Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
  • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
  • A01N25/34—Shaped forms, e.g. sheets, not provided for in any other sub-group of this main group
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
  • A23G4/00—Chewing gum
  • A23G4/06—Chewing gum characterised by the composition containing organic or inorganic compounds
  • A23G4/12—Chewing gum characterised by the composition containing organic or inorganic compounds containing microorganisms or enzymes; containing paramedical or dietetical agents, e.g. vitamins
  • A23G4/123—Chewing gum characterised by the composition containing organic or inorganic compounds containing microorganisms or enzymes; containing paramedical or dietetical agents, e.g. vitamins containing microorganisms, enzymes
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Preparation or treatment thereof
  • A23L2/52—Adding ingredients
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
  • A61K35/741—Probiotics
  • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
• • A—HUMAN NECESSITIES
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  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/02—Bacterial antigens
  • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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  • C12Y—ENZYMES
  • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
  • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
  • C12Y101/01001—Alcohol dehydrogenase (1.1.1.1)
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
  • A61K2800/74—Biological properties of particular ingredients
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K39/02—Bacterial antigens
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• Dental caries are one of the most prevalent chronic infectious diseases in the world. Over half of U.S. children age 5 - 9 have at least one cavity or filling; by age 17, nearly 80% of our young people have had a cavity.
• Tooth decay is characterized by the demineralization of enamel and dentin, eventually resulting in the destruction of the teeth. Dietary sugar is often misperceived as the cause of tooth decay; however, the immediate cause of tooth decay is lactic acid produced by microorganisms that metabolize sugar on the surface of the teeth. Studies suggest that of the approximately 700 oral microorganisms, Streptococcus mutans, a bacterium found in virtually all humans, is the principal causative agent in the development of tooth decay. Residing within dental plaque on the surface of teeth, S.
• mutans derives energy from carbohydrate metabolism as it converts dietary sugar to lactic acid which, in turn, promotes demineralization in enamel and dentin, eventually resulting in a cavity.
• the rate at which mineral is lost depends on several factors, including the number of S. mutans cells that are present and the frequency and amount of sugar that is consumed.
• Therapeutic regimens that take advantage of bacterial interference to replace a pathogenic bacterial strain such as S. mutans with a non-pathogenic, effector strain are known as replacement therapies.
• Successful replacement therapy requires an effector strain that: 1 ) is non-pathogenic, 2) alters the microenvironment to prevent colonization or outgrowth of a pathogenic organism, and 3) persistently colonizes the host at risk to prevent reinfection by the target pathogenic organism, and aggressively displaces the pathogenic organism from the tissues at risk in the case where the pathogen is part of the host's indigenous flora.
• Non-cariogenic effector strain of S. mutans e.g., an S. mutans strain deficient in lactic acid synthesis that can outcompete native S. mutans in the oral cavity of the host.
• S. mutans e.g., an S. mutans strain deficient in lactic acid synthesis that can outcompete native S. mutans in the oral cavity of the host.
• the mutants were used in a rat model to correlate lantibiotic production to colonization potential. It was found that the ability of these strains to preemptively colonize the host and aggressively displace indigenous strains of S. mutans increased significantly as the amount of MU1 140 produced increased.
• strains of S. mutans that produce elevated amounts of MU1 140 or produce variants of this molecule with increased specific activity. Such strains would reduce the period required for the effector strain to eliminate indigenous, lactic acid-producing strains and thereby achieve full effectiveness. Such strains are also more likely to overcome any inherent resistance to colonization, which, while not currently known, may exist in certain individuals in the population being treated. See, e.g., Hillman, Antonie van Leeuwenhoek 82: 361-366, 2002.
• the strain can further comprise a Trp4insAla mutation or a ATrp4 mutation.
• the following amino acid substitutions can also be present: Abu8Ala, or Dhb14Ala, or both Abu8Ala and Dhb14Ala.
• the strain can further comprise a mutation in a polynucleotide involved in ComE, ComC, or both ComE and ComC synthesis such that expression of ComE, ComC, or both ComE and ComC is diminished by about 80% or more as compared to a wild-type S. mutans strain.
• the strain can further comprising a mutation in a polynucleotide involved in D-amino acid synthesis such that expression of the D-amino acid is diminished by about 80% or more as compared to a wild-type Streptococcus mutans strain.
• the polynucleotide involved in D-amino acid synthesis can be dal or a promoter for dal.
• the recombinant alcohol dehydrogenase polynucleotide can be a Zymomonas mobilis alcohol dehydrogenase polynucleotide or a Streptococcus mutans alcohol dehydrogenase polynucleotide.
• Another embodiment of the invention provides a method of reducing the incidence or severity of dental caries in a dental caries-susceptible host comprising administering orally to the host an isolated recombinant S. mutans strain of the invention in an amount effective for replacement of dental caries-causing S. mutans host strains in the oral cavity of the host.
• the isolated recombinant S. mutans strain can be contained in a mouthwash, toothpaste, chewing gum, floss, chewable tablet, food, or beverage.
• Still another embodiment of the invention provides a pharmaceutical composition for reducing the incidence or severity of dental caries comprising an isolated recombinant S. mutans strain of the invention and a pharmaceutically acceptable carrier.
• the invention provides strains of S. mutans that are stable, lactic acid-deficient, and non-cariogenic that can aggressively outcompete native S. mutans due to, inter alia, the expression of a variant MU1 140 lantibiotic that has improved biological activity as compared to a wild-type MU1 140 lantibiotic.
• Figure 1A shows the wild-type MU1 140 structure (SEQ ID NO:1 ).
• Figure 1 B shows mutation sites of MU1 140 (SEQ ID NO:2).
• Figure 2 shows the sequence of chromosomal DNA highlighting mutations of variant MU1 140 lanA polynucleotide sequences with the wild type MU1 140 lanA polynucleotide sequence.
• Figure 3 shows the primers used for mutagenesis of lanA, the MU1 140 structural gene.
• Figure 4A-B shows the results of the zone of inhibition plate assays.
• Figure 5 shows the means and standard deviations for the bioactivity of strains producing variants of MU 1 140 compared to wild-type MU1 140.
• Figure 6 shows the biological activity of strains producing variants of MU1 140 (Phel lle and Phel Gly) compared to wild-type MU1 140.
• Streptococcus mutans can be recombinantly manipulated to produce no lactic acid or substantially reduced amounts of lactic acid. Hillman et al. J. Appl. Microbiol. 102:1209 (2007).
• Viable, lactic acid-deficient S. mutans strains can be generated by transforming the strains with nucleic acid encoding a recombinant alcohol dehydrogenase (ADH) such that a recombinant alcohol dehydrogenase is expressed, and introducing a mutation in the lactic acid synthesis pathway to render the recombinant ADH-producing strain lactic acid deficient.
• ADH recombinant alcohol dehydrogenase
• S. mutans strains can be recombinantly engineered to express a variant MU1 140 lantibiotic that has greater biological activity than wild-type MU1 140 lantibiotics. These strains can outcompete and replace dental caries-causing wild-type, native S. mutans strains in the oral cavity of hosts.
• any S. mutans strains can be used to construct the recombinant S. mutans strains of the invention.
• Recombinant S. mutans strains of the invention have a selective advantage over wild-type S. mutans strains that normally colonize the oral cavity.
• the selective advantage can be conferred by any of a variety of characteristics (e.g., production of an antibacterial compound, reduced or advantageous relative metabolic needs, greater relative growth rate, production of scavengers for metabolites) that promote oral cavity colonization by the strain and replacement of the resident strain colonizing the oral cavity.
