The invention features methods and compositions for treatment or prevention of infection by, or disease caused by infection with, certain species of bacteria, including in particular bacteria in which a RAP-type and/or TRAP-type molecule plays a role in pathogenesis. This includes Staphylococcus species.

Description of the Invention

SUMMARY OF THE INVENTION

The invention features methods and compositions for treatment and/or prevention of infection by, or disease caused by infection with bacteria expressing RAP or TRAP or RAP-like or TRAP-like molecules (for example Staphylococcus spp., including S. aureus and S. epidermidis, Bacillus spp., including B. subtilus, B. cereus, B. anthracis, Listeria spp., including L. innocua and L. monoctogenes, Streptococcus pyogenes, Lactococcus lactis, Enterococcus faecalis, Escherichia coli, and Clostridium acetobtylicum).

The invention features treatment methods including coating devices, injecting systemically (IV IP IM or SQ), applying topically, or taking orally.

One aspect of the invention is a composition containing a polypeptide containing an amino acid sequence comprising all or parts of the general formula Y(K or S) PXTNF (SEQ ID NOS 21 and 22), (SEQ ID NOS: 1 and 2 in Ser. No. 09/839,695), where any of the amino acids can be modified and where X can be C, W, or I. Pharmaceutical compositions are also provided in some embodiments.

A further aspect of the invention is a composition wherein the polypeptide comprises an amino acid sequence containing the general formula IKKY(K or S)PXTN, (SEQ ID NOS 23 and 24) where X is C, W, I, or modified amino acids. Pharmaceutical compositions are also provided in some embodiments.

A further aspect of the invention is a method for treating a host for certain bacterial infections, wherein an antagonist of the RAP receptor is administered to the host. In some embodiments the host is a human patient. In further embodiments the host is an animal, such as but not limited to an experimental animal. In some embodiments the antagonist is a polypeptide, a peptidomimetic, or an antibody.

A further aspect of the invention is a nucleic acid molecule encoding a polypeptide of the invention. The nucleic acid molecule can be RNA or DNA or an antisense nucleic acid molecule. In an embodiment, the nucleic acid molecule comprises the nucleotide sequence of RIP, RAP or TRAP or their homologues.

In another aspect, the invention features an isolated native or recombinant RAP polypeptide, as well as nucleic acid encoding such RAP polypeptides.

In another aspect, the invention features an isolated native or synthetic RIP peptide, as well as nucleic acid encoding such RIP peptides.

In another aspect, the invention features an isolated TRAP polypeptide (native or recombinant, TRAP or TRAP homologues), as well as nucleic acid encoding such TRAP polypeptides.

DETAILED DESCRIPTION OF THE INVENTION

In some embodiments of the invention, antagonists of RAP or TRAP or RAP receptor are provided. Without being limited to any one theory, RIP may function by competing with RAP for binding to the RAP receptor, thus acting as an antagonist of the RAP, TRAP and/or RAP receptor. Such antagonists include but are not limited to antibodies which specifically bind to RAP or TRAP; antibodies which specifically bind to a RAP or TRAP ligand; ligands for RAP TRAP or RIP; antisense nucleic acids; aid peptide, non-peptide, and peptidomimetic analogs of RAP, RIP, and their ligands.

Antibodies can be synthetic, monoclonal, or polyclonal and can be made by techniques well known in the art. For therapeutic applications, "human" monoclonal antibodies having human constant and variable regions are often preferred so as to minimize the immune response of a patient against the antibody. Such antibodies can be generated by immunizing transgenic animals which contain human immunoglobulin genes. See Jakobovits et al. Ann NY Acad Sci 764:525-535 (1995). In connection with synthetic and semi-synthetic antibodies, such terms are intended to cover but are not limited to antibody fragments, isotype switched antibodies, humanized antibodies (e.g., mouse-human, human-mouse, and the like), hybrids, antibodies having plural specificities, fully synthetic antibody-like molecules, and the like.

As discussed below, antibodies can be screened for the ability to block the binding of a ligand to RAP, TRAP or RIP and/or for other properties, such as the ability to protect in vivo against bacterial infection.

In some embodiments of the invention, antisense nucleic acid molecules are used as antagonists of RAP or TRAP. Antisense nucleic acid molecules are complementary oligonucleotide strands of nucleic acids designed to bind to a specific sequence of nucleotides to inhibit production of a targeted protein. These agents may be used alone or in combination with other antagonists.

