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
1997-03-12
2003-06-03
Duffy, Patricia A. (Department: 1645)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S071100, C435S252300, C435S254110, C435S320100, C435S325000, C536S023700
Reexamination Certificate
active
06573066
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 novel polynucleotides and polypeptides of the ATPase family, hereinafter referred to as “FtsH”.
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 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.
FtsH, an essential membrane bound protein involved in membrane functions, cell cycle control and gene expression was initially characterised in
Escherichia coli
(Tomoyasu, T., Yuki, T., Morimura, S., Mori, H., Yamanaka, K., Niki, H., Hiraga, S. and Ogura, T. (1993) Journal of Bacteriology 175: 1344-1351). The
Escherichia coli
FtsH protein comprises 644 amino acid residues with a predicted molecular mass of 70.7 kDa. and has been shown to localise to the cytoplasmic membrane via two hydrophobic domains (Tomoyasu, T., Yamanaka, K., Murata, K., Suzaki, T., Bouloc, P., Kato, A., Niki, H., Hiraga, S. and Ogura, T. (1993) Journal of Bacteriology 175: 1352-1357). It belongs to a novel putative ATPase family known as the AAA-protein family, members of which are widely distributed among eubacteria, archaebacteria and eukaryotes (Kunau, W.H., Beyer, A., Franken, T., Gotte, K., Marzioch, M., Saidowski, J., Skaletz-Rorowski, A. and Wiebel, F. F. (1993) Biochemie 75: 209-224). FtsH demonstrates significant homology to these ATPases over a cytoplasmic region of some 200 amino acid residues which includes a putative ATP binding site, a zinc-binding motif and the adjacent C-terminal sequence. Recently,
Escherichia coli
FtsH was shown to catalyse the ATP dependent degradation of the (&sgr;32 subunit of
Escherichia coli
RNA polymerase (Tomoyasu, T., Gamer, J., Bukau, B., Kanemori, M., Mori, H., Rutman, A. J., Oppenheim, A. B., Yura, T., Yamanaka, K., Niki, H., Hiraga, S. and Ogura, T. (1995) The EMBO Journal 14 2551-2560) and as such is thought to be a key element in transcriptional control. In addition, FtsH is required for the proteolytic elimination of uncomplexed forms of SecY, important in maintaining optimal protein translocation and integrity of the membrane (Kihara, A., Akiyama, Y., Ito, K. (1995) Proceedings of the National Academy of Sciences USA 92: 4532-4536). Overproduction of SecY in FtsH mutant cells has been shown to deleteriously effect cell growth and protein export.
In addition to
Escherichia coli
(Tomoyasu, T., Yuki, T., Morimura, S., Mori, H., Yamanaka, K., Niki, H., Hiraga, S. and Ogura, T. (1993) Journal of Bacteriology 175: 1344-1351), highly conserved FtsH homologues have been identified in
Lactococcus lactis
(Nilsson, D., Lauridsen, A. A., Tomoyasu, T. and Ogura, T. (1994) Microbiology 140: 2601-2610),
Bacillus subtilis
, (Ogasawara, N., Nakai, S. and Yoshikawa, H. (1994) DNA Research 1: 1-14), and
Saccharomyces cerevisiae
(Thorsness, P. E., White, K. H. and Fox, T. D. (1993) Molecular and cellular Biology 13: 5418-5426, Schnall, R., Mannhaupt, G., Stuka, R., Ehnle, S., Schwarzlose, C., Vetter, I. and Feldmann, H. (1994) Yeast 10 1141-1155) however with the exception of
Escherichia coli
FtsH, none of these proteins have been purified and studied biochemically. The high level of identity among diverse eubacteria and eukaryotes strongly suggests commonality of function. The ftsH gene is essential for cell viability in
Escherichia coli
(Tomoyasu, T., Yuki, T., Morimura, S., Mori, H., Yamanaka, K., Niki, H., Hiraga, S. and Ogura, T. (1993) Journal of Bacteriology 175: 1344-1351). Inhibitors of FtsH proteins would prevent bacteria from establishing and maintaining infection of the host by disrupting transcription and protein translocation, resulting in arrested growth and ultimately to cell death as the bacteria become susceptible to host defences and thereby have utility in anti-bacterial therapy.
Clearly, 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
Bacillus subtilis
FtsH protein.
SUMMARY OF THE INVENTION
It is an object of the invention to provide polypeptides that have been identified as novel FtsH 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
Bacillus subtilis
FtsH protein.
It is a further object of the invention to provide polynucleotides that encode FtsH polypeptides, particularly polynucleotides that encode the polypeptide herein designated FtsH.
In a particularly preferred embodiment of the invention the polynucleotide comprises a region encoding FtsH polypeptides comprising the sequence set out in Table 1[SEQ ID NO:1], or a variant thereof.
In another particularly preferred embodiment of the invention there is a novel FtsH 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 NCIMB Deposit No. 40771.
A further aspect of the invention there are provided isolated nucleic acid molecules encoding FtsH, particularly
Staphylococcus aureus
FtsH, 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 allelic variants of FtsH and polypeptides encoded thereby.
Another aspect of the invention there are provided novel polypeptides of
Staphylococcus aureus
referred to herein as FtsH 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 FtsH polypeptide encoded by naturally occur
Hodgson John Edward
Jenkins Owen
Sarginson Gillian
Duffy Patricia A.
Fedon Jason C
Gimmi Edward R
Kinzig Charles M.
SmithKline Beecham Corporation
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