WO2004058088A2 - Bacteriophage-encoded antibacterial enzyme for the treatment and prevention of gingivitis and root surface caries - Google Patents

Abstract

A method and composition for the treatment and prevention of gingivitis and root surface caries using Actinomyces naeslundii bacteriophage Av-1 encoded anti-bacterial enzyme (Av-1 lysin) to remove and inhibit establishment of cariogenic and pathogenic bacteria in the oral cavity is disclosed, along with the amino acid sequence of the enzyme used therein and the isolated DNA molecule encoding the same.

Description

BACTERIOPHAGE-ENCODED ANTIBACTERIAL

ENZYME FOR THE TREATMENT AND PREVENTION

OF GINGIVITIS AND ROOT SURFACE CARIES

The work described herein was supported by a grant from the NIH (Grant No. DE13181). The Federal Government has certain rights in this disclosure.

This application claims priority to U.S. Provisional Application No. 60/435,301 filed December 23, 2002, the entirety of which is hereby incorporated by reference.

FIELD OF THE DISCLOSURE A genetic and amino acid sequence and a method of use is disclosed for the treatment of oral diseases caused by Actinomyces naeslundii

BACKGROUND OF THE DISCLOSURE

I. Actinomyces naeslundii and Root Surface Caries

Actinomyces species are Gram-positive, homofermentative lactic acid-producing, facultative anaerobic rods which are found only in the mouths of animals and man. Human isolates of this genus are classified into six species (A. georgiae, A. gerencseriae, A. israelii, A. meyeri, A. naeslundii and odontolyticus).

A. naeslundii (which now includes strains formerly classified as A. viscosus) is believed to be the most common species contributing to the initial stages of periodontal disease (gingivitis) and in the initiation of root surface (cementum) caries. This species has a strong predilection for colonizing cementum and, in monoinfected animals, is known to cause extensive periodontal disease with massive alveolar bone loss. While the causative agent(s) of human root surface caries has not yet been definitively established, most studies implicate S. mutans and A. naeslundii (and occasionally lactobacilli) as the major causes of caries in this region of the tooth (Van Houte et al, J. Dent. Res., 73:1727-1734, incorporated herein by reference).

II. Cell Walls of A. naeslundii

The Gram-positive cell walls of A. naeslundii have a classical peptidoglycan backbone composed of alternating subunits of N-acetylglucosamine and N-acetylmuramic acid ( urein), which is attached via amide bonds to short, cross-linking peptides containing D- and L-alanine. Lysine serves as the link between the peptides rather than the more common m-diaminopimelic acid (Schleifer et al, J. Dent. Res., 63:1047-1050 (1984); and Schleifer et al, Meth. Microbial., 18:126-156 (1985) incorporated herein by reference).

The cell wall of A. naeslundii is comprised of a sugar, D-tagatose, and has two types of surface fimbriae (which are involved in various adhesion and specific, interspecies coaggregation reactions). The wall structure is not known to contain teichoic acids or LTA (Kolenbrander et al, "Use of Coaggregation-Defective Mutants to Study the Relationship of Cell-to-Cell Interactions and Oral Microbial Ecology", pages 164-171, In: S.E. Mergenhagen and B. Rosan (eds.), Molecular Basis of Oral Microbial Adhesion, American Society for Microbiology, Washington D.C. (1985); and Maiden et al, "Characteristics of Oral Gram- Positive Bacteria", Chapter 20, pages 342-372, In: J. Slots and M. A. Taubman, eds., Contemporary Oral Microbiology and Immunology, Mosby-Year Book, Inc., St. Louis, Missouri (1992) all incorporated herein by reference). III. Lysozyme resistance of A. naeslundii

Structurally, the peptidoglycans of A. naeslundii are similar to those of many other Gram-positive bacteria which are sensitive to cleavage by conventional (animal) lysozymes (Schliefer et al (1985), supra, incorporated herein by reference); however, this species of oral bacteria is notoriously resistant to conventional lysozymes, although growth in the presence of elevated levels of threonine (Chassy, Biochem. Biophys. Res. Commun., 68:603-608 (1976) incorporated herein by reference) or brief exposure to 1-5 % glycine (Macrina et al, Infect. Immun., 17:215-226 (1977) incorporated herein by reference), and treatment with the Streptomyces enzyme mutanolysin can be used to cause partial lysis of log-phase cells. The natural resistance of this and other oral bacteria to lysozyme is presumably the result of its evolution to tolerate the presence of lysozyme in saliva (9-12 μg/ml). It is hypothesized that the cell walls of these bacteria have some unusual, possibly unique structural features that are responsible for this resistance. The molecular basis for the resistance of this organism to animal lysozymes is unknown. The lysozyme resistance of many other Gram- positive and Gram-negative bacterial cell walls has been previously ascribed to (a) additional cross-linking of peptide sub-units, (b) different degrees of N- and O-acetylation of amino sugars, or (c) barriers to substrate binding by wall polysaccharides, teichoic acids or lipoteichoic acids.

O-acetyl groups were first described in the cell wall of S. faecalis (Abrams, J. Biol. Chem., 230:949-959 (1958) incorporated herein by reference) and have been shown to be responsible for the lysozyme resistance of several Gram-positive species (Brumfϊtt, Brit. J. Exp. Pathol, 40:441-451 (1959); and Zipperle et al, Canad. J. Microbiol., 30:553-559 (1984) incorporated herein by refernce). It is believed in the present disclosure that the lysozyme resistance of cariogenic oral bacteria is due to the presence of O-acetyl groups in their muramic acid moieties, which presumably interferes with substrate recognition and binding, based on the following two lines of evidence: (a) cell walls of A. naeslundii are sensitive to lysis by mutanolysin, an enzyme which attacks peptidoglycans containing both N-acetylated and O-acetylated muramic acid, and (b) removal of O-acetyl groups by mild alkali treatment makes whole cells of this specie sensitive to lysis by chicken lysozyme, as demonstrated in lysoplate type assays. Whether such treatment also removes or alters wall LTA or polysaccharides, which could potentially affect lysozyme binding, remains to be determined.

IV. Mechanisms of Phage Lysis the Holin-lysin Paradigm A unified mechanism of bacteriophage-mediated host cell lysis was proposed which was consistent with all the then-known information on dsDNA phages of Gram-negative bacteria (Young, Microbiol. Rev., 56:430-481 (1992) incorporated herein by reference). The same mechanism is probably utilized by phages of Gram-positive bacteria.

It is believed that this mechanism involves two gene products. One gene product is a peptidoglycan-degrading enzyme (classically referred to as endolysin or simply "lysin") which is responsible for lysis of the cell wall; in most cases the lysin proteins lack a signal sequence and accumulate in an active, fully folded form in the cytosol. The second gene product is a membrane-spanning protein that upon insertion into the cytoplasmic membrane forms a pore or "hole" which allows the lysin to gain access to the peptidoglycan layer. In smaller, less complex dsDNA phages, the holin and lysin genes are usually transcribed late during the intracellular stage of growth, from a distant, upstream promoter, with the lysin gene being adjacent to and immediately downstream of the holin gene. In the case of large dsDNA phages having a complex genome like T4, although they encode lysozymes and holin-like proteins, the encoded genes may be widely separated in the genome and may be expressed at different times in different operons. The holin-lysin model does not apply to filamentous ssDNA phages, which do not lyse their hosts, or to RNA or other ssDNA phages, which employ a different type of lysis-effecting protein.

The holin-lysin paradigm was soon confirmed for the Gram-positive Bacillus phage φ29 (Steiner et al, J. Bacteriol., 175:1038-1042 (1993) incorporated herein by reference) and, more recently, for a number of phages infecting other Gram-positive species of Bacillus, Lactobacillus, Lactococcus and Listeria and of S. pneumoniae (Arendt et al, Appl. Environ. Microbiol., 60:1875-1883 (1994); Birkeland et al, Canad. J. Microbiol., 40:658-665 (1994); Bousque et al, J. Dent. Res., 67:394, Abstract No. 2253 (1988); Boizet et al, Gene, 94-61-67 (1990); Boyce et al, Appl. Environ. Microbiol., 61:4089-4098 (1995); Buist et al, J. Bacteriol., 177:1554-1563 (1995); Diaz et al, Molec. Microbiol., 19:667-681 (1996); Garcia et al, Proc. Natl. Acad. Sci., 85:914-918 (1988); Garcia et al, J. Viol., 61:2573-2580 (1987); Garvey et al, Nucleic Acids Res., 14:10001-10008 (1986); Henrich et al, J. Bacteriol., 177:723-732 (1995); Loessner et al, Molec. Microbiol., _16:1231-1241 (1995a); Longchamp et al, Microbiology, 140:1855-1867 (1994); Shearman et al, Mol. Gen. Genet, 218:214-221 (1989); Shearman et al, Appl. Environ. Microbiol., 60:3063-3073 (1994); Trautwetter et al, J. Virol., 59:551-555 (1986); Vasala et al, Appl. Environ. Microbiol., 6J,:4004-4011 (1995); and Ward et al, Can. J. Microbiol., 39:767-774 (1993) all incorporated by reference).

