Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1997-09-26
2001-10-30
Minnifield, Nita (Department: 1645)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C536S023600, C536S023400, C536S023200, C536S023700, C435S183000, C435S069100, C435S252330, C435S320100, C435S252300
Reexamination Certificate
active
06310192
ABSTRACT:
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 “SigB”.
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 enzyme and an additional protein, or sigma factor, which permits the whole enzyme to recognize 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, 1995]. 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). See Boylan, et al., Journal of Bacteriology, 175, 3957-63 (1993a); Boylan, et al., Journal of Bacteriology, 175, 7931-7 (1993b).
S. 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 and Haldenwang, Journal of Bacteriology 176, 1813-20 (1995). RsbU, directly or indirectly, facilitates the RsbV-dependent release of sigma-B from RsbW. Voelker et al., Journal of Bacteriology 177, 114-22 (1995).
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 sigma factor. See, for example, Haldenwang, W.G., Microbiological Reviews, 59, 1-30 (1995).
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 SigB 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
SigB.
It is a further object of the invention, moreover, to provide polynucleotides that encode SigB polypeptides, particularly polynucleotides that encode the polypeptide herein designated SigB.
In a particularly preferred embodiment of this aspect of the invention the polynucleotide comprises the region encoding SigB 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 SigB, particularly Staphylococcus SigB, 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 SigB and polypeptides encoded thereby.
In accordance with this aspect of the invention there are provided novel polypeptides of Staphylococcus referred to herein as SigB 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 SigB polypeptide encoded by naturally occurring alleles of the SigB gene.
In a preferred embodiment of this aspect of the invention there are provided methods for producing the aforementioned SigB 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 SigB 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 SigB 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 SigB polynucleotide sequences.
In certain additional preferred embodiments of this aspect of the invention there are provided antibodies against SigB polypeptides.
In accordance with another aspect of the present invention, there are provided SigB agonists which are also preferably bacteriostatic or bactericidal.
In accordance with yet another aspect of the present invention, there are provided SigB antagonists which are also preferably bacteriostatic or bactericidal.
In a further aspect of the invention there are provided compositions comprising a SigB polynucleotide or a SigB polypeptide for administration to a cell or to a multicellular organism. Various changes and modifications within the spirit and scope of the disclosed invention will become readily a
Fosberry Andrew P
Lawlor Elizabeth J
Nicholas Richard O
Deibert Thomas S.
Gimmi Edward R.
King William T.
Minnifield Nita
SmithKline Beecham Corporation
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