Inhibition of Infection
FIELD OF INVENTION
This invention relates to the inhibition of infection of a host site by a micro-organism and particularly provides use of an isolated polypeptide having one or more of at least one of the amino acid motifs (Glycine-Proline), (Proline-Glycine), (Glycine-Hydroxyproline) or (Hydroxyproline-Glycine) and consisting of from 2 to 100 amino acid residues, or use of a chemical compound comprising such a polypeptide associated with a non-polypeptide chemical entity, in the preparation of a medicament for the at least partial inhibition of a host site by a micro-organism. The invention particularly relates to inhibition of infection of dentine by bacteria.
BACKGROUND
A wide variety of oral bacteria are found associated with infections of the tooth roots, pulp, and root canal systems (Sundqvist, (1994) Oral Surg. Oral Med. Oral Pathol. 78 522-530). These infections are generally long-term chronic conditions that manifest in inflammation, tissue necrosis and deep pain. They usually require specialist endodontic or periodontal treatment, often surgery, with the aim of removing bacterial contamination and providing effective barriers to re-infection. The infamous root canal treatment involves physically removing the regions of bacterial contamination and damaged tissue, irrigation and disinfection of the root canals, filling of these with a suitable biologically inert compound such as gutta-percha, and then sealing (occluding) the dentinal tubules and completing the tooth restoration with proprietary biomaterials.
Collagen types I-IV make up a family of fibrous proteins which are the most abundant proteins in mammals. Each protein contains three polypeptide chains, each of which has a very regular amino acid sequence, with nearly every third residue being glycine. The unusual amino acids, hydroxyproline and hydroxylysine, are also present. There are
multiple sequence similarities between the various types of collagen. Collagen type I is found associated with bone, dentine, tendons, ligaments and skin. It is made up of polypeptide chains CoIIAl (Fig. IA; GenBank Accession No. NP_000079) and ColIA2 (Fig. IB; GenBank Accession No. NP_000080). These precursor chains are modified by cleavage and chemical cross-linking to generate mature collagen type I.
The bacteria that invade dentinal tubules are primarily streptococci (Love & Jenkinson, (2002) Crit. Rev. Int. Endod. J. 13 171-183) and enterococci. Streptococcus gordonii, Streptococcus sanguis, Streptococcus mutans, Streptococcus oralis, Streptococcus sobrinus and Enterococcus faecalis have all been shown to invade dentinal tubules. Streptococci bind to collagen type I present within the tubules via the activity of cell surface proteins designated the antigen VH family proteins (Jenkinson & Demuth, (1997) MoI. Microbiol. 23 183-190). It has been shown that these proteins are also able to bind other oral bacteria (Demuth et al, (1996) MoI Microbiol. 20 403-413) and may be responsible not only for promoting initial dentine infection, but also for establishing co- infections with other bacteria leading to mixed microbial communities (Love et al., (2000) Infect. Immun. 68 1359-1365). Such mixed microbial infection results in inflammation, tissue destruction and much pain for the host. Thus, inhibitors of antigen I/π functions may in future be important for the control of infections by oral streptococci, and for controlling poly-microbial diseases that are driven by primary streptococcal infections. In addition, inhibitors of antigen I/H interactions with collagen type I could be useful in preventing dentine invasion.
Patients with severe oral bacterial infections are almost always prescribed antibiotics. The topical application of tetracycline derivatives is an established practice in periodontal disease treatment and control, sometimes in combination with systemic antibiotics given in association with surgery. While many of these treatments are quite effective, there is an increasing awareness of higher treatment failure rates, probably related directly to the increasing incidence of antibiotic resistant bacteria. Recent surveys have suggested a growing build up in the numbers of oral bacterial isolates that are resistant to the most frequently used antibiotics in dentistry. For bacteria such as streptococci, penicillin
derivatives are no longer certain to be effective, and in enterococci there is major concern that new virulent isolates are now resistant to vancomycin, previously considered as the agent to use when all else has failed. In addition to the resistance problem, the use of broad-range antibiotics in the context of mixed microbial communities is now thought less desirable, because of the appreciation of how a balanced microflora is essential for the well-being of the host. Thus, side effects of prolonged antibiotic treatment, which kills components of the resident microflora, include inflammatory reactions, the acquisition of potential pathogens, and the overgrowth of opportunistic pathogens such as Candida yeasts (Jenkinson & Douglas, (2002) Polymicrobial Infections and Disease. Washington DC, ASM Press, pp357-373). There is much to be desired, therefore, with developing alternative approaches to broad-spectrum antibiotic usage, or new protocols to augment antibiotic function and therefore reduce usage.
