JP2002543776A - Method using action mechanism of aroA - Google Patents

Abstract

  (57) [Summary] The invention provides aroA polynucleotides and DNA (RNA) encoding aroA polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods of utilizing aroA for screening antimicrobial compounds.

Description

DETAILED DESCRIPTION OF THE INVENTION

TECHNICAL FIELD The present invention relates to newly identified polynucleotides and polypeptides, and their manufacture and use, as well as their variants, agonists and antagonists, and their uses. In particular, in these and other respects, the present invention relates to novel polynucleotides and polypeptides of the aro (5-enolpyruvylshikimate-3-phosphate synthase) family (hereinafter "aroA").

[0002] The genus Streptococci forms a medically important microbial genus and in humans, for example, otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pyothorax and It is known to cause several types of diseases, including endocarditis, most particularly meningitis such as, for example, cerebrospinal fluid infection. Streptococcus pneumoniae is one of the microorganisms that have been studied in more detail since more than 100 years have passed since the isolation of the genus Streptococcus. For example, in fact, much of the early view that DNA is the genetic material was stated in Griffith and Avery, MacLeod and McCarthy studies using this microorganism. Despite the vast amount of research on Streptococcus pneumoniae, many questions remain regarding the toxicity of this microorganism. It is particularly preferred to use Streptococcus genes and gene products as targets for the development of antibiotics. The frequency of Streptococcus pneumoniae 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 compromised immune systems. It is no longer rare to isolate Streptococcus pneumoniae strains that are resistant to some or all of the standard antibiotics. This phenomenon has created a need for new antimicrobial agents, vaccines and diagnostic tests for this organism. Clearly, there is a need for factors such as novel compounds of the present invention that have the benefit of being useful for screening compounds for antibiotic activity. Such factors are also useful for examining their role in the development of infection, dysfunction and disease. There is also a need for such factors and their antagonists and agonists that can play a role in preventing, ameliorating or correcting infection, dysfunction or disease. The polypeptide of the present invention may comprise Lactococcus lactis.
5-enolpyruvoylshikimate-3-phosphate synthase (aroA
) Has amino acid sequence homology to proteins.

DISCLOSURE OF THE INVENTION [0003] The amino acid sequence shown in Table 1 (SEQ ID NO: 2) and known amino acid sequences or other amino acids such as Lactococcus lactis 5-enolpyruvoylshikimate-3-phosphate synthase (aroA) protein Due to the homology between the protein sequences,
It is an object of the present invention to provide a polypeptide identified as a roA polypeptide. It is a further object of the present invention to provide a polynucleotide encoding an aroA polypeptide, particularly a polynucleotide encoding a polypeptide herein designated aroA. In a particularly preferred embodiment of the present invention, the polynucleotide comprises a region encoding an aroA 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 invention, there is a novel aroA protein from Streptococcus pneumoniae comprising the amino acid sequence of Table 1 (SEQ ID NO: 2), or a variant thereof. 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. pneumoniae 0100993 contained in the deposited strain.

[0004] A further aspect of the invention provides an isolated nucleic acid molecule encoding aroA, in particular aroA of Streptococcus pneumoniae, comprising mRNA,
cDNA and genomic DNA are included. Further embodiments of the invention are biological,
Variants thereof useful diagnostically, prophylactically, clinically or therapeutically, and compositions comprising them. According to another aspect of the present invention there is provided the use of a polynucleotide of the present invention for therapeutic or prophylactic purposes, particularly for genetic immunity. Particularly preferred embodiments of the present invention include naturally occurring allelic variants of aroA and the polypeptides encoded thereby.

[0005] In another aspect of the invention, a novel polypeptide of Streptococcus pneumoniae, referred to herein as aroA, and its biologically, diagnostically, prophylactically, clinically or therapeutically useful polypeptides Fragments, variants and derivatives, and variants and derivatives of the foregoing fragments and analogs, and compositions comprising them, are provided. Particularly preferred embodiments of the invention include variants of the aroA polypeptide encoded by a naturally occurring allele of the aroA gene. In a preferred embodiment of the present invention, there is a method for producing the aroA polypeptide described above. According to yet another aspect of the present 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, assessing aroA expression, treating a disease,
For example, treatment of otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pyothorax and endocarditis, most particularly meningitis such as cerebrospinal fluid infection, genetic mutation assays , And products, compositions and methods for the administration of an aroA polypeptide or polynucleotide to an organism to elicit an immunological response against bacteria, especially Streptococcus pneumoniae bacteria. 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 an aroA polypeptide sequence. In certain preferred embodiments of the present invention, there are provided antibodies against aroA polypeptides.

[0007] In another embodiment of the present invention, there is provided a method of identifying a compound that binds to or interacts with a polypeptide or polynucleotide of the present invention to inhibit or activate their activity. The method comprises contacting a polypeptide or polynucleotide of the present invention with a compound to be screened under conditions that allow binding or other interaction between the compound and the polypeptide or polynucleotide, and contacting the compound with the compound. Assess binding or other interaction (such binding or interaction is associated with a second compound capable of providing a detectable signal in response to the binding or interaction of the compound with the polypeptide or polynucleotide). are doing),
The compound then binds 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 to reduce their activity. Deciding whether to activate or inhibit.

In accordance with yet another aspect of the present invention, there are provided aroA agonists and antagonists, preferably bacteriostatic or bactericidal agonists and antagonists. In a further aspect of the invention, for administration to a single or multicellular organism,
Compositions comprising an aroA polynucleotide or an aroA polypeptide are provided. The present invention is selected from the group consisting of a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2 or 4, and a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 or 4. Provided are antagonists or agonists that inhibit the activity of the polypeptide to be administered.

As used herein, in a method that uses or refers to the activity of AroA or a polypeptide of the invention, the activity may include synthesizing p-aminobenzoate, synthesizing ubiquinone, phospho (enol) pyruvate ( PEP) and shikimate 3
-Transformation of phosphate (S3P) into EPSP and inorganic phosphate (Pi), transformation of EPSP and inorganic phosphate (Pi) into phospho (enol) pyruvate (PEP) and shikimate 3-phosphate (S3P), Ar
Binding of oA to phospho (enol) pyruvate, AroA to phospho (enol) pyruvate-pyruvate kinase complex, AroA to phospho (enol)
Binding to pyruvate-lactate dehydrogenase complex, binding of AroA to shikimate 3-phosphate, glyphosate of AroA's forward reaction ("GLP
Antagonistic inhibition by phospho (enol) pyruvate, non-antagonistic inhibition of the forward reaction of AroA by glyphosate versus shikimate 3-phosphate, Aro
Competitive inhibition of the forward response of A by EPSP versus phospho (enol) pyruvate, A
competitive inhibition of the forward response of roA by EPSP versus shikimate 3-phosphate,
Non-antagonistic inhibition of the reverse reaction of AroA by glyphosate versus EPSP, non-antagonistic inhibition of the reverse reaction of AroA by glyphosate versus Pi, antagonistic inhibition of the reverse reaction of AroA by shikimate 3-phosphate versus EPSP, and reverse reaction of AroA Is selected from the group consisting of shikimate 3-phosphate versus antagonistic inhibition by Pi.

Further, according to the present invention, there is provided a method of treating an individual in need of inhibition or activation of an AroA polypeptide, wherein the antimicrobial effective amount of the amino acid sequence of SEQ ID NO: 2 or 4 is An antagonist that inhibits or activates a polypeptide selected from the group consisting of a polypeptide comprising an amino acid sequence that is at least 90% identical thereto, and a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 or 4. A method is provided that comprises administering an agonist to the individual. Also, a method of treating an individual infected with a bacterium, comprising an antimicrobial effective amount of a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2 or 4, and SEQ ID NO: A method is provided which comprises administering to the individual an antagonist or an agonist that inhibits the activity of a polypeptide selected from the group consisting of polypeptides comprising the amino acid sequence shown in 2 or 4.

In any of the methods of the present invention, the bacterium is preferably selected from the group consisting of bacteria of the genus Staphylococcus, Staphylococcus aureus, bacteria of the genus Streptococcus, and Streptococcus pneumoniae. Further, according to the present invention, there is provided a method of treating an individual in need of inhibition or activation of an AroA polypeptide, comprising an antimicrobial effective amount of an antagonist inhibiting the activity of AroA, or activating the activity thereof. A method comprising administering to the individual an agonist that produces The present invention also provides a method of treating an individual infected with a bacterium, the method comprising:
A method comprising administering to the individual an antagonist that inhibits the activity of AroA, or an agonist that activates the activity. The method is also a method for treating an individual infected with a bacterium, the method comprising:
A method comprising administering to the individual a compound that is a competitive inhibitor of shikimate 3-phosphate substrate utilization by AroA. In a preferred embodiment, a compound of the invention, such as an antagonist or agonist, binds to or interacts with the active site of AroA, or interacts with, or prevents the binding of shikimate 3-phosphate.

[0012] A method of treating an individual infected with Streptococcus pneumoniae, comprising administering to the individual an antibacterial effective amount of an antagonist that inhibits the activity of AroA of Streptococcus pneumoniae, or an agonist that activates the activity. Provided by the present invention. Furthermore, it is selected from the group consisting of a polypeptide comprising an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO: 2 or 4, and a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 or 4. Also provided are antagonists that inhibit the activity of the polypeptide. A method of treating an individual in need of inhibition of an AroA polypeptide, comprising: an antimicrobial effective amount of a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2 or 4. Also provided by the present invention is a method comprising administering to the individual an antagonist that inhibits the activity of a polypeptide selected from the group consisting of a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 or 4. .

[0013] Still further provided is a method of inhibiting the activity of an AroA polypeptide, comprising contacting a composition comprising the polypeptide with an effective amount of an antagonist that inhibits the activity of AroA. . Another embodiment of the present invention is a method of inhibiting the growth of bacteria, comprising contacting a composition comprising bacteria with an antimicrobial effective amount of an antagonist that inhibits the activity of AroA. . Yet a further embodiment of the present invention is a method of inhibiting an AroA polypeptide, comprising contacting a composition comprising bacteria with an antimicrobial effective amount of an antagonist that inhibits the activity of AroA. 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.

Terminology The following description is provided to facilitate understanding of certain terms that are commonly used herein, particularly in the examples. "Physical substance (s)" refers to, but is not limited to, cells, tissues and waste, such as bone, blood, from an individual or from a biologically infected, parasitic or inhabited individual. Any substance, including, serum, cerebrospinal fluid, semen, saliva, muscle, cartilage, organ tissue, skin, urine, feces or autopsy material. "Disease (s)" include upper respiratory tract infections (eg, otitis media, bacterial tracheitis, acute pharyngeal inflammation, thyroiditis), lower respiratory tract infections (eg, pyometra, pulmonary abscess), heart infections (eg,
For example, infective endocarditis, gastrointestinal infection (eg, secretory diarrhea, spleen abscess, retroperitoneal abscess), CNS infection (eg, cerebral abscess), eye infection (eg, blepharitis, conjunctivitis,
Keratitis, endophthalmitis, anterior septum and orbital cellulitis, lacrimal inflammation), renal and ureteral infections (eg, epididymitis, intrarenal and perirenal abscess, toxic shock syndrome), skin infections (eg, impetigo) Rash, folliculitis, skin abscess, cellulitis, wound infection, bacterial myositis), and diseases such as bone and joint infections (eg, septic arthritis, osteomyelitis)
It also means diseases caused by or associated with bacterial infections. A “host cell” is a cell that has been introduced (eg, transformed or transfected), or is capable of being introduced (eg, transformed or transfected) by an exogenous polynucleotide sequence. is there.

