JP2000139475A - NEW FabH - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new polynucleotide which has high homology to a nucleic acid coding for FabH polypeptide containing a specific amino acid sequence, and can be used for screening antibacterial compounds, treating and/or diagnosing the infection by staphylococci, and so on. SOLUTION: A newly isolated polynucleotide is provided which contains a polynucleotide having homology at 70% or higher to a polypeptide coding for FabH polypeptide containing the amino acid sequence of the formula, a polynucleotide having homology at 70% or higher to a nucleic acid coding for the same mature polypeptide as one which is expressed by FabH gene contained in a stipulated strain of Staphylococcus aureus, and can be used for screening antibacterial compounds by FabH polypeptide, treating and/or diagnosing the infection by S. aureus, and so on. This polynucleotide is obtained by screening a typical library of a chromosomal DNA close derived from S. aureus WCVH29 using a radiolabelled oligonucleotide preferably having 17 bp or more derived from a partial sequence as a probe.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates, in part, to newly identified polynucleotides and polypeptides, and their production and use, and variants thereof,
Agonists and antagonists and their use. In particular, in these and other respects, the present invention relates to novel polynucleotides and polypeptides of the Fab family (hereinafter FabH).

[0002]

2. Description of the Related Art Staphylococc spp.
It is particularly preferred to use the genes and gene products of i) for the development of antibiotics. Staphylococci form a medically important microbial genus. They are known to cause two types of diseases: invasive and toxogenic. In general, invasive infections are characterized by abscess formation that affects both skin surface and deep tissues. S. aureus
Is the second leading cause of bacteremia in cancer patients. Osteomyelitis, septic arthritis, septic thrombophlebitis and acute bacterial endocarditis are also relatively common. There are three clinical symptoms caused by the toxogenic properties of Staphylococcus. Obvious signs of these diseases result from the action of endotoxin as opposed to tissue invasion and bacteremia. These conditions include Staphylococcus food contamination, burn skin syndrome and toxin shock syndrome.

[0003] The frequency of Staphylococcus aureus infections has increased dramatically over the past two decades. This has been attributed to the emergence of multiple antibiotic resistant strains and an increasing population of people with a compromised immune system. It is no longer uncommon to isolate Staphylococcus aureus strains that are resistant to some or all of the standard antibiotics. This phenomenon is a new antibacterial agent for this organism,
A need has been formed for vaccines and diagnostic tests.

[0004] The biosynthetic pathway of saturated fatty acids is very similar between prokaryotes and eukaryotes. However, although there may be no difference in chemical reactions, the mechanisms of biosynthesis are very different. Vertebrates and yeast have type I fatty acid synthetases (FASs), all of which are encoded on one or two polypeptide chains, respectively. Acyl carrier protein (ACP) is an essential part of the complex. In contrast, in most bacterial and plant FASs (type II), each reaction is catalyzed by a separate monofunctional enzyme, and ACP is a separate protein. Mycobacteria are unique in having both type I and type II FASs, the former being involved in basic fatty acid biosynthesis, while the latter being involved in the synthesis of complex cell envelope lipids such as mycolic acid. are doing. Therefore, there appears to be considerable promise for selective inhibition of bacterial systems by broad spectrum antibacterial agents (Rock, C. & Cronan, J. 1996. Biochimica e).
t Biophysica Acts 1302,1-16; Jackowski, S. 1992.I
n Emerging Targets in Antibacterial and Antifungal
Chemotherapy. Ed. J. Sutcliffe & N. Georgopapadak
ou. Chapman & Hall, New York; Jackowski, S. et al.
(1989). J. Biol. Chem. 264, 7624-7629).

[0005] The first step in the biosynthesis cycle is the condensation of malonyl-ACP with acetyl-CoA by FabH. Prior to this, malonyl-ACP was purchased from Malonyl Co.
A: The ACP transacylase FabD
It is synthesized from CP and malonyl-CoA. In the next round, malonyl-ACP is an extended-chain acyl-AC
Condenses with P (FabB and FabF, synthetases I and II, respectively). The second step of the elongation cycle is
Ketoester reduction by NADPH-dependent β-ketoacyl-ACP reductase (FabG). Subsequent dehydration with β-hydroxyacyl-ACP dehydrase (either FabA or FabZ) results in trans-
2-Enoyl-ACP, which in turn is NADH-
It is converted to acyl-ACP by dependent enoyl-ACP reductase (FabI). In a further round of this cycle, two carbon atoms are added per cycle, resulting in palmitoyl-ACP, where the cycle arrests primarily due to feedback inhibition of FabH and I by palmitoyl-ACP (Heath. , et al, (1996), J. Biol. Chem. 271, 1833.
-1836).

[0006] Cerulenin and thiolactomycin are effective and selective inhibitors of bacterial fatty acid biosynthesis. Further studies of these inhibitors in Gram-negative bacteria have demonstrated that this biosynthetic pathway is essential for survival. To a lesser extent, there have been studies in Gram-positive bacteria. In general, mutants lacking FabD activity are not described. A mammalian homolog of FabD has not yet been identified. Commercial antibiotics do not target fatty acid biosynthesis. Therefore, it is possible that new antibiotics may be inactivated by known antibiotic resistance mechanisms. FabD
There is a need to develop a new class of antibiotics that would be legal.

[0007] Clearly, there is a need for factors such as the FabH embodiments of the present invention which currently have useful benefits in screening compounds for antibiotic activity. Such factors are also useful for examining their role in the pathogenesis of infection, dysfunction and disease. There is also a need for the identification and characterization of such agents and their antagonists and agonists to prevent, ameliorate or ameliorate such infections, dysfunctions and diseases.

[0008] Certain polypeptides of the present invention have the known F
Has amino acid sequence homology to abH protein.

Clearly, there is a need for factors that can be used to screen compounds for antibiotic activity and that can be used to determine their role in the pathogenesis of infection, dysfunction and disease. Therefore, there is a need for the identification and characterization of such factors that can play a role in preventing, ameliorating or correcting infection, dysfunction or disease.

[0010] The polypeptide of the present invention has a known malonyl C activity.
oA: has amino acid homology to ACP transacylase.

[0011]

Means for Solving the Problems and Embodiments of the Invention
Due to the homology between the amino acid sequence shown in Table 1 (SEQ ID NO: 2) and the known amino acid sequence such as d-alanine transamidase protein or the sequence of other proteins, the novel Fa
It is an object of the present invention to provide a polypeptide identified as a bH polypeptide. It is a further object of the present invention to provide a polynucleotide encoding a FabH polypeptide, particularly a polynucleotide encoding a polypeptide designated herein as FabH. In a particularly preferred embodiment of the present invention, the polynucleotide comprises a region encoding a FabH polypeptide comprising the sequence shown in Table 1 (SEQ ID NO: 1) and includes the full length gene or a variant thereof. In another particularly preferred embodiment of the present invention, Table 1 (SEQ ID NO: 2)
And a novel FabH protein derived from Staphylococcus aureus comprising the amino acid sequence of According to another aspect of the present invention there is provided an isolated nucleic acid molecule encoding a mature polypeptide expressible by the strain S. aureus WCUH29 contained in the deposited strain.

[0012] A further aspect of the invention provides an isolated nucleic acid molecule encoding FabH, in particular FabH of Staphylococcus aureus, comprising mRN.
A, cDNA and genomic DNA are included. Further embodiments of the present invention include biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comprising them. According to another aspect of the present invention,
There is provided use of the polynucleotide of the present invention for the purpose of treatment or prevention, particularly for the purpose of genetic immunity.
Particularly preferred embodiments of the present invention include naturally occurring allelic variants of FabH and the polypeptides encoded thereby.

In another aspect of the present invention, a novel Staphylococcus aureus polypeptide, referred to herein as FabH, and biologically, diagnostically, prophylactically, clinically or therapeutically useful Also provided are fragments, variants and derivatives thereof, and variants and derivatives of the foregoing fragments and analogs, and compositions comprising them. Particularly preferred embodiments of the invention include variants of the FabH polypeptide encoded by the natural allele of the FabH gene. In a preferred embodiment of the present invention, there is a method for producing the above FabH polypeptide. According to yet another aspect of the invention, there are provided inhibitors of such polypeptides, including, for example, antibodies, which are useful as antibacterial agents.

According to certain preferred embodiments of the present invention,
For assessing FabH expression, treating disease, assaying for genetic variation, and administering FabH polypeptides or polynucleotides to an organism to produce an immunological response against bacteria, especially Staphylococcus aureus bacteria , Products, compositions and methods are provided.

