Def1

Details Def1 Def1

1999-08-13

2002-06-25

Minnifield, Nita (Department: 1645)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

C530S300000, C530S324000, C424S184100, C424S185100, C424S192100, C424S237100, C424S234100, C424S243100, C424S094500, C424S094100, C435S183000, C514S002600

Reexamination Certificate

active

06410688

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to newly identified polynucleotides and polypeptides, and heir 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 novel polynucleotides and polypeptides of the Polypeptide deformylase (pdf, def, fms) family, hereinafter to as “Def1”.
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 issues.
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.
The frequency of
Staphylococcus aureus
infections has risen dramatically in the past 20 years. 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 has created a demand for both new anti-microbial agents and diagnostic tests for this organism.
Bacterial protein syntheses is initiated with an N-terminal formyl-methionine. The formate group and methionine are subsequently removed from most proteins in a two step process. Polypeptide deformylase catalyses the first step in this process, removal of the N-terminal formyl group. In
E. coli
this activity is essential for growth (Mazel, D., Pochet, S., and Marliere, P. (1994). EMBRO J. 13, 914-923). S. pneumonia has two homologs of this gene, Def1 is most similar to an unidentified orf, orf211 of Genbank entry L36907 (Cancilla, M. R., Hillier, A. J. and Davidson, B. E. (1995) Microbiology 141:1027-1036) which we have identified as peptide deformylase on the basis of sequence similarity, including presence of all of the active site motifs (Meinnel, T., Blanquet, S., and Dardel, F. (1996). Journal Of Molecular Biology 262, 375-386.). Def2 is most similar to the peptide deformylase enzyme from
Thermus aquaticus
(subsp. thermophilus, Swiss Protein entry DEF
13
THETH, Meinnel T. and Blanquet, S. (1994) J Bacteriol 176: 7387-7390).
Cleary, there is a need for factors, such as the novel compounds 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 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 Def1
Bacillus subtilis
protein. (NCBI non-redundant database (National Center for Biotechnology, U.S.A.), gi 1377833 (U51911) (without identification or characterization, but likely to be a polypeptide deformylase based on similarity to entry gi 806487 (46% identity; 64% similarity) which has been shown to code for a polypeptide deformylase (D. Mazel et al., J.Mol.Biol., 266, 939 (1997)).)
SUMMARY OF THE INVENTION
It is an object of the invention to provide polypeptides that have been identified as novel Def1 polypeptides by homology between the amino acid sequence set out in Table 1 [SEQ ID NO: 2] and a known amino acid sequence or sequences of other proteins such as Def1
Bacillus subtilis
protein.
It is a further object of the invention to provide polynucleotides that encode Def1 polypeptides, particularly polynucleotides that encode the polypeptides herein designated Def1.
In a particularly preferred embodiment of the invention, the polynucleotide comprises a region encoding Def1 polypeptides comprising the sequence set out in Table 1 [SEQ ID NO: 1] which includes a full length gene, or a variant thereof.
In another particularly preferred embodiment of the invention, there is a novel Def1 protein from
Staphylococcus aureus
comprising the amino acid sequence of Table 1 [SEQ ID NO:2], or a variant thereof.
In accordance with another 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 the deposited strain.
As a further aspect of the invention, there are provided isolated nucleic acid molecules encoding Def1 , particularly
Staphylococcus aureus
Def1 , 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 allergic variants of Def1 and polypeptides encoded thereby.
As another aspect of the invention, there are provided novel polypeptides of
Staphylococcus aureus
referred to herein as Def1 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 Def1 polypeptide encoded by naturally occurring alleles of the Def1 gene.
In a preferred embodiment of the invention, there are provided methods for producing the aforementioned Def1 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 Def1 expression, treating disease, for example, disease, such as, infections of the 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 absces, retroperitoneal abscess), CNS (e.g., cerebral abscess), eye (e.g., blepharitis, conjunctivitis, keratitis, endophtalmitis, preseptal and orbital cellulitis, darcryocystitis), kidney and urinary tract (e.g., epididymitis, intrarenal and perinephric absces, 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 Def1 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 Def1 polynucleotide sequences, particularly under stringent conditions.
In certain preferred embodiments of the invention, there are provided antibodies against Def1 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 comp

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