Polynucleotides encoding GTP cyclohydrolase II (RIBA)

Details Polynucleotides encoding GTP cyclohydrolase II (RIBA) Polynucleotides encoding GTP cyclohydrolase II (RIBA)

1999-01-11

2001-08-28

Kunz, Gary L. (Department: 1647)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S320100, C435S325000, C435S069700, C536S023100, C536S023200, C536S023400, C536S023700, C536S024100, C536S024320

Reexamination Certificate

active

06280971

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 GTP cyclohydrolase II family, hereinafter referred to as “ribA”.
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.
Staphylococcus 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.
Riboflavin (vitamin B2) is a member of the B complex of vitamins which function as coenzymes in metabolic reactions. Riboflavin has two coenzyme forms, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) which act in oxidation-reduction reactions such as the cytochrome system of electron transport and the oxidative degradation of pyruvate, fatty acids and amino acids. The first committed step in the biosynthesis of riboflavin is the opening of the imidazole ring of GTP. In the presence of 3 H
2
O and Mg
++
, the C-8 of GTP is released as formate accompanied by the release of pyrophosphate by the action of GTP cyclohyrolase II (GCH2; EC 3.5.4.25). This enzyme function is encoded by ribA in bacteria and rib1 in yeast species. Through a series of steps, involving 3,4-dihydroxy-2-butanone 4 phosphate synthase (ribA), 6,7-dimethyl-8-ribityllumazine synthase (ribH), riboflavin synthase (ribB), pyrimidine deaminase and pyrimidine reductase (ribG), enzymes encoded by genes within the riboflavin biosynthesis operon, riboflavin is formed. Because GCH2 catalyzes the first committed step in the biosynthesis of riboflavin (Richter G, et al. (1993) Biosynthesis of riboflavin: cloning, sequencing, mapping, and expression of the gene coding for GTP cyclohydrolase II in
Escherichia coli.
J Bacteriol. 175:4045-4051), is present in many pathogenic microorganisms, and since riboflavin biosynthesis has shown to be required for virulence in the swine pathogen
Actinobacillus pleuropneumoniae
(Fuller, T E, et al. (1996) A riboflavin auxotroph of
Actinobacillus pleuropneumoniae
is attenuated in swine. Infect. Immun. 64:4659-4664), it represents a broad spectrum antibacterial as well as antifungal target.
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.
Clearly, there exists a need for factors, such as the ribA 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 Bacillus amyloliquefaciens protein. (Swiss Prot. Accession no. P51695; GenBank Accession no. U27202; Perkins, J. B. et al., In: Bacillus subtilis and Other Gram-Positive Bacteria. Eds: Sonenshein, A. L. et al., 1993. American Society for Microbiology.)
SUMMARY OF THE INVENTION
It is an object of the invention to provide polypeptides that have been identified as novel ribA 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 Bacillus amyloliquefaciens protein.
It is a further object of the invention to provide polynucleotides that encode ribA polypeptides, particularly polynucleotides that encode the polypeptide herein designated ribA.
In a particularly preferred embodiment of the invention, the polynucleotide comprises a region encoding ribA polypeptides comprising a sequence set out in Table 1 [SEQ ID NO:1, 3 or 7] which includes a full length gene, or a variant thereof.
In another particularly preferred embodiment of the invention, there is a novel ribA protein from
Staphylococcus aureus
comprising the amino acid sequence of Table 1 [SEQ ID NO:2 or 4], or a variant thereof.
As a further aspect of the invention, there are provided isolated nucleic acid molecules encoding ribA, particularly
Staphylococcus aureus
ribA, 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 ribA and polypeptides encoded thereby.
As another aspect of the invention, there are provided novel polypeptides of
Staphylococcus aureus
referred to herein as ribA 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 ribA polypeptide encoded by naturally occurring alleles of the ribA gene.
In a preferred embodiment of the invention, there are provided methods for producing the aforementioned ribA 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 ribA expression, treating disease, assaying genetic variation, and administering a ribA 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 ribA polynucleotide sequences, particularly under stringent conditions.
In certain preferred embodiments of the invention, there are provided antibodies against ribA 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 bin

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