According to certain preferred embodiments of this and other aspects of the invention, there are provided, in particular, polynucleotides that hybridize under stringent conditions to FabH polynucleotide sequences. In certain preferred embodiments of the present invention, antibodies to FabH polypeptides are provided. In another embodiment of the present invention, there is provided a method of identifying a compound that binds or interacts with a polypeptide or polynucleotide of the invention to inhibit or activate their activity, the method comprising: Alternatively, a polypeptide or polynucleotide of the present invention is contacted with the compound to be screened under conditions that allow binding or other interaction with the polynucleotide to evaluate binding or other interaction with the compound. (Such binding or interaction may be a secondary binding or interaction between the polypeptide or polynucleotide and the compound that is capable of providing a detectable signal in response to a second signal.
In which the compound binds to or interacts with the polypeptide or polynucleotide by detecting the presence or absence of a signal resulting from the binding or interaction of the compound with the polypeptide or polynucleotide. And deciding whether to activate or inhibit their activity.

According to yet another aspect of the present invention,
Provided are FabH agonists and antagonists, preferably bacteriostatic or bactericidal agonists and antagonists. In a further aspect of the present invention there is provided a composition comprising a FabH polynucleotide or a FabH polypeptide for administration to a single or 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 description and from reading the remainder of the specification. .

The present invention relates to novel FabH polypeptides and polynucleotides, as described in greater detail below. In particular, the invention relates to novel Staphylococcus aureus FabH polypeptides and polynucleotides, which are related by amino acid sequence homology to FabH polypeptides. In particular, the present invention relates to FabH having the nucleotide and amino acid sequences shown in Table 1 (SEQ ID NO: 1) and Table 1 (SEQ ID NO: 2), respectively, and furthermore, the FabH nucleotide sequence of the DNA in the deposited strain and encoded thereby. Amino acid sequence.

Table 1 FabH Polynucleotide and Polypeptide Sequences (A) Sequence derived from the Staphylococcus aureus FabH polynucleotide sequence [SEQ ID NO: 1] The start codon is shown in bold and underlined. The stop codon is underlined.

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(B) Staphylococcus aureus FabH polypeptide sequence deduced from the polynucleotide sequences in this table [SEQ ID NO: 2]

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Deposited Materials Deposits containing Staphylococcus aureus strain WCUH29 include the National Collection of Industrial and Marine Bacteria Limited (referred to as NCIMB), 23 Street, McDrough Drive, Aberdeen AB21RY, Scotland Deposited on September 11, 1995, with NCIMB accession number 40
771 was assigned. Due to the deposit, the deposited strain is referred to as Staphylococcus aureus WCUH29 strain. In the present specification, the deposit of Staphylococcus aureus strain is referred to as “deposited strain” or “DNA of the deposited strain”. The deposited strain contains the full-length FabH gene. The polynucleotide sequence contained in the deposited strain, as well as the amino acid sequence of the polypeptide encoded thereby, will dominate in events that conflict with any description of the sequences herein. Deposits of the deposited strains have been made under the terms of the Budapest Treaty on the International Recognition of Microbial Deposits for Patent Procedure.
Once the patent is issued, the shares will eventually be sold without any restrictions or conditions. The deposit is provided solely for the convenience of the skilled artisan and does not acknowledge that the deposit is a feasible requirement, as required under 35 USC 112. A license is required to make, use, or sell the deposit, but no such license has been granted here.

Polypeptides The polypeptides of the present invention include the polypeptides of Table 1 [SEQ ID NO: 2] (specifically, mature polypeptides) and polypeptides and fragments, particularly those having the biological activity of FabH. And Table 1 [SEQ ID NO:
Polypeptides and fragments having at least 70%, preferably at least 80%, identity to the polypeptide of Table 2 [SEQ ID NO: 2], more preferably Table 1 At least 90% relative to the polypeptide of [SEQ ID NO: 2]
(More preferably, at least 90% identity), and even more preferably, at least 95% similarity to the polypeptides of Table 1 [SEQ ID NO: 2] (additional More preferably at least 95%
Polypeptides and fragments)
It also includes portions of such polypeptides that usually comprise at least 30, more preferably at least 50, amino acids.

The present invention also relates to a compound represented by the formula: X- (R ₁ ) _m- (R ₂ )-(R ₃ ) _n -Y wherein X is hydrogen at the amino terminal and Y is hydrogen or Is a metal, R ₁ and R ₃ are amino acid residues, m is n, is an integer from 1 to 1000 or zero, and R 2 is an amino acid sequence of the present invention,
Which is an amino acid sequence selected from Table 1]. In the above formula, R ₂ is arranged such that its amino terminal residue is on the left and binds to R ₁ and its carboxy terminal residue is on the right and binds to R ₃ . m
And / or n may be a heteropolymer or a homopolymer in the arrangement of amino acid residues represented by any R group greater than 1, but is preferably a heteropolymer.

A fragment is a variant polypeptide having an amino acid sequence that is entirely identical to some, but not all, of the amino acid sequences of the aforementioned polypeptides. Fa
For bH polypeptides, the fragments may be “free standing,” or may be included within a larger polypeptide by forming a portion or region, most preferably a single polypeptide. As a large single polypeptide. Preferred fragments are listed in Table 1 [SEQ ID NO: 2]
Truncated polypeptides having a portion of the amino acid sequence of, or variants thereof, such as truncating a series of residues including the amino terminus, or truncating a series of residues including the carboxyl terminus There is something. Also preferred are degradation forms of the polypeptides of the present invention in a host, especially Staphylococcus aureus.
Also, fragments characterized by structural or functional attributes, such as alpha helices and alpha helix forming regions, beta sheets and beta sheet forming regions, turns and turn forming regions, coils and coil forming regions, hydrophilic regions, hydrophobic Also preferred are regions, alpha-amphiphilic regions, beta amphiphilic regions, variable regions, surface-forming regions, substrate-binding regions, and fragments containing high antigenic index regions.

Also preferred are biologically active fragments that are those that mediate or improve FabH activity, and have reduced undesired activity. Also included are fragments that are antigenic or immunogenic in animals, especially humans. Fragments containing a receptor or domain of an enzyme that confers a function essential for the survival of Staphylococcus aureus in an individual, particularly a human, or the ability to initiate or maintain a disease, are particularly preferred. A variant which is a fragment of the polypeptide of the invention,
The variants may be used for the production of corresponding full-length polypeptides by peptide synthesis, and thus these variants may be used as intermediates for the production of full-length polypeptides. A variant that is a fragment of a polynucleotide of the invention may be used to synthesize a full-length polynucleotide of the invention.

In addition to the standard one-letter and three-letter codes for amino acids, the terms "X" or "Xaa" are also used to describe certain polypeptides of the present invention. "X" and "Xaa" mean that any of the twenty naturally occurring amino acids can be at such designated positions in the polypeptide sequence.

Polynucleotides Another aspect of the present invention relates to isolated polynucleotides, including the full length gene encoding the FabH polypeptide having the deduced amino acid sequence of Table 1 [SEQ ID NO: 2] and closely related thereto. And variants thereof. Using the information provided herein, for example, using the polynucleotide sequence shown in Table 1 [SEQ ID NO: 1], the starting material Staphylococcus.
Using standard cloning and screening techniques such as those used to clone and sequence chromosomal DNA fragments from Aureus WCUH29 cells, a polynucleotide of the invention encoding a FabH polypeptide is obtained, and then a full-length clone May be obtained. For example, to obtain a polynucleotide sequence of the invention, such as the sequence set forth in Table 1 [SEQ ID NO: 1], of Staphylococcus aureus WCUH29 in E. coli or some other suitable host. Chromosome DN
A typical library of clones of A is probed with a radiolabeled oligonucleotide, preferably 17-mer or longer, derived from the partial sequence. Clones carrying DNA identical to the probe DNA can be distinguished using stringent conditions. By sequencing the individual clones thus identified using sequencing primers designed from the original sequence, the sequence can be extended in both directions and the entire gene sequence determined. Such sequencing is conveniently performed using denatured double-stranded DNA prepared from a plasmid clone. For suitable techniques, see Maniatis, T., Fitsch, FF and Sambrook.
Et al., Molecular Cloning, A Laboratory Manual, No. 2
Edition; Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, New York (1989) (Screening By Hybridiza
tion 1.90 and Sequencing Denatured Double-Strande
d DNA Templates 13.70). In a typical example of the present invention, the polynucleotide shown in Table 1 [SEQ ID NO: 1] is:
DNA from Staphylococcus aureus WCUH29
Found in the library.

The DNA sequence shown in Table 1 [SEQ ID NO: 1] contains an open reading frame encoding a protein consisting of approximately the same number of amino acids as the number of amino acid residues shown in Table 1 [SEQ ID NO: 2]. The estimated molecular weight of a protein can be calculated using the molecular weight of amino acids well known in the art. SEQ ID NO:
The polynucleotide of SEQ ID NO: 1 between nucleotide number 1 of 1 and the stop codon starting at the third nucleotide from the 3 ′ end of SEQ ID NO: 1 encodes the polypeptide of SEQ ID NO: 2. The FabH proteins of the present invention are structurally related to other proteins of the Fab family, as indicated by the results of sequencing the DNA encoding FabH of the deposited strain.

