U.S. patent application number 09/917382 was filed with the patent office on 2003-01-23 for ndp.
Invention is credited to Biswas, SanJoy, Brown, James Raymond, Burnham, Martin Karl Russel, Chalker, Alison Francis, Holmes, David John, Ingraham, Karen Anne, Mathie, Thomas B., Warren, Richard Lloyd, Zalacain, Magdalena.
Application Number | 20030017532 09/917382 |
Document ID | / |
Family ID | 22284433 |
Filed Date | 2003-01-23 |
United States Patent
Application |
20030017532 |
Kind Code |
A1 |
Biswas, SanJoy ; et
al. |
January 23, 2003 |
ndp
Abstract
The invention provides ndp polypeptides and polynucleotides
encoding ndp polypeptides and methods for producing such
polypeptides by recombinant techniques. Also provided are methods
for utilizing ndp polypeptides to screen for antibacterial
compounds.
Inventors: |
Biswas, SanJoy; (Paoli,
PA) ; Brown, James Raymond; (Berwyn, PA) ;
Burnham, Martin Karl Russel; (Barto, PA) ; Chalker,
Alison Francis; (Trappe, PA) ; Holmes, David
John; (West Chester, PA) ; Ingraham, Karen Anne;
(Auburn, PA) ; Mathie, Thomas B.; (Eagleville,
PA) ; Warren, Richard Lloyd; (Blue Bell, PA) ;
Zalacain, Magdalena; (West Chester, PA) |
Correspondence
Address: |
Attn: Teresa O. Bittenbender, Esq.
DECHERT
4000 Bell Atlantic Tower
1717 Arch Street
Philadelphia
PA
19103-2793
US
|
Family ID: |
22284433 |
Appl. No.: |
09/917382 |
Filed: |
July 27, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09917382 |
Jul 27, 2001 |
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09285576 |
Apr 2, 1999 |
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6268177 |
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60101396 |
Sep 22, 1998 |
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Current U.S.
Class: |
435/69.1 ;
424/190.1; 435/183; 435/252.3; 435/320.1; 514/44R; 536/23.7 |
Current CPC
Class: |
A61P 31/04 20180101;
A61P 31/00 20180101; A61P 27/02 20180101; A61P 43/00 20180101; A61P
11/00 20180101; C12N 9/1077 20130101; A61P 27/16 20180101 |
Class at
Publication: |
435/69.1 ;
435/183; 435/252.3; 435/320.1; 536/23.7; 514/44; 424/190.1 |
International
Class: |
A61K 048/00; C12P
021/02; C12N 001/21; C07H 021/04; C12N 009/00; A61K 039/02; C12N
015/74 |
Claims
What is claimed is:
1. An isolated polypeptide selected from the group consisting of:
(i) an isolated polypeptide comprising an amino acid having at
least 95% identity to the amino acid sequence of SEQ ID NO:2 over
the entire length of SEQ ID NO:2; (ii) an isolated polypeptide
comprising the amino acid sequence of SEQ ID NO:2, (iii) an
isolated polypeptide that is the amino acid sequence of SEQ ID
NO:2, and (iv) a polypeptide that is encoded by a recombinant
polynucleotide comprising the polyncleotide sequence of SEQ ID
NO:1.
2. An isolated polynucleotide selected from the group consisting
of: (i) an isolated polynucleotide comprising a polynucleotide
sequence encoding a polypeptide that has at least 95% identity to
the amino acid sequence of SEQ ID NO:2, over the entire length of
SEQ ID NO:2; (ii) an isolated polynucleotide comprising a
polynucleotide sequence that has at least 95% identity over its
entire length to a polynucleotide sequence encoding the polypeptide
of SEQ ID NO:2; (iii) an isolated polynucleotide comprising a
nucleotide sequence that has at least 95% identity to that of SEQ
ID NO:1 over the entire length of SEQ ID NO:1; (iv) an isolated
polynucleotide comprising a nucleotide sequence encoding the
polypeptide of SEQ ID NO:2; (v) an isolated polynucleotide that is
the polynucleotide of SEQ ID NO: 1; (vi) an isolated polynucleotide
of at least 30 nucleotides in length obtainable by screening an
appropriate library under stringent hybridization conditions with a
probe having the sequence of SEQ ID NO: 1 or a fragment thereof of
of at least 30 nucleotides in length; (vii) an isolated
polynucleotide encoding a mature polypeptide expressed by the ndp
gene comprised in the Streptococcus pneumoniae; and (viii) a
polynucleotide sequence complementary to said isolated
polynucleotide of (i), (ii), (iii), (iv), (v), (vi) or (vii).
3. A method for the treatment of an individual: (i) in need of
enhanced activity or expression of or immunological response to the
polypeptide of claim 1 comprising the step of: administering to the
individual a therapeutically effective amount of an antagonist to
said polypeptide; or (ii) having need to inhibit activity or
expression of the polypeptide of claim 1 comprising: (a)
administering to the individual a therapeutically effective amount
of an antagonist to said polypeptide; or (b) administering to the
individual a nucleic acid molecule that inhibits the expression of
a polynucleotide sequence encoding said polypeptide; (c)
administering to the individual a therapeutically effective amount
of a polypeptide that competes with said polypeptide for its
ligand, substrate, or receptor; or (d) administering to the
individual an amount of a polypeptide that induces an immunological
response to said polypeptide in said individual.
4. A process for diagnosing or prognosing a disease or a
susceptibility to a disease in an individual related to expression
or activity of the polypeptide of claim 1 in an individual
comprising the step of: (a) determining the presence or absence of
a mutation in the nucleotide sequence encoding said polypeptide in
an organism in said individual; or (b) analyzing for the presence
or amount of said polypeptide expression in a sample derived from
said individual.