The antisense antagonist may be provided as an antisense oligonucleotide such as RNA (see, for example, Murayama et al. Antisense Nucleic Acid Drug Dev. 7:109-114 (1997)). Antisense sequences may also be provided in a viral vector, such as, for example, in hepatitis B virus (see, for example, Ji et al., J. Viral Hepat. 4:167-173 (1997)); in adeno-associated virus (see, for example, Xiao et al. Brain Res. 756:76-83 (1997)); or in other systems including but not limited to an HVJ(Sendai virus)-liposome gene delivery system (see, for example, Kaneda et al Ann. N.Y. Acad. Sci. 811:299-308 (1997)); a "peptide vector" (see, for example, Vidal et al. CR Acad. Sci. IU 32):279-287 (1997)); as a gene in an episomal or plasmid vector (see, for example, Cooper et al Proc. Natl. Acad. Sci. U.S.A. 94:6450-6455 (1997), Yew et al. Hum Gene TJier. 8:575-584 (1997)); as a gene in a peptide-DNA aggregate (see, for example, Niidome et al. J. Biol. Chem. 272:1530.7-15312 (1997)); as "naked DNA" (see, for example, U.S. Pat. No. 5,580,859 and U.S. Pat. No. 5,589,466); and inlipidic vector systems (see, for example, Lee et al. Crit. Rev Ther Drug Carrier Syst. 14:173-206 (1997)).

Candidate antagonists of the RAP, TRAP or RAP receptor can be screened for function by a variety of techniques known in the art and/or disclosed within the instant application, such as protection against S. aureus infection in a mouse model. A multitude of appropriate formulations of the antagonists of the invention can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, (15th Edition, Mack Publishing Company, Easton, Pa. (1975)), particularly Chapter 87, by Blaug, Seymour, therein. These formulations include for example, powders, pastes, ointments, jelly, waxes, oils, lipids, anhydrous absorption bases, oil-in-water or water-in-oil emulsions, emulsions carbowax (polyethylene glycols of a variety of molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax.

The quantities of active ingredient necessary for effective therapy will depend on many different factors, including means of administration, target site, physiological state of the patient, and other medicaments administered. Thus, treatment dosages should be titrated to optimize safety and efficacy. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of the active ingredients. Animal testing of effective doses for treatment of particular disorders will provide further predictive indication of human dosage. Various considerations are described, for example, in Goodman and Oilman's The Plumnacological Basis of Therapeutics, 7th Edition (1985), MacMillan Publishing Company, New York, and Remington's Pharmaceutical Sciences 18th Edition, (1990) Mack Publishing Co. Easton Perm. Methods for administration are discussed therein, including oral, intravenous, intraperitoneal, intramuscular, transdermal, nasal, iontophoretic administration, and the like.

The compositions of the invention may be administered in a variety of unit dosage forms depending on the method of administration. For example, unit dosage forms suitable for oral administration include solid dosage forms such as powder, tablets, pills, and capsules, and liquid dosage forms, such as elixirs, syrups, and suspensions. The active ingredients may also be administered parenterally in sterile liquid dosage forms. Gelatin capsules contain the active ingredient and as inactive ingredients powdered carriers, such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate and the like.

Examples of additional inactive ingredients that may be added to provide desirable color, taste, stability, buffering capacity, dispersion or other known desirable features are red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide, edible white ink and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric-coated for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.

The concentration of the compositions of the invention in the pharmaceutical formulations can vary widely, i.e., from less than about 0.1%, usually at or at least about 2% to as much as 20% to 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.

The compositions of the invention may also be administered via liposomes. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations the composition of the invention to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which binds to a desired target, such as antibody, or with other therapeutic or immunogenic compositions. Thus, liposomes either filled or decorated with a desired composition of the invention of the invention can delivered systemically, or can be directed to a tissue of interest, where the liposomes then deliver die selected therapeutic/immunogenic polypeptide compositions.

Liposomes for use in the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of, e.g., liposome size, acid lability and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in, e.g., Szokaet al. Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369, incorporated herein by reference.

A liposome suspension containing a composition of the invention may be administered intravenously, locally, topically, etc. in a dose which varies according to, inter alia, the manner of administration, the composition of the invention being delivered, and the stage of the disease being treated.

For solid compositions, conventional nontoxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, that is, one or more compositions of the invention of the invention, and more preferably at a concentration of 25%-75%.

For aerosol administration, the compositions of the invention are preferably supplied in finely divided form along with a surfactant and propellant. Typical percentages of compositions of the invention are 0.01%-20% by weight, preferably 1%-10%. The surfactant must, of course, be nontoxic, and preferably soluble in the propellant. Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. The surfactant may constitute 0.1%-20% by weight of the composition, preferably 0.25-5%. The balance of the composition is ordinarily propellant. A carrier can also be included, as desired, as with, e.g., lecithin for intranasal delivery.

The constructs of the invention can additionally be delivered in a depot-type system, an encapsulated form, or an implant by techniques well-known in the art. Similarly, the constructs can be delivered via a pump to a tissue of interest.

Any of the foregoing formulations may be appropriate in treatments and therapies in accordance with the present invention, provided that the active agent in the formulation is not inactivated by the formulation and the formulation is physiologically compatible. Polyclonal and/or monoclonal antibodies to the polypeptides of the present invention may be prepared. The polypeptides of the invention thereof may be prepared as described herein, and coupled to a carrier molecule, for example keyhole limpet hemocyanin, and injected into rabbits at selected limes over several months. The rabbit sera may be tested for immunoreactivity to the polypeptides thereof. Monoclonal antibodies may be made by injecting mice with the polypeptides. Monoclonal antibodies may be screened by methods known in the art, as are described, for example, in Harlow and Lane (1988) Antibodies: A laboratory manual. Cold Spring Harbor Press, New York, and Coding (1986) Monoclonal antibodies: Principles and Practice (2d ed.) Academic Press, New York. The antibodies will be tested for specific immunoreactivity with an epilope of the polypeptides. These antibodies will find use in diagnostic assays or as an active ingredient in a pharmaceutical composition.

