Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1998-12-04
2001-07-10
Navarro, Mark (Department: 1645)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S820000, C530S825000, C435S069100, C435S069700, C435S071100, C435S243000, C435S252100, C435S252300
Reexamination Certificate
active
06258936
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, in these and in other regards, the invention relates to polynucleotides and polypeptides of the spo family, hereinafter referred to as “spoIIIE”.
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.
SpoIIIE is a membrane bound protein involved in chromosome partitioning during sporulation and vegetative replication in a wide variety of bacteria. The SpoIIIE gene was initially characterised in
Bacillus subtilis
(Butler P. D. and Mandelstam J. (1987) Journal of General Microbiology 133:2359-2370). SpoIIIE protein has an ATP binding site and is membrane-bound, and appears to form a pore in the nascent spore septum, through which the prespore chromosome is driven in a conjugation-like mechanism (Wu L. J., Lewis P. J., Allmansberger R., Hauser P. M. and Errington J. (1995) Genes and Development 9:1316-1326). spoIIIE mutants cannot sporulate as they are unable to partition the prespore chromosome into the polar prespore compartment. Instead a specific chromosomal segment comprising approximately 30% of the chromosome enters the prespore, while the rest remains in the mother cell, trapped by the septum (Wu L. J. and Errington J. (1994) Science 264:572-575). In wild-type cells SpoIIIE is membrane-bound, and appears to form a pore in the nascent spore septum, through which the prespore chromosome is driven in a conjugation-like mechanism (Wu L. J., Lewis P. J., Allmansberger R., Hauser P. M. and Errington J. (1995) Genes and Development 9:1316-1326).
It has been shown that SpoIIIE is also required for correct partitioning of the
B.subtilis
chromosome during vegetative cell division. spoIIIE- Mutants in which replication has been artificially delayed are unable to separate the replicated chromosomes before septum formation, resulting in a trapped nucleoid similar to that formed at the start of sporulation (Sharpe M. E. and Errington J. (1995) Proceedings of the Natural Academy of Sciences USA 92:8630-8634).
SpoIIIE has been shown to be essential in
Escherichia coli
(Begg K. J., Dewar, S. J. and Donachie W. D. (1995) Journal of Bacteriology 177:6211-6222). Highly conserved SpoIIIE homologues are found in diverse members of the eubacteria such as
Campylobacter jejuni
(Miller, S., Pesci E. C. and Pickett C. L. (1994) Gene 146:31-38),
Coxiella burnetii
(Oswald W. and Thiele D. (1993) Journal of Veterinary Medecine B40:366-370),
Eshcerichia coli
(Begg 1995 above) and
Haemophilus influenzae
(Fleischmann, R. D., Adams, M. D., White, O., Clayton, R. A., Kirkness, E. F., Kerlavage, A. R., Bult, C. J., Tomb, J.-F., Dougherty, B. A., Merrick, J. M., McKenney, K., Sutton, G., FitzHugh, W., Fields, C. A., Gocayne, J. D., Scott, J. D., Shirley, R., Liu, L.-I., Glodek, A., Kelley, J. M., Weidman, J. F., Phillips, C. A., Spriggs, T., Hedblom, E., Cotton, M. D., Utterback, T. R., Hanna, M. C., Nguyen, D. T., Saudek, D. M., Brandon, R. C., Fine, L. D., Fritchman, J. L., Fuhrmann, J. L., Geoghagen, N. S. M., Gnehm, C. L., McDonald, L. A., Small, K. V., Fraser, C. M., Smith, H. O. and Venter, J. C. (1995) Science 269:496-512). All of these proteins are 36-55% identical at the amino acid level overall. Their N-terminal 200 amino acids are hydrophobic and not conserved, so if the C-terminal 500 or so amino acids are considered alone the level of conservation rises to 42-67% identical amino acids. This high level of identity among diverse eubacteria strongly suggests commonality of function.
Inhibitors of SpoIIIE proteins would prevent the bacterium from establishing and maintaining infection of the host by preventing it from correctly partitioning the chromosome in the manner described above and thus arresting cell division and growth, rendering the bacterium susceptible to host defences and leading ultimately to cell death 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 factors may also be used to determine their roles in pathogenesis of infection, dysfunction and disease. There is also a a need for identification and characterization of such factors and their antagonists and agonists which can play a role in preventing, ameliorating or correcting infections, dysfunctions or diseases.
The polypeptides of the invention have amino acid sequence homology to a known
B. subtilis
spoIIIE protein.
SUMMARY OF THE INVENTION
It is an object of the invention to provide polypeptides that have been identified as novel spoIIIE polypeptides by homology between the amino acid sequence set out in
FIG. 2 and a
known amino acid sequence or sequences of other proteins such as
B. subtilis
spoIIIE protein.
It is a further object of the invention to provide polynucleotides that encode spoIIIE polypeptides, particularly polynucleotides that encode the polypeptide herein designated spoIIIE.
In a particularly preferred embodiment of this aspect of the invention the polynucleotide comprises a region encoding spoIIIE polypeptides comprising the sequence set out in
FIG. 1
[SEQ ID NO:1 and SEQ ID NO:3], or a variant thereof.
In another particularly preferred embodiment of the invention there is a novel spoIIIE protein from
Staphylococcus aureus
comprising the amino acid sequence of
FIG. 2
[SEQ ID NO:2 and SEQ ID NO:4], or a variant thereof.
In accordance with this 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 NCIMB Deposit No. 40771.
In accordance with this aspect of the invention there are provided isolated nucleic acid molecules encoding spoIIIE, particularly
Staphylococcus aureus
spoIIIE, including mRNAs, cDNAs, genomic DNAs. Further embodiments of this aspect 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 this aspect of the invention are naturally occurring alelic variants of spoIIIE and polypeptides encoded thereby.
In accordance with this aspect of the invention there are provided novel polypeptides of
Staphylococcus aureus
referred to herein as spoIIIE as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comprising the same.
Among the particularly preferred embodiments of this aspect of the invention are variants of spoIIIE polypeptide encoded by naturally occurring alleles of the spoIIIE gene.
In a preferred embodiment of this aspect of the invention there are provided methods for producing the aforementioned spoIIIE polypeptides.
In accordance with yet another aspect of the invention, there are provided inhibitors to such polypeptides, usefu
Chalker Alison Frances
Hodgson John Edward
Deibert Thomas S.
Gimmi Edward R.
King William T.
Lee Li
Navarro Mark
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