# Patent

## Title
Nucleic acids and polypeptides of C1 bacteriophage and uses thereof

## Abstract

The full-length nucleic acid sequence of the C1 bacteriophage is disclosed in the present application. The specific regions of the C1 genome encoding the PlyC lysin have also been identified and sequenced. The invention relates to the pharmaceutical and diagnostic utility of these sequences and provides for development of pharmaceutical compositions for treating or preventing streptococcal infections in mammals, for compositions for decontamination of inanimate surfaces and for diagnosis of streptococcal infections.

## Background
FIELD OF THE INVENTION
The invention relates to novel nucleic acids and proteins isolated from C1 bacteriophage and therapeutic and diagnostic uses of these nucleic acids and polypeptides and related pharmaceutical compositions useful in treating Streptococcal infections. The invention further relates to the identification, isolation and cloning of specific genes from the C1 bacteriophage, specifically the C1 bacteriophage lysin, termed PlyC, and the use of these genes and gene products for prophylactic and therapeutic use to treat or prevent Streptococcal infections, and to aid in diagnosis of infections caused by Streptococcus.
BACKGROUND OF THE INVENTION
Group C streptococci are a common cause of infection in several animal species but are generally considered to be a rare cause of infection in humans (Ghoneim, A. T. et al. (1980), J. Clin. Pathol. 33:188-190; Feldman, W. E. Postgrad. Med. (1993), 93(3): 141-145). Of the four species of group C streptococci, S. equisimilis has been reported to cause most human illnesses, including bacteremia, endocarditis, meningitis, pneumonia, epiglottitis, puerperal sepsis, and wound infections. However, S. zooepidemicus has been associated with two outbreaks of pharyngitis and nephritis in Europe (Duca, E. et al. (1969), J. Hyg. Camb. 67: 691-698; Barnham, M. et al. (1983), Lancet 8331: 945-948). In both of the European outbreaks, unpasteurized milk was suspected as the source of infection. More recently, Group C streptococci was identified as a causative agent in a prosthetic joint infection (Kleshinski, J. et al. (2000) Southern Medical Journal 93:1217-1220). This is the first reported outbreak in the United States and is the first such reported outbreak in which the vehicle, ie. cheese made from unpasteurized cows' milk, has been epidemiologically implicated. Although S. zooepidemicus and S. equisimilis are rarely reported causes of mastitis in cows, the cause of this outbreak was contaminated milk from cows with mammary infections due to S. zooepidemicus. 
Group C streptococci are also the causative agent of strangles, a highly contagious and serious infection of horses and other equids. The disease is characterized by severe inflammation of the mucosa of the head and throat, with extensive swelling and often rupture of the lymph nodes, which produces large amounts of thick, creamy pus. The organism can be isolated from the nose or lymph nodes of affected animals, and is usually readily identified in the laboratory by simple sugar tests.
Control of the disease is complicated by the development of long-term carriers that outwardly appear healthy and are frequently difficult to detect because swab samples from them often do not yield cultures of S. equi. The organism resides in the guttural pouches and resumption of active shedding can recur sporadically for unknown reasons. Previously, endoscopy was the only way to reliably detect most carriers but this is too impractical in most circumstances. The outbreak prevalence of these carriers combined with movement and mixing with susceptible animals probably accounts for the high incidence of strangles. Although a “PCR test” to detect the DNA of the organism in nasopharyngeal swabs is now available, enabling sensitive carrier detection, there are still practical difficulties and expense with multiple nasal sampling and some endoscopy, although much less than previously. For the large majority of horses the most effective means of controlling strangles would be a good vaccine or other immunogenic compositions that are effective in eliminating the causative agent.
Group A Streptococcus (Streptococcus pyogenes), the primary etiologic agent of bacterial pharyngitis, is one of few human pathogens that remain uniformly sensitive to penicillin (Macris, M. H., et al. (1998) Pediatr. Infect. Dis. J. 17:377-381). Additionally, the advent of rapid group A streptococcal diagnostic test kits over the last decade has allowed early initiation of antibiotic treatment. Despite these factors, streptococcal-mediated pharyngitis is reported in over 2.5 million people annually in the United States with >80% of these cases occurring in children under 15 years of age (Schappert, S. M., et al. (1999) Vital Health Stat. 13). However, streptococcal pharyngitis classically is not a reportable disease and it has been speculated that the documented number of these pharyngitis cases may be considerably underestimated. Additionally, penicillin fails to completely eradicate streptococci in up to 35% of patients treated for pharyngitis (Pichichero, M. E. (1998) Pediatr. Rev. 19:291-302) and carriage rates as high as 50% have been reported in close contact areas such as day care centers (Feldman, S., et al. (1987) J. Pediatr. 110:783-787). This high carriage rate contributes to the spread of streptococcal pharyngitis (Nguyen, L., et al. (1997) J. Clin. Microbiol. 35:2111-2114) and correlates with outbreaks of rheumatic fever (Oliver, C. (2000) J. Antimicrob. Chemother. 45 Topic T1:13-21). While eradication of the carrier state would reduce the pool of streptococci in the population, and thus streptococcal-related diseases, to date the only treatment is an extensive regimen of antibiotics (Tanz, R. R., et al. (1998) Pediatric Annals 27:281-285) that may increase streptococcal resistance to macrolides, which are often prescribed for patients with penicillin allergies (York, M. K., et al. (1999) J. Clin. Microbiol. 37:1727-1731).
Bovine mastitis is an inflammation of a cow's mammary gland, usually due to a microbial infection originating from contaminated teats. Experimental bovine mastitis can be induced with as little as 100 organisms, so a few chronic infections within a herd can maintain a persistent bacterial reservoir. Several bacterial species have the ability to cause bovine mastitis, including Staphylococcus aureus, Streptococcus uberis, Streptococcus agalactiae (Group B strep), and Escherichia coli. Of these, S. aureus, which causes acute conditions, and S. uberis, which causes chronic conditions, are responsible for the bulk of bovine mastitis cases. The persistence and economic impact of bovine mastitis is alarming. Wilson et al. (Wilson, D. J., et al. (1997) J. Dairy Sci. 80:2592-2598) recently published the results of a retrospective study of milk samples collected from more than 100,000 cows in New York and northern Pennsylvania between 1991 and 1995. They found that intramammary infections were present in 36% of cows enrolled in the Dairy Herd Improvement Association. This disease is estimated to cost the producer approximately $200/cow/year, which corresponds to a U.S. total of $1.7 billion annually.
Current therapies for bovine mastitis rely heavily on the use of β-lactam antibiotics such as penicillins and cephalosporins. These agents have had a beneficial impact on dairy-animal health and milk production. However, the cure rate for treatment of some infections, particularly S. aureus, is often less than 15%. This is attributed to incomplete penetration of the antibiotics throughout the mammary gland (Yancey, R. J., et al. (1991) Eur. J. Clin. Microbiol. Infec. Dis. 10:107-113). Additionally, concerns of accidental exposure of susceptible consumers to residual antibiotics resulting in anaphylaxis has necessitated the imposition of a post-treatment milk discard period and strict industry surveillance of all milk shipments. While infections can be cleared in days with antibiotic treatment, the discard period can often last weeks until residual antibiotic levels fall within acceptable parameters. Finally, there is growing concern that the agricultural use of antibiotics contributes to the emergence of antibiotic resistance in human pathogens (Smith, D. L., et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99:6434-5439). Taken together, these concerns suggest alternative therapies are needed for the therapeutic management of bovine mastitis.
Tailed bacteriophages are the most populous “organism” on Earth with roughly 1030 inhabitants in the biosphere (Brussow, H. et al. (2002) Cell 108:13-16). However, we are just beginning to appreciate the role they play in bacterial diversity (Hendrix, R. W. et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96:2192-2197) and more recently, bacterial pathogenesis (Broudy, T. B. et al. (2003) Infect. Immun. 71:3782-3786; Wagner, P. L. et al. (2002) Infect. Immun. 70:3985-3993). Indeed, whole genome sequencing of two different strains of group A streptococci reveals that polylysogenic phage represent the only diversity between the two strains (Beres, S. B. et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99:10078-10083; Ferretti, J. J. et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663). Recent advances have allowed whole bacteriophage genomes of evolutionary or biological interest to be rapidly sequenced for comparison to known genomes.
The streptococcal C1 bacteriophage has roots at the forefront of bacteriophage research. Shortly after the discovery of bacteriophages by Twort and d'Herelle (Twort, F. W. (1915) Lancet ii.: 1241-1243; d'Herelle, F. H. (1917) C. R. Acad. Sci. (Paris) 165:373-375), the C1 bacteriophage was isolated by Clark in 1925 from a sewage plant in Milwaukee, Wis. and represents the first documented bacteriophage found to be active on any type of streptococci (Clark, P. F. et al. (1926) J. Bacteriol. 11:89). Initially known as the “sludge” phage or “Clark” phage, it infected streptococci isolated from animals (which we now know to be group C streptococci), but not streptococci found in humans (now known to be group A streptococci) (Shwartzman, G. (1927) J. Exp. Med. 46-497-509; Lancefield, R. C. (1932) Proc. Soc. Exp. Biol. Med. 30:169-171). In a hallmark 1934 paper, Alice Evans, using the “Clark” phage which she renamed B563, was the first person to utilize phage in order to classify bacterial strains, thus founding the analytical field of phage typing (Evans, A. C. (1934) Public Health Reports 49:1386-1401). Additionally, Evans noticed that phage lysates had lytic activity on streptococci that were not sensitive to the phage itself. She called this phenomenon “nascent lysis” and attributed it to a “lysin” activity originally defined by Twort (Twort, F. W. (1925) Lancet ii.:642-644). In 1957, Krause renamed the Evans B563 phage, C1, to imply an exquisite specificity for group C streptococci (Krause, R. M. (1957) J. Exp. Med. 106:365-384). Krause also noted that the C1 cell wall hydrolase, or lysin, had a less restrictive range given that groups A, C, and E streptococci were rapidly lysed by this enzyme.
On the genetic level, the C1 phage has not been studied in detail. Two published restriction maps of this genome exist (Pomrenke, M. E. et al. (1989) J. Basic Microbiol. 6:395-398; Totolian, A. A. et al. (1981) Reedbooks Ltd., Surrey), but to date, no sequence data was available. The majority of interest in this phage involves its lysin, which has been used extensively as a tool to dissolve the streptococcal cell wall in order to make protoplasts, extract genomic DNA, or to study surface proteins (van de Rijn, I. et al. (1981) Infect. Immun. 32:86-91; Wheeler, J. et al. (1980) J. Gen. Microbiol. 120:27-33).
At the end of a bacteriophage lytic cycle in a sensitive bacterial host, all double stranded DNA bacteriophages produce a lytic system that consists of a holin and at least one peptidoglycan hydrolase, or “lysin”, capable of degrading the bacterial cell wall. Lysins can be endo-β-N-acetylglucosaminidases or N-acetylmuramidases (lysozymes), which act on the sugar moiety, endopeptidases which cleave the peptide cross bridge, or more commonly, an N-acetylmuramoyl-L-alanine amidase, which hydrolyzes the amide bond connecting the sugar and peptide constituents. Typically, the holin is expressed in the late stages of phage infection forming a pore in the cell membrane allowing the lysin(s) to gain access to the cell wall peptidoglycan resulting in release of progeny phage (for review, see (Young, R. (1992) Microbiol. Rev. 56:430-481)). Lysin, added to sensitive organisms in the absence of bacteriophage, lyses the cell wall producing a phenomenon known as “lysis from without”.
The C1 bacteriophage specifically infect group C streptococci and produce a lysin (termed PlyC) that has been partially purified and characterized (Fischetti, V. A., et al. (1971) J. Exp. Med. 133:1105-1117; Raina, J. L. (1981) J. Bacteriol. 145:661-663). C1 phage lysin can cause “lysis from without” in groups A and E streptococci as well as group C streptococci (Maxted, W. R. (1957) J. Gen. Microbiol. 16:584-594; Krause, R. M. (1957) J. Exp. Med. 106:365-384). Additionally, PlyC works on Streptococcus equi and Streptococcus uberis. This unique activity has been exploited as a tool in group A streptococcal studies to isolate surface molecules including M proteins (Fischetti, V. A., et al. (1985) J. Exp. Med. 161:1384-1401), to lyse cells for DNA extraction, and to make protoplasts when used in a hypertonic environment (Wheeler, J., et al. (1980) J. Gen. Microbiol. 120:27-33).
The present invention provides alternate means for the prevention and/or treatment of pathogenic streptococcal infections in humans and animals such as streptococcal pharyngitis, equine Strangles disease, bovine mastitis, and other disease states associated with groups A, C, and E streptococci as well as S. equi and S. uberis. Furthermore, the present invention provides the means for diagnosis of such pathogenic infections.
SUMMARY OF THE INVENTION
The present invention relates to the isolation and elucidation of the nucleic acid and protein sequence of C1 bacteriophage, which is specific for Group C streptococci, and the therapeutic and diagnostic uses of these nucleic acid and polypeptide sequences. Furthermore, several open reading frames have been identified for which functions have been assigned. In a more particular aspect of the invention, the open reading frames associated with the two subunits of the PlyC (lysin) enzyme have been identified. Previous studies had demonstrated that the lytic properties associated with an isolated lysin had therapeutic potential to eliminate streptococcal colonization (Nelson et al. (2001) Proc. Nat. Acad. Sci. USA. 98:4107-4112). The available sequence data now provides the means by which such therapeutic and diagnostic utility can be carried out.
Accordingly, a first aspect of the invention provides for the isolation and elucidation of the full length nucleic acid sequence of C1 bacteriophage, provided herein as SEQ ID NO: 21(GenBank accession No.AY212251). More specifically, the C1 bacteriophage contains a double-stranded, linear sequence of DNA with 16,687 base pairs, and a G+C content of 34.6%. Also present are 143 base pair inverted terminal repeats. Furthermore, 20 predicted open reading frames (ORFs) have been identified, described herein in Table III as SEQ ID NOs: 1-20, with the first 11 on the positive strand and the remaining 9 on the negative strand. The majority of the ORF-encoded proteins are dissimilar to known phage proteins and have no homology to any proteins contained in GenBank. Further analysis of the bacteriophage C1 DNA revealed a terminal protein (TP) covalently linked to the 5′ terminus of the DNA. The scientific evidence for the novel sequences and potential therapeutic and diagnostic utility of these sequences is provided below.