[0015] As is well known in the art, "identity" refers to the sequence between two or more polypeptide sequences or between two or more polynucleotide sequences, as determined by comparing the sequences. The relationship between In the art, "identity" also means
Sometimes, it also means the degree of relatedness between two polypeptide or polynucleotide sequences, as determined by the match between the two chains of such sequences. "identity"
Can be easily calculated by the known method described below (Computational Molecu
lar Biology, Lesk, AM, Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, S
mith, DW, Academic Press, New York, 1993; Computer Analys
is of Sequence Data, Part I, edited by Griffin, AM and Griffin, HG, Humana Press, New Jersey, 1994; Sequence Analysis in Molecul
ar Biology, von Heinje, G., Academic Press, 1987; and Seque
nce Analysis Primer, Ed. Gribskov, M. and Devereux, J., M. Stockton Press, New York, 1991; and 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. Moreover, methods for determining identity are codified in publicly available computer programs. A preferred computer program method for determining the identity between two sequences is the GCG program package (Devere
ux, J. et al., Nucleic Acids Research 12 (1): 387 (1984), BLASTP,
BLASTN and FASTA (Atschul, SF et al., J. Molec. Biol. 215: 403 (199).
0))), but not limited thereto. The BLAST X program is available from NCBI and other sources (BLAST Manual, Altshul, S. et al., NCBI NLM NIH Bethesda
Altschul, S., MD 20894; J. et al. Mol. Biol., 215: 403-410 (199).
0)) is publicly available. In addition, well-known Smith Waterman
The identity can also be measured using the (rman) algorithm.

The parameters for comparing polypeptide sequences include the following parameters: Algorithm: Needleman and Wunsch, J. et al. Mol Biol. 48: 443-45
3 (1970) Comparative matrix: Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA
89: 10915-10919 (1992) BLOSSUM62 Gap penalty: 12 Gap length penalty: 4 A useful program with these parameters is Geneti, Madison, Wiscosin.
Available publicly as the "gap" program from cs Computer Group. The above parameters are the default parameters for comparing peptides (no penalty for end gaps).

The parameters for comparing polynucleotides include the following parameters: Algorithm: Needleman and Wunsch, J. et al. Mol Biol. 48: 443-45
3 (1970) Comparison matrix: match = + 10, mismatch = 0 Gap penalty: 50 Gap length penalty: 3 Available as the "gap" program from the Genetics Computer Group, Madison, Wiscosin. These are the default parameters for comparing nucleic acids.

In some cases, preferred means for "identifying" polynucleotides and polypeptides are obtained in (1) and (2) below. (1) Specific examples of the polynucleotide further include an isolated polynucleotide having a polynucleotide sequence having at least 95, 97 or 100% identity to the control sequence of SEQ ID NO: 1 or 3. Here, the polynucleotide sequence may be identical to the control sequence of SEQ ID NO: 1 or 3, or may have changed by up to an integer number of nucleotides when compared to the control sequence. Is selected from the group consisting of substitutions or insertions involving deletions, transpositions and inversions of at least one nucleotide, and variations thereof are at or between the 5 ′ or 3 ′ terminal positions of the control nucleotide sequence. It may occur anywhere, may be interspersed individually between nucleotides in the control sequence, or at one or more adjacent groups in the control sequence, and the number of nucleotide variations is SEQ ID NO: 1 or 3 Multiplied by the number determined by dividing the percent identity by 100 and then subtracting the total number of nucleotides of SEQ ID NO: 1 or 3 By subtracting the product, or the formula:

[0019] _{n n ≦ X n - (X} n · y) [ wherein, _{n n} is the number of nucleotide variations, _{X n} is SEQ ID NO: is the total number of 1 or 3 nucleotide, y is 95% 0.97, 0.97 or 10 at 97%
Is the symbol of the multiplication operator, and if the product of _Xn and y is not an integer, round it down to the nearest integer before subtracting it from _Xn ]. You. Variations in the polynucleotide sequence encoding the polypeptide of SEQ ID NO: 2 or 4 may result in non-sense, missense or frameshift mutations in the coding sequence, and thereby the polypeptide encoded by the polynucleotide so modified Changes.

(2) Specific examples of the polynucleotide further include an isolated polypeptide comprising a polypeptide having at least 95, 97 or 100% identity to the control sequence of the polypeptide of SEQ ID NO: 2 or 4. And peptides. Here, the polypeptide sequence may be the same as the control sequence of SEQ ID NO: 2 or 4, or may have a different number of amino acids up to an integer number when compared to the control sequence. Is selected from the group consisting of substitutions or insertions, including deletions, conservative and non-conservative substitutions of at least one nucleotide, and variations thereof are at or between the amino or carboxy terminal positions of the control polypeptide sequence. And may be interspersed individually between amino acids in the control sequence, or at one or more adjacent groups in the control sequence, and the number of amino acid variants is determined by SEQ ID NO: 2 or 4 is multiplied by the number determined by dividing the percent identity by 100 and then subtracting this product from the total number of amino acids in SEQ ID NO: 2. Ri,
Or the formula:

[0021] n _a ≦ _{X a} - in _(X a · y) [wherein, _{n a} is the number of amino acid variations, _{X a} is SEQ ID NO: is the total number of 2 or 4 amino acids, y is 95% 0.97 at 0.95, 97% or 1.100 at 100%.
It is 00, and • is the symbol for the multiplication operator, product of X _a and y is not an integer, rounded down prior to subtracting it from X _a, is the nearest integer.

“Individual” means a multicellular eukaryote, including, but not limited to, metazoans, mammals, cows, monkeys, primates, and humans. “Isolated” means altered “by the hand of man” from its natural state, ie, in the case of natural products, altered or removed from its original environment, or both. 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". Moreover, a polynucleotide or polypeptide introduced into an organism by transformation, genetic manipulation, or by other recombinant methods, even if it is in the organism and the organism is alive or dead As well as being “isolated”.

“Organism (s)” includes (i) Streptococcus, Staphylococcus, Bordete
lla, Corynebacterium, Mycobacterium, Neisseia, Haemophilus, Actinomyces
, Streptomyces, Nocardia, Enterobacter, Yersinia, Fancisella, Pasturella
, Moraxella, Acinetobacter, Erysipelothrix, Branhamella, Actinobacillus
, Streptobacillus, Listeria, Calymmatobacterium, Brucella, Bacillus, Clo
sterdium, Treponema, Escherichia, Salmonella, Kleibsiella, Vibrio, Prote
us, Erwinia, Borrelia, Leptospira, Spirillum, Campylobacter, Shigella, L
egionella, Pseudomonas, Aeromonas, Rickettsia, Chlamydia, Borrelia and
Members of the genus (but not limited to) Mycoplasma, and St
reptococcus, Streptococcus of group B, Streptococcus of group C, Streptococcus of group D, Streptococcus of group G, Streptococcus pneumoni
ae, Streptococcus pyrogenes, Streptoxoxxus agalactiae, Streptococcus fae
calis, Streptococcus faecium, Streptococcus durans, Neisseria gonorrheae
, Neisseria meningitidis, Staphylococcus aureus, Staphylococcus epidermi
dis, Corynebacterium diptheriae, Garnella vaginalis, Mycobacterium tuber
culosis, Mycobacterium bovis, Mycobacterium ulcerans, Mycobacterium lepr
ae, Actinomyces israelli, Listeria monocytogenes, Bordetella pretusis, B
ordetella parapretusis, Bordetella bronchiseptica, Esherichia coli, Shig
ella dysenteriae, Haemophilus influenzae, Haemophilus aegyptius, Haemoph
ilus parainfluenzae, Haemophilus ducreyi, Bordetella, Salmonella typhi,
Citrobacter freundii, Proteus mirabilis, Proteus vulgaris, Yersinia pest
is, Klebsiella pneumoniae, Serratia marcessens, Serratia liquefaciens, V
ibrio cholera, Shigella dysenterii, Shigella flexneri, Pseudomonas aerug
inosa, Franscisella tularensis, Brucella abortis, Bacillus anthracis, Ba
cillus cereus, Clostridium perfringens, Clostridium tetani, Clostridium
butulinum, Treponema pallidum, Rickettsia rickettsii and Chlamydia trac
prokaryotes, including, but not limited to, members of a species or group that are homitis; (ii) archaebacteria, including, but not limited to, Archaebacter;
iii) Protozoa, fungi, members of, but not limited to, the genus Saccharomyces, Kluveromyces or Candida, and Saccharomyces cerevisiae, Kluveromyces
A single cell or filamentous eukaryote, including but not limited to members of the lactis or Candida albicans species.

The “bacteria (s)” are (i) Streptococcus, Staphylococcus, Bordetell
a, Corynebacterium, Mycobacterium, Neisseia, Haemophilus, Actinomyces, S
treptomyces, Nocardia, Enterobacter, Yersinia, Fancisella, Pasturella, M
oraxella, Acinetobacter, Erysipelothrix, Branhamella, Actinobacillus, St
reptobacillus, Listeria, Calymmatobacterium, Brucella, Bacillus, Closter
dium, Treponema, Escherichia, Salmonella, Kleibsiella, Vibrio, Proteus,
Erwinia, Borrelia, Leptospira, Spirillum, Campylobacter, Shigella, Legio
nella, Pseudomonas, Aeromonas, Rickettsia, Chlamydia, Borrelia and Myco
members of the genus (but not limited to) plasma, as well as Strept of group A
ococcus, Streptococcus of group B, Streptococcus of group C, Streptococcus of group D, Streptococcus of group G, Streptococcus pneumoniae,
Streptococcus pyrogenes, Streptoxoxxus agalactiae, Streptococcus faecali
s, Streptococcus faecium, Streptococcus durans, Neisseria gonorrheae, Ne
isseria meningitidis, Staphylococcus aureus, Staphylococcus epidermidis
, Corynebacterium diptheriae, Garnella vaginalis, Mycobacterium tubercul
osis, Mycobacterium bovis, Mycobacterium ulcerans, Mycobacterium leprae
, Actinomyces israelli, Listeria monocytogenes, Bordetella pretusis, Bor
detella parapretusis, Bordetella bronchiseptica, Esherichia coli, Shigel
la dysenteriae, Haemophilus influenzae, Haemophilus aegyptius, Haemophil
us parainfluenzae, Haemophilus ducreyi, Bordetella, Salmonella typhi, Ci
trobacter freundii, Proteus mirabilis, Proteus vulgaris, Yersinia pestis
, Klebsiella pneumoniae, Serratia marcessens, Serratia liquefaciens, Vib
rio cholera, Shigella dysenterii, Shigella flexneri, Pseudomonas aerugin
osa, Franscisella tularensis, Brucella abortis, Bacillus anthracis, Baci
llus cereus, Clostridium perfringens, Clostridium tetani, Clostridium bo
tulinum, Treponema pallidum, Rickettsia rickettsii and Chlamydia tracho
mitis means prokaryotes, including but not limited to members of a species or group, and (ii) archaebacteria, including but not limited to Archaebacter.