It is now apparent that virtually all dsDNA phages of both Gram-negative and Gram- positive bacteria employ this mechanism of host cell lysis. There appears to be a wide variety of different, sequence-unrelated but structurally similar phage holin families encoded by different phages (Young et al, FEMS Microbiol. Rev., 17:191-205 (1995) incorporated herein by reference), whereas most phage lysins show some primary amino acid sequence relatedness. All of the phage lysins described to date exhibit peptidoglycan-hydrolyzing ability and can be classified as glycosylases (rnura idases or "true" lysozymes), amidases, or peptidases or. as in the case of the lambda R protein, transglycosylases. In most cases, the species-specificity of phage lysins involves substrate binding domains which recognize wall constituents other than, or in addition to, the peptidoglycan itself. Several pneumococcal phages, for example, encode lysins which, although they can degrade E. coli peptidoglycan when expressed inside this host, require the presence of choline in pneumococcal cell walls for external lysis of S. pneumoniae.

V. Phage Tail Enzymes

There is an extensive body of literature on phage tail enzymes, but in most cases the substrates hydrolyzed by tail-associated enzymes are cell surface carbohydrate or LPS moieties.

Such enzymes are thought to be involved in recognizing or adsorbing to cell wall receptors and their action does not lead to peptidoglycan cleavage and cell lysis.

There are only a few examples of phage tail enzymes which have cell wall-lytic activity. The most studied tail enzyme of this type is the gp5 tail protein of phage T4, which has been extensively characterized by Fumio Arisaka and shown to be a true lysozyme. This type of activity is rare among lytic Gram-negative phages and has not been reported in any phages which infect Gram-positive bacteria.

SUMMARY OF THE DISCLOSURE

Lytic phages infecting A. naeslundii (A. viscosus) have been studied for a number of years (Delisle et al, Infect. Immun., 20:303-306 (1978); Delisle, Microbios Lett., 33:107-113 (1986); Delisle et al, Infect. Immun., 56:54-59 (1988); and Delisle et al, Microbial Ecol. Health Dis., 8: 121-127 (1995) incorporated herein by reference. ). The present disclosure is directed to a representative phage of this species, Av-1 (a small, group I phage) which has a wide host range. This phage encodes a lysin that specifically attacks the cell walls of only this species, includmg phage-resistant strains. Based on genome size (about 17 kb) and the presence of proteins attached to the 5' ends of its DNA, it is disclosed that phage Av-1 (and all other group I phages) resembles the small Bacillus phages, like φ29. Further characterization of phage Av-1, and results of the cloning of sequences from Av-1 DNA, are presented herein.

Accordingly, an object of the present disclosure is to provide a method for the treatment or prevention of gingivitis or root surface caries. Another object of the present disclosure is to provide a method for inhibiting growth of A. naeslundii.

Still another object of the present disclosure is to provide isolated and purified Av-1 lysin, and to disclose the sequence of the DNA molecule encoding the same, a vector containing the same and a host cell transformed with the same. Yet another object of the present disclosure is to provide an antibody which binds specifically to Av-1 lysin.

These and other objects of the present disclosure, have been met by use of lysin comprising SEQ ID NO:2 or SEQ ID NO:3, a composition comprising the same and a DNA molecule encoding the same. In one embodiment of this disclosure, the DNA encoding the lytic enzyme or holin protein, including their isozymes, analogs, or variants, has been genetically altered. In another embodiment, the lytic enzyme or holin protein, including their isozymes, analogs, or variants, has been chemically altered. In yet another embodiment, the lytic enzyme or holin protein, including their isozymes, analogs, or variants, are used in a combination of natural and modified (genetically or chemically altered) forms. The altered forms of lytic enzymes and holin proteins are made synthetically by chemical synthesis and/or DNA recombinant techniques. The enzymes are made synthetically by chimerization and/or shuffling.

Another embodiment of the present invention also provides for chimeric proteins or peptides fragments which include fusion proteins for the aforesaid uses.

A definition of terms used and their applicability to the disclosure are provided as follows: Phage enzymes or proteins, as disclosed herein, include phage polypeptides, peptide fragments, nucleic acid molecules encoding phage protein or protein peptides fragments, antibody and antibody fragments, having biological activity either alone or with combination of other molecules. When reference is made to lytic enzymes, the enzyme may include any form of the peptide that allows for the destruction of the cell wall under the specified conditions.

Nucleic acid molecules, as disclosed herein, include genes, gene fragments polynucleotides, oligonucleotides, DNA, RNA, DNA-RNA hybrids, EST, SNIPs, genomic DNA, cDNA, mRNA, antisense RNA, ribozyme vectors containing nucleic acid molecules, regulatory sequences, and signal sequences. Nucleic acid molecules of this disclosure include any nucleic acid-based molecule that either alone or in combination with other molecules produces an oligonucleotide molecule capable or incapable of translation into a peptide.

In this context of the embodiments, the term "lytic enzyme genetically coded for by a bacteriophage" means a polypeptide having at least some lytic activity against the host bacteria. The polypeptide has a sequence that encompasses a native sequence of a lytic enzyme and variants thereof. The polypeptide may be isolated from a variety of sources, such as from phage, or prepared by recombinant or synthetic methods, such as those by Garcia et al. Every phage lysin has two domains. One domain is a substrate (cell wall) binding portion at the carboxyl terminal side and the other domain contains the catalytic site (amidase) whose activity acts upon amide bonds in the peptidoglycan at the amino terminal side. Generally speaking, a lytic enzyme according to the disclosure is between 25,000 and 35,000 daltons in molecular weight and comprises a single polypeptide chain; however, this can vary depending on the enzyme chain. The molecular weight most conveniently is determined by assay on denaturing sodium dodecyl sulfate gel electiophoresis and comparison with molecular weight markers.

The term "isolated" means at least partially purified from a starting material. The term "purified" means that the biological material has been measurably increased in concentration by any purification process, including by not limited to, column chromatography, HPLC, precipitation, electiophoresis, etc., thereby partially, substantially or completely removing impurities such as precursors or other chemicals involved in preparing the material. Hence, material that is homogenous or substantially homogenous (e.g., yields a single protein signal in a separation procedure such as electrophoresis or chromatography) is included within the meanings of isolated and purified. Skilled artisans will appreciated that the amount of purification necessary will depend upon the use of the material. For example, compositions intended for administration to humans ordinarily must be highly purified in accordance with regulatory standards.

"A native sequence phage associated lytic enzyme" is a polypeptide having the same amino acid sequence as an enzyme derived from nature. Such native sequence enzyme can be isolated from nature or can be produced by recombinant or synthetic means. The term "native sequence enzyme" specifically encompasses naturally occurring forms (e.g., alternatively spliced or modified forms) and naturally-occurring variants of the enzyme. In one embodiment of the disclosure, the native sequence enzyme is a mature or full-length polypeptide that is genetically coded for by a gene from a bacteriophage specific for a specific bacteria. Of course, a number of variants are possible and known, as acknowledged in publications such as Lopez et al., Microbial Drug Resistance 3: 199-211 (1997); Garcia et al., Gene 86: 81-88 (1990); Garcia et al., Proc. Natl. Acad. Sci. USA 85: 914-918 (1988); Garcia et al., Proc. Natl. Acad. Sci. USA 85: 914-918 (1988); Garcia et al., Streptococcal Genetics (J.J. Ferretti and Curtis eds., 1987); Lopez et al., FEMS Microbiol. Lett. 100: 439- 448 (1992); Romero et al., J. Bacteriol. 172: 5064-5070 (1990); Ronda et al., Eur. J. Biochem. 164: 621-624 (1987) and Sanchez et al., Gene 61: 13-19 (1987)( all incorporated herein by reference). The contents of each of these references, particularly the sequence listings and associated text that compares the sequences, including statements about sequence homologies, are specifically incorporated by reference in their entireties.

