Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1999-08-23
2001-10-02
McKelvey, Terry (Department: 1636)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S324000
Reexamination Certificate
active
06297358
ABSTRACT:
FIELD OF THE INVENTION
This invention relates, in part, to newly identified polynucleotides and polypeptides; variants and derivatives of these polynucleotides and polypeptides; processes for making these polynucleotides and these polypeptides, and their variants and derivatives; agonists and antagonists of the polypeptides; and uses of these polynucleotides, polypeptides, variants, derivatives, agonists and antagonists. In particular, in these and in other regards, the invention relates to polynucleotides and polypeptides of SigB operon, hereinafter referred to as “RsbV-1”.
BACKGROUND OF THE INVENTION
Regulation of gene expression in bacteria occurs frequently at the level of transcription. RNA polymerases which transcribe these genes are composed of a multi-subunit core enzyne and an additional protein, or sigma factor, which permits the whole enzyme to recognine promoter elements and initiate transcription at these specific sites. Cells contain multiple sigma factors and their relative levels in the cell provide a fundamental control of gene expression.
Bacillus subtilis
has at least 10 different sigma factors. Haldenwang, W. G. (1995) The sigma factors of
Bacillus subtilis
. Microbiological Reviews 59, 1-30. Sigma-B is activated to direct the transcription of a subset of genes when
B. subtilis
stops exponential growth or is subjected to a number of environmental stresses (e.g., heat, salt, ethanol and peroxide). Boylan, S. A., Redfield, A. R. & Price, C. W. (1993) Transcription factor sigma B of
Bacillus subtilis
controls a large stationary-phase regulon. Journal of Bacteriology 175, 3957-63; Boylan, S. A., Redfield, A. R., Brody, M. S. & Price C. W. (1993) Stress-induced activation of the sigma B transcription factor of
Bacillus subtilis
. Journal of Bacteriology 175, 7931-7.
Staphylococcus aureus
, is a pathogen related to Bacillus species. In the host,
S. aureus
cells are exposed to a range of environmental stresses, some analogous to those mentioned above. Furthermore, bacterial populations in infection loci are likely to contain slow or non-growing bacteria. Thus an equivalent of sigma-B is likely to play a crucial role in the adaptation of
S. aureus
to the host environment.
At least four proteins are known to regulate sigma-B. Three (RsbV, RsbW and RsbX) are the products of genes which are co-transcribed with the structural gene for sigma-B (sigB). The fourth (RsbU) lies immediately upstream of the RsbV gene. RsbW binds to sigma-B, blocking sigma-B-dependent transcription. RsbV can form a complex with RsbW and reduce the sequestration of sigma-B by RsbW. Additionally RsbW is able to phosphorylate RsbV to a form which is unable to interact with RsbW. Dufour, A. & Haldenwang, W. G. (1995) Interactions between a
Bacillus subtilis
anti-sigma factor (RsbW) and its antagonist (RsbV) Journal of Bacteriology 176, 1813-20. RsbU, directly or indirectly, facilitates the RsbV-dependent release of sigma-B from RsbW. Voelker, U., Dufour, A. & Haldenwang, W. G. (1995) The
Bacillus subtilis
rsbU gene product is necessary for RsbX-dependent regulation of sigma B. Journal of Bacteriology 177, 114-22.
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.
The polypeptide of the present invention has amino acid sequence homology to known regulator of a sigma factor.
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 RsbV-1 peptides by homology between the amino acid sequence set out in FIG.
2
and known amino acid sequences of other proteins such as
Bacillus subtillis
RsbV.
It is a further object of the invention, moreover, to provide polynucleotides that encode RsbV-1 polypeptides, particularly polynucleotides that encode the polypeptide herein designated RsbV-1.
In a particularly preferred embodiment of this aspect of the invention, the polynucleotide comprises the region encoding RsbV-1 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 sigma factor 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
bacterial strain,
Staphylococcus aureus
WCUH 29 contained in NCIMB Deposit No. 40771.
In accordance with this aspect of the invention, there are provided isolated nucleic acid molecules encoding RsbV-1, particularly Staphylococcus RsbV-1, 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 compostions compnising 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 RsbV-1 and polypeptides encoded thereby.
In accordance with this aspect of the invention, there are provided novel polypeptides of Staphylococcus referred to herein as RsbV-1 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 compostions comprising same.
Among the particularly preferred embodiments of this aspect of the invention are variants of RsbV-1 polypeptide encoded by naturally occurring alleles of the RsbV-1 gene.
In a preferred embodiment of this aspect of the invention, there are provided methods for producing the aforementioned RsbV-1 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 RsbV-1 expression; to treat upper respiratory tract (e.g. otitis media, bacterial tracheitis, acute epiglottitis, thyroiditis), lower respiratory (e.g. empyema, lung abscess), cardiac (e.g. infective endocarditis), gastrointestinal (e.g. secretory diarrhoea, splenic abscess, retroperitoneal abscess), CNS (e.g. cerebral abscess), eye (e.g. blepharitis, conjunctivitis, keratitis, endophthalmitis, preseptal & orbital cellulitis, darcryocystitis), kidney and urinary tract (e.g. epididymitis, intrarenal and perinephric abscess, toxic shock syndrome), skin (e.g. impetigo, folliculitis , cutaneous abscesses, cellulitis, wound infection, bacterial myositis) bone and joint (e.g. septic arthritis, osteomyelitis).; assaying genetic variation; and administering a RsbV-1 polypeptide or polynucleotide to an organism to raise an immunological response against a bacteria, especially a Staphylococcus.
In accordance with certain preferred embodiments of this and other aspects of the invention, there are provided polynucleotides that hybridize to RsbV-1 polynucleotide sequences.
In certain additional preferred embodiments of this aspect of the invention, there are provided antibodies against RsbV-1 polypeptides.
In accordance with another aspect of the presen
Fosberry Andrew P
Lawlor Elizabeth J
Nicholas Richard O
Deibert Thomas S.
Gimmi Edward R.
King William T.
McKelvey Terry
SmithKline Beecham Corporation
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