Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing
Reexamination Certificate
1999-07-15
2001-04-17
Navarro, Albert (Department: 1645)
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
C424S190100, C424S193100, C424S197110, C424S234100, C424S243100, C530S350000
Reexamination Certificate
active
06217862
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to newly identified polynucleotides and polypeptides in the family of the PhoP response regulatory protein from
Bacillus subtilis.
BACKGROUND OF THE INVENTION
Many two component signal transduction systems (TCSTS) have been identified in bacteria (Stock, J. B. et al., (1989)
Microbiol. Rev
., 53, 450-490). These are involved in the bacterium's ability to monitor its surroundings and adapt to changes in its environment. Several of these bacterial TCSTS are involved in virulence and bacterial pathogenesis within the host. Response regulators are components of the TCSTS. These proteins are phosphorylated by histidine kinases and once phosphorylated effect the response, often through a DNA binding domain becoming activated. The response regulators are characterized by a conserved N-terminal domain of approximately 100 amino acids. The N-terminal domains of response regulators as well as retaining five functionally important residues, corresponding to the residues D12, D13, D57, T87, K109 in CheY (Matsumura, P. et al., (1984)
J. Bacteriol
. 160, 36-41), have conserved structural features (Volz, K. (1993)
Biochemistry
32, 11741-11753). The 3-dimensional structures of CheY from
Salmonella typhimurium
(Stock, A. M. et al., (1989)
Nature
, 337, 745-749) and
Escherichia coli
(Volz, K. & Matsumura, P. (1991)
J. Biol. Chem
. 266, 15511-15519) and the N-terminal domain of nitrogen regulatory protein C from
S. typhimurium
(Volkman, B. F. et al., (1995)
biochemistry
, 34, 1413-1424), are available, as well as the secondary structure of SpoOF from
Bacillus subtilis
(Feher, V. A. et al., (1995)
Protein Science
, 4, 1801-1814). These structures have a (a/b)
5
fold. Several structural residues are conserved between different response regulator sequences, specifically hydrophobic residues within the b-sheet hydrophobic core and sites from the a-helices. This family of response regulators includes PhoP. PhoP is the response regulator of the Pho TCSTS which is involved in the regulation of alkaline phosphatase genes (Seki, T. et al., (1987)
J. Bacteriol
, 169, 2913-2916).
Histidine kinases are components of the TCSTS which autophosphorylate at a histidine residue. The phosphate group is then transferred to the cognate response regulator. The histidine kinases have five short conserved amino acid sequences (Stock, J. B. et al.,
Microbiol. Rev
. 53, 450-490; Swanson, R. V. et al., (1994)
TIBS
, 19, 485-491). These are the histidine residue, which is phosphorylated, followed by a conserved asparagine residue after approximately 100 residues. After another 15 to 45 residues a DXGXG motif is found, followed by a FXXF motif after another 10-20 residues. Ten to twenty residues further on another glycine motif, GXG is found. The two glycine motifs are thought to be involved in nucleotide binding.
Among the processes regulated by TCSTS are production of virulence factors, motility, antibiotic resistance and cell replication. Inhibitors of TCSTS proteins would prevent the bacterium from establishing and maintaining infection of the host by preventing it from producing the necessary factors for pathogenesis and thereby have utility in anti-bacterial therapy.
Clearly, there is a need for factors that may be used to screen compounds for antibiotic activity and which may also be used to determine their roles in pathogenesis of infection, dysfunction and disease. There is a need, therefore, for identification and characterization of such factors which can play a role in preventing, ameliorating or correcting infections, dysfunctions or diseases.
SUMMARY OF THE INVENTION
Toward these ends, and others, it is an object of the present invention to provide polypeptides, inter alia, that have been identified as novel peptides by comparison between the amino acid sequence set out in FIG.
2
and known amino acid sequences of other proteins such as PhoP protein.
It is a further object of the invention, moreover, to provide polynucleotides that encode the novel polypeptides of the invention.
In a particularly preferred embodiment of this aspect of the invention, the polynucleotide comprises the region encoding the novel polypeptides in the sequence set out in
FIG. 1
[SEQ ID NO:1], or a fragment, analogue or derivative thereof.
In another particularly preferred embodiment of the present invention there is a novel protein from
Staphylococcus aureus
comprising the amino acid sequence of
FIG. 2
[SEQ ID NO:2], or a fragment, analogue or derivative thereof.
In accordance with this aspect of the present invention there is provided an isolated nucleic acid molecule encoding a mature polypeptide expressible by the
Staphylococcus aureus
DNA contained in the National Collection of Industrial and Marine Bacteria Ltd. (NCIMB), Aberdeen, Scotland under number NCIMB 40771 on Sep. 11, 1995.
In accordance with this aspect of the invention there are provided isolated nucleic acid molecules encoding novel polypeptides, including mRNAs, cDNAs, genomic DNAs and, in further embodiments of this aspect of the invention include biologically, diagnostically, prophylactically, clinically or therapeutically useful variants, analogs or derivatives thereof, or fragments thereof, including fragments of the variants, analogs and derivatives, and compositions comprising same.
In accordance with another aspect of the present 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 this aspect of the invention are naturally occurring allelic variants of the novel nucleic acid sequences and polypeptides encoded thereby.
In accordance with this aspect of the invention there are provided a novel polypeptide characterized by the amino acid sequence of SEQ ID NO:2, as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful fragments, variants and derivatives thereof, variants and derivatives of the fragments, and analogs of the foregoing, and compositions comprising same.
Among the particularly preferred embodiments of this aspect of the invention are variants of the polypeptide encoded by naturally occurring alleles of the gene of the invention, which is characterized by the nucleic acid sequences of SEQ ID NO:1.
In a preferred embodiment of this aspect of the invention there are provided methods for producing the aforementioned polypeptides.
In accordance with yet another aspect of the present invention, there are provided inhibitors to such polypeptides, useful as antibacterial agents, including, for example, antibodies.
In accordance with certain preferred embodiments of this aspect of the invention, there are provided products, compositions and methods, inter alia: assessing expression; to treat and prevent bacterial infections; assaying genetic variation; and administering a novel polypeptide or polynucleotide of the invention to an organism to raise an immunological response against bacteria, especially Staphylococcus.
In accordance with certain preferred embodiments of this and other aspects of the invention there are provided polynucleotides that hybridize to the novel polynucleotide sequences of the invention, including SEQ ID NO:1.
In certain additional preferred embodiments of this aspect of the invention there are provided antibodies against the polypeptides of the invention.
In accordance with another aspect of the present invention, there are provided agonists to the polynucleotides and/or polypeptides of the invention which are also preferably bacteriostatic or bacteriocidal.
In accordance with yet another aspect of the present invention, there are provided antagonists to the polynucleotides and/or polypeptides of the invention which are also preferably bacteriostatic or bacteriocidal.
In a further aspect of the invention there are provided compositions comprising a polynucleotide or a polypeptide of the invention for administration to a cell or to a multicel
Hodgson John Edward
Wallis Nicola Gail
Deibert Thomas S.
Gimmi Edward R.
King William T.
Navarro Albert
SmithKline Beecham Plc
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