"A variant sequence phage associated lytic enzyme" means a functionally active lytic enzyme genetically coded for by a bacteriophage specific for a specific bacteria, as defined below, having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%o, or even at least 99.5% amino acid sequence identity with the sequences shown in some of the figures. Of course a skilled artisan readily will recognize portions of this sequence that are associated with functionalities such as binding, and catalyzing a reaction. Accordingly, polypeptide sequences and nucleic acids that encode these sequences are contemplated that comprise at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more of each functional domain of some of the sequences. Such portions of the total sequence are very useful for diagnostics as well as therapeutics/prophylaxis. In fact, sequences as short as 5 amino acids long have utility as epitopic markers for the phage. More desirable, larger fragments or regions of protein having a size of at least 8, 9, 10, 12, 15 or 20 amino acids, and homologous sequences to these, have epitopic features and may be used either as small peptides or as sections of larger proteins according to embodiments. Nucleic acids corresponding to these sequences also are contemplated.

Such phage associated lytic enzyme variants include, for instance, lytic enzyme polypeptides wherein one or more amino acid residues are added, or deleted at the N or C terminus of the sequences shown. In one embodiment one or more amino acids are substituted, deleted, and/or added to any position(s) in the sequence, or sequence portion. Ordinarily, a phage associated lytic enzyme will have at least about (e.g. exactly) 50%, 55%, 60%, 65%, 70%, 75%, amino acid sequence identity with native phage associated lytic enzyme sequences, more preferably at least about (e.g. exactly) 80%, 85%, 90%, 95%, 97%, 98%, 99%) or 99.5% amino acid sequence identity. In other embodiments a phage associated lytic enzyme variant will have at least about 50% (e.g. exactly 50%) , 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or even at least 99.5% amino acid sequence identity with the sequences shown.

"Percent amino acid sequence identity" with respect to the phage associated lytic enzyme sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the phage associated lytic enzyme sequence, after aligning the sequences in the same reading frame and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, such as using publicly available computer software such as blast software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the whole length of the sequences being compared. In each case, of course conservative amino acid substitutions also may be made simultaneously in determining percent amino acid sequence identity. For example, a 15 amino acid long region of protein may have 50%, 55%, 60%, 65%>, 70%, 75%, 80%, 85%, 90%), 95%o, 97%>, 98%), or 99% sequence homology with a region of the sequences shown. At the same time, the 15 amino acid long region of the protein may also have up to 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 65%, 75%, or more amino acids replaced with conservative substitutions. Preferably the region will have fewer than 30%, 20%, 10% or even less conservative substitutions. The "percent amino acid sequence identity" calculation in such cases will be higher than the actual percent sequence identity when conservative amino acid substitutions have been made. "Percent nucleic acid sequence identity" with respect to the phage associated lytic enzyme sequences identified herein is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in the phage associated lytic enzyme sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the scope of those skilled in the art, including but not limited to the use of publicly available computer software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. "Polypeptide" refers to a molecule comprised of amino acids which correspond to those encoded by a polynucleotide sequence which is naturally occurring. The polypeptide may include conservative substitutions wherein the naturally occurring amino acid is replaced by one having similar properties, where such conservative substitutions do not alter the function of the polypeptide (see, for example, Lewin "Genes V" Oxford University Press Chapter 1, pp. 9-13 1994).

A "chimeric protein" or "fusion protein" comprises all or (preferably a biologically active) part of a polypeptide of the disclosure operably linked to a heterologous polypeptide. Chimeric proteins or peptides are produced, for example, by combining two or more proteins having two or more active sites. Chimeric protein and peptides can act independently on the same or different molecules, and hence have a potential to treat two or more different bacterial infections at the same time. Chimeric proteins and peptides also are used to treat a bacterial infection by cleaving the cell wall in more than one location.

The term "operably linked" means that the polypeptide of the disclosure and the heterologous polypeptide are fused in-frame. The heterologous polypeptide can be fused to the N-terminus or C-terminus of the polypeptide of the disclosure. Chimeric proteins are produced enzymatically by chemical synthesis, or by recombinant DNA technology. A number of chimeric lytic enzymes have been produced and studied. Gene E-L, a chimeric lysin constructed from bacteriophages phi XI 74 and MS2 lysis proteins E and L, respectively, was subjected to internal deletions to create a series of new E-L clones with altered lysis or killing properties. The lytic activities of the parental genes E, L, E-L, and the internal truncated forms of E-L were investigated in this study to characterize the different lysis mechanism, based on differences in the architecture of the different membranes spanning domains. Electron microscopy and release of marker enzymes for the cytoplasmic and periplasmic spaces revealed that two different lysis mechanisms can be distinguished depending on penetration of the proteins of either the inner membrane or the inner and outer membranes of the E. coli. FEMS Microbiol. Lett. 1998 Jul 1, 164(1); 159-67 (incorporated herein by reference). In another experiment, an active chimeric cell wall lytic enzyme (TSL) was constructed by fusing the region coding for the N-terminal half of the lactococcal phage Tuc2009 lysin and the region coding for the C-terminal domain of the major pneumococcal autolysin. The chimeric enzyme exhibited a glycosidase activity capable of hydrolysing choline-containing pneumococcal cell walls. One example of a useful fusion protein is a GST fusion protein in which the polypeptide of the disclosure is fused to the C-terminus of a GST sequence. Such a chimeric protein can facilitate the purification of a recombinant polypeptide of the disclosure.

In another embodiment, the chimeric protein or peptide contains a heterologous signal sequence at its N-terminus. For example, the native signal sequence of a polypeptide of the disclosure can be removed and replaced with a signal sequence from another protein. Examples of eukaryotic heterologous signal sequences include the secretory sequences of melittin and human placental alkaline phosphatase (Stratagene; La Jolla, California). In yet another example, useful prokaryotic heterologous signal sequences include the phoA secretory signal (Sambrook et al., supra) and the protein A secretory signal (Pharmacia Biotech; Piscataway, New Jersey).

Another embodiment discloses an immunoglobulin fusion protein in which all or part of a polypeptide of the disclosure is fused to sequences derived from a member of the immunoglobulin protein family. An immunoglobulin fusion protein can be incorporated into a pharmaceutical composition and administered to a subject to inhibit an interaction between a ligand (soluble or membrane-bound) and a protein on the surface of a cell (receptor), to thereby suppress signal transduction in vivo. The immunoglobulin fusion protein can alter bioavailability of a cognate ligand of a polypeptide of the disclosure. Inhibition of ligand/receptor interaction may be useful therapeutically, both for treating 5 bacterial-associated diseases and disorders for modulating (i.e. promoting or inhibiting) cell survival. Moreover, an immunoglobulin fusion protein of the disclosure can be used as an immunogen to produce antibodies directed against a polypeptide of the disclosure in a subject, to purify ligands and in screening assays to identify molecules which inhibit the interaction of receptors with ligands. Chimeric and fusion proteins and peptides of the l o disclosure can be produced by standard recombinant DNA techniques.

In another embodiment, the fusion gene can be synthesized by conventional techniques, including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which subsequently can be annealed and

15 reamplified to generate a chimeric gene sequence (see, i.e., Ausubel et al., supra). Moreover, many expression vectors are commercially available that already encode a fusion moiety (i.e., a GST polypeptide). A nucleic acid encoding a polypeptide of the disclosure can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the polypeptide of the disclosure.

