Compounds

Details Compounds Compounds

1997-07-01

2001-05-08

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, C435S193000, C435S252300, C435S254110, C435S320100, C435S325000, C536S023200

Reexamination Certificate

active

06228612

ABSTRACT:

FIELD OF THE INVENTION
This invention relates, in part, to newly identified polynucleotides and polypeptides; variants and derivatives of these polynucleotides and polypeptides; processes for making these polynucleotides and these polypeptides, and their variants and derivatives; agonists and antagonists of the polypeptides; and uses of these polynucleotides, polypeptides, variants, derivatives, agonists and antagonists. In particular, in these and in other regards, the invention relates to polynucleotides and polypeptides of and homologous to UDP-N-acetylglucosamine enolpyruvyltransferase enzymes, hereinafter referred to as “Mur A-1”.
BACKGROUND OF THE INVENTION
The Streptococci make up a medically important genera of microbes known to cause several types of disease in humans, including otitis media, pneumonia and meningitis. 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.
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. Further 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 enzyme UDP-N-acetylglucosamine enolpyruvyltransferase, encoded by the gene MurA (MurZ), catalyses the first committed step of peptidoglyean biosynthesis. The gene has been cloned and sequenced from
Escherichia coli
and the corresponding enzyme (419 amino acids) has been over-expressed and purified (Marquardt, J. L., Siegele, D. A., Kolter, R. & Walsh, C. T. (1992) J. Bacteriol. 174, 5748-5752). The kinetic mechanism of this enzyme has been well characterized. Enolpyruvate is transferred from phosphoenolpyruvate (PEP) to UDPN-acetylglucosamine (UDPGlcNAc) with the release of inorganic phosphate. A cysteine residue (115) at the active site of the enzyme has been shown to be important in the enzyme mechanism and is the residue to which phosphomycin, an irreversible inhibitor of the enzyme, binds (Wanke, C. & Amrhein, N., (1993) Eur J. Biochem. 218 861-870; Marquardt, J. L., Brown, E. D., Lane, W. S., Haley, T. M., Ichikawa, Y., Wong, C-H., Walsh, C. T. (1994) Biochemistry 33 10646-10651.; Kim, D. H., Lees, W. J., Kempsell, K. E., Lane, W. S., Duncan, K. & Walsh, C. T. (1996) Biochemistry 35 4923-4928.) The MurA gene has been shown to be essential in
E.coli
(Brown, E. D., Vivas, E. I., Walsh, C. T. & Kolter, R. (1995) J. Bacteriol. 177, 4194-4197.) and has also been cloned from
Bacillus subtilis, Enterobacter cloacae
(Wanke, C. Falchetto, R. & amrhein, N. (1992) FEBS Lett 301, 271-276),
Mycobacterium tuberculosis
and
Acinetobacter calcoaceticus
(Ehrt, S. & Hillen, W. (1994) FEMS Microbiol. Lett, 117, 137142). The discovery of a MurA homologue in the human pathogen
Streptococcus pneumoniae
allows production of UDP-N-acetylglucosamine enolpyruvyl transferase 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 that may be used to screen compounds for antibiotic activity and which may also be used to determine their roles in pathogenesis of infection, dysfunction and disease. There is a need, therefore, for identification and characterization of such factors which can play a role in preventing, ameliorating or correcting infections, dysfunctions or diseases.
The polypeptide of the present invention has amino acid sequence homology to known UDP-N-acetylglucosamine enolpyruvyl transferase. The amino acid sequence of SEQ ID NO: 2 is the translated open reading frame sequence of SEQ ID NO:1 and displays homology (51% identity) to the UDP-N-acetylglucosamine enolpyruvyl transferase from
E. coli.
SUMMARY OF THE INVENTION
Toward these ends, and others, it is an object of the present invention to provide polypeptides, inter alia, that have been identified as novel Mur A-1 peptides by homology between the amino acid sequence set out in FIG.
2
and known amino acid sequences of other proteins such as
Escherichia coli.
It is a further object of the invention, moreover, to provide polynucleotides that encode Mur A-1 polypeptides, particularly polynucleotides that encode the polypeptide herein designated Mur A-1.
In a particularly preferred embodiment of this aspect of the invention the polynucleotide comprises the region encoding Mur A-1 polypeptides in the sequence set out in
FIG. 1
[SEQ ID NO:1], or a fragment, analogue or derivative thereof.
In another particularly preferred embodiment of the present invention there is a novel UDP-N-acetylglucosamine enolpyruvyl transferase protein from
Streptococcus pneumoniae
comprising the amino acid sequence of
FIG. 2
[SEQ ID NO:2], or a fragment, analogue or derivative thereof.
In accordance with this aspect of the present invention there is provided an isolated nucleic acid molecule encoding a mature polypeptide expressible by the
Streptococcus pneumoniae S. pneumoniae
010093 clone,
Streptococcus pneumoniae
0100993 contained in NCIMB Deposit No. 40794.
In accordance with this aspect of the invention there are provided isolated nucleic acid molecules encoding Mur A-1, particularly Streptococcus Mur A-1, including mRNAs, cDNAs, genomic DNAs and, in further embodiments of this aspect of the invention include biologically, diagnostically, prophylactically, clinically or therapeutically useful variants, analogs or derivatives thereof, or fragments thereof, including fragments of the variants, analogs and derivatives, and compositions comprising same.
In accordance with another aspect of the present 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 this aspect of the invention are naturally occurring allelic variants of Mur A-1 and polypeptides encoded thereby.
In accordance with this aspect of the invention there are provided novel polypeptides of Streptococcus referred to herein as Mur A-1 as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful fragments, variants and derivatives thereof, variants and derivatives of the fragments, and analogs of the foregoing, and compositions comprising same.
Among the particularly preferred embodiments of this aspect of the invention are variants of Mur A-1 polypeptide encoded by naturally occurring alleles of the Mur A-1 gene.
In a preferred embodiment of this aspect of the invention there are provided methods for producing the aforementioned Mur A-1 polypeptides.
In accordance with yet another aspect of the present invention, there are provided inhibitors to such polypeptides, useful as antibacterial agents, including, for example, antibodies.
In accordance with certain preferred embodiments of this aspect of the invention, there are provided products, compositions and methods, inter alia: assessing Mur A-1 expression; to treat otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusit

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