Inhibitors of oral bacterial colonization that have been successfully tested include antibodies that block adhesin functions (Lehner et ah, (1985) Infect. Immun. 50 796-799) and adhesin-derived peptides that probably block colonization sites in the host (Kelly et al., (1999) Nature Biotechnol. 17 42-47). Neither of these strategies has been thought of as applicable to preventing dentine infections because they are designed to target the micro-organisms rather than the specific host site of infection, which may also result in an imbalance in the host microflora, as mentioned above.
Other anti-microbial strategies include the introduction of competitive micro-organisms that interfere with the growth and survival of the target pathogens. These competitors either block colonization of the undesirable bacteria, or they secrete inhibitory compounds called bacteriocins that inactivate the undesirable bacteria (Upton et al., (2000) J. Bacteriol. 183 3931-3938). These methods suffer at present from the uncertainties and potential risks of introducing foreign bacteria into humans.
An important area of anti-microbial research is to develop ways to augment the natural host defences. Antibacterial peptides are important for microbial clearance at boundaries susceptible to infection. The peptide LL-37 has been isolated from neutrophils and is also
found in the lung and in keratinocytes. LL-37 may act in concert with α-defensins, a family of anti-microbial peptides, produced mainly by neutrophils and epithelial cells. However, pathogens can overcome the innate defence systems. Various proteinases of Pseudomonas aeruginosa, Enterococcus faecaϊis and Streptococcus pyogenes can directly degrade LL-37 (Schmidtchen et al, (2002) MoI. Microbiol. 46 157-168). Also, proteinases may indirectly inactivate LL-37 or α-defensins through the generation of dermatan sulphate or heparan sulphate, respectively, which block the anti-bacterial peptide activities. Other resistance mechanisms may include modification of cell wall components or modulation of efflux pumps. Thus, a problem with utilizing these compounds as therapeutic agents is that they are relatively indiscriminate in their actions and decreased bacterial susceptibility can be acquired.
Speziale et al (J. Bacteriol. (1986) 167 77-81) discusses the peptide sequences (Proline- Proline-Glycine)io, (Proline-OH-Proline-Glycine)^ and (Proline-Glycine-Proline)n and their ability to inhibit the binding of Staphylococcus aureus to 125I-labelled collagen. Although there is no indication as to the size range encompassed by "n", the authors indicate that larger peptides generally have a greater inhibitory activity than smaller peptides.
Ascencio & Wadstrόm (J. Med. Microbiol. (1998) 47 417-425) discloses that a collagen- homologous peptide, which contains (Glycine-Proline-D-Arginine-OH), can inhibit binding of the human gastrointestinal pathogen Aeromonas hydrophilia to collagen. However, similar peptides, which contained (Glycine-Proline-Leucine), (Glycine- Proline-Glutamate) and/or (Glycine-Proline-Lysine), did not inhibit binding of this organism to collagen.
JP2001131084, published on 15th May 2001, discloses means of promoting collagen production in the body, the promoter for collagen synthesis containing a (Glycine-X-Y) motif (where X and Y are any amino acid), especially (Glycine-Proline-Hydroxyproline). There is no disclosure of peptides containing this motif inhibiting the binding of microorganisms to collagen.
There are no products commercially available that work selectively at a molecular level to inhibit or prevent bacterial invasion of dentinal tubules. If it is necessary to seal dentine clinically then materials are used to block the tubules. These materials include glass ionomer cement, resin, or in the case of sensitive dentine something like Sensodyne™ to precipitate mineral in the tubule to block it. Topical fluoride application is still the best method for re-mineralizing dentine, though vital non-infected dentine will re-mineralize itself.
Cleaning of dentine is mainly effected by using non-specific anti-microbial agents. In clinical endodontic treatment the agent employed is principally sodium hypochlorite (NaOCl). A mixture of NaOCl and calcium hydroxide is often used as an irrigant and is very effective. However it is toxic and destructive and, if it penetrates the tissues surrounding the tooth root (periapical bone, etc), it causes severe lesions that cause the death of bone, alveolar mucosa or gingiva tissues. Calcium hydroxide, Ca(OH)2, is used as an intra-canal medicament and is somewhat inhibitory to growth of intra-tubule bacteria. However, extended treatment dressing times (at least 1 week) are required for it to be clinically effective and there are resistant species of bacteria e.g. Enterococcus faecalis. Synthetic corticosteroid/tetracycline antibiotic (Ledermix™) is less effective than Ca(OH)2 at disinfecting the root canal, probably because at the site of application it functions only as a bacteristatic agent. Thus, new inhibitors that can target bacterial dentine invasion mechanisms and be very easily incorporated into a medicament would be highly desirable.
