NrdF from Staphylococcus aureus

Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...

Reexamination Certificate

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C435S070100, C435S071100, C435S183000, C435S252300, C435S254110, C435S320100, C435S325000, C536S023400, C536S023700

Reexamination Certificate

active

06555338

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, the invention relates to polynucleotides and polypeptides of the nrd family, as well as their variants, hereinafter referred to as “nrdF,” “nrdF polynucleotide(s),” and “nrdF polypeptide(s).”
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.
The frequency of
Staphylococcus aureus
infections has risen dramatically in the past few decades. 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 phenomenon has created an unmet medical need and demand for new anti-microbial agents, vaccines, drug screening methods, and diagnostic tests for this organism.
Moreover, the drug discovery process is currently undergoing a fundamental revolution as it embraces “functional genomics,” that is, high throughput genome- or gene-based biology. This approach is rapidly superseding earlier approaches based on “positional cloning” and other methods. Functional genomics relies heavily on the various tools of bioinformatics to identify gene sequences of potential interest from the many molecular biology databases now available as well as from other sources. There is a continuing and significant need to identify and characterize further genes and other polynucleotides sequences and their related polypeptides, as targets for drug discovery.
Clearly, there exists a need for polynucleotides and polypeptides, such as the nrdF embodiments of the invention, that have a present benefit of, among other things, 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 to find ways to prevent, ameliorate or correct such infection, dysfunction and disease.
Certain of the polypeptides of the invention possess significant amino acid sequence homology to a known Bacillus subtilis NrdF protein, see Swissprot accession number P50621 and Genbank accession number Z68500.
SUMMARY OF THE INVENTION
The present invention relates to nrdF, in particular nrdF polypeptides and nrdF polynucleotides, recombinant materials and methods for their production. In another aspect, the invention relates to methods for using such polypeptides and polynucleotides, including the treatment of microbial diseases, amongst others. In a further aspect, the invention relates to methods for identifying agonists and antagonists using the materials provided by the invention, and for treating microbial infections and conditions associated with such infections with the identified compounds. In a still further aspect, the invention relates to diagnostic assays for detecting diseases associated with microbial infections and conditions associated with such infections, such as assays for detecting nrdF expression or activity.
Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following descriptions and from reading the other parts of the present disclosure.
DESCRIPTION OF THE INVENTION
The invention relates to nrdF polypeptides and polynucleotides as described in greater detail below. In particular, the invention relates to polypeptides and polynucleotides of a nrdF of
Staphylococcus aureus
, which is related by amino acid sequence homology to Bacillus subtilis NrdF polypeptide. The invention relates especially to nrdF having the nucleotide and amino acid sequences set out in Table 1 as SEQ ID NO: 1 and SEQ ID NO: 2 respectively.
TABLE 1
NrdF Polynucleotide and Polypeptide Sequences
(A)
Staphylococcus aureus
 nrdF
polynucleotide sequence [SEQ ID NO:1].
5′-ATGATAGCTGTTAATTGGAACACACAAGAAGATATGACGAATATGTTTTGGAGACAAAAT
ATATCTCAAATGTGGGTTGAAACAGAATTTAAAGTATCAAAAGACATTGCAAGTTGGAAG
ACTTTATCTGAAGCTGAACAAGACACATTTAAAAAAGCATTAGCTGGTTTAACAGGCTTA
GATACACATCAAGCAGATGATGGCATGCCTTTAGTTATGCTACATACGACTGACTTAAGG
AAAAAAGCAGTTTATTCATTTATGGCGATGATGGAGCAAATACACGCGAAAAGCTATTCA
CATATTTTCACAACACTATTACCATCTAGTGAAACAAACTACCTATTAGATGAATGGGTT
TTAGAGGAACCCCATTTAAAATATAAATCTGATAAAATTGTTGCTAATTATCACAAACTT
TGGGGTAAAGAAGCTTCGATATACGACCAATATATGGCCAGAGTTACGAGTGTATTTTTA
GAAACATTCTTATTCTTCTCAGGTTTCTATTATCCACTATATCTTGCTGGTCAAGGGAAA
ATGACGACATCAGGTGAAATCATTCGTAAAATTCTTTTAGATGAATCTATTCATGGTGTA
TTTACCGGTTTAGATGCACAGCATTTACGAAATGAACTATCTGAAAGTGAGAAACAAAAA
GCAGATCAAGAAATGTATAAATTGCTAAATGACTTGTATTTAAATGAAGAGTCATACACA
AAAATGTTATACGATGATCTTGGAATCACTGAAGATGTGCTAAACTATGTTAAATATAAT
GGAAACAAAGCACTTTCAAACTTAGGCTTTGAACCTTATTTTGAGGAACGTGAATTTAAC
CCAATCATTGAGAATGCCTTAGATACAACAACTAAAAACCATGACTTCTTCTCAGTAAAA
GGTGATGGTTATGTATTAGCATTAAACGTAGAAGCATTACAAGATGATGACTTTGTATTT
GACAACAAA-3′
(B)
Staphylococcus aureus
 nrdF
polypeptide sequence deduced from a
polynucleotide sequence in this table [SEQ ID NO:2].
NH
2
-MIAVNWNTQEDMTNMFWRQNISQMWVETEFKVSKDIASWKTLSEAEQDTFKKALAGLTGL
DTHQADDGMPLVMLHTTDLRKKAVYSFMAMMEQIHAKSYSHIFTTLLPSSETNYLLDEWV
LEEPHLKYKSDKIVANYHKLWGKEASIYDQYMARVTSVFLETFLFFSGFYYPLYLAGQGK
MTTSGEIIRKILLDESIHGVFTGLDAQHLRNELSESEKQKADQEMYKLLNDLYLNEESYT
KMLYDDLGITEDVLNYVKYNGNKALSNLGFEPYFEEREFNPIIENALDTTTKNHDFFSVK
GDGYVLALNVEALQDDDFVFDNK-COOH
Deposited Materials
A deposit containing a
Staphylococcus aureus
WCUH 29 strain has been deposited with the National Collections of Industrial and Marine Bacteria Ltd. (herein “NCIMB”), 23 St. Machar Drive, Aberdeen AB2 1RY, Scotland on Sep. 11, 1995 and assigned NCIMB Deposit No. 40771, and referred to as
Staphylococcus aureus
WCUH29 on deposit. The
Staphylococcus aureus
strain deposit is referred to herein as “the deposited strain” or as “the DNA of the deposited strain.”
The deposited strain contains the full length nrdF gene. The sequence of the polynucleotides contained in the deposited strain, as well as the amino acid sequence of any polypeptide encoded thereby, are controlling in the event of any conflict with any description of sequences herein.
The deposit of the deposited strain has been made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for Purposes of Patent Procedure. The strain will be irrevocably and without restriction or condition released to the public upon the issuance of a patent. The deposited strain is provided merely as convenience to those of skill in the art and is not an admission that a deposit is required for enablement, such as that required under 35 U.S.C. §112.
A license may be required to make, use or sell the deposited strain, and compounds derived therefrom, and no such license is hereby granted.
In one aspect of the invention there is provided an isolated nucleic acid molecule encoding a mature polypeptide expressible by the
Staphylococcus aureus
WCUH 29 strain, which polypeptide is contained in the depo

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