A second aspect of the invention provides for identification and elucidation of the nucleic acid and protein sequence of the multimeric PlyC (lysin). In a specific embodiment, the PlyC has two open reading frames (ORF 9 and ORF 11) and comprises a light chain (ORF 9) and heavy chain (ORF 11) subunits, the amino acid sequences both of which are provided herein as SEQ ID NOs: 9 and 11, respectively. Note, the light chain is now referred to as PlyC-B, although it was formerly called the PlyC alpha subunit (Nelson, D., et al. (2003) J. Bacteriol. 185:3325-3332). Likewise, the heavy chain is now referred to as PlyC-A, although it was formerly called the PlyC beta subunit. In a yet further embodiment, the PlyC may further comprise the polypeptide of SEQ ID NO: 10 (ORF 10). In a yet further embodiment, the PlyC orf 9 codes for a protein that is 8 kDa in size and the PlyC orf 11 codes for a protein that is 50 kDa in size. In a particular embodiment, the polypeptide comprises at least two subunits, the subunits comprising a PlyC lysin light chain (PlyC-B) and a PlyC lysin heavy chain (PlyC-A). In a further embodiment, the polypeptide comprises multiple copies of one or both subunits and fragments, mutants, variants, analogs or derivatives thereof. Particularly, since the native protein has an apparent molecular weight of 100 kDa as measured by gel filtration, a further embodiment of the invention provides for native PlyC to be a “multimer”, composed of multiple copies of the heavy subunit (ORF 11), or the light subunit (ORF 9), or both light and heavy subunits. More specifically, this embodiment provides for PlyC to be preferably composed, but not limited to one heavy subunit (ORF 11) and 5 or 6 light subunits (ORF 9). A further embodiment provides that the operon containing the PlyC gene(s) is referred to as plyC (SEQ ID: 25). plyC is a 2.2 kb sequence containing the genes plyCA (SEQ ID: 23), lil (SEQ ID: 24), plyCB (SEQ ID: 22), and  ̃200 bp of untranslated sequence on both ends (See FIG. 2). It is envisioned that the multimeric PlyC may be used for treating or preventing bacterial infections, comprising administering a therapeutically effective amount of the PlyC multimer. In a yet further embodiment of the invention, the PlyC multimer is prepared as a pharmaceutical composition with a pharmaceutically acceptable carrier for use in treating bacterial infections, including infections caused by pathogenic streptococci of Groups A, C and E, as well as S. uberis, and S. equi. It is envisioned that the composition comprising the multimeric PlyC may be useful in treating streptococcal infections in mammals, including, but not limited to, horses and cows. A further embodiment provides for use of the pharmaceutical compositions for treatment of bovine mastitis in cows and strangles in horses. A yet further embodiment includes the use of the pharmaceutical compositions comprising multimeric PlyC for treatment of humans. A yet further embodiment provides for the use of the pharmaceutical compositions for treatment of streptococcal pharyngitis. A yet further embodiment provides for the use of the polypeptides of the present invention, that is, the C1 bacteriophage PlyC lytic enzyme, including the PlyC heavy and light chain subunits and multiple copies of one or the other subunits and fragments, mutants, variants, analogs or derivatives thereof for the preparation of a medicament for the treatment of a bacterial infection. In another preferred embodiment, the invention provides for the use of the polypeptides of the present invention, that is, the C1 bacteriophage PlyC lytic enzyme, including the PlyC heavy and light chain subunits and multiple copies of one or the other subunits and fragments, mutants, variants, analogs or derivatives thereof for the preparation of a medicament for the treatment of streptococcal infections selected from the group consisting of group A, E, C, S. uberis and S. Equi. 
A third aspect of the invention provides for the generation of antibodies specific for the PlyC multimer. In a preferred embodiment, the antibodies are monoclonal antibodies specific for the PlyC multimer, or subunits or fragments thereof. In a yet further embodiment, the antibodies are polyclonal antibodies prepared in mice, rats, guinea pigs, rabbits, goats, sheep, horses, pigs, cows, or any other mammal generally used in the art for generation of polyclonal antibodies. In another embodiment, the antibodies may be chimeric antibodies, humanized antibodies, single chain antibodies or fragments thereof. A further embodiment provides for an immortal cell line that produces a monoclonal antibody that is specific for the multimeric PlyC or subunits or fragments thereof.
A fourth aspect of the invention provides for methods of diagnosing the presence of a pathogenic streptococcal infection. In a preferred embodiment, the binding domain of PlyC is labeled with a fluorescent chemical or protein using methods known to those skilled in the art and the labeled PlyC is then incubated directly with a sample taken from a subject suspected of harboring a pathogenic streptococci. The observation of fluorescence in the sample is indicative of the presence of a pathogenic streptococcal infection. A yet further embodiment provides for the use of the PlyC multimer in a luciferin-luciferase assay to aid in diagnosing pathogenic streptococcal infections. In this assay format, a sample suspected of harboring a pathogenic streptococcus is incubated directly with the PlyC multimer. If the sample contains a pathogenic streptococcus, the PlyC will bind to the bacteria, resulting in lysis of the bacteria and subsequent release of ATP or other components normally present in the cytoplasm of the bacterial cell, such as enzymes. The lysate is then tested in a luciferin-luciferase assay. In another embodiment, the sample suspected of harboring a pathogenic streptococcus may be added directly to the PlyC multimer concurrently with luciferin-luciferase without the need to collect the cell lysate first before adding it to the luciferin-luciferase. If a pathogenic streptococcus is present in the sample, the release of ATP from the lysed bacteria will trigger a positive reaction in the luciferin-luciferase system, resulting in release of measurable light from the reaction mixture.
One further embodiment of the invention may use an antibody to the PlyC multimer or subunits or fragments thereof, or antibodies may be prepared to any of the proteins listed in Table III as SEQ ID NOs: 1-20, and the antibody may be labeled (eg. with fluorescein or other known fluorescent proteins or chemicals), coupled to the bacteriophage protein and used to monitor binding of the specific protein to the bacteria in a patient sample, thus aiding in detection of pathogenic streptococci. Alternatively, any one of the proteins from Table III may be fluorescein labeled directly and used to detect the presence of pathogenic streptococci in a patient sample.
Further diagnostic testing formats, including ELISA assays or radioimmunoassays may also be contemplated for use with the present invention. In these formats, one can utilize the proteins identified in Table III directly or one may prepare antibodies to these proteins as noted herein for use in kits to monitor the presence of pathogenic streptococci in a patient sample. The procedures for ELISA or radioimmunoassays are known to those skilled in the art.
A fifth aspect of the invention provides for methods of preventing or treating bacterial infections comprising administering a therapeutically effective dose of a composition comprising a therapeutically effective amount of the PlyC multimer having a sequence as set forth in SEQ ID NOs: 9 and 11. A particular embodiment comprises methods for preventing or treating bacterial infections comprising administering a therapeutically effective dose of a composition containing the amino acid sequences as set forth in SEQ ID NOs: 9 and 11, further comprising a therapeutically effective amount of the polypeptide having the amino acid sequence as set forth in SEQ ID NO: 10. A specific embodiment provides that the PlyC multimer is composed of gene products of the plyC operon (SEQ ID NO: 25).
A sixth aspect of the invention provides for pharmaceutical compositions comprising a therapeutically effective amount of the PlyC multimer as set forth is SEQ ID NOs: 9 and 11 and a pharmaceutically acceptable carrier. A further embodiment provides for a pharmaceutical composition comprising a therapeutically effective amount of the PlyC multimer as set forth is SEQ ID NOs: 9 and 11 and further comprising a therapeutically effective amount of the polypeptide having the amino acid sequence as set forth in SEQ ID NO: 10. A specific embodiment may include a pharmaceutical composition designed for use in treatment of infections caused by streptococcus groups A, E and C, as well as S. uberis, and S. equi. Another embodiment may include a pharmaceutical composition designed for use in treatment of topical or systemic infections, or infections that are non-responsive to other antibiotic modalities. A yet further embodiment provides for veterinary use of the pharmaceutical compositions of the present invention. A preferred embodiment is for use in treating infections in mammals, including but not limited to, horses or cows. A yet further embodiment is use of the pharmaceutical compositions for treatment of human subjects. Another embodiment provides for a composition comprising the polypeptides of the present invention, that is, the C1 bacteriophage PlyC lytic enzyme, including the PlyC heavy and light chain subunits and multiple copies of one or the other subunits and fragments, mutants, variants, analogs or derivatives thereof for use in decontaminating inanimate surfaces to eliminate possible contamination with streptococci from Groups A, E, and C, as well as S. uberis and S. equi. In a particular embodiment, the composition may be used to decontaminate milking, dairy, and agricultural equipment from streptococci.
A seventh aspect of the invention provides for the identification and use of a particular polypeptide comprising the amino acid sequence for the holin polypeptide of bacteriophage C1. In a particular embodiment, the amino acid sequence for the holin is identified as SEQ ID NO: 8. It is envisioned that the holin may be utilized to better understand the biochemical aspects of bacterial lysis and may be used either to identify agents useful for antimicrobial therapy or for use in the diagnosis of streptococcal infections.
An eighth aspect of the invention provides for the identification and use of a particular polypeptide comprising the amino acid sequence for the major tail polypeptide of bacteriophage C1. In a particular embodiment, the amino acid sequence for the major tail polypeptide is identified as SEQ ID NO: 12. It is envisioned that the major tail polypeptide may be utilized to better understand the biochemical aspects of binding of the bacteriophage to the bacterial cell wall. Thus, this polypeptide may be used either to aid in identification of agents useful for antimicrobial therapy or for use in the diagnosis of streptococcal infections.
A ninth aspect of the invention provides for the identification and use of a particular polypeptide comprising the amino acid sequence for the major capsid polypeptide of bacteriophage C1. In a particular embodiment, the amino acid sequence for the major capsid polypeptide is identified as SEQ ID NO: 16. It is envisioned that the major capsid polypeptide may be utilized to better understand the biochemical aspects of binding of the bacteriophage to the bacterial cell wall. Thus, this polypeptide may be used either to aid in identification of agents useful for antimicrobial therapy or for use in the diagnosis of streptococcal infections.
Other advantages of the present invention will become apparent from the ensuing detailed description taken in conjunction with the following illustrative drawings.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. Elucidation of subunits for PlyC. (A) Native-PAGE of purified PlyC shows a single, homogeneous band. (B) The native-PAGE was placed on an group A streptococcal-embedded agarose and allowed to incubate for 2 hr before the native gel was removed. The clearing zone on the agarose indicated that the PlyC activity corresponds to the single band on the native-PAGE. (C) An SDS-PAGE of the purified PlyC used in figure A shows that PlyC is composed of 2 subunits. The 50 kDa heavy chain is now termed PlyC-A and the 8 kDa light chain is termed PlyC-B. N-terminal sequencing of the native-PAGE gave a double sequence, which corresponded to the two chains sequenced in the SDS-PAGE (see text for details).
FIG. 2. Gene structure and analysis of the PlyC gene(s). (A) A clone displaying PlyC activity was found to contain 2.2 kb of sequence comprising 3 open reading frames (ORFs) and  ̃100 bp of untraslated region on both the 5′ and 3′ ends. This operon is called plyC (SEQ ID NO: 25). The first ORF contains the sequence for the PlyC light chain, PlyC-B (SEQ ID NO: 9), and its gene is called plyCB (SEQ ID NO: 22). The second ORF, positioned between the genes for the two chains of PlyC, contains a putative HNH endonuclease (SEQ ID NO: 10), suggestive of an intron, and hence its gene is called lil (SEQ ID NO: 24), for lysin intergenic locus. The third ORF contains the sequence for the PlyC heavy chain, PlyC-A (SEQ ID NO: 11), and its gene is called plyCA (SEQ ID NO: 23). (B) Expression of plyCB by itself contained no activity. (C) Expression of lil by itself contained no activity. (D) Expression of plyCA by itself contained no activity. (E) Expression of a chimera containing plyCB and plyCA possessed wild-type activity, indicating that lil is not necessary for formation of the active enzyme. (F) A double transformant, containing plyCB in pBAD33 (Crm+) and plyCA in pBAD24 (Amp+) contained PlyC activity, indicating that PlyC is composed of two separate gene products, PlyC-A and PlyC-B.
FIG. 3. Denaturation and refolding of PlyC subunits. Lane 1, activity of the native PlyC clone normalized to 100%. Lanes 2 and 3, individual activity of expressed PlyC-B and PlyC-A, respectively. Lane 4, activity of PlyC-B and PlyC-A mixed together, but expressed individually. Inactive samples from lane 4 were denatured in 8M urea and allowed to refold by dialyzing against decreasing concentrations of urea, and finally PBS, which reconstituted  ̃25% of the wild-type activity (lane 5).
FIG. 4. Crosslinking of PlyC. SDS-PAGE on a 4-20% gradient gel. Lane 1, native PlyC showing the 50 and 8 kDa subunits. Lane 2, PlyC treated with the non-cleavable crosslinker, BS3 (arrow shows the diffuse band  ̃100 kDa).
FIG. 5. PlyC activity on various streptococci. Representative streptococcal strains were exposed to 250 U of purified PlyC and the OD650 was monitored. The activity of PlyC for each strain was reported as the initial velocity of lysis, in OD650/min, based on the time it took to decrease the starting OD by half (i.e. from an OD650 of 1.0 to 0.5). All assays were performed in triplicate and the data are expressed as means±standard deviations.
FIG. 6. PlyC activity on Streptococcus uberis strains. Representative streptococcal strains were exposed to 250 U of purified PlyC and the OD650 was monitored. The activity of PlyC for each strain was reported as the % lysis that occurred in 3 minutes. S. uberis strains were clinical isolates or ATCC samples. Group A and C streptococci were control strains. All assays were performed in triplicate and the data are expressed as means±standard deviations
FIG. 7. PlyC activity on Streptococcus equi strains. Two S. equi strains and the control Group A streptococcus strain (S. pyogenes) were exposed to 250 U of purified PlyC and the OD650 was monitored. The activity of PlyC for each strain was reported as the initial velocity of lysis, in −OD650/min, based on the time it took to decrease the starting OD by half (i.e. from an OD650 of 1.0 to 0.5). All assays were performed in triplicate and the data are expressed as means±standard deviations.
FIG. 8. Luminometer assay shows PlyC can correlate Relative Light Units to colony forming units of Group A streptococci as effectively as detergent. Group A streptococci were serially diluted, 100 ul was aliquoted in the NHD microluminometer, and 50 U of PlyC lysin was added for 10 min, after which the luciferin/luciferase was added. Alternately, a bacterial releasing agent (detergent) was used to identify the total microbial ATP. The PlyC treatment consistently yielded  ̃50% of the total ATP and was linear (r2=0.912) down to  ̃300 organisms.
FIG. 9. Utility of PlyC as a diagnostic. Staphylococcus aureus, E. coli, and Streptococcus salivarius ( ̃106 organisms) or group A strep ( ̃104 organisms) were treated with 500 U of PlyC for 2 min and assayed in the microluminometer for a 10 sec integration. Mixing experiments contained the same proportion of CFU's.



