US20020019515A1 - Novel spoIIIE - Google Patents
Novel spoIIIE Download PDFInfo
- Publication number
- US20020019515A1 US20020019515A1 US09/775,978 US77597801A US2002019515A1 US 20020019515 A1 US20020019515 A1 US 20020019515A1 US 77597801 A US77597801 A US 77597801A US 2002019515 A1 US2002019515 A1 US 2002019515A1
- Authority
- US
- United States
- Prior art keywords
- spoiiie
- polypeptide
- seq
- sequence
- leu
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
- C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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- A61P17/00—Drugs for dermatological disorders
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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Definitions
- This invention relates to newly identified polynucleotides and polypeptides, and their production and uses, as well as their variants, agonists and antagonists, and their uses.
- the invention relates to novel polynucleotides and polypeptides of the spoIIIE/ftsK (putative chromosomal partitioning function) family, hereinafter referred to as “spoIIIE”.
- Streptococci make up a medically important genera of microbes kwown to cause several types of disease in humans, including, for example, otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid.
- Streptococcus pneumoniae Since its isolation more than 100 years ago, Streptococcus pneumoniae has been one of the more intensively studied microbes. For example, much of our early understanding that DNA is, in fact, the genetic material was predicated on the work of Griffith and of Avery, Macleod and McCarty using this microbe.
- Streptococcal genes and gene products as targets for the development of antibiotics.
- polypeptides of the invention have amino acid sequence homology to a known Staphylococcus aureus spoIIIE protein.
- the polynucleotide comprises a region encoding spoIIIE polypeptides comprising the sequence set out in Table 1 [SEQ ID NO:1] which includes a full length gene, or a variant thereof.
- an isolated nucleic acid molecule encoding a mature polypeptide expressible by the Streptococcus pneumoniae 0100993 strain contained in the deposited strain.
- isolated nucleic acid molecules encoding spoIIIE particularly Streptococcus pneumoniae spoIIIE, including mRNAs, cDNAs, genomic DNAs.
- Further embodiments of the mention include biologically, diagnostically, prophylactically, clinically or therapeutically useful variant thereof, and compositions comprising the same.
- a polynucleotide of the invention for therapeutic or prophylactic purposes, in particular genetic immunization.
- particularly preferred embodiments of the invention are naturally occurring allelic variants of spoIIIE and polypeptides encoded thereby.
- novel polypeptides of Streptococcus pneumoniae referred to herein as spoIIIE as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comprising the same.
- inhibitors to such polypeptides useful as antibacterial agents, including, for example, antibodies.
- products, compositions and methods for assessing spoIIIE expression treating disease, for example, otitis media, cojunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid, assaying genetic variation, and administering a spoIIIE polypeptide or polynucleotide to an organism to raise an immunological response against a bacteria, especially a Streptococcus pneumoniae bacteria.
- polynucleotides that hybridize to spoIIIE polynucleotide sequences, particularly under stringent conditions.
- antibodies against spoIIIE polypeptides there are provided antibodies against spoIIIE polypeptides.
- methods for identifying compounds which bind to or otherwise interact with and inhibit or activate an activity of a polypeptide or polynucleotide of the invention comprising a polypeptide or polynucleotide of the invention with a compound to be screened under conditions to permit binding to or other interaction between the compound and the polypeptide or polynucleotide to assess the binding to or other interaction with the compound, such binding or interaction being associated with a second component capable of providing a detectable signal in response to the binding or interaction of the polypeptide or polynucleotide with the compound; and determining whether the compound binds to or otherwise interacts with and activates or inhibits an activity of the polypeptide or polynucleotide by detecting the presence or absence of a signal generated from the binding or interaction of the compound with the polypeptide or polynucleotide.
- spoIIIE agonists and antagonists preferably bacteriostatic or bacteriocidal agonists and antagonists.
- compositions comprising a spoIIIE polynucleotide or a spoIIIE polypeptide for administration to a cell or to a multicellular organism.
- “Host cell” is a cell which has been transformed or transfected, or is capable of transformation or transfection by an exogenous polynucleotide sequence.
- Identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences.
- identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences.
- Identify and similarity can be readily calculated by known methods, including but not limited to those described in ( Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.
- Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990).
- the BLAST X program is publicly available from NCBI and other sources ( BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990).
- a polynucleotide having a nucleotide sequence having at least, for example, 95% “identity” to a reference nucleotide sequence of SEQ ID NO: 1 it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence of SEQ ID NO: 1.
- a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
- These mutations of the reference sequence may occur at the 5 or 3 terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
- a polypeptide having an amino acid sequence having at least, for example, 95% identity to a reference amino acid sequence of SEQ ID NO:2 is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of SEQ ID NO: 2.
- up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence.
- These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
- isolated means altered “by the hand of man” from its natural state, i.e., if it occurs in nature, it has been changed or removed from its original environment, or both.
- a polynucleotide or a polypeptide naturally present in a living organism is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein.
- Polynucleotide(s) generally refers to any polyribonucleotide or polydeoxiribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
- Polynucleotide(s)” include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double- and triple-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or triple-stranded regions, or a mixture of single- and double-stranded regions.
- polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
- the strands in such regions may be from the same molecule or from different molecules.
- the regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules.
- One of the molecules of a triple-helical region often is an oligonucleotide.
- the term “polynucleotide(s)” also includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are “polynucleotide(s)” as that term is intended herein.
- DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art.
- the term “polynucleotide(s)” as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including, for example, simple and complex cells. “Polynucleotide(s)” also embraces short polynucleotides often referred to as oligonucleotide(s).
- Polypeptide(s) refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds. “Polypeptide(s)” refers to both short chains, commonly referred to as peptides, oligonucleotides and oligomers and to longer chains generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene encoded amino acids. “Polypeptide(s)” include those modified either by natural processes, such as processing and other post translational modifications, but also by chemical modification techniques. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature, and they are well known to those of skill in the art.
- Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini.
- Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalant attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosilation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation,
- Polypeptides may be branched or cyclic, with or without branching. Cyclic, branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well.
- Variant(s) is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties.
- a typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below.
- a typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical.
- a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination.
- a substituted or inserted amino acid residue may or may not be one encoded by the genetic code.
- a variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to skilled artisans.
- the invention relates to novel spoIIIE polypeptides and polynucleotides as described in greater detail below.
- the invention relates to polypeptides and polynucleotides of a novel spoIIIE of Streptococcus pneumoniae, which is related by amino acid sequence homology to Staphylococcus aureus spoIIIE polypeptide.
- the invention relates especially to spoIIIE having the nucleotide and amino acid sequences set out in Table 1 [SEQ ID NO: 1] and Table 1 [SEQ ID NO: 2] respectively, and to the spoIIIE nucleotide sequences of the DNA in the deposited strain and amino acid sequences encoded thereby.
- a deposit containing a Streptococcus pneumoniae 0100993 strain has been deposited with the National Collections of Industrial and Marine Bacteria Ltd. (herein “NCIMB”), 23 St. Machar Drive, Aberdeen AB2 1RY, Scotland on Apr. 11, 1996 and deposit number 40794. The deposit was described as Streptococcus pneumoniae 0100993 on deposit. On Apr. 17, 1996 a Streptococcus pneumoniae 0100993 DNA library in E. coli was similarly deposited with the NCIMB and assigned deposit number 40800.
- the Streptococcus pneumoniae strain deposit is referred to herein as “the deposited strain” or as “the DNA of the deposited strain.”
- the deposited strain contains the full length spoIIIE gene.
- the sequence of the polynucleotides contained in the deposited strain, as well as the amino acid sequence of the polypeptide encoded thereby, are controlling in the event of any conflict with any description of seqences herein.
- a license may be required to make, use or sell the deposited strain, and compounds derived therefrom, and no such license is hereby granted.
- polypeptides of the invention include the polypeptide of Table 1 [SEQ ID NO:2] (in particular the mature polypeptide) as well as polypeptides and fragments, particularly those which have the biological activity of spoIIIE, and also those which have at least 70% identity to the polypeptide of Table 1 [SEQ ID NO:2] or the relevant portion, preferably at least 80% identity to the polypeptide of Table 1 [SEQ ID NO:2], and more preferably at least 90% similarity (more preferably at least 90% identity) to the polypeptide of Table 1 [SEQ ID NO:2] and still more preferably at least 95% similarity (still more preferably at least 95% identity) to the polypeptide of Table 1 [SEQ ID NO:2] and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids.
- the invention also includes polypeptides of the formula set forth in Table 1 (D) wherein, at the amino terminus, X is hydrogen, and at the carboxyl terminus, Y is hydrogen or a metal, R 1 and R 2 is any amino acid residue, and n is an integer between 1 and 1000. Any stretch of amino acid residues denoted by either R group, where R is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer.
- a fragment is a varient polypeptide having an amino acid sequence that entirely is the same as part but not all of the amino acid sequence of the aforementioned polypeptides.
- fragments may be “free-standing,” or comprised with a larger polypeptide of which they form a part or region, most preferably as a single continuous region, a single larger polypeptide.
- Preferred fragments include, for example, truncation polypeptides having a portion of the amino acid sequence of Table 1 [SEQ ID NO:2], or of variants thereof such as a continuous series of residues that includes the amino terminus, or a continuous series of residues that includes the carboxyl terminus.
- Degradation forms of the polypeptides of the invention in a host cell, particularly a Streptococcus pneumoniae, are also preferred.
- fragments characterized by structural or functional attributes such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions.
- biologically active fragments which are those fragments that mediate activities of spoIIIE, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also included are those fragments that are antigenic or immunogenic in an animal, especially in a human. Particularly preferred are fragments comprising receptors or domains of enzymes that confer a function essential for viability of Streptococcus pneumoniae or the ability to initiate, or maintain cause disease in an individual, particularly a human.
- Variants that are fragments of the polypeptides of the invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, these variants may be employed as intermediates for producing the full-length polypeptides of the invention.
- Another aspect of the invention relates to isolated polynucleotides, including the full length gene, that encode the spoIIIE polypeptide having the deduced amino acid sequence of Table 1 [SEQ ID NO:2] and polynucleotides closely related thereto and variants thereof.
- a polynucleotide of the invention encoding spoIIIE polypeptide may be obtained using standard cloning and screening methods, such as those for cloning and sequencing chromosomal DNA fragments from bacteria using Streptococcus pneumoniae 0100993 cells as starting material, followed by obtaining a full length clone.
- standard cloning and screening methods such as those for cloning and sequencing chromosomal DNA fragments from bacteria using Streptococcus pneumoniae 0100993 cells as starting material, followed by obtaining a full length clone.
- a polynucleotide sequence of the invention such as the sequence given in Table 1 [SEQ ID NO:1]
- coli or some other suitable host is probed with a radiolabeled oligonucleotide, preferably a 17-mer or longer, derived from a partial sequence.
- Clones carrying DNA identical to that of the probe can then be distinguished using stringent conditions.
- sequencing primers designed from the original sequence it is then possible to extend the sequence in both directions to determine the full gene sequence.
- sequencing is performed using denatured double stranded DNA prepared from a plasmid clone. Suitable techniques are described by Maniatis, T., Fritsch, E. F.
- the DNA sequence set out in Table 1 [SEQ ID NO:1] contains an open reading frame encoding a protein having about the number of amino acid residues set forth in Table 1 [SEQ ID NO:2] with a deduced molecular weight that can be calculated using amino acid residue molecular weight values well known im the art.
- the polynucleotide of SEQ ID NO: 1, between nucleotide number 1 through number 2349 encodes the polypeptide of SEQ ID NO:2.
- the stop codon begins at nucleotide number 2350 of SEQ ID NO:1.
- the spoIIIE polypeptide of the invention is structurally related to other proteins of the spoIIIE/ftsK (putative chromosomal partitioning function) family, as shown by the results of sequencing the DNA encoding spoIIIE of the deposited strain.
- the protein exhibits greatest homology to Staphylococcus aureus spoIIIE protein among known proteins.
- the spoIIIE protein of Table 1 [SEQ ID NO:2] has about 42.237% identity to Staphylococcus aureus spoIIIE protein over its entire length and about 63.947% similarity to Staphylococcus aureus spoIIIE protein over its entire length with the amino acid sequence of Staphylococcus aureus spoIIIE polypeptide.
- the invention provides a polynucleotide sequence identical over its entire length to the coding sequence in Table 1 [SEQ ID NO:1]. Also provided by the invention is the coding sequence for the mature polypeptide or a fragment thereof, by itself as well as the coding sequence for the mature polypeptide or a fragment in reading frame with other coding sequence, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence.
- the polynucleotide may also contain non-coding sequences, including for example, but not limited to non-coding 5′ and 3′ sequences, such as the transcribed, non-translated sequences, termination signals, ribosome binding sites, sequences that stabilize mRNA, introns, polyadenylation signals, and additional coding sequence which encode additional amino acids.
- non-coding sequences including for example, but not limited to non-coding 5′ and 3′ sequences, such as the transcribed, non-translated sequences, termination signals, ribosome binding sites, sequences that stabilize mRNA, introns, polyadenylation signals, and additional coding sequence which encode additional amino acids.
- a marker sequence that facilitates purification of the fused polypeptide can be encoded.
- the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al., Proc. Natl. Acad.
- Polynucleotides of the invention also include, but are not limited to, polynucleotides comprising a structural gene and its naturally associated sequences that control gene expression.
- a preferred embodiment of the invention is the polynucleotide of comprising nucleotide 1 to 2349 set forth in SEQ ID NO:1 of Table 1 which encodes the spoIIIE polypeptide.
- the invention also includes polynucleotides of the formula set forth in Table 1 (C) wherein, at the 5′ end of the molecule, X is hydrogen, and at the 3′ end of the molecule, Y is hydrogen or a metal, R 1 and R 2 is any nucleic acid residue, and n is an integer between 1 and 1000. Any stetch of nucleic acid residues denoted by either R group, where R is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer.
- polynucleotide encoding a polypeptide encompasses polynucleotides that include a sequence encoding a polypeptide of the invention, particularly a bacterial polypeptide and more particularly a polypeptide of the Streptococcus pneumoniae spoIIIE having the amino acid sequence set out in Table 1 I[SEQ ID NO:2].
- the term also encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (for example, interrupted by integrated phage or an insertion sequence or editing) together with additional regions, that also may contain coding and/or non-coding sequences.
- the invention further relates to variants of the polynucleotides described herein that encode for variants of the polypeptide having the deduced amino acid sequence of Table 1 [SEQ ID NO:2]. Variants that are fragments of the polynucleotides of the invention may be used to synthesize full-length polynucleotides of the invention.
- spoIIIE variants that have the amino acid sequence of spoIIIE polypeptide of Table 1 [SEQ ID NO:2] in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, deleted or added, in any combination. Especially preferred among these are silent substitutions, additions and deletions, that do not alter the properties and activities of spoIIIE.
- polynucleotides that are at least 70% identical over their entire length to a polynucleotide encoding spoIIIE polypeptide having the amino acid sequence set out in Table 1 [SEQ ID NO:2], and polynucleotides that are complementary to such polynucleotides.