5. A process for producing a polypeptide selected from the group
consisting of: (i) an isolated polypeptide comprising an amino acid
sequence selected from the group having at least 95% identity to
the amino acid sequence of SEQ ID NO:2 over the entire length of
SEQ ID NO:2; (ii) an isolated polypeptide comprising the amino acid
sequence of SEQ ID NO:2; (iii) an isolated polypeptide that is the
amino acid sequence of SEQ ID NO:2, and (iv) a polypeptide that is
encoded by a recombinant polynucleotide comprising the
polynucleotide sequence of SEQ ID NO:1, comprising the step of
culturing a host cell under conditions sufficient for the
production of the polypeptide.
6. A process for producing a host cell comprising an expression
system or a membrane thereof expressing a polypeptide selected from
the group consisting of: (i) an isolated polypeptide comprising an
amino acid sequence selected from the group having at least 95%
identity to the amino acid sequence of SEQ ID NO:2 over the entire
length of SEQ ID NO:2; (ii) an isolated polypeptide comprising the
amino acid sequence of SEQ ID NO:2; (iii) an isolated polypeptide
that is the amino acid sequence of SEQ ID NO:2, and (iv) a
polypeptide that is encoded by a recombinant polynucleotide
comprising the polynucleotide sequence of SEQ ID NO:1, said process
comprising the step of transforming or transfecting a cell with an
expression system comprising a polynucleotide capable of producing
said polypeptide of (i), (ii), (iii) or (iv) when said expression
system is present in a compatible host cell such the host cell,
under appropriate culture conditions, produces said polypeptide of
(i), (ii), (iii) or (iv).
7. A host cell or a membrane expressing a polypeptide selected from
the group consisting of: (i) an isolated polypeptide comprising an
amino acid sequence selected from the group having at least 95%
identity to the amino acid sequence of SEQ ID NO:2 over the entire
length of SEQ ID NO:2; (ii) an isolated polypeptide comprising the
amino acid sequence of SEQ ID NO:2; (iii) an isolated polypeptide
that is the amino acid sequence of SEQ ID NO:2, and (iv) a
polypeptide that is encoded by a recombinant polynucleotide
comprising the polynucleotide sequence of SEQ ID NO:1.
8. An antibody immunospecific for the polypeptide of claim 1.
9. A method for screening to identify compounds that agonize or
that inhibit the function of the polypeptide of claim 1 that
comprises a method selected from the group consisting of: (a)
measuring the binding of a candidate compound to the polypeptide
(or to the cells or membranes bearing the polypeptide) or a fusion
protein thereof by means of a label directly or indirectly
associated with the candidate compound; (b) measuring the binding
of a candidate compound to the polypeptide (or to the cells or
membranes bearing the polypeptide) or a fusion protein thereof in
the presence of a labeled competitor; (c) testing whether the
candidate compound results in a signal generated by activation or
inhibition of the polypeptide, using detection systems appropriate
to the cells or cell membranes bearing the polypeptide; (d) mixing
a candidate compound with a solution comprising a polypeptide of
claim 1, to form a mixture, measuring activity of the polypeptide
in the mixture, and comparing the activity of the mixture to a
standard; or (e) detecting the effect of a candidate compound on
the production of mRNA encoding said polypeptide and said
polypeptide in cells, using for instance, an ELISA assay.
10. An agonist or antagonist to the polypeptide of claim 1.
Description
RELATED APPLICATIONS
[0001] This application claims benefit to US Provisional Patent
Application Number 60/101,396 filed Sept. 22, 1998.
FIELD OF THE INVENTION
[0002] 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
nucleotide pyrophosphorylase family, as well as their variants,
herein referred to as "ndp," "ndp polynucleotide(s)," and "ndp
polypeptide(s)" as the case may be.
BACKGROUND OF THE INVENTION
[0003] 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 reman It is particularly preferred to employ Streptococcal
genes and gene products as targets for the development of
antibiotics.
[0004] 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 that 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.
[0005] 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.
[0006] Clearly, there exists a need for polynucleotides and
polypeptides, such as the ndp embodiments of the invention, that
have a present benefit of, among other things, being useful to
screen compounds for antimicrobial 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.
SUMMARY OF THE INVENTION
[0007] The present invention relates to ndp, in particular ndp
polypeptides and ndp polynucleotides, recombinant materials and
methods for their production. In another aspect, the invention
relates to methods for using such polypeptides and polynucleotides,
including 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 agonist or
antagonist 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 ndp expression or
activity.
[0008] 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
[0009] The invention relates to ndp polypeptides and
polynucleotides as described in greater detail below. In
particular, the invention relates to polypeptides and
polynucleotides of a ndp of Streptococcus pneumoniae, that is
related by amino acid sequence homology to H. influenzae CDP
ribitol pyrophosphorylase polypeptide. The invention relates
especially to ndp having a nucleotide and amino acid sequences set
out in Table 1 as SEQ ID NO:1 and SEQ ID NO:2 respectively. Note
that sequences recited in the Sequence Listing below as "DNA"
represent an exemplification of the invention, since those of
ordinary skill will recognize that such sequences can be usefully
employed in polynucleotides in general, including
ribopolynucleotides.
1TABLE 1 ndp Polynucleotide and Polypeptide Sequences (A)
Streptococcus pneumoniae ndp polynucleotide sequence [SEQ ID NO:1].