DETAILED DESCRIPTION
Before the present methods and treatment methodology are described, it is to be understood that this invention is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only in the appended claims.
As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “the method” includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference in their entirety.
Definitions
“Treatment” refers to the administration of medicine or the performance of medical procedures with respect to a patient, for either prophylaxis (prevention) or to cure the infirmity or malady in the instance where the patient is afflicted.
The term “antibody” as used herein includes intact molecules as well as fragments thereof, such as Fab and F(ab′)2, which are capable of binding the epitopic determinant. The antibodies may be monoclonal, polyclonal, chimeric, humanized, or single chain antibodies, or fragments thereof. Antibodies that bind multimeric PlyC, or subunits or fragments thereof, can be prepared using intact dimers, polypeptides or fragments-containing small peptides of interest as the immunizing antigen attached to a carrier molecule. Commonly used carriers that are chemically coupled to peptides include bovine serum albumin and thyroglobulin. The coupled peptide is then used to immunize the animal (e.g, a mouse, rat or rabbit).
A “therapeutically effective amount” or “therapeutically effective dose” is an amount or dose sufficient to decrease, prevent or ameliorate the symptoms associated with the bacterial infection.
“Fragment” refers to either a protein or polypeptide comprising an amino acid sequence of at least 5 amino acid residues (preferably, at least 10 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 125 amino acid residues, at least 150 amino acid residues, at least 175 amino acid residues, at least 200 amino acid residues, or at least 250 amino acid residues) of the amino acid sequence of a parent protein or polypeptide, or a nucleic acid comprising a nucleotide sequence of at least 10 base pairs (preferably at least 20 base pairs, at least 30 base pairs, at least 40 base pairs, at least 50 base pairs, at least 50 base pairs, at least 100 base pairs, at least 200 base pairs) of the nucleotide sequence of the parent nucleic acid. Any given fragment may or may not possess a functional activity of the parent nucleic acid or protein or polypeptide.
“Analog” as used herein, refers to a nucleotide, a protein, or a polypeptide that possesses similar or identical activity or function(s) as the nucleotide, protein or polypeptide having the desired activity and therapeutic effect of the present invention (eg. having the ability to prevent or treat streptococcal infections or to aid in the diagnosis of streptococcal infections), but need not necessarily comprise a sequence that is similar or identical to the sequence of the preferred embodiment, such as that of SEQ ID NOS: 21 or 9 or 11, or possess a structure that is similar or identical to that of SEQ ID NOS: 21 or 9 or 11. As used herein, a nucleic acid or nucleotide sequence, or an amino acid sequence of a protein or polypeptide is “similar” to that of a nucleic acid, nucleotide or protein or polypeptide having the desired activity if it satisfies at least one of the following criteria: (a) the nucleic acid, nucleotide, protein or polypeptide has a sequence that is at least 30% (more preferably, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99%) identical to the nucleic acid, nucleotide, protein or polypeptide sequences having the desired activity as described herein (b) the polypeptide is encoded by a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence encoding at least 5 amino acid residues (more preferably, at least 10 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 125 amino acid residues, or at least 150 amino acid residues) of the AAPI; or (c) the polypeptide is encoded by a nucleotide sequence that is at least 30% (more preferably, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99%) identical to the nucleotide sequence encoding the polypeptides of the present invention having the desired therapeutic effect. As used herein, a polypeptide with “similar structure” to that of the preferred embodiments of the invention refers to a polypeptide that has a similar secondary, tertiary or quarternary structure as that of the preferred embodiment. The structure of a polypeptide can determined by methods known to those skilled in the art, including but not limited to, X-ray crystallography, nuclear magnetic resonance, and crystallographic electron microscopy.
“Derivative” refers to either a protein or polypeptide that comprises an amino acid sequence of a parent protein or polypeptide that has been altered by the introduction of amino acid residue substitutions, deletions or additions, or a nucleic acid or nucleotide that has been modified by either introduction of nucleotide substitutions or deletions, additions or mutations. The derivative nucleic acid, nucleotide protein or polypeptide possesses a similar or identical function as the parent polypeptide.
“Diagnosis” refers to diagnosis, prognosis, monitoring, characterizing, selecting patients, including participants in clinical trials, and identifying patients at risk for or having a particular disorder or clinical event or those most likely to respond to a particular therapeutic treatment, or for assessing or monitoring a patient's response to a particular therapeutic treatment.
A “variant” (v) of polynucleotides or polypeptides, as the term is used herein, are polynucleotides or polypeptides that are different from a reference polynucleotide or polypeptide, respectively. Variant polynucleotides are generally limited so that the nucleotide sequence of the reference and the variant are closely related overall and, in many regions, identical. Changes in the nucleotide sequence of the variant may be silent. That is, they may not alter the amino acid sequence encoded by the polynucleotide. Where alterations are limited to silent changes of this type a variant will encode a polypeptide with the same amino acid sequence as the reference. Alternatively, changes in the nucleotide sequence of the variant may alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Such nucleotide changes may result in amino acid substitutions, additions, deletions, fusions, and truncations in the polypeptide encoded by the reference sequence. Variant polypeptides are generally limited so that the sequences of the reference and the variant are that are closely similar overall and, in many regions, identical. For example, a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions, fusions, and truncations, which may be present or absent in any combination. Such variants can differ in their amino acid composition (e.g. as a result of allelic or natural variation in the amino acid sequence, e.g. as a result of alternative mRNA or pre-mRNA processing, e.g. alternative splicing or limited proteolysis) and in addition, or in the alternative, may arise from differential post-translational modification (e.g., glycosylation, acylation, phosphorylation, isoprenylation, lipidation).
A nucleic acid which is “hybridizable” to a nucleic acid of the present invention or to its reverse complement, or to a nucleic acid encoding a derivative, or to its reverse complement under conditions of low stringency can be used in the methods of the invention to detect the presence of a C1 bacteriophage gene and/or presence or expression level of a C1 gene product. By way of example and not limitation, procedures using such conditions of low stringency are as follows (see also Shilo and Weinberg, 1981, Proc. Natl. Acad. Sci. U.S.A. 78, 6789-6792). Filters containing DNA are pretreated for 6 h at 40° C. in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denatured salmon sperm DNA. Hybridizations are carried out in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20×106 cpm 32P-labeled probe is used. Filters are incubated in hybridization mixture for 18-20 h at 40° C., and then washed for 1.5 h at 55° C. in a solution containing 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution is replaced with fresh solution and incubated an additional 1.5 h at 60° C. Filters are blotted dry and exposed for autoradiography. If necessary, filters are washed for a third time at 65-68° C. and re-exposed to film. Other conditions of low stringency that may be used are well known in the art (e.g., as employed for cross-species hybridizations).
A nucleic acid which is “hybridizable” to an C1 bacteriophage nucleic acid (e.g., having a sequence as set forth in SEQ ID NO: 21 or to its reverse complement, or to a nucleic acid encoding a derivative thereof, or to its reverse complement under conditions of high stringency) is also provided for use in the methods of the invention. By way of example and not limitation, procedures using such conditions of high-stringency are as follows. Prehybridization of filters containing DNA is carried out for 8 h to overnight at 65° C. in buffer composed of 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA. Filters are hybridized for 48 h at 65° C. in prehybridization mixture containing 100 μg/ml denatured salmon sperm DNA and 5-20×106 cpm of 32P-labeled probe. Washing of filter is done at 37° C. for 1 h in a solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This is followed by a wash in 0.1×SSC at 50° C. for 45 min before autoradiography. Other conditions of high stringency that may be used are well known in the art.
A nucleic acid which is “hybridizable” to a C1 bacteriophage nucleic acid (e.g., having a sequence as set forth in SEQ ID NO: 21 or to its reverse complement, or to a nucleic acid encoding a derivative thereof, or to its reverse complement under conditions of moderate stringency) is also provided for use in the methods of the invention. For example, but not limited to, procedures using such conditions of moderate stringency are as follows: filters comprising immobilized DNA are pretreated for 6 hours at 55° C. in a solution containing 6×SSC, 5× Denhardt's solution, 0.5%. SDS and 100 μg/ml denatured salmon sperm DNA. Hybridizations are carried out in the same solution with 5-20×106 cpm 32P-labeled probe. Filters are incubated in hybridization mixture for 18-20 hours at 55° C., and then washed twice for 30 minutes at 60° C. in a solution containing 1×SSC and 0.1% SDS. Filters are blotted dry and exposed for autoradiography. Washing of filters is done at 37° C. for 1 hour in a solution containing 2×SSC, 0.1% SDS. Other conditions of moderate stringency that may be used are well known in the art. (see, e.g., Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; see also, Ausubel et al., eds., in the Current Protocols in Molecular Biology series of laboratory technique manuals, 1987-1997 Current Protocols,® 1994-1997 John Wiley and Sons, Inc.).
General Description
The C1 phage is currently classified as a member of the Podoviridae family of bacteriophage based on its physical appearance of short, non-contractile tails. This designation makes the C1 phage a tempting choice to sequence for several reasons. First, the Podoviridae represent a diverse set of phage where only a few sequenced genomes exist and even fewer have been studied in detail. Additionally, both the historical interest of the C1 phage and the current medical implications of its lysin warrant further investigation. Taken together, the C1 genome is an ideal candidate for genomic sequencing and characterization presented herein;
Accordingly, the present invention provides methods for the elucidation of the nucleic acid and protein sequence of C1 bacteriophage, which is specific for Group C streptococci, and the therapeutic and diagnostic uses of these nucleic acid and polypeptide sequences. The invention further provides for the full-length nucleic acid sequence of C1 bacteriophage, as set forth in SEQ ID NO: 21. More specifically, the C1 bacteriophage contains a double-stranded, linear sequence of DNA with 16,687 base pairs, and a G+C content of 34.6%. Also present are 143 base pair inverted terminal repeats. Furthermore, 20 open reading frames (orfs), have been identified and some of the functions of the polypeptides encoded by these nucleic acids have been determined. These are described herein in Table III as SEQ ID NOs: 1-20, with the first 11 on the positive strand and the remaining 9 on the negative strand. The majority of the ORF-encoded proteins are dissimilar to known phage proteins and have no homology to any proteins contained in GenBank. Further analysis of the bacteriophage C1 DNA revealed a terminal protein (TP) covalently linked to the 5′ terminus of the DNA.
Previous studies demonstrated that the lytic properties associated with an isolated lysin had therapeutic potential to eliminate streptococcal colonization (Nelson et al. (2001) Proc. Nat. Acad. Sci. USA. 98:4107-4112). The available sequence data now provides the means by which such therapeutic and diagnostic utility can be carried out. Accordingly, in a more particular aspect of the invention, the open reading frames associated with the two subunits of the PlyC (lysin) enzyme have been identified. The amino acid sequences of the two subunits have been elucidated and are described herein as SEQ ID NOs: 9 and 11. The DNA sequences encoding the light chain (plyCB) and heavy chain (plyCA) are set forth as SEQ ID NOs: 22 and 23, respectively. The PlyC orf9 codes for a protein that is 8 kDa in size (light chain) and the PlyC orf 11 codes for as protein that is 50 kDa in size (heavy chain). Furthermore, since the native protein has an apparent molecular weight of 100 kDa as measured by gel filtration, a further embodiment of the invention provides for a PlyC multimer composed of multiple copies of the heavy subunit (ORF 11), the light subunit (ORF 9), or both subunits; More preferably, this embodiment provides, but is not limited to, one heavy subunit (ORF 11) and 5 or 6 light subunits (ORF 9). The DNA sequences contained in the plyC operon (SEQ ID NO: 25) include both of the PlyC genes, plyCB and plyCA, as well as untranslated regions thought be involved in the transcriptional regulation. It is envisioned that the multimeric PlyC may be used for treating or preventing bacterial infections, comprising administration of a therapeutically effective amount of the PlyC multimer.
In a yet further embodiment of the invention, the PlyC multimer is prepared as a pharmaceutical composition with a pharmaceutically acceptable carrier for use in treating bacterial infections, including infections caused by pathogenic streptococci of Groups A, C and E, as well as S. uberis, and S. equi. It is envisioned that the composition comprising the multimeric PlyC may be useful in treating streptococcal infections in mammals, including, but not limited to, horses and cows. A further embodiment provides for use of the pharmaceutical compositions for treatment of bovine mastitis in cows and strangles in horses. A yet further embodiment includes the use of the pharmaceutical compositions comprising multimeric PlyC for treatment of humans. A yet further embodiment provides for the use of the pharmaceutical compositions for treatment of streptococcal pharyngitis.
A further aspect of the invention provides for the generation of antibodies specific for the PlyC multimer. In a preferred embodiment, the antibodies are monoclonal antibodies specific for the PlyC multimer, or subunits or fragments thereof. In a yet further embodiment, the antibodies are polyclonal antibodies prepared in mice, rats, guinea pigs, rabbits, goats, sheep, horses, pigs, cows, or any other mammal generally used in the art for generation of polyclonal antibodies. In another embodiment, the antibodies may be chimeric antibodies, humanized antibodies, single chain antibodies, or fragments thereof. A further embodiment provides for an immortal cell line that produces a monoclonal antibody that is specific for the multimeric PlyC or subunits or fragments thereof.
A yet further aspect of the invention provides for methods of diagnosing the presence of a pathogenic streptococcal infection. In a preferred embodiment, the binding domain of PlyC is labeled with a fluorescent chemical or protein and the labeled PlyC is then incubated directly with a sample taken from a subject suspected of harboring a pathogenic streptococci. The observation of fluorescence in the sample is indicative of the presence of a pathogenic streptococcal infection. A yet further embodiment provides for the use of the PlyC in a luciferin-luciferase assay to aid in diagnosing pathogenic streptococcal infections. In this assay format, a sample suspected of harboring a pathogenic streptococcus is incubated directly with the PlyC multimer. If the sample contains a pathogenic streptococcus, the PlyC will bind to the bacteria, resulting in lysis of the bacteria and subsequent release of ATP or other components normally present in the cytoplasm of the bacterial cell, such as enzymes. The lysate is then tested in a luciferin-lucifersase assay. In another embodiment, the sample suspected of harboring a pathogenic streptococcus may be added directly to the PlyC multimer concurrently with luciferin-luciferase without the need to collect the cell lysate first before adding it to the luciferin-luciferase. If a pathogenic streptococcus is present in the sample, the release of ATP from the lysed bacteria will trigger a positive reaction in the luciferin-luciferase system, resulting in release of measurable light from the reaction mixture.
Therapeutic Uses of the Invention
Another aspect of the invention provides for the use of the PlyC (lysin) multimer in treatment of bacterial infections or in prevention of bacterial cell growth in vitro and in vivo. One embodiment of the invention features use of the PlyC (lysin) multimer to treat infections caused by streptococci or to prevent growth of streptococci, in particular streptococci from group A, E or C as well as S. uberis, and S. equi. A further aspect of this invention provides for use of the PlyC lysin as a decontamination agent. A specific embodiment of this invention is to use PlyC to decontaminate milking equipment and teet dip cups of S. uberis. 
The invention provides for treatment or prevention of various diseases and disorders by administration of PlyC multimer. The administration of PlyC multimer would be by way of a pharmaceutically acceptable carrier. The administration of PlyC multimer may be by way of the oral cavity or it may be delivered parenterally. The PlyC multimer may be administered for use as an anti-infective and may be delivered topically, mucosally or sublingually. For systemic infections, it may be delivered intravenously, intramuscularly, or subcutaneously.
In a further embodiment, treatment of infections of the upper respiratory tract can be prophylactically or therapeutically treated with a composition comprising an effective amount of the PlyC multimer, and a carrier for delivering the PlyC multimer to a mouth, throat, or nasal passage. It is preferred that the PlyC multimer is in an environment having a pH that allows for activity of multimeric PlyC. If an individual has been exposed to someone with an upper respiratory disorder, the PlyC multimer will reside in the mucosal lining and prevent any colonization of the infecting bacteria.
Means of application include, but are not limited to direct, indirect, carrier and special means or any combination of means. Direct application of the PlyC multimer may be by nasal sprays, nasal drops, nasal ointments, nasal washes, nasal injections, nasal packings, bronchial sprays and inhalers, or indirectly through use of throat lozenges, or through use of mouthwashes or gargles, or through the use of ointments applied to the nasal nares, the bridge of the nose, or the face or any combination of these and similar methods of application. The forms in which PlyC may be administered include but are not limited to lozenges, troches, candies, injectants, chewing gums, tablets, powders, sprays, liquids, ointments, and aerosols.
The lozenge, tablet, or gum into which the PlyC multimer is added may contain sugar, corn syrup, a variety of dyes, non-sugar sweeteners, flavorings, any binders, or combinations thereof. Similarly, any gum based products may contain acacia, carnauba wax, citric acid, corn starch, food colorings, flavorings, non-sugar sweeteners, gelatin, glucose, glycerin, gum base, shellac, sodium saccharin, sugar, water, white wax, cellulose, other binders, and combinations thereof.
Lozenges may further contain sucrose, corn starch, acacia, gum tragacanth, anethole, linseed, oleoresin, mineral oil, and cellulose, other binders, and combinations thereof. In another embodiment of the invention, sugar substitutes are used in place of dextrose, sucrose, or other sugars.
The PlyC multimer may also be placed in a nasal spray, wherein the nasal spray is the carrier. The nasal spray can be a long acting or timed release spray, and can be manufactured by means well known in the art. An inhalant may also be used, so that the PlyC multimer may reach further down into the bronchial tract, including into the lungs.
Another composition and use of the PlyC multimer is for the therapeutic or prophylactic treatment of bacterial infections of burns and wounds of the skin. The composition comprises an effective amount of the PlyC multimer and a carrier for delivering the PlyC multimer to the wounded skin. The mode of application for the PlyC multimer includes a number of different types and combinations of carriers which include, but are not limited to an aqueous liquid, an alcohol base liquid, a water soluble gel, a lotion, an ointment, a nonaqueous liquid base, a mineral oil base, a blend of mineral oil and petrolatum, lanolin, liposomes, protein carriers such as serum albumin or gelatin, powdered cellulose carmel, and combinations thereof. A mode of delivery of the carrier containing the therapeutic agent includes but is not limited to a smear, spray, a time-release patch, a liquid absorbed wipe, and combinations thereof. The PlyC multimer may be applied to a bandage either directly or in one of the other carriers. The bandages may be sold damp or dry, wherein the PlyC multimer is in a lyophilized form on the bandage. This method of application is most effective for the treatment of burns.
In a further embodiment wherein a bacteriocidal activity is desirable, the PlyC multimer is administered alone or in combination with one or more additional therapeutic compounds or treatments. In a preferred embodiment, the PlyC multimer can be administered to a human subject for therapy (e.g. to ameliorate symptoms or to retard onset or progression) of bacterial infections. One embodiment provides for treatment of streptococcal pharyngitis. A further embodiment provides for the PlyC lysin to be administered to non-human mammals, including but not limited to horses and cows. One embodiment provides for treatment of bovine mastitis or strangles in horses. Specific embodiments provide for pharmaceutical compositions comprising the PlyC multimer for administration to human subjects or non-human mammals, including but not limited to cows and horses. It is also envisioned that one embodiment may provide for treatment of mammals, including human subjects and non-human mammals, suffering from streptococcal infections and who are not responsive to more traditional modes of anti-microbial therapy. It is also envisioned that the PlyC multimer may be used for decontamination purposes, such as to decontaminate milking, dairy, and agricultural equipment from streptococci. It is also envisioned that the PlyC multimer may be administered along with other lytic enzymes or with other antibiotics or anti-microbial forms of therapy.
The PlyC multimers for use in therapy can be tested in suitable animal model systems prior to testing in humans, including but not limited to rats, mice, chicken, cows, horses, monkeys, rabbits, etc. For in vivo testing, prior to administration to humans, any animal model system known in the art may be used. In one embodiment, PlyC multimers are tested in non-human animals (e.g., mice, rats, monkeys, rabbits, and guinea pigs), preferably non-human animal models for streptococcal infectious diseases. In accordance with this embodiment, PlyC multimer is administered to the animals, and the effect of the PlyC multimers on microbial levels is determined in the infected animal. Active PlyC multimers can be identified by comparing the level of bacteria in a culture obtained from an animal or group of animals treated with PlyC multimers with the level of the bacteria in a culture obtained from an animal or group of animals treated with a control nucleic acid or protein.
In yet another embodiment, test compounds that modulate the activity of PlyC multimers are identified in human subjects having an infection associated with streptococcal bacteria. In accordance with this embodiment, a test compound or a control compound is administered to the human subject in conjunction with the PlyC multimer, and the effect of a test compound on either reduction in spread of the microbial infection, elimination of the bacterial infection or amelioration of symptoms associated with the
Therapeutic and Prophylactic Compositions and Their Use
The invention provides methods of treatment comprising administering to a subject an effective amount of the PlyC multimer. In a preferred aspect, the PlyC multimer is substantially purified (e.g., substantially free from substances that limit its effect or produce undesired side-effects). The subject is preferably an animal, including but not limited to animals such as monkeys, cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human. In one specific embodiment, a non-human mammal is the subject. In another specific embodiment, a human mammal is the subject.
Various delivery systems are known and can be used to administer the PlyC multimer, e.g., encapsulation in liposomes, microparticles, or microcapsules. Methods of introduction can be enteral or parenteral and include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, topical and oral routes. The compounds may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. In a specific embodiment, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment, such as topical use on the skin; any suitable method known to the art may be used.
Another aspect of the invention provides for pharmaceutical compositions comprising the PlyC multimer for therapeutic use in treatment of bacterial infections. Moreover, a further embodiment may include a pharmaceutical composition designed for use in topical treatment of bacterial infections. Another embodiment may include a pharmaceutical composition designed for use in treatment of systemic infections, or infections that are non-responsive to other antibiotic modalities.
Such compositions comprise a therapeutically effective amount of an agent, and a pharmaceutically acceptable carrier. In a particular embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier”refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositions will contain a therapeutically effective amount of the nucleic acid of C1 bacteriophage or PlyC multimer, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject. The formulation should suit the mode of administration.
In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
The amount of the PlyC multimer which will be effective in the treatment of infectious diseases, can be determined by standard clinical techniques based on the present description. In addition, its vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each subject's circumstances. However, suitable dosage ranges for intravenous administration are generally about 20-500 micrograms of active compound per kilogram body weight. Suitable dosage ranges for intranasal administration are generally about 0.01 pg/kg body weight to 1 mg/kg body weight. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects (a) approval by the agency of manufacture, use or sale for human administration, (b) directions for use, or both.
In a specific embodiment, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment; this may be achieved, for example, and not by way of limitation, by local infusion during surgery, by topical application, by injection, by means of a catheter, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers or co-polymers such as Elvax (see Ruan et al, 1992, Proc Natl Acad Sci USA, 89:10872-10876). In one embodiment, administration can be by direct injection by aerosol inhaler.
In another embodiment, the PlyC multimer can be delivered in a vesicle, in particular a liposome (see Langer (1990) Science 249:1527-1533; Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)
In yet another embodiment, the PlyC multimer can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al. (1980) Surgery 88:507; Saudek et al. (1989) N. Engl. J. Med. 321:574). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, N.Y. (1984); Ranger and Peppas, J. (1983) Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al. (1985) Science 228:190; During et al. (1989) Ann. Neurol. 25:351; Howard et al. (1989) J. Neurosurg. 71:105). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, i.e., the airways, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release (1984) supra, vol. 2, pp. 115-138). Other suitable controlled release systems are discussed in the review by Langer (1990) Science 249:1527-1533.
Diagnostic Uses
A further aspect of the invention provides for methods of diagnosing a pathogenic streptococcal infection. In a preferred embodiment, the binding domain of PlyC is labeled with a fluorescent chemical or protein and the labeled PlyC is then incubated directly with a sample taken from a subject suspected of harboring a pathogenic streptococci. The observation of fluorescence in the sample is indicative of the presence of a pathogenic streptococcal infection. A yet further embodiment provides for the use of the PlyC in a luciferin-luciferase assay to aid in diagnosing pathogenic streptococcal infections. In this assay format, a sample suspected of harboring a pathogenic streptococcus is incubated directly with the PlyC multimer. If the sample contains a pathogenic streptococcus, the PlyC multimer will bind to the bacteria, resulting in lysis of the bacteria and subsequent release of ATP or other components normally present in the cytoplasm of the bacterial cell, such as enzymes. The lysate is then tested in a luciferin-luciferase assay. In another embodiment, the sample suspected of harboring a pathogenic streptococcus may be added directly to the PlyC multimer concurrently with luciferin-luciferase without the need to collect the cell lysate first before adding it to the luciferin-luciferase. If a pathogenic streptococcus is present in the sample, the release of ATP from the lysed bacteria will trigger a positive reaction in the luciferin-luciferase system, resulting in release of measurable light from the reaction mixture. In another embodiment, any other cytoplasmic markers, enzymes, proteins, cell wall fragments, or carbohydrates liberated by PlyC from streptococci could also be detected by any methodologies common to the diagnostic art.
Alternatively, one embodiment of the invention may use an antibody to the PlyC multimer or subunits or fragments thereof, or antibodies may be prepared to any of the proteins listed in Table III as SEQ ID NOs: 1-20, and the antibody may be labeled (eg. with fluorescein or other known fluorescent proteins or chemicals), coupled to the bacteriophage protein and used to monitor binding of the specific protein to the bacteria in a patient sample, thus aiding in detection of pathogenic streptococci. Alternatively, any one of the proteins from Table III may be fluorescein labeled directly and used to detect the presence of pathogenic streptococci in a patient sample.
Further diagnostic testing formats, including ELISA assays or radioimmunoassays may also be contemplated for use with the present invention. In these formats, one can utilize the proteins identified in Table III directly or one may prepare antibodies to these proteins as noted herein for use in kits to monitor the presence of pathogenic streptococci in a patient sample. The procedures for ELISA or radioimmunoassays are known to those skilled in the art.
A further aspect of the invention provides for a method of diagnosing a pathogenic streptococcal infection, comprising:

    a) collecting a patient sample suspected of harboring a streptococcus; 
    b) contacting the sample with a fluoresceinated PlyC multimer; and
    c) measuring the amount of fluoresceinated multimer bound to the sample, wherein the detection of binding indicates the presence of streptococci in the sample.


A yet further aspect of the invention provides for a method for detecting the presence of streptococci in a sample, comprising:

    a) collecting a patient sample suspected of harboring a streptococcus; 
    b) incubating the sample with the PlyC multimer;
    c) collecting the cell lysate;
    d) incubating the cell lysate with luciferin-luciferase; and
    e) measuring the amount of light produced, wherein an increase in the amount of light produced is indicative of the presence of streptococci in the sample.


A yet further aspect of the invention provides for a method for detecting the presence of streptococci in a sample, comprising:

    a) collecting a patient sample suspected of harboring a streptococcus; 
    b) incubating the sample in the presence of luciferin-luciferase along with the PlyC multimer; and
    c) measuring the amount of light produced, wherein an increase in the amount of light produced is indicative of the presence of streptococci in the sample.


A yet further aspect of the invention provides for generation of antibodies to the PlyC multimer or subunits or fragments thereof. The antibodies may be polyclonal, monoclonal, chimeric, humanized, or single chain antibodies. They may be prepared in animals such as mice, rats, guinea pigs, rabbits, goats, sheep, horses, and pigs. These antibodies may be used for identification and isolation of the components of the streptococcal cell wall to which the PlyC multimer binds. An additional use of these antibodies may be for mobilizing the PlyC multimer to a Biacore chip to perform studies on the affinity or kinetics of binding of the PlyC multimer to its binding site on the streptococcal cell wall.
A yet further aspect of the invention would be to use the PlyC multimer to lyse the streptococcus in the infection, which will release the DNA of the streptococcus. This released DNA can then be utilized for PCR analysis to identify the streptococcus. A more particular embodiment of the invention is a method for detection of pathogenic streptococci in a sample, comprising:

    
    
        a) collecting a sample from a patient suspected of having a streptococcal infection;
        b) adding the PlyC multimer into the sample until lysis of bacteria is observed;
        c) isolating the DNA from the lysed bacteria;
        d) utilizing the isolated DNA for preparation of a probe which can be utilized for analysis and identification of the presence of streptococcus in a patient sample.
    
    


