Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Reexamination Certificate
1997-11-20
2001-05-01
Nashed, Nashaat T. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
C435S252330, C435S253400, C435S320100, C536S023200, C536S023700
Reexamination Certificate
active
06225098
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to newly identified polynucleotides and polypeptides, and their production and uses, as well as their variants, agonists and antagonists, and their uses. In particular, the invention relates to novel polynucleotides and polypeptides of the MurB family, hereinafter referred to as “MurB”.
BACKGROUND OF THE INVENTION
It is particularly preferred to employ Staphylococcal genes and gene products as targets for the development of antibiotics. The Staphylococci make up a medically important genera of microbes. They are known to produce two types of disease, invasive and toxigenic. Invasive infections are characterized generally by abscess formation effecting both skin surfaces and deep tissues.
S. aureus
is the second leading cause of bacteremia in cancer patients. Osteomyelitis, septic arthritis, septic thrombophlebitis and acute bacterial endocarditis are also relatively common. There are at least three clinical conditions resulting from the toxigenic properties of Staphylococci. The manifestation of these diseases result from the actions of exotoxins as opposed to tissue invasion and bacteremia. These conditions include: Staphylococcal food poisoning, scalded skin syndrome and toxic shock syndrome.
The frequency of
Staphylococcus aureus
infections has risen dramatically in the past few decades. This has been attributed to the emergence of multiply antibiotic resistant strains and an increasing population of people with weakened immune systems. It is no longer uncommon to isolate
Staphylococcus aureus
strains which are resistant to some or all of the standard antibiotics. This phenomenon has created a demand for both new anti-microbial agents, vaccines, and diagnostic tests for this organism.
The enzyme UDP-N-acetylenolpyruvylglucosamine reductase, encoded by the gene MurB catalyses the reduction of UDP-N-acetylpyruvylglucosamine to UDP-N-acetyl muramate, with the concommitant oxidation of NADPH. N-acetyl muramate is a precusor for peptidoglycan biosynthesis.
The gene has been sequenced from
Escherichia coli
(Pucci, M. J., Discotto, L. F. & Dougherty T. J., 1992, J. Bacteriol., 174, 1690-1693) and the enzyme over-expressed, purified and kinetically characterized (Benson, T. E., Marquardt, J. L., Marquardt, A. C., Etzkorn, F. A. & Walsh, C. T. (1993) Biochemistry, 32, 2024-2030). There is also a crystal structure of this enzyme (Benson, T. E., Walsh, C. T., & Hogle, J. M., (1996) Structure, 4, 47-54).
The gene has also been sequenced from
Bacillus subtilis
and shown to be essential (Rowland, S. L., Errington, J. & Wake, R. G., (1995) Gene 164, 113-116). The discovery of a MurB homologue in the human pathogen Staphylococcus aureus allows the production of UDP-N-acetlyenolpyruvylglucosamine reductase enzyme which can then be used to screen for antibiotics. Inhibitors of this enzyme can be used in anti-bacterial therapy as they will prevent the construction of the bacterial cell wall.
Clearly, there exists a need for factors, such as the MurB embodiments of the invention, that have a present benefit of being useful to screen compounds for antibiotic activity. Such factors are also useful to determine their role in pathogenesis of infection, dysfunction and disease. There is also a need for identification and characterization of such factors and their antagonists and agonists to find ways to prevent, ameliorate or correct such infection, dysfunction and disease.
Certain of the polypeptides of the invention possess amino acid sequence homology to a known MurB from
Bacillus subtilis
protein.
SUMMARY OF THE INVENTION
It is an object of the invention to provide polypeptides that have been identified as novel MurB polypeptides by homology between the amino acid sequence set out in Table 1 [SEQ ID NO: 2 or 4] and a known amino acid sequence or sequences of other proteins such as MurB from
Bacillus subtilis
protein.
It is a further object of the invention to provide polynucleotides that encode MurB polypeptides, particularly polynucleotides that encode the polypeptide herein designated MurB.
In a particularly preferred embodiment of the invention the polynucleotide comprises a region encoding MurB polypeptides comprising a sequence set out in Table 1 [SEQ ID NO: 1 or 3] which includes a full length gene, or a variant thereof.
In another particularly preferred embodiment of the invention there is a novel MurB protein from Staphylococcus aureus comprising the amino acid sequence of Table 1 [SEQ ID NO:2 or 4], or a variant thereof.
In accordance with another aspect of the invention there is provided an isolated nucleic acid molecule encoding a mature polypeptide expressible by the
Staphylococcus aureus
WCUH 29 strain contained in the deposited strain.
A further aspect of the invention there are provided isolated nucleic acid molecules encoding MurB, particularly
Staphylococcus aureus
MurB, including mRNAs, cDNAs, genomic DNAs. Further embodiments of the invention include biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comprising the same.
In accordance with another aspect of the invention, there is provided the use of a polynucleotide of the invention for therapeutic or prophylactic purposes, in particular genetic immunization. Among the particularly preferred embodiments of the invention are naturally occurring allelic variants of MurB and polypeptides encoded thereby.
Another aspect of the invention there are provided novel polypeptides of Staphylococcus aureus referred to herein as MurB as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comprising the same.
Among the particularly preferred embodiments of the invention are variants of MurB polypeptide encoded by naturally occurring alleles of the MurB gene.
In a preferred embodiment of the invention there are provided methods for producing the aforementioned MurB polypeptides.
In accordance with yet another aspect of the invention, there are provided inhibitors to such polypeptides, useful as antibacterial agents, including, for example, antibodies.
In accordance with certain preferred embodiments of the invention, there are provided products, compositions and methods for assessing MurB expression, treating disease, assaying genetic variation, and administering a MurB polypeptide or polynucleotide to an organism to raise an immunological response against a bacteria, especially a
Staphylococcus aureus
bacteria.
In accordance with certain preferred embodiments of this and other aspects of the invention there are provided polynucleotides that hybridize to MurB polynucleotide sequences, particularly under stringent conditions.
In certain preferred embodiments of the invention there are provided antibodies against MurB polypeptides.
In other embodiments of the invention there are provided methods for identifying compounds which bind to or otherwise interact with and inhibit or activate an activity of a polypeptide or polynucleotide of the invention comprising: contacting a polypeptide or polynucleotide of the invention with a compound to be screened under conditions to permit binding to or other interaction between the compound and the polypeptide or polynucleotide to assess the binding to or other interaction with the compound, such binding or interaction being associated with a second component capable of providing a detectable signal in response to the binding or interaction of the polypeptide or polynucleotide with the compound; and determining whether the compound binds to or otherwise interacts with and activates or inhibits an activity of the polypeptide or polynucleotide by detecting the presence or absence of a signal generated from the binding or interaction of the compound with the polypeptide or polynucleotide.
In accordance with yet another aspect of the invention, there are provided MurB agonists and antagonists, preferably bacteriostatic or bactericidal agonists and antagonists.
In a furthe
Fedon Jason Craig
Palmer Leslie Marie
Shilling Lisa Kathleen
Wallis Nicola Gail
Deibert Thomas S.
Fronda Christian
Gimmi Edward R.
King William T.
Nashed Nashaat T.
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