Pth

Details Pth Pth

1998-05-18

2001-10-16

Martinell, James (Department: 1633)

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

Organic compounds

Carbohydrates or derivatives

C435S320100, C435S252300, C536S023100

Reexamination Certificate

active

06303771

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 polynucleotides and polypeptides of the peptidyl-tRNA hydrolases family, as well as their variants, hereinafter referred to as “pth,” “pth polynucleotide(s),” and “pth polypeptide(s)” as the case may be.
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 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 an unmet medical need and demand for new anti-microbial agents, vaccines, drug screening methods, and diagnostic tests for this organism.
Moreover, the drug discovery process is currently undergoing a fundamental revolution as it embraces “functional genomics,” that is, high throughput genome- or gene-based biology. This approach is rapidly superseding earlier approaches based on “positional cloning” and other methods. Functional genomics relies heavily on the various tools of bioinformatics to identify gene sequences of potential interest from the many molecular biology databases now available as well as from other sources. There is a continuing and significant need to identify and characterize further genes and other polynucleotides sequences and their related polypeptides, as targets for drug discovery.
Clearly, there exists a need for polynucleotides and polypeptides, such as the pth embodiments of the invention, that have a present benefit of, among other things, 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.
Peptidyl tRNA hydrolase plays a pivotal role in mRNA translation, regenerating tRNA from peptidyl tRNA intermediates. Pth activity is apparently ubiquitous and pth homologs have been identified in a wide range of bacteria. Analogous activity in mammals is mediated by a quite different set of enzymes. Inhibition of Pth activity in
E. coli
results in the inhibition of protein synthesis and ultimately cell death. We have shown that Pth is essential for in vitro growth in
S. pneumoniae
and
S. aureus
and that the gene encoding for this protein is expressed during infection of both pathogenic strains. Therefore, inhibitors of this protein could prevent the bacterium from establishing or maintaining infection of the host and thereby have utility in anti-bacterial therapy.
Certain of the polypeptides of the invention possess significant amino acid sequence homology to a known
B. subtilis
pth protein. (Dutka S, et al., Role of the 1-72 base pair in tRNAs for the activity of
Escherichia coli
peptidyl-tRNA hydrolase. Nucleic Acids Res Aug. 25, 1993; 21 (17):4025-4030; Heurgue-Hamard V, et al., The growth defect in
Escherichia coli
deficient in peptidyl-tRNA hydrolase is due to starvation for Lys-tRNA(Lys). EMBO J Jun. 3, 1996; 15 (11):2826-2833; Schmitt E, et al., Crystal structure at 1.2 A resolution and active site mapping of
Escherichia coli
peptidyl-tRNA hydrolase. EMBO J Aug. 1, 1997; 16 (15):4760-4769; Swiss-prot Accession number: P37470 Peptidyl tRNA hydrolase (pth)
B. subtilis;
Ogasawara, N., et al. Systematic sequencing of the 180 kilobase region of the
Bacillus subtilis
chromosome containing the replication origin; DNA Res. 1 (1), 1-14 (1994); TIGR contig 4125.)
SUMMARY OF THE INVENTION
The present invention relates to pth, in particular pth polypeptides and pth polynucleotides, recombinant materials and methods for their production. In another aspect, the invention relates to methods for using such polypeptides and polynucleotides, including the treatment of microbial diseases, amongst others. In a further aspect, the invention relates to methods for identifying agonists and antagonists using the materials provided by the invention, and for treating microbial infections and conditions associated with such infections with the identified compounds. In a still further aspect, the invention relates to diagnostic assays for detecting diseases associated with microbial infections and conditions associated with such infections, such as assays for detecting pth expression or activity.
Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following descriptions and from reading the other parts of the present disclosure.
DESCRIPTION OF THE INVENTION
The invention relates to pth polypeptides and polynucleotides as described in greater detail below. In particular, the invention relates to polypeptides and polynucleotides of a pth of
Streptococcus pneumoniae,
which is related by amino acid sequence homology to
B. subtilis
pth polypeptide. The invention relates especially to pth having the nucleotide and amino acid sequences set out in Table 1 as SEQ ID NO: 1 or 3 and SEQ ID NO: 2 or 4 respectively.
TABLE 1
pth Polynucleotide and Polypeptide Sequences
(A)
Streptococcus pneumoniae
pth polynucleotide sequence [SEQ ID NO:1].
5′-

ATGACCAAATTACTTGTAGGCTTGGGAAATCCAGGGGATAAATATTTTGAAACAAAACACAATGTTGGTTTT

ATGTTGATTGATCAACTAGCGAAGAAACAGAATGTCACTTTTACACACGATAAGATATTTCAAGCTGACCTA

GCATCCTTTTTCCTAAATGGAGAAAAAATTTATCTGGTTAAACCAACGACCTTTATGAATGAAAGTGGAAAA

GCAGTTCATGCTTTATTAACTTACTATGGTTTGGATATTGACGATTTACTTATCATTTACGATGATCTTGAC

ATGGAAGTTGGGAAAATTCGTTTAAGAGCAAAAGGCTCAGCAGGTGGTCATAATGGTATCAAGTCTATTATT

CAACATATAGGAACTCAGGTCTTTAACCGTGTTAAGATTGGAATTGGAAGACCTAAAAATGGTATGTCAGTT

GTTCATCATGTTTTGAGTAAGTTTGACAGGGATGATTATATCGGTATTTTACAGTCTGTTGACAAAGTTGAC

GATTCTGTAAACTACTATTTACAAGAGAAAAAATTTTGAGAAAACAATGCAGAGGTATAACGGATAA-3′

(B)
Streptococcus pneumoniae
pth polypeptide sequence deduced from a
polynucleotide sequence in this table [SEQ ID NO:2].
NH
2
-

MTKLLVGLGNPGDKYFETKHNVGFMLIDQLAKKQNVTFTHDKIFQADLASFFLNGEKIYLVKPTTFMNESGK

AVAALLTYYGLDIDDLLIIYDDLDMEVGKIRLRAKGSAGGHNGIKSIIQHIGTQVFNRVKIGIGRFKNGMSV

VHHVLSKFDRDDYIGILQSVDKVDDSVNYYLQEKNFEKTMQRYNG-COOH

(C)
Streptococcus pneumoniae
pth ORF sequence [SEQ ID NO:3].
5′-

ATGACCAAATTACTTGTAGGCTTGGGAAATCCAGGGGATAAATATTTTGAAACAAAACACAATGTTGGTTTT

ATGTTGATTGATCAACTAGCGAAGAAACAGAATGTCACTTTTACACACGATAAGATATTTCAAGCTGACCTA

GCATCCTTTTTCCTAAATGGAGAAAAAATTTATCTGGTTAAACCAACGACCTTTATGAATGAAAGTGGAAAA

GCAGTTCATGCTTTATTAACTTACTATGGTTTGGATATTGACGATTTACTTATCATTTACGATGATCTTGAC

ATGGAAGTTGGGAAAATTCGTTTAAGAGCAAAAGGCTCAGCAGGTGGTCATAATGGTATCAAGTCTATTATT

CAACATATAGGAACTCAGGTCTTTAACCGTGTTAAGATTGGAATTGGAAGACCTAAAAATGGTATGTCAGTT

GTTCATCArGTTTTGAGTAAGTTTGACAGGGATGATTATATCGGTATTTTACAGTCTATTGACAAAGTTGAC

GATTC

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