For production of polyclonal antibodies, an appropriate target immune system is selected, typically a mouse or rabbit, although other species such as goats, sheep, cows, guinea pigs, and rats may be used. The substantially purified antigen is presented to the immune system according to methods known in the art. The immunological response is typically assayed by an immunoassay. Suitable examples include ELISA, RIA, fluorescent assay, or the like. These antibodies will find use in diagnostic assays or as an active ingredient in a pharmaceutical composition.

Rap Nucleic Acid and Proteins

The present invention also provides a protein (RAP) isolated and purified from a non-pathogenic Staphylococcus spp. The RAP protein has a molecular weight of about 33 kDa. In one embodiment, RAP is the protein encoded by a polynucleotide comprising the sequence of SEQ ID NO: 12 in Ser. No. 09/839,695 and comprising an amino acid sequence of SEQ ID NO: 13 in Ser. No. 09/839,695. These sequences are provided in the Sequence Listing below.

RAP Nucleic Acid

The term "RAP gene" is used generically to designate RAP genes and their alternate forms. "RAP gene" is also intended to mean the open reading frame encoding specific RAP proteins, and adjacent 5' and 3' non-coding nucleotide sequences involved in the regulation of expression (e.g., promoter region). The gene may be introduced into an appropriate vector for extrachromosomal maintenance or for integration into the host, in one embodiment the RAP gene comprises the sequence of SEQ ID NO: 12 in Ser. No. 09/839,695.

RAP regulatory sequences may be used to identify cis acting sequences required for transcriptional or translational regulation of RAP expression, especially at different stages of growth (e.g., early, mid, and late log phase), and to identify cis acting sequences and trans acting factors that regulate or mediate RAP expression. Such transcriptional or translational control regions may be operably linked to a RAP coding sequence or other coding sequence.

The nucleic acid compositions used in the subject invention may encode all or a part of the RAP protein as appropriate. Fragments may be obtained of the DNA sequence by chemically synthesizing oligonucleotides in accordance with conventional methods, by restriction enzyme digestion, by PCR amplification, etc. For the most part, DNA fragments will be of at least about ten contiguous nucleotides, usually at least about 15 nt, more usually at least about 18 nt to about 20 nt, more usually at least about 25 nt to about 50 nt. Such small DNA fragments are useful as primers for PCR, hybridization screening, etc. Larger DNA fragments, i.e. greater than 100 nt are useful for production of the encoded polypeptide. For use in amplification reactions, such as PCR, a pair of primers will be used.

The exact composition of the primer sequences is not critical to the invention, but for most applications the primers will hybridize to the subject sequence under stringent conditions, as known in the art. It is preferable to choose a pair of primers that will generate an amplification product of at least about 50 nt, preferably at least about 100 nt. Algorithms for the selection of primer sequences are generally known, and are available in commercial software packages. Amplification primers hybridize to complementary strands of DNA, and will prime towards each other.

The RAP gene and RAP coding sequence are isolated and obtained in substantial purity, generally as other than an intact bacterial chromosome. Usually, the DNA will be obtained substantially free of other nucleic acid sequences that do not include a RAP sequence or fragment thereof, generally being at least about 50%, usually at least about 90% pure and are typically "recombinant", i.e. flanked by one or more nucleotides with which it is not normally associated on a naturally occurring chromosome.

The DNA sequences are used in a variety of ways. They may be used as probes for identifying RAP coding sequences of other strains of Staphylococcus or of other bacteria. Homologues isolated from other stains, species, or genera generally have substantial sequence similarity to one another, i.e. at least 75%, usually at least 90%, more usually at least 95% sequence identity. In general, RAP-encoding sequences of the invention (including homologoues, variants, etc) are characterized by having a sequence identity greater than at least about 65%, preferably at least about 75%, more preferably at least about 85%, and can be greater than at least about 90% or more as determined by the Smith-Waterman homology search algorithm as implemented in MPSRCH program (Oxford Molecular). For the purposes of this invention, a sequence identity is calculated using the Smith-Waterman algorithm as follows: Global DNA sequence identity must be greater than 65% as determined by the Smith-Waterman homology search algorithm as implemented in MPSRCH program (Oxford Molecular) using an affine gap search with the following search parameters: gap open penalty, 12; and gap extension penalty, 1.

Nucleic acids having sequence similarity can also be detected by hybridization under low stringency conditions, for example, at 50.degree. C. and 6.times.SSC (0.9 M saline/0.09 M sodium citrate) and remain bound when subjected to washing at 55.degree. C. in 1.times.SSC (0.15 M sodium chloride/0.015 M sodium citrate). In addition, sequence identity may also be determined by hybridization under high stringency conditions, for example, at 50.degree. C. or higher and 0.1.times.SSC (15 in M salme/0.15 mM sodium citrate). By using probes, particularly labeled probes of DNA sequences, one can isolate homologous or related genes. It may also be possible to identify homologs of RAP from mammalian sources.