The present invention provides a polynucleotide sequence that is identical over its entire length to the coding sequence in Table 1 [SEQ ID NO: 1]. The coding sequence of the mature polypeptide or fragments thereof, as well as the coding sequence of the mature polypeptide in reading frame with other coding sequences or fragments thereof, e.g., sequences encoding leader or secretory sequences, pre, pro, preproprotein sequences Provided by the present invention. Polynucleotides include, for example, transcribed untranslated sequences, termination signals, ribosome binding sites, sequences that stabilize mRNA, introns, transcribed untranslated sequences such as polyadenylation signals, and additional coding sequences that encode additional amino acids. It may also contain non-coding sequences such as, but not limited to, non-coding 5 'and 3' sequences. For example, it may encode a marker sequence that facilitates purification of the fusion polypeptide. In certain preferred embodiments of the invention, the marker sequence is a hexa-histidine peptide (Gentz et al., Proc. Natl. Acad. Sci., US) as provided in a pQE vector (Qiagen, Inc.).
A, 86: 821-824 (1989)) or HA tag (Wilson et al., Cell, 37: 767 (1984)). The polynucleotides of the present invention also include, but are not limited to, structural genes and native sequences that regulate gene expression.

The present invention also relates to a compound represented by the formula: X- (R ₁ ) _m- (R ₂ )-(R ₃ ) _n -Y wherein X is hydrogen at the 5 'end of the molecule and 3' At the terminus, Y is hydrogen or metal, R ₁ and R ₃ are nucleic acid residues, m is an integer from 1 to 300 or zero, n is an integer from 1 to 300 or zero, and R 2 is Nucleic acid sequence, in particular, a nucleic acid sequence selected from Table 1]. In the polynucleotide of the above formula, R ₂ is
Its 5 'terminal residue is in the left bound to R _1, Part 3'
Terminal residues are arranged to be coupled to R ₃ be in the right. any R wherein m and / or n is greater than 1
The arrangement of the nucleic acid residues represented by the groups may be a heteropolymer or a homopolymer, but is preferably a heteropolymer. In a preferred embodiment, m and / or
Or n is an integer of 1 to 1000.

Most preferably, the polynucleotide of the present invention is derived from Staphylococcus aureus, but is also preferably derived from other organisms of the same taxonomic genus. Further, the polypeptide of the present invention may be obtained from, for example, the same family or order.

The term "polynucleotide encoding a polypeptide" as described herein includes a polynucleotide comprising a sequence encoding the polypeptide sequence of the present invention, and more specifically, a bacterial polypeptide, Specifically, it includes a polynucleotide comprising a sequence encoding a FabH polypeptide of Staphylococcus aureus having the amino acid sequence shown in Table 1 [SEQ ID NO: 2]. The term refers to a single contiguous or discontinuous region encoding a polypeptide (eg, an integrated phage or sequence of phage or sequence), with additional regions that may include coding and / or non-coding sequences. (Interrupted by insertion or editing of the sequence). Furthermore, the present invention also relates to variants of the above polynucleotide that encode variants of the polypeptide having the deduced amino acid sequence of Table 1 [SEQ ID NO: 2]. A variant that is a fragment of the polynucleotide of the present invention may be used to synthesize a full-length polynucleotide of the present invention.

More particularly preferred embodiments are polynucleotides encoding FabH variants having the amino acid sequence of the FabH polypeptide of Table 1 [SEQ ID NO: 2], including some, a few, 10, 1
It has an amino acid sequence obtained by substituting, deleting, or adding 5 to 1, 1 to 3, 2, 1 or 0 amino acid residues in any combination. Especially preferred among these are silent substitutions, additions and deletions, that do not alter the properties and activities of FabH.

A more preferred embodiment of the present invention is shown in Table 1.
FabH having the amino acid sequence shown in [SEQ ID NO: 2]
Polynucleotides that are at least 70% identical over their entire length to a polynucleotide encoding a polypeptide, and polynucleotides that are complementary to such polynucleotides. Alternatively,
At least 80% over the entire length of the polynucleotide encoding the FabH polypeptide of the deposited strain
Most highly preferred are polynucleotides comprising regions that are identical, and polynucleotides complementary thereto. In this regard, polynucleotides that are at least 90% identical over their entire lengths are particularly preferred, especially those with at least 95% identity, and more preferably at least 97% among those with at least 95% identity. It is more preferred, especially at least 98% and at least 99%, even more preferably at least 99%.
A particularly preferred embodiment is a polynucleotide encoding a polypeptide that retains substantially the same biological function or activity as the mature polypeptide encoded by Table 1 [SEQ ID NO: 1].

The present invention further relates to polynucleotides that hybridize to the herein above-described sequences. In this regard, the present invention particularly relates to polynucleotides that hybridize under stringent conditions to the herein above-described polynucleotides. As used herein, the terms "stringent conditions" and "stringent hybridization conditions" refer to at least 95%, preferably at least 97%, identity between sequences.
Means hybridization that occurs only when An example of stringent hybridization conditions is 50% formamide. 5xSSC (150 mM N
aCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution,
10% dextran sulfate, and 20 micrograms /
Incubate overnight at 42 ° C in a solution containing ml of denatured, sheared salmon sperm DNA, followed by about 6
Wash the filter in 0.1 × SSC at 5 ° C. Hybridization and washing conditions are well known and are described in Sambrook et al., Molecular Cloning, A Laboratory.
Manual, 2nd ed .; Cold Spring Harbor, New York (1989), and especially chapter 11 thereof.

The present invention also provides, under stringent hybridization conditions, a suitable library containing the complete gene for the polynucleotide sequence set forth in SEQ ID NO: 1, comprising the sequence of the polynucleotide sequence or a fragment thereof as set forth in SEQ ID NO: 1. Also provided is a polynucleotide essentially comprising a polynucleotide sequence obtainable by screening with a probe having the following and isolating the DNA sequence. Fragments useful for obtaining such polynucleotides include, for example, probes and primers described elsewhere herein.

As further discussed herein with respect to the polynucleotide assays of the present invention, for example, the above-described polynucleotides of the present invention may be used to isolate full-length and genomic clones of FabH-encoding cDNAs and have a high degree of sequence similarity to the FabH gene. CD of other genes having sex
RNA for isolating NA and genomic clones,
It can be used as a hybridization probe for cDNA and genomic DNA. Such probes usually contain at least 15 bases. Preferably such probes have at least 30 bases and may have at least 50 bases. Particularly preferred probes have at least 30 bases and are 50 bases or less. For example, the coding region of the FabH gene can be isolated by screening by synthesizing an oligonucleotide probe using the DNA sequence shown in Table 1 [SEQ ID NO: 1]. The labeled oligonucleotide having a sequence complementary to the sequence of the gene of the present invention is then used to screen a library of cDNA, genomic DNA or mRNA to determine to which member of the library the probe will hybridize. .

The polynucleotides and polypeptides of the present invention can be used, for example, as research reagents and materials for the discovery of therapeutics and diagnostics for diseases, especially humans, and are described herein in particular in connection with polynucleotide assays. Will be discussed further. Table 1 "SEQ ID NO: 1 or 2]
The polynucleotide of the present invention, which is an oligonucleotide derived from the sequence of the present invention, may be used in the method described herein, but is preferably used for PCR to infect all or a part of the polynucleotide identified herein. To determine whether it is transcribed into the isolated tissue. It is understood that such sequences are also useful in diagnosing the stage and type of infection achieved by the pathogen.

The present invention also provides polynucleotides that can encode a polypeptide that is a mature protein with additional amino or carboxyl terminal amino acids, or amino acids present in the mature polypeptide (eg, one or more of the mature forms of the polypeptide). Having a peptide chain). Such sequences play a role in the processing of the protein from the precursor to the mature form, allowing for the transport of the protein, extending or shortening the half-life of the protein, or otherwise manipulating the protein for assay or production. Can be easier. Generally, in vivo, additional amino acids are processed by cellular enzymes and removed from the mature protein. A precursor protein having the mature form of the polypeptide fused to one or more prosequences can be an inactive form of the polypeptide. Removal of the prosequence usually activates such inactive precursors. Some or all of the prosequence can be removed prior to activation. Usually such precursors are called proproteins.

Standard symbols A, G, C, T for nucleic acid bases
In addition to / U, the term "N" can be used to describe certain polynucleotides of the present invention.
"N" is when read in reading the correct reading frame when acting together with adjacent nucleotide positions, and N
Is preferably not a base that has the effect of forming a premature stop codon in such an open reading frame,
Any of the four DNA bases or RNA bases is DNA
Or means at the designated position in the RNA sequence. In short, the polynucleotide of the present invention is a mature protein,
A mature protein to which a leader sequence has been added (which may be referred to as a preprotein), a precursor of a mature protein having one or more prosequences that are not the leader sequence of the preprotein, or a leader sequence and one or more prosequences May encode a preproprotein which is a precursor of a proprotein having the following sequence: the prosequence is usually removed in a processing step that produces an active and mature form of the polypeptide.