“Polynucleotide (s)” also generally means polyribonucleotides or polydeoxyribonucleotides, which may be unmodified RNA or DNA, or modified RNA or DNA. "Polynucleotide" refers to single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions or DNA that is a mixture of single-stranded, double-stranded and triple-stranded regions, single-stranded and double-stranded. RNA, which is a mixture of single-stranded and double-stranded regions, as well as single-stranded or more typically double- or triple-stranded, or even a mixture of single- and double-stranded regions Including but not limited to hybrid molecules including good DNA and RNA. 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" refers to a DNA or RNA as described above containing one or more modified bases.
NA. 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, DNA or RNA containing only unusual bases such as inosine or modified bases such as tritylated bases (only two examples are shown) is also the term polypeptide herein. DNA and RN that provide many useful purposes known to those skilled in the art
It will be apparent that there are numerous modifications to A. 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" embraces short polynucleotides, often referred to as oligonucleotide (s).

“Polypeptide” refers to a peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified 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 in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. 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, ADP-ribosylation, amidation, flavin covalent bonding, heme moiety covalent bonding, nucleotide or nucleotide derivative covalent bonding, lipid or lipid derivative covalent bonding, phosphotidyl Inositol covalent, cross-link, cyclization, disulfide bond formation, demethylation, covalent cross-link formation, cystine formation, pyroglutamate formation, formylation, gamma-carboxylation, sugar chain formation, GPI anchor formation, hydroxylation Transfer RNA-mediated amino acid addition to proteins, such as, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulphation, arginylation, and ubiquitination There is. For example, Proteins-Structure and Molecular Properties, 2nd edition, TECreighton, W.
H. Freeman and Company, New York (1993). For example, Posttranslational Covalent Modification of Proteins, edited by BC Johnson, Academic Press, New York (1983), Wold, F., Posttranslational.
Protein Modifications: Perspective and Prospects, pp. 1-12; Seifter et al., Meth. Enzymol. 182: 626-646 (1990) and Rattan et al., Protei.
n 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.

“Recombinant expression system (s)” refers to an expression system, or a portion or book thereof, introduced or transformed into a host cell or host cell lysate for the production of the polynucleotides and polypeptides of the invention. Refers to a polynucleotide of the invention. The term "variant" as 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 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, differences are limited so that the sequences of the control polypeptide and the variant are closely similar overall and, in many regions, identical. Variant and control polypeptides can have an altered amino acid sequence by one or more substitutions, additions, deletions occurring in any combination. The substituted or inserted amino acid residue may or may not be one encoded by the genetic code. The invention also relates to variants of each polypeptide of the invention, i.e., those that differ from a reference standard by conservative amino acid substitutions, whereby a residue is replaced with another residue having similar properties. Include. Typical such substitutions are between Ala, Val, Leu and Ile; between Ser and Thr; between acidic residues Asp and Glu; between Asn and Gln; between basic residues Lys and Arg; or between aromatic residues Phe and It is between Tyr. Variants in which several, five to ten, one to five, one to three, one to two or one or more amino acids are substituted, deleted or added in any combination are particularly preferred. A variant of a polynucleotide or polypeptide may be naturally occurring, such as an allelic variant, or may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis methods or by direct synthesis or other recombinant methods known to those skilled in the art.

BEST MODE FOR CARRYING OUT THE INVENTION The present invention relates, inter alia, to novel aroA polypeptides and polynucleotides as described in greater detail below. In particular, the present invention relates to 5-enolpyruvoylshikimate-3-phosphate synthase of Lactococcus lactis (aroA
) Amino acid sequence homology to polypeptides (similarity 79.673%, identity 6)
8.224%) of the novel aroA polypeptides and polynucleotides of Streptococcus pneumoniae. The present invention particularly relates to aroA having the nucleotide sequence and amino acid sequence shown in Table 1 (SEQ ID NO: 1) and Table 1 (SEQ ID NO: 2), respectively.
A nucleotide sequence and the amino acid sequence encoded thereby.

Table 1 aroA polynucleotide and polypeptide sequences (A) Sequence from the aroA polynucleotide sequence of Streptococcus pneumoniae [SEQ ID NO: 1]

[0030]

[Table 1] (B) aroA polypeptide sequence deduced from the polynucleotide sequences in this table [SEQ ID NO: 2]

[Table 2]

(C) Specific example of polynucleotide sequence [SEQ ID NO: 1]

[Table 3]

(D) Specific Example of Polypeptide Sequence [SEQ ID NO: 2]

[Table 4]

(E) aroA polynucleotide ORF of Streptococcus pneumoniae
Sequence derived from the sequence [SEQ ID NO: 3]

[Table 5]

(F) aroA polypeptide sequence deduced from the polynucleotide ORF sequences in this table [SEQ ID NO: 4]

[Table 6]

Deposited Materials The deposit containing Streptococcus pneumoniae strain 0100993 was deposited at National Collection of Industrial and Marine Bacteria Limited (NCIMB), 23 Street, McDrough Drive, Aberdeen AB21RY, Scotland in 1996. Deposited on April 11, 2014 and assigned NCIMB accession number 40794. After the deposit, the deposited strain is referred to as Streptococcus pneumoniae 0100993 strain. 1996
On April 17, 1998, the Streptococcus pneumoniae 0100993 DNA library in E. coli was similarly deposited with the NCIMB, and accession number 4
0800 was obtained. The S. pneumoniae strain deposit is referred to herein as the "deposited strain" or "DNA of the deposited strain." The deposited strain contains the full length aroA 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. When the patent is issued, the shares are eventually sold without any restrictions or conditions. Deposits are provided only for the convenience of those skilled in the art and are subject to 35 US
C. Deposit does not acknowledge that it is a feasible requirement, as required under section 112. A license is required to make, use or sell the deposit, but such a license has not been granted here.

Polypeptides The polypeptides of the present invention include the polypeptides of Table 1 [SEQ ID NO: 2] (specifically, the mature polypeptides) and polypeptides and fragments, particularly those having the biological activity of aroA. And at least 70% identity to the polypeptide of Table 1 [SEQ ID NO: 2 and 4] or a significant portion thereof, preferably at least 80% to the polypeptide of Table 1 [SEQ ID NO: 2 and 4]. And more preferably Table 1 [SEQ ID NOs: 2 and 4]
Has at least 90% similarity to the polypeptide (more preferably,
With at least 90% identity), and even more preferably from Table 1 [SEQ ID NO: 2
And 4] polypeptides and fragments having at least 95% similarity (even more preferably at least 95% identity) to the polypeptides, and also generally at least 30, more preferably at least 50
Encompasses such portions of the polypeptide, including the amino acids.

The invention also includes a polypeptide of the formula shown in Table 1 (D) (SEQ ID NO: 2),
In the formula, X is hydrogen at the amino terminus, Y is hydrogen or metal at the carboxyl terminus, R ₁ and R ₂ are amino acid residues, and n is an integer from 1 to 1000. The chain portion of the amino acid residue represented by any of the R groups (R is greater than one) may be a heteropolymer or a homopolymer, and is preferably a heteropolymer. Fragments are variant polypeptides that have an amino acid sequence that is entirely identical to some, but not all, of the amino acid sequences of the aforementioned polypeptides. aroA
For polypeptides, fragments are "free standing"
Or by forming a portion or region, it may be contained within a larger polypeptide, most preferably as a single contiguous region, ie, a large single polypeptide.

Preferred fragments are truncated polypeptides having a portion of the amino acid sequence of Table 1 [SEQ ID NOs: 2 and 4], or variants thereof, eg, truncating a series of residues including the amino terminus, or Includes truncations of a series of residues including the carboxyl terminus. Also preferred are degradation forms of the polypeptides of the present invention in a host, especially Streptococcus pneumoniae. 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 regions 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 aroA activity or have similar activity or have improved activity and 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 Streptococcus pneumoniae in an individual, particularly a human, or the ability to initiate or maintain a disease, are particularly preferred. "X" or "Xaa" can also be used to describe a particular polypeptide of the invention. “X” and “Xaa” refer to any of the 20 naturally occurring amino acids, which can be located at predetermined positions in a polypeptide sequence. Variants that are fragments of the polypeptides of the present invention may be used for the production of the corresponding full-length polypeptide by peptide synthesis, and therefore these variants may be used as intermediates for the production of full-length polypeptides. Good.

Polynucleotides Another aspect of the present invention relates to a full-length gene encoding an aroA polypeptide having the deduced amino acid sequence of Table 1 [SEQ ID NOs: 2 and 4] and polynucleotides closely related thereto and variants thereof. Including isolated polynucleotides. Using information such as the polynucleotide sequences shown in Table 1 [SEQ ID NOs: 1 and 3] provided herein, bacterial cloning using standard cloning and screening methods, eg, using Streptococcus pneumoniae 0100993 cells as starting material. Using the method of cloning and sequencing the chromosomal DNA fragment of the present invention, a polynucleotide of the invention encoding an aroA polypeptide may be obtained, followed by obtaining a full-length clone. For example, Streptococcus pneumoniae 0100993 in E. coli or some other suitable host to obtain a polynucleotide sequence of the invention, such as the sequences shown in Table 1 [SEQ ID NOs: 1 and 3]. A typical library of chromosomal DNA clones is probed with a radiolabeled oligonucleotide, preferably 17-mer or longer, derived from the partial sequence. D that is identical to the probe DNA
Clones carrying NA 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 appropriate techniques, see Maniatis, T., F.
itsch, FF and Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed.
Edition; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989) (Screening By
Hybridization 1.90 and Sequencing Denatured Double-Stranded DNA Templa
tes 13.70). The polynucleotide shown in Table 1 [SEQ ID NO: 1], which is a typical example of the present invention, was found in a DNA library derived from Streptococcus pneumoniae 0100993.

The DNA sequence shown in Table 1 [SEQ ID NO: 1] is a reading frame encoding a protein consisting of approximately the same number of amino acid residues as the amino acid residues shown in Table 1 [SEQ ID NO: 2] having the estimated molecular weight. And its estimated molecular weight can be calculated using the molecular weight of amino acids well known in the art. SEQ ID NO: between nucleotide numbers 1 to 1281:
One polynucleotide encodes the polypeptide of SEQ ID NO: 2. The stop codon starts at nucleotide number 1284 of SEQ ID NO: 1 (1-1281). The aroA protein of the present invention is structurally different from other proteins of the aro (5-enolpyruvylshikimate-3-phosphate synthase) family, as indicated by the results of sequencing the DNA encoding aroA of the deposited strain. Related. The protein has the greatest homology to Lactococcus lactis 5-enolpyruvoylshikimate-3-phosphate synthase (aroA) protein among known proteins (similarity 79.7%, identity 68). .2%). Other related sequences are as follows:

Accession No. Species X78413 L. Lactis aroA M80245 B. subtilis aroA Z29339 D. nodosus aroA I19982 Sequence No. 14 L05004 of US Pat. No. 5,512,466. jejuni aroA L46372 Yersinia pestis aroA

The relevant sequences are described in Sharps, Analytical Biochem 140: 183-189, 198.
4; Majumder et al., Eur. J. Biochem. 229: 99-106, 1995; O'Connell.
139: 1449-1460, 1993; Oyston et al., "Im.
munization with live recombinant Salmonelle typhimurium aroA producing F
1 antigen protects against plaque "1995; and Rogers et al.," Amplificat
ion of the aroA gene from Escherichia coli results in tolerance to the h
erbicide glyphosphate "1983.