• colonization by the recombinant S. mutans strains of the invention will not substantially disrupt colonization by other, non-S.
• mutans strains e.g., normal bacterial flora not associated with cariogenesis.
• infection with a recombinant strain of S. mutans that produces a variant MU1 140 lantibiotic with enhanced lantibiotic activity can result in replacement of the resident, cariogenic S. mutans strains without effect upon other resident microbial species of the oral cavity.
• a recombinant S. mutans strain is a non-naturally occurring strain of S. mutans that has been generated using any of a variety of recombinant nucleic acid techniques (i.e., techniques involving the manipulation of DNA or RNA).
• a recombinant S. mutans strain of the invention has a deficiency in lactic acid production; expresses a recombinant alcohol dehydrogenase (ADH) polypeptide; and expresses recombinant polypeptides sufficient to produce a variant MU1 140 lantibiotic that has greater biological activity than wild-type MU 1 140.
• ADH alcohol dehydrogenase
• mutans can optionally be deficient in ComE, ComC, or both ComE and ComC expression and/or can optionally be auxotrophic for an organic substance not normally present in the oral cavity or diet of a particular host (e.g., a D-amino acid).
• Variant MU 1140 can optionally be deficient in ComE, ComC, or both ComE and ComC expression and/or can optionally be auxotrophic for an organic substance not normally present in the oral cavity or diet of a particular host (e.g., a D-amino acid).
• MU1 140 has an overall horseshoe-like shape kinked at the "hinge region" between rings B and C. Smith et al. (2003) Biochem. 42:10372-10384. This shape is the result of a turn-like motif in the hinge region that folds the amino-terminal AB rings (the lipid II binding domain) towards the carboxy-terminal overlapped rings CD.
• the flexibility of the hinge region is believed to be important in promoting lateral assembly of MU1 140, enabling it to abduct and sequester lipid II.
• the ⁇ angle of Trp4 and ⁇ angle of Dha5 in ring A help contribute to its flexibility.
• sAla7 (a residue that is not confined by the thioether ring) rotates 360° allowing ring A to spin freely with respect to ring B. This flexibility is thought to be important in orienting rings A and B during lipid II binding.
• the hinge region also contains a potentially enzymatically susceptible arginine at residue 13. Mutations in the structural gene (lanA) for MU1 140 were generated to determine the effect of the following amino acid alterations: Phel lle, Phel Gly, Trp4Ala, Trp4insAla, ATrp4, Dha5Ala, Ala s 7insAla, and Arg13Asp. Figure 1 B.
• Trp4Ala substitution resulted in a statistically significant (p ⁇ .05) increase in bioactivity when compared to the wild-type. Since both amino acids are uncharged and hydrophobic, it can be speculated that the difference in bioactivity was due to the size difference between the two amino acids.
• Replacement of Dha5 with Ala also resulted in a statistically significant (p ⁇ .05) increase in bioactivity. Insertion of alanine after s Ala at position 7 resulted in a significant (p ⁇ .05) reduction of bioactivity.
• an effector strain producing a variant MU1 140 possessing one or more of these site-directed changes has the potential to be superior to an effector strain producing wild type MU1 140 by improving its ability to colonize the oral cavity and aggressively displace disease-causing, indigenous strains of S. mutans.
• Variants of the lantibiotic MU1 140 of the invention are polypeptides comprising post-translational modifications.
• Post-translational modifications are chemical modifications of a polypeptide after it has been translated.
• a polypeptide is a polymer of two or more amino acids covalently linked by amide bonds.
• a purified polypeptide is a polypeptide preparation that is substantially free of cellular material, other types of polypeptides, chemical precursors, chemicals used in synthesis of the polypeptide, or combinations thereof.
• a purified polypeptide does not include unpurified or semi-purified cell extracts or mixtures of polypeptides that are less than 70% pure.
• Wild-type MU1 140 is shown in Figure 1A.
• One embodiment of the invention provides one or more of the following variants of the lantibiotic mutacin, MU1 140, shown in Figure 1 B (SEQ ID NO:2). That is, the invention includes variants of the wild-type lantibiotic MU1 140 (SEQ ID NO:1 ) with one or more of the following mutations:
• Phel lle or Phel Gly that is the phenylalanine at position 1 is changed to isoleucine or glycine.
• Trp4Ala that is, the tryptophan at position 4 is changed to alanine.
• a variant of the lantibiotic MU1 140 comprises a Phel lle or Phel Gly amino acid substitution; a Trp4Ala amino acid substitution; a Dha5Ala amino acid substitution; an Arg13Asp amino acid substitution; or combinations thereof.
• An MU1 140 variant of the invention can also comprise, e.g., a Trp4insAla in which an alanine is inserted after the fourth tryptophan residue; or a ATrp4 in which there is a deletion of the tryptophan at position 4; or both of these changes in the primary amino acid sequence.
• Biologically active equivalents of MU1 140 lantibiotic polypeptides can have one or more conservative amino acid variations or other minor modifications and retain biological activity.
• a biologically active equivalent has substantially equivalent function when compared to the corresponding lantibiotic MU1 140.
• a lantibiotic mutacin has about 1 , 2, 3, 4, or 5 or less conservative amino acid substitutions.
• a conservative substitution is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and general nature of the polypeptide to be substantially unchanged.
• the following groups of amino acids represent conservative changes: (1 ) ala, pro, gly, glu, asp, gin, asn, dha, abu, dhb, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, gly, dha, abu, dhb, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.
• Biologically active equivalent lantibiotic mutacins or other lantibiotic polypeptides can generally be identified by modifying one of the variant lantibiotic mutacin sequences of the invention, and evaluating the properties of the modified lantibiotic mutacin to determine if it is a biological equivalent.
• a lantibiotic is a biological equivalent if it reacts substantially the same as a lantibiotic mutacin of the invention in an assay such as a zone of inhibition assay, e.g. has 90-1 10% of the activity of the original lantibiotic mutacin.
• Recombinant S. mutans strains of the invention comprise a polynucleotide that expresses a functional variant MU1 140.
• Biological activity of a variant MU1 140 can be assayed using, e.g., zone of inhibition assays (see Example 2).
• Recombinant S. mutans strains produce enough variant MU1 140 to outcompete and substantially eliminate wild-type, cariogenic S. mutans from the oral cavity of a host (e.g., reduce the number of wild-type S. mutans by about 5, 10, 25, 50, 75, 90, 95, 99, or 100% (or any range between about 5 % and about 100%)).
• a lantibiotic of the invention can be covalently or non-covalently linked to an amino acid sequence to which the lantibiotic is not normally associated with in nature, i.e., a heterologous amino acid sequence.
• a heterologous amino acid sequence can be from a non-Streptococcus mutans organism, a synthetic sequence, or an S. mutans sequence not usually located at the carboxy or amino terminus of a lantibiotic of the invention.
• a lantibiotic of the invention can be covalently or non-covalently linked to compounds or molecules other than amino acids such as indicator reagents.
• a lantibiotic of the invention can be covalently or non-covalently linked to an amino acid spacer, an amino acid linker, a signal sequence, a stop transfer sequence, TMR stop transfer sequence, a transmembrane domain, a protein purification ligand, or a combination thereof.
• a polypeptide can also be linked to a moiety (i.e., a functional group that can be a polypeptide or other compound) that facilitates purification (e.g., affinity tags such as a six-histidine tag, trpE, glutathione-S-transferase, maltose binding protein, staphylococcal Protein A or com), or a moiety that facilitates polypeptide stability (e.g., polyethylene glycol; amino terminus protecting groups such as acetyl, propyl, succinyl, benzyl, benzyloxycarbonyl or t-butyloxycarbonyl; carboxyl terminus protecting groups such as amide, methylamide, and ethylamide).