Vectors, Host Cells, Expression The present invention also relates to polynucleotides or vectors containing the polynucleotides of the present invention, host cells genetically engineered with the vectors of the present invention, and the production of polypeptides of the present invention by recombinant techniques. Related. Using RNA derived from the DNA construct of the present invention, such a protein can be produced using a cell-free translation system. To produce a recombinant, the host cell can be genetically engineered to incorporate an expression system or a portion thereof, or a polynucleotide of the present invention.
Introduction of a polynucleotide into a host cell can be performed, for example, by using Davi
s et al., Basic Methods in Molecular Biology (1986); S
ambrook et al., Molecular Cloning; A Laboratory Manua
1, second edition; can be performed by methods described in many standard laboratory manuals, such as Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989), for example, calcium phosphate Examples include transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid mediated transfection, electroporation, transduction, scrape loading, ballistic transfer and infection, and the like.

Representative of suitable hosts include bacterial cells, such as Streptococcus, Staphylococci, Enterococci, E. coli, Streptomyces ( Streptomyces and Bacillus subtiis 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, C127,3
T3, BHK, 293 and Bows melanoma cells; and plant cells.

Numerous expression systems can be used to produce the polypeptides of the present invention. Such vectors include chromosomal, episomal and viral derived vectors, such as bacterial plasmid derived, bacteriophage derived, transposon derived, yeast episomal derived, insertion element derived, yeast chromosomal element derived such as baculovirus, papovavirus such as SV40, Vectors derived from viruses such as vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus and retrovirus, and vectors derived from combinations thereof, such as vectors derived from genetic elements of plasmids and bacteriophages, such as cosmids And phagemids. The expression system constructs may contain regulatory regions that control and cause expression. In this regard, in general, any system or vector suitable for retaining, extending or expressing a polynucleotide in a host and / or expressing a polypeptide can be used for expression. Appropriate DNA sequences may be inserted into the expression system by any of a variety of well-known and conventional techniques, for example, see Sambrook et al., Molecular Cloning, A.
Laboratory Manual (described above).

In order to secrete the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, it can be incorporated into a polypeptide expressing an appropriate secretion signal. These signals may be intrinsic to the polypeptide or they may be heterologous signals.

The polypeptides of the present invention can be recovered and purified from recombinant cell culture by well-known methods, including, for example, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose. Examples include chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. Most preferably, high performance liquid chromatography is used for purification. If the polypeptide is denatured during isolation and / or purification, well-known techniques for protein regeneration can be used to regain an active conformation.

Diagnostic Assays The present invention also provides the F of the invention for use as a diagnostic reagent.
It also relates to the use of abH polynucleotides. Detection of FabH in eukaryotes, particularly mammals, and especially humans,
A diagnostic method for diagnosis of a disease is provided. Eukaryotes (also referred to herein as "individuals"), particularly mammals, and especially humans, infected with an organism containing the FabH gene can be detected at the DNA level by a variety of methods. Diagnostic nucleic acids include cells and tissues of infected individuals, such as bone, blood,
It can be obtained from muscle, cartilage and skin. Genome D
NA can be used directly for detection, or can be amplified enzymatically by using PCR or other amplification methods prior to analysis. RNA or cDNA can also be used in the same way. Using amplification methods, prokaryotic strains present in eukaryotes, particularly mammals, and especially humans, can be characterized by genotyping prokaryotic genes. Deletions and insertions can be detected by changes in the size of the amplification product when compared to the genotype of the control sequence. Point mutations can be identified by hybridizing the amplified DNA to a labeled FabH polynucleotide sequence. Perfectly matched sequences are digested by RNase
Alternatively, it can be distinguished from mismatched duplexes by differences in melting temperatures. By detecting changes in electrophoretic mobility of DNA fragments in gels with or without denaturing agents,
Alternatively, DNA sequence differences may be detected by direct DNA sequencing. For example, Meyers et al., Science, 230: 1.
242 (1985). Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and S1 protection or chemical cleavage methods. For example, Cotton et al., Proc. Natl. Acad. Sci., US
A, 85: 4397-4401 (1985).

Cells carrying a mutation or polymorphism in the gene of the present invention can be prepared at the DNA level by various techniques.
For example, it may be detected by serotyping.
For example, mutations can be detected using RT-PCR. RT-PCR is an automatic detection system such as GeneScan
It is particularly preferable to use them in combination with the like. RNA
Or cDNA or RT-PCR for the same purpose
May be used. By way of example, PCR primers complementary to the nucleic acid encoding FabH can be used to identify and analyze mutations. These primers may be used to amplify a gene isolated from an infected individual, and then subject the gene to various techniques for examining DNA sequences. In this way, mutations in the DNA sequence can be detected and used to diagnose infection and serotype and / or classify infectious agents.

The present invention also provides a method of diagnosing a disease, preferably a bacterial infection, more preferably an infection by Staphylococcus aureus, comprising a polynucleotide having the sequence of Table 1 [SEQ ID NO: 1]. Is characterized by detecting an increase in the expression level of, from a sample derived from an individual. Increasing or decreasing FabH polynucleotide expression can be determined by any of the methods well known in the art for quantifying polynucleotides, such as amplification, PCR, RT-P.
It can be measured using CR, RNase protection, Northern blotting and other hybridization methods.

In addition, a diagnostic assay of the invention for detecting overexpression of FabH protein compared to normal control tissue samples may be used to detect, for example, the presence of an infection. Assay techniques that can be used to determine levels of FabH protein, in a sample derived from a host are well-known to those of skill in the art. Such assays include radioimmunoassays, competitive binding assays, western blot analysis, and ELISA assays.

Antibodies Antibodies immunospecific for such polypeptides can be obtained using the polypeptides of the present invention or variants thereof, or cells expressing them, as an immunogen. As used herein, “antibody” includes monoclonal and polyclonal antibodies, chimeras, single chains, monated and humanized antibodies, and Fab fragments, as well as Fab fragments, such as the products of an immunoglobulin expression library.
Fragments are also included. Antibodies raised against the polypeptides of the invention can be obtained by administering fragments, analogs or cells bearing the polypeptides or epitopes, preferably to non-human animals, using conventional laboratory methods. Monoclonal antibodies can be prepared using techniques well known to those skilled in the art, which provide antibodies produced by continuous cell line cultures. Examples include Kohler, G. and Milstein, C., Nature, 256: 49.
5-497 (1975); Kozbor et al., Immunology Today, 4:72.
(1983); Cole et al., Monoclonal Antibodies and Canc
er Therapy, Alan R Liss, Inc., pages 77-96 (1985). Techniques described for the production of single chain antibodies (US Pat. No. 4,946,778)
Can be applied to obtain a single-chain antibody against the polypeptide of the present invention. In addition, antibodies such as humanized antibodies may be expressed using transgenic mice or other organisms, for example, other mammals.

Alternatively, using a phage display technique, a repertoire of PCR amplified v genes of human lymphocytes screened for having anti-polypeptide was screened for having anti-FabH. Antibody genes having binding activity to polypeptides can also be selected from human or untreated libraries (McCafferty,
J. et al., Nature 348: 552-554 (1990); Marks, J. et al., B
iotechnology 10: 779-783 (1992)). The affinity of these antibodies can also be improved by chain shuffling (Clackson, T. et al., Nature 3
52: 624-628 (1991)).

When there are two antigen binding domains,
Each domain is directed against a different epitope, termed a "bispecific" antibody. Clones expressing polypeptides may be isolated or identified using the above antibodies, and the antibodies can be purified by affinity chromatography.

Thus, antibodies, especially against FabH, may be used to treat infections, especially bacterial infections.

[0054] Polypeptide variants include antigenically, epitopically or immunologically equivalent variants and are particular embodiments of the present invention. As used herein, the term "antigenically equivalent derivative" refers to a specific product that, when formed on a protein or polypeptide in accordance with the present invention, interferes with the immediate physical interaction between a pathogen and a mammalian host. And polypeptides specifically recognized by the antibodies of the above. As used herein, the term "immunologically equivalent derivative" refers to an antibody that, when used in a formulation suitable for producing antibodies in vertebrates, causes immediate physical contact between the pathogen and the mammalian host. Includes peptides or equivalents that act to interfere with the interaction.

Polypeptides, such as antigenically and immunologically equivalent derivatives, or fusion proteins thereof, can be used as antigens to immunize mice or other animals, such as rats or chickens. Fusion proteins can confer stability on a polypeptide. The antigen can bind to an immunogenic carrier protein, such as, for example, by conjugation, to bovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH). Alternatively, a multi-antigen peptide comprising multiple copies of a protein or polypeptide, or a polypeptide antigenically or immunologically equivalent thereto, has sufficient antigenicity to improve immunogenicity. No need to use carrier.