The RAP-encoding DNA may also be used to detect expression of the gene in a biological specimen. Methods and materials for probing a sample for the presence of particular nucleotide sequences are well established in the literature and do not require elaboration here. mRNA is isolated from a cell sample. mRNA may be amplified by RT-PCR, using reverse transcriptase to form a complementary DNA strand, followed by polymerase chain reaction amplification using primers specific for the subject DNA sequences. Alternatively, mRNA sample is separated by gel electrophoresis, transferred to a suitable support, e.g. nitrocellulose, nylon, etc., and then probed with a fragment of the subject DNA as a probe. Other techniques, such as oligonucleotide ligation assays, in situ hybridizations, and hybridization to DNA probes arrayed on a solid chip may also find use. Detection of mRNA hybridizing to an RAP sequence is indicative of RAP gene expression in the sample.

The RAP nucleic acid sequence may be modified for a number of purposes, particularly where they will be used intracellularly, for example, by being joined to a nucleic acid cleaving agent, e.g. a chelated metal ion, such as iron or chromium for cleavage of the gene; or the like.

The RAP coding sequence and/or promoter sequence may be mutated in various ways known in the art to generate targeted changes in promoter strength, sequence of the encoded protein, etc. The DNA sequence or product of such a mutation will be substantially similar to the sequences provided herein, i.e. will differ by at least one nucleotide or amino acid, respectively, and may differ by at least two but not more than about ten nucleotides or amino acids. The sequence changes may be substitutions, insertions or deletions. Deletions may further include larger changes, such as deletions of a domain. Other modifications of interest include production of fusion proteins (e.g., with green fluorescent proteins (GFP), luciferase, and the like).

Techniques for in vitro mutagenesis of cloned genes are known. Examples of protocols for scanning mutations may be found in Gustin et al., 1993 Biotechniques 14:22; Barany, 1985 Gene 37:111-23; Colicelli et al., 1985 Mol Gen Genet. 199:537-9; and Prentki et al., 1984 Gene 29:303-13. Methods for site specific mutagenesis can be found in Sambrook et al., 1989 Molecular Cloning: A Laboratory Manual, CSH Press, pp. 15.3-15.108; Weiner et al., 1993 Gene 126:35-41; Sayers et al., 1992 Biotechniques 13:592-6; Jones and Winistorfer, 1992 Biotechniques 12:528-30; Barton et al., 1990 Nucleic Acids Res 18:7349-55; Marotti and Tomich, 1989 Gene Anal Tech 6:67-70; and Zhu 1989 Anal Biochem 177:120-4.

RAP Protein

RAP protein can be produced by any suitable means, e.g., by isolated from a bacteria that naturally expresses RAP, by recombinant means (e.g., by expression of a polynucleotide having a sequence of SEQ ID NO: 12 in Ser. No. 09/839,695), by synthetic means, and the like.

In one embodiment, RAP is isolated directly from a strain of Staphylococcus producing RAP, e.g., S. aureus. Typically, wild type cells are collected from postexponential culture broth. Cells are then centrifuged and the supernatant subjected to purification by, for example, filtration followed by lyophilization, resuspension in water, and further purification.

The staphylococci bacterium from which RAP may be isolated may include, but is not necessarily limited to, S. aureus, S. capitus, S. warneri S. capitis, S. caprae, S. carnosus, S. saprophyticus, S. chronii, S. simulans, S. caseolyticus, S. epidermidis, S. haemolyticus, S. hominis, S. hyicus, S. kloosii, S. lentus, S. lugdunensis, S. scruri, S. simulans, and S. xylosus. Preferably RAP is isolated from S. aureus.

In another embodiment, RAP-encoding nucleic acid is employed to synthesize full-length RAP protein or fragments thereof, particularly fragments corresponding to functional domains (e.g., phosphorylation sites that interact with RAP, etc.), and including fusions of the subject polypeptides to other proteins or parts thereof. For expression, an expression cassette may be employed, providing for a transcriptional and translational initiation region, which may be inducible or constitutive, where the coding region is operably linked under the transcriptional control of the transcriptional initiation region, and a transcriptional and translational termination region. Various transcriptional initiation regions may be employed that are functional in the expression host.

The polypeptides may be expressed in prokaryotes or eukaryotes in accordance with conventional ways, depending upon the purpose for expression. For large scale production of the protein, a unicellular organism, such as E. coli, B. subtilis, S. cerevisiae, or cells of a higher organism such as vertebrates, particularly mammals, e.g. COS 7 cells, may be used as the expression host cells. Alternatively, RAP fragments can be synthesized.

With the availability of the polypeptides in large amounts, by employing an expression host, RAP protein can be isolated and purified in accordance with conventional ways, e.g., using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. The purified protein will generally be at least about 80% pure, preferably at least about 90% pure, and may be up to and including 100% pure.

