WO1995031556A1

WO1995031556A1 - Glucan-binding proteins, and use thereof

Info

Publication number: WO1995031556A1
Application number: PCT/GB1995/001070
Authority: WO
Inventors: Howard Kikuo Kuramitsu; Kurt Matthew Schilling
Original assignee: Unilever Nv; Unilever Plc
Priority date: 1994-05-11
Filing date: 1995-05-11
Publication date: 1995-11-23
Also published as: EP0759081A1; JPH10500127A; GB9409387D0

Abstract

Polypeptides with specific binding affinity for glucan - especially the glucan-binding domain of glycosyl transferase enzyme - is utilised in a composition for oral care. The polypeptide may block the binding sites in dental plaque where glycosyl transferase would bind and generate more plaque, or it may be conjugated to - and provide targeted delivery of - an antiplaque or antistain agent.

Description

GLUCAN-BINDING PROTEINS. AND USE THEREOF

Field of the Invention The present invention relates to polypeptides with glucan-binding properties, to hybrid materials containing such polypeptides, and to novel active systems targeted to dental plaque and oral care compositions containing same.

Background of the Invention

The initiation and accumulation of dental plaque results from the adherent attachment of bacteria and their products to the teeth. Attachment of many plaque bacteria is mediated through the production of adhesive extracellular polysaccharides from dietary sucrose. The main two such polysaccharides synthesised from sucrose in dental plaque are glucans and fructans [Hamada S and Slade HD 1980 Biology, immunology and cariogenicity of

Streptococcus mutans Microbiol. Rev. 44: 331-384]. These adhesive polysaccharides are known to increase attachment of plaque bacteria by providing stereochemically specific binding sites, and also through non-stereospecific trapping of bacteria. The bacterial enzymes which synthesise glucans and fructans, referred to as glucosyltransferases (GTFs) and fructosyltransferases (FTFs) respectively, are secreted by many plaque bacteria. They are found associated with bacterial surfaces, adsorbed to tooth surfaces, and in the saliva which bathes the oral cavity [Rolla G, Ciardi JE, Eggen K, Bowen WHandAfseth J (1983) Free glycosyl- and fructosyltransferase in human saliva and adsorption of these enzymes to teeth in vivo. In: R J Doyle andJE Ciarid (ed) Glucosyltransferase, glucans, sucrose and dental caries. Spec. Suppl. Chem. senses. IRL Press Washington DCp 21-30].

Glucans in plaque are composed predominantly of alpha 1,6-linked and alpha 1,3 -linked glucose residues.

Polysaccharide-mediated bacterial attachment is considered to be crucial to the accumulation of pathogenic dental plaque [Hamada andSlade, 1980]. Several species of oral bacteria produce proteins which recognise and bind to glucans. These proteins include the glucosyltransferases (GTFs) which are involved in forming plaque and also non- enzymatic glucan-binding proteins (GBPs). GTFs and GBPs specifically recognise glucans containing alpha 1,6-linked glucose residues, and mediate bacterial binding to extracellular plaque matrix [Schilling K M and Bowen WH 1992 Glucans synthesized in situ in experimental salivary pellicle function as specific binding sites for Streptococcus mutans Infect. Immun. 60; 284-295].

Molecular analysis of a GBP and several GTFs from oral streptococci has shown that, these proteins have distinct domains (GBDs) which are responsible for stereospecific binding to glucans [Wong C, Hefta SA, Paxton R J, ShivelyJE andMooser G (1990) Size and subdomain Architecture of the glucan binding domain of sucrose; 3 -D glucosyltransferase from Streptococcus sobrinus. Infect. Immum. 58; 2165-2170; FerrettiJ J, Gilpin ML and Russell R B (1987) Nucleotide sequence of a glucosyltransferase gene from Streptococcus sobrinus MEF28, Journal Bact. 169, 4271-4278; Koto C and Kuramitsu HK (1990) Carboxyl-terminal deletion analysis of the Streptococcus mutans glucosyltransferase-I enzyme. FEMS Microbiol. Lett 72, 299-302] Studies with glucan- binding domains have shown that these bind to glucans with affinities similar to that observed for intact GTFs.

Other polysaccharide-binding domains are known and have been conjugated to other proteins and peptides to aid in downstream processing of recombinant fermentation products. For instance, researchers have made fusion proteins containing starch-binding domains [Chen L, Ford C and Nikolov Z (1991) Adsorption to starch of a β-galatosidase fusion protein containing the starch-binding region of Aspergϊllus glucoamylase Gene 991, 121-126] and cellulose-binding domains [OngE, Greenwood J M, Gilkes NR, Kilburn D G, Miller R C and Warren R A J (1989) The cellulose-binding domains of celluloses; tools for biotechnology TIBTech 7; 239-243] for the purpose of purifying the proteins on starch and cellulose resins. One of the major technical problems associated with the development of effective active systems for oral care benefits is obtaining substantive delivery of the agents to the desired site in the mouth (eg: plaque). For the most part, substantive anti-plaque agents currently in use are positively charged microbicides, such as bis-biguanides and quaternary ammonium compounds, which bind to oral surfaces through electrostatic interactions.

However, binding of cationic microbicides is non-specific in nature (as it is for hydrophobic agents as well); these agents bind to all oral tissues. Furthermore, use of cationic molecules such as chlorhexidine and cetyl pyridinium chloride in dentifrices and oral rinses has been associated with tooth staining and undesirable product taste.

