Method for the treatment and the prevention of dental caries and peridontal diseases using bacterio phage-encoded enzymes

Abstract

A method for the treatment and prevention of dental caries and periodontal diseases using bacteriophages and phage-encoded anti-bacterial enzymes to inhibit establishment of bacteria in the oral cavity is provided. Also provided are methods for studying the cell wall of an oral bacterium, a method for preventing spoilage of perishable items and a method for removing dextrans from surfaces utilized in sugar manufacture. Purified enzymes and the isolated DNA fragments encoding them are also provided.

What is claimed is:  
     
         1 . A method for the treatment and prevention of dental caries and periodontal diseases comprising providing a phage-encoded anti-bacterial enzyme to an organism in an amount sufficient to kill bacteria in an oral cavity of said organism.  
     
     
         2 . The method of    claim 1   , wherein the enzyme is a lysozyme.  
     
     
         3 . The method of    claim 2   , wherein the lysozyme is active at low pH.  
     
     
         4 . The method of    claim 1   , wherein the enzyme is present in a bacteriophage lysate.  
     
     
         5 . The method of    claim 1   , wherein the enzyme is purified.  
     
     
         6 . The method of    claim 1   , wherein the bacteria are Actinobacillus, Actinomyces, Bacteroides, Capnocytophaga, Eikenella, Eubacterium, Fusobacterium, Haemophilus, Lactobacillus, Peptostreptococcus, Porphyromonas, Prevotella, Rothia, Selenomonas, Streptococcus, Treponema, Wolinella.  
     
     
         7 . A method for studying a cell wall of an oral bacterium comprising treating said cell wall with a phage-encoded cell wall-lytic enzyme.  
     
     
         8 . The method of    claim 7   , wherein the enzyme is a lysozyme.  
     
     
         9 . The method of    claim 8   , wherein the lysozyme is active at low pH.  
     
     
         10 . The method of    claim 7   , wherein the enzyme is present in a bacteriophage lysate.  
     
     
         11 . The method of    claim 7   , wherein the enzyme is purified.  
     
     
         12 . The method of    claim 7   , wherein the bacterium is Actinobacillus, Actinomyces, Bacteroides, Capnocytophaga, Eikenella, Eubacterium, Fusobacterium, Haemophilus, Lactobacillus, Peptostreptococcus, Porphyromonas, Prevotella, Rothia, Selenomonas, Streptococcus, Treponema, Wolinella.  
     
     
         13 . A method for the treatment and prevention of dental caries and periodontal diseases comprising providing a phage-encoded anti-bacterial enzyme to an organism, wherein the enzyme digests extracellular polysaccharides of the bacteria in an oral cavity of said organism.  
     
     
         14 . The method of    claim 13   , wherein the bacteria are Actinobacillus, Actinomyces, Bacteroides, Capnocytophaga, Eikenella, Eubacterium, Fusobacterium, Haemophilus, Lactobacillus, Peptostreptococcus, Porphyromonas, Prevotella, Rothia, Selenomonas, Streptococcus, Treponema, Wolinella.  
     
     
         15 . A method for preventing spoilage of fresh, refrigerated or pasteurized perishable items comprising treating said items with an amount of a phage-encoded anti-bacterial enzyme sufficient to inhibit growth of a gram-positive spoilage bacteria on said items.  
     
     
         16 . The method of    claim 15   , wherein the enzyme is a lysozyme.  
     
     
         17 . The method of    claim 15   , wherein the lysozyme is active at low pH.  
     
     
         18 . The method of    claim 15   , wherein the enzyme is present in a bacteriophage lysate.  
     
     
         19 . The method of    claim 15   , wherein the enzyme is purified.  
     
     
         20 . An isolated and purified phage-encoded anti-bacterial enzyme which inhibits the establishment of oral bacteria.  
     
     
         21 . The enzyme of    claim 20   , wherein the enzyme is a lysozyme.  
     
     
         22 . The lysozyme of    claim 21   , wherein the lysozyme is active at low pH.  
     
     
         23 . The enzyme of    claim 20   , wherein the bacterium is Actinobacillus, Actinomyces, Bacteroides, Capnocytophaga, Eikenella, Eubacterium, Fusobacterium, Haemophilus, Lactobacillus, Peptostreptococcus, Porphyromonas, Prevotella, Rothia, Selenomonas, Streptococcus, Treponema, Wolinella.  
     