• a functional group i.e., a functional group that can be a polypeptide or other compound
• affinity tags such as a six-histidine tag, trpE, glutathione-S-transferase, maltos
• a protein purification ligand can be one or more amino acid residues at, for example, the amino terminus or carboxy terminus of a polypeptide of the invention.
• An amino acid spacer is a sequence of amino acids that are not associated with a polypeptide of the invention in nature.
• An amino acid spacer can comprise about 1 , 5, 10, 20, 100, or 1 ,000 amino acids.
• a lantibiotic of the invention can be part of a fusion protein, which can contain heterologous amino acid sequences. Heterologous amino acid sequences can be present at the C or N terminus of a lantibiotic of the invention to form a fusion protein. More than one lantibiotic of the invention can be present in a fusion protein. Fragments of lantibiotics of the invention can be present in a fusion protein of the invention.
• a fusion protein of the invention can comprise one or more lantibiotic of the invention, fragments thereof, or combinations thereof.
• a recombinant S. mutans strain of the invention is ATCC 55676 (deposited under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations thereunder (Budapest Treaty)), which has been genetically engineered to express a variant MU1 140 as described herein.
• Production of a mutant MU1 140 lantibiotic with enhanced biological activity as compared to a wild-type MU1 140 lantibiotic can therefore provide an S. mutans with a selective advantage over non-MU1 140-producing S. mutans strains present in the oral cavity of a host.
• the variant MU1 140 when expressed by a recombinant S. mutans strain of the invention, eliminates the resident, MU1 140-susceptible S. mutans strains, thus interfering with colonization of MU1 140-susceptible strains and promoting recombinant S. mutans colonization of the oral cavity. Since the wild-type, native S. mutans is displaced from the oral cavity, the incidence and/or severity of dental caries is reduced.
• the effector strain can additionally express lanB, lanC, lanE, lanF, lanG, lanK, lanM, lanP, lanR, lanT or combinations of two or more of these S. mutans polypeptides.
• “Lactic acid deficient” or “deficiency in lactic acid production” means that a recombinant S. mutans strain produces substantially decreased amounts of lactic acid relative to wild-type S. mutans. Substantially decreased amounts of lactic acid are about 40, 50, 60, 70, 80, 90, 95, or 100% (or any range between about 40% and about 100%) less lactic acid than is produced by a wild-type S. mutans strain (e.g. S. mutans strain UA159 (ATCC 700610)) or other species belonging to the mutans streptococcus group including Streptococcus sobrinus (e.g. S.
• a wild-type S. mutans strain e.g. S. mutans strain UA159 (ATCC 700610)
• Streptococcus sobrinus e.g. S.
• sobrinus strain SL1 ATCC 334708
• a lactic acid- deficient S. mutans effector strain produces no detectable lactic acid. Lactic acid expression can be detected as described in, e.g., Hillman et al., Infect. Immun. 62:60 (1994); Hillman et al., Infect. Immun. 64:4319 (1996); Hillman et al., 1990, Infect. Immun., 58:1290-1295.
• Recombinant S. mutans strains of the invention can be lactic acid deficient as a result of a non-functional, inactivated, partially functional, or partially inactivated regulatory region, translational signal, transcriptional signal, or structural sequence in the lactic acid synthesis pathway.
• Regulatory regions, translational signals, and transcriptional signals include, e.g., promoters, enhancers, ribosome binding sites, CAAT box, CCAAT box, Pribnow box, TATA box, etc.
• Nonfunctional or inactivated means that the known wild-type function or activity of the polynucleotide, gene, polypeptide or a protein has been eliminated or highly diminished by about 80, 90, 95, or 100% (or any range between about 80% and about 100%) as compared to a wild-type polynucleotide, gene, polypeptide or protein.
• Partially functional or partially inactivated means that the known wild-type function or activity of the polynucleotide, gene, polypeptide or a protein has been partially diminished by about 20, 30, 40, 50, 60, 70, 79% (or any range between about 20% and about 79%) as compared to a wild-type polynucleotide, gene, polypeptide or protein.
• Inactivation or partial inactivation which renders the polynucleotide, gene, polypeptide, or protein non-functional or partially functional, can be accomplished by methods such as incorporating mutations (e.g., point mutations, frame shift mutations, substitutions, deletions (part of or an entire signal, region or structural polynucleotide), interruptions, and/or insertions) in polynucleotides involved in the lactic acid synthetic pathway.
• a mutation in a polynucleotide involved in lactic acid synthesis can affect expression of lactic acid such that the expressed amount of lactic acid is diminished by about 20, 30, 40, 50, 60, 70, 80, 90, 95% or more as compared to a wild-type S. mutans strain.
• inactivation or partial inactivation of lactic acid expression can be effected by inactivating or partially inactivating, e.g., the lactate dehydrogenase (Idh) gene by deleting part of or the entire Idh structural polynucleotide or part of or the entire Idh promoter.
• inactivation or partial inactivation of lactic acid expression can be effected by inactivating or partially inactivating genes encoding enzymes involved in carbohydrate transport, e.g., the phosphoenolpyruvate phosphotransferase system (pfs) gene(s), by deleting part of or the entire pts structural polynucleotide or part of or the entire pts promoter.
• pfs phosphoenolpyruvate phosphotransferase system
• Inactivation or partial inactivation of lactic acid expression can be effected by inactivating or partially inactivating genes encoding enzymes involved in intracellular and extracellular polysaccharide storage, e.g., the glycogen synthase (glgA) gene (see e.g., Spatafora et al., Infect. Immun. 63:2556 (1995)) and the fructosyltransferase (ftf) gene (see e.g., Schroeder et al., Infect. Immun. 57:3560 (1989)), by deleting part of or the entire glgA or ftf structural polynucleotide or part of or the entire glgA or ftf promoter.
• glgA glycogen synthase
• ftf fructosyltransferase
• One or more defects in the lactic acid synthesis pathway can be introduced by mutagenesis (i.e., exposure of S. mutans to a mutagen), selection of spontaneous mutants, or genetic manipulation using recombinant techniques. These techniques are well known in the art (see, e.g., Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
• the lactic acid synthesis pathway defect is introduced using recombinant techniques, e.g., introduction of a defective Idh structural gene into the bacterium and subsequent site-specific recombination to replace the wild-type Idh with the defective Idh.
• mutans Idh gene has been cloned, its nucleotide sequence determined (GenBank accession number M72545), and the recombinant Idh gene expressed in Escherichia coli (Hillman et al., 1990, Infect. Immun., 58:1290-1295; Duncan et al., 1991 , Infect. Immun., 59:3930-3934). Hillman et al. deleted essentially the entire open reading frame of Idh from a S. mutans strain (J. Appl. Microbiol. 102:1209 (2007)).
• ADH alcohol dehydrogenase
• An S. mutans strain can be genetically engineered to express a recombinant alcohol dehydrogenase for example, alcohol dehydrogenase B, alcohol dehydrogenase II, or iron-containing alcohol dehydrogenase from Zymomonas mobilis (see e.g., GenBank Accession No. M15394; Conway et al., 1987, J.