5'-
ATGATTTATGCAGGAATTCTTGCCGGTGGAACTGGCACACGCATGGGGATCAGTAACTTGCCAAAACAATTT
TTAGAGCT AGGTGATCGACCTATTTTGATTCATACAATTG-
AAAAATTTGTCTTGGAGCCAAGTATTGAAAAAATTGTAGT TGGTGTTC
ATGGAGACTGGGTTTCTCATGCAGAAGATCTTGTAGATAAATATCTTCCTCTTTATAAGGAACGTA-
TCATCA TTACAAAG GGTGGTGCTGACCGCAATACAAGTAT-
TAAGAAAATCATTGAAGCCATTGATGCTTATCGTCCGCTTACTCCA GAGGATAT
CGTTGTTACCCACGATTCTGTTCGTCCATTTATTACACTTCGCATGATTCAGGACAATAT-
CCAACTTGCCCA AAATCATG
ACGCAGTGGACACAGTGGTAGAAGCGGTTGATACTATCGTTGAAAGTACCAATGGTCAATTTATTACAGATA
TTCCAAAT CGTGCTCACCTTTATCAAGGACAAACACCTCA-
AACATTCCGTTGCAAGGACTTCATGGACCTTTATGGATCT CTTTCTGA
TGAAGAGAAGGAAATCTTGACAGATGCATGTAAAATCTTTGTGATCAAAGGAAAAGATGTGGCCTT-
GGCCAA AGGTGAAT ACTCAAATCTGAAGATTACAACCGTA-
ACAGATTTGAAGATTGCAAAAAGTATGATTGAGAAAGACTAG-3' (B) Streptococcus
pneumoniae ndp polypeptide sequence deduced from a polynucleotide
sequence in this table [SEQ ID NO:2]. NH.sub.2-
MIYAGILAGGTGTRMGISNLPKQFLELGDRPILIHTIEKFVLEPSIE-
KIVVGVHGDWVSHAEDLVDKYLPLY KERIIITK
GGADRNTSIKKIIEAIDAYRPLTPEDIVVTHDSVRPFITLRMIQDNIQLAQNHDAVDTVVEAVDTIVESTNG
QFITDIPN RAHLYQGQTPQTFRCKDFMDLYGSLSDEEKEI-
LTDACKIFVIKGKDVALAKGEYSNLKITTVTDLKIAKSMI EKD*-COOH
[0010] Deposited Materials
[0011] A deposit comprising a Streptococcus pneumoniae 0100993
strain has been deposited with the National Collections of
Industrial and Marine Bacteria Ltd. (herein "NCIMB"), 23 St. Machar
Drive, Aberdeen AB2 1RY, Scotland on Apr. 11, 1996 and assigned
deposit number 40794. The deposit was described as Streptococcus
pneumoniae 0100993 on deposit.
[0012] On Apr. 17, 1996 a Streptococcus pneumoniae 0100993 DNA
library in E. coli was similarly deposited with the NCIMB and
assigned deposit number 40800. The Streptococcus pneumoniae strain
deposit is referred to herein as "the deposited stain" or as "the
DNA of the deposited strain."
[0013] The deposited strain comprises a full length ndp gene. The
sequence of the polynucleotides comprised in the deposited strain,
as well as the amino acid sequence of any polypeptide encoded
thereby, are controlling in the event of any conflict with any
description of sequences herein.
[0014] The deposit of the deposited strain has been made under the
terms of the Budapest Treaty on the International Recognition of
the Deposit of Micro-organisms for Purposes of Patent Procedure.
The deposited strain will be irrevocably and without restriction or
condition released to the public upon the issuance of a patent. The
deposited strain is provided merely as convenience to those of
skill in the art and is not an admission that a deposit is required
for enablement, such as that required under 35 U.S.C. .sctn.112. A
license may be required to make, use or sell the deposited strain,
and compounds derived therefrom, and no such license is hereby
granted.
[0015] In one aspect of the invention there is provided an isolated
nucleic acid molecule encoding a mature polypeptide expressible by
the Streptococcus pneumoniae 0100993 strain, which polypeptide is
comprised in the deposited strain. Further provided by the
invention are ndp polynucleotide sequences in the deposited strain,
such as DNA and RNA, and amino acid sequences encoded thereby. Also
provided by the invention are ndp polypeptide and polynucleotide
sequences isolated from the deposited strain.
[0016] Polypeptides
[0017] Ndp polypeptide of the invention is substantially
phylogenetically related to other proteins of the nucleotide
pyrophosphorylase family.
[0018] In one aspect of the invention there are provided
polypeptides of Streptococcus pneumoniae referred to herein as
"ndp" and "ndp polypeptides" as well as biologically,
diagnostically, prophylactically, clinically or therapeutically
useful variants thereof, and compositions comprising the same.
[0019] Among the particularly preferred embodiments of the
invention are variants of ndp polypeptide encoded by naturally
occurring alleles of a ndp gene.
[0020] The present invention further provides for an isolated
polypeptide that: (a) comprises or consists of an amino acid
sequence that has at least 95% identity, most preferably at least
97-99% or exact identity, to that of SEQ ID NO:2 over the entire
length of SEQ ID NO:2; (b) a polypeptide encoded by an isolated
polynucleotide comprising or consisting of a polynucleotide
sequence that has at least 95% identity, even more preferably at
least 97-99% or exact identity to SEQ ID NO:1 over the entire
length of SEQ ID NO: 1; (c) a polypeptide encoded by an isolated
polynucleotide comprising or consisting of a polynucleotide
sequence encoding a polypeptide that has at least 95% identity,
even more preferably at least 97-99% or exact identity, to the
amino acid sequence of SEQ ID NO:2, over the entire length of SEQ
ID NO:2.