The RAP proteins (native, recombinant or synthetic) can be used for the production of antibodies, where short fragments provide for antibodies specific for the particular polypeptide, and larger fragments or the entire protein allow for the production of antibodies over the surface of the polypeptide. Antibodies may be raised to the wild-type or variant forms of RAP. Antibodies may be raised to isolated peptides corresponding to these domains, or to the native protein, e.g. by immunization with cells expressing RAP, immunization with liposomes having RAP protein inserted in the membrane, etc.

Anti-RAP Antibodies

The present invention also provides an antibody that specifically binds and is immunoreactive with RAP. The antibody may be monoclonal, polyclonal or humanized, and is prepared using methods well known in the art. In general, antibodies are prepared in accordance with conventional ways, where the protein or an antigenic portion thereof is used as an immunogen, by itself or conjugated to known immunogenic carriers, e.g. KLH, pre-S HBsAg, other viral or eukaryotic proteins, or the like. Various adjuvants may be employed, with a series of injections, as appropriate. For monoclonal antibodies, after one or more booster injections, the spleen is isolated, the lymphocytes immortalized by cell fusion, and then screened for high affinity antibody binding. In a preferred embodiment, the spleen or lymph node cells and myeloma cells are mixed in about 20:1 to about 1:1 ratio, but preferably in about 2:1 ratio. It is preferred that the same species of animal serve as the source of somatic and myeloma cells used in the fusion procedure, where the animal is chosen from rat, mouse, rabbit, cow, chicken, turkey, or man. The fusion of the somatic and myeloma cells produces a hybridoma, which is grown in culture to produce the desired monoclonal antibody by standard procedures. For further description, see, for example, Monoclonal Antibodies: A Laboratory Manual, Harlow and Lane eds., Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 1988. If desired, the mRNA encoding the heavy and light chains may be isolated and mutagenized by cloning in E. coli, and the heavy and light chains mixed to further enhance the affinity of the antibody. Alternatives to in vivo immunization as a method of raising antibodies include binding to phage "display" libraries, usually in conjunction with in vitro affinity maturation.

The polyclonal antibodies of the present invention may be produced by injecting a rat, a mouse, a rabbit, a cow, a chicken, or a turkey with RAP to initiate an immunogenic response. They can be polyclonal or monoclonal, and can be engineered or synthesized. RAP may be coupled to a protein carrier such as deyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA). An adjuvant may also be used. After a suitable amount of time to establish a high-titer of anti-RAP antibodies, the serum or eggs are collected. The presence of antibody in the serum or eggs may be tested by radioimmunoassay (RIA), by enzyme-linked immunosorbent assay (ELISA), or by immunoprecipitation. The immunoglobulins may be isolated by the sequential precipitation methods, by conventional methods of "salting out" the protein fractions from a salt solution, or by chromalographical methods well known to those skilled in the art.

RAP

RAP (RplB) Sequences in Bacteria

RAP was purified from culture supernatants of S. aureus RN6390B, and the NH2-terminal sequence of RAP was determined to be IKKYKPITN (SEQ ID NO: 29) (Balaban N, et al. Science 2000, 287: 391a [18]. This sequence was compared (TblastN algorithm) to several S. aureus databases where only one ORF was found to match strongly to the above peptide sequence. This ORF encodes a putative 277 amino acid protein, which is an ortholog of L2 (or RplB) sequences (Ban N, et al., Science 2000, 289: 905-920 [19].

The corresponding gene in S. aureus RN6390B was amplified by PCR and sequenced (GenBank accession number AF205220). S. aureus putative RplB is highly homologous to RplB in other bacteria (FIG. 1, see Original Patent). Furthermore, its sequence was compared to that of other S. aureus genome sequences available, and it was found to be totally conserved among strains (data not shown).

Production of Recombinant RAP (Termed Either rRAP or rL2)

To produce rRAP, forward and reverse primers corresponding to the 5' and 3' ends of rap (rplB) gene with added restriction sites were designed. These primers were used to amplify the complete rplB gene by PCR, using S. aureus RN6390B chromosomal DNA as a template. Amplified DNA was digested and ligated into the corresponding sites of pET14b vector (Novagen, Wis.) that possess a six histidine tag at the 5' end of the inserted gene. Plasmid containing rap (pET2-5) was used to transform E. coli BL-21(DE3)/pLysS. Cells were induced and harvested, and recombinant His-rRAP protein was isolated using a nickel column and his tag removed by thrombin.