It is an object of the current invention to enable target-specific control of dentine infection by bacteria. This involves the application of non-toxic peptides that would have minimal side effects to the patients. The peptides have been shown to inhibit infection of dentine by bacteria such as Streptococcus gordonii, Enterococcus faecalis and Streptococcus mutans.
SUMMARY OF THE INVENTION
According to a first aspect of the invention, there is provided use of an isolated polypeptide having one or more of at least one of the amino acid motifs (Glycine- Proline), (Proline-Glycine), (Glycine-Hydroxyproline) or (Hydroxyproline-Glycine) and consisting of from 2 to 100 amino acid residues, or use of a chemical compound comprising such a polypeptide associated with a non-polypeptide chemical entity, in the preparation of a medicament for the at least partial inhibition of microbial infection of a host site. Throughout this specification, the term "amino acid" is intended to encompass both L- and D-stereo isomers of amino acids and amino acid analogues, e.g. amino acids which have been modified, for example to improve stability by increasing their resistance to peptidases whilst retaining their microbial infection inhibition properties. The isolated polypeptide may consist of from 2 to 80 amino acid residues, preferably from 2 to 60 amino acid residues, more preferably from 2 to 40 amino acid residues, yet more preferably from 2 to 20 amino acid residues, even more preferably from 2 to 10 amino acid residues and most preferably from 2 to 5 amino acid residues.
The microbial infection may be bacterial infection or fungal infection, particularly members of the genera Streptococcus or Enterococcus, including Streptococcus gordonii, Streptococcus sanguis, Streptococcus mutans, Streptococcus oralis, Streptococcus sobrinus and Enterococcus faecalis. Alternatively, the microbial infection may be a fungal infection. For example, the fungus may be a Candida species. The infection may also be caused by a combination of two or more types of micro-organism, for example, different species of bacteria, or different species of bacteria and fungi.
The infection may be infection of mammalian body tissue, for example, human body tissue, particularly infection associated with invasive surgery, insertion of a surgical implant or other medical, veterinary or dental procedure. Most particularly, the infection may be infection, preferably invasion, of dentine. The microbial infection may be inhibited by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or by 100%, where the greater the percentage inhibition, the more preferred the embodiment of this aspect of the invention. Such inhibition of
infection may be assessed by comparison of a treated host site with an untreated host site, each being exposed to the microbial infective agent, as outlined in Example 2 of this specification and as shown in Figure 9.
The polypeptide may comprise at least one copy of the tripeptide motif (Glycine-Proline- X) where X is any L- or D-amino acid or modified amino acid and where, in the case that two or more copies of the motif are present, X need not be identical in each copy of the motif. X may be selected from: alanine, asparagine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, tyrosine, hydroxyproline. The term "hydroxyproline" as used throughout this specification may mean 3 -hydroxyproline or 4- hydroxyproline. X may also be a modified amino acid, as defined above. In a preferred embodiment of this aspect of the invention, at least one copy of the tripeptide motif is (Glycine-Proline-Alanine), (Glycine-Proline-Hydroxyproline), (Glycine-Proline- Proline), or (Glycine-Proline-Threonine).
The polypeptide may consist of a di-tripeptide, examples of which include (Glycine- Proline-Alanine)2, (Glycine-Proline-Hydroxyproline)2, (Glycine-Proline-Threonine)2, (Glycine-Proline-Proline)2, (Glycine-Proline-Hydroxyproline-Glycine-Proline-Alanine), (Glycine-Proline-Hydroxyproline-Glycine-Proline-Threonine). The polypeptide may consist of three or more copies of the tripeptide motif; each copy may be identical or different.
Most preferably, the polypeptide may be Glycine-Proline-Alanine, Glycine-Proline- Hydroxyproline, Glycine-Proline-Proline, Glycine-Proline-Arginine-Proline, Glycine- Proline-Alanine-Glycine, Glycine-Proline-Threonine, Glycine-Proline, Glycine- Hydroxyproline or Glycine-Proline-Glycine-Glycine.
