Compounds

Details Compounds Compounds

2001-01-09

2002-02-19

Duffy, Patricia A. (Department: 1645)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C424S185100, C424S190100, C424S192100, C424S234100, C424S243100

Reexamination Certificate

active

06348342

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 secA (AThase subunit of preprotein translocase) family, hereinafter referred to as “secA2”.
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.
SecA is an essential component of the bacterial protein secretion apparatus. This enzyme has been found in a wide variety of Gram-negative and Gram-positive bacteria. SecA proteins from different bacterial species are highly homologous and there is no known mammalian homologue making SecA an ideal target for therapeutic intervention. The proposed model of SecA activity (1,2) is based on the fact that SecA binds open-folded preproteins and interacts with a specific receptor complex at the cytoplasmic membrane. ATP hydrolysis then provides the driving force for translocation of the preprotein by a unique mechanism involving transient insertion of SecA across the cytoplasmic membrane. This mechanism of bacterial translocation requires interaction of SecA with itself (SecA is functional as a dimer (3)), ATP (4), preprotein, membrane phospholipids (5,6,7), SecB (5) and the integral membrane components of the bacterial secretion apparatus (8). These numerous interactions provide us with many opportunities to inhibit the activity of this enzyme. From the above discussion we would expect inhibitors of SecA-mediated protein secretion to be broad spectrum and bactericidal without affecting eukaryotic protein secretion. For recent reviews of Sec dependent preprotein translocation see references 9 and 10.
Information on this translocation process and SecA can be found for example in: 1. Economou, A. and Wickner, W. (1994) Cell 78, 835-843; 2. Economou, A. et al. (1995) Cell 83, 1171-1181; 3. Driessen, A. J. M. (1993)Biochemistry 32, 13190-13197; 4. van der Wolk, J. P. W. et al. (1995) Journal of Biological Chemistry 270, 18975-18982; 5. Breukink, E. et al. (1995) Journal of Biological Chemistry 270, 7902-7907; 6. Chen, X et al. (1996) Journal of Biological Chemistry 271, 29698-29706; 7. van der Does, C. (1996) Molecular Microbiology 22, 619-629; 8. Snyders, S. et al. (1997) Journal of Biological Chemistry 272, 11302-11306; 9. Wickner, W and Leonard, M. R. (1996) Journal of Biological Chemistry 271,29514-29516; 10. den Blaauwen, T. and Driessen, J. M. (1996) Arch Microbiol 165, 1-8; 11. WPI Accession No: 96-425087, WO 9626276 A; and 12. WPI Accession No: 95-190181, JP 7107981 A.
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
secA protein.
SUMMARY OF THE INVENTION
It is an object of the invention to provide polypeptides that have been identified as novel secA2 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
secA protein.
It is a further object of the invention to provide polynucleotides that encode secA2 polypeptides, particularly polynucleotides that encode the polypeptide herein designated secA2.
In a particularly preferred embodiment of the invention the polynucleotide comprises a region encoding secA2 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 secA2 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.
A further aspect of the invention there are provided isolated nucleic acid molecules encoding secA2, particularly
Staphylococcus aureus
secA2, 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 secA2 and polypeptides encoded thereby.
Another aspect of the invention there are provided novel polypeptides of
Staphylococcus aureus
referred to herein as secA2 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 secA2 polypeptide encoded by naturally occurring alleles of the secA2 gene.
In a preferred embodiment of the invention there are provided methods for producing the aforementioned secA2 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 secA2 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, endophthalmitis, 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 a

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