Specifically, to produce rL2, forward and reverse primers corresponding to the 5' and 3' ends of rap gene with added 5' Ndel and 3' BamHI restriction sites were designed based on the sequence of rap (underlined). These primers, 5' GAA TTC CAT ATG GCT ATT AAA AAG TAT AAG 3' (SEQ ID NO: 30), (nucleotides 1-21 (SEQ ID NO: 14 in Ser. No. 09/839,695)) and 5'CGC GCG GAT CCT TAT TTT TTC TTA CGT CCACG 3' (SEQ ID NO: 31), (complement of nucleotides 840-819 (SEQ ID NO: 15 in Ser. No. 09/839,695)), were used amplify the complete rap gene by PCR, using S. aureus chromosomal DNA as a template. Amplified DNA was digested by Ndel and BamHI and ligated into the corresponding sites of pET14b vector (Novagen, Wis.) that possess a six histidine tag at the 5' end of the inserted gene. Plasmid containing rap (pET2-5) was used to transform E. coli BL-21 (DE3)pLysS (SBpET2-5). Induction of synthesis of recombinant protein was carried out by addition of 1 mM IPTG to the culture and incubation for 3 hours. Cells were harvested and washed once with 50 mM Tris buffer pH 7.9. Recombinant His-rRAP protein was isolated using a nickel column according to the manufacturer's instructions with some modifications (Xpress Systems Protein Purification, Invitrogen, CA). Cell pellet of 50 ml was resuspended in 10 ml binding buffer (20 mM sodium phosphate pH 7.8+0.5M NaCl) and sonicated (for 2 cycles of 15 sec pulses at the maximal level with 30 sec intervals) and then spun in a microcentrifuge. The supernatant was loaded onto the pre-equilibrated nickel column.

Prior to loading, the column containing chelated Sepharose beads was loaded with a charging buffer containing 50 mM NiCl, and equilibrated with binding buffer. The column was washed three times with five volumes of binding buffer, followed by three washes with five volumes of 20 mM sodium phosphate+0.5M NaCl pH 7.8, then with buffer adjusted to pH 6. Recombinant protein was sequentially eluted from the column using 0-5M imidazole. His tag was removed by thrombin.

Monoclonal Anti-rRAP Antibodies Recognize Native RAP

Monoclonal antibodies were produced from mice that were primed by rRAP. Hybridoma supernatants were tested by ELISA for the presence of specific antibodies against injected antigen. Positive hybridomas were cloned and used to raise ascites. To test if antibodies to recombinant protein recognize the native molecule, rRAP and partially purified native RAP (postexponential supernatants of wild type S. aureus RN6390>10 kDa) were applied to SDS 12.5% PAGE, western blotted, and membrane stained in ponceau (FIG. 2 lanes 1,2 , see Original Patent). The membrane was blocked, and incubated with ascites made of a positive anti rRAP hybridoma (diluted 1:1000). Bound antibody was detected using peroxidase-conjugated anti mouse IgG, and visualized by chemiluminescence (FIG. 2 lanes 3,4, see Original Patent). As shown in FIG. 2 (see Original Patent), monoclonal antibodies raised against rRAP specifically recognize the native molecule that is secreted to the supernatant (FIG. 2 lane 4, see Original Patent).

Recombinant RAP is Active and Induces RNAIII Synthesis

To test if rRAP is active and can induce RNAIII synthesis, S. aureus cells containing rnaiii::blaZ fusion construct were incubated with increasing amounts of rRAP. As controls, cells were incubated only with growth medium (negative control). RNAIII (as .beta.-lactamase activity) was detected by the addition of nitrocefin, a yellow substrate that turns pink in the presence of .beta.-lactamase. This method is useful when the difference between experimental and control groups is at least twice, and one group turns pink while the other remains yellow. As shown in FIG. 3A (see Original Patent), rRAP activates RNAIII synthesis in a concentration dependent manner, reaching threshold levels at 2.25 nmole/10.sup.7 bacteria. Activation of RNAIII by recombinant RAP was similar to that of partially purified RAP. The increase in RNAIII synthesis (.beta.-lactamase activity) in the presence of 2.25 nmole rRAP is significant (P<0.00169) as compared to no addition of rRAP. We also tested induction of RNAIII synthesis by Northern blotting, where cells were grown for 30 min with or without rRAP, cells collected, Northern blotted, and the presence of RNAIII was detected using RNAIII-specific radiolabeled DNA. Membrane was autoradiographed and density of bands determined. As shown in FIG. 3B, rRAP induced the synthesis of RNAIII.

TRAP is Necessary for S. aureus Pathogenesis

RAP has been shown to induce and RIP has been shown to inhibit TRAP phosphorylation [5]. To show that TRAP is important for S. aureus pathogenesis, the traP gene was disrupted in S. aureus 8325-4 and the parent strain (TRAP+) and the mutant strain (TRAP.sup.-) were tested for growth, RNAIII synthesis, toxin production and pathogenesis. To test for cell growth and RNAIII synthesis, cells were grown from the early to the post exponential phase of growth. At time intervals cell density was determined and cell samples from each growth phase were collected, Northern blotted, and the presence of RNAIII was detected using RNAIII-specific radiolabeled DNA. As shown in FIG. 6A (see Original Patent), no difference in cell growth was observed between TRAP+ and TRAP.sup.- strains. The synthesis of RNAIII in the TRAP+ strain was observed from the midexponential phase but was absent in the TRAP-strain, confirming that TRAP is an important factor in the induction of RNAIII synthesis.