• alcohol dehydrogenase from Streptococcus rattus iron-containing alcohol dehydrogenase from Commensalibacter intestini, iron-containing alcohol dehydrogenase from Azotobacter vinelandii, iron-containing alcohol dehydrogenase from Enterobacteriaceae bacterium, alcohol dehydrogenase from Pseudomonas fluorescens, iron-containing alcohol dehydrogenase from Dickeya zeae, alcohol dehydrogenase from Proteus mirabilis, iron-containing alcohol dehydrogenase from Rhodoferax ferriredcuens, iron-containing alcohol dehydrogenase from Rhodospirillum rubrum, alcohol dehydrogenase from Pseudomonas brassicacearum, alcohol dehydrogenase II from Pseudomonas syringae, alcohol dehydrogenase from Dick
• a polynucleotide encoding a bacterial alcohol dehydrogenase or iron-containing alcohol dehydrogenase has at least about 60, 65, 75, 80, 90, 95, 98, 99, or 100% (or any range between about 65% and 100%) homology to Zymomonas mobilis alcohol dehydrogenase B.
• an ADH-encoding polynucleotide can be derived from S. mutans, so that introduction of the ADH-encoding polynucleotide, in combination with the native S. mutans adh gene, provides for multiples copies of ADH-encoding polynucleotides in the S. mutans genome.
• the recombinant ADH polynucleotide can be generated by introducing a mutation in the regulatory mechanism of the S. mutans adh gene to upregulate the production of ADH (e.g., a mutation in the adh promoter to provide increased transcription of the adh gene).
• An adh polynucleotide can be introduced into a S. mutans strain of the invention using well-known recombinant techniques, for example, transforming the S. mutans strain with polynucleotides encoding an ADH polypeptide.
• Transforming or transformation means that a S. mutans has a non-native nucleic acid sequence integrated into its genome or as a plasmid that is maintained through multiple generations.
• the adh polynucleotide expresses a functional ADH polypeptide such that an S. mutans strain of the invention is viable despite the inactivation of lactic acid expression.
• polynucleotides encoding e.g., ADH
• isolation of polynucleotides encoding ADH can be performed by PCR amplification of the molecule from genomic DNA or from a preexisting clone of the gene.
• Expression of recombinant ADH can be accomplished by operably linking the adh structural polynucleotide to a promoter that facilitates expression in S. mutans (e.g., spaP or the native Idh promoter).
• Production of a functional ADH can be assayed by, for example, using conventional ADH activity assays (e.g., assays for NAD-dependent oxidation of ethanol) that are well known in the art (Neal et al., 1986, Eur. J. Biochem., 154:1 19- 124). Hillman et al. constructed a strain of S. mutans that expressed a functional, recombinant ADH. See e.g., Hillman et al, Infect. Immun. 68:543 (2000).
• ADH activity assays e.g., assays for NAD-dependent oxidation of ethanol
• Hillman et al. constructed a strain of S. mutans that expressed a functional, recombinant ADH. See e.g., Hillman et al, Infect. Immun. 68:543 (2000).
• Recombinant S. mutans strains of the invention can optionally be genetically engineered to be auxotrophic for an organic substance not normally present in the oral cavity or diet of a host so that the oral cavity colonization by the recombinant S. mutans strains can be controlled. That is, the recombinant S. mutans strains can optionally be genetically engineered so that they are unable to synthesize a particular organic compound required for growth.
• the strains of the invention can be auxotrophs for a D-amino acid, such as a D-alanine. Colonization of the auxotrophic strains can then be controlled by regulating the amount of the organic substance in the oral cavity. For example, colonization can be promoted by providing the organic compound periodically to the oral cavity and colonization can be terminated by withholding administration of the organic substance to the oral cavity.
• D-alanine is not normally produced or present in the oral cavity or diet of mammals above trace amounts. Therefore, if a recombinant S. mutans of the invention was auxotrophic for D-alanine, then D-alanine would need to be periodically delivered to the oral cavity of the mammal to maintain the colonization of a recombinant S. mutans of the invention in the oral cavity. In the absence of delivery of D-alanine to the oral cavity, the recombinant S. mutans strains of the invention will eventually die out.
• a recombinant S. mutans is alanine racemase deficient.
• Alanine racemase is required for D-alanine metabolism.
• "Alanine racemase deficient" or “deficiency in alanine racemase production” means that a recombinant S. mutans strain produces substantially decreased amounts of alanine racemase relative to wild-type S. mutans.
• Substantially decreased amounts of alanine racemase are about 40, 50, 60, 70, 80, 90, 95, or 100% (or any range between about 40% and about 100%) less alanine racemase than is produced by a wild-type S. mutans strain.
• an alanine racemase deficient recombinant S. mutans strain produces no detectable alanine racemase.
• Alanine racemase can be assayed as described in, e.g., Wantanabe et al., J. Biochem. 126:781 (1999).
• Inactivation or partial inactivation which renders the polynucleotide, gene, polypeptide, or protein non-functional or partially functional, can be accomplished by methods such as incorporating mutations (e.g., point mutations, frame shift mutations, substitutions, deletions (part of or an entire signal, region or structural polynucleotide), interruptions, and/or insertions in genes involved in the alanine racemase synthesis.
• a mutation in a polynucleotide involved in alanine racemase synthesis can effect expression of alanine racemase such that the expressed amount of alanine racemase is diminished by about 20, 30, 40, 50, 60, 70, 80, 90, 95% or more as compared to a wild-type S. mutans strain.
• inactivation or partial inactivation of alanine racemase expression can be effected by inactivating or partially inactivating, e.g., the dal gene by deleting part or all of the dal structural polynucleotide or part or the entire dal promoter.
• Bacterial auxotrophs can be generated using a variety of techniques well known in the art, such as chemical mutagenesis, selection of spontaneous mutants, and/or recombinant techniques (e.g., transposon mutagenesis, replacement by recombination with a defective or non-functional gene).
• D-alanine auxotrophic S. mutans strains can be generated by introduction of a defect in the gene encoding alanine racemase (dal), the enzyme that converts L-alanine to D- alanine. Such strains have been generated. See, e.g., Hillman et al., J. Appl. Microbiol. 102: 1209-1219 (2007).
• a recombinant S. mutans strain of the invention can comprise an inactivated or non-functional comE gene.
• a strain with an inactivated or nonfunctional comE gene would be less prone to transformation because ComE is important in the uptake of environmental DNA. Furthermore, comE cannot be complemented.
• ComE deficient or “deficiency in ComE production” means that a recombinant S. mutans strain produces substantially decreased amounts of ComE protein relative to wild-type S. mutans. Substantially decreased amounts of ComE are about 40, 50, 60, 70, 80, 90, 95, or 100% (or any range between about 40% and about 100%) less ComE protein than is produced by a wild-type S. mutans strain. In one embodiment of the invention, a ComE deficient recombinant S. mutans strain produces no detectable ComE protein. ComE expression can be assayed as described in, e.g., Chen & Gotschlich, J. Bact. 183:3160 (2001 ).
• Recombinant S. mutans strains of the invention can be ComE deficient as a result of a non-functional, inactivated, partially functional, or partially inactivated regulatory region, translational signal, transcriptional signal, or structural sequence in ComE synthesis.
• Inactivation or partial inactivation which renders the polynucleotide, gene, polypeptide, or protein non-functional or partially functional includes methods such as incorporating mutations (e.g., point mutations, frame shift mutations, substitutions, deletions (part of or an entire signal, region or structural polynucleotide), interruptions, and/or insertions) in polynucleotides involved in ComE synthesis.
• a mutation in a polynucleotide involved in ComE synthesis can effect expression of ComE such that the expressed amount of ComE is diminished by about 20, 30, 40, 50, 60, 70, 80, 90, 95% or more as compared to a wild-type S. mutans strain.