[0021] The polypeptides of the invention include a polypeptide of
Table 1 [SEQ ID NO:2] (in particular a mature polypeptide) as well
as polypeptides and fragments, particularly those that has a
biological activity of ndp, and also those that have at least 95%
identity to a polypeptide of Table 1 [SEQ ID NO:2] and also include
portions of such polypeptides with such portion of the polypeptide
generally comprising at least 30 amino acids and more preferably at
least 50 amino acids.
[0022] The invention also includes a polypeptide consisting of or
comprising a polypeptide of the formula:
X--(R.sub.1).sub.m--(R.sub.2)--(R.sub.3).sub.n--Y
[0023] wherein, at the amino terminus, X is hydrogen, a metal or
any other moiety described herein for modified polypeptides, and at
the carboxyl terminus, Y is hydrogen, a metal or any other moiety
described herein for modified polypeptides, R.sub.1 and R.sub.3 are
any amino acid residue or modified amino acid residue, m is an
integer between 1 and 1000 or zero, n is an integer between 1 and
1000 or zero, and R.sub.2 is an amino acid sequence of the
invention, particularly an amino acid sequence selected from Table
1 or modified forms thereof. In the formula above, R.sub.2 is
oriented so that its amino terminal amino acid residue is at the
left, covalently bound to R.sub.1, and its carboxy terminal amino
acid residue is at the right, covalently bound to R.sub.3. Any
stretch of amino acid residues denoted by either R.sub.1 or
R.sub.3, where m and/or n is greater than 1, may be either a
heteropolymer or a homopolymer, preferably a heteropolymer. Other
preferred embodiments of the invention are provided where m is an
integer between 1 and 50, 100 or 500, and n is an integer between 1
and 50, 100, or 500.
[0024] It is most preferred that a polypeptide of the invention is
derived from Streptococcus pneumoniae, however, it may preferably
be obtained from other organisms of the same taxonomic genus. A
polypeptide of the invention may also be obtained, for example,
from organisms of the same taxonomic family or order.
[0025] A fragment is a variant polypeptide having an amino acid
sequence that is entirely the same as part but not all of any amino
acid sequence of any polypeptide of the invention. As with ndp
polypeptides, fragments may be "free-standing," or comprised within
a larger polypeptide of which they form a part or region, most
preferably as a single continuous region in a single larger
polypeptide.
[0026] Preferred fragments include, for example, truncation
polypeptides having a portion of an amino acid sequence of Table 1
[SEQ ID NO:2], or of variants thereof, such as a continuous series
of residues that includes an amino- and/or carboxyl-terminal amino
acid sequence. Degradation forms of the polypeptides of the
invention produced by or in a host cell, particularly a
Streptococcus pneumoniae, are also preferred. Further preferred are
fragments characterized by structural or functional attributes such
as fragments that comprise alpha-helix and alpha-helix forming
regions, beta-sheet and beta-sheet-forming regions, turn and
turn-forming regions, coil and coil-forming regions, hydrophilic
regions, hydrophobic regions, alpha amphipathic regions, beta
amphipathic regions, flexible regions, surface-forming regions,
substrate binding region, and high antigenic index regions.
[0027] Further preferred fragments include an isolated polypeptide
comprising an amino acid sequence having at least 15, 20, 30, 40,
50 or 100 contiguous amino acids from the amino acid sequence of
SEQ ID NO:2, or an isolated polypeptide comprising an amino acid
sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino
acids truncated or deleted from the amino acid sequence of SEQ ID
NO:2.
[0028] Fragments of the polypeptides of the invention may be
employed for producing the corresponding full-length polypeptide by
peptide synthesis; therefore, these variants may be employed as
intermediates for producing the full-length polypeptides of the
invention.
[0029] Polynucleotides
[0030] It is an object of the invention to provide polynucleotides
that encode ndp polypeptides, particularly polynucleotides that
encode a polypeptide herein designated ndp.
[0031] In a particularly preferred embodiment of the invention the
polynucleotide comprises a region encoding ndp polypeptides
comprising a sequence set out in Table 1 [SEQ ID NO:1] that
includes a full length gene, or a variant thereof. The Applicants
believe that this full length gene is essential to the growth
and/or survival of an organism that possesses it, such as
Streptococcus pneumoniae.
[0032] As a further aspect of the invention there are provided
isolated nucleic acid molecules encoding and/or expressing ndp
polypeptides and polynucleotides, particularly Streptococcus
pneumoniae ndp polypeptides and polynucleotides, including, for
example, unprocessed RNAs, ribozyme RNAs, mRNAs, cDNAs, genomic
DNAs, B- and Z-DNAs. Further embodiments of the invention include
biologically, diagnostically, prophylactically, clinically or
therapeutically useful polynucleotides and polypeptides, and
variants thereof, and compositions comprising the same.
[0033] Another aspect of the invention relates to isolated
polynucleotides, including at least one full length gene, that
encodes a ndp polypeptide having a deduced amino acid sequence of
Table 1 [SEQ ID NO:2] and polynucleotides closely related thereto
and variants thereof.
[0034] In another particularly preferred embodiment of the
invention there is a ndp polypeptide from Streptococcus pneumoniae
comprising or consisting of an amino acid sequence of Table 1 [SEQ
ID NO:2], or a variant thereof.