20 As used herein, shuffled proteins or peptides, gene products, or peptides for more than one related phage protein or protein peptide fragments have been randomly cleaved and reassembled into a more active or specific protein. Shuffled oligonucleotides, peptides or peptide fragment molecules are selected or screened to identify a molecule having a desired functional property. This method is described, for example, in Sterømer, US Patent No. 6,132,970.(Method of shuffling polynucleotides) ; Kauffman, U.S. Patent No 5, 976,862 (Evolution via Condon-based Synthesis) and Huse, U.S. Patent No. 5,808,022 (Direct Codon Synthesis) (all incorporated herein by reference). The contents of these patents are incorporated herein by reference. Shuffling is used to create a protein that is 10 to 100 fold more active than the template protein. The template protein is selected among different varieties of lysin or holin proteins. The shuffled protein or peptides constitute, for example, one or more binding domains and one or more catalytic domains. Each binding or catalytic domain is derived from the same or a different phage or phage protein. The shuffled domains are either oligonucleotide based molecules, as gene or gene products, that either alone or in combination with other genes or gene products are translatable into a peptide fragment, or they are peptide based molecules. Gene fragments include any molecules of DNA, RNA, DNA-RNA hybrid, antisense RNA, Ribozymes, ESTs, SNIPs and other oligonucleotide-based molecules that either alone or in combination with other molecules produce an oligonucleotide molecule capable or incapable of translation into a peptide. As noted above, the present disclosure discusses the use of holin proteins. Holin proteins produce holes in the cell membrane. More specifically, holins form lethal membrane lesions. Like the lytic proteins, holin proteins are coded for and carried by a phage. In fact, it is quite common for the genetic code of the holin protein to be next to or even within the code for the phage lytic protein. Most holin protein sequences are short, and overall, hydrophobic in nature, with a highly hydrophilic carboxy-terminal domain. In many cases, the putative holin protein is encoded on a different reading frame within the enzymatically active domain of the phage. In other cases, holin protein is encoded on the DNA next or close to the DNA coding for the cell wall lytic protein. Holin proteins are frequently synthesized during the late stage of phage infection and found in the cytoplasmic membrane where they cause membrane lesions.

Holins can be grouped into two general classes based on primary structure analysis. Class I holins are usually 95 residues or longer and may have three potential transmembrane domains. Class II holins are usually smaller, at approximately 65-95 residues, with the distribution of charged and hydrophobic residues indicating two TM domains (Young, et al. Trends in Microbiology v. 8, No. 4, March 2000). At least for the phages of gram-positive hosts, however, the dual-component lysis system may not be universal.

The modified or altered form of the protein or peptides and peptide fragments, as disclosed herein, includes protein or peptides and peptide fragments that are chemically synthesized or prepared by recombinant DNA techniques, or both. These techniques include, for example, chimerization and shuffling. When the protein or peptide is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. Accordingly such preparations of the protein have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the polypeptide of interest.

In one embodiment of the disclosure, a signal sequence of a polypeptide can facilitate transmembrane movement of the protein and peptides and peptide fragments of the disclosure to and from mucous membranes, as well as by facilitating secretion and isolation of the secreted protein or other proteins of interest. Signal sequences are typically characterized by a core of hydrophobic amino acids which are generally cleaved from the mature protein during secretion in one or more cleavage events. Such signal peptides contain processing sites that allow cleavage of the signal sequence from the mature proteins as they pass through the secretory pathway. Thus, the disclosure can pertain to the described polypeptides having a signal sequence, as well as to the signal sequence itself and to the polypeptide in the absence of the signal sequence (i.e., the cleavage products). In one embodiment, a nucleic acid sequence encoding a signal sequence of the disclosure can be operably linked in an expression vector to a protein of interest, such as a protein which is ordinarily not secreted or is otherwise difficult to isolate. The signal sequence directs secretion of the protein, such as from an eukaryotic host into which the expression vector is transformed, and the signal sequence is subsequently or concurrently cleaved. The protein can then be readily purified from the extracellular medium by art recognized methods. Alternatively, the signal sequence can be linked to a protein of interest using a sequence which facilitates purification, such as with a GST domain.

In another embodiment, a signal sequence can be used to identify regulatory sequences, i.e., promoters, enhancers, repressors. Since signal sequences are the most amino-terminal sequences of a peptide, it is expected that the nucleic acids which flank the signal sequence on its amino-terminal side will be regulatory sequences that affect transcription. Thus, a nucleotide sequence which encodes all or a portion of a signal sequence can be used as a probe to identify and isolate the signal sequence and its flanking region, and this flanking region can be studied to identify regulatory elements therein. The present disclosure also pertains to other variants of the polypeptides of the disclosure. Such variants have an altered amino acid sequence which can function as either agonists (mimetics) or as antagonists. Variants can be generated by mutagenesis, i.e., discrete point mutation or truncation. An agonist can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of the protein. An antagonist of a protein can inhibit one or more of the activities of the naturally occurring form of the protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the protein of interest. Thus, specific biological effects can be elicited by treatment with a variant of limited function. Treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein can have fewer side effects in a subject relative to treatment with the naturally occurring form of the protein. Variants of a protein of the disclosure which function as either agonists (mimetics) or as antagonists can be identified by screening combinatorial libraries of mutants, i.e., truncation mutants, of the protein of the disclosure for agonist or antagonist activity. In one embodiment, a variegated library of variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential protein sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (i.e., for phage display). There are a variety of methods which can be used to produce libraries of potential variants of the polypeptides of the disclosure from a degenerate oligonucleotide sequence. Methods for synthesizing degenerate oligonucleotides are lαiown in the art (see, i.e., Narang (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11 :477, all herein incorporated by reference).

In addition, libraries of fragments of the coding sequence of a polypeptide of the disclosure can be used to generate a variegated population of polypeptides for screening and subsequent selection of variants. For example, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of the coding sequence of interest with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with SI nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes N-terminal and internal fragments of various sizes of the protein of interest. Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. The most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify variants of a protein of the disclosure (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering 6(3):327-331) (all incorporated herein by reference).

Immunologically active portions of a protein or peptide fragment include regions that bind to antibodies that recognize the phage enzyme. In this context, the smallest portion of a protein (or nucleic acid that encodes the protein) according to embodiments is an epitope that is recognizable as specific for the phage that makes the lysin protein. Accordingly, the smallest polypeptide (and associated nucleic acid that encodes the polypeptide) that can be expected to bind antibody and is useful for some embodiments may be 8, 9, 10, 11, 12, 13, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 75, 85, or 100 amino acids long. Although small sequences as short as 8, 9, 10, 11, 12 or 15 amino acids long reliably comprise enough structure to act as epitopes, shorter sequences of 5, 6, or 7 amino acids long can exhibit epitopic structure in some conditions and have value in an embodiment. Thus, the smallest portion of the protein or nucleic acid sequence described by specific sequences includes polypeptides as small as 5, 6, 7, 8, 9, or 10 amino acids long. Homologous proteins and nucleic acids can be prepared that share functionality with such small proteins and/or nucleic acids (or protein and/or nucleic acid regions of larger molecules) as will be appreciated by a skilled artisan. Such small molecules and short regions of larger molecules, that may be homologous specifically are intended as embodiments. Preferably the homology of such valuable regions is at least 50%, 65%>, 75%, 85%, and more preferably at least 90%, 95%, 97%, 98%, or at least 99% compared to the specific sequences. These percent homology values do not include alterations due to conservative amino acid substitutions.

Of course, an epitope as described herein may be used to generate an antibody and also can be used to detect binding to molecules that recognize the lysin protein. Another embodiment is a molecule such as an antibody or other specific binder that may be created through use of an epitope such as by regular immunization or by a phase display approach where an epitope can be used to screen a library if potential binders. Such molecules recognize one or more epitopes of lysin protein or a nucleic acid that encodes lysin protein. An antibody that recognizes an epitope may be a monoclonal antibody, a humanized antibody, or a portion of an antibody protein. Desirably the molecule that recognizes an epitope has a specific binding for that epitope which is at least 10 times as strong as the molecule has for serum albumin. Specific binding can be measured as affinity (Km). More desirably the specific binding is at least 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, or even higher than that for serum albumin under the same conditions. In a desirable embodiment the antibody or antibody fragment is in a form useful for detecting the presence of the lysin protein. A variety of forms and methods for their synthesis are known as will be appreciated by a skilled artisan. The antibody may be conjugated (covalently complexed) with a reporter molecule or atom such as a fluor, an enzyme that creates an optical signal, a chemilumiphore, a microparticle, or a radioactive atom. The antibody or antibody fragment may be synthesized in vivo, after immunization of an animal, for example, The antibody or antibody fragment may be synthesized via cell culture after genetic recombination. The antibody or antibody fragment may be prepared by a combination of cell synthesis and chemical modification. Biologically active portions of a protein or peptide fragment of the embodiments, as described herein, include polypeptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the phage protein of the disclosure, which include fewer amino acids than the full length protein of the phage protein and exhibit at least one activity of the corresponding full-length protein. Typically, biologically active portions comprise a domain or motif with at least one activity of the corresponding protein. A biologically active portion of a protein or protein fragment of the disclosure can be a polypeptide which is, for example, 10, 25, 50, 100 less or more amino acids in length. Moreover, other biologically active portions, in which other regions of the protein are deleted, or added can be prepared by recombinant techniques and evaluated for one or more of the functional activities of the native form of a polypeptide of the embodiments.