The present invention provides a polynucleotide sequence identical over its entire length to the coding sequence in Table 1 [SEQ ID NO: 1 or SEQ ID NO: 2]. 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 embodiments of the invention, the marker sequence is a pQE vector (Qiagen, Inc.
Hex-histidine peptide as provided in (Gentz et al., Proc. Natl. Aca
d. Sci., USA, 86: 821-824 (1989)) or HA tag (Wilson et al., Cell 37: 767 (1984)). The polynucleotides of the present invention also include structural genes and native sequences that regulate gene expression,
It is not limited to these.

A preferred embodiment of the present invention is a polynucleotide comprising nucleotides 1 to 1281 or 1284 shown in SEQ ID NO: 1 of Table 1, encoding an aroA polypeptide. The invention also encompasses a polynucleotide of the formula shown in Table 1 (C) [SEQ ID NO: 1], wherein X is hydrogen at the 5 'end and Y at the 3' end of the molecule.
Is hydrogen or a metal, R ₁ and R ₂ are amino acid residues, n is 1 to 100
It is an integer of 0. The chain portion of the nucleic acid residue represented by any of the R groups (R is greater than one) may be either a heteropolymer or a homopolymer, and is preferably a heteropolymer.

As used herein, the term “polynucleotide encoding a polypeptide” encompasses a polynucleotide comprising a sequence encoding a polypeptide of the invention,
In particular, it includes bacterial polypeptides, and more particularly, aroA polypeptides of Streptococcus pneumoniae 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), with additional regions that may include coding and / or non-coding sequences. (Separated by insertion or sequence editing). 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 a polynucleotide of the invention may be used to synthesize a full-length polynucleotide of the invention.

A further particularly preferred embodiment is a polynucleotide encoding the aroA variant, having the amino acid sequence of the aroA polypeptide of Table 1 [SEQ ID NO: 2],
Some of them, a little, 5-10, 1-5, 1-3, 2,
It has an amino acid sequence in which 1 or 0 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 aroA.

A further preferred embodiment of the present invention provides a polynucleotide encoding an aroA polypeptide having the amino acid sequence shown in Table 1 [SEQ ID NOs: 2 and 4] that has at least 70% identity over its entire length. And polynucleotides complementary to such polynucleotides. Also,
Most highly preferred are polynucleotides comprising a region that is at least 80% identical over its entire length to a polynucleotide encoding the aroA polypeptide of the deposited strain, 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 preferred embodiment is a polynucleotide that encodes a polypeptide that retains substantially the same biological function or activity as the mature polypeptide encoded by the DNA in Table 1 [SEQ ID NO: 1].

The 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 polynucleotides provided herein. As used herein, the terms "stringent conditions" and "stringent hybridization conditions" refer to hybridization that occurs only when the identity between the sequences is at least 95%, preferably at least 97%. . Examples of stringent hybridization conditions include 50% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate),
Incubate overnight at 42 ° C. in a solution containing 0 mM sodium phosphate (pH 7.6), 5 × denhardt's solution, 10% dextran sulfate and 20 μg / ml denatured, sheared salmon sperm DNA, followed by about 65 ° C. Washing the hybridization support in 0.1 × SSC. Hybridization and washing conditions are well known and described in Sambrook et al., Molecular Cloning, AL.
aboratory Manual, 2nd edition; Cold Spring Harbor, New York (
1989), especially in 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 or SEQ ID NO: 3, comprising the polynucleotide described in SEQ ID NO: 1. Also provided is a polynucleotide essentially comprising the polynucleotide sequence obtainable by screening with a probe having the sequence of the sequence or a fragment thereof 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 invention, for example, the polynucleotides of the invention described above may be used to isolate full length cDNA and genomic clones encoding aroA, and to a high degree of sequence similarity to the aroA gene. RNA for isolating cDNA and genomic clones of other genes having sex
, CDNA and genomic DNA. Such probes generally contain at least 15 bases. Preferably such probes will have at least 30 bases and at least 50
It may have a base. Particularly preferred probes have at least 30 bases and are 50 bases or less. For example, the coding region of the aroA gene can be isolated by screening by synthesizing an oligonucleotide probe using the DNA sequence shown in 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 human diseases, and further described herein in connection with polynucleotide assays. Will be discussed further. Polynucleotides of the invention that are oligonucleotides from the sequences of SEQ ID NOs: 1 and / or 2 may be used in the methods described herein, but are preferably
Used for CR to determine whether all or a portion of the polynucleotides identified herein are transcribed into infected tissues in bacteria. 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, the mature form of one or more polymorphic polypeptides). 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 a mature form of the polypeptide fused to one or more prosequences can be an inactive form of the polypeptide. Removal of the prosequence generally activates such inactive precursors. Some or all of the prosequence can be removed prior to activation. Generally, such precursors are called proproteins.

In addition to the standard symbols A, G, C, T / U for nucleotides, the word “N” can also be used to describe a particular polynucleotide of the invention. "
"N", when acting together with adjacent nucleotide positions, reads the correct reading frame, and preferably is not a nucleic acid that has the effect of forming a premature terminal codon in such reading frame. Except where otherwise, it means that any of the four DNA or RNA nucleotides is at such designated position in the DNA or RNA sequence. In short, the polynucleotide of the present invention is a mature protein, a mature protein to which a leader sequence is added (which may be referred to as a preprotein), a precursor of a mature protein having one or more prosequences which are not the leader sequence of the preprotein, Alternatively, it may encode a preproprotein which is a precursor of a proprotein having a leader sequence and one or more prosequences, which are 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. Such a protein can be produced using a cell-free translation system using RNA derived from the DNA construct of the present invention. 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 is described, for example, in Davis et al., Basic Methods in Molecula.
r Biology (1986); Sambrook et al., Molecular Cloning; A Laboratory Manua.
1, second edition; can be performed by methods described in many standard laboratory manuals, such as the 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 Streptococci, Staphylococci, Enterococci, E. coli, Streptomyces ( Streptomyce
s) 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, 3T3, BHK
, 293 and Bows melanoma cells; and plant cells.

[0057] 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, generally, any system or vector suitable for holding, 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, and are described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual (supra).

In order to secrete the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space or the extracellular environment, it can be incorporated into a polypeptide that expresses an appropriate secretion signal. These signals may be native 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 chromatography, Examples include 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 relates to the use of the aroA polynucleotides of the present invention for use as diagnostic reagents. Detection of aroA in a eukaryote, particularly a mammal, and especially a human, will provide a diagnostic method for diagnosis of a disease. Eukaryotes (also referred to herein as "individuals"), particularly mammals, and especially humans, infected with an organism containing the aroA gene can be detected at the nucleic acid level by various methods. Diagnostic nucleic acids can be obtained from cells and tissues of infected individuals, 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 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 a change in 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 aroA polynucleotide sequence. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures.
DNA sequence differences may be detected by detecting changes in electrophoretic mobility of DNA fragments in gels with or without denaturing agents, or by direct DNA sequencing. For example, Meyers et al., Science, 230: 1242 (1985).
)checking. Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and S1 protection or chemical cleavage methods. See, eg, Cotton et al., Proc. Natl. Acad. Sci., USA, 85: 4397.
-4401 (1985).

Cells carrying mutations or polymorphisms in the gene of the invention may be detected at the DNA level by various techniques, for example by serotyping. For example, mutations can be detected using RT-PCR. It is particularly preferred to use RT-PCR in combination with an automatic detection system, such as GeneScan. RN
A or cDNA may be used for PCR or RT-PCR for the same purpose. As an example, PCR primers complementary to the nucleic acid encoding aroA can be used to identify and analyze mutations.

The present invention also provides these primers with 1, 2, 3 or 4 nucleotides removed from the 5 'and / or 3' end. Using these primers,
AroA DNA isolated from a sample derived from an individual may be amplified. The primers may be used to amplify a gene isolated from an infected individual, and the gene may then be subjected to various techniques for examining DNA sequences. Thus, DN
Mutations in the A sequence can be detected and used to diagnose infection and serotype and / or classify infectious agents.

The invention also relates to diseases, preferably bacterial infections, more preferably infections by Streptococcus pneumoniae, and most preferably otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, empyema and heart. Provided is a method of diagnosing endometritis, most particularly a disease such as meningitis, such as an infection of the cerebrospinal fluid, comprising the expression of a polynucleotide having the sequence of Table 1 [SEQ ID NO: 1]. An increase in the level is detected from a sample derived from an individual. Increasing or decreasing aroA polynucleotide expression can be determined by any method known in the art for quantifying polynucleotides, such as amplification, PCR, RT-PCR, RNase protection, Northern blotting, and other hybridization methods. Can be measured.

In addition, a diagnostic assay of the invention for detecting overexpression of aroA protein, as compared to a normal control tissue sample, may be used, for example, to detect the presence of an infection. Assay techniques that can be used to determine levels of aroA 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 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. 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. Included. Antibodies raised against the polypeptides of the present invention can be obtained by administering the polypeptides or epitope-tagged fragments, analogs or cells, 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. For example, Kohler, G. and Milstein, C., Nature, 2
56: 495-497 (1975); Kozbor et al., Immunology Today, 4:72 (
1983); Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R Lis.
s, Inc., pages 77-96 (1985).

The techniques described for the production of single-chain antibodies (US Pat. No. 4,946,778) can be applied to obtain single-chain antibodies to the polypeptides of the invention. Also,
Transgenic mice or other organisms, such as other mammals, may be used to express antibodies such as humanized antibodies. Alternatively, using phage display technology, the binding activity to the polypeptide from a repertoire of PCR-amplified v genes of human-derived lymphocytes screened for possession of anti-aroA, or from an intact library. Antibody genes can be selected (McCafferty, J. et al., Nature 3).
48: 552-554 (1990); Marks, J. et al., Biotechnology 10: 779.
-783 (1992)). The affinity of these antibodies is determined by chain shuffling (chai
n shuffling) (Clackson, T. et al., Nature 352:
624-628 (1991)).

If there are two antigen binding domains, each domain is directed against a different epitope and is referred to as a “bispecific” antibody. Clones expressing the polypeptide may be isolated or identified using the above antibodies, and the antibodies may be purified by affinity chromatography. Thus, in particular, antibodies against aroA may be infected, especially bacterial infections, especially otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pyothorax and endocarditis, most particularly for example cerebrospinal fluid infection It may be used to treat diseases such as meningitis.

[0067] Polypeptide variants include antigenically, epitopically or immunologically equivalent variants, and are particular embodiments of the present invention. "Antigenically equivalent derivative" as used herein
The term refers to a polypeptide or polypeptide thereof specifically produced by a particular antibody that, when produced against a protein or polypeptide according to the present invention, interferes with the immediate physical interaction between the pathogen and the mammalian host. Includes equivalents. 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.