Summary of the Present Invention

The present invention uses glucan-binding polypeptides to target and bind to glucans either in plaque matrix or associated with bacterial surfaces. The glucan-binding polypeptides can be conjugated to an anti-plaque or other agent and used to deliver this to plaque, providing substantivity and often reducing the amount of agent needed. They can avoid the negative aspects associated with cationics and other non-specific oral care active agents.

Conjugates can be formed with a variety of materials, either chemically or through the tools of molecular biology (i.e. fusion proteins).

Broadly, in a first aspect, the present invention provides a polypeptide with specific binding affinity for glucan, which is not incorporated within a glycosyltransferase enzyme and desirably is covalently chemically bound to a further material which does not display glycosyltransferase enzymic activity.

The present invention's use of a glucan-binding polypeptide conjugated to another moiety is distinctive in several ways. First, conjugates described herein are derived from recognition systems involving alpha 1,6-linked glucans. Second, the conjugates described herein are used to provide a benefit in the oral environment. Third, the glucan-binding conjugates are intended to target to a biofilm where they deliver secondary physiological effects in a biochemically hostile environment, as opposed to the described use of other polysaccharide-binding domain fusions to bind to simple chromatographic resins in vitro.

It is also a possibility within this invention to use glucan-binding polypeptides to block binding sites on existing glucan. Polymer synthesis by GTF requires glucan-binding by the enzyme. Glucan-binding polypeptides will compete with GTFs for binding sites and thus act as stereospecific inhibitors of GTF-catalysed glucan synthesis in plaque. This will decrease the build-up and tenacity of plaque. An analogous possibility is that glucan- binding polypeptides will block sites in plaque matrix to which oral bacteria will bind, and in this way inhibit the build-up of plaque. If a glucan-binding polypeptide is used as a competitive inhibitor in this way, rather than as a means of targeting some other agent, it is nevertheless likely that the glucan-binding polypeptide will be fused to some other peptide residue which was needed for the synthesis step.

In a second aspect this invention provides a composition for topical application in the mouth, comprising a polypeptide with specific binding affinity for glucan, in a carrier vehicle which is acceptable for use in the mouth.

Detailed Description of the Invention

It is a purpose of the present invention to utilise GBDs or other glucan-binding polypeptides or other glucan-binding polypeptides as targeting groups for the substantive delivery of oral care active agents, especially anti-plaque and anti-stain agents to the plaque on human teeth. Conjugates can be formed through chemical conjugation between glucan- binding polypeptide and a large number of agents by ester, sulftiydryl, peptide, isopeptide, amide and other types of chemical bonds. Entities which may be conjugated in one way or another, include organic compounds, inorganic complexes, proteins, enzymes, peptides, antibodies and various ligands. The conjugates can also be fusion or hybrid proteins produced by recombinant DNA technology. Enzymes to be conjugated to glucan-binding polypeptides include but are not limited to oxidases, peroxidases, proteases, glycosidases, Upases, esterases, amidases, deaminases, ureases and polysaccharide hydrolases. In particular, oxidases can function as cytotoxic agents acting against the microbial species in dental plaque. Glucose and galactose oxidases produce hydrogen peroxide which is cytotoxic. Peroxidases can correct this to hypohalite, which is even more toxic. Both hydrogen peroxide and hypohalite are short¬ lived in vivo, and are made more effective by creation at their intended point of action, as provided by this invention.

Such enzymes can also function as anti-stain agents, since their products are bleaching species. Non-enzymatic anti-microbial proteins and peptides such as antibodies, antibody fragments, histatins, lactoferrin, defensins, magainins, cecropins, other cationic antibacteriocins and bacteriocins can also be conjugated to glucan-binding polypeptides. Microbicides including but not limited to triclosan, chlorhexidine, quaternary ammonium compounds, chloroxylenol, chloroxyethanol, thymol and fluoride can also be chemically conjugated to glucan-binding polypeptide. Anti-microbial cations such as Zn, Sn, Cu and others can be complexed to glucan-binding polypeptide by forming conjugates with appropriate chelating agents such as (poly) carboxylic acids, amino acids and so on. Targeting systems can also be produced by biotin-avidin conjugates. For instance biotin can be chemically conjugated to GBD which targets it to plaque where the biotin acts as a specific binding site for avidin-conjugates. Similarly, avidin-GBD conjugates can be used as plaque-specific binding sites for biotin conjugates.

It is also possible within this invention to use glucan-binding polypeptides as targeted release agents by conjugating anti-plaque or other oral care active agents to glucan-binding polypeptides with bonds which are sensitive to hydrolase, pH or oxidation/reduction. Fusions of a protein to glucan-binding polypeptide can be linked by protease-sensitive linker peptides which will be hydrolysed by endogenous or exogenous protases in plaque. Non-proteinaceous microbicides can be conjugated by esterase, amidase, lipase or other hydrolase-sensitive bonds. Release can also come by the use of pH sensitive bonds or by conjugating proteins, peptides or other agents through S-S bonds which are sensitive to chemical reduction as the redox potential drops in plaque or as reducing agents accumulate.

Oral care products containing glucan-binding polypeptides can be in a variety of forms including toothpastes, gels, mouthwashes, powders, gargles, solutions, lozenges, chewing gum and dental floss.

The oral composition may furthermore comprise conventional ingredients, such as pharmaceutically acceptable carriers like starch, sucrose, polyols, surfactants, water or water/alcohol systems etc. When formulated into a dentifrice, such formulation may contain usual dentifrice ingredients. Thus, they may comprise particulate abrasive materials such as silicas, aluminas, calcium carbonates, dicalciumphosphates, hydroxyapatites, calcium pyrophosphates, trimetaphosphates, insoluble hexametaphosphates and so on, usually in amounts between 5 and 60% by weight.