- polynucleotides that comprise a region that is at least 80% identical over its entire length to a polynucleotide encoding spoIIIE polypeptide of the deposited strain and polynucleotides complementary thereto.
- polynucleotides at least 90% identical over their entire length to the same are particularly preferred, and among these particularly preferred polynucleotides, those with at least 95% are especially preferred. Furthermore, those with at least 97% are highly preferred among those with at least 95%, and among these those with at least 98% and at least 99% are particularly highly preferred, with at least 99% being the more preferred.
- Preferred embodiments are polynucleotides that encode polypeptides that retain substantially the same biological function or activity as the mature polypeptide encoded by the DNA of Table 1 [SEQ ID NO:1].
- the invention further relates to polynucleotides that hybridize to the herein above-described sequences.
- the invention especially relates to polynucleotides that hybridize under stringent conditions to the herein above-described polynucleotides.
- stringent conditions and “stringent hybridization conditions” mean hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences.
- An example of stringent hybridization conditions is overnight incubation at 42° C.
- the invention also provides a polynucleotide consisting essentially of a polynucleotide sequence obtainable by screening an appropriate library containing the complete gene for a polynucleotide sequence set forth in SEQ ID NO:1 under stringent hybridization conditions with a probe having the sequence of said polynucleotide sequence set forth in SEQ ID NO:1 or a fragment thereof, and isolating said DNA sequence. Fragments useful for obtaining such a polynucleotide include, for example, probes and primers described elsewhere herein.
- polynucleotides of the invention may be used as a hybridization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding spoIIIE and to isolate cDNA and genomic clones of other genes that have a high sequence similarity to the spoIIIE gene.
- Such probes generally will comprise at least 15 bases.
- such probes will have at least 30 bases and may have at least 50 bases.
- Particularly preferred probes will have at least 30 bases and will have 50 bases or less.
- the codig region of the spoIIIE gene may be isolated by screening using the DNA sequence provided in SEQ ID NO: 1 to synthesize an oligonucleotide probe.
- a labeled oligonucleotide having a sequence complementary to that of a gene of the invention is then used to screen a library of cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.
- polynucleotides and polypeptides of the invention may be employed, for example, as research reagents and materials for discovery of treatments of and diagnostics for disease, particularly human disease, as further discussed herein relating to polynucleotide assays.
- Polynucleotides of the invention that are oligonucleotides derived from the sequences of SEQ ID NOS:1 and/or 2 may be used in the processes herein as described, but preferably for PCR, to determine whether or not the polynucleotides identified herein in whole or in part are transcribed in bacteria in infected tissue. It is recognized that such sequences will also have utility in diagnosis of the stage of infection and type of infection the pathogen has attained.
- the invention also provides polynucleotides that may encode a polypeptide that is the mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mature polypeptide (when the mature form has more than one polypeptide chain, for instance).
- Such sequences may play a role in processing of a protein from precursor to a mature form, may allow protein transport, may lengthen or shorten protein half-life or may facilitate manipulation of a protein for assay or production, among other things.
- the additional amino acids may be processed away from the mature protein by cellular enzymes.
- a precursor protein, having the mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide.
- inactive precursors When prosequences are removed such inactive precursors generally are activated. Some or all of the prosequences may be removed before activation. Generally, such precursors are called proproteins.
- a polynucleotide of the invention may encode a mature protein, a mature protein plus a leader sequence (which may be referred to as a preprotein), a precursor of a mature protein having one or more prosequences that are not the leader sequences of a preprotein, or a preproprotein which is a precursor to a proprotein, having a leader sequence and one or more prosequences, which generally are removed during processing steps that produce active and mature forms of the polypeptide.
- a leader sequence which may be referred to as a preprotein
- a precursor of a mature protein having one or more prosequences that are not the leader sequences of a preprotein or a preproprotein which is a precursor to a proprotein, having a leader sequence and one or more prosequences, which generally are removed during processing steps that produce active and mature forms of the polypeptide.
- the invention also relates to vectors that comprise a polynucleotide or polynucleotides of the invention, host cells that are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
- Cell-free translation can also be employed to produce such proteins using RNAs derived from the DNA constructs of the invention.
- host cells can be genetically engineered to incorporate expression systems or portions thereof or polynucleotides of the invention.
- Introduction of a polynucleotide into the host cell can be effected by methods described in many standard laboratory manuals, such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction and infection.
- Representative examples of appropriate hosts include bacterial cells, such as streptococci, staphylococci, enterococci E. coli, streptomyces and Bacillus subtilis cells; fungal cells, such as yeast cells and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 313, BHK, 293 and Bowes melanoma cells; and plant cells.
- bacterial cells such as streptococci, staphylococci, enterococci E. coli, streptomyces and Bacillus subtilis cells
- fungal cells such as yeast cells and Aspergillus cells
- insect cells such as Drosophila S2 and Spodoptera Sf9 cells
- animal cells such as CHO, COS, HeLa, C127, 313, BHK, 293 and Bowes melanoma cells
- plant cells include bacterial cells, such as streptococci, staphylococci
- vectors include, among others, chromosomal, episomal and virus-derived vectors, e.g., vectors derived from baterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
- vectors include, among others, chromosomal, episomal and virus-derived vectors, e.g., vectors derived from baterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruse
- the expression system constructs may contain control regions that regulate as well as gender expression.
- any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide in a host may be used for expression in this regard.
- the appropriate DNA sequence may be inserted into the expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, (supra).
- secretion signals may be incorporated into the expressed polypeptide. These signals may be endogenous to the polypeptide or they may be heterologous signals.
- Polypeptides of the invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. Most preferably, high performance liquid chromatography is employed for puification. Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification.
- This invention is also related to the use of the spoIIIE polynucleotides of the invention for use as diagnostic reagents. Detection of spoIIIE in a eukaryote, particularly a mammal, and especially a human, will provide a diagnostic method for diagnosis of a disease. Eukaryotes (herein also “individual(s)”), particularly mammals, and especially humans, infected with an organism comprising the spoIIIE gene may be detected at the nucleic acid level by a variety of techniques.
- Nucleic acids for diagnosis may be obtained from an infected individual's cells and tissues, such as bone, blood, muscle, cartilage, and skin. Genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification technique prior to analysis. RNA or cDNA may also be used in the same ways. Using amplification, characterization of the species and strain of prokaryote present in an individual, may be made by an analysis of the genotype of the prokaryote gene. Deletions and insertions can be detected by a change in size of the amplified product in comparison to the genotype of a reference sequence.
- Point mutations can be identified by hybridizing amplified DNA to labeled spoIIIE polynucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures. DNA sequence differences may also be detected by alterations in the electrophoretic mobility of the DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing. See, e.g., Myers et al., Science, 230: 1242 (1985). Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase and S1 protection or a chemical cleavage method. See, e.g., Cotton et al., Proc. Natl. Acad. Sci., USA, 85: 4397-4401 (1985).
- Cells carrying mutations or polymorphisms in the gene of the invention may also be detected at the DNA level by a variety of techniques, to allow for serotyping, for example.
- RT-PCR can be used to detect mutations. It is particularly preferred to used RT-PCR in conjunction with automated detection systems, such as, for example, GeneScan.
- RNA or cDNA may also be used for the same purpose, PCR or RT-PCR.
- PCR primers complementary to a nucleic acid encoding spoIIIB can be used to identify and analyze mutations.
- the invention further provides these primers with 1, 2, 3 or 4 nucleotides removed from the 5′ and/or the 3′ end.
- These primers may be used for, among other things, amplifying spoIIIE DNA isolated from a sample derived from an individual.
- the primers may be used to amplify the gene isolated from an infected individual such that the gene may then be subject to various techniquLes for elucidation of the DNA sequence. In this way, mutations in the DNA sequence may be detected and used to diagnose infection and to serotype and/or classify the infectious agent.
- the invention further provides a process for diagnosing, disease, preferably bacterial infections, more preferably infections by Streptococcus pneumoniae, and most preferably otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid, comprising determining from a sample derived from an individual a increased level of expression of polynucleotide having the sequence of Table 1 [SEQ ID NO: 1].
- Increased or decreased expression of spoIIIE polynucleotide can be measured using any on of the methods well known in the art for the quantation of polynucleotides, such as, for example, amplification, PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods.
- a diagnostic assay in accordance with the invention for detecting over-expression of spoIIIE protein compared to normal control tissue samples may be used to detect the presence of an infection, for example.
- Assay techniques that can be used to determine levels of a spoIIIE protein, in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays.
- polypeptides of the invention or variants thereof, or cells expressing them can be used as an immunogen to produce antibodies immunospecific for such polypeptides.
- Antibodies as used herein includes monoclonal and polyclonal antibodies, chimeric, single chain, simianized antibodies and humanized antibodies, as well as Fab fragments, including the products of an Fab immunoglobulin expression library.
- Antibodies generated against the polypeptides of the invention can be obtained by administering the polypeptides or epiptope-bearing fragments, analogues or cells to an animal, preferably a nonhuman, using routine protocols.
- an animal preferably a nonhuman
- any technique known in the art that provides antibodies produced by continuous cell line cultures can be used. Examples include various techniques, such as those in Kohler, G. and Milstein, C., Nature 256: 495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).
- phage display technology may be utilized to select antibody genes with binding activities towards the polypeptide either from repertoires of PCR amplified v-genes of Iymphocytes from humans screened for possessing anti-spoIIIE or from naive libraries (McCafferty, J. et al., (1990), Nature 348, 552-554; Marks, J. et al., (1992) Biotechnology 10, 779-783).
- the affinity of these antibodies can also be improved by chain shuffling (Clackson, T. et al., (1991) Nature 352, 624-628).
- each domain may be directed against a different epitope-termed ‘bispecific’ antibodies.
- the above described antibodies may be employed to isolate or to identify clones expressing the polypeptides to purify the polypeptides by affinity chromatography.
- antibodies against spoIIIE polypeptide may be employed to treat infections, particularly bacterial infections and especially otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid.
- Polypeptide variants include antigenically, epitopically or immunologically equivalent variants that form a particular aspect of this invention.
- the term “antigenically equivalent derivative” as used herein encompasses a polypeptide or its equivalent which will be specifically recognized by certain antibodies which, when raised to the protein or polypeptide according to the invention, interfere with the immediate physical interaction between pathogen and mammalian host.
- the term “immunologically equivalent derivative” as used herein encompasses a peptide or its equivalent which when used in a suitable formulation to raise antibodies in a vertebrate, the antibodies act to interfere with the immediate physical interaction between pathogen and mammalian host.
- the polypeptide such as an antigenically or immunologically equivalent derivative or a fusion protein thereof is used as an antigen to immunize a mouse or other animal such as a rat or chicken.
- the fusion protein may provide stability to the polypeptide.
- the antigen may be associated, for example by conjugation, with an immunogenic carrier protein for example bovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH).
- BSA bovine serum albumin
- KLH keyhole limpet haemocyanin
- a multiple antigenic peptide comprising multiple copies of the protein or polypeptide, or an antigenically or immunologically equivalent polypeptide thereof may be sufficiently antigenic to improve immunogenicity so as to obviate the use of a carrier.
- the antibody or variant thereof is modified to make it less immunogenic in the individual.
- the antibody may most preferably be “humanized ”; where the complimentary determining region(s) of the hybridoma-derived antibody has been transplanted into a human monoclonal antibody, for example as described in Jones, P. et al. (1986), Nature 321,522-525 or Tempest et al.,(1991) Biotechnology 9,266-273.
- a polynucleotide of the invention in genetic immunization will preferably employ a suitable delivery method such as direct injection of plasmid DNA into muscles (Wolff et al., Hum Mol Genet 1992, 1:363, Manthorpe et al., Hum. Gene Ther. 1963.4, 419), delivery of DNA complexed with specific protein carriers (Wu et al., J Biol Chem.
- Antagonists and Agonists Assays and Molecules
- Polypeptides of the invention may also be used to assess the binding of small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures.
- substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics. See, e.g., Coligan et al., Current Protocols in Immunology 1(2): Chapter 5 (1991).
- the invention also provides a method of screening compounds to identify those which enhance (agonist) or block (antagonist) the action of spoIIIE polypeptides or polynucleotides, particularly those compounds that are bacteriostatic and/or bacteriocidal.
- the method of screening may involve high throughput techniques. For example, to screen for agonists or antagonists, a synthetic reaction mix, a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof comprising spoIIIE polypeptide and a labeled substrate or ligand of such polypeptide is incubated in the absence or the presence of a candidate molecule that may be a spoIIIE agonist or antagonist.
- the ability of the candidate molecule to agonize or antagonize the spoIIIE polypeptide is reflected in decreased binding of the labeled ligand or decreased production of product from such substrate.
- Molecules that bind gratuitously, i.e., without inducing the effects of spoIIIE polypeptide are most likely to be good antagonists.
- Molecules that bind well and increase the rate of product production from substrate are agonists. Detection of the rate or level of production of product from substrate may be enhanced by using a reporter system.
- Reporter systems that may be useful in this regard include but are not limited to colorimetric labeled substrate converted into product, a reporter gene that is responsive to changes in spoIIIE polynucleotide or polypeptide activity, and binding assays known in the art.
- an assay for spoIIIE antagonists is a competitive assay that combines spoIIIE and a potential antagonist with spoIIIE-binding molecules, recombinant spoIIIE binding molecules, natural substrates or ligands, or substrate or ligand mimetics, under appropriate conditions for a competitive inhibition assay.
- the spoIIIE molecule can be labeled, such as by radioactivity or a colorimetric compound, such that the number of spoIIIE molecules bound to a binding molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist.
- Potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to a polynucleotide or polypeptide of the invention and thereby inhibit or extinguish its activity. Potential antagonists also may be small organic molecules, a peptide, a polypeptide such as a closely related protein or antibody that binds the same sites on a binding molecule, such as a binding molecule, without inducing spoIIIE-induced activities, thereby preventing the action of spoIIIE by excluding spoIIIE from binding.
- Potential antagonists include a small molecule that binds to and occupies the binding site of the polypeptide thereby preventing binding to cellular binding molecules, such that normal biological activity is prevented.
- small molecules include but are not limited to small organic molecules, peptides or peptide-like molecules.
- Other antagonists include antisense molecules (see Okano, J. Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988), for a description of these molecules).
- Preferred postal antagonists include compounds related to and variants of spoIIIE.
- Each of the DNA sequences provided herein may be used in the discovery and development of antibacterial compounds.
- the encoded protein upon expression, can be used as a target for the screening of antibacterial drugs.
- the DNA sequences encoding the amino terminal regions of the encoded protein or Shine-Delgarno or other translation facilitating sequences of the respective mRNA can be used to construct antisense sequences to control the expression of the coding sequence of interest.
- the invention also provides the use of the polypeptide, polynucleotide or inhibitor of the invention to interfere with the initial physical interaction between a pathogen and mammalian host responsible for sequence of infection.
- the molecules of the invention may be used: in the prevention of adhesion of bacteria, in particular gram positive bacteria, to mammalian extracellular matrix proteins on in-dwelling devices or to extracellular matrix proteins in wounds; to block spoIIIE protein-mediated mammalian cell invasion by, for example, initiating phosphorylation of mammalian tyrosine kinases (Rosenshine et al., Infect. Immun.