A polypeptide, such as an antigenically or immunologically equivalent derivative, or a fusion protein thereof can be used as an antigen to immunize a mouse or other animal, such as a rat or chicken. Fusion proteins can confer stability on a polypeptide. The antigen may be, for example, by conjugation, to produce an immunogenic carrier protein, such as bovine serum albumin (BSA) or keyhole limpet hemocyanin (keyhole limp).
et 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 So you don't have to use a carrier.

[0069] Preferably, the antibodies or variants thereof are 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 region of the antibody from the hybridoma is human.
It has been transplanted into monoclonal antibodies and is described, for example, in Jones, P. et al., Nature 321:
522-525 (1986) or Tempest et al., Biotechnology 9: 266-2.
73 (1991). Use of the polynucleotides of the present invention in genetic immunization includes plasmid DNA
Injection directly into muscle (Wolff et al., Hum. Mol. Genet. 1: 363 (1992); Mant
Horpe et al., Hum. Gene Ther. 4: 419 (1963)), Delivery of complexes of DNA with specific protein carriers (Wu et al., J. Biol. Chem. 264: 16985 (1989).
)), DNA co-precipitation with calcium phosphate (Benvenisty & Reshef, PNAS 83: 9).
551 (1986)), DNA encapsulation in various forms of liposomes (Kaneda et al.,
Science 243: 375 (1989)), particle bombardment (Tang et al., Nature 356).
: 152 (1992); Eisenbraun et al., DNA Cell Biol. 12: 791 (199
3)) and in vivo infection using a cloned retroviral vector (Seeger
Preferably, a suitable delivery method such as PNAS 81: 5849 (1984)) is used.

Mechanisms of Action and Methods of Use The polypeptides of the invention may be used to assess 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 can be natural substrates and ligands, and can be structural or functional mimetics. For example, Coligan et al., Curre
nt Protocols in Immunology 1 (2): See Chapter 5 (1991).

The present invention also enhances the action of aroA polypeptides or polynucleotides (
Also provided are methods of screening compounds to identify compounds that are agonists) or block (antagonists), particularly bacteriostatic and / or bactericidal compounds. The screening method includes a high-throughput method. For example, to screen for agonists or antagonists, aroA polypeptides and cell compartments, such as membranes, cell envelopes or cell walls, or preparations thereof, comprising aroA polypeptides and labeled substrates or ligands of such polypeptides may be treated with aroA agonists. Alternatively, incubate in the absence or presence of candidate molecules that may be antagonists. The ability of a candidate molecule to agonize or antagonize an aroA polypeptide is reflected in reduced binding of the labeled ligand or reduced production of the product from such a substrate. Molecules that bind and have no effect, ie, that do not elicit the effects of aroA, are likely to be good antagonists. Molecules that bind well and increase the rate of product formation from the substrate are agonists. The detection of the rate or level of product from the substrate can be enhanced by using a reporter system.
Useful reporter systems in this regard include colorimetrically labeled substrates that are converted to products, reporter genes that respond to changes in aroA polynucleotide or polypeptide activity, and binding assays that are well known in the art. However, the present invention is not limited to these.

Another example of an assay for aroA antagonists is a competition assay, in which aroA and a potential antagonist are prepared under conditions suitable for a competition inhibition assay.
It is mixed with a roA binding molecule, a recombinant aroA binding molecule, a natural substrate or ligand, or a substrate or ligand mimetic. AroA is labeled, for example with a radioactive or colorimetric compound, and bound to a binding molecule or converted to product.
The number of roA molecules can be accurately determined to assess the effects of potential antagonists. The agonists and antagonists of the present invention preferably act by modifying the reversible transformation of phospho (enol) pyruvate (PEP) and shikimate 3-phosphate (S3P) into EPSP and inorganic phosphate (Pi).

[0073] Potential antagonists include small organic molecules, peptides, polypeptides, and antibodies that bind to a polynucleotide or polypeptide of the invention, thereby inhibiting and eliminating its activity. Also, potential antagonists do not elicit aroA-induced activity, and thus eliminate aroA from binding to aroA
May be small organic molecules, peptides, polypeptides, such as closely related proteins or antibodies, that bind to the same site as the binding molecule, such as a binding molecule that interferes with the action of the binding molecule. Preferably, the small organic molecules of the present invention have a molecular weight of less than 2000 Daltons, more preferably between 300 and 1000 Daltons, most preferably between 400 and 700 Daltons. One specific example is a compound that has not been published before the filing date of the present application. Another specific example includes compounds that exclude glyphosate (N-phosphonomethylglycine, GLP) and / or transition state inhibitors or transition state mimetics.

Potential antagonists include small molecules that bind to and occupy the binding site of a polypeptide, thereby preventing binding to a cellular binding molecule and thereby disrupting normal biological activity. There is. Examples of small molecules include small organic molecules, peptides,
Examples include, but are not limited to, peptide-like molecules. Other potential antagonists include antisense molecules and the like (for a description of these molecules, see Okano, J., Neurochem. 56: 560 (1991); Oligodeoxy-nucleotides).
See tides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)). Preferred potential antagonists include aroA related compounds and aroA variants.

Each of the DNA sequences provided herein may be used in the discovery and development of antimicrobial compounds. The encoded protein, when expressed, can be used as a target for antimicrobial screening. In addition, an antisense sequence is constructed using a DNA sequence encoding the amino-terminal region of the encoded protein or the Shine-Dalgarno sequence or other translation-enhancing sequence of each mRNA, and expressing 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 sequela of infection and the initial physical interaction between mammalian hosts. In particular, the molecules of the present invention may 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; eg, phosphorylation of mammalian tyrosine kinases. Blockade of mammalian cells mediated by the aroA protein by initiating (Rosenshine et al., Infect. Immunol. 60: 2211 (1
992)); blockage of bacterial adhesion mediating tissue damage between mammalian extracellular matrix protein and bacterial aroA protein; common pathology of infection, initiated by other than implantation of an indwelling device or other surgical method Can be used to block the progress of the The antagonists and agonists of the present invention, for example, otitis media, conjunctivitis, pneumonia,
It may be used to inhibit and treat bacteremia, meningitis, sinusitis, empyema and endocarditis, most particularly meningitis such as, for example, cerebrospinal fluid infection.

Vaccine Another aspect of the invention is a method of eliciting an immunological response in an individual, especially a mammal, which protects the individual from infection, particularly a bacterial infection, most particularly a Streptococcus pneumoniae infection. Inoculating an individual with an antibody and / or a fragment or variant thereof sufficient to generate a T cell immune response. Also provided is a method wherein such an immunological response slows bacterial replication. Yet another aspect of the invention is a method of eliciting an immunological response in an individual, wherein aroA, or a fragment or variant thereof, is expressed in vivo, whether or not the disease has already been established in the individual. A nucleic acid vector that directs the expression of aroA or a fragment or variant thereof to such an individual is delivered, for example, to an antibody and / or a T cell immune response (eg, to elicit a cytokine producing T cell or a cytotoxic T cell). Inducing an immunological response that produces antibodies that produce antibodies that protect the individual from disease. Methods for hastening the administration of the gene into the desired cells include coding on the particles. Such nucleic acid vectors may include DNA, RNA, modified nucleic acids, or DNA / RNA hybrids.

A further aspect of the present invention is directed to immunological response to aroA or a protein encoded thereby when introduced into an individual having the ability to elicit an immunological response in an individual. An immunological composition that elicits a response in such an individual, comprising a recombinant aroA comprising DNA that encodes and expresses an antigen against aroA or a protein encoded thereby, or a composition comprising a protein encoded thereby. About things. The immunological response may be used therapeutically or prophylactically,
The immunological response may also be in the form of antibody immunity or cell-mediated immunity, such as that arising from CTL or CD4 ⁺ T cells.

An aroA polypeptide or a fragment thereof may be a co-localized 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 can be obtained by solubilizing an antigen coprotein such as lipoprotein D derived from Hemophilus influenzae, a coexisting protein such as glutathione-S-transferase (GST) or beta-galactosidase, Includes relatively large co-proteins that facilitate production and purification.
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 may be linked to either the amino or carboxy terminus of the first protein. The present invention relates to a polypeptide or polynucleotide of the invention and an immunostimulatory DN.
Provided are compositions, especially vaccine compositions, and methods comprising the A sequence, for example, the sequences described in Sato, Y. et al., Science 273: 352 (1966).

Further, the present invention provides an animal model infected with Streptococcus pneumoniae using the above-described polynucleotide or a specific fragment thereof,
A method which has been found to encode a constant region of a bacterial cell surface protein in a DNA construct used in such a genetic immunization experiment, in particular a protein capable of stimulating a prophylactic or therapeutic immune response Methods are provided that are useful for identifying epitopes. This study is particularly valuable from the essential organs of animals that have successfully resisted and cleared infections for the development of prophylactic or therapeutic treatments of bacterial infections, particularly Streptococcus pneumoniae infections in mammals, especially humans. It will be possible to prepare monoclonal antibodies successfully.

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 present invention also includes a vaccine formulation comprising the immunogenic recombinant protein of the present invention together with a suitable carrier. Since the protein can be broken down in the stomach, it can be parenterally, for example, subcutaneously,
It is preferably administered intramuscularly, intravenously or intradermally. Formulations suitable for parenteral administration include aqueous or non-aqueous sterilization which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the body fluids of the individual, preferably blood. Injectable solutions and aqueous or non-aqueous sterile suspensions which may contain suspending or thickening agents, and the like. 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 also include adjuvant systems that increase the immunogenicity of the formulation, such as oil-in-water systems 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 respect to particular aroA 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 polynucleotides or polypeptides described above, or agonists or antagonists thereof. The polypeptides of the present invention can be used in combination with non-sterile or sterile carriers for use in cells, tissues or organisms, for example, in combination 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 invention, and a pharmaceutically acceptable carrier or excipient. Such carriers include saline, buffered saline, dextrose, water, glycerol,
Examples include, but are not limited to, 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 composition components of the present invention.

The polypeptides and other compounds of the present invention may be used alone or in combination with other compounds such as therapeutic compounds. Pharmaceutical compositions may be prepared in any effective and convenient manner, for example,
Administration may be by the oral, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal, or intradermal route. 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 compositions may be formulated for topical application, for example, in the form of ointments, creams, lotions, eye ointments, eye drops, ear drops, mouthwashes, impregnated bandages and sutures, and aerosols. Occasionally, suitable conventional additives such as preservatives, solvents to aid penetration of the drug, and ointments and creams may contain emollients.
Such topical formulations may contain compatible conventional carriers, such as cream or ointment bases,
And the lotion may contain ethanol or oleyl alcohol. Such carriers may range from about 1% to about 98% by weight of the formulation, more usually up to about 80% by weight of the formulation. For administration to mammals, especially humans, the daily dose of active substance is:
It 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, prostheses, 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, and continuous ambulatory peritoneal dialysis.
ambulatory peritoneal dialysis (CAPD) catheter. 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 Streptococcus pneumoniae.