Furthermore, the dentifrice formulations may comprise humectants such as glycerol, sorbitol, propyleneglycol, lactitol and so on.

Surface-active agents may also be included such as anionic, nonionic, amphoteric and zwitterionic synthetic detergents. Examples thereof are sodiumlaurylsulphate, sodium dodecylbenzenesulphonate, sodium mono- and dioctyl-phosphate, sodiumlauroylsarcosinate, cocamidopropylbetain

Binders and thickeners such as sodium carboxymethyl-cellulose, xanthan gum, gum arabic etc. may also be included, as well as synthetic polymers such as polyacrylates and carboxyvinyl polymers such as Carbopol®.

Flavours such as peppermint and spearmint oils may also be included, as well as preservatives, opacifying agents, colouring agents, pH-adjusting agents, sweetening agents and so on.

Additional anti-bacterial agents may also be included such as Triclosan, chlorhexidine, copper-, zinc- and stannous salts, such as copper sulphate, zinc citrate and stannous pyrophosphate, sanguinarine extract, metronidazole. Further examples of additional anti¬ bacterial agents are quaternary ammonium compounds such as cetylpyridinium chloride; bis-guanides such as chlorhexidine digluconate, hexetidine, octenidine, alexidine; halogenated bisphenolic compounds such as 2,2' methylenebis-(4-chloro-6-bromophenol).

Polymeric compounds which can enhance the delivery of active ingredients such as the anti-bacterial agents can also be included. Examples of such polymers are copolymers of polyvinylmethylether with maleic anhydride and other similar delivery enhancing polymers, e.g. those described in DE-A-3, 942,643 (Colgate)

Furthermore anti-inflammatory agents such as ibuprofen, flurbiprofen, aspirin, indomethacin etc. may also be included.

Anti-caries agents such as sodium- and stannous fluoride, aminefluorides, sodium monofluorophosphate, calcium lactate and/or calcium glycerophosphates, strontium salts and strontium polyacrylates, casein and casein digests and phosphoproteins may also be included.

Other optional ingredients include vitamins such as Vitamin C, plant extracts, potassium salts such as potassium citrate, potassium chloride and potassium nitrate.

Other optional ingredients include enzymes such as dextranase and/or mutanase, amyloglucosidase, glucose-oxidase with lactoperoxidase, neuraminidases, and hydrogen peroxide generating compounds such as potassiumperoxydiphosphate.

Furthermore, the oral compositions may comprise anti-calculus agents such as alkalimetal pyrophosphates, hypophosphite-containing polymers, organic phosphonates, phosphocitrates etc.

Other optional ingredients that may be included are e.g. bacteriocins, bacteriophages, tissue respiratory factors, antibodies, bleaching agents such as peroxy compounds, effervescing systems such as sodium bicarbonate/citric acid systems, colour change systems, and so on.

Brief description of the Drawings

Fig. 1 diagrammatically illustrates the structures of the plasmid pUC18 38Hind and derivatives constructed from it. Restriction sites (where relevant) are shown in this and some subsequent figures with abbreviated identities as follows: B=-5αmHI; Bgl=_9gTI;

S=Scal, and X=Xbal. Restriction sites without abbreviation denote a BstYI boundary site of two DNA fragments created following BamWBglll ligation.

Fig. 2 illustrates the general strategy of the "heterodimer" system used to make intermediates for use in transformation.

Fig. 3 illustrates structure of the plasmids used for introduction of the integration anchor sites into the S.gordonii chromosome. Restriction sites are shown with abbreviated identities as described under Fig. 1 ; also Bs=B.stBI; C=Clal; and Xh=.Λ7zoI.

Fig. 4 is a series of restriction maps which illustrate integration of the hybrid GBD gene into the S.gordonii chromosome. The topmost map shows chromosomal structure around the gtfG gene in S.gordonii. The subsequent maps, which are on a larger scale, show chromosomal structures of wild type (A), and primary (C), secondary (E), and GBD⁺ (G) integrants. DNA fragments (B) and (D) were prepared by digesting the heterodimer plasmids of Figs. 3E and 3H with Hindill and Notl respectively. Restriction fragment (F) was the PvwII digest of the plasmid 38HGBDEm^r of Fig. IC. Fig. 5 is a diagram of a preliminary experiment demonstrating resident plasmid integration in S.gordonii. Restriction sites are indicated with abbreviations as for Figs. 1 and 3. Also

Fig. 6A,B and C show the structures of plasmids and illustrate integration of the hybrid GBD gene into resident plasmids harbored in S.gordonii cells.

Fig 6D shows in more detail the DNA sequences which recombine.

Fig 6E shows the result of agarose gel electrophoresis in which the lanes are:

M, a mixture of λDNA EcoRl-HindUl double digests and λDNA Hindlll digest; 1 , initial resident plasmid pPIOZ' 18Spec^r;

2 and 3, and 4 and 5, primary and secondary transformants, respectively; 2 and 4, and 3 and 5, positive and negative control integrants, respectively.

Fig. 7 shows (on successive lines) restriction maps of the S.mutans GS-5 chromosomal structure containing the gtfD gene, the gtfD gene (as used for probe DNA) and the 5'- and 3'-flanking regions of the gtfD gene. Restriction sites shown are as described under Figs. 1, 3, and 5 above; in addition (D) denotes a non unique Dral site; and

Fig 8 is a bar chart of results from Example 3.

Fig 9 illustrates a slide with an array of wells, used in Example 4.

Fig 10 shows a restriction map of the galactose oxidase gene of Dactylium dendroides, and the incorporation of a gene fragment into a plasmid.