[0035] Using the information provided herein, such as a
polynucleotide sequence set out in Table 1 [SEQ ID NO:1], a
polynucleotide of the invention encoding ndp polypeptide may be
obtained using standard cloning and screening methods, such as
those for cloning and sequencing chromosomal DNA fragments from
bacteria using Streptococcus pneumoniae 0100993 cells as starting
material, followed by obtaining a full length clone. For example,
to obtain a polynucleotide sequence of the invention, such as a
polynucleotide sequence given in Table 1 [SEQ ID NO:1], typically a
library of clones of chromosomal DNA of Streptococcus pneumoniae
0100993 in E. coli or some other suitable host is probed with a
radiolabeled oligonucleotide, preferably a 17-mer or longer,
derived from a partial sequence. Clones carrying DNA identical to
that of the probe can then be distinguished using stringent
hybridization conditions. By sequencing the individual clones thus
identified by hybridization with sequencing primers designed from
the original polypeptide or polynucleotide sequence it is then
possible to extend the polynucleotide sequence in both directions
to determine a fall length gene sequence. Conveniently, such
sequencing is performed, for example, using denatured double
stranded DNA prepared from a plasmid clone. Suitable techniques are
described by Maniatis, T., Fritsch, E. F. and Sambrook et al.,
MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. (1989). (see in
particular Screening By Hybridization 1.90 and Sequencing Denatured
Double-Stranded DNA Templates 13.70). Direct genomic DNA sequencing
may also be performed to obtain a full length gene sequence.
Illustrative of the invention, each polynucleotide set out in Table
1 [SEQ ID NO:1] was discovered in a DNA library derived from
Streptococcus pneumoniae 0100993.
[0036] Moreover, each DNA sequence set out in Table 1 [SEQ ID NO:1]
contains an open reading frame encoding a protein having about the
number of amino acid residues set forth in Table 1 [SEQ ID NO:2]
with a deduced molecular weight that can be calculated using amino
acid residue molecular weight values well known to those skilled in
the art. The polynucleotide of SEQ ID NO:1, between nucleotide
number 1 and the stop codon that begins at nucleotide number 706 of
SEQ ID NO:1, encodes the polypeptide of SEQ ID NO:2.
[0037] In a further aspect, the present invention provides for an
isolated polynucleotide comprising or consisting of: (a) a
polynucleotide sequence that has at least 95% identity, even more
preferably at least 97%, still more preferably at least 99%, yet
still more preferably at least 99.5% or exact identity to SEQ ID
NO:1 over the entire length of SEQ ID NO:1, or the entire length of
that portion of SEQ ID NO:1 which encodes SEQ ID NO:2; (b) a
polynucleotide sequence encoding a polypeptide that has at least
95% identity, even more preferably at least 97-99% or 100% exact,
to the amino acid sequence of SEQ ID NO:2, over the entire length
of SEQ ID NO:2.
[0038] A polynucleotide encoding a polypeptide of the present
invention, including homologs and orthologs from species other than
Streptococcus pneumoniae, may be obtained by a process that
comprises the steps of screening an appropriate library under
stringent hybridization conditions with a labeled or detectable
probe consisting of or comprising the sequence of SEQ ID NO:1 or a
fragment thereof; and isolating a full-length gene and/or genomic
clones comprising said polynucleotide sequence.
[0039] The invention provides a polynucleotide sequence identical
over its entire length to a coding sequence (open reading frame) in
Table 1 [SEQ ID NO:1]. Also provided by the invention is a coding
sequence for a mature polypeptide or a fragment thereof, by itself
as well as a coding sequence for a mature polypeptide or a fragment
in reading frame with another coding sequence, such as a sequence
encoding a leader or secretory sequence, a pre-, or pro- or
prepro-protein sequence. The polynucleotide of the invention may
also comprise at least one non-coding sequence, including for
example, but not limited to at least one non-coding 5' and 3'
sequence, such as the transcribed but non-translated sequences,
termination signals (such as rho-dependent and rho-independent
termination signals), ribosome binding sites, Kozak sequences,
sequences that stabilize mRNA, introns, and polyadenylation
signals. The polynucleotide sequence may also comprise additional
coding sequence encoding additional amino acids. For example, a
marker sequence that facilitates purification of a fused
polypeptide can be encoded. In certain embodiments of the
invention, the marker sequence is a hexa-histidine peptide, as
provided in the pQE vector (Qiagen, Inc.) and described in Gentz et
al., Proc. Natl. Acad. Sci., USA 86: 821-824 (1989), or an HA
peptide tag (Wilson et al., Cell 37: 767 (1984), both of that may
be useful in purifying polypeptide sequence fused to them.
Polynucleotides of the invention also include, but are not limited
to, polynucleotides comprising a structural gene and its naturally
associated sequences that control gene expression.
[0040] A preferred embodiment of the invention is a polynucleotide
of consisting of or comprising nucleotide 1 to the nucleotide
immediately upstream of or including nucleotide 706 set forth in
SEQ ID NO:1 of Table 1, both of that encode a ndp polypeptide.
[0041] The invention also includes a polynucleotide consisting of
or comprising a polynucleotide of the formula:
X--(R.sub.1).sub.m--(R.sub.2)--(R.sub.3).sub.n--Y
[0042] wherein, at the 5' end of the molecule, X is hydrogen, a
metal or a modified nucleotide residue, or together with Y defines
a covalent bond, and at the 3' end of the molecule, Y is hydrogen,
a metal, or a modified nucleotide residue, or together with X
defines the covalent bond, each occurrence of R.sub.1 and R.sub.3
is independently any nucleic acid residue or modified nucleic acid
residue, m is an integer between 1 and 3000 or zero, n is an
integer between I and 3000 or zero, and R.sub.2 is a nucleic acid
sequence or modified nucleic acid sequence of the invention,
particularly a nucleic acid sequence selected from Table 1 or a
modified nucleic acid sequence thereof. In the polynucleotide
formula above, R.sub.2 is oriented so that its 5' end nucleic acid
residue is at the left, bound to R.sub.1, and its 3' end nucleic
acid residue is at the right, bound to R.sub.3. Any stretch of
nucleic acid residues denoted by either R.sub.1 and/or R.sub.2,
where m and/or n is greater than 1, may be either a heteropolymer
or a homopolymer, preferably a heteropolymer. Where, in a preferred
embodiment, X and Y together define a covalent bond, the
polynucleotide of the above formula is a closed, circular
polynucleotide, that can be a double-stranded polynucleotide
wherein the formula shows a first strand to which the second strand
is complementary. In another preferred embodiment m and/or n is an
integer between 1 and 1000. Other preferred embodiments of the
invention are provided where m is an integer between 1 and 50, 100
or 500, and n is an integer between 1 and 50, 100, or 500.