A large variety of isolated cDNA sequences that encode phage associated lysing enzymes and partial sequences that hybridize with such gene sequences are useful for recombinant production of the lysing enzyme. Representative nucleic acid sequences in this context are the sequences shown in the figures and sequences that hybridize, under stringent conditions, with complementary sequences of the DNA of the sequences. Still further variants of these sequences and sequences of nucleic acids that hybridize with those shown in the figures also are contemplated for use in production of lysing enzymes according to the disclosure, including natural variants that may be obtained. Many of the contemplated variant DNA molecules include those created by standard

DNA mutagenesis techniques, such as Ml 3 primer mutagenesis. Details of these techniques are provided in Sambrook et al. (1989) In Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y. (incorporated herein by reference). By the use of such techniques, variants may be created which differ in minor ways from those disclosed. DNA molecules and nucleotide sequences which are derivatives of those specifically disclosed herein and which differ from those disclosed by the deletion, addition or substitution of nucleotides while still encoding a protein which possesses the functional characteristic of the BSMR protein are contemplated by the disclosure. Also included are one small DNA molecules which are derived from the disclosed DNA molecules. Such small DNA molecules include oligonucleotides suitable for use as hybridization probes or polymerase chain reaction (PCR) primers. As such, these small DNA molecules will comprise at least a segment of a lytic enzyme genetically coded for by a bacteriophage specific for a specific bacteria and, for the purposes of PCR, will comprise at least a 10-15 nucleotide sequence and, more preferably, a 15-30 nucleotide sequence of the gene. DNA molecules and nucleotide sequences which are derived from the disclosed DNA molecules as described above may also be defined as DNA sequences which hybridize under stringent conditions to the DNA sequences disclosed, or fragments thereof.

Hybridization conditions corresponding to particular degrees of stringency vary depending upon the nature of the hybridization method of choice and the composition and length of the hybridizing DNA used. Generally, the temperature of hybridization and the ionic strength (especially the sodium ion concentration) of the hybridization buffer will determine the stringency of hybridization. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed by Sambrook et al. (1989), In Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., chapters 9 and 11, (herein incorporated by reference).

An example of such calculation is as follows. A hybridization experiment may be performed by hybridization of a DNA molecule (for example, a natural variation of the lytic enzyme genetically coded for by a bacteriophage specific for A. naeslundii) to a target DNA molecule. A target DNA may be, for example, the corresponding cDNA which has been electrophoresed in an agarose gel and transferred to a nitrocellulose membrane by Southern blotting (Southern (1975). J. Mol. Biol. 98:503), a technique well known in the art and described in Sambrook et al. (1989) In Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y. (incorporated herein by reference). Hybridization with a target probe labeled with isotopic P (32) labeled-dCTP is carried out in a solution of high ionic strength such as 6 times SSC at a temperature that is 20 -25 degrees Celsius below the melting temperature, Tm, (described infra). For such Southern hybridization experiments where the target DNA molecule on the Southern blot contains 10 ng of DNA or more, hybridization is carried out for 6-8 hours using 1-2 ng/ml radiolabeled probe (of specific activity equal to 109 CPM/mug or greater). Following hybridization, the nitrocellulose filter is washed to remove background hybridization. The washing conditions are as stringent as possible to remove background hybridization while retaining a specific hybridization signal. The term "Tm" represents the temperature above which, under the prevailing ionic conditions, the radiolabeled probe molecule will not hybridize to its target DNA molecule. The Tm of such a hybrid molecule may be estimated from the following equation: Tm =81.5 degrees C -16.6(logl0 of sodium ion concentration)+0.41(%G+C)-0.63(% formamide)-(600/l) where l=the length of the hybrid in base pairs. This equation is valid for concentrations of sodium ion in the range of 0.01M to 0.4M, and it is less accurate for calculations of Tm in solutions of higher sodium ion concentration (Bolton and McCarthy (1962). Proc. Natl. Acad. Sci. USA 48:1390) (incorporated herein by reference). The equation also is valid for DNA having G+C contents within 30%> to 75%, and also applies to hybrids greater than 100 nucleotides in length. The behavior of oligonucleotide probes is described in detail in Ch. 11 of Sambrook et al. (1989), In Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y. (incorporated herein by reference). The preferred exemplified conditions described here are particularly contemplated for use in selecting variations of the lytic gene.

Thus, by way of example, of a 150 base pair DNA probe derived from the first 150 base pairs of the open reading frame of a cDNA having a % GO=45%, a calculation of hybridization conditions required to give particular stringencies may be made as follows:

Assuming that the filter will be washed in 0.3 X SSC solution following hybridization, sodium ion =0.045M; % GC=45%; Formamide concentration^ 1=150 base pairs (see equation in Sambrook et al.) and so Tm =74.4 degrees C. The Tm of double- stranded DNA decreases by 1-1.5 degrees C with every 1% decrease in homology (Bonner et al. (1973). J. Mol. Biol. 81:123) (incorporated herein by reference). Therefore, for this given example, washing the filter in 0.3 times SSC at 59.4-64.4 degrees C will produce a stringency of hybridization equivalent to 90%; DNA molecules with more than 10% sequence variation relative to the target BSMR cDNA will not hybridize. Alternatively, washing the hybridized filter in 0.3 times SSC at a temperature of 65.4-68.4 degrees C will yield a hybridization stringency of 94%; DNA molecules with more than 6% sequence variation relative to the target BSMR cDNA molecule will not hybridize. The above example is given entirely by way of theoretical illustration. One skilled in the art will appreciate that other hybridization techniques may be utilized and that variations in experimental conditions will necessitate alternative calculations for stringency.

In preferred embodiments of the present disclosure, stringent conditions may be defined as those under which DNA molecules with more than 25%) sequence variation (also termed "mismatch") will not hybridize. In a more preferred embodiment, stringent conditions are those under which DNA molecules with more than 15% mismatch will not hybridize, and more preferably still, stringent conditions are those under which DNA sequences with more than 10% mismatch will not hybridize. Preferably, stringent conditions are those under which DNA sequences with more than 6% mismatch will not hybridize.

The degeneracy of the genetic code further widens the scope of the embodiments as it enables major variations in the nucleotide sequence of a DNA molecule while maintaining the amino acid sequence of the encoded protein. For example, a representative amino acid residue is alanine. This may be encoded in the cDNA by the nucleotide codon triplet GCT. Because of the degeneracy of the genetic code, three other nucleotide codon triρlets~GCT, GCC and GCA~also code for alanine. Thus, the nucleotide sequence of the gene could be changed at this position to any of these three codons without affecting the amino acid composition of the encoded protein or the characteristics of the protein. The genetic code and variations in nucleotide codons for particular amino acids are well lαiown to the skilled artisan. Based upon the degeneracy of the genetic code, variant DNA molecules may be derived from the cDNA molecules disclosed herein using standard DNA mutagenesis techniques as described above, or by synthesis of DNA sequences. DNA sequences which do not hybridize under stringent conditions to the cDNA sequences disclosed by virtue of sequence variation based on the degeneracy of the genetic code are herein comprehended by this disclosure.

One skilled in the art will recognize that the DNA mutagenesis techniques described here can produce a wide variety of DNA molecules that code for a bacteriophage lysin specific for a specific bacteria yet that maintain the essential characteristics of the lytic protein. Newly derived proteins may also be selected in order to obtain variations on the characteristic of the lytic protein, as will be more fully described below. Such derivatives include those with variations in amino acid sequence including minor deletions, additions and substitutions.

While the site for introducing an amino acid sequence variation is predetermined, the mutation per se does not need to be predetermined. For example, in order to optimize the performance of a mutation at a given site, random mutagenesis may be conducted at the target codon or region and the expressed protein variants screened for the optimal combination of desired activity. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence as described above are well known.

