GlmU

Details GlmU GlmU

1999-05-10

2001-03-20

Low, Christopher S. F. (Department: 1653)

Chemistry: molecular biology and microbiology

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

Transferase other than ribonuclease

C435S252300, C435S320100, C435S023000, C435S006120, C435S348000, C435S325000, C536S023200

Reexamination Certificate

active

06204042

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 novel polynucleotides and polypeptides of the GlmU family, hereinafter referred to as “GlmU”.
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 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
Streptococcus pneumoniae
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.
N-Acetylglucosamine-1-Phosphate Uridyltransferase (GlmU) catalyses the formation of UDP-N-acetylglucosamine, an essential precursor for cell wall peptidoglycan in all bacteria and of lipopolysaccharide and enterobacterial common antigen in gram negatives. The enzyme has been purified from
Escherichia coli
and is bifunctional, also catalyzing the preceeding step of N-acetylation of glucosamine-1-phosphate (Mengin-Lecreulx, D. and van Heijenoort, J, J.Bacteriol. 176: 5788-5795 [1994]). It is possible to block the acetyltransferase activity but not the uridyl transferase activity with thiol inhibitors, suggesting that the enzyme may have two domains. The gene, glmU, encoding the enzyme has been cloned from
E. coli
(Mengin-Lecreulx, D. and van Heijenoort, J, J.Bacteriol. 175: 6150-6157 [1993]) and its counterpart in
Bacillus subtilis
(gcaD) has also been identified (Hove-Jensen B, J.Bacteriol. 174: 6852-6 [1992]).
The essential nature of the gene product of gcaD is demonstrated by temperature sensitive mutants of
Bacillus subtilis
which are unable to make active enzyme and stop growing at the restrictive temperature (Hove-Jensen [1992]). Inhibitors of these proteins therefore have utility in anti-bacterial therapy. The discovery of the gene from the human pathogen
Streptococcus pneumoniae
corresponding to gcaD/GlmU permits production of the enzyme which can be used to screen for novel antibiotics.
Clearly, there exists a need for factors, such as the GlmU 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 GlmU protein from
Bacillus subtilis.
See PIR database S66050; Genembl D26185; and Swissprot P14192. Also see NILSSON D., HOVE-JENSEN B., ARNVIG K. MOL. GEN. GENET. 218:565-571 (1989); OGASAWARA N., NAKAI S., YOSHIKAWA H. DNA RES. 1:1-14 (1994).
SUMMARY OF THE INVENTION
It is an object of the invention to provide polypeptides that have been identified as novel GlmU polypeptides by homology between the amino acid sequence set out in Table 1 [SEQ ID NO: 2 or 4] and a known amino acid sequence or sequences of other proteins such as GlmU from
Bacillus subtilis
protein.
It is a further object of the invention to provide polynucleotides that encode GlmU polypeptides, particularly polynucleotides that encode the polypeptide herein designated GlmU.
In a particularly preferred embodiment of the invention the polynucleotide comprises a region encoding GlmU polypeptides comprising a sequence set out in Table 1 [SEQ ID NO:1 or 3] which includes a full length gene, or a variant thereof.
In another particularly preferred embodiment of the invention there is a novel GlmU protein from
Streptococcus pneumoniae
comprising the amino acid sequence of Table 1 [SEQ ID NO:2 or 4], 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 GlmU, particularly
Streptococcus pneumoniae
GlmU, 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 GlmU and polypeptides encoded thereby.
Another aspect of the invention there are provided novel polypeptides of
Streptococcus pneumoniae
referred to herein as GlmU 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 GlmU polypeptide encoded by naturally occurring alleles of the GlmU gene.
In a preferred embodiment of the invention there are provided methods for producing the aforementioned GlmU 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 GlmU expression, treating disease, assaying genetic variation, and administering a GlmU 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 GlmU polynucleotide sequences, particularly under stringent conditions.
In certain preferred embodiments of the invention there are provided antibodies against GlmU 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 d

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