     
         24 . A DNA fragment isolated from a bacteriophage which encodes an anti-bacterial enzyme which inhibits the establishment of an oral bacterium.  
     
     
         25 . The DNA fragment of    claim 24   , wherein the enzyme is a lysozyme.  
     
     
         26 . The DNA fragment of    claim 25   , wherein the lysozyme is active at low pH.  
     
     
         27 . The DNA fragment of    claim 24   , wherein the bacterium is Actinobacillus, Actinomyces, Bacteroides, Capnocytophaga, Eikenella, Eubacterium, Fusobacterium, Haemophilus, Lactobacillus, Peptostreptococcus, Porphyromonas, Prevotella, Rothia, Selenomonas, Streptococcus, Treponema, Wolinella.  
     
     
         28 . An expression vector comprising the DNA fragment of    claim 24   , operatively linked in proper reading to control elements which direct expression of said DNA fragment.  
     
     
         29 . A host cell comprising the expression vector of    claim 24   .  
     
     
         30 . A method for the removal of insoluble dextran polysaccharides synthesized by bacteria comprising providing a phage-encoded enzyme in an amount sufficient remove said insoluble dextran polysaccharides.  
     
     
         31 . The method of    claim 30   , wherein the insoluble dextran polysaccharides are produced in industrial sugar refining processes.  
     
     
         32 . The method of    claim 30   , wherein the enzyme is a dextranase.  
     
     
         33 . The method of    claim 30   , wherein the enzyme is present in a bacteriophage lysate.  
     
     
         34 . The method of    claim 30   , wherein the enzyme is purified.  
     
     
         35 . An isolated and purified phage-encoded enzyme which can digest insoluble dextran polysaccharides.  
     
     
         36 . The enzyme of    claim 35   , wherein the enzyme is a dextranase.  
     
     
         37 . A DNA fragment isolated from a bacteriophage which encodes the enzyme of    claim 30   .  
     
     
         38 . An expression vector comprising the DNA fragment of    claim 37   , operatively linked in proper reading to control elements which direct expression comprising of said DNA fragment.  
     
     
         39 . A host cell comprising the expression vector of    claim 38   .  
     
     
         40 . A DNA fragment isolated from a bacteriophage which encodes an enzyme which can remove dental plaque.  
     
     
         41 . A method for the removal of dental plaque comprising providing a phage-encoded enzyme in an amount sufficient remove said dental plaque.  
     
     
         42 . The method of    claim 41   , wherein the enzyme is a dextranase.  
     
     
         43 . The method of    claim 41   , wherein the enzyme is present in a bacteriophage lysate.  
     
     
         44 . The method of    claim 41   , wherein the enzyme is purified.  
     
     
         45 . An isolated and purified phage-encoded enzyme which can remove dental plaque.  
     
     
         46 . The enzyme of    claim 45   , wherein the enzyme is a dextranase.  
     
     
         47 . A DNA fragment isolated from a bacteriophage which encodes the enzyme of    claim 45   .  
     
     
         48 . An expression vector containing the DNA fragment of    claim 47   .  
     
     
         49 . A host cell transfected with the expression vector of    claim 47   .  
     
     
         50 . An antibody directed to the enzyme of    claim 20   .  
     
     
         51 . The antibody of    claim 50   , wherein the antibody is monoclonal.  
     
     
         52 . The antibody of    claim 50   , wherein the antibody is polyclonal.  
     
     
         53 . An antibody directed to the enzyme of    claim 30   .  
     
     
         54 . The antibody of    claim 53   , wherein the antibody is monoclonal.  
     
     
         55 . The antibody of    claim 53   , wherein the antibody is polyclonal.  
     
     
         56 . A vehicle for the delivery of the enzyme of    claim 20   , comprising a mouthwash, mouthrinse, topical gel, topical ointment, toothpaste, powder, slow release implant, slow release coating or chewing gum.  
     
     
         57 . A vehicle for the delivery of the enzyme of    claim 45   , comprising a mouthwash, mouthrinse, topical gel, topical ointment, toothpaste, powder, slow release implant, slow release coating or chewing gum.  
     
     
         58 . A genetically engineered, non-cariogenic organism which colonizes dental plaque and produces phage-encoded enzymes that inhibit establishment of a cariogenic organism.  
     
     
         59 . The genetically engineered organism of    claim 58   , wherein the non-cariogenic organism is  S. sanguis.    
     
     
         60 . The genetically engineered organism of    claim 58   , wherein the cariogenic organism it inhibits is  S. mutans.