[0043] It is most preferred that a polynucleotide of the invention
is derived from Streptococcus pneumoniae, however, it may
preferably be obtained from other organisms of the same taxonomic
genus. A polynucleotide of the invention may also be obtained, for
example, from organisms of the same taxonomic family or order.
[0044] The term "polynucleotide encoding a polypeptide" as used
herein encompasses polynucleotides that include a sequence encoding
a polypeptide of the invention, particularly a bacterial
polypeptide and more particularly a polypeptide of the
Streptococcus pneumoniae ndp having an amino acid sequence set out
in Table 1 [SEQ ID NO:2]. The term also encompasses polynucleotides
that include a single continuous region or discontinuous regions
encoding the polypeptide (for example, polynucleotides interrupted
by integrated phage, an integrated insertion sequence, an
integrated vector sequence, an integrated transposon sequence, or
due to RNA editing or genomic DNA reorganization) together with
additional regions, that also may comprise coding and/or non-coding
sequences.
[0045] The invention further relates to variants of the
polynucleotides described herein that encode variants of a
polypeptide having a deduced amino acid sequence of Table 1 [SEQ ID
NO:2]. Fragments of polynucleotides of the invention may be used,
for example, to synthesize full-length polynucleotides of the
invention.
[0046] Further particularly preferred embodiments are
polynucleotides encoding ndp variants, that have the amino acid
sequence of ndp polypeptide of Table 1 [SEQ ID NO:2] in which
several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid
residues are substituted, modified, deleted and/or added, in any
combination. Especially preferred among these are silent
substitutions, additions and deletions, that do not alter the
properties and activities of ndp polypeptide.
[0047] Preferred isolated polynucleotide embodiments also include
polynucleotide fragments, such as a polynucleotide comprising a
nuclic acid sequence having at least 15, 20, 30, 40, 50 or 100
contiguous nucleic acids from the polynucleotide sequence of SEQ ID
NO: 1, or an polynucleotide comprising a nucleic acid sequence
having at least 15, 20, 30, 40, 50 or 100 contiguous nucleic acids
truncated or deleted from the 5' and/or 3' end of the
polynucleotide sequence of SEQ ID NO:1.
[0048] Further preferred embodiments of the invention are
polynucleotides that are at least 95% or 97% identical over their
entire length to a polynucleotide encoding ndp polypeptide having
an amino acid sequence set out in Table 1 [SEQ ID NO:2], and
polynucleotides that are complementary to such polynucleotides.
Most highly preferred are polynucleotides that comprise a region
that is at least 95% are especially preferred. Furthermore, those
with at least 97% are highly preferred among those with at least
95%, and among these those with at least 98% and at least 99% are
particularly highly preferred, with at least 99.5% being the more
preferred.
[0049] Preferred embodiments are polynucleotides encoding
polypeptides that retain substantially the same biological function
or activity as a mature polypeptide encoded by a DNA of Table 1
[SEQ ID NO:1].
[0050] In accordance with certain preferred embodiments of this
invention there are provided polynucleotides that hybridize,
particularly under stringent conditions, to ndp polynucleotide
sequences, such as those polynucleotides in Table 1.
[0051] The invention further relates to polynucleotides that
hybridize to the polynucleotide sequences provided herein. In this
regard, the invention especially relates to polynucleotides that
hybridize under stringent conditions to the polynucleotides
described herein. As herein used, the terms "stringent conditions"
and "stringent hybridization conditions" mean hybridization
occurring only if there is at least 95% and preferably at least 97%
identity between the sequences. A specific example of stringent
hybridization conditions is overnight incubation at 42.degree. C.
in a solution comprising: 50% formamide, 5.times.SSC (150 mM NaCl,
15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6),
5.times.Denhardt's solution, 10% dextran sulfate, and 20
micrograms/ml of denatured, sheared salmon sperm DNA, followed by
washing the hybridization support in 0.1.times.SSC at about
65.degree. C. Hybridization and wash conditions are well known and
exemplified in Sambrook, et al., Molecular Cloning: A Laboratory
Manual, Second Edition, Cold Spring Harbor, N.Y., (1989),
particularly Chapter 11 therein. Solution hybridization may also be
used with the polynucleotide sequences provided by the
invention.
[0052] The invention also provides a polynucleotide consisting of
or comprising a polynucleotide sequence obtained by screening an
appropriate library comprising a complete gene for a polynucleotide
sequence set forth in SEQ ID NO:1 under stringent hybridization
conditions with a probe having the sequence of said polynucleotide
sequence set forth in SEQ ID NO: 1 or a fragment thereof, and
isolating said polynucleotide sequence. Fragments useful for
obtaining such a polynucleotide include, for example, probes and
primers fully described elsewhere herein.