Amino acid substitutions are typically of single residues; insertions usually will be on the order of about from 1 to 10 amino acid residues; and deletions will range about from 1 to 30 residues. Deletions or insertions may be in single form, but preferably are made in adjacent pairs, i.e., a deletion of 2 residues or insertion of 2 residues. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final construct. Obviously, the mutations that are made in the DNA encoding the protein must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure (EP 75, 444 A). Substitutional variants are those in which at least one residue in the amino acid sequence has been removed and a different residue inserted in its place. Such substitutions may be made in accordance with the following Table 1 when it is desired to finely modulate the characteristics of the protein. Table 1 shows amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative substitutions. Table 1

Original Residue Conservative Substitutions

Ala ser

Arg lys

Asn gin, his

Asp glu

Cys ser

Gin asn

Glu asp

Gly pro

His asn; gin

He leu, val

Leu ile; val

Lys arg; gin; glu

Met leu; ile Phe met; leu; tyr

Ser thr

Thr ser

Tip tyr

Tyr tip; phe

Val ile; leu

Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than in Table 1, i.e., selecting residues that differ more significantly in their effect on maintaining: (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation; (b) the charge or hydrophobicity of the molecule at the target site; or (c) the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in protein properties will be those in which: (a) a hydrophilic residue, e.g., seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histadyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine. The effects of these amino acid substitutions or deletions or additions may be assessed for derivatives of the lytic protein by analyzing the ability of the derivative proteins to complement the sensitivity to DNA cross-linking agents exhibited by phages in infected bacteria hosts. These assays may be performed by transfecting DNA molecules encoding the derivative proteins into the bacteria as described above. Having herein provided nucleotide sequences that code for lytic enzyme genetically coded for by a bacteriophage specific for a specific bacteria and fragments of that enzyme, correspondingly provided are the complementary DNA strands of the cDNA molecule and DNA molecules which hybridize under stringent conditions to the lytic enzyme cDNA molecule or its complementary strand. Such hybridizing molecules include DNA molecules differing only by minor sequence changes, including nucleotide substitutions, deletions and additions. Also contemplated by this disclosure are isolated oligonucleotides comprising at least a segment of the cDNA molecule or its complementary strand, such as oligonucleotides which may be employed as effective DNA hybridization probes or primers useful in the polymerase chain reaction. Hybridizing DNA molecules and variants on the lytic enzyme cDNA may readily be created by standard molecular biology techniques.

The detection of specific DNA mutations may be achieved by methods such as hybridization using specific oligonucleotides (Wallace et al. (1986). Cold Spring Harbor Symp. Quant. Biol. 51:257-261), direct DNA sequencing (Church and Gilbert (1988). Proc. Natl. Acad. Sci. USA 81:1991-1995), the use of restriction enzymes (Flavell et al. (1978). Cell 15:25), discrimination on the basis of electrophoretic mobility in gels with denaturing reagent (Myers and Maniatis (1986). Cold Spring Harbor Symp. Quant. Biol. 51:275-284), RNase protection (Myers et al. (1985). Science 230:1242), chemical cleavage (Cotton et al. (1985). Proc. Natl. Acad. Sci. USA 85:4397-4401) (incorporated herein by reference), and the ligase-mediated detection procedure (Landegren et al., 1988)(incorporated herein by reference).

Oligonucleotides specific to normal or mutant sequences may be chemically synthesized using commercially available machines, labeled radioactively with isotopes (such as .sup.32 P) or non-radioactively (with tags such as biotin (Ward and Langer et al. Proc. Natl. Acad. Sci. USA 78:6633-6657 1981) (incorporated herein by reference), and hybridized to individual DNA samples immobilized on membranes or other solid supports by dot-blot or transfer from gels after electiophoresis. The presence or absence of these specific sequences are visualized by methods such as autoradiography or fluorometric or colorimetric reactions (Gebeyehu et al. Nucleic Acids Res. 15:4513-4534 1987) (incorporated herein by reference). Sequence differences between normal and mutant forms of the gene may also be revealed by the direct DNA sequencing method of Church and Gilbert (1988) (incorporated herein by reference). Cloned DNA segments may be used as probes to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined with PCR (Stoflet et al. Science 239:491-494, 1988) (incorporated herein by reference). In this approach, a sequencing primer which lies within the amplified sequence is used with double- stranded PCR product or single-stranded template generated by a modified PCR. The sequence determination is performed by conventional procedures with radiolabeled nucleotides or by automatic sequencing procedures with fluorescent tags. Such sequences are useful for production of lytic enzymes according to embodiments of the disclosure.

Additional objects and advantages embodiments found in the disclosure will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the embodiments. The objects and advantages of the disclosure may be realized and obtained by means of the instrumentalities and combinations particularly pointed out in the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the complete nucleotide sequence of the bacteriophage Av-1 genome; Figure 2 shows the nucleotide sequence of the CDS of the phage Av-1 endolysin gene, which is located at bp 15246-16061 of SEQ ID NO: 1 ;

Figure 3 shows the deduced amino acid sequence of Av-1 lysin, which consists of 272 amino acids (SEQ ID NO:3); Figure 4 shows the amino acid sequence of 6x-His-tagged Av-1 lysin (SEQ ID NO:4);

Figure 5 shows SDS-PAGE gel of 6x-His-tagged Av-1 lysin expressed in E. coli DH5

Figure 6 shows lysoplate detection of lytic activity of 6x-His-tagged Av-1 lysin.

DETAILED DESCRIPTION OF THE DISCLOSURE The complete sequence of the bacteriophage Av-1 genome was obtained by first cloning EcoRI fragments of purified Av-1 phage DNA into the EcoRI site of plasmid vector pIBI76 (International Biotechnologies, Inc.; Eastman Kodak). Thereafter, DNA inserts in several recombinant plasmids were sequenced using standard forward and reverse vector primers, and one of the sequenced fragments (about 500 bp - the sequence of both strands was obtained) was used to design appropriate sequencing primers to extend the sequence in both directions, using whole phage DNA as template. The primer walking approach was followed to eventually obtain the complete genome sequence, which is shown in Figure 1 (SEQ ID NO:l), which consists of 17,171 bp of dsDNA.

Computer analyses of SEQ ID NO:l were preformed to identify potential open reading frames, whose deduced amino acid sequences were then compared with known proteins in the GenBank database by standard BLAST searches. In this manner, a putative Av-1 phage endolysin gene was identified, based on similarities to several known phage lysin enzymes. The putative Av-1 phage endolysin gene is located on the genome at bp 15246-16061. The CDS of this enzyme is shown in Figure 2 (SEQ ID NO:2). The deduced amino acid sequence of this lysin gene, which consists of 272 amino acids, is shown in Figure 3 (SEQ ID NO:3). The nucleotide sequence and amino acid sequence of the Av-1 lysin gene are unique and differ from all known phage lysin genes and proteins.

Next, the Av-1 lysin gene was cloned into the E. coli plasmid expression vector pQE80L (QIAGEN, Inc.) as follows: Forward and reverse PCR primers were synthesized, and the Av-1 lysin gene amplified by PCR using purified whole phage DNA as template. The primers which contained extensions that included restriction sites for BamHI (forward primer) and Bglll (reverse primer) are shown below:

P295 (forward primer) :

5'-GCCGGATCCGCAACTCGCGCAGACATCATT-3'(SEQ ID NO:5)

P296 (reverse primer): 5'-GCGAGATCTAACTGTTTATGGGCGCTCTGC-3'(SEQ ID NO:6).

The 840 bp resulting PCR-amplified DNA fragment was isolated by gel electrophoresis, extracted and purified from the gel, restricted with BamHI and Bglll, and then ligated into BamHI-restricted, dephosphorylated pQE80L. The resulting ligation product was electroporated into E. coli DH5ct Transformants were selected on ampicillin-containing medium, and several clones were then grown in broth culture and their plasmids extracted, purified and analyzed by restriction analysis to identify recombmants having inserts in the correct orientation. One plasmid, designated pAv-lLl, was selected for further study and sequenced to confirm that it has the expected nucleotide sequence.

The active enzyme produced from the insert in pAv-lLl is actually a N-terminal 6x- His-tagged fusion protein of the Av-1 lysin gene, which resulted from inserting it in-frame immediately downstream of the 6x-His codons of the vector. In this construct, the vector's promoter, ribosomal binding site and ATG start codon are used to drive transcription and translation. The amino acid sequence of the resulting tagged protein (deduced from its actual DNA sequence) is shown in Figure 4 (SEQ ID NO:4). The additional eleven amino acids after the first Met at the N-terminal end of the Av-1 lysin gene do not affect the protein's enzymatic activity, indicating that some sequence changes in this region of the protein can be tolerated. The advantage in using this construct is that the 6x-His tag can be used to quickly purify the enzyme to near homogeneity by employing standard nickel affinity matrix chromatography technology. Expression of Av-1 lysin in E. coli was achieved by growing host cells containing plasmid pAv-lLl to mid-log phase and inducing transcription by adding the lad inducer, IPTG. Cultures were grown for 4 hours at 37°C in the absence or presence of IPTG (1.0 mM), centrifuged and the cell pellets sonicated to obtain cell extracts and to release soluble proteins. Samples were then electrophoresed in a 12% (w/v) polyacrylamide-SDS gel and stained with Coomasie Blue. The results are shown in Figure 5. α. Lanes 1 and 8 are protein MW

standards; lane 2, uninduced DH5α cells; lane 3, DH5c- cells induced with IPTG; lane 4, uninduced cells containing pQE80L vector; lane 5, pQE80L-containing cells induced with IPTG; lane 6, uninduced cells containing Av-1 lysin-pQE80L construct; and lane 7, Av-1 lysin- pQE80L construct induced with IPTG. The arrow indicates 30 MDa standard As shown in Figure 5, expression of a protein of the expected size (30 kDa) was observed (arrow). 6x-His-tagged Av-1 lysin is weakly expressed in uninduced cells (lane 6) and overexpressed in the presence of IPTG (lane 7).

