert to suspension and become more susceptible to killing by antimicrobials and host defenses (2). However, it is worth noting that biofilm dispersal also contributes to transmissible transmission, mediating intra-host spread/persistence of pathogens (33), leading to risk of recolonization or bacteremia. Therefore, EPS degradation approaches need to be co-administered with antibacterial agents to reinforce the notion of synergistic killing of bacteria and preventing bacterial colonization in situ. These findings suggest the potential use of EDA as an anti-biofilm therapeutic approach with both dispersive and prophylactic effects.

Bacterial species within mixed-species biofilms exhibit extensive and complex competition under the influence of a multifactorial microenvironment (34). Previous work on matrix targeting strategies has largely been done using a single species biofilm model. Here, we evaluate the effectiveness of this approach for the first time using a mixed-species biofilm model. Unexpectedly, the EDA approach has resulted in the EPS-producing oral pathogen S. It has been discovered that mutans can be selectively targeted to prevent colonization and overgrowth in a mixed-species ecological biofilm model, thus providing symbionts with a competitive advantage. Glucanohydrolase disrupts S. cerevisiae by disrupting the glucan-dependent binding mechanism. It was found that it is possible to target colonization of mutans. This is advantageous for the attachment and accumulation of pathogens. Unlike attachment of symbionts (such as S. oralis and A. naeslundii), which are largely dependent on the interaction between salivary adhesins and surface-immobilized receptors (35), S. Mutans significantly promotes adhesion by glucan-dependent mechanisms such as surface-adsorbed Gtf and glucan-binding proteins (Gbps) (36). Using a single cell-based assay, it was discovered that glucanohydrolase selectively interferes with colonization of EPS-producing pathogens by disrupting glucan production in situ. The sHA surface coated with glucan is S. It was found to promote adhesion and further accumulation of mutans cells. Conversely, degradation of EPS on glucan-coated surfaces reestablished symbiotic colonization, which may be due to exposing the pellicle receptor for adhesin-mediated binding by symbiotic bacteria.

Due to the heterogeneous architecture and diverse species distribution, certain species can develop protective microenvironments for killing (2). In our model, S. mutans can create EPS-microcolony complexes that confer greater resistance to antimicrobial load than symbionts. Our data indicate that the EDA approach can spatially target EPS-producing pathogens by locally degrading the protective matrix and promoting bacterial killing in situ. Such a synergistic effect is evident in comparison to conventional antimicrobial monotherapy, where the drug fails to eradicate the preformed biofilm matrix, leaving the entrapped pathogens in the matrix. Provide significant benefits. Collectively, our combined approach has a community-level impact on mixed-species biofilms, preventing pathogen colonization and overgrowth from an ecological perspective.

While certain preferred embodiments of the invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the invention as set forth in the appended claims.

Claims (25)

A composition, one or more essential oils that act synergistically on a biologically acceptable carrier for the delivery of said composition to degrade biofilm structure and inhibit biofilm deposition. And a composition having at least one biofilm-degrading enzyme. The composition of claim 1, wherein the essential oil comprises 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. Composition. The composition according to claim 2, wherein the essential oils are menthol, thymol, eucalyptol, and methyl salicylate, and the biofilm-degrading enzymes are mutanase and dextranase. The composition of claim 2, wherein the biofilm-degrading enzyme is produced in plant plastids. The composition of claim 3, further comprising:
A composition comprising glucoamylase. The composition of claim 1, wherein the biofilm-degrading enzyme has a dextranase to mutanase ratio of 5:1. A composition comprising an effective amount of one or more biofilm degrading enzymes selected from mutanases, dextranases, and glucoamylases, and menthol, thymol, eucalyptol, methyl salicylate, and two or more thereof. Having one or more essential oils selected from the group consisting of combinations in a carrier suitable for oral delivery, said carrier being a mouthwash, mouthrinse, mouth spray, toothpaste, chewing gum, tooth gel, gingiva A composition selected from the group consisting of lower gels, mousses, foams, chewable tablets, dentifrices, lozenges, and dissolvable strips. The composition of claim 7, wherein the carrier is a mouthwash, mouthrinse, or mouthspray. 8. The composition of claim 7, wherein the carrier is chewing gum, chewable tablets, lozenges, or dissolvable strips. The composition of claim 7, wherein the carrier is chewing gum. The composition of claim 7, wherein the essential oil comprises a combination of menthol, thymol, eucalyptol, and methyl salicylate. A method for degrading and/or removing unwanted biofilms from the oral cavity, wherein one or more biofilm degradations are selected from the group consisting of mutanases, dextranases, glucoamylases, and combinations of two or more thereof. Administering an effective amount of an enzyme 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 the surface of the oral cavity having the biofilm. A method comprising the steps of: the enzyme degrading the film and the essential oil exert a bactericidal effect, thereby synergistically reducing or eliminating the undesirable biofilm. 13. The method of claim 11, wherein the one or more essential oils comprises at least two essential oils selected from menthol, thymol, eucalyptol, and methyl salicylic acid. 14. The method of claim 13, wherein the essential oil comprises a combination of menthol, thymol, eucalyptol, and methyl salicylate. The method according to any one of claims 12, 13 or 14, wherein the symbiotic bacterium A. naeslundii and S. pathogenic S. oralis without adversely affecting Oralis. A method that is effective in selectively killing mutans. 13. The method of claim 12, wherein the administering step comprises first administering the biofilm-degrading enzyme and subsequently administering the essential oil. A method for suppressing the deposition of biofilm on the surface of the oral cavity, which comprises the surface of the oral cavity susceptible to biofilm deposition, mutanase, dextranase, glucoamylase, and a combination of two or more thereof. One or more selected from the group consisting of menthol, thymol, eucalyptol, methyl salicylate, and combinations of two or more thereof, contacted with an effective amount of one or more biofilm degrading enzymes selected from the group Of the essential oil of the biofilm having a step of contacting the surface of the oral cavity, the enzyme for degrading the biofilm and the essential oil exert a bactericidal effect, thereby, the deposition of the undesirable biofilm. How to suppress synergistically. 18. The method of claim 17, wherein the antimicrobial oral rinse comprises at least two essential oils selected from menthol, thymol, eucalyptol, and methyl salicylate. 19. The method of claim 18, wherein the essential oil comprises a combination of menthol, thymol, eucalyptol, and methyl salicylate. 20. The method of any one of claims 17, 18, or 19 wherein the symbiotic bacterium A. naeslundii and S. pathogenic S. oralis without adversely affecting Oralis. A method that is effective in selectively killing mutans. 18. The method of claim 17, wherein the administering step comprises first administering the biofilm-degrading enzyme and subsequently administering the essential oil. The composition of claim 4 further comprising:
A composition having plant debris. 21. The composition of claim 20, wherein the plant is a tobacco or lettuce plant. The composition of claim 1, wherein the enzyme is recombinant, is expressed as a fusion protein in bacteria, and is purified therefrom. The composition of claim 1, wherein the enzyme is purified from a non-recombinant enzyme expressing bacterium.

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