Figs 11 and 12 diagramatically illustrate further manipulation to construct a plasmid containing the whole of the gene. Figs 13 and 14 diagramatically illustrate the construction of a plasmid incorporating both the GBD gene and the galactose oxidase gene.

Fig 15 shows the results of a galactose oxidase activity assay.

Fig 16 shows the results of a glucan binding assay.

Example 1 - Preparation of Fusion Proteins Containing GBPs

As shown by the restriction maps of Fig 7, the gtfD gene within the chromosome of S.mutans GS-5 contains the gene for a glucose binding domain (GBD) within a 1.67-kb Xbal-BamHi fragment near to its 3' terminal, [see Hondo,0., Kato,C, and Kuramitsu, H.K. (1990) Nucleotide sequence of the Streptococcus mutans gtfD Gene encoding the Glucosyltransferase-S Enzyme. J Gen Microbiol 136:2099-2105]

a) Preparation of the hybrid GBD gene. A secretion domain from the S.mutans GS- 5 gtfB gene which specifies the first 38 amino acid residues from the initiator Met as the signal peptide for secretion of the GTF-I enzyme had been isolated previously (Infect Immun 61:3745).

A plasmid designated pUC18 38Hind (Fig 1A) was constructed which contains this domain (800-bp Hindlll fragment) oriented in the opposite direction relative to the lacZ' gene of pUC18. The 3'portion of the Xbάl-BamRl fragment (1.67-kb) of the gtfD gene (5),containing the GBD gene (Fig.7), was then cloned into Xbal-BamHi cleaved plasmid pUCl 8 38Hind. In the resulting plasmid (Fig IB) designated 38HGBD, the reading frames of both the 3' -Hindlll site in the signal sequence coding region and the 5'-Xbάl site in the GBD gene are the same as that of the multiple cloning site (MCS) of pUC18. Consequently, the GBD molecule is expressed fused to the GTF-I protein leader sequence. For a negative control, the unique Xbal site was digested, filled-in and recircularized to create a plasmid in which the reading frame of the GBD was out of frame with the signal sequence. The presence or absence of the GBD protein in crude extracts oϊE.coli transformants harbouring each plasmid was confirmed by Western blot analysis (data not shown).

The plasmid of Fig IB was further modified to incorporate an Em^r gene in its unique

BgRl site (38HGBDEm^r, 6.3-kb, Fig.lC). This was subsequently used in transformation of S.gordonii as mentioned below.

b) Integration of the hybrid GBD gene into the S.gordonii chromosome. Attempts were made to clone a fragment containing the hybrid GBD gene into an E. coli- streptococcus shuttle plasmid designated pResEm749. This was unsuccessful, making it necessary to devise an alternative cloning strategy. The approach which was adopted sought to introduce the hybrid GBD gene directly into S.gordonii without utilizing E.cali- streptococcus shuttle plasmid.

The internal 1.5-kb Hindlll fragment of the S.gordonii gtfG gene was chosen as a target site for integration of the hybrid gene into the S.gordonii chromosome. As shown in Fig 4, the gtfG gene in wild type S.gordonii encompasses three Hindlll fragments. The middle one of these was chosen as the target fragment. This fragment and one adjoining Hindlll fragment have been cloned in the known plasmid pAM5010 [Sulavik,M., Tardof,G., and Clewell, D.D. (1992) Identification of a Gene rgg which regulates expression of glucosyltransferase and influences the SPP Phenotype of S. Gordonii Challis, J Bacteriol 174, 3577-3586]. The plasmid pAM-S15 contains the 3'-end of the 1.5-kb Hindlll fragment of the pAM5010 insert.

As an initial step, prior to the intended transformation of the S. Gordonii chromosome, it was necessary to introduce appropriate integration anchor sites into the chosen target fragment. In order to construct intermediates for this task, a novel "heterodimer" system was designed. This allows DNA fragments to be placed between the 5'- and 3'-flanking regions of a target site. This "heterodimer" system is explained in general terms with reference to Fig.2. A plasmid referred to as the "acceptor" is shown at Fig 2A. It has a single Pvull site designated in Fig 2 as site A. Initially, the target gene containing the anchor sites (Fig 2F) is cloned into the acceptor plasmid following appropriate conversion of the single Pvull site, yielding the plasmid shown at Fig.2B. Next, the vwII site of another plasmid is modified by ligating a linker DNA so that the resulting "rescue" plasmid shown in Fig 2C has a site compatible with a unique site for restriction by enzyme B, which is present in the insertcloned into the acceptor plasmid. The plasmids of Figs 2B and 2C are both digested to completion with the restriction enzyme B. The resulting two DNA fragments are ligated without any phosphatase treatment and transformed into E. coli JM 109. The plasmid DNA purified from these transformants selected on LB agar plates containing a combination of both antibiotics would be a dimer containing two identical replication regions (pl5Aori) as well as two different drag resistance markers (Fig.2D). Therefore, this plasmid is designated as a "heterodimer". Finally, the DNA fragment for integration is readily prepared by digesting the heterodimer plasmid of Fig 2D with restriction enzyme A. This leaves a moiety termed pRes located between the correctly oriented 5'- and 3'- flanking regions of the target gene. "Rescued" plasmid (E) would be isolated following recircularization, and transformation into E. coli. The structure of the insert in the rescued plasmid (Fig.2E) has a different arrangement, 5'-B-A-B-3', compared to that of the target site, 5'-A-B-A-3'.

This procedure, just described in general terms, was used as shown by Fig 3.