RNAIII is known to upregulate the production of many of the toxins produced by S. aureus, some of which are hemolysins [2]. To test for the production of hemolysins, the TRAP+ and TRAP.sup.- strains were grown on a sheep blood agar plate overnight at 37.degree. C. and then at 4.degree. C. (to test for both .alpha. and .beta. hemolysins). As shown in FIG. 7A (see Original Patent), no hemolysis was observed in the TRAP.sup.- strain, confirming that TRAP is an important factor in toxin production.

To test whether TRAP is important for biofilm formation, the TRAP+ and TRAP.sup.- strains were grown in polystyrene wells and adherent bacteria was stained by safranin. As shown in FIG. 7B, safranin staining was greatly reduced in the TRAP.sup.- strain, suggesting that TRAP is an important factor in cell adhesion and biofilm formation.

To test the importance of TRAP in S. aureus pathogenesis in vivo, the TRAP+ and TRAP.sup.- strains were injected subcutaneously into mice and animals followed for mortality, the development of lesion and overall health. As shown in FIG. 8 (see Original Patent), all animals (n=10) that were injected with 4.times.10.sup.8 CFU (of TRAP+ that were grown on a plate) developed a lesion with an average size of 1.74 cm.sup.2. All animals (n=5) that were injected with 6.times.10.sup.9 CFU (of TRAP+ that were grown on a plate) developed a lesion with an average size of 9.4 cm.sup.2. Of 8 animals (n=8) that were injected with 1.3.times.10.sup.9 CFU (TRAP+ that were grown in culture (*)), 4 died within the first 24 hrs and the rest developed a large lesion, with an average size of 8.63 cm.sup.2. On the other hand, all animals that were injected with the TRAP.sup.- strain seemed perfectly healthy, including the few that developed a very small lesion. Specifically, none of the animals (n=10) that were injected with 3.times.10.sup.8 CFU of TRAP.sup.- that were grown on a plate, developed a lesion. All animals (n=5) that were injected with 2.6.times.10.sup.9 CFU of TRAP.sup.- that were grown on a plate developed a small lesion with an average size of 0.62 cm.sup.2. Of the animals (n=8) that were injected with 1.5.times.10.sup.9 CFU of TRAP.sup.- that were grown in culture (*), 7 animals developed no lesion at all and one animal developed a small lesion of 0.4 cm.sup.2 (average size of 0.05 cm.sup.2). The differences in lesion size between the animals that were injected with the TRAP+ or TRAP.sup.- strains is significant (p<0.0008). These results confirm that the expression of TRAP is important for S. aureus pathogenesis and opens the field further for the development of novel drugs to prevent or treat staphylococcal infections.

Amino Acid Sequence Analysis of TRAP

The traP gene in various clinical isolates of S. aureus and S. epidermidis was amplified by PCR, and its sequence determined. Comparison of these sequences to BLAST searches in different databases (including the NCBI Microbial Genomes Databases [http://www.ncbi.nlm.nih.gov/Microb_blast/unfinishedgenome.html]) indicates that TRAP is unique to staphylococci. Comparison of the deduced amino acid sequences of the S. aureus and S. epidermidis TRAP proteins shows that they are highly conserved among staphylococci. Multi sequence alignment analysis (ClustalW) of S. aureus TRAP protein sequences from the various strains indicates that its sequence is divided into two sub-groups. Group I includes TRAP in 8325, which is identical to TRAP in COL, MSSA476, Mu50/ATCC700699 and N315 (16) and our clinical nare-isolates # 7 and 11 (see Original Patent). Group II includes TRAP in our clinical nare-isolates 12 and 15 and MRSA252 (Sanger Centre Database). TRAP of Group I is .about.97% identical to that of Group II (an E-value determined with the blastP algorithm is 2e-84). Similarity of both group I TRAP and Group II TRAP sequences to that of S. epidermidis is approximately 86% (an E-value is 9e-65).

S. aureus clinical isolate #12 has an insertion of IS1181 exactly in front of the TRAP stop codon (GenBank accession number AJ489-447). Insertion of IS1811 shifted the native traP stop codon but introduced another stop codon 27 bp downstream, that elongated TRAP from 167 to 176 AA (TRAP+GSSSFMVGR) (SEQ ID NO: 16). Like other clinical or lab strains, TRAP is phosphorylated and RNAIII is expressed in strain isolate # 12, suggesting that the insertion element does not disrupt its function (not shown).

Secondary Structure Predictions of TRAP

TRAP is highly unique to staphylococci, but has some sequence similarity to the hypothetical protein YhgC protein in Bacillus subtilis (GenBank Accession Number Z99109) (an E-value is 9e-15). Interestingly, in addition to Bacillus subtilis, among more than 160 eubacterial genomes, only in Bacillus anthracis which is 97% identical to that of B. cereus (TIGR database), Listeria innocua and L. monocytogenes ORFs could be identified which have some sequence similarity to TRAP (an E-values are 0.005, 0.035 and 0.1, respectively). Like TRAP, all ORFs are of exactly 167 AA except for YhgC, which is 166 AA.