- the antagonists and agonists of the invention may be employed, for instance, to inhibit and treat otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly menigitis, such as for example infection of cerebrospinal fluid.
- Another aspect of the invention relates to a method for inducing an immunological response in an individual, particularly a mammal which comprises inoculating the individual with spoIIIE, or a fragment or variant thereof, adequate to produce antibody and/ or T cell immune response to protect said individual from infection, particularly bacterial infection and most particularly Streptococcus pneumoniae infection. Also provided are methods whereby such immunological response slows bacterial replication.
- Yet another aspect of the invention relates to a method of inducing immunological response in an individual which comprises delivering to such individual a nucleic acid vector to direct expression of spoIIIE, or a fragment or a variant thereof, for expressing spoIIIE, or a fragment or a variant thereof in vivo in order to induce an immunological response, such as, to produce antibody and/ or T cell immune response, including, for example, cytokine-producing T cells or cytotoxic T cells, to protect said individual from disease, whether that disease is already established within the individual or not.
- an immunological response such as, to produce antibody and/ or T cell immune response, including, for example, cytokine-producing T cells or cytotoxic T cells, to protect said individual from disease, whether that disease is already established within the individual or not.
- One way of administering the gene is by accelerating it into the desired cells as a coating on particles or otherwise.
- Such nucleic acid vector may comprise DNA, RNA, a modified nucleic acid, or a DNA/RNA hybrid.
- a further aspect of the invention relates to an immunological composition which, when introduced into an individual capable or having induced within it an immunological response, induces an immunological response in such individual to a spoIIIE or protein coded therefrom, wherein the composition comprises a recombinant spoIIIE or protein coded therefrom comprising DNA which codes for and expresses an antigen of said spoIIIE or protein coded therefrom.
- the immunological response may be used therapeutically or prophylactically and may take the form of antibody immunity or cellular immunity such as that arising from CTL or CD4+T cells.
- a spoIIIE polypeptide or a fragment thereof may be fused with co-protein which may not by itself produce antibodies, but is capable of stabilizing the first protein and producing a fused protein which will have immunogenic and protective properties.
- fused recombinant protein preferably further comprises an antigenic co-protein, such as lipoprotein D from Hemophilus influenzae, Glutathione-S-transferase (GST) or beta-galactosidase, relatively large co-proteins which solubilize the protein and facilitate production and purification thereof.
- the co-protein may act as an adjuvant in the sense of providing a generalized stimulation of the immune system.
- the co-protein may be attached to either the amino or carboxy terminus of the first protein.
- compositions particularly vaccine compositions, and methods comprising the polypeptides or polynucleotides of the invention and immunostimulatory DNA sequences, such as those described in Sato, Y. et al. Science 273: 352 (1996).
- the polypeptide may be used as an antigen for vaccination of a host to produce specific antibodies which protect against invasion of bacteria, for example by blocking adherence of bacteria to damaged tissue.
- tissue damage include wounds in skin or connective tissue caused, e.g., by mechanical, chemical or thermal damage or by implanation of indwelling devices, or wounds in the mucous membranes, such as the mouth, mammary glands, urethra or vagina.
- the invention also includes a vaccine formulation which comprises an immunogenic recombinant protein of the invention together with a suitable carrier. Since the protein may be broken down in the stomach, it is preferably administered parenterally, including, for example, administration that is subcutaneous, intramuscular, intravenous, or intradermal.
- Formulations suitable for parenteral administration include aqueous and nonaqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation insotonic with the bodily fluid, preferably the blood, of the individual; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents.
- the formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use.
- the vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
- the invention also relates to compositions comprising the polynucleotide or the polypeptides discussed above or their agonists or antagonists.
- the polypeptides of the invention may be employed in combination with a non-sterile or sterile carrier or carriers for use with cells, tissues or organisms, such as a pharmaceutical carrier suitable for administration to a subject.
- Such compositions comprise, for instance, a media additive or a therapeutically effective amount of a polypeptide of the invention and a pharmaceutically acceptable carrier or excipient.
- Such carriers may include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof.
- the formulation should suit the mode of administration.
- the invention further relates to diagnostic and pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.
- Polypeptides and other compounds of the invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
- compositions may be administered in any effective, convenient manner including, for instance, administration by topical, oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes among others.
- the active agent may be administered to an individual as an injectable composition, for example as a sterile aqueous dispersion, preferably isotonic.
- the composition may be formulated for topical application for example in the form of ointments, creams, lotions, eye ointments, eye drops, ear drops, mouthwash, impregnated dressings and sutures and aerosols, and may contain appropriate conventional additives, including, for example, preservatives, solvents to assist drug penetration, and emollients in ointments and creams.
- Such topical formulations may also contain compatible conventional carriers, for example cream or ointment bases, and ethanol or oleyl alcohol for lotions.
- Such carriers may constitute from about 1% to about 98% by weight of the formulation; more usually they will constitute up to about 80% by weight of the formulation.
- the daily dosage level of the active agent will be from 0.01 mg/kg to 10 mg/g, typically around 1 mg/kg.
- the physician in any event will determine the actual dosage which will be most suitable for an individual and will vary with the age, weight and response of the particular individual.
- the above dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
- In-dwelling devices include surgical implants, prosthetic devices and catheters, i.e., devices that are introduced to the body of an individual and remain in position for an extended time.
- Such devices include, for example, artificial joints, heart valves, pacemakers, vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinary catheters, continuous ambulatory peritoneal dialysis (CAPD) catheters.
- CAD continuous ambulatory peritoneal dialysis
- composition of the invention may be administered by injection to achieve a systemic effect against relevant bacteria shortly before insertion of an in-dwelling device. Treatment may be continued after surgery during the in-body time of the device.
- the composition could also be used to broaden perioperative cover for any surgical technique to prevent bacterial wound infections, especially Streptococcus pneumoniae wound infections.
- compositions of this invention may be used generally as a wound treatment agent to prevent adhesion of bacteria to matrix proteins exposed in wound tissue and for prophylactic use in dental treatment as an alternative to, or in conjunction wit, antibiotic prophylaxis.
- composition of the invention may be used to bathe an indwelling device immediately before insertion.
- the active agent will preferably be present at a concentration of 1 ⁇ g/ml to 10 mg/ml for bathing of wounds or indwelling devices.
- a vaccine composition is conveniently in injectable form. Conventional adjuvants may be employed to enhance the immune response.
- a suitable unit dose for vaccination is 0.5-5 microgram/kg of antigen, and such dose is preferably administered 1-3 times and with an interval of 1-3 weeks. With the indicated dose range, no adverse toxicological effects will be observed with the compounds of the invention which would preclude their administration to suitable individuals.
- the polynucleotide having the DNA sequence given in SEQ ID NO:1 was obtained from a library of clones of chromosomal DNA of Streptococcus pneumoniae in E. coli.
- the sequencing data from two or more clones contain overlapping Streptococcus pneumoniae DNAs was used to construct the contiguous DNA sequence in SEQ ID NO:1.
- Libraries may be prepared by routine methods, for example:
- Total cellular DNA is isolated from Streptococcus pneumoniae 0100993 according to standard procedures and size-fractionated by either of two methods.
- Total cellular DNA is mechanically sheared by passage through a needle in order to size fractionate according to standard procedures.
- DNA fragments of up to 11 kbp in size are rendered blunt by treatment with exonuclease and DNA polymerase, and EcoRI linkers added. Fragments are ligated into the vector Lambda ZapII that has been cut with EcoRI, the library packaged by standard procedures and E. coli infected with the packaged library.
- the library is amplified by standard procedures.
- Total cellular DNA is partially hydrolyzed with a one or a combination of restriction enzymes appropriate to generate a series of fragments for cloning into library vectors (e.g., RsaI, PalI, AluI, Bsh1235I), and such fragments are size-fractionated according to standard procedures.
- EcoRI linkers are ligated to the DNA and the fragments then ligated into the vector Lambda ZapII that have been cut with EcoRI, the library packaged by standard procedures, and E.coli infected with the packaged library.
- the library is amplified by standard procedures.
- SpoIIIE is a membrane bound protein involved in chromosome partitioning during sporulation and vegetative replication in a wide variety of bacteria.
- the spoIIIE gene was initially characterized in Bacillus subtilis (Butler P. D. and Mandelstam J. (1987) Journal of General Microbiology 133:2359-2370).
- SpoIIIE protein has an ATP binding site and is membrane-bound, and appears to form a pore in the nascent spore septum, through which the prespore chromosome is driven in a conjugation-like mechanism (Wu L. J., Lewis P. J., Allmansberger R., Hauser P. M. and Errington J. (1995) Genes and Development 9:1316-1326).
- spoIIIE mutants cannot sporulate as they are unable to partition the prespore chromosome into the polar prespore compartment. Instead a specific chromosomal segment comprising approximately 30% of the chromosome enters the prespore, while the rest remains in the mother cell, trapped by the septum (Wu L. J. and Errington J. (1994) Science 264:572-575).
- SpoIIIE is membrane-bound, and appears to form a pore in the nascent spore septum, through which the prespore chromosome is driven in a conjugation-like mechanism (Wu L. J., Lewis P. J., Allmansberger R., Hauser P. M. and Errington J. (1995) Genes and Development 9:1316-1326).
- SpoIIIE is also required for correct partitioning of the B.subtilis chromosome during vegetative cell division. SpoIIIE-mutants in which replication has been artificially delayed are unable to separate the replicated chromosomes before septum formation, resulting in a trapped nucleoid similar to that formed at the start of sporulation (Sharpe M. E. and Errington J. (1995) Proceedings of the Natural Academy of Sciences USA 92:8630-8634).
- SpoIIIE has been shown to be essential in Escherichia coli (Begg K. J., Dewar, S. J. and Donachie W. D. (1995) Journal of Bacteriology 177:6211-6222). Highly conserved SpoIIIE homologues are found in diverse members of the eubacteria such as Campylobacter jejuni (Miller, S., Pesci E. C. and Pickett C. L. (1994) Gene 146:31-38), Coxiella burnetii (Oswald W. and Thiele D. (1993) Journal of Veterinary Medecine B40:366-370), Escherichia coli (Begg 1995 above) and Haemophilus influenzae (Fleischmann, R.
- Streptococcus pneumoniae spoIIIE in infection is demonstrated by the fact that the gene has been shown to be expressed in vivo in a mouse model using the following methodology: Streptococcus pneumoniae 0100993 cultures resuspended in phosphate-buffered saline are administered intranasally to mice. After 48 hours, the infected mouse lungs are aseptically removed, snap-frozen in liquid nitrogen and treated to release total RNA. Bacterial cDNA is made from the mRNA component by reverse transcription, then subjected to PCR using primers specific to spoIIIE. The spoIIIE transcript was detected, demonstrating that this gene is expressed during the infection.
- Inhibitors of SpoIIIE proteins would prevent the bacterium from establishing and maintaining infection of the host by preventing it from correctly partitioning the chromosome in the manner described above and thus arresting cell division and growth, rendering the bacterium susceptible to host defences and leading ultimately to cell death and thereby have utility in anti-bacterial therapy.
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Abstract
The invention provides spoIIIE polypeptides and DNA (RNA) encoding spoIIIE polypeptides and methods for producing such polypeptides by recomombinant techniques. Also provided are methods for utilizing spoIIIE polypeptides to screen for antibacterial compounds.
Description
- This invention relates to newly identified polynucleotides and polypeptides, and their production and uses, as well as their variants, agonists and antagonists, and their uses. In particular, in these and in other regards, the invention relates to novel polynucleotides and polypeptides of the spoIIIE/ftsK (putative chromosomal partitioning function) family, hereinafter referred to as “spoIIIE”.
- The Streptococci make up a medically important genera of microbes kwown to cause several types of disease in humans, including, for example, otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid. Since its isolation more than 100 years ago,Streptococcus pneumoniae has been one of the more intensively studied microbes. For example, much of our early understanding that DNA is, in fact, the genetic material was predicated on the work of Griffith and of Avery, Macleod and McCarty using this microbe. Despite the vast amount of research with Streptococcus pneumoniae, many questions concerning the virulence of this microbe remain. It is particularly preferred to employ Streptococcal genes and gene products as targets for the development of antibiotics.
- The frequency ofStreptococcus pneumoniae infections has risen dramatically in the past 20 years. This has been attributed to the emergence of multiply antibiotic resistant strains and an increasing population of people with weakened immune systems. It is no longer uncommon to isolate Streptococcus pneumoniae strains which are resistant to some or all of the standard antibiotics. This has created a demand for both new anti-microbial agents and diagnostic tests for this organism.
- Clearly, there is a need for factors, such as the novel compounds of the invention, that have a present benefit of being useful to screen compounds for antibiotic activity. Such factors are also useful to determine their role in pathogenesis of infection, dysfunction and disease. There is also a need for identification and characterization of such factors and their antagonists and agonists which can play a role in preventing, ameliorating or correcting infections, dysfunctions or diseases.
- The polypeptides of the invention have amino acid sequence homology to a knownStaphylococcus aureus spoIIIE protein.
- It is an object of the invention to provide polypeptides that have been identified as novel spoIIIE polypeptides by homology between the amino acid sequence set out in Table 1 [SEQ ID NO: 2] and a known amino acid sequence or sequences of other proteins such asStaphylococcus aureus spoIIIE protein.
- It is a further object of the invention to provide polynucleotides that encode spoIIIE polypeptides, particularly polynucleotides that encode the polypeptide herein designated spoIIIE.
- In a particularly preferred embodiment of the invasion, the polynucleotide comprises a region encoding spoIIIE polypeptides comprising the sequence set out in Table 1 [SEQ ID NO:1] which includes a full length gene, or a variant thereof.
- In another particularly preferred embodiment of the invention, there is a novel spoIIIE protein fromStreptococcus pneumoniae comprising the amino acid sequence of Table 1 [SEQ ID NO:2], or a variant thereof.
- In accordance with another aspect of the invention, there is provided an isolated nucleic acid molecule encoding a mature polypeptide expressible by theStreptococcus pneumoniae 0100993 strain contained in the deposited strain.
- As a further aspect of the invention, there are provided isolated nucleic acid molecules encoding spoIIIE, particularlyStreptococcus pneumoniae spoIIIE, including mRNAs, cDNAs, genomic DNAs. Further embodiments of the mention include biologically, diagnostically, prophylactically, clinically or therapeutically useful variant thereof, and compositions comprising the same.
- In accordance with another aspect of the invention, there is provided the use of a polynucleotide of the invention for therapeutic or prophylactic purposes, in particular genetic immunization. Among the particularly preferred embodiments of the invention are naturally occurring allelic variants of spoIIIE and polypeptides encoded thereby.
- As another aspect of the invention, there are provided novel polypeptides ofStreptococcus pneumoniae referred to herein as spoIIIE as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comprising the same.
- Among the particularly preferred embodiments of the invention are variants of SpoIIIE polypeptide encoded by naturally occurring alleles of the spoIIIE gene.
- In a preferred embodiment of the invention, there are provided methods for producing the aforementioned spoIIIE polypeptides.
- In accordance with yet another aspect of the invention, there are provided inhibitors to such polypeptides, useful as antibacterial agents, including, for example, antibodies.