To provide the appropriate integration anchor sites, two plasmids analogous to that of Fig 2B, designated pResAmpdBC (2.1 -kb) and pResKmHindS- 15 (3.5-kb) were constructed (Figs 3A and D, respectively). The former plasmid contains both 250-bp 5'- and 3'- flanking regions of the gtfB and gtfC genes, respectively, from S.mutans while the latter contains the 1.5-kb internal Hindll fragment of the gtfG gene from S.gordonii (18). The unique Seal site present in this insert within the latter plasmid was further converted to a Pstl site by introducing the Pstl linker DNA. The structure within the MCS of the plasmid pResAmpdBC shown in Fig 3 A was:

Not-Bcl-Hind-Sph-Pst-5' end-Bgl-3' end-Pst— Bam/Bgl-Not.

Into the unique BgRl site of the insert, the BgRl cleaved rescue plasmid, pResEmBgl (1.6- kb, Fig.3B) was ligated. The resultant heterodimer plasmid pResAmpdBC:pResEmBgl (3.7-kb, Fig.3C) was isolated from E.coli JM109 transformants following selection of colonies on LB agar plates supplemented with both Amp and Em. The structure within the MCS of this plasmid would be:

Not-Bcl-Hind-Sph-Pst-S' end-Bg pResEmBgkBgl-S' end-Pst— Bam/Bgl-Not.

This heterodimer plasmid was digested with Pstl and a 2.1-kb fragment containing pResEmBgl flanked by both the 5'- and 3'-end fragments was gel purified. This fragment • was ligated with the Pstl cleaved plasmid, pResKmHindS- 15P (3.5-kb, Fig.3D) and the heterodimer plasmid, pResKmHindS- 15P:pResEmdBC (5.6-kb, Fig.3E) was isolated. Digestion of this heterodimer plasmid with Hindlll leads to the fragment shown at Fig 4B.

S.gordonii was transformed using the Hindlll digested plasmid. The common regions between the chromosome and DNA fragments where double cross over can occur are the 5'- and 3'- fragments of the pAM-S15 insert shown with thick lines in Figs 4A and 4B. S.gordonii primary integrants (Em^r, GTF", Fig.4C) which contain an integration anchor site replacing the Seal site of the gtfG gene were isolated following transformation.

The 1.2-kb HindlU-BamHl fragment (containing the 3'-portion of the gtfD gene, Fig.7) was introduced into the Hindlll-Bglll cleaved plasmid pResAmpdBC (Fig 3A) to yield pResAmp3'GBDdC (3.0-kb, Fig.3F). The structure of the insert in this plasmid was:

Not-Bcl-Hind-3' GBD-Bam/Bgl-3' end-Pst— Bam/Bgl-Not. Subsequently, pResSpecHind (1.8-kb, Fig.3G) was cloned into the unique Hindlll site of pResAmp3'GBDdC and the heterodimer pResAmp3'GBDdC:ρResSpecHind (4.8-kb, Fig.3H) was isolated. The structure around the insert is:

Not-Bcl-Hind:pResSpecHind:Hind-3' GBD-Bam/Bgl-3' end-Pst— Bam/Gbl-Not.

Notl digestion of this plasmid leads to the fragment illustrated at Fig 4D.

The S.gordonii primary integrants containing the 5'- and 3'-flanking regions of the gtβ and gtfC genes (Fig.4C) were transformed with this heterodimer plasmid following Notl digestion (Fig.3D) and secondary integrants (Em^s, Spec^r, 3'GBD, Fig.4E) were isolated.

The common regions between the chromosome and DNA fragments where double cross over can occur are pl5Aori and the 3'-end of the S.mutans gtfC gene.

Finally, this integrant was transformed with the plasmid 38HGBDEm^r (Fig.lC) following PvwII digestion to yield the fragment shown at Fig 4D. The common regions between the chromosome and DNA fragments where double cross over can occur are the 5'-end of the

S.mutans gtfB gene and 3'-end of the GBD gene. Following transformation Spec^s, Em^r,

GBD⁺ transformants were isolated (Fig.4G).

The corresponding GBD" transformants were constructed in the same way, using the negative control hybrid gene created, as described above, by change at the Xbάl site in the 38HGBD plasmid shown at FiglB.

c) Resident plasmid integration. A preliminary experiment was carried out to been mentioned above, the hybrid GBD gene could not be cloned into the shuttle plasmid pResEm749 in E.coli. Since S.gordonii is a strain, chromosomal integration could be readily accomplished with an appropriate linear DNA fragment following transformation. This approach was examined by determining whether a DNA fragment residing on a plasmid replicating in S.gordonii (which is a recombination proficient strain) could be replaced following integration events. This is illustrated by Fig.5. Em^r and Spec' genes were (separately) cloned into the BamHl site of plasmid KmOZ'18, a pUC-type Km' vector (Infect Immun 61 :3745). Since the Km' gene of this plasmid is flanked by Xhol sites, this drug resistance gene was eliminated by Xhol digestion and a BgRl linker was introduced following a filling-in reaction. In the shuttle plasmid pResEm749, the Clal and one of the Seal sites were converted to BamHl sites following several DNA manipulations. The basic replicon from pV A380-1 (2.5-kb BamHl fragment, ref.7) active in streptococci was isolated from this pResEm749 derivative and gel purified. It was used to construct E.coli- streptococcus shuttle plasmids pPIOZ'18Em^r (4.9-kb) and pPIOZ"18Spec^r (5.1-kb) (pPI:resident Plasmid Integration). S.gordonii competent cells were then transformed with each shuttle plasmid and Em' and Spec' strains were isolated. An Em' strain harbouring pPIOZ'18Em' was next transformed with the BgRl linearized plasmid dKmOZ'lδSpec'. These have two regions (pUCori and downstream oflacZ') in common and double crossover can occur via these two homologous regions. As a result, an Em' gene was replaced with a Spec' gene following selection of S.gordonii transformants on TSB containing Spec. Plasmid DNA was isolated from the Spec' transformants which was indistinguishable from the pPIOZ'18Spec' shuttle plasmid previously constructed using E.coli cells. Similar results were obtained when the Spec' S.gordonii was transformed with the linear dKmOZ'18Em' DNA (as illustrated at the right hand side of Fig5).