• inactivation or partial inactivation of ComE expression can be effected by inactivating or partially inactivating, e.g., the comE gene by deleting part of or the entire comE structural gene or part of or the entire comE promoter.
• Other genes involved in DNA uptake such as comA, comB, comC, and comD, can also or alternatively be inactivated or partially inactivated.
• the defect in ComE synthesis can be introduced by mutagenesis (i.e., exposure of the bacterium to a mutagen), selection of spontaneous mutants, or genetic manipulation using recombinant techniques.
• a S. mutans strain with a mutated comE gene has been constructed. See, e.g., Hillman et al., J. Appl. Microbiol. 102: 1209-1219 (2007).
• Polynucleotides of the invention contain less than an entire microbial genome and can be single- or double-stranded nucleic acids.
• a polynucleotide can be RNA, DNA, cDNA, genomic DNA, chemically synthesized RNA or DNA or combinations thereof.
• the polynucleotides can be purified free of other components, such as proteins, lipids and other polynucleotides.
• the polynucleotide can be 50%, 75%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% purified.
• a nucleic acid molecule existing among hundreds to millions of other nucleic acid molecules within, for example, cDNA or genomic libraries, or gel slices containing a genomic DNA restriction digest are not to be considered an isolated polynucleotide.
• the polynucleotides of the invention encode the polypeptides of the invention described above (e.g., MU1 140 polypeptides, ADH polypeptides, ComE polypeptides, D-amino acid synthesis polypeptides, and lactic acid synthesis polypeptides).
• the polynucleotides encode a variant mutacin 1 140 polypeptides shown in SEQ ID NOs:20-27, combinations thereof, or fragments thereof.
• the effector strain can additionally express lanB, lanC, lanE, lanF, lanG, lanK, lanM, lanP, lanR, lanT or combinations of two or more of these S. mutans polynucleotides.
• Polynucleotides of the invention can consist of less than about 600, 500, 400, 300, 200, 100, 66, 60, 50, 45, 30, 15 (or any range between about 600 and 15) contiguous polynucleotides.
• the purified polynucleotides can comprise additional heterologous nucleotides and/or additional homologous polynucleotides.
• Polynucleotides of the invention can comprise other nucleotide sequences, such as sequences coding for linkers, signal sequences, TMR stop transfer sequences, transmembrane domains, or ligands useful in protein purification such as glutathione-S-transferase, histidine tag, and Staphylococcal protein A.
• One embodiment of the invention provides a purified polynucleotide comprising at least about 6, 10, 15, 20, 25, 30, 40, 45, 50, 60, 66, or more contiguous nucleotides of encoding SEQ ID NOs:20-27
• Polynucleotides of the invention can be isolated.
• An isolated polynucleotide is a naturally-occurring polynucleotide that is not immediately contiguous with one or both of the 5' and 3' flanking genomic sequences that it is naturally associated with.
• An isolated polynucleotide can be, for example, a recombinant DNA molecule of any length. Isolated polynucleotides also include non-naturally occurring nucleic acid molecules.
• Polynucleotides of the invention can encode full-length polypeptides, polypeptide fragments, and variant or fusion polypeptides.
• Degenerate nucleotide sequences encoding polypeptides of the invention, as well as homologous nucleotide sequences that are at least about 80, or about 90, 95, 96, 97, 98, or 99% identical to the polynucleotide sequences of the invention and the complements thereof are also polynucleotides of the invention.
• Degenerate nucleotide sequences are polynucleotides that encode a polypeptide of the invention or fragments thereof, but differ in nucleic acid sequence from the given polynucleotide sequence due to the degeneracy of the genetic code.
• Percent sequence identity has an art recognized meaning and there are a number of methods to measure identity between two polypeptide or polynucleotide sequences. See, e.g., Lesk, Ed., Computational Molecular Biology, Oxford University Press, New York, (1988); Smith, Ed., Biocomputing: Informatics And Genome Projects, Academic Press, New York, (1993); Griffin & Griffin, Eds., Computer Analysis Of Sequence Data, Part I, Humana Press, New Jersey, (1994); von Heinje, Sequence Analysis In Molecular Biology, Academic Press, (1987); and Gribskov & Devereux, Eds., Sequence Analysis Primer, M Stockton Press, New York, (1991 ).
• Methods for aligning polynucleotides or polypeptides are codified in computer programs, including the GCG program package (Devereux et al. (1984) Nuc. Acids Res. 12:387), BLASTP, BLASTN, FASTA (Atschul et al. (1990) J. Molec. Biol. 215:403), and Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wl 5371 1 ) which uses the local homology algorithm of Smith and Waterman ((1981 ) Adv. App. Math., 2:482-489).
• the computer program ALIGN which employs the FASTA algorithm can be used, with an affine gap search with a gap open penalty of -12 and a gap extension penalty of -2.
• Polynucleotides of the invention can be isolated from nucleic acid sequences present in, for example, a bacterial sample. Polynucleotides can also be synthesized in the laboratory, for example, using an automatic synthesizer. An amplification method such as PCR can be used to amplify polynucleotides from either genomic DNA or cDNA encoding the polypeptides.
• Polynucleotides of the invention can comprise coding sequences for naturally occurring polypeptides or can encode altered sequences that do not occur in nature. If desired, polynucleotides can be cloned into an expression vector comprising expression control elements, including for example, origins of replication, promoters, enhancers, or other regulatory elements that drive expression of the polynucleotides of the invention in host cells.
• An expression vector can be, for example, a plasmid.
• Minichromosomes such as MC and MC1 , bacteriophages, phagemids, yeast artificial chromosomes, bacterial artificial chromosomes, virus particles, virus-like particles, cosmids (plasmids into which phage lambda cos sites have been inserted) and replicons (genetic elements that are capable of replication under their own control in a cell) can also be used.
• a polynucleotide of the invention is operably linked when it is positioned adjacent to or close to one or more expression control elements, which direct transcription and/or translation of the polynucleotide.
• compositions Comprising Recombinant S. mutans of the Invention
• Recombinant S. mutans strains of the invention can be characterized by: 1 ) a lactic acid deficiency, and 2) production of a recombinant ADH, 3) variant MU1 140 production, 4) optionally, an auxotrophy for a specific organic substance (e.g., a D- amino acid such as D-alanine), 5) optionally, a deficiency in ComE expression, or combinations thereof.
• a specific organic substance e.g., a D- amino acid such as D-alanine
• compositions of the invention can comprise one or more strains of recombinant S. mutans strains as described herein and a pharmaceutically acceptable or nutritionally acceptable carrier.
• the carrier is physiologically compatible with the area of the subject to which it is administered.
• Carriers can be comprised of solid-based, dry materials for formulation into tablet, capsule, lozenge, or powdered form.
• a carrier can also be comprised of liquid or gel-based materials for formulations into liquid, gel, and chewing gum forms.
• the composition of the carrier can be varied so long as it does not interfere significantly with the therapeutic activity of the bacterial strains of the invention. .
• a composition can be formulated to be suitable for oral administration in a variety of ways, for example in a solid, semi-solid, liquid (including, e.g., a viscous liquid, a paste, a gel, or a solution), a dried mass, a dentifrice, a mouth wash, an oral rinse, a liquid suspension, a beverage, a topical agent, a powdered food supplement, a paste, a gel, a solid food, an oral rinse, a packaged food, a wafer, lozenge, chewing gum and the like.
