MurC from Streptococcus pneumoniae

Details MurC from Streptococcus pneumoniae MurC from Streptococcus pneumoniae

1997-09-30

2001-10-30

Minnifield, Anita (Department: 1645)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S023600, C536S023700, C435S069300, C435S320100, C435S325000, C435S362000, C435S365000, C435S367000, C435S252300, C435S252330

Reexamination Certificate

active

06310193

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 MurC family, hereinafter referred to as “MurC”.
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
Streptococcus 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.
The enzyme UDP-N-acetylmuramate:L-alanine ligase, encoded by the gene MurC catalyzes the addition of the first amino acid (L-alanine) of the peptide moiety in peptidoglycan biosynthesis. L-alanine is added to UDP-N-acetyl muramate with the concomittant hydrolysis of ATP to yield UDP-N-acetylmuramyl-L-alanine, ADP and phosphate. The gene has been cloned and sequenced from
Escherichia coli
and the corresponding protein has been over-expressed, purified and kinetically characterized (Liger, D., Masson, A., Blanot, D., van Heijenoort, J. & Parquet, C., Eur. J. Biochem. 230, 80-87). The kinetic mechanism of this enzyme has been investigated. (Falk, P. J., Ervin, K. M.,Volk, K. S. & Ho, H. T. (1996) Biochemistry, 35, 1417-1422). The gene sequences of MurC from
Bacillus subtilis
and
Haemophilus influenzae
are also known.
The discovery of a MurC homologue in the human pathogen
Streptococcus pneumoniae
will allow us to produce UDP-N-acetylmuramate:L-alanine ligase enzyme which can then be used to screen for novel antibiotics as described below. Inhibitors of this protein have utility in anti-bacterial therapy as they will prevent the construction of the bacterial cell wall.
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 MurC from
Bacillus subtilis
protein (Genembl database accession number AF008220).
SUMMARY OF THE INVENTION
It is an object of the invention to provide polypeptides that have been identified as novel MurC 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 MurC from
Bacillus subtilis
protein.
It is a further object of the invention to provide polynucleotides that encode MurC polypeptides, particularly polynucleotides that encode the polypeptide herein designated MurC.
In a particularly preferred embodiment of the invention, the polynucleotide comprises a region encoding MurC 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 MurC 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.
As a further aspect of the invention, there are provided isolated nucleic acid molecules encoding MurC, particularly
Streptococcus pneumoniae
MurC, 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 MurC and polypeptides encoded thereby.
As another aspect of the invention, there are provided novel polypeptides of
Streptococcus pneumoniae
referred to herein as MurC 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 MurC polypeptide encoded by naturally occurring alleles of the MurC gene.
In a preferred embodiment of the invention, there are provided methods for producing the aforementioned MurC 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 MurC 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 MurC 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 MurC polynucleotide sequences, particularly under stringent conditions.
In certain preferred embodiments of the invention, there are provided antibodies against MurC polypeptides.
In other embodiments of the invention, there are provided methods for identifying compounds which bind to or otherwise interact with and inhibit or activate an activity of a polypeptide or polynucleotide of the invention comprising: contacting a polypeptide or polynucleotide of the invention with a compound to be screened under conditions to permit binding to or other interaction between the compound and the polypeptide or polynucleotide to assess the binding to or other interaction with the compound, such binding or interaction being associated with a second component capable of providing a detectable signal in response to the binding or interaction of the polypeptide or polynucleotide with the compound; and determining whether the compound binds to or otherwise interacts with and activates or inhibits an activity of the polypeptide or polynucleotide by detecting the presence or absence of a signal generated from the b

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