It is an advantage of the current invention that the use of a medicament containing such polypeptides does not perturb the general composition or activities of the host microflora, except at the target site for disease prevention. The polypeptides are found naturally within components of human tissue proteins and are products to which the host does not normally have any adverse response. In a further advantage, the polypeptides are not
bactericidal and thus will not induce development of bacterial resistance. These novel peptides do not generally inhibit bacterial growth in the oral cavity, but work at the site of potential infection. The peptides have the potential to block mixed species bacterial infection because inhibiting streptococci, which are primary colonizers, will prevent a more pathogenic community from developing. Advantageously, this alleviates the subsequent need to use broad-spectrum antibiotics to combat an established multi- organism infection. In a yet further advantage, small tripeptides according to the invention are inexpensive and simple to produce in large quantities. It is relatively easy to prepare a medicament which can deliver such small polypeptides to a host target site.
According to a second aspect of the invention, there is provided an isolated polypeptide comprising one or more copies of at least one of the amino acid motifs (Glycine-Proline), (Proline-Glycine), (Glycine-Hydroxyproline) or (Hydroxyproline-Glycine), the polypeptide consisting of from 2 to 100 amino acid residues, wherein any section of the polypeptide which contains one or more copies of the at least one of the motifs contains between 2 and 29 contiguous amino acids selected from: Glycine, Proline and Hydroxyproline.
The isolated polypeptide may consist of from 2 to 80 amino acid residues, preferably from 2 to 60 amino acid residues, more preferably from 2 to 40 amino acid residues, yet more preferably from 2 to 20 amino acid residues, even more preferably from 2 to 10 amino acid residues and most preferably from 2 to 5 amino acid residues.
The polypeptide may comprise at least one copy of the tripeptide motif (Glycine-Proline- X) where X is any L- or D-amino acid or modified amino acid and where, in the case that two or more copies of the motif are present, X need not be identical in each copy of the motif. X may be selected from: alanine, asparagine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, tyrosine, hydroxyproline. X may also be a modified amino acid, as defined above. In a preferred embodiment of this aspect of the invention, at least one copy of the tripeptide motif may be (Glycine-Proline-
Alanine), (Glycine-Proline-Hydroxyproline), (Glycine-Proline-Proline), or (Glycine- Proline-Threonine).
The polypeptide may consist of a di-tripeptide, examples of which include (Glycine- Proline-Alanine)2, (Glycine-Proline-HydroxyproHne)2, (Glycine-Proline-Threonine)2, (Glycine-Proline-Proline)2, (Glycine-Proline-Hydroxyproline-Glycine-Proline-Alanine), (Glycine-Proline-Hydroxyproline-Glycine-Proline-Threonine). The polypeptide may consist of three or more copies of the tripeptide motif; each copy may be identical or different.
Most preferably, the polypeptide may be Glycine-Proline-Alanine, Glycine-Proline- Hydroxyproline, Glycine-Proline-Proline, Glycine-Proline-Arginine-Proline, Glycine- Proline-Alanine-Glycine, Glycine-Proline-Threonine, Glycine-Proline, Glycine- Hydroxyproline or Glycine-Proline-Glycine-Glycine.
Polypeptides according to this aspect of the invention may also promote maintenance of healthy oral tissues, for example, gum, mouth and/or teeth tissues.
According to a third, related, aspect of the invention, there is provided an isolated polypeptide comprising one or more copies of at least one of the motifs (Glycine- Proline), (Proline-Glycine), (Glycine-Hydroxyproline) or (Hydroxyproline-Glycine), the polypeptide consisting of from 2 to 100 amino acid residues, wherein the polypeptide at least partially inhibits infection of a host site by a micro-organism and wherein any section of the polypeptide which contains one or more copies of the at least one of the motifs contains between 2 and 29 contiguous amino acid residues selected from: Glycine, Proline and Hydroxyproline.
The isolated polypeptide may consist of from 2 to 80 amino acid residues, preferably from 2 to 60 amino acid residues, more preferably from 2 to 40 amino acid residues, yet more preferably from 2 to 20 amino acid residues, even more preferably from 2 to 10 amino acid residues and most preferably from 2 to 5 amino acid residues.
The polypeptide may inhibit binding of the micro-organism to a collagen, preferably collagen type I, present at the host site. The infection of a host site by a micro-organism
may be inhibited by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or by 100%, where the greater the percentage inhibition, the more preferred the embodiment of this aspect of the invention.