These observations indicated that integration events following transformation could occur not only on the chromosome but also within resident plasmids in S.gordonii cells. Therefore, certain plasmid structures which are unstable in E.coli cells might be constructed directly in a S.gordonii system without prior ligation of component DNA fragments. To put this novel strategy into effect, S.gordonii Spec' transformants harbouring pPIOZ'18Spec' (as shown in Fig5 and also FigόA) were transformed with EcoO109 linearized 38HGBDEm' (FiglC also Fig6B) which contained the hybrid GBD gene. Resident plasmid pPIOZ' 18Spec^r and EcoO 109 linearized 38HGBDEm' share two homologous domains (pUCori and downstream of lacZ¹) and the hybrid GBD gene was integrated following recombination. Analysis of plasmid DNA from primary Em' transformants indicated the presence of two types of plasmids within a single transformant: initial and integrant plasmids lanes 2 and 3). Secondary Em' isolates transformed using these plasmids indicated the presence of only one plasmid, pPI38HGBDEm^r (7.5-kb, lanes 4 and 5). Transformation of E.coli JM109 with the integrant plasmid from the latter transformants yielded no Em' colonies again indicating that this plasmid cannot be maintained in E.coli cells.

d) Preparation of GBD fusion proteins. Following growth of S. gordonii transformants (containing either a plasmid or chromosomal copy of the GBD-fusion gene), the culture supernatant fluids were concentrated 50 times by acetone precipitation and assayed for the presence of GBD. Western blot analysis using anti GTF-S antibody as described previously (Gene 69:101-109) confirmed secretion of the protein bands of the expected size (60 kd) as well as smaller degradation products.

e) Expression of GBD hybrid genes in E. coli. GBD can be expressed in E. coli as a fusion protein containing peptides from other proteins.

A Xbάl fragment from the gtfD gene was inserted in frame into the plasmid pGD103X. This then expressed a fusion of the GBD with the first thirteen amino acids of β- galactosidase. The fusion protein readily binds glucans as determined by: binding of biotinylated-dextran by the fusion protein or after attachment of the fusion protein to dextran-Sepharose beads followed by elution and detection on Western blots with anti- GTF-S sera. This strategy also incorporates a cysteine residue into the fusion protein derived from the β-galactosidase peptide. This allows for the covalent attachment of biotin or other detection molecules to the GBD.

In addition, the GBD has been fused to the Tag peptide of the pTOPE system (Novagen, Madison, Wisconsin) for expression in E. coli. The resultant fusion protein is naturally biotinylated in E. coli and can be readily purified following absorption to avidin columns. Following elution of the fusion protein with biotin the GBD can be released from the fusion protein following Factor Xa cleavage and passage of the mixture through a second avidin column.

GBD-β galactosidase peptide fusion proteins were purified from transformed E.coli by applying cell extracts to dextran-Sepharose columns and eluting with a gradient of guanidine HC1 in 10 mMol acetate buffer (pH=6.0). This was followed by dialysis into buffer devoid of guanidine HC1.

Example 2 - Binding of 6BD fusion proteins to dextrans

a) Binding to biotinylated-dextran. An enzyme linked immunoassay (ELIS A) for measuring dextran binding activity was devised utilizing biotinylated-dextran (Pharmacia). Briefly:

Protein samples (50 uL of culture supernatant from S. Gordonii transformants) were added to wells in microtiter plates and incubated for 18 h at 4°C. The supernatant fluids were discarded and the absorbed proteins washed three times with water. Each well was then filled with 200 uL of blocking buffer (0.5% BSA in acetate buffer, pH 6) and incubated for one hour at 37°C and washed as described above. The wells can then be filled with various concentrations of biotinylated-dextran (Pharmacia) and incubated for 10 min at room temperature. Each well was then washed as described above with buffer. Streptavidin- peroxidase was then added and incubated for 5 mins at RT. The contents of each well was removed and the absorbed material washed as described above. The colour reaction was then developed by adding the peroxidase substrate and incubated for 10-30 min and the reaction terminated by adding 100 uL of 3N sulfuric acid. The absorbance of each solution was then measured at 410 nm in an ELIS A plate reader.

Using this assay, culture supernatant fluids were compared from a S.gordonii strain either harbouring the GBD fusion chromosomal integrant and a control strain with no integrated gene for the hybrid protein. The results in Table 1 clearly show that the strain harbouring the GBD fusion protein gene secreted a protein which binds to biotinylated-dextran in a dose dependent manner. In contrast, the negative control strain does not produce this activity.

Table 1 Glucan binding by S.gordonii transformants A_4]0

Biotin-dextran rug/mil 0 1 10 100

S.gordonii dXba (neg.control) .047 .053 .049 .050

S.gordonii 38HGBD (integrant) .051 .076 .101 .201

Example 3 - Binding of GBD-biotin conjugates to bacteria

a) Preparation of glucan-binding domain-biotin conjugates. In order to demonstrate that GBDs can be used to target other molecules to dental plaque, conjugates of GBD and biotin were produced and tested for binding to glucan-coated bacteria, and for targeting to plaque. To produce GBD-biotin the amino acid cysteine can be engineered genetically into the structure of the protein (GBD is normally devoid of cysteine) at either the C or N terminus. This can be done by adding the appropriate codon to the sequence of the GBD gene or by adding cysteine codons in the peptide sequences fused to GBD. In the experiments described here, cysteine was added to GBD as part of the sequence of the β- galactosidase peptide in the GBD fusion produced in E.coli.