- In accordance with certain preferred embodiments of the invention, there are provided products, compositions and methods for assessing spoIIIE expression, treating disease, for example, otitis media, cojunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid, assaying genetic variation, and administering a spoIIIE polypeptide or polynucleotide to an organism to raise an immunological response against a bacteria, especially aStreptococcus pneumoniae bacteria.
- In accordance with cetain preferred embodiments of this and other aspects of the invention, there are provided polynucleotides that hybridize to spoIIIE polynucleotide sequences, particularly under stringent conditions.
- In certain preferred embodiments of the invention, there are provided antibodies against spoIIIE polypeptides.
- In other embodiments of the invention, there are provided methods for identifying compounds which bind to or otherwise interact with and inhibit or activate an activity of a polypeptide or polynucleotide of the invention comprising a polypeptide or polynucleotide of the invention with a compound to be screened under conditions to permit binding to or other interaction between the compound and the polypeptide or polynucleotide to assess the binding to or other interaction with the compound, such binding or interaction being associated with a second component capable of providing a detectable signal in response to the binding or interaction of the polypeptide or polynucleotide with the compound; and determining whether the compound binds to or otherwise interacts with and activates or inhibits an activity of the polypeptide or polynucleotide by detecting the presence or absence of a signal generated from the binding or interaction of the compound with the polypeptide or polynucleotide.
- It accordance with yet another aspect of the invention, there are provided spoIIIE agonists and antagonists, preferably bacteriostatic or bacteriocidal agonists and antagonists.
- In a further aspect of the invention, there are provided compositions comprising a spoIIIE polynucleotide or a spoIIIE polypeptide for administration to a cell or to a multicellular organism.
- Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following descriptions and from reading the other parts of the present disclosure.
- The following definitions are provided to facilitate understanding of certain terms used frequently herein.
- “Host cell” is a cell which has been transformed or transfected, or is capable of transformation or transfection by an exogenous polynucleotide sequence.
- “Identity,” as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, “identify” also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. “Identify” and “similarity” can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990). As an illustration, by a polynucleotide having a nucleotide sequence having at least, for example, 95% “identity” to a reference nucleotide sequence of SEQ ID NO: 1 it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence of SEQ ID NO: 1. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5 or 3 terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. Analogously, by a polypeptide having an amino acid sequence having at least, for example, 95% identity to a reference amino acid sequence of SEQ ID NO:2 is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of SEQ ID NO: 2. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
- “Isolated” means altered “by the hand of man” from its natural state, i.e., if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living organism is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein.
- “Polynucleotide(s)” generally refers to any polyribonucleotide or polydeoxiribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. “Polynucleotide(s)” include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double- and triple-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or triple-stranded regions, or a mixture of single- and double-stranded regions. In addition, “polynucleotide” as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. As used herein, the term “polynucleotide(s)” also includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are “polynucleotide(s)” as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term “polynucleotide(s)” as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including, for example, simple and complex cells. “Polynucleotide(s)” also embraces short polynucleotides often referred to as oligonucleotide(s).
- “Polypeptide(s)” refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds. “Polypeptide(s)” refers to both short chains, commonly referred to as peptides, oligonucleotides and oligomers and to longer chains generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene encoded amino acids. “Polypeptide(s)” include those modified either by natural processes, such as processing and other post translational modifications, but also by chemical modification techniques. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature, and they are well known to those of skill in the art. It will be appreciated that the same type of modification may be present in the same or varying degree at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini. Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalant attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosilation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins, such as arginylation, and ubiquitination. See, for instance,PROTEINS-STRUCTURE AnD MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. R Freeman and Company, New York (1993) and Wold, F., Posttranslational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed, Academic Press, New York (1983); Seifter et al., Meth. Enzymol. 182:626-646 (1990) and Rattan et al., Protein Synthesis: Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663: 48-62 (1992). Polypeptides may be branched or cyclic, with or without branching. Cyclic, branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well.
- “Variant(s)” as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties. A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to skilled artisans.
- The invention relates to novel spoIIIE polypeptides and polynucleotides as described in greater detail below. In particular, the invention relates to polypeptides and polynucleotides of a novel spoIIIE ofStreptococcus pneumoniae, which is related by amino acid sequence homology to Staphylococcus aureus spoIIIE polypeptide. The invention relates especially to spoIIIE having the nucleotide and amino acid sequences set out in Table 1 [SEQ ID NO: 1] and Table 1 [SEQ ID NO: 2] respectively, and to the spoIIIE nucleotide sequences of the DNA in the deposited strain and amino acid sequences encoded thereby.
TABLE 1 spoIIIE Polynueleolide and Polypeptide Sequences (A) Sequences from Strepococcus pneumoniae spoIIIE polynucleotide sequence [SEQ ID NO:1]. 5′- GTCTTTTTCTCTTCCCGAAATGCCTGTGAAATATGGTATAATAGAAGAATGGCAAACAAGAATACAAGTA CAACAAGACGGAGACCGTCTAAAGCAGAACTGGAAAGAAAAGAAGCGATTCAACGAATGTTGATTTCGTT AGGAATTGCGATTTTATTGATTTTCGCAGCCTTCAAATTAGGGGCTGCAGGTATAACCCTTTATAATTTA ATTCGCTTGCTAGTGGGTAGCCTAGCTTATCTGGCGATATTCGGCCTATTAATCTATCTCTTCTTTTTCA AGTGGATACGAAAACAGGAAGGACTCTTATCTGGCTTTTTCACCATATTTGCTGGCTTACTCTTGATTTT TGAGGCCTACTTGGTTTGGAAATATGGTTTGGACAAGTCCGTTCTAAAAGGGACCATGGCTCAGGTTGTG ACAGATCTGACTGGTTTTCGAACGACTAGCTTTGCTGGAGGGGGCTTGATCGGGGTCGCTCTTTATATTC CAACAGCCTTTCTCTTTTCAAATATCGGAACTTACTTTATTGGTTCTATCTTGATTTTAGTGGGTTCTCT CCTAGTCAGCCCTTGGTCTGTTTACGATATTGCTGAATTTTTCAGTAGAGGCTTTGCCAAATGGTGGGAA GGGCACGAGCGTCGAAAAGAGGAACGCTTTGTCAAACAAGAAGAAAAAGCTCGCCAAAAGGCTGAGAAAG AGGCTAGATTAGAACAA AGAGACTGAAAAAGCCTTACTCGATTTGCC CTGTTGATATGGAAACGGG TGAAATTCTGACAGAGGAAGCTGTTCAAAATCTTCCACCTATTCCAGAAGAAAAGTGGGTGGAACCAGAA ATCATCCTGCCTCAAGCTGAACTTAAATTCCCTGAACAGGAAGATGACTCAGATGACGAAGATGTTCAGG TCGATTTTTCAGCCAAAGAAGCCCTTGAATACAAACTTCCAAGCTTACAACTCTTTGCACCAGATAAACC AAAAGATCAGTCTAAAGAGAAGAAAATTGTCAGAGAAAATATCAAAATCTTAGAAGCAACCTTTGCTAGC TTTGGTATTAAGGTAACAGTTGAACGGGCCGAAATTGGGCCATCAGTGACCAAGTATGAAGTCAAGCCGG CTGTTGGTGTAAGGGTCAACCGCATTTCCAATCTATCAGATGACCTCGCTCTAGCCTTGGCTGCCAAGGA TGTCCGAATTGAAGCACCAATCCCTGGGAAATCTCTAATCGGAATTGAAGTGCCCAACTCCGATATTGCC ACTGTATCTTTCCGAGAACTATGGGAACAATCGCAAACGAAAGCAGAAAATTTCTTGGAAATTCCTTTAG GGAAGGCTGTTAATGGAACCGCAAGAGCTTTTGACCTTTCTAAAATGCCCCACTTGCTAGTTGCAGGTTC AACGGGTTCAGGGAAGTCAGTAGCAGTTAACGGCATTATTGCTAGCATTCTCATGAAGGCGAGACCAGAT CAAGTTAAATTTATGATGGTCGATCCCAAGATGGTTGAGTTATCTGTTTACAATGATATTCCCCACATAA AGATTCCAGTCGTGACCAATCCACGCAAAGCCAGCAAGGCTCTGCAAAAGGTTGTGGATGAAATGGAAAA CCGTTATGAACTCTTTGCCAAGGTGGGAGTTCGGAATATTGCAGGTTTTAATGCCAAGGTAGAAGAGTTC AATTCCCAGTCTGAGTACAAGCAAATTCCGCTACCATTCATTGTCGTGATTGTGGATGAGTTGGCTGACC TCATGATGGTGGCCAGCAAGGAAGTGGAAGATGCTATCATCCGTCTTGGGCAGAAGGCGCGTGCTGCAGG TATCCACATGATTCTTGCAACTCAGCGTCCATCTGTTGATGTCATCTCTGGTTTGATTAAGGCCAATGTT CCATCTCGTGTAGCATTTGCGGTTTCATCAGGAACAGACTCCCGTACGATTTTGGATGAAAATGGAGCAG AAAAACTTCTTGGTCGAGGAGACATGCTCTTTAAACCGATTAATGAAAATCATCCAGTTCGTCTCCAAGG CTCCTTTATCTCGGATGACGATGTTGAGCGCATTGTGAACTTCATCAAGACTCAGGCAGATGCAGACTAC GATGAGAGTTTTGATCCAGGTGAGGTTTCTGAAAATGAAGGAGAATTTTCGGATGGAGATGCTGGTGGTG ATCCGCTTTTTGAAGAAGCTAAGTCTTTGGTTATCGAAACACAGAAAGCCAGTGCATCCATGATTCAGCG TCGTTTGTCAGTTGGATTTAACCGTGCGACCCGTCTCATGGAAGAGCTTGAGATGGCAGGTGTCATTGGT CCAGCTGAAGGTACCAAGCCAAGAAAAGTATTGCAGCAATAA-3′ (B) spoIIIE polypeptide sequence deduced from the polynuoleotide sequence in this table [SEQ ID NO:2]. NH2-VFFSSRNACE IWYNRRMANK NTSTTRRRPS KAELERKEAI QRMLISLGIA ILLIFAAFKL GAAGITLYNL IRLLVGSLAY LAIFGLLIYL FFFKWIRKQE GLLSGFFTIF AGLLLIFEAY LVWKYGLDKS VLKGTMAQVV TDLTGFRTTS FAGGGLIGVA LYIPTAFLFS NIGTYFIGSI LILVGSLLVS PWSVYDIAEF FSRGFAKWWE GHERRKEERF VKQEEKARQK AEKEARLEQE ETEKALLDLP PVDMETGEIL TEEAVQNLPP IPEEKWVEPE IILPQAELKF PEQEDDSDDE DVQVDFSAKE ALEYKLPSLQ LFAPDKPKDQ SKEKKIVREN IKILEATFAS FGIKVTVEPA EIGPSVTKYE VKPAVGVRVN RISNLSDDLA LALAAKDVRI EAPIPGKSLI GIEVPNSDIA TVSFRELWEQ SQTKAENFLE IPLGKAVNGT ARAFDLSKMP HLLVAGSTGS GKSVAVNGII ASILMKARPD QVKFMMVDPK MVELSVYNDI PHIKIPVVTN PRKASKALQK VVDEMENRYE LFAKVGVRNI AGFNAKVEEF NSQSEYKQIP LPFIVVIVDE LADLMMVASK EVEDAIIPIG QKARAAGIHM ILATQ VD VISGLIKANV PSRVAFAVSS GTDSRTILDE NGAEKLLGRG DMLFKPINEN HPVRLQGSFI SDDDVERIVN FIKTQADADY DESFDPGEVS ENEGEFSDGD AGGDPLFEEA KSLVIETQKA SASMIQRRLS VGFNRATRLM EELEMAGVIG PAEGTKPRKV LQQ -COOH (C) Polynucleotide sequence embodiments [SEQ ID NO:1]. X-(R1)n- GTCTTTTTCTCTTCCCGAAATGCCTGTGAAATATGGTATAATAGAAGAATGGCAAACAAGAATACAAGTA CAACAAGACGGAGACCGTCTAAAGCAGAACTGGAAAGAAAAGAAGCGATTCAACGAATGTTGATTTCGTT AGGAATTGCGATTTTATTGATTTTCGCAGCCTTCAAATTAGGGGCTGCAGGTATAACCCTTTATAATTTA ATTCGCTTGCTAGTGGGTAGCCTAGCTTATCTGGCGATATTCGGCCTATTAATCTATCTCTTCTTTTTCA AGTGGATACGAAAACAGGAAGGACTCTTATCTGGCTTTTTCACCATATTTGCTGGCTTACTCTTGATTTT TGAGGCCTACTTGGTTTGGAAATATGGTTTGGACAAGTCCGTTCTAAAAGGGACCATGGCTCAGGTTGTG ACAGATCTGACTGGTTTTCGAACGACTAGCTTTGCTGGAGGGGGCTTGATCGGGGTCGCTCTTTATATTC CAACAGCCTTTCTCTTTTCAAATATCGGAACTTACTTTATTGGTTCTATCTTGATTTTAGTGGGTTCTCT CCTAGTCAGCCCTTGGTCTGTTTACGATATTGCTGAATTTTTCAGTAGAGGCTTTGCCAAATGGTGGGAA GGGCACGAGCGTCGAAAAGAGGAACGCTTTGTCAAACAAGAAGAAAAAGCTCGCCAAAAGGCTGAGAAAG AGGCTAGATTAGAACAAGAAGAGACTGAAAAGCCTTACTCGATTTGCCTCCTGTTGATATGGAAACGGG TGAAATTCTGACAGAGGAAGCTGTTCAAAATCTTCCACCTATTCCAGAAGAAAAGTGGGTGGAACCAGAA ATCATCCTGCCTCAAGCTGAACTTAAATTCCCTGAACAGGAAGATGACTCAGATGACGAAGATGTTCAGG TCGATTTTTCAGCCAAAGAAGCCCTTGAATACAAACTTCCAAGCTTACAACTCTTTGCACCAGATAAACC AAAAGATCAGTCTAAAGAGAAGAAAATTGTCAGAGAAAATATCAAAATCTTAGAAGCAACCTTTGCTAGC TTTGGTATTAAGGTAACAGTTGAACGGGCCGAAATTGGGCCATCAGTGACCAAGTATGAAGTCAAGCCGG CTGTTGGTGTAAGGGTCAACCGCATTTCCAATCTATCAGATGACCTCGCTCTAGCCTTGGCTGCCAAGGA TGTCCGAATTGAAGCACCAATCCCTGGGAAATCTCTAATCGGAATTGAAGTGCCCAACTCCGATATTGCC ACTGTATCTTTCCGAGAACTATGGGAACAATCGCAAACGAAAGCAGAAAATTTCTTGGAAATTCCTTTAG GGAAGGCTGTTAATGGAACCGCAAGAGCTTTTGACCTTTCTAAAATGCCCCACTTGCTAGTTGCAGGTTC AACGGGTTCAGGGAAGTCAGTAGCAGTTAACGGCATTATTGCTAGCATTCTCATGAAGGCGAGACCAGAT CAAGTTAAATTTATGATGGTCGATCCCAAGATGGTTGAGTTATCTGTTTACAATGATATTCCCCACATAA AGATTCCAGTCGTGACCAATCCACGCAAAGCCAGCAAGGCTCTGCAAAAGGTTGTGGATGAAATGGAAAA CCGTTATGAACTCTTTGCCAAGGTGGGAGTTCGGAATATTGCAGGTTTTAATGCCAAGGTAGAAGAGTTC AATTCCCAGTCTGAGTACAAGCAAATTCCGCTACCATTCATTGTCGTGATTGTGGATGAGTTGGCTGACC TCATGATGGTGGCCAGCAAGGAAGTGGAAGATGCTATCATCCGTCTTGGGCAGAAGGCGCGTGCTGCAGG TATCCACATGATTCTTGCAACTCAGCGTCCATCTGTTGATGTCATCTCTGGTTTGATTAAGGCCAATGTT CCATCTCGTGTAGCATTTGCGGTTTCATCAGGAACAGACTCCCGTACGATTTTGGATGAAAATGGAGCAG AAAAACTTCTTGGTCGAGGAGACATGCTCTTTAAACCGATTAATGAAAATCATCCAGTTCGTCTCCAAGG CTCCTTTATCTCGGATGACGATGTTGAGCGCATTGTGAACTTCATCAAGACTCAGGCAGATGCAGACTAC GATGAGAGTTTTGATC GTGAGGTTTCTGAAAATGAAGGAGAATTTTCGGATGGAGATGCTGGTGGTG ATCCGCTTTTTGAAGAAGCTAAGTCTTTGGTTATCGAAACACAGAAAGCCAGTGCATCCATGATTCAGCG TCGTTTGTCAGTTGGATTTAACCGTGCGACCCGTCTCATGGAAGAGCTTGAGATGGCAGGTGTCATTGGT CCAGCTGAAGGTACCAAGCCAAGAAAAGTATTGCAGCAATAA-(R2)n-Y (D) Polypeptide sequence embodiments [SEQ ID NO:2]. X-(R1)n-VFFSSRNACE IWYNRRMANK NTSTTRRRPS KAELERKEAI QRMLISLGIA ILLIFAAFKL GAAGITLYNL IRLLVGSLAY LAIFGLLIYL FFFKWIRKQE GLLSGFFTIF AGLLLIFEAY LVWKYGLDKS VLKGTMAQVV TDLTGFRTTS FAGGGLIGVA LYIPTAFLFS NIGTYFIGSI LILVGSLLVS PWSVYDIAEF FSRGFAKWWE GHERRKEERF VKQEEKARQK AEKEARLEQE ETEKALLDLP PVDMETGEIL TEEAVQNLPP IPEEKWVEPE IILPQAELKF PEQEDDSDDE DVQVDFSAKE ALEYKLPSLQ LFAPDKPKDQ SKEKKIVREN IKILEATFAS FGIKVTVERA EIGPSVTKYE VKPAVGVRVN RISNLSDDLA LALAAKDVRI EAPIPGKSLI GIEVPNSDIA TVSFRELWEQ SQTKAENFLE IPLGKAVNGT ARAFDLSKMP HLLVAGSTGS GKSVAVHGII ASILMKARPD QVKFMMVDPK MVELSVYNDI PHIKIPVVTN PRKASKALQK VVDEMENRYE LFAKVGVRNI AGFNAKVEEF NSQSEYKQIP LPFIVVIVDE LADLMMVASK EVEDAIIRLG QKARAAGIHM ILATQRPSVD VISGLIKANV PSRVAFAVSS GTDSRTILDE NGAEKLLGRG DMLFKPINEN HPVRLQGSFI SDDDVERIVN FIKTQADADY DESFDPGEVS ENEGEFSDGD AGGDPLFEEA KSLVIETQKA SASMIQRRLS VGFNRATRLM EELEMAGVIG PAEGTKPRKV LQQ-(R2)n-Y - Deposited Materials
- A deposit containing aStreptococcus pneumoniae 0100993 strain has been deposited with the National Collections of Industrial and Marine Bacteria Ltd. (herein “NCIMB”), 23 St. Machar Drive, Aberdeen AB2 1RY, Scotland on Apr. 11, 1996 and deposit number 40794. The deposit was described as Streptococcus pneumoniae 0100993 on deposit. On Apr. 17, 1996 a Streptococcus pneumoniae 0100993 DNA library in E. coli was similarly deposited with the NCIMB and assigned deposit number 40800. The Streptococcus pneumoniae strain deposit is referred to herein as “the deposited strain” or as “the DNA of the deposited strain.”