The polypeptide may comprise at least one copy of the tripeptide motif (Glycine-Proline- X) where X is any L- or D-amino acid or modified amino acid and where, in the case that two or more copies of the motif are present, X need not be identical in each copy of the motif. X may be selected from: alanine, asparagine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, tyrosine, hydroxyproline. X may also be a modified amino acid, as defined above. In a preferred embodiment of this aspect of the invention, at least one copy of the tripeptide motif may be (Glycine-Proline- Alanine), (Glycine-Proline-Hydroxyproline), (Glycine-Proline-Proline), or (Glycine- Proline-Threonine).
The polypeptide may consist of a di-tripeptide, examples of which include (Glycine- Proline-Alanine)2, (Glycine-Proline-Hydroxyproline)2, (Glycine-Proline-Threonine)2, (Glycine-Proline-Proline)2, (Glycine-Proline-Hydroxyproline-Glycine-Proline-Alanine), (Glycine-Proline-Hydroxyproline-Glycine-Proline-Threonine). The polypeptide may consist of three or more copies of the tripeptide motif; each copy may be identical or different.
Most preferably, the polypeptide may be Glycine-Proline-Alanine, Glycine-Proline- Hydroxyproline, Glycine-Proline-Proline, Glycine-Proline-Arginine-Proline, Glycine- Proline-Alanine-Glycine, Glycine-Proline-Threonine, Glycine-Proline, Glycine- Hydroxyproline or Glycine-Proline-Glycine-Glycine.
The infection may be infection of mammalian body tissue, for example, human body tissue, particularly infection associated with invasive surgery, insertion of a surgical implant or other medical, veterinary or dental procedure. Most particularly, the infection may be infection, preferably invasion, of dentine. The infection may be bacterial infection or fungal infection, particularly by members of the genera Streptococcus or Enterococcus, including Streptococcus gordonii, Streptococcus sanguis, Streptococcus
mutans, Streptococcus oralis, Streptococcus sobήnus and Enterococcus faecalis. The micro-organism may be a fungus. For example, the fungus may be a Candida species. . The infection may also be caused by a combination of two or more types of microorganism, for example, different species of bacteria, or different species of bacteria or fungi.
Polypeptides according to this aspect of the invention may also promote maintenance of healthy oral tissues, for example, gum, mouth and/or teeth tissues.
The polypeptide may be associated with a non-polypeptide chemical moiety to form a chemical compound. The peptide may be covalently linked to the non-polypeptide chemical moiety or may be non-covalently bound to the non-polypeptide chemical moiety. The chemical moiety may itself be an anti-bacterial or bacteriostatic molecule, or a molecule with other microbial inhibiting properties, or a molecule which aids retention, stability or longevity of the peptide at a host site, or a molecule which has host site binding properties, or a molecule which increases the efficiency of delivery of the peptide to the host site, or a molecule which enables continuous release of the peptide to the host site over a period of time.
According to a fourth aspect of the invention there is provided a composition containing a polypeptide according to the second or third aspects of the invention, or a chemical compound comprising a polypeptide according to the second or third aspects of the invention. The composition may preferably be a paste, a toothpaste, a mouthwash, a chewing gum, a gel, an oral spray, a dental fissure sealant, a dental root filling composition, a dental filling material, a dental intra-root canal material or a periodontal medicament The term "periodontal medicament" as used throughout this specification includes periodontal pack and/or periodontal dressing. The composition may more specifically be a composite resin dental fissure sealant, a glass ionomer dental fissure sealant, a zinc oxide/eugenol dental root filling composition or a gutta-percha dental intra-root canal material.
According to a fifth aspect of the invention there is provided a method of at least partially inhibiting the infection of a host site by microbial infection, comprising applying to the host site a composition containing a polypeptide according to the second or third aspects of the invention, or a chemical compound comprising a polypeptide according to the second or third aspects of the invention. Infection may be inhibited by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or by 100%, where the greater the percentage inhibition, the more preferred the embodiment of this aspect of the invention. The infection may be a bacterial infection, particularly infection by members of the genera Streptococcus or Enterococcus, including Streptococcus gordonii, Streptococcus sanguis, Streptococcus mutans, Streptococcus oralis, Streptococcus sobrinus and Enterococcus faecalis. The infection may be caused by a fungus, for example a Candida species. The infection may be caused by a combination of two or more types of micro-organism, for example different species of bacteria, or different species of bacteria and fungi. The infection may be infection of mammalian body tissue, for example, human body tissue, particularly infection associated with invasive surgery, insertion of a surgical implant or other medical, veterinary or dental procedure. Most particularly the infection may be infection, preferably invasion, of dentine.