• a solid, semi-solid, liquid including, e.g., a viscous liquid, a paste, a gel, or a solution
• a dried mass including, e.g., a viscous liquid, a paste, a gel, or a solution
• a dried mass including, e.g., a dentifrice, a mouth wash, an oral rinse, a liquid suspension, a beverage, a
• a composition of the invention can include a nutrient supplement component and can include any of a variety of nutritional agents, as are well known, including vitamins, minerals, essential and non-essential amino acids, carbohydrates, lipids, foodstuffs, dietary supplements, and the like.
• compositions of the invention can also include natural or synthetic flavorings and food-quality coloring agents, all of which are compatible with maintaining viability of the bacterial strains of the invention.
• a composition of the invention can include one or more gelling agents that can act as an adhesive agent to adhere the composition to the teeth or mouth.
• the concentration of the gelling agent may be greater than about 2, 4, 6, 8, 10, 15, 20, 30, 40, 50, 60, 70, 80 or less than about 80, 70, 60, 50, 40, 30, or 20 percent by weight of the composition.
• Suitable gelling agents and adhesion agents useful in the present invention include, for example, silicone, polyethylene oxide, polyvinyl alcohol, polyalkyl vinyl ether-maleic acid copolymer (PVM/MA copolymer) such as, Gantrez AN 1 19, AN 139, and S-97, polyvinyl alcohol, polyacrylic acid, Poloxamer 407 (Pluronic), polyvinyl pyrrolidone-vinyl acetate copolymer (PVP/VA copolymer), such as Luviskol VA, and Plasdone S PVP/VA, polyvinyl pyrrolidone (PVP, e.g., K-15 to K-120), Polyquaterium-1 1 (Gafquat 755N), Polyquaterium-39 (Merquat plus 3330), carbomer or carboxypolymethylene (Carbopol), hydroxypropyl methylcellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, corn starch, carb
• a humectant or plasticizer can be present in compositions of the invention.
• Humectants or plasticizers include, for example, glycerin, glycerol, sorbitol, polyethylene glycol, propylene glycol, and other edible polyhydric alcohols.
• the humectants or plasticizers can be present between at about 1 % to about 99%, about 10% to about 95%, or at between about 50% and about 80% (or any range between 1 % and 99%) by weight of a composition.
• Bacteria of the invention can be prepared in, for example, a fermenter.
• the bacteria can be harvested from the fermenter and can be, for example, concentrated.
• Bacteria of the invention can be prepared for use by, for example, dehydration, air drying, lyophilizing, freezing, and spray-drying.
• Bacteria can also be prepared for use by microencapsulation (see e.g., U.S. Pat. No. 6,251 ,478) or by coating with a protective substance such as, for example, lipid material such as triacylglycerols, waxes, organic esters, soybean oil, cottonseed oil, palm kernel oil, and esters of long-chain fatty acids and alcohols.
• lipid material such as triacylglycerols, waxes, organic esters, soybean oil, cottonseed oil, palm kernel oil, and esters of long-chain fatty acids and alcohols.
• the coated or encapsulated bacteria of the invention are released in the oral cavity of the host.
• the recombinant S. mutans of the invention can be present in a composition of the invention in a therapeutically effective amount.
• Therapeutically effective means effective to prevent or reduce the number or incidence (e.g., 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% fewer cavities than controls that did not receive the composition) and/or reduce the severity (e.g., 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% less severe cavities than controls that did not receive the composition) of cavities.
• a therapeutically effective amount or dosage is an amount or dosage of a composition of the invention at high enough levels to prevent caries and/or reduce caries number and/or caries severity, but low enough to avoid serious side effects (at a reasonable benefit/risk ratio), within the scope of sound medical/dental judgment.
• the therapeutically effective amount or dosage of a composition of the invention may vary with the particular condition being treated, the age and physical condition of the patient being treated, the severity of the condition, the duration of treatment, the nature of concurrent therapy, the specific form of the source employed, and the particular vehicle from which the composition is applied.
• the compositions of the invention can be applied in a therapeutically effective amount to the oral cavity of a host for the treatment and/or prevention of cavities.
• a composition of the invention may be swallowed or may be rinsed around the oral cavity and then spit out, such that it is not substantially delivered to the gastrointestinal tract. That is, less than about 10, 5, 4, 3, 2, or 1 , 0.5, or 0.1 % (or any range or value between about 10 and 0.1 %) of the delivered bacteria are delivered to the gastrointestinal tract. Treatment means inducing a reduction in the amount or intensity (or combination thereof) of cavities.
• the bacterial strains of the invention can form at least a part of the transient or indigenous flora of the oral cavity and exhibit beneficial prophylactic and/or therapeutic effects.
• Treatment means reducing the amount of wild-type S. mutans in the oral cavity of a host such that remineralization of small carious lesions can occur and that further damage to larger carious lesions is stopped or slowed.
• the amount of wild- type S. mutans in the oral cavity can be reduced by about 20, 30, 40, 50, 60, 70, 80, 90, or 100% (or any range between about 10 and 100%).
• prevention means prevention in a population of subjects. That is, given a population of subjects, the treatment can prevent dental caries in about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90% or more of the subjects as compared to a control population that did not receive the treatment.
• a composition can comprise one or more isolated recombinant strains of the invention along with one or more isolated Streptococcus oralis strains and/or one or more isolated Streptococcus uberis strains.
• Streptococcus oralis (previously known as Streptococcus sanguis Type II) and S. uberis are important components in maintaining the normal, healthy balance of microorganisms that compose the periodontal flora. See, Socransky et al., Oral Microbiol. Immunol. 3:1-7 (1988); Hillman and Shivers, Arch. Oral. Biol., 33:395-401 (1988); Hillman, et al., Arch. Oral. Biol., 30:791 -795 (1985).
• S. oralis produces hydrogen peroxide, which can inhibit periodontal pathogens such as Actinobacillus actinomycetemcomitans (Aa), Bacteroides forsythus, and P.
• compositions of the invention can comprise one or more isolated strains of S. oralis, for example, ATCC 35037, ATCC 55229, ATCC 700233, ATCC 700234 and ATCC 981 1.
• Other strains of S. oralis include KJ3 and KJ3sm.
• KJ3sm is a naturally occurring genetic variant of KJ3 that is resistant to streptomycin. The streptomycin resistance is advantageous because it provides a marker for easy isolation of the bacteria. Additionally, streptomycin resistant strains are slightly attenuated and do not survive as long in an oral cavity as wild-type strains. This property is useful where the goal is to non-persistently colonize the oral cavity of an animal with the bacteria.
• compositions of the invention can comprise one or more isolated strains of S. uberis, for example, ATCC 13386, ATCC 13387, ATCC 19435, ATCC 27958, ATCC 35648, ATCC 700407, ATCC 9927, strain KJ2 or strain KJ2sm.
• KJ2sm is a naturally occurring genetic variant of KJ2. That is streptomycin resistant and provides the same advantages as for streptomycin-resistant strains of S. oralis.
• oralis or one or more isolated strains of S. uberis, or both can be used in compositions and methods of the invention.
• Additional oral care benefits of these compositions of the invention include, for example, the treatment and/or prevention of periodontitis, oral bacterial infections and diseases, oral wounds, Candida or fungal overgrowth, halitosis, or xerostomia-induced dental caries and associated periodontal diseases, the promotion of wound healing, teeth whitening or a combination thereof to a subject.
• One embodiment of the invention provides a method for treating dental caries comprising administering a composition comprising one or more recombinant S. mutans strains of the invention to the oral cavity of a subject in need thereof. That is, the subject has one or more dental caries.
• One embodiment of the invention provides for the prevention of dental caries in normal, healthy subjects.
• Another embodiment of the invention provides for treatment and/or prevention of dental caries in subjects having an increased susceptibility to dental caries as compared to normal, healthy subjects.