Biotin was attached to the GBD fusion protein by a previously described method (Anal. Biochem.149:529; 1985). To do this, a solution of the GBD (purified from E.coli clone extracts on dextran-Sepharose columns) was mixed with 0.3 mg of biotin-maleimide (Sigma). The solution was incubated for 5 hr at 37°C and dialysed against phosphate- buffered saline for 18 hr to remove unreacted biotin. The resultant biotinylated-GBD could be used to quantitate binding of the complex to bacterial cells or dextran by mixing the GBD with beads or cells and filtering the mixtures on Ultra-free MC filters (Millipore). The samples could be washed directly on the filters and incubated with streptavidin- peroxidase to quantitate the amount of GBD bound to the cells or resins following determination of the absorbance of the coloured conjugates in an ELISA reader.

b) Measurement of binding

To measure binding of GBD-biotin to glucans on bacterial surfaces, Streptococcus mutans GS-5, a producer of glucans from dietary sucrose, was grown in Todd Hewitt Broth with either glucose or sucrose as carbohydrate source. Growth of S.mutans in sucrose results in the production of cell associated glucans (Microbiol. Rev. 44:331-384). In contrast, growth of the bacteria in glucose results in no glucan production.

.After growth in suspension at 37°C, the bacteria were washed with buffer and resuspended in the absence or presence of GBD-biotin. The bacteria were washed again with buffer and incubated with strepavidin peroxidase (Gibco/BRL) dissolved in 0.1 M Tris HC1 pH=7.5 containing 0.1 M NaCl, 2 mM MgCl₂, and 0.05% Triton α-100. After washing, the cells were incubated with colour reagent solution (1 mg/ml o-phenylenediamine HC1 and 0.012% hydrogen peroxide in citrate buffer; pH=4.5). The cell binding by GBD-biotin was thus measured as absorbance at 410 nm following colour development. The results are shown in Fig. 8, and it is clear that incubation of S.mutans in sucrose-medium results in enhanced binding of GBD-biotin due to production of the natural binding site for GBD, bacterial glucan. Glucose grown cells did not provide significant amounts of binding sites for GBD-biotin (ie. they did not produce glucan).

Example 4 - Binding of GBD-biotin to human plaque

This example demonstrates that GBD will bind to plaque, and this binding may be detected by an anti-GTF antibody, which in turn may be detected by an anti-species antibody conjugated to a fluorescent marker. Binding can then be visualised on a slide using a fluorescent microscope. Method

Sample Preparation

Human plaque was obtained from volunteers who had used 10 ml of a 5% sucrose rinse three times on the day before plaque sampling. They had also not brushed their teeth for 24 hours prior to plaque sampling. The plaque was frozen immediately after collection and stored at -20°C until used. The plaque was intensively sonicated (in 1 second cycles: 0.5 seconds on and 0.5 seconds off) for 30 seconds in Ringer's solution containing 2% formalin. Aliquots of 10 μl of plaque suspension, diluted to 1 mg/ml, were pipetted into glass slides bearing 8 sample "wells" as shown by Fig 9. The wells were allowed to air dry, then briefly flamed to fix.

Sample Treatment

The slides were blocked to reduce non-specific binding by immersion in phosphate- buffered saline pH 7.2 containing 1% BSA and 0.05% Tween 20 (blocking buffer) for 20 minutes. The solution was rinsed off by immersing in two successive Ringer's solution baths. Subsequently, solutions containing other reagents were added in this buffer using the same procedure. GBD at 20 μg/ml was added, followed by rabbit polyclonal anti-GTF- S antibody at dilutions from x 50 to x 2000 as indicated in Fig. 9. A GBD-free well and a blank well were used as controls.

Detection of Bound GBD

The fluorescence of the prepared slide was examined using a mercury lamp and epi- illumination. Photographs of representative areas of the slides were taken using HS-400 Ektachrome film.

Results

The photographs of the fluorescence of plaque samples after treatment showed that intense fluorescence was present in these samples. There was a strong dependence of this fluorescence on the GBD concentration used, showing that GBD binds to plaque, even in the presence of a blocking protein. Conclusions

These experiments demonstrate that GBD binds strongly and in considerable amounts to plaque, in a concentration-dependent manner. However, some fluorescence was still present on the GBD-free control. This is most likely to be due to binding of anti-GTF-S antibody to GTF naturally present in plaque. These results are the first evidence that GBD, devoid of the remainder of the parent GTF molecule, retains the ability to bind to glucans and target to human plaque.

Example 5

An experiment was carried out to demonstrate binding to biofilms, analogues of plaque on teeth. The procedure was as follows:

(a) An overnight culture of S.mutans was added to cells of a sterile microtitre plate, incubated for 1 hour, then poured off.

(b) After a rinse with phosphate buffered saline (PBS) the plate was incubated for 2V- hours with a 1% solution of sucrose in PBS. In both cases the bacteria were expected to form a film, but with sucrose the film will include glucan.

(d) The plate was incubated for 30 minutes with a suspension of rabbit polyclonal antibodies to GTF, then rinsed with PBS.