EXAMPLES
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to isolate and use the nucleic acid of C1 bacteriophage or the PlyC multimer or the other proteins and nucleic acids described herein, and to provide a suitable means for development of pharmaceutical compositions for therapeutic or diagnostic use, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.
Example 1
Determination of the Genomic Sequence of C1 Bacteriophage
Preparation and Purification of Phage
The lytic bacteriophage, C1, and its host bacteria, group C streptococcus 26RP66, are both part of The Rockefeller University collection. For preparation of the C1 bacteriophage, 26RP66 was grown at 37° C. in chemically defined media (CDM) for streptococci (JRH Biosciences) (27.13 g/L) and supplemented with 2.5 g/L sodium bicarbonate and 0.5 g/L cysteine. During early log phase (OD650 ̃0.25) 1/10 to 1/2 (v/v) of pre-warmed C1 phage was added and allowed to incubate until complete lysis occurred (approximately 40 minutes). The lysate was clarified by centrifugation (10,000×g, 10 min), passed through a 0.45-micron filter (Amicon) and final phage purification was achieved by ultracentrifugation (100,000×g, 2 hr) with the phage pellet resuspended in PBS and stored at 4° C.
Purification of Phage DNA
To purified phage particles, RNase and DNase (10 μg each) was added and allowed to incubate for 30 min at 37° C., after which, 50 μl of 0.5 M EDTA was supplemented to inhibit the endonucleases. Protease K (200 μg) and SDS (0.5% final concentration) were added and the mixture and incubated for 1 hr at 65° C. Final DNA purification was achieved through common phenol/chloroform extraction protocols as previously described (Sambrook, J. et al. (1989) Cold Spring Harbor Laboratory Press, New York).
Receptor Studies
For C1 infection studies, phage were added to an exponential growth of group C streptococci or group A-variant streptococci and monitored for either a clearing of a liquid culture as described above or plaque formation in a soft agar overlay. Alternately, group C streptococci were pretreated with pronase (100 μg/ml) or trypsin and chymotrypsin (100 μg/ml, each) for 30 minutes prior to exposure to the C1 phage. For adsorption studies, group C cell walls were isolated as described (Fischetti et al. (1968) J. Exp. Med. 127:489-505), and the group C carbohydrate was isolated by the nitrous acid extraction method as described (Swanson, J. et al. (1969) J. Exp. Med. 130:1063-1091) Briefly, 100 μl of 108 pfu/ml C1 phage was mixed with isolated group C cell walls (5 mg/ml), group C carbohydrate (5 mg/ml), or 20 mM GalNAc in a final volume of 0.5 ml. After 10 min incubation at 37° C., log phase group C streptococci were added to a final volume of 1.0 ml, incubated 5 min, centrifuged to pellet the streptococci, and plated according to the soft agar layer technique. Adsorption is quantified a corresponding decrease in residual PFU/ml.
Terminal Protein Studies
To purify the DNA-protein complex, the same procedure used to purify the phage DNA from above was followed except a phenol extraction step with gentle shaking was used instead of multiple phenol/chloroform extractions. The DNA-protein complex, found at the interface between the aqueous and phenol layer, was extracted and precipitated by ethanol. Half of the DNA-protein complex was digested with Protease K (10 μg) for 30 min at 37° C. and re-purified by ethanol precipitation. Aliquots (10 μg each) of the DNA-protein complex or Protease K digested DNA (PK-DNA) were treated with either 2 μl (130 U) Exonuclease III or 2 μl (11 U) Lambda exonuclease (both from GibcoBRL), at 37° C. according to the manufacturer's instructions. Reactions were stopped with the addition of 10 mM EDTA at the indicated times prior to electrophoresis. Alternatively, PK-DNA (10 μg) was pretreated with 0.5 M piperidine for 2 h at 37° C. and then subjected to Lambda exonuclease treatment.
DNA Sequencing
For the library construction, genomic DNA was hydrodynamically sheared in an HPLC and separated on a 1% agarose gel. 3000-3500 bp fragments were excised, purified by the GeneClean procedure (Bio101, Inc.), blunt-ended using T4 DNA polymerase and ligated to unique BstX1-linker adapters. The linker-adapted inserts were separated from the unincorporated linkers by a second gel purification using GeneClean and ligated to BstX1-cut vector to form subclone libraries which were transformed into DH10β competent cells (Gibco/BRL, DH5α transformation protocol). DNA was purified from positive transformants using the PerfectPrep384® system (Brinkmann Instruments) and then sequenced using ABI dye-terminator chemistry on automated MegaBaceTM 1000 (Amersham) machines. Base calls and quality scores were determined using the program PHRED (Ewing, B. et al. (1998) Genome Res. 8:186-194). Reads were assembled using PHRAP with default program parameters and quality scores. Closure of gaps was accomplished by using primer-directed sequencing directly from purified phage DNA.
Bioinformatics
The LASERGENE suite of programs from DNASTAR was used for analysis, annotation, and assembly of the nucleotide and amino acid sequences. Putative open reading frames (ORFs) were identified by either the ORF Finder, available through the National Center for Biotechnology Information (http://www.ncbi/nlm.nih.gov), or the heuristic approach of gene prediction from GeneMarkTM (Besemer, J. et al. (1999) Nucleic Acid Res. 27:3911-3920). The BLAST algorithms, (Altschul, S. F. et al. (1997) Nucleic Acid Res. 25:3389-3402) also available through NCBI, were used for similarity searches of putative ORFs. Sequence alignments were performed with CLUSTAL W and visualized with BOXSHADE.
Electron Microscopy
Purified phage were applied to a carbon film and fixed to a copper grid before being negatively stained with 1% uranyl acetate. Electron micrographs of the phage were taken using a JEOL 100 CXII transmission electron microscope. For phage infection images, C1 bacteriophage were incubated with early log phase group C streptococci 26RP66 for 20 minutes, pelleted by centrifugation, and suspended in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4). The samples were then postfixed in 1% osmium tetroxide, block stained with uranyl acetate and processed according to standard procedures. All microscopy was performed by The Rockefeller University Bio-Imaging Resource Center or in collaboration with Dr. John Swanson. The electron microscopy studies demonstrated the binding of C1 bacteriophage to the cell wall of a group C streptococcus at 15 minutes post infection. The electron opaque capsid indicated that the phage DNA had not yet been injected. The honeycomb structure showed the progeny virions.
Nucleotide Sequence Accession Number
The DNA sequences of the genome reported herein appear in GenBank under accession number AY212251.
Results
C1 Bacteriophage Characterization
In agreement with a previously published report (Moynet, D. J. et al. (1985) Virology 142:263-269), the C1 phage has a small polyhedral head ( ̃50×50 nm) and a very short tail and tail fibers. A small collar or base plate was noted with three protruding appendages. The wider and longer central appendage is presumed to be the tail, but it is not clear if the side appendages are minor tail fibers similar to those seen in phage T7 or P22, or collar spikes as observed in the φ29 phage (Ackerman, H. et al. (1999), Adv. Vir. Res. 51: 135-201). Upon infection, complete lysis of susceptible group C streptococci was achieved by 40 minutes; however, mature phage particles could be observed emerging from infected streptococci by electron microscopy as soon as 10 minutes post infection (data not shown). We were able to manually count>100 progeny particles in one thin section micrograph of an infected streptococcus, which is consistent with a relatively high burst size noted previously (Fischetti, V. A. et al. (1968) J. Exp. Med. 127:475-488)
Elucidation of the C1 Phage Receptor
Consistent with previous reports (Krause, R. M. (1957) J. Exp. Med. 106:365-384), it was determined that only group C streptococci were susceptible to infection by the C1 bacteriophage. When these same streptococci were pretreated with pronase or trypsin and chymotrypsin, no inhibition of infection was noted indicating that the binding receptor is not of proteinaceous origin (Table I). The surface carbohydrate of group C streptococci is composed of a polyrhamnose backbone with side chains of two N-acetyl-galactosamine (GalNAc) residues whereas the group A-variant carbohydrate contains only the polyrhamnose backbone (Coligan, J. E. et al. (1978) Immunochemistry 15:755-760). Therefore, our data demonstrating that the C1 phage is unable to infect group A-variant streptococci implicates GalNAc as a potential phage receptor.
For adsorption studies, we found that isolated group C streptococcal cell walls were very efficient in adsorbing the C1 phage, reducing 107 pfu/ml to less than 102 pfu/ml (Table II). Additionally, we found that the chemically extracted group C carbohydrate also retained the ability to adsorb C1 phage. This is in contrast to Fischetti and Zabriskie's earlier findings (Fischetti, V. A. (1968) J. Exp. Med. 127:489-505); however a method of nitrous acid extraction of the carbohydrate layer was used, which is more efficient than the hot formamide method utilized by the former authors. Finally, when C1 phage were treated in the presence of 20 mM GalNAc monosaccharide, no adsorption was observed, strongly suggesting that the disaccharide rather than the monosaccharide of GalNAc, serves as the receptor for the C1 bacteriophage, although we cannot rule out possible contributions of other epitopes to the infection process at this time.
Organization of the C1 Genome and Identification of ORFs
A double stranded, linear sequence of 16,687 bp was established with a mean redundancy of six, with each region being sequenced a minimum of at least once on each strand. A G+C content of 34.6% is similar to the host and other “low GC” streptococci. 200 bp inverted terminal repeats were present which are characteristic of the “φ29-like” Podoviridae (Ackermann, H. W. (1999) Adv. Virus Res. 51:135:201).
The criteria for the characterization of a potential open reading frame (ORF) were the existence of a start codon (ATG, GTG, or TTG) and a minimum coding size of 50 amino acids. Using these criteria, 20 predicted ORFs were identified by both ORF Finder and GeneMarkTM, labeled 1-20 from the left end of transcription (Table III). The first eleven ORFs are on the positive strand and the remaining 9 ORFs are all on the negative strand. Unexpectedly, the majority of ORFs were not only dissimilar to known phage proteins, but had no homology to any proteins contained in GenBank. Therefore, we only assigned putative function to ORFs with significant homology or experimental proof. Included in this group are the following:

    
    
        (i) Orf6. Although orf6 did not have a high E value (1.2), it did have  ̃20% identity to neck appendage proteins (late protein GP12) from Bacillus phages φ29 and PZA.
        (ii) Orf7. Orf7 had high homology to DNA polymerases from Bacillus phages φ29 and GA-1. Significantly, these phage polymerases utilize a protein-primed mechanism of replication (see below for evidence of a terminal protein).
        (iii) Orf8. Orf8 is a putative holin with similarity to a Listeria prophage holin and the Bacillus φ-105 phage holin. Additionally, with 108 amino acids and 3 predicted transmembrane domains, this sequence fits the classic type I holin, as do holins from the φ-29, φ-105, and Cp-1 Podoviridae (Wang, I. N. et al. (2000) Annu. Rev. Micrbiol. 54:799-825.
        (iv) Orfs 9,10, and 11. The 72 amino acid orf9 has no homology to any known protein. Yet, sequencing of the purified C1 lysin yielded an N-terminal sequence that corresponded to Orf9 (data not shown). However, the native C1 lysin has a predicted MW of  ̃100 kDa, significantly larger than ORF9 (Nelson, D. et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98:4107-4112). This may be explained by investigating ORFs 10 and 11. ORF 10 has noteworthy homology to the HNH family of homing endonucleases found in many phages (specifically, LambdaSa2 from Streptococcus agalactiae and bIL170 from Lactococcus spp.). These endonucleases are often part of bacteriophage intron systems that give rise to modular enzymes. Orf11 has the highest identity with a putative amidase (lysin) from the LambdaSa1 phage infecting group B streptococci. However, the LambdaSa1protein is over 1200 amino acids and the “amidase” region comprises less than 100 amino acids, none of which share identity with the orf11 gene product. The remaining 1100 amino acids of the LambdaSa1 protein resemble a phage tail protein. As such, the C1 orf11 also has high homology to a putative tail protein from phage 315.5 infecting group A streptococci. Although no typical lysin or amidase regions are present on either orf9 or 11, it is inviting to speculate that if orf10 is an intron, gene products of orf9 and 11 could be spliced together to form an active lysin. The combined size of Orfs9 and 11 would approximate the size of the native C1 lysin, and remarkably, introns have been found in the middle of modular lysin genes for multiple streptococcal phage (Foley, S. et al. (2000) J. Virol. 74:611-618). Work is in progress to elucidate the exact interactions between Orfs 9, 10, and 11 to yield a functional lysin.
        (v) Orf12. Orf12 is the major tail protein based on homology to the GP9 protein from Bacillus phage in the Podoviridae family (B103, φ29, and GA-1).
        (vi) Orf15. Orf15 is a head tail connector (collar) protein based on homology to the GP10 protein from Bacillus phage (GA-1 and φ29).
        (vii) Orf16. Orf16 does not have homology to known proteins. Yet, we experimentally determined this 44-kDa protein to be the major structural protein (see below) and believe it is the head or capsid protein.
    
    