- The deposited strain contains the full length spoIIIE gene. The sequence of the polynucleotides contained in the deposited strain, as well as the amino acid sequence of the polypeptide encoded thereby, are controlling in the event of any conflict with any description of seqences herein.
- The deposit of the deposited strain has been made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for Purposes of Patent Procedure. The strain will be irrevocably and without restriction or condition released to the public upon the issuance of a patent. The deposited strain is provided merely as convenience to those of skill in the art and is not an admission that a deposit is required for enamblement, such as that required under 35 U.S.C. §112.
- A license may be required to make, use or sell the deposited strain, and compounds derived therefrom, and no such license is hereby granted.
- Polypeptides
- The polypeptides of the invention include the polypeptide of Table 1 [SEQ ID NO:2] (in particular the mature polypeptide) as well as polypeptides and fragments, particularly those which have the biological activity of spoIIIE, and also those which have at least 70% identity to the polypeptide of Table 1 [SEQ ID NO:2] or the relevant portion, preferably at least 80% identity to the polypeptide of Table 1 [SEQ ID NO:2], and more preferably at least 90% similarity (more preferably at least 90% identity) to the polypeptide of Table 1 [SEQ ID NO:2] and still more preferably at least 95% similarity (still more preferably at least 95% identity) to the polypeptide of Table 1 [SEQ ID NO:2] and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids.
- The invention also includes polypeptides of the formula set forth in Table 1 (D) wherein, at the amino terminus, X is hydrogen, and at the carboxyl terminus, Y is hydrogen or a metal, R1 and R2 is any amino acid residue, and n is an integer between 1 and 1000. Any stretch of amino acid residues denoted by either R group, where R is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer.
- A fragment is a varient polypeptide having an amino acid sequence that entirely is the same as part but not all of the amino acid sequence of the aforementioned polypeptides. As with spoIIIE polypeptides fragments may be “free-standing,” or comprised with a larger polypeptide of which they form a part or region, most preferably as a single continuous region, a single larger polypeptide.
- Preferred fragments include, for example, truncation polypeptides having a portion of the amino acid sequence of Table 1 [SEQ ID NO:2], or of variants thereof such as a continuous series of residues that includes the amino terminus, or a continuous series of residues that includes the carboxyl terminus. Degradation forms of the polypeptides of the invention in a host cell, particularly aStreptococcus pneumoniae, are also preferred. Further preferred are fragments characterized by structural or functional attributes such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions.
- Also preferred are biologically active fragments which are those fragments that mediate activities of spoIIIE, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also included are those fragments that are antigenic or immunogenic in an animal, especially in a human. Particularly preferred are fragments comprising receptors or domains of enzymes that confer a function essential for viability ofStreptococcus pneumoniae or the ability to initiate, or maintain cause disease in an individual, particularly a human.
- Variants that are fragments of the polypeptides of the invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, these variants may be employed as intermediates for producing the full-length polypeptides of the invention.
- Polynucleotides
- Another aspect of the invention relates to isolated polynucleotides, including the full length gene, that encode the spoIIIE polypeptide having the deduced amino acid sequence of Table 1 [SEQ ID NO:2] and polynucleotides closely related thereto and variants thereof.
- Using the information provided herein, such as the polynucleotide sequence set out in Table 1 [SEQ ID NO:1], a polynucleotide of the invention encoding spoIIIE polypeptide may be obtained using standard cloning and screening methods, such as those for cloning and sequencing chromosomal DNA fragments from bacteria usingStreptococcus pneumoniae 0100993 cells as starting material, followed by obtaining a full length clone. For example, to obtain a polynucleotide sequence of the invention, such as the sequence given in Table 1 [SEQ ID NO:1], typically a library of clones of chromosomal DNA of Streptococcus pneumoniae 0100993 in E. coli or some other suitable host is probed with a radiolabeled oligonucleotide, preferably a 17-mer or longer, derived from a partial sequence. Clones carrying DNA identical to that of the probe can then be distinguished using stringent conditions. By sequencing the individual clones thus identified with sequencing primers designed from the original sequence it is then possible to extend the sequence in both directions to determine the full gene sequence. Conveniently, such sequencing is performed using denatured double stranded DNA prepared from a plasmid clone. Suitable techniques are described by Maniatis, T., Fritsch, E. F. and Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). (see in particular Screening By Hybridization 1.90 and Sequencing Denatured Double-Stranded DNA Templates 13.70). Illustrative of the invention, the polynucleotide set out in Table 1 [SEQ ID NO:1] was discovered in a DNA library derived from Streptococcus pneumoniae 0100993.
- The DNA sequence set out in Table 1 [SEQ ID NO:1] contains an open reading frame encoding a protein having about the number of amino acid residues set forth in Table 1 [SEQ ID NO:2] with a deduced molecular weight that can be calculated using amino acid residue molecular weight values well known im the art. The polynucleotide of SEQ ID NO: 1, between nucleotide number 1 through number 2349 encodes the polypeptide of SEQ ID NO:2. The stop codon begins at nucleotide number 2350 of SEQ ID NO:1.
- The spoIIIE polypeptide of the invention is structurally related to other proteins of the spoIIIE/ftsK (putative chromosomal partitioning function) family, as shown by the results of sequencing the DNA encoding spoIIIE of the deposited strain. The protein exhibits greatest homology toStaphylococcus aureus spoIIIE protein among known proteins. The spoIIIE protein of Table 1 [SEQ ID NO:2] has about 42.237% identity to Staphylococcus aureus spoIIIE protein over its entire length and about 63.947% similarity to Staphylococcus aureus spoIIIE protein over its entire length with the amino acid sequence of Staphylococcus aureus spoIIIE polypeptide.
- The invention provides a polynucleotide sequence identical over its entire length to the coding sequence in Table 1 [SEQ ID NO:1]. Also provided by the invention is the coding sequence for the mature polypeptide or a fragment thereof, by itself as well as the coding sequence for the mature polypeptide or a fragment in reading frame with other coding sequence, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence. The polynucleotide may also contain non-coding sequences, including for example, but not limited to non-coding 5′ and 3′ sequences, such as the transcribed, non-translated sequences, termination signals, ribosome binding sites, sequences that stabilize mRNA, introns, polyadenylation signals, and additional coding sequence which encode additional amino acids. For example, a marker sequence that facilitates purification of the fused polypeptide can be encoded. In certain embodiments of the invention, the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al.,Proc. Natl. Acad. Sci., USA 86: 821-824 (1989), or an HA tag (Wilson et al., Cell 37: 767 (1984). Polynucleotides of the invention also include, but are not limited to, polynucleotides comprising a structural gene and its naturally associated sequences that control gene expression.
- A preferred embodiment of the invention is the polynucleotide of comprising nucleotide 1 to 2349 set forth in SEQ ID NO:1 of Table 1 which encodes the spoIIIE polypeptide.
- The invention also includes polynucleotides of the formula set forth in Table 1 (C) wherein, at the 5′ end of the molecule, X is hydrogen, and at the 3′ end of the molecule, Y is hydrogen or a metal, R1 and R2 is any nucleic acid residue, and n is an integer between 1 and 1000. Any stetch of nucleic acid residues denoted by either R group, where R is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer.
- The term “polynucleotide encoding a polypeptide” as used herein encompasses polynucleotides that include a sequence encoding a polypeptide of the invention, particularly a bacterial polypeptide and more particularly a polypeptide of theStreptococcus pneumoniae spoIIIE having the amino acid sequence set out in Table 1 I[SEQ ID NO:2]. The term also encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (for example, interrupted by integrated phage or an insertion sequence or editing) together with additional regions, that also may contain coding and/or non-coding sequences.
- The invention further relates to variants of the polynucleotides described herein that encode for variants of the polypeptide having the deduced amino acid sequence of Table 1 [SEQ ID NO:2]. Variants that are fragments of the polynucleotides of the invention may be used to synthesize full-length polynucleotides of the invention.
- Further particularly preferred embodiments are polynucleotides encoding spoIIIE variants, that have the amino acid sequence of spoIIIE polypeptide of Table 1 [SEQ ID NO:2] in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, deleted or added, in any combination. Especially preferred among these are silent substitutions, additions and deletions, that do not alter the properties and activities of spoIIIE.
- Further preferred embodiments of the invention are polynucleotides that are at least 70% identical over their entire length to a polynucleotide encoding spoIIIE polypeptide having the amino acid sequence set out in Table 1 [SEQ ID NO:2], and polynucleotides that are complementary to such polynucleotides. Alternatively, most highly preferred are polynucleotides that comprise a region that is at least 80% identical over its entire length to a polynucleotide encoding spoIIIE polypeptide of the deposited strain and polynucleotides complementary thereto. In this regard, polynucleotides at least 90% identical over their entire length to the same are particularly preferred, and among these particularly preferred polynucleotides, those with at least 95% are especially preferred. Furthermore, those with at least 97% are highly preferred among those with at least 95%, and among these those with at least 98% and at least 99% are particularly highly preferred, with at least 99% being the more preferred.
- Preferred embodiments are polynucleotides that encode polypeptides that retain substantially the same biological function or activity as the mature polypeptide encoded by the DNA of Table 1 [SEQ ID NO:1].
- The invention further relates to polynucleotides that hybridize to the herein above-described sequences. In this regard, the invention especially relates to polynucleotides that hybridize under stringent conditions to the herein above-described polynucleotides. As herein used, the terms “stringent conditions” and “stringent hybridization conditions” mean hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences. An example of stringent hybridization conditions is overnight incubation at 42° C. in a solution comprising: 50% formamide, 5× SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 micrograms/ml denatured, sheared salmon sperm DNA, followed by washing the hybridization support in 0.1× SSC at about 65° C. Hybridization and wash conditions are well known and exemplified in Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), particularly Chapter 11 therein.
- The invention also provides a polynucleotide consisting essentially of a polynucleotide sequence obtainable by screening an appropriate library containing the complete gene for a polynucleotide sequence set forth in SEQ ID NO:1 under stringent hybridization conditions with a probe having the sequence of said polynucleotide sequence set forth in SEQ ID NO:1 or a fragment thereof, and isolating said DNA sequence. Fragments useful for obtaining such a polynucleotide include, for example, probes and primers described elsewhere herein.
- As discussed additionally herein regarding polynucleotide assays of the invention, for instance, polynucleotides of the invention as discussed above, may be used as a hybridization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding spoIIIE and to isolate cDNA and genomic clones of other genes that have a high sequence similarity to the spoIIIE gene. Such probes generally will comprise at least 15 bases. Preferably, such probes will have at least 30 bases and may have at least 50 bases. Particularly preferred probes will have at least 30 bases and will have 50 bases or less.