(e) The plate was incubated for 30 minutes with a suspension of a conjugate consisting of goat anti-rabbit polyclonal antibodies attached to horse radish peroxidase (HRP) enzyme, then washed 10 times with distilled deionised water. If the films on the on the cell contained glucan, this should be anchoring on the plate some of the GBD, and through this the antibodies of steps (d) and (e). The resulting complex would be

substrate: GBD: rabbit anti GTF: anti-rabbit-HRP.

The cells were supplied with substrates which can be converted by HRP enzyme to generate a blue colour. The rate of change of absorbance at 630 nm was measured.

If the GBD, or anti GTF antibody was omitted, there was very little colour formation. If the whole system was complete, colour formation was six times greater. If glucose was used in place of sucrose in step (b), there was very little colour formation.

This indicates that GBD was binding successfully to the film (a model of plaque) made by S. mutans in the presence of sucrose. With glucose in place of sucrose there was no glucan formation and nothing for the GBD to bind to, as was demonstrated by comparative experiments.

If dextran, at a concentration of 1 mg/ml or more was included in the GBD solution used at step (c), it provided an alternative site for binding by GBD. It suppressed colour formation, indicating that GBD had bound to the dextran and subsequently been washed away with it.

Example 6

The galactose oxidase gene from the fungus Dactylium dendroides was contained in the plasmid pGAO [McPherson et al (1992) J. Biol. Chem. 267, 8146-8152]. The BamHl- EcoRl fragment shown enlarged in Fig 10 and containing a portion of the leader sequence was cloned into the plasmid pUCl 18 to produce the plasmid pUCGAOEB (Figl 1) . A Ddel fragment was then taken from this plasmid, treated with Klenow fragment to give it blunt ends, and inserted at the Smal site in a plasmid pUCl 18Nar(W) which has an inactived lacZ gene. The resulting plasmid p05 contains the fragment of the galactose oxidase gene under control of the lacZ promoter.

A Narl-Xbal fragment was next taken from the galactose oxidase gene and inserted between corresponding sites in plasmid p05, as shown by Fig 12, thus constructing plasmid pR3 which contains the complete galactose oxidase gene under control of the lacZ promoter. This plasmid was used to express galactose oxidase in E.coli.

A Ddel fragment from the 3' end of the galactose oxidase gene was treated with Klenow fragment to give it blunt ends and then inserted into the Hindi site in plasmid pUCl 19, as shown by Fig 13, leading to plasmid pP4. The GBD gene constructed in part (a) of Example 1 was cut, as an Xbal-EcoRl fragment, from a plasmid analogous to that of Fig IB. It was inserted into plasmid pP4 thus creating plasmid pS2 which contained the GBD gene fused to a sequence from the 3' end of the galactose oxidase gene. The GBD gene fused to the 3' terminus of the galactose oxidase gene was then cut out as a BamHl- Eagl fragment and , as shown by Fig 14, inserted into plasmid pR3 which had been cut with BcR and Eagl. The result was plasmid pU4 which was used in E.coli to express a fusion protein consisting of the glucan binding domain fused to galactose oxidase.

E.coli strains containing the plasmids pS2, pR3 and pU4 respectively were cultured and the culture supernatants were assayed for galactose oxidase activity. Controls were provided by solutions containing various concentrations of galactose oxidase and a solution containing buffer only. The results are shown in Fig 15 from which it can be seen that the fusion protein expressed by E.coli cells with plasmid pU4 possessed galactose oxidase activity which was greater than the activity of the control with lOug/ml of galactose oxidase.

These E.coli strains were also assayed for glucan binding activity by the procedure of Example 2. These results show that a fusion of galactose oxidase and glucan binding domain is able to exhibit the enzyme function and also to bind to glucans.

Claims

1. A polypeptide with specific binding affinity for glucan, covalently chemically bound to a further material which is not a glycosyltransferase enzyme.

2. A polypeptide according to claim 1 wherein the further material is effective to inhibit the formation of dental plaque.

3. A polypeptide according to claim 2 wherein the further material is an antimicrobial agent.

4. ^■ A polypeptide according to claim 2 wherein the further material is active against the staining of teeth.

5. A polypeptide according to claim 2 wherein the further material is selected from the group consisting of antibodies, antibody fragments, histatins, lactoferrin, defensins, magainins, cecropins, cationic antibacteriocins, bacteriocins, triclosan, chlorhexidine, quaternary ammonium compounds, chloroxylenol, chloroxyethanol, thymol and chelating agents for zinc, tin or copper ions.

6. A polypeptide according to claim 2 wherein the further material is selected from the group consisting of biotin and avidin.

7. A polypeptide according to claim 2 wherein the further material is a second polypeptide, connected to the first said polypeptide through a peptide bond.

8. A polypeptide according to claim 7 wherein the second polypeptide is an enzyme

9. A polypeptide according to claim 8 wherein the enzyme is an oxidase or a peroxidase

SUBSTITUTE SHEET (RULE 25)

10. A polypeptide according to claim 1 wherein the polypeptide is a clone of a glucan binding domain of glycosyltransferase.

11. A composition for topical application in the oral cavity, comprising a polypeptide as defined in claim 1 , in a carrier vehicle which is acceptable for use in the mouth

12. A composition according to claim 1 containing from 0.01% to 1% by weight of the polypeptide.

13. A product comprising a pair of cooperating compositions for topical application in the oral cavity, the first composition comprising a polypeptide according to claim 1, covalently chemically bound to a target material which is not a glycosyltransferase enzyme; the second composition comprising a an agent which is effective against dental plaque or tooth stain, covalently chemically bound to a material with specific binding affinity for the said target material, in a carrier vehicle which is acceptable for use in the mouth.

14. A method of inhibiting dental plaque, comprising topical application of a polypeptide as defined in claim 1 in the mouth.