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
1998-12-04
2003-05-06
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...
C435S070100, C435S071100, C435S071200, C435S212000, C435S252300, C435S254110, C435S320100, C435S325000, C536S023200, C536S023700
Reexamination Certificate
active
06558919
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 aminopeptidases family, hereinafter referred to as “ampS”.
BACKGROUND OF THE INVENTION
The Streptococci make up a medically important genera of microbes known to cause several types of disease in humans, including, for example, otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid. Since its isolation more than 100 years ago,
Streptococcus pneumoniae
has been one of the more intensively studied microbes. For example, much of our early understanding that DNA is, in fact, the genetic material was predicated on the work of Griffith and of Avery, Macleod and McCarty using this microbe. Despite the vast amount of research with
S. pneumoniae
, many questions concerning the virulence of this microbe remain. It is particularly preferred to employ Streptococcal genes and gene products as targets for the development of antibiotics.
The frequency of
Streptococcus pneumoniae
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
Streptococcus pneumoniae
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.
While certain Streptococcal factors associated with pathogenicity have been identified, e.g., capsule polysaccharides, peptidoglycans, pneumolysins, PspA Complement factor H binding component, autolysin, neuraminidase, peptide permeases, hydrogen peroxide, IgA1 protease, the list is certainly not complete. Moreover, very little is known concerning the temporal expression of such genes during infection and disease progression in a mammalian host. Discovering the sets of genes the bacterium is likely to be expressing at the different stages of infection, particularly when an infection is established, provides critical information for the screening and characterization of novel antibacterials which can interrupt pathogenesis. In addition to providing a fuller understanding of known proteins, such an approach will identify previously unrecognised targets.
The hydrolysis of peptide bonds is a central metabolic activity in bacterial cells.
E. coli
contains at least 40 enzymes that catalyze this reaction with representatives in every cellular compartment (Maurizi, M. R. (1992) Experientia, 48, 178-201; Gottesman, S. and Maurizi, M. R. (1992) Microbiol. Rev., 56, 592-621). These enzymes participate in a diverse array of processes. In growing cells, some substrates are degraded to rid the cell of damaged or nonfunctional proteins or peptides, whereas in other cases degradation is used to regulate the level of functional proteins. In starving cells, functional (although perhaps not functioning) proteins that are not normally degraded become susceptible to degradation. In both circumstances, proteases and peptidases play an indispensable role on the survival of the cell and therefore, inhibitors of these activities would prevent the bacterium from establishing and maintaining infection of the host 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
B. subtilis
aminopeptidase protein. See Winters et al., “The ampS-nprE (124-127 degrees) region of the
Bacillus subtilis
168 chromosome: Sequencing of a 27 kb segment and identification of several genes in the area”, Microbiology 142, 3033-3037 (1996); Genbank, Accession U51911. Also see Motoshima et al., “Molecular cloning and nucleotide sequence of the aminopeptidase T gene of Thermus aquaticus YT-1 and its high-level expression in
Escherichia coli
”, Agric. Biol. Chem., 54(9):2385-2392 (1990).
SUMMARY OF THE INVENTION
It is an object of the invention to provide polypeptides that have been identified as novel ampS 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 proteins such as
B. subtilis
aminopeptidase protein.
It is a further object of the invention to provide polynucleotides that encode ampS polypeptides, particularly polynucleotides that encode the polypeptide herein designated ampS.
In a particularly preferred embodiment of the invention the polynucleotide comprises a region encoding ampS 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 ampS protein from
Streptococcus pneumoniae
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
Streptococcus pneumoniae
0100993 strain contained in the deposited strain.
A further aspect of the invention there are provided isolated nucleic acid molecules encoding ampS, particularly
Streptococcus pneumoniae
ampS, 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 ampS and polypeptides encoded thereby.
Another aspect of the invention there are provided novel polypeptides of
Streptococcus pneumoniae
referred to herein as ampS 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 ampS polypeptide encoded by naturally occurring alleles of the ampS gene.
In a preferred embodiment of the invention there are provided methods for producing the aforementioned ampS 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 ampS expression, treating disease, for example, otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid, assaying genetic variation, and administering a ampS polypeptide or polynucleotide to an organism to raise an immunological response against a bacteria, especially a
Streptococcus pneumoniae
bacteria.
In accordance with certain preferred embodiments of this and other aspects of the invention there are provided polynucleotides that hybridize to amps polynucleotide sequences, p
Brown James Raymond
Zalacain Magdalena
Duffy Patricia A.
Fedon Jason C.
Gimmi Edward R.
Kinzig Charles M.
SmithKline Beecham Corporation
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