- For example, the codig region of the spoIIIE gene may be isolated by screening using the DNA sequence provided in SEQ ID NO: 1 to synthesize an oligonucleotide probe. A labeled oligonucleotide having a sequence complementary to that of a gene of the invention is then used to screen a library of cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.
- The polynucleotides and polypeptides of the invention may be employed, for example, as research reagents and materials for discovery of treatments of and diagnostics for disease, particularly human disease, as further discussed herein relating to polynucleotide assays.
- Polynucleotides of the invention that are oligonucleotides derived from the sequences of SEQ ID NOS:1 and/or 2 may be used in the processes herein as described, but preferably for PCR, to determine whether or not the polynucleotides identified herein in whole or in part are transcribed in bacteria in infected tissue. It is recognized that such sequences will also have utility in diagnosis of the stage of infection and type of infection the pathogen has attained.
- The invention also provides polynucleotides that may encode a polypeptide that is the mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mature polypeptide (when the mature form has more than one polypeptide chain, for instance). Such sequences may play a role in processing of a protein from precursor to a mature form, may allow protein transport, may lengthen or shorten protein half-life or may facilitate manipulation of a protein for assay or production, among other things. As generally is the case in vivo, the additional amino acids may be processed away from the mature protein by cellular enzymes.
- A precursor protein, having the mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide. When prosequences are removed such inactive precursors generally are activated. Some or all of the prosequences may be removed before activation. Generally, such precursors are called proproteins.
- In sum, a polynucleotide of the invention may encode a mature protein, a mature protein plus a leader sequence (which may be referred to as a preprotein), a precursor of a mature protein having one or more prosequences that are not the leader sequences of a preprotein, or a preproprotein which is a precursor to a proprotein, having a leader sequence and one or more prosequences, which generally are removed during processing steps that produce active and mature forms of the polypeptide.
- Vectors, Host Cells, Expression
- The invention also relates to vectors that comprise a polynucleotide or polynucleotides of the invention, host cells that are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques. Cell-free translation can also be employed to produce such proteins using RNAs derived from the DNA constructs of the invention.
- For recombinant production, host cells can be genetically engineered to incorporate expression systems or portions thereof or polynucleotides of the invention. Introduction of a polynucleotide into the host cell can be effected by methods described in many standard laboratory manuals, such as Davis et al.,BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction and infection.
- Representative examples of appropriate hosts include bacterial cells, such as streptococci, staphylococci, enterococciE. coli, streptomyces and Bacillus subtilis cells; fungal cells, such as yeast cells and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 313, BHK, 293 and Bowes melanoma cells; and plant cells.
- A great variety of expression systems can be used to produce the polypeptides of the invention. Such vectors include, among others, chromosomal, episomal and virus-derived vectors, e.g., vectors derived from baterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. The expression system constructs may contain control regions that regulate as well as gender expression. Generally, any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide in a host may be used for expression in this regard. The appropriate DNA sequence may be inserted into the expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al.,MOLECULAR CLONING, A LABORATORY MANUAL, (supra).
- For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the expressed polypeptide. These signals may be endogenous to the polypeptide or they may be heterologous signals.
- Polypeptides of the invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. Most preferably, high performance liquid chromatography is employed for puification. Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification.
- Diagnostic Assays
- This invention is also related to the use of the spoIIIE polynucleotides of the invention for use as diagnostic reagents. Detection of spoIIIE in a eukaryote, particularly a mammal, and especially a human, will provide a diagnostic method for diagnosis of a disease. Eukaryotes (herein also “individual(s)”), particularly mammals, and especially humans, infected with an organism comprising the spoIIIE gene may be detected at the nucleic acid level by a variety of techniques.
- Nucleic acids for diagnosis may be obtained from an infected individual's cells and tissues, such as bone, blood, muscle, cartilage, and skin. Genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification technique prior to analysis. RNA or cDNA may also be used in the same ways. Using amplification, characterization of the species and strain of prokaryote present in an individual, may be made by an analysis of the genotype of the prokaryote gene. Deletions and insertions can be detected by a change in size of the amplified product in comparison to the genotype of a reference sequence. Point mutations can be identified by hybridizing amplified DNA to labeled spoIIIE polynucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures. DNA sequence differences may also be detected by alterations in the electrophoretic mobility of the DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing. See, e.g., Myers et al.,Science, 230: 1242 (1985). Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase and S1 protection or a chemical cleavage method. See, e.g., Cotton et al., Proc. Natl. Acad. Sci., USA, 85: 4397-4401 (1985).
- Cells carrying mutations or polymorphisms in the gene of the invention may also be detected at the DNA level by a variety of techniques, to allow for serotyping, for example. For example, RT-PCR can be used to detect mutations. It is particularly preferred to used RT-PCR in conjunction with automated detection systems, such as, for example, GeneScan. RNA or cDNA may also be used for the same purpose, PCR or RT-PCR. As an example, PCR primers complementary to a nucleic acid encoding spoIIIB can be used to identify and analyze mutations.
- The invention further provides these primers with 1, 2, 3 or 4 nucleotides removed from the 5′ and/or the 3′ end. These primers may be used for, among other things, amplifying spoIIIE DNA isolated from a sample derived from an individual. The primers may be used to amplify the gene isolated from an infected individual such that the gene may then be subject to various techniquLes for elucidation of the DNA sequence. In this way, mutations in the DNA sequence may be detected and used to diagnose infection and to serotype and/or classify the infectious agent.
- The invention further provides a process for diagnosing, disease, preferably bacterial infections, more preferably infections byStreptococcus pneumoniae, and most preferably otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid, comprising determining from a sample derived from an individual a increased level of expression of polynucleotide having the sequence of Table 1 [SEQ ID NO: 1]. Increased or decreased expression of spoIIIE polynucleotide can be measured using any on of the methods well known in the art for the quantation of polynucleotides, such as, for example, amplification, PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods.
- In addition, a diagnostic assay in accordance with the invention for detecting over-expression of spoIIIE protein compared to normal control tissue samples may be used to detect the presence of an infection, for example. Assay techniques that can be used to determine levels of a spoIIIE protein, in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays.
- Antibodies
- The polypeptides of the invention or variants thereof, or cells expressing them can be used as an immunogen to produce antibodies immunospecific for such polypeptides. “Antibodies” as used herein includes monoclonal and polyclonal antibodies, chimeric, single chain, simianized antibodies and humanized antibodies, as well as Fab fragments, including the products of an Fab immunoglobulin expression library.
- Antibodies generated against the polypeptides of the invention can be obtained by administering the polypeptides or epiptope-bearing fragments, analogues or cells to an animal, preferably a nonhuman, using routine protocols. For preparation of monoclonal antibodies, any technique known in the art that provides antibodies produced by continuous cell line cultures can be used. Examples include various techniques, such as those in Kohler, G. and Milstein, C.,Nature 256: 495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).
- Techniques for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce single chain antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized antibodies.
- Alternatively phage display technology may be utilized to select antibody genes with binding activities towards the polypeptide either from repertoires of PCR amplified v-genes of Iymphocytes from humans screened for possessing anti-spoIIIE or from naive libraries (McCafferty, J. et al., (1990),Nature 348, 552-554; Marks, J. et al., (1992) Biotechnology 10, 779-783). The affinity of these antibodies can also be improved by chain shuffling (Clackson, T. et al., (1991) Nature 352, 624-628).
- If two antigen binding domains are present each domain may be directed against a different epitope-termed ‘bispecific’ antibodies.
- The above described antibodies may be employed to isolate or to identify clones expressing the polypeptides to purify the polypeptides by affinity chromatography.
- Thus, among others, antibodies against spoIIIE polypeptide may be employed to treat infections, particularly bacterial infections and especially otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid.
- Polypeptide variants include antigenically, epitopically or immunologically equivalent variants that form a particular aspect of this invention. The term “antigenically equivalent derivative” as used herein encompasses a polypeptide or its equivalent which will be specifically recognized by certain antibodies which, when raised to the protein or polypeptide according to the invention, interfere with the immediate physical interaction between pathogen and mammalian host. The term “immunologically equivalent derivative” as used herein encompasses a peptide or its equivalent which when used in a suitable formulation to raise antibodies in a vertebrate, the antibodies act to interfere with the immediate physical interaction between pathogen and mammalian host.
- The polypeptide, such as an antigenically or immunologically equivalent derivative or a fusion protein thereof is used as an antigen to immunize a mouse or other animal such as a rat or chicken. The fusion protein may provide stability to the polypeptide. The antigen may be associated, for example by conjugation, with an immunogenic carrier protein for example bovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH). Alternatively a multiple antigenic peptide comprising multiple copies of the protein or polypeptide, or an antigenically or immunologically equivalent polypeptide thereof may be sufficiently antigenic to improve immunogenicity so as to obviate the use of a carrier.
- Preferably, the antibody or variant thereof is modified to make it less immunogenic in the individual. For example, if the individual is human the antibody may most preferably be “humanized ”; where the complimentary determining region(s) of the hybridoma-derived antibody has been transplanted into a human monoclonal antibody, for example as described in Jones, P. et al. (1986),Nature 321,522-525 or Tempest et al.,(1991) Biotechnology 9,266-273.
- The use of a polynucleotide of the invention in genetic immunization will preferably employ a suitable delivery method such as direct injection of plasmid DNA into muscles (Wolff et al.,Hum Mol Genet 1992, 1:363, Manthorpe et al., Hum. Gene Ther. 1963.4, 419), delivery of DNA complexed with specific protein carriers (Wu et al., J Biol Chem. 1989: 264,16985), coprecipitation of DNA with calcium phosphate (Benvenisty & Reshef, PNAS USA, 1986:83,9551), encapsulation of DNA in various forms of liposomes (Kaneda et al., Science 1989:243,375), particle bombardment (Tang et al., Nature 1992, 356:152, Eisenbraun et al., DNA Cell Biol 1993, 12:791) and in vivo infection using cloned retroviral vectors (Seeger et al., PNAS USA 1984:81,5849).
- Antagonists and Agonists—Assays and Molecules
- Polypeptides of the invention may also be used to assess the binding of small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures. These substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics. See, e.g., Coligan et al.,Current Protocols in Immunology 1(2): Chapter 5 (1991).
- The invention also provides a method of screening compounds to identify those which enhance (agonist) or block (antagonist) the action of spoIIIE polypeptides or polynucleotides, particularly those compounds that are bacteriostatic and/or bacteriocidal. The method of screening may involve high throughput techniques. For example, to screen for agonists or antagonists, a synthetic reaction mix, a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof comprising spoIIIE polypeptide and a labeled substrate or ligand of such polypeptide is incubated in the absence or the presence of a candidate molecule that may be a spoIIIE agonist or antagonist. The ability of the candidate molecule to agonize or antagonize the spoIIIE polypeptide is reflected in decreased binding of the labeled ligand or decreased production of product from such substrate. Molecules that bind gratuitously, i.e., without inducing the effects of spoIIIE polypeptide are most likely to be good antagonists. Molecules that bind well and increase the rate of product production from substrate are agonists. Detection of the rate or level of production of product from substrate may be enhanced by using a reporter system. Reporter systems that may be useful in this regard include but are not limited to colorimetric labeled substrate converted into product, a reporter gene that is responsive to changes in spoIIIE polynucleotide or polypeptide activity, and binding assays known in the art.
- Another example of an assay for spoIIIE antagonists is a competitive assay that combines spoIIIE and a potential antagonist with spoIIIE-binding molecules, recombinant spoIIIE binding molecules, natural substrates or ligands, or substrate or ligand mimetics, under appropriate conditions for a competitive inhibition assay. The spoIIIE molecule can be labeled, such as by radioactivity or a colorimetric compound, such that the number of spoIIIE molecules bound to a binding molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist.
- Potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to a polynucleotide or polypeptide of the invention and thereby inhibit or extinguish its activity. Potential antagonists also may be small organic molecules, a peptide, a polypeptide such as a closely related protein or antibody that binds the same sites on a binding molecule, such as a binding molecule, without inducing spoIIIE-induced activities, thereby preventing the action of spoIIIE by excluding spoIIIE from binding.
- Potential antagonists include a small molecule that binds to and occupies the binding site of the polypeptide thereby preventing binding to cellular binding molecules, such that normal biological activity is prevented. Examples of small molecules include but are not limited to small organic molecules, peptides or peptide-like molecules. Other antagonists include antisense molecules (see Okano,J. Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988), for a description of these molecules). Preferred postal antagonists include compounds related to and variants of spoIIIE.
- Each of the DNA sequences provided herein may be used in the discovery and development of antibacterial compounds. The encoded protein, upon expression, can be used as a target for the screening of antibacterial drugs. Additionally, the DNA sequences encoding the amino terminal regions of the encoded protein or Shine-Delgarno or other translation facilitating sequences of the respective mRNA can be used to construct antisense sequences to control the expression of the coding sequence of interest.
- The invention also provides the use of the polypeptide, polynucleotide or inhibitor of the invention to interfere with the initial physical interaction between a pathogen and mammalian host responsible for sequence of infection. In particular the molecules of the invention may be used: in the prevention of adhesion of bacteria, in particular gram positive bacteria, to mammalian extracellular matrix proteins on in-dwelling devices or to extracellular matrix proteins in wounds; to block spoIIIE protein-mediated mammalian cell invasion by, for example, initiating phosphorylation of mammalian tyrosine kinases (Rosenshine et al.,Infect. Immun. 60:2211 (1992); to block bacterial adhesion between mammalian extracellular matrix proteins and bacterial spoIIIE proteins that mediate tissue damage and; to block the normal progression of pathogenesis in infections initiated other than by the implantation of in-dwelling devices or by other surgical techniques.
- The antagonists and agonists of the invention may be employed, for instance, to inhibit and treat otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly menigitis, such as for example infection of cerebrospinal fluid.
- Vaccides
- Another aspect of the invention relates to a method for inducing an immunological response in an individual, particularly a mammal which comprises inoculating the individual with spoIIIE, or a fragment or variant thereof, adequate to produce antibody and/ or T cell immune response to protect said individual from infection, particularly bacterial infection and most particularlyStreptococcus pneumoniae infection. Also provided are methods whereby such immunological response slows bacterial replication. Yet another aspect of the invention relates to a method of inducing immunological response in an individual which comprises delivering to such individual a nucleic acid vector to direct expression of spoIIIE, or a fragment or a variant thereof, for expressing spoIIIE, or a fragment or a variant thereof in vivo in order to induce an immunological response, such as, to produce antibody and/ or T cell immune response, including, for example, cytokine-producing T cells or cytotoxic T cells, to protect said individual from disease, whether that disease is already established within the individual or not. One way of administering the gene is by accelerating it into the desired cells as a coating on particles or otherwise.
- Such nucleic acid vector may comprise DNA, RNA, a modified nucleic acid, or a DNA/RNA hybrid.
- A further aspect of the invention relates to an immunological composition which, when introduced into an individual capable or having induced within it an immunological response, induces an immunological response in such individual to a spoIIIE or protein coded therefrom, wherein the composition comprises a recombinant spoIIIE or protein coded therefrom comprising DNA which codes for and expresses an antigen of said spoIIIE or protein coded therefrom. The immunological response may be used therapeutically or prophylactically and may take the form of antibody immunity or cellular immunity such as that arising from CTL or CD4+T cells.