• the method consists of administering a composition comprising one or more recombinant S. mutans strains to the oral cavity of a subject.
• Subjects have an increased susceptibility to dental caries when they are more likely than a normal, healthy host to develop dental caries.
• Such hosts may have, for example, decreased saliva production (e.g., patients undergoing radiation therapy on the head or neck, patients having Sjogren's syndrome, diabetes mellitus, gastroesophageal reflux disease, diabetes insipidus, or sarcoidosis, patients taking antihistamines and antidepressants or other medications that cause "dry mouth”), smokers, smokeless tobacco users, patients having a genetic predisposition (Shuler, J. Dent. Ed. 65:1038 (2001 )), or are infants (0 to 2 years old or 6 months to 2 years old), children (3 years to 18 years old), or elderly (older than 65).
• saliva production e.g., patients undergoing radiation therapy on the head or neck, patients having Sjogren's syndrome, diabetes mellitus, gastroesophageal reflux disease, diabetes insipidus, or sarcoidosis, patients taking
• the invention also provides a method of reducing the amount in a subject of bacteria that can cause dental caries.
• the method comprises administering a composition comprising one or more recombinant S. mutans strains of the invention to the oral cavity of a subject having one or more strains or species of bacteria that can cause dental caries.
• the compositions can be administered just once or on a regular basis.
• the number of the one or more strains or species of bacteria that can cause dental caries in the subject is reduced.
• the reduction can be about a 5, 10, 25, 50, 75, 90, 95, 99, or 100% (or any range between about 5% and about 100%) reduction in numbers.
• one or more bacteria that can cause dental caries can be detected and/or quantitated using any detection/quantitation method known in the art. Those of skill in the art are aware of methods of detection of bacteria that cause dental caries.
• one or more dental caries can be diagnosed in the subject using any methodology known in the art.
• Another embodiment of the invention provides a method of preventing dental caries in a subject.
• the method comprises obtaining data regarding a therapeutically effective dosage range for prevention of dental caries in a particular type of subject and determining the effective dosage range of recombinant S. mutans for the particular type of subject.
• a particular type of subject can be, for example, a subject with decreased saliva production (e.g., patients undergoing radiation therapy on the head or neck, patients having Sjogren's syndrome, diabetes mellitus, gastroesophageal reflux disease, diabetes insipidus, or sarcoidosis, patients taking antihistamines and antidepressants or other medications that cause "dry mouth"), smokers, smokeless tobacco users, patients having a genetic predisposition, or are infants (0 to 2 years old or 6 months to 2 years old), children (3 years to 18 years old), or elderly (older than 65).
• the determined therapeutically effective dosage range for the particular type of subject of one or more recombinant S. mutans strains of the invention are administered to the oral cavity of the particular type of subject.
• compositions can be administered to the oral cavity of a host or subject such as an animal, including a mammal, for example, a human, a non-human primate, a dog, a cat, a horse, a bovine, a goat, or a rabbit.
• a host or subject such as an animal, including a mammal, for example, a human, a non-human primate, a dog, a cat, a horse, a bovine, a goat, or a rabbit.
• compositions of the invention can be orally administered in for example, food, water, a dentifrice, a gel, a paste, an emulsion, aerosol spray, chewing gum, lozenge, tablet, capsule, or a liquid suspension.
• the bacteria can either be already formulated into food, water, gel or other carrier or can be a composition (e.g., powder, tablet or capsule) that is added to the carrier (e.g., food, water, dentifrice, gel, paste, emulsion, aerosol spray, or liquid suspension) by the user prior to consumption.
• One embodiment of the invention provides a method of non-persistently colonizing an oral cavity of a subject with therapeutically-effective bacteria comprising administering to the oral cavity of a subject a composition of the invention.
• the administered bacterial strains do not permanently colonize the oral cavity, rather the strains are present in the oral cavity for about 1 day, about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 3 months or about 12 months after administration of the bacteria.
• recombinant strains of S. mutans persistently colonize an oral cavity of a host for a long term period, e.g., 2 weeks, 1 month, 3 months, 6 months, 1 year, 5 years, or more or for the life of the host.
• compositions of the invention can be administered at a dose of about 1x10 3 , 1 x10 5 , 1 x10 7 , 1 x10 8 1x10 9 , or 1x10 11 CFU (or any range or value between about 1 x10 3 and about 1 x10 11 ) of viable bacteria.
• a dose of a composition of the invention can be administered at four times a day, three times a day, twice a day, once a day, every other day, two times a week, weekly, biweekly, monthly, or yearly.
• One, two, or more doses of a composition of the invention can be administered per day for about 1 day, about 1 week, about 2 weeks, about 1 month, about 2 months, about 3 months, about a year or more.
• a composition of the invention is administered one time and is effective for a long term period.
• a composition of the invention can comprise bacterial strains at a concentration between about 0.01 % and about 50%, or about 0.1 % to about 25%, or about 1 .0% to about 10% or any ranges or values in between 0.01 % and 50% by weight of the composition.
• a kit of the invention can contain a single dose, a one week, one month, two month, three month, four month, five month, six month, or 12 month supply of a composition of the invention.
• a composition of the invention can be packaged and, in turn, a plurality of the packaged compositions can be provided in a storage container or outer package or carton.
• the kit can include a bacterial auxotroph-maintaining amount of an organic substance, e.g., a composition comprising a D-amino acid such as D- alanine.
• a composition of the invention comprises one or more strains of S. mutans that are auxotrophic for an organic substance
• a bacterial auxotroph- maintaining amount of an organic substance can be administered to hosts to maintain the recombinant S. mutans in the oral cavity.
• a "bacterial auxotroph- maintaining amount” is an amount of an organic substance sufficient to maintain viability of the recombinant S. mutans auxotroph in the oral cavity.
• a D-alanine bacterial auxotroph-maintaining amount is an amount of D-alanine sufficient for survival of the D-alanine auxotrophic strain in the host's oral cavity.
• a single dose of a D- alanine bacterial auxotroph-maintaining amount of D-alanine contains about 1 , 5, 10, 20, 25, 50, 75 or 100 mg (or any range between about 1 and about 100 mg).
• the concentration of D-alanine in a composition in the form of a solution is about 0.01 , 1 , 10, 25, 50, 75, 100, or 167 mg/ml (the latter being a saturated solution of D-alanine in water at 25°C) (or any range between about 0.01 and about 167 mg/ml).
• the concentrations of D-alanine in a composition can vary according to the carrier used and the saturation point of D-alanine in that specific carrier.
• the organic substance, e.g., D-alanine, required for maintenance of the auxotrophic, recombinant S. mutans in the oral cavity can be formulated as a mouthwash, chewing gum, dental floss, toothpaste, chewable tablet, food, beverage or any other formulation suitable for oral administration to the host's oral cavity.
• the composition can additionally contain flavoring agents, coloring agents, fragrances, or other compounds that increase the palatability of the composition and/or enhance patient compliance without compromising the effectiveness of the organic substance contained in the composition.
• the cloned insert in p190 was derived by PCR amplification of chromosomal DNA of S. mutans strain JH1 140 (ATCC 55676) using the primer sequences of SRWIanA_1 and SRWIanA_2 (see Figure 3). Reagents and media were purchased from Fisher Scientific, and enzymes were purchased from New England BioLabs (Ipswich, MA).
• the result of this step was the production of two fragments, one that included 5' flanking DNA and a portion of lanA, including the site directed base alterations.
• the second fragment contained the remainder of lanA plus 3' flanking DNA.