To prove that the expressed protein has enzymatic (lytic) activity, lysoplate detection of lytic activity of 6x-His-tagged Av-1 lysin was carried out. Specifically, stationary phase cells of A. naeslundii strain MG-1 were washed with distilled water and concentrated 100-fold by centrifugation. 2.0 ml of the concentrated cells were then added to 7.0 ml of 1.0% (w/v) molten agarose (in PBS), and the mixture was poured into a 50 mm petri plate. After solidification, 5 mm wells were made in the gel by using a #2 cork borer. The wells were filled with 40 μl of the cell extracts described above, and the plate was photographed after 5 hours of incubation at 37°C. The results are shown in Figure 6. The wells contained: 1, uninduced DH5α cells; 2, IPTG-induced cells; 3, uninduced cells containing the pQE80L vector; 4, pQE80L-containing cells induced with IPTG; 5 and 6, uninduced and IPTG-induced cells containing the pQE80L- Av-1 lysin construct, respectively. As shown in Figure 6, the protein expressed from the cloned Av-1 lysin gene extensively degraded the cell wall of A. naeslundii, as evidenced by the lysis zones surrounding the wells containing extracts of E. coli cells carrying only this gene on a plasmid. Furthermore, Figure 6 shows that the enzyme can lyse the cells by degrading the well from the outside of the cell. The clear zones surrounding wells 5 and 6 (which were visibly noticeable after only one hour) are due to lysis of the A. jiaeslundii cells imbedded in the agarose.

Purification of Av-1 lysin from natural or recombinant sources can be accomplished by conventional purification means such as ammonium sulfate precipitation, gel filtration chromatography, ion exchange chromatography, adsorption chromatography, affinity chromatography, chromatafocusing, HPLC, FPLC, and the like. Where appropriate, purification steps can be done in batch or in columns. Fractions containing Av-1 lysin can be identified by enzymatic activity.

Peptide fragments can be prepared by proteolysis or by chemical degradation. Typical proteolytic enzymes are trypsin, chymotiypsin, V8 protease, subtilisin and the like; the enzymes are commercially available, and protocols for performing proteolytic digests are well-known. Peptide fragments can be purified by conventional means, as described above. Peptide fragments can often be identified by amino acid composition or sequence. Peptide fragments are useful as immunogens to obtain antibodies against Av-1 lysin.

It has been found in the present disclosure that Av-1 lysin is not only novel but unique, with respect to both its molecular sequence and it activity against the bacterial species A. naeslundii, i.e., it does not lyse the cell walls of any other Gram-positive or Gram-negative organisms in the oral cavity, including those which are considered beneficial to good oral health, e.g., S. sanguis and S. gordonii. Av-1 lysin of the presen disclosure degrades the cell walls of both stationary and actively growing target cells so that it should be effective in removing cells which have already colonized the oral cavity and become established in this econiche, and in preventing re-colonization. Thus, Av-1 lysin and the sequences thereof are useful for both treatment and prevention of gingivitis and root surface caries.

Prophylactic and therapeutic compositions containing as an active ingredient, one or more bacteria-associated phage proteins or protein peptides fragments, including isozymes, analogs, or variants of phage enzymes or phage peptides and peptide fragments thereof in a natural or altered form as active drugs and the method in therapeutic, diagnostic, and drug screening of use of such compositions may be used in the treatment of A naeslundii. The lytic enzyme may be in a natural, chimeric or shuffled, form.

A. naeslundii is thought to be the etiological agent of gingivitis and root surface caries.

The bacteriolytic enzyme (lysin) encoded on the genome of bacterial vims (bacteriophage) Av-1 which infects A. naeslundii, as well as the chimeric and shuffled versions of the lysine, can be used in topical treatment preparations to treat gingivitis and to kill cariogenic bacteria on tooth enamel and root surfaces, in order to halt their destructive effects. Av-1 lysin, based on it sequence or the chimeric or shuffled forms of the sequence can thus be used both for prophylactic and therapeutic treatment of these and other dental diseases where A. naeslundii to be treated.

It is most preferable that a phannaceutically acceptable carrier deliver this phage associated lytic enzyme to the possible site of the infection. Pharmaceutically effective carriers for delivering the Av-1 lysin can include mouthwashes/rinses, topical gels/ointments, toothpastes/powders, slow release implants/coatings, chewing gums and the like.

Application of Av-1 lysin facilitates plaque removal both in the home and in the dentist's office. Physical removal of dental plaque can be carried out using any known topical means, including dental floss, toothpaste (including abrasive toothpastes), plaque-loosening mouthwashes and professional cleaning by a dentist or dental hygienist. The enzyme can be in or on any one these topical means listed. Other preventive measures include pit and fissure sealants (for children) and various fluoride-containing toothpastes and gels (to reduce the acid-solubility of enamel). The pit and fissure sealants may be coated with the enzyme or it may be included in the sealant as well.

Other uses of Av-1 lysin include the cleaning or disinfecting of dental appliances, including fixed and removable bridges, partial and full dentures, caps and crowns; veterinary applications; orthodontic and surgical appliances; implant materials; temporary crowns, caps and bridges; endodontic uses (root canals); periodontal treatments (root scaling, cleaning); preparation of enamel, dentinal and cemental surfaces for restorations. The lysin can be delivered in some form of pharmaceutically acceptable carrier, such as in the form of a disinfectant carrier.

Av-1 lysin of the present disclosure, being a protein, adheres to dental plaque well, and so acts over considerable periods of time. Continued, intermittent applications of the enzyme can thus, be used to prevent the re-establishment of the target bacteria in the oral cavity.

It is most prefened that no alcohol be in the mouthwash or a topical cleaner in the presence of the enzyme. Additionally, in all uses described, a shuffled and/or chimeric enzyme may be used individually, or with the unaltered lytic enzyme, or the unaltered lytic , and a holin protein, may be used.

The effective amount of Av-1 lysin employed in the present disclosure will vary depending upon the carrier in which it is dissolved or suspended. Generally, the amount of Av-1 lysin employed in the present disclosure will be 0.01 to 10 mg/ml, preferably 0.1 to 1.0 mg/ml. Larger or smaller amounts of the enzyme may be used. Additionally, the amount of enzyme in the pharmaceutical carrier may be measured in active enzyme units. The enzyme unit dosage may be in an amount ranging from The concentration of the active units of enzyme(s) believed to provide for an effective amount or dosage of enzyme may be in the range of about 100 units/ml to about 100,000 units/ml of fluid in the wet or damp environment of the nasal and oral passages, and possibly in the range of about 100 units/ml to about 10,000 units/ml. In some circumstances, the units/ml of enzyme may be as high as 500,000 units/ml, and possibly several million units/ml

It is preferred that the enzyme be in a stabilizing buffer environment for maintaining a pH range between about 4.0 and about 9.0, with preferred range being from about 4.0 to about 7.0

Av-1 lysin is a "natural" product, and therefore does not have any harmful effect on oral tissues. Chicken lysozyme is presently given GRAS status (generally recognized as safe) by the FDA for use in food products. Ingestion of Av-1 lysin would therefore be harmless. The enzyme should not harm tissues or beneficial microorganisms in the gastrointestinal tract.

Av-1 lysin is not normally synthesized in large amounts during phage growth. Thus, cloning the Av-1 lysin gene into an appropriate vector allows Av-1 lysin to be produced in large quantities for purification. This disclosure therefore includes the construction and use of such recombinant DNA vectors and their appropriate hosts.