C1 Phage Structural Proteins
In order to examine C1 structural proteins, purified phage particles were subjected to SDS-PAGE. Although several bands could be distinguished, two notable bands comprised>90% of the visualized protein. N-terminal sequencing of the two most prominent bands revealed the 44 kDa and 36 kDa bands correspond to orfs16 and orf15, respectively. Whereas orf15 is consistent with head/tail connector base plate proteins from several phage systems, orf16 does not share homology with any known proteins. N-terminal sequencing of the smaller, 36-kDa band gave the sequence MQITSGIK (amino acids 1-8 of SEQ ID NO: 15), which corresponds to Orf15 (SEQ ID NO: 15), a putative 35.9 kDa protein with significant homology to the upper collar proteins (GP-10) from Bacillus phages GA-1, B103, and φ29 (Table III). The larger and more abundant 44-kDa protein had an N-terminal sequence of ADETTNVA (amino acids 1-8 of SEQ ID NO: 16). This sequence corresponds to Orf16 (SEQ ID NO: 16), a putative 43.7-kDa protein that does not share similarity with any known protein in GenBank. Because this band accounts for  ̃75% of the total phage structural proteins by scanning densitometry, we believe that it represents the major capsid or head protein despite any homology with similar proteins. This is partially supported by a lack of an identified capsid protein in Table III, but presence of other expected structural proteins such as neck appendage (Orf6), major tail (Orf12), and collar protein (Orf15).
Evidence of a Covalently Linked 5′ Terminal Protein
Bacteriophages that utilize a protein-primed mechanism of replication have a terminal protein (TP) covalently linked to the 5′ termini of the DNA. One characteristic of this DNA-protein complex is a noted lack of migration in a standard agarose gel (Garcia, E., et. al (1983) Virology 128:92-104). We observed this trait for the C1 bacteriophage DNA (data not shown). The migrating band presumably represents DNA that had the TP sheared off during the phenol step in purification. Protease K (PK) treatment of the complex results in complete conversion of all non-migrating complex to migrating DNA with a size of  ̃17 kb. Exonuclease III, which is specific for unblocked 3′ termini, degrades both the DNA-protein complex and PK-DNA. Although we demonstrate that the 3′ termini is free in both the DNA-protein complex and the PK-DNA, Exonuclease III has slower activity on the complex, most likely due to steric hindrance of the TP near the 3′ termini.
In contrast to Exonuclease III, both the DNA-protein complex and the PK-DNA are insensitive to the effects of the 5′ specific Lambda exonuclease. However, pretreatment of the PK-DNA with 0.5 M piperidine, which has been shown to specifically hydrolyze the bond between the DNA and TP in the φ29 bacteriophage (Penalva, M. S. et. al (1982) Proc. Natl. Acad. Sci. U.S.A. 79:5522-5526) renders the 5′ termini unprotected.
Analysis of the DNA Polymerase
The data noted above confirms that the C1 bacteriophage DNA contains protein covalently linked to the 5′ termini. Further evidence supporting the existence of a terminal protein can be found by examination of the putative C1 DNA polymerase. Bioinformatic analysis suggests the C1 polymerase belongs to family B of DNA polymerases (also referred to as eukaryotic or α-like), which comprises eukaryotic, viral, and protein-primed polymerases. This family has consensus sequences known to be important for proofreading and strand displacement functions (ExoI, ExoII, ExoIII, cross-talk) as well as several consensus motifs (A,B,C) involved in initiation and polymerization. Significantly, polymerases that utilize a protein-primed mechanism of replication have two insertion motifs, called terminal protein regions (TPR), which lie between motif A and B (TPR-1) or motif B and C (TPR-2) (Blasco, M. A. et al. (Nucleic Acid Res. (18:4763-4770). The C1 polymerase contains all necessary conserved elements, including both TPR regions.
Example 2
Cloning of PlyC
Bacterial strains, phage, and growth conditions. Streptococcus pyogenes D471 (group A strep), Streptococcus equisimilis 26RP66 (group C strep), and the C1 bacteriophage are part of the Rockefeller University collection and were grown and maintained as previously described (Nelson, D., et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98:4107-4112; Nelson, D., et al. (2003) J. Bacteriol. 185:3325-3332). E. coli XL-1 Blue (Stratagene) and E. coli B834 (Novagen) were grown in Luria-Bertani (LB) broth at 37° C. in a shaking incubator (250 r.p.m.) unless otherwise stated. When needed, ampicillin (100 μg ml−1) or chloremphenical (34 μg ml−1) was added to the growth medium.
DNA manipulation. Phage DNA was isolated as previously described (Nelson, D., et al. (2003) J. Bacteriol. 185:3325-3332) and plasmid DNA was isolated using the Qiaprep kit (Qiagen). DNA polymerase, restriction, and modification enzymes were all purchased from New England Biolabs and used according to the manufactures instructions. Oligonucleotides were obtained from Sigma Genosys and all PCR was performed with the Eppendorf Mastercycler.
Cloning plyC. 5 μg aliquots of C1 phage DNA were digested with Tsp509I and fragments ranging in size from 0.5-3.0 kb were cloned into the EcoRI site of the arabinose-inducible expression vector pBAD24 (Guzman, L. M., et al. (1995) J. Bacteriol. 177:4121-4130). The resulting expression library was then transformed into E. coli XL1-Blue and screened for lytic activity on glass LB plates containing 100 μg ml−1 ampicillin and 0.25% arabinose. The induced library was permeabilized with chloroform vapors and overlaid with exponential phase Streptococcus pyogenes D471 in 0.75% LB agar. After 4 h incubation, distinct clearing, or lytic, zones were identified over library members. Corresponding plasmid DNA was prepared and sequenced at the Rockefeller University DNA Sequencing Resource Center. DNA sequence analysis and manipulations required the BLASTP (NCBI), ORF finder (NCBI), and SeqMan 5.0 (Dnastar Inc.) programs.
Biochemical techniques. PlyC was induced from XL1-Blue/pBAD24::plyC with 0.5% arabinose in overnight LB cultures. Cells were washed in 20 mM phosphate buffer at pH 7.0, and lysed with chloroform to yield crude PlyC. Purification of the recombinant enzyme and the plate assay to follow activity were identical to previously described methods used for the phage-produced enzyme (Nelson, D., et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98:4107-4112). The purified enzyme was routinely stored in phosphate buffered saline (PBS) at 4° C. and was stable for several months. Purification of the individually expressed heavy and light chains utilized the same column chromatography methods as the native enzyme, but purification was followed by SDS-PAGE rather than activity assays. Native and SDS-PAGE analysis were performed according to the method of Schagger (Schagger, H., et al. (1987) Anal. Biochem. 166:368-379) and blotting to polyvinylidene difluoride membranes was according to Matsudaira (Matsudaira, P. (1987) J. Biol. Chem. 262:10035-10038). N-terminal sequencing was performed at Rockefeller University Proteomics Resource Center. For the denaturation and refolding studies, urea was added to 100 μg each of PlyC-A and PlyC-B until the final concentration was 8M. Urea was slowly removed over 72 hours by successive dialysis against 6M, 4M, 2M urea, and finally PBS. Standard activity assays, normalized to control PlyC, were performed on equal masses of PlyC-B, PlyC-A, a mixture of heavy and light chains before denaturation, and after refolding. In the cross-linking experiments, we employed bis(sulfosuccinimidyl)suberate (BS3), a water soluble, non-cleavable, homo-bifunctional cross-linker with a chain length of 11.4 Å and reactivity toward amino groups (purchased from Pierce). A fresh stock of BS3 (1 mg ml−1) was made in 20 mM phosphate buffer, pH 7.4, and 55 μl of this solution (5 μM final concentration) was added to 100 μg of purified PlyC in a final volume of 1 ml and allowed to react for 45 minutes at room temperature. The reaction was quenched by the addition of 25 mM Tris for 15 min at room temperature and then SDS-PAGE reduced sample buffer. Analytical gel filtration for size estimation of the native and the cross-linked enzymes employed a Superose 12 column (Amersham Biosciences) calibrated with gel filtration standards (Bio-Rad).
Expression of individual ORFs, chimeras, and co-transformants. Plasmid DNA was purified from XL1-Blue/pBAD24::plyC and individual ORFs were amplified by PCR as follows: for the light chain (plyCB), the primers Light-F (5′-GTACCCGGGGAAGTAATTTCCATTCTTGAA-3′) (SEQ ID NO: 26) and Light-R (5′-CCCAAGCTTTTACTTTTTCATAGCCTTTCT-3′) (SEQ ID NO: 27); for the intergenic region (lil), the primers LIL-F (5′-GTACCGGGGAGGAGGAATTCATGATTGAGGAGTGGGTC-3′) (SEQ ID NO: 28) and LIL-R (5′-GGGAAGCTTTTACTCATTAAATAAATTCTCCCTTTC-3′) (SEQ ID NO: 29); and for the heavy chain (plyCA), the primers Heavy-F (5′-GTACCCGGGAAAGGGAGAATTTATTTAATG-3′) (SEQ ID NO: 30) and Heavy-R (5′-CCCAAGCTTTGGGTTCAATTCAAGGGAATA-3′) (SEQ ID NO: 31). All forward primers contained a SmaI site and all reverse primers contained a HindIII site. PCR products were digested by SmaI and HindIII and ligated into a SmaI/HindIII digested pBAD24 (Ampr) vector or the pBAD33 (Crmr) vector. For the PlyC heavy chain and light chain chimera, plyCB was PCR amplified by Light-F and Light-R primers, blunt ended, and ligated to pBAD24::plyCA which had previously been linearized by digestion with SmaI. A 1.6 kb PCR product using the primers Light-F and Heavy-R confirmed the proper insert size for this chimera. Finally, XL1-Blue cells were co-transformed with pBAD24::plyCA and pBAD33::plyCB, and cells displaying a Cmr/Ampr phenotype were selected for further study. Positive PCR products were obtained using either primer sets Light-F/Light-R or Heavy-F/Heavy-R, but no product was observed using Light-F/Heavy-R, thus verifying that plyCB and plyCA were on separate plasmids.
35S-methionine labeling of PlyC. E. coli B834 cells were transformed with pBAD24::plyC and methionine auxotrophy was confirmed by observing growth in M9 minimal media supplemented with 50 μg ml−1 methionine, while no growth was noted in M9 media alone. A fresh 20 ml overnight culture of B834/pBAD24::plyC was washed twice with M9 media, and used to inoculate a 2 L flask of M9 minimal media supplemented with 2 mCi 35S-methionine (New England Nuclear). Expression of 35S-Methionine-PlyC was induced with 0.5% arabinose and the enzyme was purified as described above. The heavy and light chains were resolved by SDS-PAGE using 15 μg of labeled PlyC loaded each lane (n=20). Individual bands were cut out of the gel, suspended in 1 ml of SigmaFluor scintillation fluid (Sigma), and counts per minute (cpm) of β-activity was assessed on a Beckman LS5000TD counter.
Results
PlyC is composed of 2 sub-units. In 2001, we reported that the streptococcal C1 bacteriophage lysin, now termed PlyC, was  ̃50 kDa by SDS-PAGE (Nelson, D., et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98:4107-4112). Nonetheless, we did note that gel filtration of the enzyme suggested a mass of  ̃100 kDa, which was consistent with previous reports (Fischetti, V. A., et al. (1971) J. Exp. Med. 133:1105-1117). At the time, it was speculated that the native enzyme exists as a homodimer or possesses additional subunits that could not be visualized. Here we have purified larger quantities of phage produced PlyC, as well as a recombinant PlyC, and shown that the enzyme behaves as a homogeneous preparation on native gel electrophoresis (FIG. 1A). Moreover, this band is responsible for the lytic activity as observed on an overlay of streptococci-embedded agarose (FIG. 1B). However, an SDS-PAGE of the same material revealed the presence of two bands, a 50 kDa heavy chain and an 8 kDa light chain (FIG. 1C), neither of which retain any lytic activity (data not shown). Furthermore, N-terminal sequencing of the heavy and light chains resulted in two unique sequences, SKKYTQQQYE (amino acids 9-18 of SEQ ID: 11) and SKINVNVENV (amino acids 2-11 of SEQ ID: 9), respectively, and sequencing of the native enzyme resulted in a double sequence, which corresponds exactly to both chains. Thus, we conclude that PlyC is composed of at least one heavy chain, which we now call PlyC-A, and at least one light chain, which we now call PlyC-B.
In the 2001 manuscript (Nelson, D., et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98:4107-4112), we never observed the light chain in an SDS-PAGE, however, this can be explained by several factors. In the original publication, a 10% polyacrylamide gel was used for electrophoresis, which allowed the 8 kDa light chain to migrate in the dye front and was subsequently not detected. Additionally, we were only able to purify ng quantities of enzyme necessitating the need for silver staining, which is now known not to effectively label the 8 kDa light chain. In this report, we utilized a 4-20% gradient gel, which allows for visualization of proteins<10 kDa. Finally, we purified sufficient quantities of enzyme to stain the gel in FIG. 1 by Coomassie stain, which does resolve the 8 kDa band.
Cloning and sequencing the PlyC gene(s). To further investigate PlyC, we attempted to identify the gene(s) responsible for its activity in an expression library of the C1 bacteriophage genome. Screening a Tsp509I expression library revealed only a single clone containing a 2.2 kb insert (SEQ ID: 25) possessed lytic activity toward group A streptococci. This insert included three open reading frames (ORFs) in addition to  ̃100 bp of non-coding sequence on both the 5′ and 3′ ends (FIG. 2A). Significantly, these ORFs correspond to ORFs 9, 10, and 11 of the recently sequenced C1 phage genome (Nelson, D., et al. (2003) J. Bacteriol. 185:3325-3332) (SEQ. ID: 9, 10, and 11, respectively). Furthermore, ORF8 (SEQ ID: 8) of the genomic sequence is a holin and the majority of bacteriophage lysis systems exhibit a gene arrangement of a holin immediately prior to the lysins gene.