[0086] Many orthopedic surgeons consider that humans with prosthetic joints should be considered for antibiotic prophylaxis before dental treatment that can result in bacteremia. Delayed serious infection is a serious complication sometimes leading to loss of prosthetic joints, with significant morbidity and mortality. Therefore, in this context, it is also possible 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 are generally used as therapeutic agents for wounds,
Bacteria may be prevented from adhering to the exposed matrix proteins in the wound tissue, and may be used prophylactically in dental treatment instead 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 immersion of a wound or indwelling device, the active substance is 1 μg / ml to 10 m
Preferably, the concentration is g / 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 μm.
g / kg of antigen, and 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 their administration to appropriate individuals in the dosage ranges indicated. The contents of each of the documents disclosed in the present specification are explicitly incorporated by reference. The contents of any patent application for which this application claims priority is hereby incorporated by reference.

EXAMPLES 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 Preparation and Sequencing A polynucleotide having the DNA sequence shown in SEQ ID NO: 1 was obtained from a library of clones of chromosomal DNA of Streptococcus pneumoniae in E. coli. Was. 2 containing overlapping Streptococcus pneumoniae DNA
Using the sequencing data from one or more clones, a contiguous DNA sequence of SEQ ID NO: 1 was constructed. The library can be produced by a conventional method, for example, the following methods 1 and 2. Total cellular DNA is isolated from Streptococcus pneumoniae 0100993 according to standard methods and size fractionated by one of two methods.

Method 1 Whole cell DNA is mechanically sheared through an injection needle to size fractionate according to standard methods. Fragments up to 11 kbp in size are blunt-ended by treatment with exonuclease and DNA polymerase and an EcoRI linker is added. The fragment is ligated into the vector Lambda ZapII that has been cut with EcoRI, the library is packaged by standard methods, and E. coli is then infected with the packaged library. The library is amplified by standard methods.

Method 2 Whole cell DNA was prepared using a library vector (eg, RsaI, PalI, Al
uI, Bshl235I), is subjected to partial hydrolysis with a restriction enzyme or combination of restriction enzymes suitable for producing a series of fragments for cloning into Bshl235I), and such 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 then infected with the packaged library. The library is amplified by standard methods.

Example 2 Characterization of aroA: Steady-rate experiments of 5-enolpyruvylshikimate-3-phosphate synthase from Streptococcus pneumoniae 5-enolpyruvirshikimate-3-phosphate (EPSP) synthase (EC2) .5.1.19) is the aromatic amino acid biosynthetic pathway ("shikimate pathway")
It is an important enzyme in (1). This enzyme is phospho (enol) pyruvate (
PEP) and shikimate 3-phosphate (S3P) catalyze the unusual reversible transformation of EPSP and inorganic phosphate (Pi) (Scheme 1). Glyphosate (N-phosphonomethylglycine, GLP), a broad-spectrum post-emergence herbicide, functions as a transition state inhibitor (3) to inhibit this single enzyme (2) (Scheme 2). Strong interest in this enzyme has arisen.

Provided herein is the identification of an EPSP synthase in Streptococcus pneumoniae and purification of the enzyme to homogeneity (4). Furthermore, a comprehensive steady-state kinetic study of this enzyme herein is aimed at assuming a kinetic mechanism, especially assuming the dimensions of substrate binding in both forward and reverse reactions. To provide the results. The pattern of inhibition of the enzyme by GLP and the product of each enzyme reaction are described. Because the EPSP synthase reaction is a free and reversible reaction, the enzyme can be assayed in both forward and reverse directions. In the forward reaction, the formation of Pi when coupled to nucleoside phosphorylase and 7-methylguanosine was monitored using a change in fluorescence (5); in the reverse reaction, PEP was converted to pyruvate kinase and lactate. Coupling with dehydrogenase detected a change in absorbance at 340 nm (NADH) (6).

In the forward reaction, GLP is a competitive inhibitor for PEP, but S3
It is an antagonistic inhibitor of P (FIG. 1). Product inhibition in the forward reaction by EPSP indicates that EPSP
It shows that it is a competitive inhibitor for both 3P (FIG. 2). In the reverse reaction, GLP provides antagonistic inhibition of EPSP,
Causes non-competitive inhibition of Pi. Product inhibition in the reverse reaction by S3P indicates that S3P antagonistically inhibits the enzyme against EPSP, but is clearly an antagonistic inhibitor for Pi (FIG. 4).

Table I summarizes the pattern of inhibition of S. pneumoniae EPSP synthase and the corresponding rate constants. Based on these results, we propose a random sequential mechanism for the forward reaction of S. pneumoniae EPSP synthase (Scheme 3); however, the present data accurately describes the mechanism of the reverse reaction. Not enough to point out. The herbicide glyphosate is an inhibitor of S. numoniae EPSP synthase. Inhibition of EPSP synthase by glyphosate in both forward and reverse reactions is characterized by a steady state rate.

Product inhibition of the enzyme in both directions was also characterized. While the results support a random sequential mechanism in the forward reaction, assuming a mechanism in the reverse reaction requires further experimentation. A number of compounds that antagonize EPSP synthase are screened in vitro and the inhibition of the enzyme is compared and shown. The MIC against S. numoniae and E. coli also showed that these compounds were active antimicrobial compounds. Analysis of these compounds revealed that the compounds were site-specifically active and certain compounds antagonized shikimate-3-phosphate substrates.

Inhibition pattern and rate constant of S. pneumoniae EPSP synthase Reaction direction Inhibitor versus substrate Inhibition method ^a Ki (μM) Forward GLP vs. PEP antagonistic inhibition 2.8 ± 0.3 GLP vs. S3P antagonistic inhibition 65.6 ± 2.7 EPSP vs. PEP antagonistic inhibition 329 ± 26 EPSP vs. S3P antagonistic inhibition 44.0 ± 2.4 Reverse GLP vs. EPSP non-antagonistic inhibition 625 ± 23 GLP vs. Pi non-antagonistic inhibition 1136 ± 60 S3P vs. EPSP antagonistic inhibition 12 0.8 ± 2.1 S3P vs. Pi non-antagonistic inhibition 4936 ± 158 a: Data was fitted to GraFit equation (v4.06, Erithacus Software Ltd.) to correct errors and determine the inhibition method. The inhibition pattern indicates the best fit for the data for each model.

References in Example 2 1. Haslam, E. (1993) Shikimic Acid: Metabolism and Metabolites, Joh
n Wiley, Chickester 2. Amrhein, N. and Steinrucken, HC (1980) Biochem. Biophys. Resm
., Commun. 94, 1207-1212 3. Steinrucken, HC and Amrhein, N. (1984) Eur. J. Biochem.
, 351-357 4. Du, W., Wallis, NG, Mazzulla, MJ, Chalker, AF, Zhang, L., L
iu, W.-S., Kallender, H., and Payne, DJ (1999) manuscript submit
ted 5. Webb, MR (1992) Proc. Natl. Acad. Sci. USA 89, 4884-4.
887 6. Boocock, MR and Coggins, JR (1983) FEBS Lett. 154, 12
7-133

[Sequence list]

[Brief description of the drawings]

FIG. 1 shows the inhibition of EPSP synthase by GLP in a forward reaction. Panel A: Competitive inhibition on PEP measured with S3P (1 mM) fixed;
[GLP] = 0 (open circles), 0.5 μM (solid circles), 3 μM (open squares) and 6 μM
(Black square). Panel B: Antagonistic inhibition on S3P measured with PEP (1 mM) fixed; [GLP] = 0 (open circles), 25 μM (solid circles), 50 μM (open squares) and 100 μM (closed squares).

FIG. 2 shows the inhibition of EPSP synthase by EPSP in a forward reaction. Panel A: Competitive inhibition of PEP measured with immobilized S3P (1 mM); [EPSP] = 0 (open circles), 50 μM (solid circles), 100 μM (open squares), 20
0 μM (solid squares) and 400 μM (open triangles). Panel B: Competitive inhibition of S3P measured with immobilized PEP (1 mM); [EPSP] = 0 (open circles), 25μ
M (closed circle), 50 μM (open square), 75 μM (closed square) and 100 μM (open triangle).

FIG. 3 shows inhibition of EPSP synthase by GLP in a reverse reaction. Panel A: Mixed inhibition on EPSP measured with immobilized Pi (10 mM); [GLP] = 0 (open circle), 0.25 mM (solid circle), 0.5 mM (open square), 0
. 75 mM (closed square) and 1 mM (open triangle). Panel B: EPSP (0.5m
M) immobilized mixed inhibition on Pi; [GLP] = 0 (open circles);
1 mM (filled circle), 0.5 mM (open square) and 1 mM (filled square).

FIG. 4 shows inhibition of EPSP synthase by S3P in a reverse reaction. Panel A: Competitive inhibition of EPS (10 mM) immobilized Pi; [S3
P] = 0 (open circles), 0.1 mM (solid circles), 0.25 mM (open squares) and 0.5
mM (solid square). Panel B: Non-antagonistic inhibition on Pi with immobilized EPSP (0.5 mM); [S3P] = 0 (open circles), 0.1 mM (closed circles), 0.25 mM (open squares) and 0.5 mM (closed squares).

FIG. 5 shows, in each of Schemes 1 and 2, the EPSP synthase catalysis reaction and the formation of PEP oxonium ions.

FIG. 6 shows the proposed mechanism for the forward reaction of S. pneumoniae EPSP synthase in Scheme 3.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 27/16 C07K 16/40 4H045 31/04 C12N 1/15 C07K 16/40 1/19 C12N 1/15 1/21 1/19 9/10 1/21 G01N 33/15 Z 5/10 33/50 Z 9/10 33/53 D G01N 33/15 M 33/50 33/566 33/53 37/00 103 C12N 15/00 ZNAA 33/566 A61K 37/02 37/00 103 C12N 5/00 A (71) Applicant SmithKline Beecham Public Limited Company SmithKline Beecham p. l. c. Tidabreu 89 UK, Middlesex, Brentford, Great West Road 980 (72) Inventor James Earl Brown United States 1912 Robins Lane 9 Birwin, PA 1972 (72) Inventor Alison EF Choker United States 19426 Trapp, PA, College Woods, Harvard Drive 137 (72) Inventor Lisa Kay Katz United States 18940 Newtown, PA, East Park Road 6 (72) Inventor Mary Jean Mazra United States 19426 Greens Way Circle 2029, Collegeville, PA (72) Inventor David Jay Payne United States 19460 Phoenix, WA, Waterfall Way 618, Phoenixville, Pennsylvania 72) Inventor Christopher M. Trainini Potter Court 50, Media, Pennsylvania, 19063, USA AA34 AA35 CA25 CA26 CB03 CB04 CB07 CB09 CB13 CB14 CB21 DA12 DA13 DA14 DA36 DA80 FB02 FB03 FB04 4B024 AA01 AA11 AA20 BA10 CA02 DA01 DA02 DA05 DA11 EA04 GA11 4B050 CC03 DD02 LL01 4B0AAAAAAAAAAAAAAAAAAX BA22 CA53 CA56 CA59 MA63 MA66 NA14 ZA331 ZA341 ZA591 ZB351 ZC781 4H045 AA10 AA11 BA10 CA40 DA75 EA20 EA31

Claims (40)