Preferably, the antibody or variant thereof is
Modified to reduce immunogenicity in the individual. For example, if the individual is a human, most preferably, the antibody is "humanized"; in this case, the complementarity-determining regions of the antibody from the hybridoma have been transplanted into human monoclonal antibodies, e.g., Jones, P. Et al., Nature 321: 522-5
25 (1986) or Tempest et al., Biotechnology 9: 266-273.
(1991).

When the polynucleotide of the present invention is used in genetic immunization, for example, direct injection of plasmid DNA into muscle (Wolff et al., Hum. Mol. Genet. 1: 363 (199)
2); Manthorpe et al., Hum. Gene Ther. 4: 419 (1963)),
Delivery of complexes of specific protein carriers and DNA (Wu
J. Biol. Chem. 264: 16985 (1989)), DNA co-precipitation with calcium phosphate (Benvenisty & Reshef, PNAS 83: 9).
551 (1986)), DNA in various forms of liposomes
Encapsulation (Kaneda et al., Science 243: 375 (1989)), Particle bombardment (Tang et al., Nature 356: 152 (1992); Eisenbraun)
Et al., DNA Cell Biol. 12: 791 (1993)) and in vivo infection with cloned retroviral vectors (Seeger
It is preferred to use a suitable delivery method such as PNAS 81: 5849 (1984).

Antagonists and Agonists-Assays and Molecules The polypeptides of the present invention may be used to evaluate the binding of small molecule substrates and ligands, for example, in cells, cell-free preparations, chemical libraries, and natural product mixtures. . These substrates and ligands may be natural substrates and ligands, and may be structural or functional mimetics. For example, Coligan et al., Current Protoco
See ls in Immunology 1 (2): Chapter 5 (1991).

The present invention also relates to compounds that enhance (agonist) or inhibit (antagonist) the action of FabH polypeptides or polynucleotides, in particular, compounds for identifying bacteriostatic and / or bactericidal compounds. Also provided are screening methods. The screening method is a high throughput method. For example, to screen for agonists or antagonists, Fab
Synthetic reaction mixtures, membranes, cell compartments such as cell surface membranes or cell walls, or preparations thereof, comprising H polypeptides, and labeled substrates or ligands of such polypeptides, can be FabH agonists or antagonists. Incubate in the presence or absence of the candidate molecule. Candidate molecule is Fa
The ability to agonize or antagonize a bH polypeptide
Reflected in reduced binding of labeled ligand or reduced production of product from such substrates. Molecules that bind and have no effect, ie, that do not induce the effects of FabH, may be the best antagonists. Molecules that bind well and increase the rate of product formation from the substrate are agonists. The production rate or level of product from the substrate can be enhanced by using a reporter system. Reporter systems useful in this regard include labeled substrates for colorimetry that are converted to products,
Examples include, but are not limited to, a reporter gene responsive to a change in FabH polynucleotide or polypeptide activity, and binding assays well known in the art.

Another example of an assay for FabH antagonists is a competition assay, in which FabH and a potential antagonist are converted to a FabH binding molecule, a recombinant FabH binding molecule, a natural substrate or ligand, under conditions suitable for a competition inhibition assay. Or mixed with a substrate or ligand mimetic. FabH can be labeled, for example, with a radioactive or colorimetric compound, and the number of FabH molecules bound to the binding molecule or converted to product can be accurately determined to assess the effect of the potential antagonist.

Potential antagonists include small organic molecules, peptides, polypeptides, and antibodies that bind to a polypeptide of the present invention and thereby inhibit or eliminate its activity. Potential antagonists may also be small organic molecules, peptides, polypeptides, such as closely related proteins or antibodies, which bind to the same site on the binding molecule but induce the activity induced by FabH. Do not interfere with the action of FabH by excluding it from binding. Potential antagonists include small molecules that bind to and occupy the binding site of the polypeptide, thereby preventing binding to a cellular binding molecule and preventing normal biological activity.
Examples of small molecules include, but are not limited to, small organic molecules, peptides, peptide-like molecules, and the like. Other potential antagonists include antisense molecules (for a description of these molecules see Ok
ano, J., Neurochem. 56: 560 (1991); Oligodeoxy-nucle
otides as Antisense Inhibitors of Gene Expressio
n, CRC Press, Boca Raton, FL (198
8)). Preferred potential antagonists include Fa
There are bH related compounds and FabH variants.

Each of the DNA sequences provided herein may be used in the discovery and development of antibacterial compounds. The encoded protein, when expressed, can be used as a target for screening antibacterial agents. In addition, constructing an antisense sequence using the amino-terminal region of the encoded protein or a DNA sequence encoding a Shine-Dalgarno sequence or other translation-enhancing sequence of each mRNA,
The expression of the coding sequence of interest can also be controlled.

The present invention provides the use of a polypeptide, polynucleotide or inhibitor of the present invention to disrupt the etiology involved in the sequelae of infection and the initial physical interaction between mammalian hosts. In particular, the molecules of the present invention can be used to prevent bacterial attachment to mammalian extracellular matrix proteins on an internal device or to extracellular matrix proteins in wounds, in particular, the attachment of Gram-positive bacteria; Initiation blocks FabH protein-mediated entry into mammalian cells (Rosenshine et al., Infect. Immunol. 6
0: 2211 (1992)); blocking bacterial adhesion between mammalian extracellular matrix proteins and bacterial FabH protein; mediating tissue damage; infection initiated by other than implantation of an indwelling device or other surgical method Can be used to block the progression of normal disease states.

The antagonists and agonists of the present invention may be used, for example, for controlling and treating diseases.

[0065] Helicobacter pylori
pylori) (also referred to herein as H. pylori) is a bacterium that affects three people worldwide who develop gastric cancer, ulcers, and gastritis.
Infects more than one in one stomach (International Agency for Research on Cancer (Inte
rnational Agency for Research on Cancer) (1994) S
chistomoses, Liver Flukes and Helicobacter Pylori
(International Agency for Research on Cancer, Lyo
n, France; http: // www. uicc. ch / ecp / ecp2904.
htm)). In addition, the International Agency for Research on Cancer has recently recognized a causal relationship between Helicobacter pylori and gastric adenocarcinoma and has classified the bacterium as a Group I (limited) carcinogen. Preferred antimicrobial compounds of the invention (gidB, found using the screening methods provided by the invention)
Agonists and antagonists of polypeptides and / or polynucleotides), especially broad spectrum antibiotics, are useful in the treatment of Helicobacter pylori infection. Such treatment reduces the appearance of Helicobacter pylori-induced cancer, for example, gastrointestinal cancer. Such treatments also cure gastric ulcers and gastritis.

Vaccine Another aspect of the present invention relates to a method for inducing an immunological response in an individual, especially a mammal, comprising the steps of infection, particularly bacterial infection, most particularly Staphylococcus aureus infection. Characterized by inoculating the individual with an antibody and / or a fragment or variant thereof sufficient to generate an antibody and / or T cell response to protect the individual. Also provided is a method wherein such an immunological response slows bacterial replication. Yet another aspect of the present invention provides
The present invention relates to a method of inducing an immunological response in an individual, the method comprising producing FabH, or a fragment or variant thereof, in vivo, whether or not the disease is already established in the individual. Delivering a nucleic acid vector that directs the expression of to an individual, for example, to induce an immunological response that produces an antibody and / or a T cell immune response (eg, a cytokine producing T cell or a cytotoxic T cell), Producing antibodies that protect the individual from disease. As a method of rapidly administering a gene into a desired cell, there is a method such as coding on a particle.
Such nucleic acid vectors may include DNA, RNA, modified nucleic acids, or DNA / RNA hybrids.

A further aspect of the invention is that an immunological response can be induced in a host, or when introduced into an induced host, an immunological response to FabH or the protein encoded thereby, is induced in the host. With respect to the immunological composition to be induced, the composition comprises the recombinant FabH or the protein encoded thereby, and comprises DNA encoding and expressing an antigen against FabH or the protein encoded thereby. The immunological response may be used therapeutically or prophylactically, and the immunological response may be CTL or C
It may be in the form of antibody immunity or cellular immunity, such as that generated from D4 ⁺ T cells.

A FabH polypeptide or a fragment thereof can be used as a co-localizing protein (co-protein) that does not itself produce antibodies, but is capable of stabilizing a first protein and producing a fusion protein having immunogenic and protective properties. protein). Preferably, such a fusion recombinant protein is
Comparison of solubilizing antigen co-proteins such as lipoprotein D from Hemophilus influenzae, co-localized proteins such as glutathione-S-transferase (GST) or beta-galactosidase, and facilitating their production and purification Including large coexisting proteins. In addition, the co-protein can act as an adjuvant in the sense that it provides a universal stimulus in the immune system. The coexisting protein can bind to either the amino or carboxyl terminus of the first protein.

The present invention relates to polypeptides or polynucleotides of the present invention and Sato Y. et al., Science 273: 352 (19).
Compositions, particularly vaccine compositions, and methods comprising immunostimulatory DNA sequences as described in 66) are provided.