The secondary structure of these proteins was predicted using the protein structure prediction server The PCIPRED v.2.4 [http://bioinf.cs.ucl.ac.uk/psiform.html/]. Interestingly, although the sequence similarity of these ORFs is very low, their predicted secondary structures are very similar to that of TRAP.

Chromosomal Organization of the S. aureus TRAP Gene Region and Comparison with Other Gram-Positive Eubacteria.

The same organization of the TRAP gene region is found in all Staphylococcus spp (in both S. aureus and S. epidermidis) genomes. The TRAP gene is flanked by two polycistronic operons; one of them (upstream of traP) encodes enzymes of the late step of the protoheme IX biosynthetic pathway (hemEHY genes), the second (downstream of trap) codes for a putative multi protein transporting system (ecsAB(C) genes). The direction of the traP gene transcription is opposite to both hem and ecs operons. The same organization is found also in yhgC region in B. subtilis, B. anthracis and in the TRAP-like ORFs in Listeria. These results suggest that TRAP represents a class of signal transducers in bacteria and that it can be a target site for therapy in many bacterial species in addition to staphylococci.

Phosphorylation of TRAP and TRAP like Molecules and Inhibition of TRAP phosphorylation by RIP

Because TRAP-like molecules are found in other bacterial species in addition to staphylocopcci, we tested whether they can undergo phosphorylation. We grew various strains of Listeria (L. monocytogenes and L. ivanovii) and various strains of Bacillus anthracis, B. subtilis and B. cereus) with P.sup.32, and tested for phosphorylation of TRAP (21 kDa) by SDS PAGE followed by autoradiography. As shown in FIG. 12 (see Original Patent) for Listeria spp, a 21 kDa protein was in vivo phosphorylated, as expected. Similar results were obtained for Bacillus spp. (not shown). These results suggest that pathogenesis may also be regulated via TRAP phosphorylation in bacteria expression TRAP-like proteins.

To test for TRAP phosphorylation in S. epidermidis, in vivo phosphorylation assays were carried out as described for S. aureus [5]. Briefly, early exponential S. epidermidis were grown in the presence of phosphate-free buffer supplemented with radiolabeled orthophosphate with or without RIP (10 .mu.g/10.sup.7 cells). After a 1 hr incubation period, the cells were collected by centrifugation, treated with lysostaphin followed by the addition of sample buffer, and total cell homogenate was applied without boiling to 15% SDS PAGE and the gel autoradiographed. The same experiment was carried out on 6390B S. aureus cells as a positive control. Results show that TRAP phosphorylation can be inhibited by RIP also in S. epidermidis. These results are not surprising in view of the fact that TRAP is found also in S. epidermidis, and they show that TRAP phosphorylation and expression is important for staphylococcal pathogenesis. Because its sequence is highly conserved among staphylococcal strains and species and because its secondary structure is similar to many TRAP-like molecules in other bacteria, TRAP can be a target site for therapy in many bacterial species in addition to staphylococci.

RIP (native or synthetic, YSPWTNF (SEQ ID NO: 26) or derivatives) has been shown not only to inhibits RNAII and RNAIII synthesis and thus to inhibit toxin production [13,18] but have also been shown to inhibits S. aureus adherence to human cells and to inhibits the formation of biofilm on plastic [16]. Virulence of S. epidermidis is also often associated with their ability to adhere to host cells and to form biofilm on medical devices. To test whether RIP can prevent S. epidermidis from colonizing host cells and therefore be a candidate for therapy and prevention, FITC-labeled S. epidermidis were incubated in the presence or absence of RIP with a confluent layer of keratinocytes (HaCat cells) for 30 min. As shown in FIG. 14A (see Original Patent), RIP significantly (p<0.05) reduced S. epidermidis adherence to HaCat cells.

To test whether RIP reduces adherence and biofilm formation of S. epidermidis to plastic, early exponential S. epidermidis were grown for 3 hrs in microtiter plates made of polystyrene, and adherent cells were stained with safranin. These experimental conditions allow for biofilm to be formed (as observed by atomic force microscopy, not shown). As shown in FIG. 14B, RIP significantly reduced the number of cells that adhered to the plastic. These results clearly demonstrate that RIP inhibits the adhesion of S. epidermidis to host cells and to plastic in vitro.

 

Claim 1 of 2 Claims

1. A method of preventing or treating a bacterial infection caused by any bacterial species in which RNAIII-activating protein (RAP) plays a role in pathogenesis in a subject, said bacterial species being selected from the group consisting of a staphylococcus bacteria, Bacillus subtilis, Bacillus anthracis, Bacillus cereus, Listeria innocua, Listeria monocytogenes, Streptococcus pyogenes, Lactocococcus lactis, Enterococcus faecalis, Escherichia coli and Clostrydium acetobtylicum, comprising: administering to the subject having or at risk of a bacterial infection caused by a bacteria in which RAP plays a role in pathogenesis an amount of RNAIII inhibiting peptide (RIP) effective to treat or prevent the bacterial infection, wherein the RIP comprises the amino acid sequence set forth in SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO: 27, or SEQ ID NO:28.

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