- A spoIIIE polypeptide or a fragment thereof may be fused with co-protein which may not by itself produce antibodies, but is capable of stabilizing the first protein and producing a fused protein which will have immunogenic and protective properties. Thus fused recombinant protein, preferably further comprises an antigenic co-protein, such as lipoprotein D fromHemophilus influenzae, Glutathione-S-transferase (GST) or beta-galactosidase, relatively large co-proteins which solubilize the protein and facilitate production and purification thereof. Moreover, the co-protein may act as an adjuvant in the sense of providing a generalized stimulation of the immune system. The co-protein may be attached to either the amino or carboxy terminus of the first protein.
- Provided by this invention are compositions, particularly vaccine compositions, and methods comprising the polypeptides or polynucleotides of the invention and immunostimulatory DNA sequences, such as those described in Sato, Y. et al.Science 273: 352 (1996).
- Also, provided by this invention are methods using the described polynucleotide or particular fragments thereof which have been shown to encode non-variable regions of bacterial cell surface proteins in DNA constructs used in such genetic immunization experiments in animal models of infection withStreptococcus pneumoniae will be particularly useful for identifying protein epitopes able to provoke a prophylactic or therapeutic immune response. It is believed that this approach will allow for the subsequent preparation of monoclonal antibodies of particular value from the requisite organ of the animal successfully resisting or clearing infection for the development of prophylactic agents or therapeutic treatments of bacterial infection, particularly Streptococcus pneumoniae infection, in mammals, particularly humans.
- The polypeptide may be used as an antigen for vaccination of a host to produce specific antibodies which protect against invasion of bacteria, for example by blocking adherence of bacteria to damaged tissue. Examples of tissue damage include wounds in skin or connective tissue caused, e.g., by mechanical, chemical or thermal damage or by implanation of indwelling devices, or wounds in the mucous membranes, such as the mouth, mammary glands, urethra or vagina.
- The invention also includes a vaccine formulation which comprises an immunogenic recombinant protein of the invention together with a suitable carrier. Since the protein may be broken down in the stomach, it is preferably administered parenterally, including, for example, administration that is subcutaneous, intramuscular, intravenous, or intradermal. Formulations suitable for parenteral administration include aqueous and nonaqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation insotonic with the bodily fluid, preferably the blood, of the individual; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use. The vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
- While the invention has been described with reference to certain spoIIIE protein, it is to be understood that this covers fragments of the naturally occurring protein and similar proteins with additions, deletions or substitutions which do not substantially affect the immunogenic properties of the recombinant protein.
- Compositions, Kits and Administration
- The invention also relates to compositions comprising the polynucleotide or the polypeptides discussed above or their agonists or antagonists. The polypeptides of the invention may be employed in combination with a non-sterile or sterile carrier or carriers for use with cells, tissues or organisms, such as a pharmaceutical carrier suitable for administration to a subject. Such compositions comprise, for instance, a media additive or a therapeutically effective amount of a polypeptide of the invention and a pharmaceutically acceptable carrier or excipient. Such carriers may include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof. The formulation should suit the mode of administration. The invention further relates to diagnostic and pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.
- Polypeptides and other compounds of the invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
- The pharmaceutical compositions may be administered in any effective, convenient manner including, for instance, administration by topical, oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes among others.
- In therapy or as a prophylactic, the active agent may be administered to an individual as an injectable composition, for example as a sterile aqueous dispersion, preferably isotonic.
- Alternatively the composition may be formulated for topical application for example in the form of ointments, creams, lotions, eye ointments, eye drops, ear drops, mouthwash, impregnated dressings and sutures and aerosols, and may contain appropriate conventional additives, including, for example, preservatives, solvents to assist drug penetration, and emollients in ointments and creams. Such topical formulations may also contain compatible conventional carriers, for example cream or ointment bases, and ethanol or oleyl alcohol for lotions. Such carriers may constitute from about 1% to about 98% by weight of the formulation; more usually they will constitute up to about 80% by weight of the formulation.
- For administration to mammals, and particularly humans, it is expected that the daily dosage level of the active agent will be from 0.01 mg/kg to 10 mg/g, typically around 1 mg/kg. The physician in any event will determine the actual dosage which will be most suitable for an individual and will vary with the age, weight and response of the particular individual. The above dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
- In-dwelling devices include surgical implants, prosthetic devices and catheters, i.e., devices that are introduced to the body of an individual and remain in position for an extended time. Such devices include, for example, artificial joints, heart valves, pacemakers, vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinary catheters, continuous ambulatory peritoneal dialysis (CAPD) catheters.
- The composition of the invention may be administered by injection to achieve a systemic effect against relevant bacteria shortly before insertion of an in-dwelling device. Treatment may be continued after surgery during the in-body time of the device. In addition, the composition could also be used to broaden perioperative cover for any surgical technique to prevent bacterial wound infections, especiallyStreptococcus pneumoniae wound infections.
- Many orthopaedic surgeons consider that humans with prosthetic joints should be considered for antibiotic prophylaxis before dental treatment that could produce a bacteremia. Late deep infection is a serious complication sometimes leading to loss of the prosthetic joint and is accompanied by significant morbidity and mortality. It may therefore be possible to extend the use of the active agent as a replacement for prophylactic antibiotics in this situation.
- In addition to the therapy described above, the compositions of this invention may be used generally as a wound treatment agent to prevent adhesion of bacteria to matrix proteins exposed in wound tissue and for prophylactic use in dental treatment as an alternative to, or in conjunction wit, antibiotic prophylaxis.
- Alternatively, the composition of the invention may be used to bathe an indwelling device immediately before insertion. The active agent will preferably be present at a concentration of 1 μg/ml to 10 mg/ml for bathing of wounds or indwelling devices.
- A vaccine composition is conveniently in injectable form. Conventional adjuvants may be employed to enhance the immune response. A suitable unit dose for vaccination is 0.5-5 microgram/kg of antigen, and such dose is preferably administered 1-3 times and with an interval of 1-3 weeks. With the indicated dose range, no adverse toxicological effects will be observed with the compounds of the invention which would preclude their administration to suitable individuals.
- Each reference disclosed herein is incorporated by reference herein in its entirety. Any patent application to which this application claims priority is also incorporated by reference herein in its entirety.
- The examples below are carried out using standard techniques, which are well known and routine to those of skill in the art, except where otherwise described in detail. The examples are illustrative, but do not limit the invention.
- The polynucleotide having the DNA sequence given in SEQ ID NO:1 was obtained from a library of clones of chromosomal DNA ofStreptococcus pneumoniae in E. coli. The sequencing data from two or more clones contain overlapping Streptococcus pneumoniae DNAs was used to construct the contiguous DNA sequence in SEQ ID NO:1. Libraries may be prepared by routine methods, for example:
- Methods 1 and 2 below.
- Total cellular DNA is isolated fromStreptococcus pneumoniae 0100993 according to standard procedures and size-fractionated by either of two methods.
- Method 1
- Total cellular DNA is mechanically sheared by passage through a needle in order to size fractionate according to standard procedures. DNA fragments of up to 11 kbp in size are rendered blunt by treatment with exonuclease and DNA polymerase, and EcoRI linkers added. Fragments are ligated into the vector Lambda ZapII that has been cut with EcoRI, the library packaged by standard procedures andE. coli infected with the packaged library. The library is amplified by standard procedures.
- Method 2
- Total cellular DNA is partially hydrolyzed with a one or a combination of restriction enzymes appropriate to generate a series of fragments for cloning into library vectors (e.g., RsaI, PalI, AluI, Bsh1235I), and such fragments are size-fractionated according to standard procedures. EcoRI linkers are ligated to the DNA and the fragments then ligated into the vector Lambda ZapII that have been cut with EcoRI, the library packaged by standard procedures, andE.coli infected with the packaged library. The library is amplified by standard procedures.
- SpoIIIE is a membrane bound protein involved in chromosome partitioning during sporulation and vegetative replication in a wide variety of bacteria. The spoIIIE gene was initially characterized inBacillus subtilis (Butler P. D. and Mandelstam J. (1987) Journal of General Microbiology 133:2359-2370). SpoIIIE protein has an ATP binding site and is membrane-bound, and appears to form a pore in the nascent spore septum, through which the prespore chromosome is driven in a conjugation-like mechanism (Wu L. J., Lewis P. J., Allmansberger R., Hauser P. M. and Errington J. (1995) Genes and Development 9:1316-1326). spoIIIE mutants cannot sporulate as they are unable to partition the prespore chromosome into the polar prespore compartment. Instead a specific chromosomal segment comprising approximately 30% of the chromosome enters the prespore, while the rest remains in the mother cell, trapped by the septum (Wu L. J. and Errington J. (1994) Science 264:572-575). In wild-type cells SpoIIIE is membrane-bound, and appears to form a pore in the nascent spore septum, through which the prespore chromosome is driven in a conjugation-like mechanism (Wu L. J., Lewis P. J., Allmansberger R., Hauser P. M. and Errington J. (1995) Genes and Development 9:1316-1326).
- It has been shown that SpoIIIE is also required for correct partitioning of theB.subtilis chromosome during vegetative cell division. SpoIIIE-mutants in which replication has been artificially delayed are unable to separate the replicated chromosomes before septum formation, resulting in a trapped nucleoid similar to that formed at the start of sporulation (Sharpe M. E. and Errington J. (1995) Proceedings of the Natural Academy of Sciences USA 92:8630-8634).
- SpoIIIE has been shown to be essential inEscherichia coli (Begg K. J., Dewar, S. J. and Donachie W. D. (1995) Journal of Bacteriology 177:6211-6222). Highly conserved SpoIIIE homologues are found in diverse members of the eubacteria such as Campylobacter jejuni (Miller, S., Pesci E. C. and Pickett C. L. (1994) Gene 146:31-38), Coxiella burnetii (Oswald W. and Thiele D. (1993) Journal of Veterinary Medecine B40:366-370), Escherichia coli (Begg 1995 above) and Haemophilus influenzae (Fleischmann, R. D., Adams, M. D., White, O., Clayton, R. A., Kirkness, E. F., Kerlavage, A. R., Bult, C. J., Tomb, J. -F., Dougherty, B. A., Merrick, J. M., McKenney, K., Sutton, G., FitzHugh, W., Fields, C. A., Gocayne, J. D., Scott, J. D., Shirley, R., Liu, L. -I., Glodek, A., Kelley, J. M., Weidman, J. F., Phillips, C. A., Spriggs, T., Hedblom, E., Cotton, M. D., Utterback, T. R., Hanna, M. C., Nguyen, D. T., Saudek, D. M., Brandon, R. C., Fine, L. D., Fritchman, J. L., Fuhrmann, J. L., Geoghagen, N. S. M., Gnehm, C. L., McDonald, L. A., Small, K. V., Fraser, C. M., Smith, H. O. and Venter, J. C. (1995) Science 269:496-512). All of these proteins are 36-55% identical at the amino acid level overall. Their N-terminal 200 amino acids are hydrophobic and not conserved, so if the C-terminal 500 or so amino acids are considered alone the level of conservation rises to 42-67% identical amino acids. This high level of identity among diverse eubacteria strongly suggests commonality of function. The importance of Streptococcus pneumoniae spoIIIE in infection is demonstrated by the fact that the gene has been shown to be expressed in vivo in a mouse model using the following methodology: Streptococcus pneumoniae 0100993 cultures resuspended in phosphate-buffered saline are administered intranasally to mice. After 48 hours, the infected mouse lungs are aseptically removed, snap-frozen in liquid nitrogen and treated to release total RNA. Bacterial cDNA is made from the mRNA component by reverse transcription, then subjected to PCR using primers specific to spoIIIE. The spoIIIE transcript was detected, demonstrating that this gene is expressed during the infection.
- Inhibitors of SpoIIIE proteins would prevent the bacterium from establishing and maintaining infection of the host by preventing it from correctly partitioning the chromosome in the manner described above and thus arresting cell division and growth, rendering the bacterium susceptible to host defences and leading ultimately to cell death and thereby have utility in anti-bacterial therapy.