The first gene of the 2.2 kb insert, hereafter referred to as plyCB (SEQ ID: 22), codes for a 72 amino acid polypeptide (SEQ ID: 9) that matches the N-terminal sequence of PlyC-B, the PlyC light chain. The 7.8 kDa predicted mass of the plyCB gene product matches the observed size of the PlyC-B by SDS-PAGE (FIG. 1C). A BLAST search revealed no close matches for this protein in the database.
The third gene of the 2.2 kb insert, hereafter referred to as plyCA (SEQ. ED: 23), codes for a 465 amino acid polypeptide (SEQ ID: 11) with a predicted size of 50.5 kDa. This matches both the size and the N-terminal sequence of PlyC-A, the PlyC heavy chain. A BLAST search indicated the gene product had limited homology to a putative amidase from the S. agalactiae prophage Lambda Sa1 (AAM99497), a putative tail protein of the S. pyogenes prophage 315.5 (AAM79918), and a putative minor structural protein of the S. thermophilus phage Sfi11 (AAC34413).
The second gene of the PlyC clone encodes a potential 105 amino acid protein (SEQ ID: 10), which has noteworthy homology to putative endonucleases from the S. agalactiae prophage Lambda Sa2 (ANN00738), Lactococcus phage bIL170 (AAC27227), and Vibriophage VpV262 (AAM28379). All of these endonucleases belong to the HNH family of endonucleases, which are embedded within a group I intron and confer mobility to the host intervening sequence by catalyzing double-stranded breaks in cognate alleles lacking the intron (Chevalier, B. S., et al. (2001) Nuc. Acid Res. 29:3757-3774). Significantly, group I introns are not only present in many bacteriophage systems, but they have been shown to interrupt lysin genes in over half of the known S. thermophilus bacteriophage (Foley, S., et al. (2000) J. Virol. 74:611-618). As such, HNH endonucleases are thought to be involved modular evolution of phage (Crutz-Le Coq, A.-M., et al. (2002) Microbiol. 148:985-1001). Due to the unique position of this gene between plyCB and plyCA, we choose to call this region of our clone lil (SEQ ID: 24), for lysin intergenic locus.
Transcriptional and translational analysis of the PlyC genes. To determine the minimal sequence necessary for lytic activity, individual genes or chimeras were cloned into a pBAD24 expression system and evaluated for activity on group A streptococci. Neither plyCB, lil, nor plyCA alone was sufficient for activity (FIG. 2B,C,D). However, a lil− chimera, which contained full-length plyCB and plyCA, possessed wild-type PlyC activity (FIG. 2E). Therefore, neither the lil gene nor the gene product is needed for formation of active PlyC. Additional truncations of either the plyCB or plyCA genes in the lil− clone ablated enzymatic activity (data not shown).
RT-PCR results indicate that intron splicing is not a mechanism used to fuse plyCB and plyCA (data not shown). Therefore, it is possible that each gene is independently translated and the gene products, PlyC-B and PlyC-A, self-associate post-translationally. In order to investigate this theory, we constructed two separate plasmids, pBAD33::plyCB (Crmr) and pBAD24::plyCA (Ampr), and double transformed both plasmids into one host. Light-F and Heavy-R primers were used for PCR and the absence of a product indicated that the full length PlyC clone was not present (data not shown). Upon induction with 0.5% arabinose, the double transformant contained PlyC activity (FIG. 2F). Therefore, activity of native PlyC is dependant on two separate genes products, PlyC-B and PlyC-A, rather than any pre or post-translational splicing events. Additionally, the plyC is an operon composed of plyCB and plyCA, which may also be referred to as the plyCBA operon.
In order to investigate interactions of PlyC-B and PlyC-A at the level of translation, we performed several folding experiments (FIG. 3). As expected, neither PlyC-B nor PlyC-A alone had any activity against group A streptococci. When both chains were mixed together and allowed to incubate at room temperature for 1 hr, again no activity was noted. However, when both chains were mixed together, denatured in 6 M urea, and allowed to slowly refold by dialysis against PBS overnight, we were able to reconstitute  ̃30% of wild-type activity. Consequently, PlyC-B and PlyC-A, each separate gene products, must be folded together at the level of translation to form the active enzyme, PlyC.
A model for the proposed structure of PlyC. In 1971, it was suggested that the mass of the streptococcal lysin (now PlyC) was 101 kDa based on the sedimentation coefficient and gel filtration of a semi-pure preparation (Fischetti, V. A., et al. (1971) J. Exp. Med. 133:1105-1117), which was confirmed more recently with a pure preparation (Nelson, D., et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98:4107-4112). Despite the current genetic and biochemical data of a 50 kDa PlyC-A heavy chain and an 8 kDa PlyC-B light chain presented above, a simple heterodimer model of the two chains does not rationalize the apparent 100 kDa mass of the native enzyme. To corroborate the mass of native PlyC, we utilized a non-cleavable crosslinker, bis(sulfosuccinimidyl)suberate (BS3), which reacts with primary amines and the ε amine of lysine. An SDS-PAGE of the crosslinked PlyC substantiates that the native enzyme is  ̃100 kDa (FIG. 4). Furthermore, the elution profile on a Superose 12 gel filtration column of the cross-linked PlyC was super-imposable on that of native PlyC, indicating that the crosslinking process did not alter the mass of the enzyme (data not shown).
In order to achieve a mass of 100 kDa, multiple copies of one or both subunits must exist. Thus, PlyC is a multimer. Circumstantial evidence for a molar ratio of 5 light chains to one heavy chain was obtained from N-terminal sequencing of the native enzyme, which yielded a 55 pm signal for PlyC-B and a 12 pm signal for PlyC-A (data not shown). However, this observation could be mitigated if the PlyC-A had a partially blocked N-terminus. Therefore, to further investigate the ratios of PlyC-A and PlyC-B, we cloned plyC into B834 E. coli cells, which are auxotrophic for methionine. After expressing PlyC in M9 minimal media supplemented with 35S-methionine as the only methionine source, we purified the labeled enzyme and separated the heavy and light chains by SDS-PAGE. We were able to enumerate an average signal, measured in counts per minute, for each chain by placing gel-extracted bands (n=20) in scintillation fluid and measuring the β activity (Table IV). The average chain-specific cpm was subsequently divided by the number of known methionine residues for each subunit (10 for PlyC-A and 1 for PlyC-B), thus yielding an average cpm per methionine per chain. This data suggests a molar ratio of six light chains for each heavy chain in an active PlyC molecule. Significantly, this proposed model would yield an enzyme with a mass of 98 kDa (6×8 kDa+50 kDa), which is validated by the gel filtration and crosslinking experiments, both of which predicted a mass of  ̃100 kDa.
Example 3
PlyC Activity Against Various Streptococcal Strains
Methods. Measurement of lysin activity was based upon turbidimetric determination of cell lysis. Simple detection of activity during purification utilized 96 well plates (Costar) and an automated plate reader (Molecular Devices) measuring a decrease in OD650 of group A streptococcus D471 cells. All bacterial strains were grown overnight in Todd Hewitt media, washed twice in phosphate buffered saline (PBS), and the OD650 was adjusted to  ̃1.0 with PBS. Each cell suspension (225 μl) was added in triplicate to a 96 well microtiter plate just prior to addition of 250 U of purified PlyC (25 μl of a 10,000 U/ml stock). The OD650 was monitored for each well over the course of the experiment and the plates were shaken before each reading to maintain cell suspension. Controls with PBS were used for each strain to observe spontaneous lysis of cells and/or sedimentation effects that were not resolved by the shaking. Although no spontaneous lysis was observed, several strains did have  ̃10% decrease in OD650 after several hours due to sedimentation. In these cases, the OD650 decrease seen in the controls were added back to the lysin experimental values to compensate for the natural settling. The activity of PlyC for each strain was reported as the initial velocity of lysis, in −OD/min, based on the time it took to decrease the starting OD by half (i.e. from an OD650 of 1.0 to 0.5).
Results
Purified C1 phage lysin (PlyC) was tested for muralytic activity against >40 bacterial strains in a variety of species which were divided into sets (FIG. 5). Set I contained 10 different group A streptococcal strains including the serological grouping strain, an M-negative strain, 8 distinct M types (representing class I and class II streptococci (Bessen, D., et al. (1989) J. Exp. Med. 169:269-283)), and an A-variant strain. PlyC was able to equally and completely lyse every strain in this set within 5 minutes and no viable colonies were detected after plating cells exposed to PlyC for this amount of time. Set II contained 8 different Lancefield groups of streptococci. We found that PlyC exhibited activity against groups C and E, though considerably less than that seen with group A strains; however, it was unable to lyse groups B, D, F, G, L, and N streptococci. In agreement with the spectrophotometric observations, no viable colonies were detected for groups C and E streptococci when plated up to 30 minutes after exposure to PlyC. Set III contained representative oral streptococci including S. crista, S. intermedius, S. gordonii, S. mitis, S. mutans, S. oralis, S. parasanguis, and S. salivarius. Very low but reproducible activity was only noted against all the S. gordonii strains tested. However, in these bacteria, cell viability remained near starting counts even after 30 minutes exposure to PlyC (4.8×107 starting CFU and 4.6×107 CFU after 30 min for S. gordonii Blackburn). Set IV contained a mix of Gram-positive bacteria (Bacillus pumulis, Staphylococcus aureus, or Staphylococcus epidermidis) often found in the oral flora and Gram-negative (Escherichia coli, Neisseria lactamicus, Porphyromonas gingivalis, or Pseudomonas aeruginosa) bacteria. Lysin had no effect on these organisms (data not shown in FIG. 9) despite similarities in the peptidoglycan of all Gram-positive organisms.
PlyC was subsequently tested against non-groupable streptococci that are pathogentic to animal. S. uberis causes bovine mastitis in dairy cows and significantly, PlyC was able to lyse every S. uberis strain tested including clinical isolates as well as reference strains in ATCC (FIG. 6). Likewise, PlyC possessed potent activity toward every S. equi strain tested (FIG. 7). S. equi is known to cause a condition called strangles disease in horses.
Example 4
PlyC has the Potential to be Used Diagnostically for Pathogentic Streptococcal Diseases
Methods: We utilized the PROFILE-1 Model 3550i Microluminometer with the PROFILE-1 Reagent Kit, both of which are from New Horizon's Diagnostics Corporation. The microluminometer is a hand held device that uses an AC plug adaptor, but battery operated models are available for field use. For most assays, 50 ul of a bacterial sample was placed in a 0.45 micron Filtravette and positive pressure was used to remove any liquid. The sample was washed with a somatic cell releasing agent and then either treated with a bacterial releasing agent (strong detergent) or 100 U PlyC for 10 sec. 50 ul of a luciferin/luciferase mixture was added and the digital readout gave the relative light units (RLU) emitted over a 10 second integration.
Results
Traditional microbial ATP bioluminescent assays employ strong detergents to lyse bacterial cells. While this method works well to identify the presence of bacteria, it does not provide meaningful information about the type of bacteria present. Lysins are lytic enzymes encoded by bacteriophages that act on the host cell wall to release newly formed phage particles. These enzymes hydrolyze bonds common to most bacterial cell walls, yet they display unique specificity for the host organism or species, often restricted to just those organisms susceptible to the phage itself. Since phage typing has been accepted for years as a diagnostic tool, we investigated whether these lysins have the ability to provide both the specificity of phage typing and sufficient catalytic activity to lyse target cells within seconds for ATP detection in the presence of luciferin/luciferase. We show that PlyC, which has been shown to be specific for the cell walls of group A streptococci, can detect this organism in the luciferase based assay. Additionally, the relative light units (RLU) were found to be directly proportional to the amount of group A streptococci present, indicating a quantitative measurement (FIG. 8). Additionally, the PlyC produced curve was equivalent to that produced by the more traditional detergent method of extracting ATP.
Diagnostically, PlyC can be used in the luciferase based system to detect group A streptococci even when contaminating bacteria are present (FIG. 9). In a typical 10 sec assay with 100 U of PlyC, group A streptococci produced over 5,000 relative light units (RLU) (n=20) compared to less than 10 RLU produced for PlyC treated E. coli, Staphylococcus aureus, or the closely related Streptococcus salivarius (n=10 for each) (FIG. 9). Additionally, PlyC was able to detect group A streptococci in a mixture of the other organisms. These data suggest that use of PlyC may provide a rapid diagnostic tool to identify target bacteria in a variety of clinical and field applications;