[0053] As discussed elsewhere herein regarding polynucleotide
assays of the invention, for instance, the polynucleotides of the
invention, may be used as a hybridization probe for RNA, cDNA and
genomic DNA to isolate full-length cDNAs and genonic clones
encoding ndp and to isolate cDNA and genomic clones of other genes
that have a high identity, particularly high sequence identity, to
a ndp gene. Such probes generally will comprise at least 15
nucleotide residues or base pairs. Preferably, such probes will
have at least 30 nucleotide residues or base pairs and may have at
least 50 nucleotide residues or base pairs. Particularly preferred
probes will have at least 20 nucleotide residues or base pairs and
will have lee than 30 nucleotide residues or base pairs.
[0054] A coding region of a ndp gene may be isolated by screening
using a DNA sequence provided in Table 1 [SEQ ID NO: 1] to
synthesize an oligonucleotide probe. A labeled oligonucleotide
having a sequence complementary to that of a gene of the invention
is then used to screen a library of cDNA, genomic DNA or mRNA to
determine which members of the library the probe hybridizes to.
[0055] There are several methods available and well known to those
skilled in the art to obtain full-length DNAs, or extend short
DNAs, for example those based on the method of Rapid Amplification
of cDNA ends (RACE) (see, for example, Frohman, et al., PNAS USA
85: 8998-9002, 1988). Recent modifications of the technique,
exemplified by the Marathon.TM. technology (Clontech Laboratories
Inc.) for example, have significantly simplified the search for
longer cDNAs. In the Marathon.TM. technology, cDNAs have been
prepared from mRNA extracted from a chosen tissue and an `adaptor`
sequence ligated onto each end. Nucleic acid amplification (PCR) is
then carried out to amplify the "missing" 5' end of the DNA using a
combination of gene specific and adaptor specific oligonucleotide
primers. The PCR reaction is then repeated using "nested" primers,
that is, primers designed to anneal within the amplified product
(typically an adaptor specific primer that anneals further 3' in
the adaptor sequence and a gene specific primer that anneals
further 5' in the selected gene sequence). The products of this
reaction can then be analyzed by DNA sequencing and a full-length
DNA constructed either by joining the product directly to the
existing DNA to give a complete sequence, or carrying out a
separate full-length PCR using the new sequence information for the
design of the 5' primer.
[0056] The polynucleotides and polypeptides of the invention may be
employed, for example, as research reagents and materials for
discovery of treatments of and diagnostics for diseases,
particularly human diseases, as further discussed herein relating
to polynucleotide assays.
[0057] The polynucleotides of the invention that are
oligonucleotides derived from a sequence of Table 1 [SEQ ID NOS:1
or 2] may be used in the processes herein as described, but
preferably for PCR, to determine whether or not the polynucleotides
identified herein in whole or in part are transcribed in bacteria
in infected tissue. It is recognized that such sequences will also
have utility in diagnosis of the stage of infection and type of
infection the pathogen has attained.
[0058] The invention also provides polynucleotides that encode a
polypeptide that is a mature protein plus additional amino or
carboxyl-terminal amino acids, or amino acids interior to a mature
polypeptide (when a mature form has more than one polypeptide
chain, for instance). Such sequences may play a role in processing
of a protein from precursor to a mature form, may allow protein
transport may lengthen or shorten protein half-life or may
facilitate manipulation of a protein for assay or production, among
other this. As generally is the case in vivo, the additional amino
acids may be processed away from a mature protein by cellular
enzymes.
[0059] For each and every polynucleotide of the invention there is
provided a polynucleotide complementary to it. It is preferred that
these complementary polynucleotides are fully complementary to each
polynucleotide with which they are complementary.
[0060] A precursor protein, having a mature form of the polypeptide
fused to one or more prosequences may be an inactive form of the
polypeptide. When prosequences are removed such inactive precursors
generally are activated. Some or all of the prosequences may be
removed before activation. Generally, such precursors are called
proproteins.
[0061] As will be recognized, the entire polypeptide encoded by an
open reading frame is often not required for activity. Accordingly,
it has become routine in molecular biology to map the boundaries of
the primary structure required for activity with N-terminal and
C-terminal deletion experiments. These experiments utilize
exonuclease digestion or convenient restriction sites to cleave
coding nucleic acid sequence. For example, Promega (Madison, Wis.)
sell an Erase-a-base.TM. system that uses Exonuclease III designed
to facilitate analysis of the deletion products (protocol available
at www.promega.com). The digested endpoints can be repaired (e.g.,
by ligation to synthetic linkers) to the extent necessary to
preserve an open reading frame. In this way, the nucleic acid of
SEQ ID NO:1 readily provides contiguous fragments of SEQ ID NO:2
sufficient to provide an activity, such as an enzymatic, binding or
antibody-inducing activity. Nucleic acid sequences encoding such
fragments of SEQ ID NO:2 and variants thereof as described herein
are within the invention, as are polypeptides so encoded.
[0062] In sum, a polynucleotide of the invention may encode a
mature protein, a mature protein plus a leader sequence (which may
be referred to as a preprotein), a precursor of a mature protein
having one or more prosequences that are not the leader sequences
of a preprotein, or a preproprotein, that is a precursor to a
proprotein, having a leader sequence and one or more prosequences,
that generally are removed during processing steps that produce
active and mature forms of the polypeptide.
[0063] Vectors, Host Cells, Expression Systems
[0064] The invention also relates to vectors that comprise a
polynucleotide or polynucleotides of the invention, host cells that
are genetically engineered with vectors of the invention and the
production of polypeptides of the invention by recombinant
techniques. Cell-free translation systems can also be employed to
produce such proteins using RNAs derived from the DNA constructs of
the invention.
[0065] Recombinant polypeptides of the present invention may be
prepared by processes well known in those skilled in the art from
genetically engineered host cells comprising expression systems.
Accordingly, in a further aspect, the present invention relates to
expression systems that comprise a polynucleotide or
polynucleotides of the present invention, to host cells that are
genetically engineered with such expression systems, and to the
production of polypeptides of the invention by recombinant
techniques.