-
1 2 2352 base pairs nucleic acid double linear 1 GTCTTTTTCT CTTCCCGAAA TGCCTGTGAA ATATGGTATA ATAGAAGAAT GGCAAACAAG 60 AATACAAGTA CAACAAGACG GAGACCGTCT AAAGCAGAAC TGGAAAGAAA AGAAGCGATT 120 CAACGAATGT TGATTTCGTT AGGAATTGCG ATTTTATTGA TTTTCGCAGC CTTCAAATTA 180 GGGGCTGCAG GTATAACCCT TTATAATTTA ATTCGCTTGC TAGTGGGTAG CCTAGCTTAAT 240 CTGGCGATAT TCGGCCTATT AATCTATCTC TTCTTTTTCA AGTGGATACG AAAACAGGAA 300 GGACTCTTAT CTGGCTTTTT CACCATATTT GCTGGCTTAC TCTTGATTTT TGAGGCCTAC 360 TTGGTTTGGA AATATGGTTT GGACAAGTCC GTTCTAAAAG GGACCATGGC TCAGGTTGTG 420 ACAGATCTGA CTGGTTTTCG AACGACTAGC TTTGCTGGAG GGGGCTTGAT CGGGGTCGCT 480 CTTTATATTC CAACAGCCTT TCTCTTTTCA AATATCGGAA CTTACTTTAT TGGTTCTATC 540 TTGATTTTAG TGGGTTCTCT CCTAGTCAGC CCTTGGTCTG TTTACGATAT TGCTGAATTT 600 TTCAGTAGAG GCTTTGCCAA ATGGTGGGAA GGGCACGAGC GTCGAAAAGA GGAACGCTTT 660 GTCAAACAAG AAGAAAAAGC TCGCCAAAAG GCTGAGAAAG AGGCTAGATT AGAACAAGAA 720 GAGACTGAAA AAGCCTTACT CGATTTGCCT CCTGTTGATA TGGAAACGGG TGAAATTCTG 780 ACAGAGGAAG CTGTTCAAAA TCTTCCACCT ATTCCAGAAG AAAAGTGGGT GGAACCAGAA 840 ATCATCCTGC CTCAAGCTGA ACTTAAATTC CCTGAACAGG AAGATGACTC AGATGACGAA 900 GATGTTCAGG TCGATTTTTC AGCCAAAGAA GCCCTTGAAT ACAAACTTCC AAGCTTACAA 960 CTCTTTGCAC CAGATAAACC AAAAGATCAG TCTAAAGAGA AGAAAATTGT CAGAGAAAAT 1020 ATCAAAATCT TAGAAGCAAC CTTTGCTAGC TTTGGTATTA AGGTAACAGT TGAACGGGCC 1080 GAAATTGGGC CATCAGTGAC CAAGTATGAA GTCAAGCCGG CTGTTGGTGT AAGGGTCAAC 1140 CGCATTTCCA ATCTATCAGA TGACCTCGCT CTAGCCTTGG CTGCCAAGGA TGTCCGAATT 1200 GAAGCACCAA TCCCTGGGAA ATCTCTAATC GGAATTGAAG TGCCCAACTC CGATATTGCC 1260 ACTGTATCTT TCCGAGAACT ATGGGAACAA TCGCAAACGA AAGCAGAAAA TTTCTTGGAA 1320 ATTCCTTTAG GGAAGGCTGT TAATGGAACC GCAAGAGCTT TTGACCTTTC TAAAATGCCC 1380 CACTTGCTAG TTGCAGGTTC AACGGGTTCA GGGAAGTCAG TAGCAGTTAA CGGCATTATT 1440 GCTAGCATTC TCATGAAGGC GAGACCAGAT CAAGTTAAAT TTATGATGGT CGATCCCAAG 1500 ATGGTTGAGT TATCTGTTTA CAATGATATT CCCCACATAA AGATTCCAGT CGTGACCAAT 1560 CCACGCAAAG CCAGCAAGGC TCTGCAAAAG GTTGTGGATG AAATGGAAAA CCGTTATGAA 1620 CTCTTTGCCA AGGTGGGAGT TCGGAATATT GCAGGTTTTA ATGCCAAGGT AGAAGAGTTC 1680 AATTCCCAGT CTGAGTACAA GCAAATTCCG CTACCATTCA TTGTCGTGAT TGTGGATGAG 1740 TTGGCTGACC TCATGATGGT GGCCAGCAAG GAAGTGGAAG ATGCTATCAT CCGTCTTGGG 1800 CAGAAGGCGC GTGCTGCAGG TATCCACATG ATTCTTGCAA CTCAGCGTCC ATCTGTTGAT 1860 GTCATCTCTG GTTTGATTAA GGCCAATGTT CCATCTCGTG TAGCATTTGC GGTTTCATCA 1920 GGAACAGACT CCCGTACGAT TTTGGATGAA AATGGAGCAG AAAAACTTCT TGGTCGAGGA 1980 GACATGCTCT TTAAACCGAT TAATGAAAAT CATCCAGTTC GTCTCCAAGG CTCCTTTATC 2040 TCGGATGACG ATGTTGAGCG CATTGTGAAC TTCATCAAGA CTCAGGCAGA TGCAGACTAC 2100 GATGAGAGTT TTGATCCAGG TGAGGTTTCT GAAAATGAAG GAGAATTTTC GGATGGAGAT 2160 GCTGGTGGTG ATCCGCTTTT TGAAGAAGCT AAGTCTTTGG TTATCGAAAC ACAGAAAGCC 2220 AGTGCATCCA TGATTCAGCG TCGTTTGTCA GTTGGATTTA ACCGTGCGAC CCGTCTCATG 2280 GAAGAGCTTG AGATGGCAGG TGTCATTGGT CCAGCTGAAG GTACCAAGCC AAGAAAAGTA 2340 TTGCAGCAAT AA 2352 783 amino acids amino acid single linear 2 Val Phe Phe Ser Ser Arg Asn Ala Cys Glu Ile Trp Tyr Asn Arg Arg 1 5 10 15 Met Ala Asn Lys Asn Thr Ser Thr Thr Arg Arg Arg Pro Ser Lys Ala 20 25 30 Glu Leu Glu Arg Lys Glu Ala Ile Gln Arg Met Leu Ile Ser Leu Gly 35 40 45 Ile Ala Ile Leu Leu Ile Phe Ala Ala Phe Lys Leu Gly Ala Ala Gly 50 55 60 Ile Thr Leu Tyr Asn Leu Ile Arg Leu Leu Val Gly Ser Leu Ala Tyr 65 70 75 80 Leu Ala Ile Phe Gly Leu Leu Ile Tyr Leu Phe Phe Phe Lys Trp Ile 85 90 95 Arg Lys Gln Glu Gly Leu Leu Ser Gly Phe Phe Thr Ile Phe Ala Gly 100 105 110 Leu Leu Leu Ile Phe Glu Ala Tyr Leu Val Trp Lys Tyr Gly Leu Asp 115 120 125 Lys Ser Val Leu Lys Gly Thr Met Ala Gln Val Val Thr Asp Leu Thr 130 135 140 Gly Phe Arg Thr Thr Ser Phe Ala Gly Gly Gly Leu Ile Gly Val Ala 145 150 155 160 Leu Tyr Ile Pro Thr Ala Phe Leu Phe Ser Asn Ile Gly Thr Tyr Phe 165 170 175 Ile Gly Ser Ile Leu Ile Leu Val Gly Ser Leu Leu Val Ser Pro Trp 180 185 190 Ser Val Tyr Asp Ile Ala Glu Phe Phe Ser Arg Gly Phe Ala Lys Trp 195 200 205 Trp Glu Gly His Glu Arg Arg Lys Glu Glu Arg Phe Val Lys Gln Glu 210 215 220 Glu Lys Ala Arg Gln Lys Ala Glu Lys Glu Ala Arg Leu Glu Gln Glu 225 230 235 240 Glu Thr Glu Lys Ala Leu Leu Asp Leu Pro Pro Val Asp Met Glu Thr 245 250 255 Gly Glu Ile Leu Thr Glu Glu Ala Val Gln Asn Leu Pro Pro Ile Pro 260 265 270 Glu Glu Lys Trp Val Glu Pro Glu Ile Ile Leu Pro Gln Ala Glu Leu 275 280 285 Lys Phe Pro Glu Gln Glu Asp Asp Ser Asp Asp Glu Asp Val Gln Val 290 295 300 Asp Phe Ser Ala Lys Glu Ala Leu Glu Tyr Lys Leu Pro Ser Leu Gln 305 310 315 320 Leu Phe Ala Pro Asp Lys Pro Lys Asp Gln Ser Lys Glu Lys Lys Ile 325 330 335 Val Arg Glu Asn Ile Lys Ile Leu Glu Ala Thr Phe Ala Ser Phe Gly 340 345 350 Ile Lys Val Thr Val Glu Arg Ala Glu Ile Gly Pro Ser Val Thr Lys 355 360 365 Tyr Glu Val Lys Pro Ala Val Gly Val Arg Val Asn Arg Ile Ser Asn 370 375 380 Leu Ser Asp Asp Leu Ala Leu Ala Leu Ala Ala Lys Asp Val Arg Ile 385 390 395 400 Glu Ala Pro Ile Pro Gly Lys Ser Leu Ile Gly Ile Glu Val Pro Asn 405 410 415 Ser Asp Ile Ala Thr Val Ser Phe Arg Glu Leu Trp Glu Gln Ser Gln 420 425 430 Thr Lys Ala Glu Asn Phe Leu Glu Ile Pro Leu Gly Lys Ala Val Asn 435 440 445 Gly Thr Ala Arg Ala Phe Asp Leu Ser Lys Met Pro His Leu Leu Val 450 455 460 Ala Gly Ser Thr Gly Ser Gly Lys Ser Val Ala Val Asn Gly Ile Ile 465 470 475 480 Ala Ser Ile Leu Met Lys Ala Arg Pro Asp Gln Val Lys Phe Met Met 485 490 495 Val Asp Pro Lys Met Val Glu Leu Ser Val Tyr Asn Asp Ile Pro His 500 505 510 Ile Lys Ile Pro Val Val Thr Asn Pro Arg Lys Ala Ser Lys Ala Leu 515 520 525 Gln Lys Val Val Asp Glu Met Glu Asn Arg Tyr Glu Leu Phe Ala Lys 530 535 540 Val Gly Val Arg Asn Ile Ala Gly Phe Asn Ala Lys Val Glu Glu Phe 545 550 555 560 Asn Ser Gln Ser Glu Tyr Lys Gln Ile Pro Leu Pro Phe Ile Val Val 565 570 575 Ile Val Asp Glu Leu Ala Asp Leu Met Met Val Ala Ser Lys Glu Val 580 585 590 Glu Asp Ala Ile Ile Arg Leu Gly Gln Lys Ala Arg Ala Ala Gly Ile 595 600 605 His Met Ile Leu Ala Thr Gln Arg Pro Ser Val Asp Val Ile Ser Gly 610 615 620 Leu Ile Lys Ala Asn Val Pro Ser Arg Val Ala Phe Ala Val Ser Ser 625 630 635 640 Gly Thr Asp Ser Arg Thr Ile Leu Asp Glu Asn Gly Ala Glu Lys Leu 645 650 655 Leu Gly Arg Gly Asp Met Leu Phe Lys Pro Ile Asn Glu Asn His Pro 660 665 670 Val Arg Leu Gln Gly Ser Phe Ile Ser Asp Asp Asp Val Glu Arg Ile 675 680 685 Val Asn Phe Ile Lys Thr Gln Ala Asp Ala Asp Tyr Asp Glu Ser Phe 690 695 700 Asp Pro Gly Glu Val Ser Glu Asn Glu Gly Glu Phe Ser Asp Gly Asp 705 710 715 720 Ala Gly Gly Asp Pro Leu Phe Glu Glu Ala Lys Ser Leu Val Ile Glu 725 730 735 Thr Gln Lys Ala Ser Ala Ser Met Ile Gln Arg Arg Leu Ser Val Gly 740 745 750 Phe Asn Arg Ala Thr Arg Leu Met Glu Glu Leu Glu Met Ala Gly Val 755 760 765 Ile Gly Pro Ala Glu Gly Thr Lys Pro Arg Lys Val Leu Gln Gln 770 775 780
Claims (16)
1. An isolated polypeptide comprising a fragment of SEQ ID NO:2 comprising at least 50 consecutive amino acids of SEQ ID NO:2 ; wherein said fragment has chromosomal partitioning activity.
2. A composition comprising the isolated polypeptide of claim 1 and a carrier.
3. The isolated polypeptide of claim 1 , wherein the isolated polypeptide comprises a heterologous amino acid sequence fused to the at least 50 consecutive amino acids of SEQ ID NO:2.
4. A composition comprising the isolated polypeptide of claim 3 and a carrier.
5. An isolated polypeptide comprising a fragment of SEQ ID NO:2 comprising at least 30 consecutive amino acids of SEQ ID NO:2; wherein said fragment has chromosomal partitioning activity.
6. A composition comprising the isolated polypeptide of claim 5 and a carrier.
7. The isolated polypeptide of claim 5 , wherein the isolated polypeptide comprises a heterologous amino acid sequence fused to the at least 30 consecutive amino acids of SEQ ID NO:2.
8. A composition comprising the isolated polypeptide of claim 7 and a carrier.
9. An isolated polypeptide comprising a fragment of SEQ ID NO:2 comprising at least 50 consecutive amino acids of SEQ ID NO:2; wherein said fragment has the ability to generate antibodies to SEQ ID NO:2.
10. A composition comprising the isolated polypeptide of claim 9 and a carrier.
11. The isolated polypeptide of claim 9 , wherein the isolated polypeptide comprises a heterologous amino acid sequence fused to the at least 50 consecutive amino acids of SEQ ID NO:2.
12. A composition comprising the isolated polypeptide of claim 11 and a carrier.
13. An isolated polypeptide comprising a fragment of SEQ ID NO:2 comprising at least 30 consecutive amino acids of SEQ ID NO:2; wherein said fragment has the ability to generate antibodies to SEQ ID NO:2.
14. A composition comprising the isolated polypeptide of claim 13 and a carrier.
15. The isolated polypeptide of claim 13 , wherein the isolated polypeptide comprises a heterologous amino acid sequence fused to the at least 30 consecutive amino acids of SEQ ID NO:2.
16. A composition comprising the isolated polypeptide of claim 15 and a carrier.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US09/775,978 US20020019515A1 (en) | 1997-08-26 | 2001-02-02 | Novel spoIIIE |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US08/922,837 US5888770A (en) | 1997-08-26 | 1997-08-26 | Spoiiie |
US09/351,550 US6222016B1 (en) | 1997-08-26 | 1999-07-12 | spoIIIE from Steptococcus pneumoniae |
US09/775,978 US20020019515A1 (en) | 1997-08-26 | 2001-02-02 | Novel spoIIIE |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/351,550 Division US6222016B1 (en) | 1997-08-26 | 1999-07-12 | spoIIIE from Steptococcus pneumoniae |
Publications (1)
Publication Number | Publication Date |
---|---|
US20020019515A1 true US20020019515A1 (en) | 2002-02-14 |
Family
ID=25447634
Family Applications (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US08/922,837 Expired - Fee Related US5888770A (en) | 1997-08-26 | 1997-08-26 | Spoiiie |
US09/351,550 Expired - Fee Related US6222016B1 (en) | 1997-08-26 | 1999-07-12 | spoIIIE from Steptococcus pneumoniae |
US09/775,978 Abandoned US20020019515A1 (en) | 1997-08-26 | 2001-02-02 | Novel spoIIIE |
Family Applications Before (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US08/922,837 Expired - Fee Related US5888770A (en) | 1997-08-26 | 1997-08-26 | Spoiiie |
US09/351,550 Expired - Fee Related US6222016B1 (en) | 1997-08-26 | 1999-07-12 | spoIIIE from Steptococcus pneumoniae |
Country Status (4)
Country | Link |
---|---|
US (3) | US5888770A (en) |
EP (1) | EP0899336A3 (en) |
JP (1) | JPH11225776A (en) |
CA (1) | CA2241431A1 (en) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5888770A (en) * | 1997-08-26 | 1999-03-30 | Smithkline Beecham Corporation | Spoiiie |
Family Cites Families (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB9512109D0 (en) * | 1995-06-14 | 1995-08-09 | Isis Innovation | Bacillus strain and assay method |
GB9600955D0 (en) * | 1996-01-17 | 1996-03-20 | Smithkline Beecham Plc | Novel compounds |
US6346392B1 (en) * | 1996-11-27 | 2002-02-12 | Smithkline Beecham Corporation | Polynucleotides encoding a novel glutamine transport ATP-binding protein |
EP0934336A1 (en) * | 1996-05-14 | 1999-08-11 | Smithkline Beecham Corporation | Novel compounds |
EP0942983B2 (en) * | 1996-10-31 | 2014-09-10 | Human Genome Sciences, Inc. | Streptococcus pneumoniae antigens and vaccines |
USH2071H1 (en) * | 1996-12-13 | 2003-07-01 | Eli Lilly And Company | Streptococcus pneumoniae gene HI0454 |
US5888770A (en) * | 1997-08-26 | 1999-03-30 | Smithkline Beecham Corporation | Spoiiie |
-
1997
- 1997-08-26 US US08/922,837 patent/US5888770A/en not_active Expired - Fee Related
-
1998
- 1998-08-18 EP EP98306605A patent/EP0899336A3/en not_active Withdrawn
- 1998-08-20 CA CA002241431A patent/CA2241431A1/en not_active Abandoned
- 1998-08-26 JP JP10281887A patent/JPH11225776A/en not_active Withdrawn
-
1999
- 1999-07-12 US US09/351,550 patent/US6222016B1/en not_active Expired - Fee Related
-
2001
- 2001-02-02 US US09/775,978 patent/US20020019515A1/en not_active Abandoned
Also Published As
Publication number | Publication date |
---|---|
EP0899336A3 (en) | 2001-10-04 |
CA2241431A1 (en) | 1999-02-26 |
US6222016B1 (en) | 2001-04-24 |
JPH11225776A (en) | 1999-08-24 |
US5888770A (en) | 1999-03-30 |
EP0899336A2 (en) | 1999-03-03 |
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