According to a sixth aspect of the invention there is provided a method for improving the appearance of a mammal, the method comprising orally administering to the mammal a composition containing a polypeptide according to the second or third aspects of the invention, or a chemical compound comprising a polypeptide according to the second or third aspects of the invention. Preferably, the mammal is human.
According to a seventh aspect of the invention there is provided a method for maintaining the oral health of a mammal, the method comprising orally administering to the mammal a composition containing a polypeptide according to the second or third aspects of the invention, or a chemical compound comprising a polypeptide according to the second or third aspects of the invention. Preferably, the mammal is human. The term "maintaining
the oral health of a mammal" includes the promotion of healthy gums, mouth and teeth of the mammal.
According to an eighth aspect of the invention there is provided a chemical species having a surface or three dimensional profile which enables it to inhibit infection of a host site by a micro-organism by inhibiting the microbial recognition of and response to a collagen present at the host site. The chemical species may be a topographical analogue to a polypeptide according to the second or third aspects of the invention, or to a chemical compound comprising a polypeptide according to the second or third aspects of the invention. The term "topographical analogue" encompasses the term "dimensional analogue" and, as used throughout this specification, means a species which has three- dimensional characteristics substantially identical or similar to those of a polypeptide, or a chemical compound comprising a polypeptide, according to the second or third aspects of the invention (i.e. a species which is a peptidomimetic). Infection may be inhibited by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or by 100%, where the greater the percentage inhibition, the more preferred the embodiment of this aspect of the invention. The infection may be a bacterial infection, particularly infection by members of the genera Streptococcus or Enterococcus, including Streptococcus gordonii, Streptococcus sanguis, Streptococcus mutans, Streptococcus oralis, Streptococcus sobrinus and Enterococcus faecalis. The infection may be caused by a fungus, for example a Candida species. The infection may be caused by a combination of two or more types of micro-organism, for example different species of bacteria, or different species of bacteria and fungi. The infection may be infection of mammalian body tissue, for example human body tissue, particularly infection associated with invasive surgery, insertion of a surgical implant or other medical, veterinary or dental procedure. Most particularly the infection may be infection, preferably invasion, of dentine. The collagen may be collagen type I.
BRIEF DESCRIPTION OF THE DRAWINGS
Embodiments of the invention will now be described, by way of example only, and with reference to the accompanying Figures 1 to 9 in which:
Figure IA shows the amino acid sequence of CoIIAl (GenBank Accession No. NP_000079);
Figure IB shows the amino acid sequence of ColIA2 (GenBank Accession No. NP_000080);
Figure 2A shows invasion of human dentine by Streptococcus gordonii DLl;
Figure 2B shows invasion of human dentine by Streptococcus mutans NG8;
Figure 2C shows invasion of human dentine by Enterococcus faecalis JH2-2;
Figure 2D shows invasion of bovine dentine by Streptococcus gordonii DLl;
Figure 2E shows invasion of bovine dentine by Streptococcus mutans NG8;
Figure 2F shows invasion of bovine dentine by Enterococcus faecalis JH2-2;
Figure 3 shows a comparison of tubule invasion index (TI) and tubule invasion factor (TIF) for measuring invasion of bovine dentinal tubules by Streptococcus gordonii, Streptococcus mutans and Enterococcus faecalis;
Figure 4A shows inhibition of human dentine invasion by Streptococcus gordonii DLl in the presence of Glycine-Proline- Alanine tripeptide;
Figure 4B shows inhibition of human dentine invasion by Streptococcus mutans NG8 in the presence of Glycine-Proline- Alanine tripeptide;
Figure 4C shows inhibition of human dentine invasion by Enterococcus faecalis JH2-2 in the presence of Glycine-Proline- Alanine tripeptide;
Figure 4D shows inhibition of bovine dentine invasion by Streptococcus gordonii DLl in the presence of Glycine-Proline- Alanine tripeptide;
Figure 4E shows inhibition of bovine dentine invasion by Streptococcus mutans NG8 in the presence of Glycine-Proline- Alanine tripeptide;