Additional embodiments of this disclosure include genetically engineered, non- cariogenic organisms (such as S. sanguis or S. gordonii) which can colonize dental plaque, but produce phage-encoded enzymes that inhibit establishment of A. naeslundii. Such engineered "replacement therapy" strains can provide long-term protection against dental caries.

This approach, described by Hillman & Socransky (Replacement therapy for the prevention of dental disease, Adv. Dent. Res., H1Y.119-125 (1987) (incorporated herein by reference) employs implanting a non-cariogenic organism (in this case a mutant of S. mutans which cannot produce lactic acid, which causes caries) into a normal mouth to replace resident, wild-type strains of S. mutans. This "effector" strain, if it can out-compete the resident strains, for example by producing a bacteriocin, replaces them and caries can no longer occur because there is not enough acid to dissolve the tooth enamel. Since bacteriocin-resistant mutants of S. mutans are likely to occur (like phage-resistant mutants), long term protection can not be guaranteed because such mutants would not be inhibited by the effector strain.

Thus, the present disclosure can be used to develop effector strains of S. sanguis or S. gordonii, which are colonizers of teeth and are non-cariogenic. By introducing the Av-1 lysin gene into S. sanguis or S. gordonii so that it is continuously expressed at low levels, an effector strain can be obtained which prevents establishment by cariogenic A. naeslundii. This situation would persist indefinitely since A. naeslundii do not appear to be capable of developing resistance to phage lysin by simple mutation.

Cloning vectors which can be used in the present disclosure include any known vector 5 in the art, such as pBR-, pUC- and M13-based plasmids, and phage-based expression vectors, such as λgtl 1 and the like.

The present disclosure also relates to Av-1 lysin which contains mutations which allow the protein to substantially retain its enzymatic activity. In addition, Av-1 lysin may be specifically engineered to contain mutations which increase or alter its activity or o characteristics in a desired manner.

The cloned DNA molecule can be inserted into replicable expression vectors such that the coding sequence is operably linked to a nucleotide sequence element capable of effecting expression of Av-1 lysin. In particular, the nucleotide sequence elements can be a promoter, a transcription enhancer element, a termination signal, a translation signal, or a combination 5 of two or more of these elements, generally including at least a promoter element.

Replicable expression vectors are generally DNA molecules engineered for controlled expression of a desired gene, especially where it is desirable to produce large quantities of a particular gene product, or polypeptide. The vectors comprise one or more nucleotide sequences operably linked to a gene to control expression of that gene, the gene being o expressed, and an origin of replication which is operable in the contemplated host. Preferably the vector encodes a selectable marker, for example, antibiotic resistance. Replicable expression vectors can be plasmids, bacteriophages, cosmids and the like. Any expression vector comprising RNA is also contemplated. The replicable expression vectors of this disclosure can express Av-1 lysin at high levels. These vectors are preferably derived from a prokaryote.

Prokaryotic vectors include bacterial plasmids and bacteriophage vectors that can transform or infect such hosts as E. coli, B. subtilis, Streptomyces sps. and other microorganisms. Many of these vectors are based on pBR322, M13 and lambda and are well-known in the art, and employ such promoters as trp, lac, λP, and the like. The cells which serve as hosts for these vectors are well-known in the art and a suitable host for a particular vector can be readily selected by one of ordinary skill in the art. Numerous texts on recombinant DNA techniques are available which describe expression vectors, the control sequences contained therein, and general methodology for making expression constructs. Hence, one skilled in the art has available many choices of replicable expression vectors, compatible hosts, and well-known methods for making and using the vectors.

The present disclosure also relates to antibodies which specifically bind to Av-1 lysin.

Such antibodies may be monoclonal or polyclonal and are contemplated to be useful in developing detection assays (immunoassays) for proteins, monitoring the activity of Av-1 lysin and in purifying Av-1 lysin. Thus, in accordance with this disclosure, an antibody to

Av-1 lysin encompasses monoclonal or polyclonal antibodies or to antigenic parts thereof.

Both polyclonal and monoclonal antibodies are obtainable by immunization of an animal with purified enzyme, purified recombinant enzyme, fragments of the same, or purified fusion proteins of Av-1 lysin with another protein. In the case of monoclonal antibodies, partially purified proteins or fragments may serve as immunogens. The methods of obtaining both types of antibodies are well-known in the art with excellent protocols for antibody production being found in Harlow et al, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, page 726 (1988). Polyclonal sera are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of the purified enzyme, or parts thereof, collecting serum from the animal, and isolating specific antibodies by any of the known immunoadsorbent techniques. Antibodies produced by this method are useful in virtually any type of immunoassay.

Monoclonal antibodies are particularly useful because they can be produced in large quantities and with a high degree of homogeneity. Hybridoma cell lines which produce monoclonal antibodies are prepared by fusing an immortal cell line with lymphocytes sensitized against the immunogenic preparation and is done by techniques which are well-known to those who are skilled in the art (see, for example, Douillard et al, "Basic Facts About Hybridomas", in Compendium of Immunology, Vol. II, L. Schwartz (Ed.) (1981); Kohler et al, Nature, 256:495-497 (1975); Harlow et al, Eur. J. Immunol., 6:511-519 (1976); Koprowski et al, U.S. Patent No. 4,172,124; Koprowski et al, U.S. Patent No. 4,196,265; and Wands, U.S. Patent No. 4,271,145, the teachings of which are incorporated herein by reference).

The gene coding Av-1 lysin of the present disclosure can also be cloned into other organisms to facilitate and improve the production and purity of the enzyme by a variety of genetic engineering techniques. Protein engineering techniques can then employed to modify the properties of the enzyme, for example to improve its stability, i.e., at low pH. U.S. Patent Publication Nos. 2001/0014463 and 2002/0044911, directed to methods for treatment and prevention of dental caries and periodontal diseases using bacteriophage- encoded enzymes, are incorporated by reference herein in their entirety. While the disclosure has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.

Claims

WHAT IS CLAIMED IS:

Claim 1. A method for the treatment or prevention of gingivitis and root surface caries comprising administering to the oral cavity of a subject in need of such treatment or prevention, an effective amount of Av-1 lysin comprising the amino acid sequence of SEQ ID NO:3, wherein said Av-1 lysin has the ability to digest a cell wall of a specific bacteria, said bacteria being A. naeslundii.

Claim 2. A method for the treatment of prevention of gingivitis and root surface caries comprising administering to the oral cavity of a subject in need of such treatment or prevention, an effective amount of Av-1 lysin comprising the amino acid sequence of SEQ ID NO:4, wherein said Av-1 lysin has the ability to digest a cell wall of a specific bacteria, said bacteria being A. naeslundii.

Claim 3 A method for the parenteral or therapeutic treatment of A. naeslundii comprising administering to the mouth, gums or teeth of an animal an effective amount of Av-1 lysin comprising the amino acid sequence of SEQ ID NO:3, wherein said Av-1 lysin has the ability to digest a cell wall of a specific bacteria, said bacteria being A. naeslundi

Claim 4. A method for the parenteral or therapeutic treatment of A. naeslundii comprising administering to the mouth, gums or teeth of an animal an effective amount of Av-1 lysin comprising the amino acid sequence of SEQ ID NO:4, wherein said Av-1 lysin has the ability to digest a cell wall of a specific bacteria, said bacteria being A. naeslundi

Claim 5. An isolated and purified Av-1 lysin comprising the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4, said lysine having the ability to digest a cell wall of a specific bacteria, said bacteria being A. naeslundi

Claim 6. The DNA molecule for wherein said DNA molecule comprises the nucleotide sequence of SEQ ID NO:2.

Claim 7 An expression vector comprising the DNA molecule of Claim 4.

Claim 8. A host cell comprising the expression vector of Claim 6.

Claim 9. An antibody which binds specifically to Av-1 lysin comprising the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4.

Claim 10. The antibody of Claim 8, wherein said antibody is a monoclonal antibody.

Claim 11. The antibody of Claim 8, wherein said antibody is a polyclonal antibody.

Claim 12. An oral composition comprising Av-1 lysin comprising the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4; and a pharmaceutically acceptable carrier.

Claim 13. The oral composition of Claim 11, wherein said composition is a mouthwash, mouthrinse, topical gel, topical ointment, toothpaste, powder, slow release implant, slow release coating or chewing gum.

Claim 14. A genetically engineered, non-cariogenic microorganism which colonizes dental plaque and is generally engineered so as to produce Av-1 lysin comprising the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4.

Claim 15. The genetically engineered microorganism of Claim 13, wherein the non-cariogenic microorganism is S. sanguis or S. gordonii.