[Claims] 1. A polynucleotide having at least 70% identity to a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2; (b) in the deposited strain Streptococcus pneumoniae A polynucleotide having at least 70% identity to a polynucleotide encoding the same mature polypeptide expressed by the aroA gene contained in (c) at least the amino acid sequence of SEQ ID NO: 2 A polynucleotide encoding a polypeptide comprising an amino acid sequence that is 70% identical; (d) a polynucleotide that is complementary to the polynucleotide of (a), (b) or (c); and (e) ) At least 15 contiguous sequences of the polynucleotide of (a), (b), (c) or (d) An isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of a polynucleotide comprising the base was. 2. The polynucleotide according to claim 1, wherein the polynucleotide is DNA. 3. The polynucleotide according to claim 1, wherein the polynucleotide is RNA. 4. The polynucleotide according to claim 2, comprising the nucleic acid sequence shown in SEQ ID NO: 1. 5. The polynucleotide according to claim 2, comprising nucleotides 1 to 1281 shown in SEQ ID NO: 1. 6. The polynucleotide according to claim 2, which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 2. 7. A vector comprising the polynucleotide of claim 1. 8. A host cell comprising the vector of claim 7. 9. A method for producing a polypeptide, comprising expressing the polypeptide encoded by the DNA from the host cell of claim 8. 10. A method for producing an aroA polypeptide or fragment, comprising culturing the host of claim 8 under conditions sufficient to produce said polypeptide or fragment. 11. A polypeptide comprising an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO: 2. 12. A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2. 13. An antibody against the polypeptide of claim 11. 14. An antagonist that inhibits the activity or expression of the polypeptide according to claim 11. 15. A method for treating an individual in need of an aroA polypeptide comprising administering to the individual a therapeutically effective amount of the polypeptide of claim 11. 16. A method for treating an individual in need of inhibition of an aroA polypeptide, comprising administering to the individual a therapeutically effective amount of the antagonist of claim 14. 17. A method for diagnosing a disease associated with expression or activity of the polypeptide of claim 11 in an individual, comprising: (a) determining the nucleic acid sequence encoding said polypeptide; and / or b) analyzing for the presence or amount of the polypeptide in a sample from the individual. 18. A method for identifying a compound that interacts with the polypeptide of claim 11 and inhibits or activates its activity, under conditions that allow the interaction between the compound and the polypeptide. Contacting the compound to be screened with the composition comprising the polypeptide to assess the interaction of the compound (such interaction may provide a second detectable signal in response to the interaction of the polypeptide with the compound). The compound then interacts with the polypeptide to activate or inhibit its activity by detecting the presence or absence of a signal resulting from the interaction of the compound with the polypeptide. A method that includes deciding whether to do so. 19. A method for eliciting an immunological response in a mammal, comprising:
12. A method comprising inoculating a mammal with an aroA polypeptide or a fragment or variant thereof of claim 11, sufficient to raise an antibody and / or a T cell immune response to protect the animal from disease. 20. A method of eliciting an immunological response in a mammal, comprising:
Delivering the nucleic acid vector to express the aroA polypeptide or a fragment or variant thereof in vivo to elicit an antibody and / or an immunological response that produces a T cell immune response to protect the animal from disease; Claim 11 a
A method comprising directing the expression of a roA polypeptide or a fragment or variant thereof. 21. A polypeptide comprising an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO: 2 or 4, and a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 or 4. An antagonist or an activating agonist that inhibits the activity of the selected polypeptide,
Here, its activity is the synthesis of p-aminobenzoate, the synthesis of ubiquinone, the transformation of phospho (enol) pyruvate (PEP) and shikimate 3-phosphate (S3P) into EPSP and inorganic phosphate (Pi), EPSP and inorganic phosphate (Pi) phospho (enol) pyruvate (
PEP) and transformation to shikimate 3-phosphate (S3P), binding of AroA to phospho (enol) pyruvate, binding of AroA to phospho (enol) pyruvate-pyruvate kinase complex, AroA and phospho (enol) pyruvate- Binding to lactate dehydrogenase complex; binding of AroA to shikimate 3-phosphate; competitive inhibition of the forward reaction of AroA by glyphosate versus phospho (enol) pyruvate; Inhibition, Competitive inhibition of AroA forward reaction by EPSP versus phospho (enol) pyruvate, EPSP of AroA forward reaction by competitive inhibition by shikimate 3-phosphate, Glyphosate versus E of AroA reverse reaction Non-antagonistic inhibition by SP, non-antagonistic inhibition of reverse reaction of AroA by glyphosate versus Pi, shikimate 3-phosphate versus reverse reaction of AroA by EPSP, and shikimate 3-phosphate versus Pi by reverse reaction of AroA An antagonist or agonist selected from the group consisting of antagonistic inhibition. 22. A method of treating an individual in need of inhibition or activation of an AroA polypeptide, comprising an antibacterial effective amount of at least 90% identical to the amino acid sequence of SEQ ID NO: 2 or 4. An antagonist that inhibits the activity of an polypeptide selected from the group consisting of a polypeptide comprising an amino acid sequence and a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 or 4 or an agonist which activates the polypeptide is administered to the individual. Wherein the activity is the synthesis of p-aminobenzoate, the synthesis of ubiquinone, the transformation of phospho (enol) pyruvate (PEP) and shikimate 3-phosphate (S3P) into EPSP and inorganic phosphate (Pi), Phospho (enol) pyrube of EPSP and inorganic phosphate (Pi) (
PEP) and transformation to shikimate 3-phosphate (S3P), binding of AroA to phospho (enol) pyruvate, binding of AroA to phospho (enol) pyruvate-pyruvate kinase complex, AroA and phospho (enol) pyruvate- Binding to lactate dehydrogenase complex; binding of AroA to shikimate 3-phosphate; competitive inhibition of the forward reaction of AroA by glyphosate versus phospho (enol) pyruvate; Inhibition, Competitive inhibition of AroA forward reaction by EPSP versus phospho (enol) pyruvate, EPSP of AroA forward reaction by competitive inhibition by shikimate 3-phosphate, Glyphosate versus E of AroA reverse reaction Non-antagonistic inhibition by SP, non-antagonistic inhibition of reverse reaction of AroA by glyphosate versus Pi, shikimate 3-phosphate versus reverse reaction of AroA by EPSP, and shikimate 3-phosphate versus Pi by reverse reaction of AroA A method of treatment selected from the group consisting of antagonistic inhibition. 23. A method of treating a bacterially infected individual, comprising an antibacterial effective amount of a polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 2 or 4, and Administering to the individual an antagonist or an agonist that inhibits the activity of a polypeptide selected from the group consisting of polypeptides comprising the amino acid sequence shown in SEQ ID NO: 2 or 4, wherein the activity is Synthesis of p-aminobenzoate, Synthesis of ubiquinone, Transformation of phospho (enol) pyruvate (PEP) and shikimate 3-phosphate (S3P) to EPSP and inorganic phosphate (Pi), Phospho of EPSP and inorganic phosphate (Pi) (Enol) pyruvate (
PEP) and transformation to shikimate 3-phosphate (S3P), binding of AroA to phospho (enol) pyruvate, binding of AroA to phospho (enol) pyruvate-pyruvate kinase complex, AroA and phospho (enol) pyruvate- Binding to lactate dehydrogenase complex; binding of AroA to shikimate 3-phosphate; competitive inhibition of the forward reaction of AroA by glyphosate versus phospho (enol) pyruvate; Inhibition, Competitive inhibition of AroA forward reaction by EPSP versus phospho (enol) pyruvate, EPSP of AroA forward reaction by competitive inhibition by shikimate 3-phosphate, Glyphosate versus E of AroA reverse reaction Non-antagonistic inhibition by SP, non-antagonistic inhibition of reverse reaction of AroA by glyphosate versus Pi, shikimate 3-phosphate versus reverse reaction of AroA by EPSP, and shikimate 3-phosphate versus Pi by reverse reaction of AroA A method of treatment selected from the group consisting of antagonistic inhibition. 24. The method of claim 23, wherein the bacterium is selected from the group consisting of a bacterium of the genus Staphylococcus, Staphylococcus aureus, a bacterium of the genus Streptococcus, and Streptococcus pneumoniae. 25. A method of treating an individual in need of inhibition or activation of an AroA polypeptide, comprising: synthesizing an antimicrobial effective amount of p-aminobenzoate, synthesizing ubiquinone, phospho (enol) pyruvate ( PEP) and shikimate 3-phosphate (S3P) transformation into EPSP and inorganic phosphate (Pi), EPSP and inorganic phosphate (Pi) phospho (enol) pyruvate (
PEP) and transformation to shikimate 3-phosphate (S3P), binding of AroA to phospho (enol) pyruvate, binding of AroA to phospho (enol) pyruvate-pyruvate kinase complex, AroA and phospho (enol) pyruvate- Binding to lactate dehydrogenase complex; binding of AroA to shikimate 3-phosphate; competitive inhibition of the forward reaction of AroA by glyphosate versus phospho (enol) pyruvate; Inhibition, Competitive inhibition of AroA forward reaction by EPSP versus phospho (enol) pyruvate, EPSP of AroA forward reaction by competitive inhibition by shikimate 3-phosphate, Glyphosate versus E of AroA reverse reaction Non-antagonistic inhibition by SP, non-antagonistic inhibition of reverse reaction of AroA by glyphosate versus Pi, shikimate 3-phosphate versus reverse reaction of AroA by EPSP, and shikimate 3-phosphate versus Pi by reverse reaction of AroA A therapeutic method comprising administering to the individual an antagonist that inhibits the activity of AroA, or an agonist that activates the activity, selected from the group consisting of non-antagonistic inhibition. 26. A method of treating an individual infected with a bacterium, comprising the preparation of an antimicrobial effective amount of p-aminobenzoate, ubiquinone, phospho (enol) pyruvate (PEP) and shikimate 3-phosphate (S3P). ) To EPSP and inorganic phosphate (Pi), phospho (enol) pyruvate of EPSP and inorganic phosphate (Pi)