Figure 4F shows inhibition of bovine dentine invasion by Enterococcus faecalis JH2-2 in the presence of Glycine-Proline- Alanine tripeptide;
Figure 5 shows inhibition of invasion of human dentinal tubules (TI) by Streptococcus gordonii, Streptococcus mutans and Enterococcus faecalis in the presence of Glycine- Proline-Hydroxyproline or Glycine-Proline- Alanine tripeptides;
Figure 6 shows inhibition of invasion of bovine dentinal tubules (TIF) by Streptococcus gordonii, Streptococcus mutans and Enterococcus faecalis in the presence of Glycine- Proline- Alanine tripeptide;
Figure 7 shows dose-dependent inhibition of Streptococcus gordonii DLl invasion of bovine dentinal tubules (TIF) by Glycine-Proline-Alanine tripeptide;
Figure 8 compares dose-dependent inhibition of Streptococcus gordonii DLl invasion of bovine dentinal tubules (% control) by Glycine-Proline-Alanine tripeptide, with growth of Streptococcus gordonii DLl (% control) in suspension culture containing similar concentrations of Glycine-Proline- Alanine tripeptide; and
Figure 9 shows the inhibition of Streptococcus gordonii DLl invasion (% TIF of control) of bovine dentinal tubules by Glycine-Proline-Alanine (GPA), Glycine-Proline (GP),
Glycine-Proline-Arginine-Proline (GPRP), Glycine-Proline-Glycine-Glycine (GPGG) and Glycine-Hydroxyproline (GO).
MODES OF CARRYING OUT THE INVENTION
1. Bacterial strains and growth media.
Bacterial strains utilized were Streptococcus gordonii DLl wild-type, Enterococcus faecalis JH2-2 wild-type, Streptococcus mutans NG8 wild-type, and Streptococcus gordonii UB 1360 A(sspA sspB) homozygous deletion mutant. Bacteria were grown at 370C on TSBY agar (Jenkinson et a!., (1993) Infect. Immun. 61 3199-3208) in a GasPak system (BD Diagnostic Systems). Liquid cultures were grown without shaking in screw- cap tubes or bottles at 370C in brain-heart infusion broth (Difco Becton Dickinson Microbiology Systems) containing 0.5% yeast extract (BHY medium) (Jenkinson et al. (1993) Infect. Immun. 61 3199-3208). Collagen type I was purchased from BD Biosciences while dipeptides and tripeptides were obtained from Sigma.
2. Collagen tripeptides block bacterial invasion of dentine; in vitro model.
For the in vitro model of dentine invasion, two types of teeth were used: human (healthy extractions from young orthodontic patients) or bovine (extractions from young (<22 month) slaughtered animals). Roots with single and fairly straight root canals were sectioned longitudinally down the canals so that the dentinal tubules were the largest possible (being at the pulp interface) and having the highest chance of containing collagen. Between two and five prepared sections were used as replicates. Invasion was visualized microscopically in the cervical to middle region of root canal dentine since the apical dentine has smaller and more obliterated tubules, due to sclerosis. Whenever dentine was cut, the cut debris was forced into the superficial few microns of the tubule and most of the debris was burnished onto the dentine resulting in blockage of the tubule. The dentine was washed with EDTA/ NaOCl, or with acid preparations, to remove such blockage.
The root dentine invasion model utilized has been described previously (Love et ah, (1996) Int. Endodon. J. 29 2-12). Penetration of bacteria into dentine was visualized by light microscopy of sections cut from infected dentine blocks and stained histologically for bacteria. The extent of invasion was initially expressed by the tubule invasion index (TI) (Love et al, (1997) Infect. Immun. 65 5157-5164), whereby TI>2.4 indicates heavy invasion and TI<0.5 is scored as nil invasion. However, this was subsequently modified to tubule invasion factor (TIF), which takes depth of invasion of tubules into account.
The TIF was obtained by multiplying the TI by the invasion depth score: xl , where invasion did not exceed 50μm; x2 where at least 5 tubules per field of view were invaded to a depth >50μm; and x3 where at least 5 tubules per field of view were invaded to a depth of lOOμm or greater.
Using this model system Streptococcus gordonii DLl (wild type) showed heavy invasion of both human and bovine dentine after 14 days (Figures 2A and 2D respectively) as did Streptococcus mutans NG8 (Figures 2B and 2E) and Enterococcus faecalis JH2-2 (Figures 2C and 2F). Graphical representations of the TI and TIF scores are shown in Figures 3 and 5. Figure 3 also shows a graphical representation of the comparison of tubule invasion index (TI) and tubule invasion factor (TIF) for the invasion of bovine tubules by the three organisms.