The present invention has also been shown to encode invariant regions of bacterial cell surface proteins in DNA constructs used for such genetic immunization experiments in animal models infected with Staphylococcus aureus. Provided are methods of using the described polynucleotides or, especially, fragments thereof, which are useful, inter alia, for identifying protein epitopes capable of stimulating a prophylactic or therapeutic immune response. This research has been used in mammals,
Successful preparation of particularly valuable monoclonal antibodies from essential organs of animals that have successfully resisted and cleared infection, particularly for the development of prophylactic or therapeutic treatments for bacterial infections, especially Staphylococcus aureus infections in humans It seems to be possible to do.

The polypeptide may be used as an antigen for host inoculation to obtain a specific antibody that protects against bacterial invasion, for example by inhibiting bacterial attachment to damaged tissue. Examples of tissue damage include skin or connective tissue wounds caused by, for example, mechanical, chemical or thermal damage, or by implantation of an indwelling device, or mucous membranes, such as wounds of the mouth, mammary gland, urethra or vagina, etc. is there.

The invention also includes a vaccine formulation containing the immunogenic recombinant protein together with a suitable carrier. Since the protein can be degraded in the stomach, it is preferably administered parenterally, for example, subcutaneously, intramuscularly, intravenously or intradermally. Formulations suitable for parenteral administration include aqueous or non-aqueous sterile agents which may contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the body fluids of the individual, preferably blood. Injectable solutions, as well as aqueous or non-aqueous sterile suspensions, which may contain suspending agents or thickening agents.
The formulations may be presented in single-dose or multi-dose containers, for example, sealed ampules and vials, and stored in a lyophilized condition requiring only the addition of a sterile liquid carrier immediately before use. Vaccine formulations may contain adjuvant systems that increase the immunogenicity of the formulation, such as, for example, an oil-in-water system or other systems well known in the art. The dosage depends on the specific activity of the vaccine and can be readily determined by routine experimentation. Although the invention has been described with reference to particular FabH proteins, the invention relates to naturally occurring proteins and fragments of similar proteins with additions, deletions or substitutions that do not substantially affect the immunogenic properties of the recombinant protein. It will be understood that it includes.

Compositions, Kits and Administration The present invention also relates to compositions comprising the above-described polynucleotides or polypeptides, or agonists or antagonists thereof. The polypeptides of the present invention can be used in admixture with non-sterile or sterile carriers used for cells, tissues or organisms, for example, with a pharmaceutical carrier suitable for administration to a subject. Such compositions comprise, for example, a solvent additive or a therapeutically effective amount of a polypeptide of the present invention, and a pharmaceutically acceptable carrier or excipient. Such carriers 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 present invention further relates to diagnostic and pharmaceutical packs and kits comprising one or more containers filled with one or more of the above-described components of the present invention.

The polypeptides of the present invention and other compounds may be used alone or in combination with other compounds such as therapeutic compounds. The pharmaceutical composition may be administered by any effective and convenient means, for example, topically, orally, vaginally, intravenously, intraperitoneally, intramuscularly, subcutaneously, intranasally, or intradermally. . In therapy or as a prophylactic, the active substance can be administered to the individual as an injectable composition, for example, preferably as an isotonic sterile aqueous dispersion. Alternatively, the composition may be for topical application, for example, ointments, creams, lotions, eye ointments, eye drops, ear drops,
Mouthwashes, impregnated dressings and sutures, and may be formulated in a form such as an aerosol, and may contain suitable conventional additives such as preservatives, solvents to assist drug penetration, and ointments and creams. May contain a softener. Such topical formulations may contain compatible conventional carriers, such as cream or ointment bases and, for lotions, ethanol or oleyl alcohol. Such carriers may range from about 1% to about 98% of the formulation by weight.
And more usually up to about 80% of the formulation by weight. For administration to mammals, especially humans, the daily dose of active substance is from 0.01 mg / kg to 10 mg / kg, typically about 1 mg / kg. The physician will always determine the actual dosage which will be most suitable for the individual and will vary according to age, weight and especially the individual's responsiveness. The above dosages are typical of the average case. Of course, there are individual examples where the high and low dose ranges fit, and such examples are within the scope of the present invention.

Indwelling devices include surgical implants, prosthetic devices, catheters, and the like, that is, those that are introduced into the body of an individual and remain in place for an extended period of time. Such devices include, for example, artificial joints, heart valves, pacemakers, vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinary catheters, continuous ambulatory peritoneal dialysis (continuous).
Continuous ambulatory peritoneal dialysis (CAPD) catheter and the like. The compositions of the present invention may be administered by injection to achieve a systemic effect against relevant bacteria shortly before insertion of the indwelling device. After surgery, treatment may continue for as long as the device is present in the body. In addition, it can be used on covers that spread during surgical procedures to protect against bacterial wound infections, especially those of Staphylococcus aureus.

Many orthopedic surgeons consider that humans with prosthetic joints should be considered for antibiotic prophylaxis before dental treatment that can result in bacteremia. Delayed severe infection is a serious complication that sometimes leads to loss of prosthetic joints, with significant morbidity and mortality.
It is therefore possible in this context to extend the use of active substances as an alternative to prophylactic antibiotics.

In addition to the treatments described above, the compositions of the invention may generally be used as wound healing agents to prevent bacteria from adhering to matrix proteins exposed to wound tissue,
In dental treatment, prophylactic use may be used in place of or in combination with antibiotic prophylaxis.

Alternatively, the composition of the present invention may be used to bathe an indwelling device immediately prior to insertion. For soaking wounds or indwelling devices, the active substance is from 1 μg / ml to 1 μg / ml.
Preferably, the concentration is 0 mg / ml. The vaccine composition is conveniently in injectable form. Conventional immune adjuvants may be used to enhance the immune response. A unit dose suitable for vaccination is 0.5-5 μg / kg of antigen,
Such a dose is preferably administered 1 to 3 times at an interval of 1 to 3 weeks. For the compounds of the present invention, no adverse toxicological effects are observed that would prevent administration to appropriate individuals in the dosage ranges indicated. Each document disclosed in this specification,
The entirety is hereby incorporated by reference. Any patent application for which this application claims priority is deemed to be incorporated herein by reference in its entirety.

Terminology The following description is provided to facilitate understanding of certain terms that are commonly used in the specification, and particularly in the Examples.

“Disease” means a disease caused by or associated with bacterial infection, including upper respiratory tract infections (eg, otitis media, bacterial tracheitis, acute pharyngealitis, thyroiditis), lower Respiratory tract infections (eg, purulent,
Pulmonary abscess), heart infection (eg, infectious endocarditis), gastrointestinal infection (eg, secretory diarrhea, spleen abscess, retroperitoneal abscess),
CNS infections (eg, cerebral abscess), eye infections (eg, blepharitis, conjunctivitis, keratitis, endophthalmitis, anterior septum and orbital cellulitis, lacrimal sacitis), renal and ureteral infections (eg, epididymitis,
Intrarenal and perirenal abscess, toxic shock syndrome),
Includes skin infections (eg, impetigo, folliculitis, skin abscess, cellulitis, wound infections, bacterial myositis), and diseases such as bone and joint infections (eg, septic arthritis, osteomyelitis). A "host cell" has been transformed or transfected with an exogenous polynucleotide sequence,
Or a cell that can be transformed or transfected.

As is well known in the art, "identity"
Or "similarity" is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be, as determined by comparing the sequences. In the art, "identity" also sometimes means the degree of relatedness between two polypeptide or polynucleotide sequences, as determined by the match between the two strands of such sequences. "identity"
And "similarity" are the following methods (but not limited to)
(Computational Molecular Bi
ology, Lesk, AM, Oxford University Press, New York, 1988; Biocomputing:
Informatics and Genome Projects, edited by Smith, DW, Academic Press, New York, 1993; Computer
Analysis of Sequence Data, Part I, Griffin, AM
And Griffin, HG, Humana Press, New Jersey, 1994; Sequence Analysis in Molecular B
iology, von Heinje, G., Academic Press, 1987
Year; and Sequence Analysis Primer, Gribskov, M. and Devereux, J. Ed., M Stockton Press, New York, 1991; Carillo, H. and Lipman, D., SIAM
J. Applied Math., 48: 1073 (1988)). Preferred methods for determining identity are designed to give the best match between the two sequences tested. Methods for determining identity and similarity are codified in publicly available computer programs. A preferred computer program method for determining identity and similarity between two sequences is
GCG program package (Devereux, J. et al., Nucleic
Acids Research 12 (1): 387 (1984), BLASTP, BLASTN
And FASTA (Atschul, SF et al., J. Molec. Biol. 215: 403).
(1990))), but not limited thereto. As an example, a polynucleotide having a nucleotide sequence having at least 95% identity to a control nucleotide sequence of SEQ ID NO: 1, for example, is a polynucleotide whose nucleotide sequence is the nucleotide of a control nucleotide acid sequence of SEQ ID NO: 1. The nucleotide sequence of the polynucleotide is identical to the control sequence except that it can include up to 5 nucleotide changes for every 100. In other words, at least 9 relative to the control acid sequence
To obtain a polypeptide having a nucleotide sequence that is 5% identical, up to 5% of the nucleotides in the control sequence may be deleted or replaced with another nucleotide, or Up to 5% of the nucleotides may be inserted into the control sequence. These mutations in the control sequence may be at the 5 'or 3' terminal position of the control nucleotide sequence, or may be at any position between those terminal positions, It may be interspersed with nucleotides, or may be one or more contiguous groups within a control sequence. Similarly, SEQ ID NO: 2
For example, a polypeptide having an amino acid sequence having at least 95% identity to the control amino acid sequence of SEQ ID NO: 2 is up to 5 per 100 amino acids of the control amino acid sequence of SEQ ID NO: 2. The amino acid sequence of the polypeptide is identical to the control sequence, except that it may include an amino acid change of In other words, to obtain a polypeptide having an amino acid sequence that is at least 95% identical to the control amino acid sequence, up to 5% of the amino acids in the control sequence may be deleted or substituted with other amino acids. Alternatively, up to 5% of all amino acids in the control sequence may be inserted into the control sequence. These changes in the control sequence may be at the amino or carboxy terminal position of the control amino acid sequence, or may be at any position between their ends, and may be interspersed in the control sequence. Or one or more contiguous groups within a control sequence.

