Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
2000-08-03
2002-10-01
Duffy, Patricia A. (Department: 1645)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C435S183000, C424S192100, C424S094100, C424S094500
Reexamination Certificate
active
06458572
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 Phosphatidylglycerophosphate synthase family, hereinafter referred to as “pgsA”.
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 a demand for both new anti-microbial agents, vaccines, and diagnostic tests for this organism.
Three major phospholipid species are present in procaryotic cells. There is some variation in their proportions in different strains and growth conditions, but the typical contain in
Escherichia coli
is 70 to 80% of phosphatidylethanolamine (PE), 15 to 25% phosphatidylglycerol (PG) and 3 to 10% cardiolopin (CL) (Kadner, R. J. 1996. In
Escherichia coli
and Salmonella, Cellular and Molecular Biology (Neidhardt, F. C, Eds), pp.58-88., ASM Press, Washington D.C.). In the membrane of
Escherichia coli,
the ratio of zwitterionic phospholipid (PE) to acidic phospholipids (PG and CL) is kept nearly constant under any growth conditions and this is thought to be important for the proper functioning of the membrane (Kumar Saha, S., Nishijima, S., Matsuzaki, H., Shibuya, I., and Matsumoto, K. 1996. Biosc. Biotech. Biochem. 60(1), 111-116).
The biosynthetic pathway for these lipids contains a branch point at which two enzymes, phosphatidyl glycerophosphate (PGP) synthase and phosphatidyl serine (PS) synthase, compete for the same substrate, CDP-diacylglycerol. PS synthase combines CDP-diacylglycerol and serine to form PS, which is decarboxylated by PS decarboxylase to yield PE. In the other branch PGP synthase combines CDP-diacylglycerol and glycerol-3-phosphate to form PGP, which is converted to PG by various phosphatases. Finally, CL is synthetized by the condensation of two molecules of PG by CL synthase (Cronan, J. E., and Rock, C. 1996. In
Escherichia coli
and Salmonella, Cellular and Molecular Biology (Neidhardt, F. C, Eds), pp.612-636., ASM Press, Washington D.C).
PGP synthase is an integral membrane protein. Its structural gene, pgsA has been cloned and sequenced in a number of bacteria including
Escherichia coli
(Gopalakrishnan, A. S., Chen, Y. C., Temkin, M., and Dowhan, W. 1986. J. Biol. Chem. 261, 1329-1338. Usui, M., Sembongi, H., Matsuzaki, H., Matsumoto, K., and Shibuya, 1. 1994. J. Bacteriol. 176, 3389-3392) and
Bacillus subtilis
(Kontigen, V. P., and Tokuda, H. 1995. FEBS Lett. 364(2), 157-160).
The essential nature of the gene product of pgsA in
Escherichia coli
has been demonstrated by the construction of temperature sensitive mutants (Heacock, P. N., and Dowhan, W. 1987. J. Biol. Chem. 262, 13044-13049. Nishijima, M, Bulawa, C. E., and Raetz, C. R. H. 1981. J. Bacteriol. 145, 113-121. Raetz, C. R. H. 1975. Proc. Natl. Acad. Sci. USA 72, 2274-2278).
Clearly, there exists a need for factors, such as the pgsA embodiments 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 to find ways to prevent, ameliorate or correct such infection, dysfunction and disease.
Certain of the polypeptides of the invention possess amino acid sequence homology to a known
Bacillus subtilis
pgsA,
Mycobacterium leprae
pgsA,
Mycobacterium tuberculosis
pgsA,
Helicobacter pylori
pgsA,,
Escherichia coli
pgsA,
Haemophilus influenzae
pgsA, Synechocystis sp. pgsA,
Pseudomonas fluorescens
pgsA,
Mycoplasma capricolum
pgsA,
Mycoplasma genitalium
pgsA,
Mycoplasma pneumoniae
pgsA,
Rhodobacter sphaeroides
pgsA,
Borrelia burgdorferi
pgsA,
Archaeoglobus fulgidus
pgsA,
Saccharomyces cerevisiae
pgsA protein.
SUMMARY OF THE INVENTION
It is an object of the invention to provide polypeptides that have been identified as pgsA 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
pgsA,
Mycobacterium leprae
pgsA,
Mycobacterium tuberculosis
pgsA,
Helicobacter pylori
pgsA,,
Escherichia coli
pgsA,
Haemophilus influenzae
pgsA, Synechocystis sp. pgsA,
Pseudomonas fluorescens
pgsA,
Mycoplasma capricolum
pgsA,
Mycoplasma genitalium
pgsA,
Mycoplasma pneumoniae
pgsA,
Rhodobacter sphaeroides
pgsA,
Borrelia burgdorferi
pgsA,
Archaeoglobus fulgidus
pgsA,
Saccharomyces cerevisiae
pgsA protein.
It is a further object of the invention to provide polynucleotides that encode pgsA polypeptides, particularly polynucleotides that encode the polypeptide herein designated pgsA.
In a particularly preferred embodiment of the invention the polynucleotide comprises a region encoding pgsa polypeptides comprising a 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 pgsA protein from
Staphylococcus aureus
comprising the amino acid sequence of Table 1 [SEQ ID NO:2], or a variant thereof.
As a further aspect of the invention there are provided isolated nucleic acid molecules encoding pgsA, particularly
Staphylococcus aureus
pgsA, 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 pgsA and polypeptides encoded thereby.
In another aspect of the invention there are provided polypeptides of
Staphylococcus aureus
referred to herein as pgsA 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 pgsA polypeptide encoded by naturally occurring alleles of the pgsA gene.
In a preferred embodiment of the invention there are provided methods for producing the aforementioned pgsA 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 p
Holmes David John
Petit Chanal Myriam
Traini Christopher Michael
Warren Richard Lloyd
Duffy Patricia A.
Fedon Jason C.
Gimmi Edward R.
Kinzig Charles M.
SmithKline Beecham Corporation
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