Deletion of the sspA and sspB genes, encoding antigen VlI proteins, rendered Streptococcus gordonii UB1360 unable to penetrate dentinal tubules. This confirms previously published data which showed that insertional inactivation of sspA and sspB genes rendered Streptococcus gordonii unable to penetrate dentine.
Incorporation of 0.1 mg/ml of the tripeptide Glycine-Proline- Alanine into the medium blocked invasion of both human and bovine dentine by Streptococcus gordonii DLl (Figures 4A and 4D respectively). Similar inhibitory data were obtained for Streptococcus mutans NG8 (Figures 4B and 4E) and Enterococcus faecalis JH2-2
(Figures 4C and 4F), where incorporation of the tripeptide also resulted in greatly reduced invasion of dentine by these normally highly invasive organisms. Figure 5 shows a graphical representation of the extent of the inhibition seen when human dentine infected with each of the three micro-organisms was incubated in the presence of either Glycine-Proline-Hydroxyproline (GPO) or Glycine-Proline-Alanine (GPA). Similarly, Figure 6 shows the effect of GPA on invasion (TIF) of bovine dentinal tubules by Streptococcus gordonii DLl , Streptococcus mutans NG8 and Enterococcus faecalis JH2- 2.
The inhibitory effect of Glycine-Proline-Alanine tripeptide on Streptococcus gordonii DLl invasion of bovine dentinal tubules was dose dependent. The TIF decreased from 5.11 ± 0.87 with no tripeptide present to 1.66 ± 0.43 in the presence of 0.2 mg/ml GPA (Figure 7). This is equivalent to a 43.9% reduction in invasion at the lowest concentration of peptide tested (0.05mg/mi), inhibition increasing with increasing peptide concentration up to a 67.5% reduction at the maximum concentration tested (0.2mg/ml).
Figure 8 compares the dose-dependent inhibition of dentinal tubule invasion by GPA with corresponding effects of GPA on the growth of Streptococcus gordonii in suspension culture in the same medium used to infect dentine. A nearly 50% inhibition of dentinal tubule invasion was observed at 0,05mg/ml GPA tripeptide. At the same concentration there was no effect of this peptide on microbial growth rate or yield. At higher concentration (0.2mg/ml) there was < 10% reduction in growth of bacteria in suspension culture, but > 70% inhibition of dentinal tubule invasion. The GPA tripeptide does not therefore exert inhibitory effect on dentinal tubule invasion by inhibiting the normal growth of the bacteria, but rather, has a direct effect on the invasion of the dentine by these organisms.
Figure 9 shows the effects of different peptides (Glycine-Proline-Alanine (GPA), Glycine-Proline (GP), Glycine-Proline-Arginine-Proline (GPRP), Glycine-Proline- Glycine-Glycine (GPGG) and Glycine-hydroxyproline (GO)) on the invasion (TIF) of bovine dentinal tubules by Streptococcus gordonii DLl. All of the peptides tested were
effective in reducing the invasion levels compared with the control (no peptide). Glycine- Proline-Alanine (GPA) was consistently the most effective. Glycine-Proline-Glycine- Glycine was also very effective. The dipeptide Glycine-Hydroxyproline also appeared to be inhibitory to invasion of bovine dentinal tubules by Streptococcus gordonii DLl, while Glycine-Proline was only slightly inhibitory.
3. Collagen tripeptides block bacterial invasion of dentine; in vivo model.
Early in vivo studies on bacterial invasion of dentine involved cutting cavities and leaving them exposed. The depth of cut would vary from 0.5 mm to 1 mm in depth and early invasion was noted at 6 days getting heavier and deeper with time (Lundy & Stanley, (1969) Oral Surg. 27 187-201; Vojinovic et al., (1973) J. Dent. Res. 52 1189-1193). However, abraded or fractured dentine also allows for invasion in vivo (Tronstad & Langeland, (197I) J. Dent. Res. 50 17-30; Olgert et al, (191 Λ) Acta. Odontol Scand. 32 61-70).
Treatment of dentine in vivo with compositions containing the tripeptides (Glycine- Proline-Alanine) or (Glycine-Proline-Hydroxyproline) inhibited the early invasion of dentine.