"Isolated" means altered "by the hand of man" from its natural state, that is, in the case of natural products, altered and / or removed from its original environment. I do. For example, a polynucleotide or polypeptide that is naturally occurring in a living organism is not `` isolated '', but the same polynucleotide or polypeptide separated from its coexisting material in its natural state is a term used herein. Has been "isolated".

“Polynucleotide (s)” is
In general, it also refers to polyribonucleotides or polydeoxyribonucleotides, which may be unmodified RNA or DNA, or modified RNA or DNA. Thus, a “polynucleotide” is, inter alia, DNA that is a mixture of single- and double-stranded DNA, a mixture of single- and double-stranded regions or a mixture of single-, double- and triple-stranded regions, single-stranded And double-stranded RNA, and RNA that is a mixture of single- and double-stranded regions, single-stranded or more typically double- or triple-stranded, or a mixture of single- and double-stranded regions. May be DN
A and hybrid molecules including RNA,
Not limited to these. In addition, "polynucleotide" as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The chains in such regions may be from the same molecule or from different molecules. This region may include all one or more of these molecules, but more typically includes only one region of some of the molecules. One of the molecules of the triple helix region is often an oligonucleotide.
As used herein, the term "polynucleotide" includes DNAs or RNAs as described above containing one or more modified bases. Thus, DNA or RNA having a backbone that has been modified for stability or for other reasons is also a "polynucleotide" as contemplated herein. In addition, DNAs containing unusual bases such as inosine or modified bases such as tritylated bases
Alternatively, RNA (only two examples are shown) is the term polynucleotide herein. It will be apparent that numerous modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term "polynucleotide", as used herein, refers to such chemically, enzymatically or metabolically modified forms of polynucleotides and viruses, especially cells, including simple and complex cells. Includes characteristic DNA and RNA chemical forms. "Polynucleotide" often refers to oligonucleotide (s)
And short polynucleotides referred to as

[0084] As used herein, "polypeptide" is used to refer to a peptide or protein containing two or more amino acids joined together linearly by peptide bonds. "Polypeptide" refers to both short chains, commonly referred to in the art as, for example, peptides, oligopeptides and oligomers, and long chains, which come in many forms and are commonly referred to in the art as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. "Polypeptide" encompasses those resulting from natural processes, such as processing and other post-translational modifications, as well as by chemical modifications. Such modifications are well described in basic texts and in more detailed articles as well as in numerous research literatures, which are well known to those skilled in the art. It will be appreciated that the same type of modification may be present at the same or varying degrees at several sites in the polypeptide. Modifications can be anywhere in the polypeptide, including the peptide backbone, amino acid side chains, and the amino or carboxy terminus. Modifications include, for example, acetylation, acylation, A
DP-ribosylation, amidation, flavin covalent bond, heme moiety covalent bond, nucleotide or nucleotide derivative covalent bond, lipid or lipid derivative covalent bond, phosphotidylinositol covalent bond, cross-linking, cyclization,
Disulfide bond formation, demethylation, covalent crosslink formation, cystine formation, pyroglutamate formation, formylation, gamma-carboxylation, sugar chain formation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation , Proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulphation,
There are transfer RNA-mediated amino acid additions to proteins, such as arginylation, and ubiquitination. For example, Prot
eins-Structure and Molecular Properties, 2nd edition, T.
E. Creighton, WH Freeman and Company, New York (1993). For example, Posttranslationa
l Covalent Modification of Proteins, BC Johnson
Hen, Wo of Academic Press, New York (1983)
ld, F., Posttranslational Protein Modifications: Pe
rspective and Prospects, pp. 1-12; Seifter et al., Meth.
Enzymol. 182: 626-646 (1990) and Rattan et al., Prote.
in Synthesis: Posttranslational Modifications and
Aging, Ann. NY Acad. Sci. 663: 48-62 (1992). The polypeptide may be branched or cyclic, with or without branching. Cyclic, branched, and branched and cyclic polypeptides may result from post-translation natural processes, as well as may be produced by entirely synthetic methods.

As used herein, the term "variant" 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 change the amino acid sequence of the polypeptide encoded by the control polynucleotide. As discussed below, nucleotide changes result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, the difference is that the sequences of the control polypeptide and the variant are
Overall very similar, limited to the same in many areas. Variant and control polypeptides can have an altered amino acid sequence by one or more substitutions, additions, or deletions occurring in any combination. The 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 naturally occurring, for example, an allelic variant, or may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides can be made by mutagenesis techniques or by direct synthesis and other recombinant methods known to those skilled in the art.

[0086]

The following examples, except as described in detail, are performed using standard techniques well known to those skilled in the art. The examples are given for illustrative purposes only and do not limit the invention. Example 1 Strain Selection, Library Production and Sequencing A polynucleotide having the DNA sequence set forth in SEQ ID NO: 1 was isolated from a Staphylococcus in E. coli.
Obtained from a library of clones of Aureus chromosomal DNA. Duplicate Staphylococcus aureus D
In some cases, flanking DNA sequences of SEQ ID NO: 1 have been constructed using sequencing data from two or more clones containing NA. Normal method, for example, the following method 1
Libraries can be manufactured by (2) and (3). Whole cell DN
A is from Staphylococcus aureus WCUH29,
Isolate according to standard methods and size fractionate by one of two methods.

Method 1 For size fractionation according to the standard method,
Is mechanically sheared through an injection needle. Fragments up to 11 kbp in size were converted to exonuclease and D
The ends are made blunt by treatment with NA polymerase and an EcoRI linker is added. Fragment to E
The vector is ligated to the vector lambda ZapII that has been cut with coRI, the library is packaged by standard methods, and E. coli is infected with the packaged library. The library is amplified by standard methods.

Method 2 Whole cell DNA was prepared using a library vector (for example, Rs
aI, PalI, AluI, Bshl235I) are partially hydrolyzed with a single restriction enzyme or combination of restriction enzymes, suitably resulting in a series of fragments, and the fragments are size fractionated according to standard methods. An EcoRI linker is ligated to the DNA and fragment, then ligated to the vector Lambda ZapII that has been cut with EcoRI, the library is packaged by standard methods, and E. coli is infected with the packaged library. The library is amplified by standard methods.

[0089]

[Sequence List] (1) General Information: (i) Applicant: (A) Name: SmithKline Beecham Corporation (B) Street: One Franklin Plaza (C) City: Philadelphia (D) State: PA (E ) Country name: United States of America (F) ZIP: 19103 (ii) Title of the invention: New FabH (iii) Number of sequences: 2 (iv) Computer readable form: (A) Media type: diskette (B) Computer: IBM compatible (C) Operating system: Windows (D) FastSEQ version 2.0b for Windows (v) Current application data: (A) Application number:

(2) Information on SEQ ID NO: 1 (i) Sequence characteristics: (A) Sequence length: 942 base pairs (B) Sequence type: nucleic acid (C) Number of chains: 2 (D ) topology: wherein the linear (xi) SEQ: SEQ ID NO: 1: ATGAACGTGG GTATTAAAGG TTTTGGTGCA TATGCACCAG AAAAGATTAT TGACAATGCC 60 TATTTTGAGC AATTTTTAGA TACATCTGAT GAATGGATTT CTAAGATGAC TGGAATTAAA 120 GAAAGACATT GGGCAGATGA CGATCAAGAT ACTTCAGATT TAGCATATGA AGCAAGTGTA 180 AAAGCAATCG CTGACGCTGG TATTCAGCCT GAAGATATAG ATATGATAAT TGTTGCCACA 240 GCAACTGGAG ATATGCCATT TCCAACTGTC GCAAATATGT TGCAAGAACG TTTAGGGACG 300 GGCAAAGTTG CCTCTATGGA TCAACTTGCA GCATGTTCTG GATTTATGTA TTCAATGATT 360 ACAGCTAAAC AATATGTTCA ATCTGGAGAT TATCATAATA TTTTAGTTGT CGGTGCAGAT 420 AAATTATCTA AAATAACAGA TTTAACTGAC CGTTCTACTG CAGTTCTATT TGGAGATGGT 480 GCAGGTGCGG TTATCATCGG TGAAGTTTCA GAAGGCAGAG GTATTATAAG TTATGAAATG 540 GGTTCTGATG GCACTGGTGG TAAACATTTA TATTTAGATA AAGATACTGG TAAACTGAAA 600 ATGAATGGTC GAGAAGTATT TAAATTTGCT GTTAGAATTA TGGGTGATGC ATCAACACGT 660 GTAGTTGAAA AAGCGAATTT AACATCAGAT GATATAGATT TATTTATTCC TCATCAAGCT 720 AATATTAGAA TTATGGAATC AGCTAGAGAA CGCTTAGGTA TTTCAAAAGA CAAAATGAGT 780 GTTTCTGTAA ATAAATATGG AAATACTTCA GCTGCGTCAA TACCTTTAAG TATCGATCAA 840 GAATTAAAAA ATGGTAAACT CAAAGATGAT GATACAATTG TTCTTGTCGG ATTCGGTGGC 900 GGCCTAACTT GGGGCGCAAT GACAATAAAA TGGGGAAAAT AG 942

(2) Information on SEQ ID NO: 2: (i) Sequence characteristics: (A) Sequence length: 313 amino acids (B) Sequence type: amino acids (C) Number of chains: 1 (D) Topology: linear (xi) Description of sequence: SEQ ID NO: 2: Met Asn Val Gly Ile Lys Gly Phe Gly Ala Tyr Ala Pro Glu Lys Ile 1 5 10 15 Ile Asp Asn Ala Tyr Phe Glu Gln Phe Leu Asp Thr Ser Asp Glu Trp 20 25 30 Ile Ser Lys Met Thr Gly Ile Lys Glu Arg His Trp Ala Asp Asp Asp 35 40 45 Gln Asp Thr Ser Asp Leu Ala Tyr Glu Ala Ser Val Lys Ala Ile Ala 50 55 60 Asp Ala Gly Ile Gln Pro Glu Asp Ile Asp Met Ile Ile Val Ala Thr 65 70 75 80 Ala Thr Gly Asp Met Pro Phe Pro Thr Val Ala Asn Met Leu Gln Glu 85 90 95 Arg Leu Gly Thr Gly Lys Val Ala Ser Met Asp Gln Leu Ala Ala Cys 100 105 110 Ser Gly Phe Met Tyr Ser Met Ile Thr Ala Lys Gln Tyr Val Gln Ser 115 120 125 Gly Asp Tyr His Asn Ile Leu Val Val Gly Ala Asp Lys Leu Ser Lys 130 135 140 Ile Thr Asp Leu Thr Asp Arg Ser Thr Ala Val Leu Phe Gly Asp Gly 145 150 155 160 Ala Gly Ala Val Ile Ile Gly Glu Val Ser Glu Gly Arg Gly Ile Ile 165 170 175 Ser Tyr Glu Met Gly Ser Asp Gly Thr Gly Gly Lys His Leu Tyr Leu 180 185 190 Asp Lys Asp Thr Gly Lys Leu Lys Met Asn Gly Arg Glu Val Phe Lys 195 200 205 Phe Ala Val Arg Ile Met Gly Asp Ala Ser Thr Arg Val Val Glu Lys 210 215 220 Ala Asn Leu Thr Ser Asp Asp Ile Asp Leu Phe Ile Pro His Gln Ala 225 230 235 240 Asn Ile Arg Ile Met Glu Ser Ala Arg Glu Arg Leu Gly Ile Ser Lys 245 250 255 Asp Lys Met Ser Val Ser Val Asn Lys Tyr Gly Asn Thrr Ser Ala Ala 260 265 270 270 Ser Ile Pro Leu Ser Ile Asp Gln Glu Leu Lys Asn Gly Lys Leu Lys 275 280 285 Asp Asp Asp Thr Ile Val Leu Val Gly Phe Gly Gly Gly Leu Thr Trp 290 295 300 Gly Ala Met Thr Ile Lys Trp Gly Lys 305 310

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 1/00 A61P 1/00 5/00 5/00 9/00 9/00 11/00 11/00 13 / 12 13/12 17/00 17/00 19/00 19/00 19/02 19/02 25/00 25/00 27/02 27/02 27/16 27/16 31/04 31/04 43/00 111 43/00 111 C07K 2/00 C07K 2/00 16/12 16/12 C12P 21/02 C12P 21/02 C C12Q 1/00 C12Q 1/00 Z G01N 33/50 G01N 33/50 (72) Inventor Daniel Earl Gentry United States 19465 Shepp Hill Road 1378, Pottstown, PA (378) Inventor John T. Lonsdale United States 19341 Exton, PA, Edgewood Drive 407 (72) Inventor Jeffrey Erloo・ M United States 19468 Laimrick, Pennsylvania Windmill Foot Court 143 (72) Inventor David Jay Payne United States 19460 Phoenixville, Pennsylvania Waterfall Way No. 618 (72) Inventor Stuart Sea Pearson U.S.A. 9312 High Point Drive 75, Berwyn, PA, USA (72) Lisa Kay Schilling, 18940 East Park Road, Newtown, PA 18940, United States Glen Van Aller, United States 19606, PA 19606 No. 799 (72) Jibrolter Road, Reading State Min Wan United States 19422 Centennial Drive No. 302, Bru Belle, PA (72) Inventor Lee Yi Tong United States 19403 Audubon, Nsylvania, Egypt Road 2828

Claims (22)

[Claims] 1. An isolated polynucleotide comprising a polynucleotide having at least 70% identity to a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2. 2. A polynucleotide having at least 70% identity to a polynucleotide encoding the same mature polypeptide as expressed by the FabH gene contained in the deposited strain Staphylococcus aureus. An isolated polynucleotide comprising: 3. An isolated polynucleotide comprising a polynucleotide encoding a polypeptide comprising an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO: 2. 4. An isolated polynucleotide which is complementary to the polynucleotide of claim 1. 5. The method according to claim 1, wherein the polynucleotide is DNA or RNA.
The polynucleotide of claim 1, which is 6. The polynucleotide of claim 1, comprising the nucleic acid sequence set forth in SEQ ID NO: 1. 7. The polynucleotide of claim 1, comprising from nucleotide 1 shown in SEQ ID NO: 1 to a stop codon. 8. The polynucleotide of claim 1, which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 2. 9. A vector comprising the polynucleotide of claim 1. 10. A host cell comprising the vector of claim 9. 11. The DNA from the host cell of claim 10
A method for producing a polypeptide, comprising expressing a polypeptide encoded by: 12. A method for producing a FabH polypeptide or fragment, comprising culturing the host of claim 10 under conditions sufficient to produce said polypeptide or fragment. 13. A polypeptide comprising an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO: 2. 14. A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2. 15. An antibody against the polypeptide of claim 14. 16. An antagonist that inhibits the activity or expression of the polypeptide according to claim 14. 17. A method of treating an individual in need of a FabH polypeptide comprising administering to the individual a therapeutically effective amount of the polypeptide of claim 14. 18. A method of treating an individual in need of FabH polypeptide inhibition comprising administering to the individual a therapeutically effective amount of the antagonist of claim 16. 19. A method for diagnosing a disease associated with the expression or activity of the polypeptide of claim 14 in an individual, comprising: (a) determining the nucleic acid sequence encoding said polypeptide; and / or b) analyzing the presence or amount of the polypeptide in a sample from the individual. 20. A method for identifying a compound that interacts with the polypeptide of claim 14 and inhibits or activates its activity, under conditions that allow the interaction between the compound and the polypeptide. Assessing the interaction of the compound by contacting the polypeptide with the compound to be screened (such interaction being associated with a second component capable of providing a detectable signal in response to the interaction of the polypeptide with the compound). And then detecting the presence or absence of a signal resulting from the interaction of the compound with the polypeptide,
A method comprising determining whether a compound interacts with a polypeptide to activate or inhibit its activity. 21. A method for inducing an immunological response in a mammal, the method comprising producing Fa and / or T cells sufficient to protect the animal from disease.
A method comprising inoculating a mammal with a bH polypeptide or a fragment or variant thereof. 22. A method of inducing an immunological response in a mammal, comprising expressing a FabH polypeptide or a fragment or variant thereof in vivo to induce an immunological response to generate antibodies and / or T cells. Claim 14 to protect the animal from disease.
And delivering a nucleic acid vector that directs the expression of a FabH polypeptide or fragment or variant thereof.