PEP) and transformation to shikimate 3-phosphate (S3P), binding of AroA to phospho (enol) pyruvate, binding of AroA to phospho (enol) pyruvate-pyruvate kinase complex, AroA and phospho (enol) pyruvate- Binding to lactate dehydrogenase complex; binding of AroA to shikimate 3-phosphate; competitive inhibition of the forward reaction of AroA by glyphosate versus phospho (enol) pyruvate; Inhibition, Competitive inhibition of AroA forward reaction by EPSP versus phospho (enol) pyruvate, EPSP of AroA forward reaction by competitive inhibition by shikimate 3-phosphate, Glyphosate versus E of AroA reverse reaction Non-antagonistic inhibition by SP, non-antagonistic inhibition of the reverse reaction of AroA by glyphosate versus Pi, shikimate 3-phosphate of the reverse reaction of AroA by antagonistic inhibition by EPSP, and shikimate 3-phosphate of the reverse reaction of AroA by Pi A therapeutic method comprising administering to the individual an antagonist that inhibits the activity of AroA, or an agonist that activates the activity, selected from the group consisting of non-antagonistic inhibition. 27. The method of claim 26, wherein the bacterium is selected from the group consisting of a bacterium of the genus Staphylococcus, Staphylococcus aureus, a bacterium of the genus Streptococcus, and Streptococcus pneumoniae. 28. A method of treating a bacterially infected individual comprising administering to the individual an antimicrobial effective amount of a compound that is a competitive inhibitor of shikimate 3-phosphate substrate utilization by AroA. Method. 29. The method of claim 28, wherein the bacterium is selected from the group consisting of a bacterium of the genus Staphylococcus, Staphylococcus aureus, a bacterium of the genus Streptococcus, and Streptococcus pneumoniae. 30. The method of claim 28, wherein the compound binds or interacts with the active site of AroA, or interacts with shikimate 3-phosphate, or prevents binding of shikimate 3-phosphate. 31. A method of treating an individual infected with Streptococcus pneumoniae, comprising an antimicrobial effective amount of p-aminobenzoate, ubiquinone, phospho (enol) pyruvate (PEP) and shikimate 3-phosphate. (S3P) transformation into EPSP and inorganic phosphate (Pi), EPSP and inorganic phosphate (Pi) phospho (enol) pyruvate (
PEP) and transformation to shikimate 3-phosphate (S3P), binding of AroA to phospho (enol) pyruvate, binding of AroA to phospho (enol) pyruvate-pyruvate kinase complex, AroA and phospho (enol) pyruvate- Binding to lactate dehydrogenase complex; binding of AroA to shikimate 3-phosphate; competitive inhibition of the forward reaction of AroA by glyphosate versus phospho (enol) pyruvate; Inhibition, Competitive inhibition of AroA forward reaction by EPSP versus phospho (enol) pyruvate, EPSP of AroA forward reaction by competitive inhibition by shikimate 3-phosphate, Glyphosate versus E of AroA reverse reaction Non-antagonistic inhibition by SP, non-antagonistic inhibition of reverse reaction of AroA by glyphosate versus Pi, shikimate 3-phosphate of reverse reaction of AroA by antagonistic inhibition by EPSP, and shikimate 3-phosphate of reverse reaction of AroA by Pi A therapeutic method comprising administering to the individual an antagonist that inhibits the activity of Streptococcus pneumoniae AroA, or an agonist that activates the activity, selected from the group consisting of antagonistic inhibition. 32. A polypeptide comprising an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO: 2 or 4, and a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 or 4. An antagonist that inhibits the activity of a selected polypeptide, wherein the activity is the synthesis of p-aminobenzoate, the synthesis of ubiquinone, the phospho (enol) pyruvate (PEP) and the EPSP of shikimate 3-phosphate (S3P) And transformation of inorganic phosphate (Pi) into phospho (enol) pyruvate of EPSP and inorganic phosphate (Pi)
PEP) and transformation to shikimate 3-phosphate (S3P), binding of AroA to phospho (enol) pyruvate, binding of AroA to phospho (enol) pyruvate-pyruvate kinase complex, AroA and phospho (enol) pyruvate- Binding to lactate dehydrogenase complex; binding of AroA to shikimate 3-phosphate; competitive inhibition of the forward reaction of AroA by glyphosate versus phospho (enol) pyruvate; Inhibition, Competitive inhibition of AroA forward reaction by EPSP versus phospho (enol) pyruvate, EPSP of AroA forward reaction by competitive inhibition by shikimate 3-phosphate, Glyphosate versus E of AroA reverse reaction Non-antagonistic inhibition by SP, non-antagonistic inhibition of reverse reaction of AroA by glyphosate versus Pi, shikimate 3-phosphate of reverse reaction of AroA by antagonistic inhibition by EPSP, and shikimate 3-phosphate of reverse reaction of AroA by Pi An antagonist selected from the group consisting of antagonistic inhibition. 33. A method of treating an individual in need of inhibition of an AroA polypeptide, wherein said amino acid sequence is at least 90% identical to an amino acid sequence of SEQ ID NO: 2 or 4 in an antibacterial effective amount. And administering to the individual an antagonist that inhibits the activity of a polypeptide selected from the group consisting of a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 or 4, and a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 or 4. Is the synthesis of p-aminobenzoate, the synthesis of ubiquinone, the transformation of phospho (enol) pyruvate (PEP) and shikimate 3-phosphate (S3P) into EPSP and inorganic phosphate (Pi), the conversion of EPSP and inorganic phosphate (Pi) Phospho (enol) pyruvate (
PEP) and transformation to shikimate 3-phosphate (S3P), binding of AroA to phospho (enol) pyruvate, binding of AroA to phospho (enol) pyruvate-pyruvate kinase complex, AroA and phospho (enol) pyruvate- Binding to lactate dehydrogenase complex; binding of AroA to shikimate 3-phosphate; competitive inhibition of the forward reaction of AroA by glyphosate versus phospho (enol) pyruvate; Inhibition, Competitive inhibition of AroA forward reaction by EPSP versus phospho (enol) pyruvate, EPSP of AroA forward reaction by competitive inhibition by shikimate 3-phosphate, Glyphosate versus E of AroA reverse reaction Non-antagonistic inhibition by SP, non-antagonistic inhibition of reverse reaction of AroA by glyphosate versus Pi, shikimate 3-phosphate versus reverse reaction of AroA by EPSP, and shikimate 3-phosphate versus Pi by reverse reaction of AroA A method of treatment selected from the group consisting of antagonistic inhibition. 34. A method of inhibiting the activity of an AroA polypeptide, comprising contacting a composition comprising the polypeptide with an effective amount of an antagonist that inhibits the activity of AroA, wherein the activity comprises: Synthesis of p-aminobenzoate, Synthesis of ubiquinone, Transformation of phospho (enol) pyruvate (PEP) and shikimate 3-phosphate (S3P) into EPSP and inorganic phosphate (Pi), Phospho of EPSP and inorganic phosphate (Pi) Enol) pyruvate (
PEP) and transformation to shikimate 3-phosphate (S3P), binding of AroA to phospho (enol) pyruvate, binding of AroA to phospho (enol) pyruvate-pyruvate kinase complex, AroA and phospho (enol) pyruvate- Binding to lactate dehydrogenase complex; binding of AroA to shikimate 3-phosphate; competitive inhibition of the forward reaction of AroA by glyphosate versus phospho (enol) pyruvate; Inhibition, Competitive inhibition of AroA forward reaction by EPSP versus phospho (enol) pyruvate, EPSP of AroA forward reaction by competitive inhibition by shikimate 3-phosphate, Glyphosate versus E of AroA reverse reaction Non-antagonistic inhibition by SP, non-antagonistic inhibition of reverse reaction of AroA by glyphosate versus Pi, shikimate 3-phosphate versus reverse reaction of AroA by EPSP, and shikimate 3-phosphate versus Pi by reverse reaction of AroA An inhibitory method selected from the group consisting of non-antagonistic inhibition. 35. A method of inhibiting the activity of AroA, wherein the activity comprises the synthesis of p-aminobenzoate, the synthesis of ubiquinone, the synthesis of phospho (enol) pyruvate (PEP) and shikimate 3-phosphate (S3P). Transformation of EPSP and inorganic phosphate (Pi), phospho (enol) pyruvate of EPSP and inorganic phosphate (Pi)
PEP) and transformation to shikimate 3-phosphate (S3P), binding of AroA to phospho (enol) pyruvate, binding of AroA to phospho (enol) pyruvate-pyruvate kinase complex, AroA and phospho (enol) pyruvate- Binding to lactate dehydrogenase complex; binding of AroA to shikimate 3-phosphate; competitive inhibition of the forward reaction of AroA by glyphosate versus phospho (enol) pyruvate; Inhibition, Competitive inhibition of AroA forward reaction by EPSP versus phospho (enol) pyruvate, EPSP of AroA forward reaction by competitive inhibition by shikimate 3-phosphate, Glyphosate versus E of AroA reverse reaction Non-antagonistic inhibition by SP, non-antagonistic inhibition of reverse reaction of AroA by glyphosate versus Pi, shikimate 3-phosphate versus reverse reaction of AroA by EPSP, and shikimate 3-phosphate versus Pi by reverse reaction of AroA An inhibitory method selected from the group consisting of non-antagonistic inhibition. 36. The method of claim 35, wherein the bacterium is selected from the group consisting of bacteria of the genus Staphylococcus, Staphylococcus aureus, bacteria of the genus Streptococcus, and Streptococcus pneumoniae. 37. A method of inhibiting the growth of a bacterium, comprising contacting a composition comprising the bacterium with an antimicrobial effective amount of an antagonist that inhibits the activity of AroA, wherein the activity comprises: Synthesis of p-aminobenzoate, Synthesis of ubiquinone, Transformation of phospho (enol) pyruvate (PEP) and shikimate 3-phosphate (S3P) into EPSP and inorganic phosphate (Pi), Phospho of EPSP and inorganic phosphate (Pi) Enol) pyruvate (
PEP) and transformation to shikimate 3-phosphate (S3P), binding of AroA to phospho (enol) pyruvate, binding of AroA to phospho (enol) pyruvate-pyruvate kinase complex, AroA and phospho (enol) pyruvate- Binding to lactate dehydrogenase complex; binding of AroA to shikimate 3-phosphate; competitive inhibition of the forward reaction of AroA by glyphosate versus phospho (enol) pyruvate; Inhibition, Competitive inhibition of AroA forward reaction by EPSP versus phospho (enol) pyruvate, EPSP of AroA forward reaction by competitive inhibition by shikimate 3-phosphate, Glyphosate versus E of AroA reverse reaction Non-antagonistic inhibition by SP, non-antagonistic inhibition of reverse reaction of AroA by glyphosate versus Pi, shikimate 3-phosphate versus reverse reaction of AroA by EPSP, and shikimate 3-phosphate versus Pi by reverse reaction of AroA An inhibitory method selected from the group consisting of non-antagonistic inhibition. 38. The method of claim 37, wherein the bacterium is selected from the group consisting of bacteria of the genus Staphylococcus, Staphylococcus aureus, bacteria of the genus Streptococcus, and Streptococcus pneumoniae. 39. A method of inhibiting an AroA polypeptide, comprising contacting a composition comprising bacteria with an antimicrobial effective amount of an antagonist that inhibits the activity of AroA, wherein the activity comprises: Synthesis of p-aminobenzoate, Synthesis of ubiquinone, Transformation of phospho (enol) pyruvate (PEP) and shikimate 3-phosphate (S3P) into EPSP and inorganic phosphate (Pi), Phospho of EPSP and inorganic phosphate (Pi) Enol) pyruvate (
PEP) and transformation to shikimate 3-phosphate (S3P), binding of AroA to phospho (enol) pyruvate, binding of AroA to phospho (enol) pyruvate-pyruvate kinase complex, AroA and phospho (enol) pyruvate- Binding to lactate dehydrogenase complex; binding of AroA to shikimate 3-phosphate; competitive inhibition of the forward reaction of AroA by glyphosate versus phospho (enol) pyruvate; Inhibition, Competitive inhibition of AroA forward reaction by EPSP versus phospho (enol) pyruvate, EPSP of AroA forward reaction by competitive inhibition by shikimate 3-phosphate, Glyphosate versus E of AroA reverse reaction Non-antagonistic inhibition by SP, non-antagonistic inhibition of reverse reaction of AroA by glyphosate versus Pi, shikimate 3-phosphate of reverse reaction of AroA by antagonistic inhibition by EPSP, and shikimate 3-phosphate of reverse reaction of AroA by Pi An inhibitory method selected from the group consisting of non-antagonistic inhibition. 40. The method of claim 39, wherein the bacterium is selected from the group consisting of bacteria of the genus Staphylococcus, Staphylococcus aureus, bacteria of the genus Streptococcus, and Streptococcus pneumoniae.