• Primers used to produce the MU1 140 variants are found in Figure 3.
• the two fragments were then mixed in equal amounts and subjected to a second round of PCR using the two outside primers, SRWIanA_1 and SRWIanA_2, to yield the final amplicon.
• PCR reactions were performed using Taq polymerase in a final volume of 50 ⁇ _ containing 0.4 ⁇ of each primer, 50 ng of template DNA, 0.016 mMdNTP, and 1 unit of DNA polymerase in 1X polymerase buffer.
• Amplification conditions for each fragment were as follows: preheat at 95°C for 1 min, followed by 27 cycles incubation with denaturation (95°C) for 30 sec, annealing (56°C) for 30 sec and extension (72°C) for 2 min followed by a final extension (72°C) for 10 min. Both fragments were combined 50:50 and amplified using the two outside primers SRWIanA_1 and SRWIanA_2 under the same amplification conditions as mentioned above.
• the final PCR product was ligated into a TOPO-TA vector (Invitrogen, Carlsbad, CA) following kit directions and transformed into DH5a-T1 ®cells (Invitrogen) using standard methods and spread on LB plates containing 50 ⁇ g/mL of ampicillin and 40 ⁇ iL of X-gal (40mg/mL). Blue-white screening was utilized to identify colonies containing an insert. Plasmid DNA from each colony was purified using a PureYield Plasmid Miniprep System (Promega, Madison, Wl) according to the manufacturer's instructions.
• Plasmids containing the proper sized insert were sequenced using M13 Forward (-20) primer, 5 ' -GTAAAACGACGGCCAG-3 ' (SEQ ID NO:28), to confirm the proper insertion, deletion, or replacement of nucleotide bases.
• Restriction enzyme digestion was performed on purified plasmid from colonies harboring a confirmed mutation.
• the insert were separated from the TOPO plasmid by electrophoresis, excised from the gel, and purified using a Qiagen Gel Extraction Kit (Qiagen, Valencia, CA).
• the purified insert was then ligated into the S. mutans suicide vector, pVA891 , in a 3: 1 inser vector ratio using T4 DNA ligase at 16°C overnight.
• the resultant plasmid was then transformed into DH5a cells using standard methods and spread on LB plates containing 300 ⁇ g/mL of erythromycin. Colonies which arose following incubation were analyzed to verify proper insert size and sequence as described above.
• Purified pVA891 DNA containing confirmed inserts was transformed into S. mutans strain JH 1 140 (ATCC 55676) as follows: S. mutans was grown overnight then diluted 1 : 15 in fresh THyex broth (30 g/L THB, 3 g/L yeast extract), 200 ⁇ of diluted cells were added to a 96 well plate and incubated at 37°C for 2 hours. Two microliters of competence stimulating peptide (CSP, O. ⁇ g/mL; see e.g., Li et al., J. Bacteriol. 183:897 (2001 )) was added, and plates were incubated for an additional 6 hours. See Li et al., (2002) J. Bacteriol. 184:2699.
• CSP competence stimulating peptide
• Clones containing the desired lanA mutations were obtained by spontaneous resolution of the heterodiploid state as follows: several confirmed heterodiploids were grown overnight in 20 mL THyex broth that did not contain erythromycin. The cultures were subcultured (1 :20 dilution into fresh media) and again grown overnight to saturation. The cultures were then diluted 100,000 fold and spread onto large THyex agar plates and incubated at 37°C for 48 hours. Resultant colonies were replica patched onto medium with and without erythromycin to identify spontaneous recombinants in which elimination of the pVA891 plasmid (expressing the erythromycin resistance gene) and either the wild-type or mutated lanA gene had occurred.
• the parent S. mutans strain, JH1 140 (ATCC 55676), and the mutants were grown to an OD 6 oo of 0.8 and diluted to an ⁇ of 0.2.
• Samples (2 ⁇ _) of the cultures were spotted in triplicate on a pre-warmed THyex agar plate (150 X 15mm) and allowed to air dry. This assay was performed in this manner to help ensure that each sample had the same colony size for comparing zones of inhibition. The plate was incubated for 24 hours at 37°C, and then placed in an oven at 55°C for thirty minutes to kill the bacteria before the M. luteus ATCC 272 indicator strain was overlayed in molten top agar. Heat killing the bacteria prevented any further antimicrobial compound production. M.
• Figure 4 illustrates the bioactivity of strains producing variants of MU1 140 compared to wild-type MU1 140.
• the results are summarized in Figure 5, which shows that the strains producing Trp4insAla and ATrp4 had zones that were not significantly different (Student's t test, p>.05) than the wild-type.
• the strain producing Arg13Asp had the largest inhibitory zone area amounting to a 2.57-fold increase relative to wild-type (p ⁇ .001 ).
• the strains producing Trp4Ala and Dha5Ala produced significant (p ⁇ .001 ) 2.12-fold and 1 .87-fold increases, respectively, relative to the wild-type.
• the strain producing Ala s 7insAla had the smallest zone area, which amounted to a significant (p ⁇ .001 ) 2-fold reduction in zone area when compared to the wild-type.
• Figure 6 shows the biological activity of strains producing other variants of MU 1 140 (Phel lle and Phel Gly) compared to wild-type MU1 140.
• the strains producing Phel lle and Phel Gly demonstrated significant (p ⁇ .001 ) 1.82-fold and 1 .57-fold increases, respectively, relative to the wild-type.
• the mutations to MU1 140 described herein are therefore unexpected and unpredictable in view of the prior art and result in variant MU1 140 molecules that have vastly improved biological and structural characteristics as compared to wild- type MU1 140. Mutations that increase activity are important from the standpoint of improving the colonization potential of an S. mutans effector strain.
• the ability of S. mutans strains to colonize the oral cavity of rodents and humans has been previously shown to correlate with the amount and/or activity of MU1 140 produced.
• the ability of S. mutans strains to aggressively displace indigenous strains of S. mutans in the oral cavity of rodents and humans has been previously shown to correlate with the amount and/or activity of MU1 140 produced.
• an S. mutans effector strain of the invention that expresses a variant MU1 140 as described herein will have unexpected and improved characteristics as compared to effector S. mutans strains that do not express a variant MU1 140 of the invention. That is, S. mutans effector strains expressing a variant MU1 140 will have improved ability to colonize and aggressively outcompete and replace native S. mutans in the oral cavities of the hosts relative to S. mutans effector strains that do not express a variant MU1 140 as described herein.
• Wild-type mutacin 1 140, mutacin 1 140 with a F1 I mutation, mutacin 1 140 with a W4A mutation, and mutacin 1 140 with a R13D mutation was purified to about 90% purity (measured via HPLC).
• the minimum inhibitory concentration (MIC) of MU1 140 and variants of MU1 140 was determined against several bacteria. The MIC is the lowest concentration of MU1 140 that will inhibit the visible growth of a microorganism after 24 hour incubation. A lower MIC is an indication of greater inhibitory activity.
• Preparation of the antimicrobial agent and bacterial inoculum for minimum inhibitory concentrations (MICs) was performed by following the method described in Clinical Laboratory Standard Institute (CLSI) M07-8A with some minor modifications.
• Streptococcus mutans UA159 was tested overnight in a shaking incubator to maintain uniform dispersion of the bacteria.
• Clostridium difficile UK1 was tested in an anaerobic chamber at 37°C.
• the medium used was THyex. The results are shown in Table 1 .
• each mutant may still have advantages over the wild-type MU1 140 because it may, for example, be easier to produce, easier to transport, have better shelf stability, have better serum stability, or have better proteolytic stability, among other advantageous properties.

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