TABLE I







Ability of the C1 bacteriophage to cause infection.









Plaque formation














Control (Group C strep)
++++



Pronase treated Group C strep
++++



Group A strep
−



Group A-variant strep
−

















TABLE II







Adsorption of C1 bacteriophage.









Residual Pfu/ml














Buffer control
107



Group C cell walls
102



Group C CHO
103



20 mM GalNAc
107

















TABLE III







Features of C1 ORFs from genomic DNA and their putative functions















ORF
Strand
From
To
Length
aa
SEQ ID NO.
Putative function
Evalue


















1
(+2)
572
1092
522
173
1
Unknown
>1


2
(+3)
1746
1934
189
62
2
Unknown
>1


3
(+2)
1934
2470
537
178
3
Unknown
>1


4
(+3)
2601
2918
318
105
4
Unknown
>1


5
(+2)
2927
3550
624
207
5
Unknown
>1


6
(+1)
3550
4776
1227
408
6
Neck appendage
  1.2


7
(+3)
4806
7160
2355
784
7
DNA Polymerase*
3.00E−5


8
(+1)
7204
7530
327
108
8
Holin
9.00E−6


9
(+2)
7517
7735
219
72
9
Lysin*
See text


10
(+1)
7735
8052
318
105
10
HNH endonuclease
1.00E−4


11
(+2)
8024
9442
1419
472
11
Lysin*
See text









Amidase (Group B strep.)
8.00E−8









Tail (S. pyogenes 315.5)
3.00E−2


12
(−3)
11195
9471
1725
574
12
Major tail
3.00E−4


13
(−1)
12673
11381
1293
430
13
Unknown
>1


14
(−3)
13370
12660
711
236
14
Unknown
>1


15
(−1)
14320
13367
954
317
15
Head-tail connector
 2.00E−06


16
(−3)
15545
14367
1179
392
16
Major capsid*
See text


17
(−2)
15738
15583
156
51
17
Unknown
>1


18
(−1)
15913
15743
171
56
18
Unknown
>1


19
(−2)
16296
16102
195
64
19
Unknown
>1


20
(−3)
16547
16412
168
55
20
Unknown
>1





*Experimentally determined or detailed further in the text













TABLE IV





35S-methionine radiolabeling PlyC to determine subunit ratios.


















Avg. PlyC-A cpm
2583



Avg. PlyC-B cpm
1594



Avg. cpm/met. for PlyC-A
258.3



Avg. cpm/met. for PlyC-B
1594



Avg. ratio PlyC-A:PlyC-B
1:6.17



Avg. ratio standard deviation
1.66



Proposed PlyC structure
PlyC-A(PlyC-B)6

## Claims

1. An isolated polypeptide comprising an amino acid sequence of a C1 bacteriophage PlyC lytic enzyme, wherein the polypeptide is a subunit PlyC lysin heavy chain (PlyC-A) and, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 11.

2. A pharmaceutical composition for use in inhibiting or treating a bacterial infection, comprising an effective amount of the isolated polypeptide claim 1 and a pharmaceutically acceptable carrier.