[0066] For recombinant production of the polypeptides of the
invention, host cells can be genetically engineered to incorporate
expression systems or portions thereof or polynucleotides of the
invention. Introduction of a polynucleotide into the host cell can
be effected by methods described in many standard laboratory
manuals, such as Davis, et al., BASIC METHODS IN MOLECULAR BIOLOGY,
(1986) and Sambrook, et al., MOLECULAR CLONING: A LABORATORY
MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (1989), such as, calcium phosphate transfection,
DEAE-dextran mediated transfection, transvection, microinjection,
cationic lipid-mediated transfection, electroporation,
transduction, scrape loading, ballistic introduction and
infection.
[0067] Representative examples of appropriate hosts include
bacterial cells, such as cells of streptococci, staphylococci,
enterococci E. coli, streptomyces, cyanobacteria, Bacillus
subtilis, and Streptococcus pneumoniae; fungal cells, such as cells
of a yeast, Kluveromyces, Saccharomyces, a basidiomycete, Candida
albicans and Aspergillus; insect cells such as cells of Drosophila
S2 and Spodoptera Sf9; animal cells such as CHO, COS, HeLa, C127,
3T3, BHK, 293, CV-1 and Bowes melanoma cells; and plant cells, such
as cells of a gymnosperm or angiosperm.
[0068] A great variety of expression systems can be used to produce
the polypeptides of the invention. Such vectors include, among
others, chromosomal-, episomal- and virus-derived vectors, for
example, vectors derived from bacterial plasmids, from
bacteriophage, from transposons, from yeast episomes, from
insertion elements, from yeast chromosomal elements, from viruses
such as baculoviruses, papova viruses, such as SV40, vaccinia
viruses, adenoviruses, fowl pox viruses, pseudorabies viruses,
picornaviruses and retroviruses, and vectors derived from
combinations thereof, such as those derived from plasmid and
bacteriophage genetic elements, such as cosmids and phagenids. The
expression system constructs may comprise control regions that
regulate as well as engender expression. Generally, any system or
vector suitable to maintain, propagate or express polynucleotides
and/or to express a polypeptide in a host may be used for
expression in this regard. The appropriate DNA sequence may be
inserted into the expression system by any of a variety of
well-known and routine techniques, such as, for example, those set
forth in Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL,
(supra).
[0069] In recombinant expression systems in eukaryotes, for
secretion of a translated protein into the lumen of the endoplasmic
reticulum, into the periplasmic space or into the extracellular
environment, appropriate secretion signals may be incorporated into
the expressed polypeptide. These signals may be endogenous to the
polypeptide or they may be heterologous signals.
[0070] Polypeptides of the invention can be recovered and purified
from recombinant cell cultures by well-known methods including
ammonium sulfate or ethanol precipitation, acid extraction, anion
or cation exchange chromatography, phosphocellulose chromatography,
hydrophobic interaction chromatography, affinity chromatography,
hydroxylapatite chromatography, and lectin chromatography. Most
preferably, high performance liquid chromatography is employed for
purification. Well known techniques for refolding protein may be
employed to regenerate active conformation when the polypeptide is
denatured during isolation and or purification.
[0071] Diagnostic, Prognostic, Serotyping and Mutation Assays
[0072] This invention is also related to the use of ndp
polynucleotides and polypeptides of the invention for use as
diagnostic reagents. Detection of ndp polynucleotides and/or
polypeptides in a eukaryote, particularly a mamrmal, and especially
a human, will provide a diagnostic method for diagnosis of disease,
staging of disease or response of an infectious organism to drugs.
Eukaryotes, particularly mammals, and especially humans,
particularly those infected or suspected to be infected with an
organism comprising the ndp gene or protein, may be detected at the
nucleic acid or amino acid level by a variety of well known
techniques as well as by methods provided herein.
[0073] Polypeptides and polynucleotides for prognosis, diagnosis or
other analysis may be obtained from a putatively infected and/or
infected individuals bodily materials. Polynucleotides from any of
these sources, particularly DNA or RNA, may be used directly for
detection or may be amplified enzymatically by using PCR or any
other amplification technique prior to analysis. RNA, particularly
mRNA, cDNA and genomic DNA may also be used in the same ways. Using
amplification, characterization of the species and strain of
infectious or resident organism present in an individual, may be
made by an analysis of the genotype of a selected polynucleotide of
the organism. Deletions and insertions can be detected by a change
in size of the amplified product in comparison to a genotype of a
reference sequence selected from a related organism, preferably a
different species of the same genus or a different strain of the
same species. Point mutations can be identified by hybridizing
amplified DNA to labeled ndp polynucleotide sequences. Perfectly or
significantly matched sequences can be distinguished from
imperfectly or more significantly mismatched duplexes by DNase or
RNase digestion, for DNA or RNA respectively, or by detecting
differences in melting temperatures or renaturation kinetics.
Polynucleotide sequence differences may also be detected by
alterations in the electrophoretic mobility of polynucleotide
fragments in gels as compared to a reference sequence. This may be
carried out with or without denaturing agents. Polynucleotide
differences may also be detected by direct DNA or RNA sequencing.
See, for example, Myers et al., Science, 230: 1242 (1985). Sequence
changes at specific locations also may be revealed by nuclease
protection assays, such as RNase, V1 and S1 protection assay or a
chemical cleavage method. See, for example, Cotton et al., Proc.
Natl. Acad. Sci., USA, 85: 4397-4401 (1985).
[0074] In another embodiment, an array of oligonucleotides probes
comprising ndp nucleotide sequence or fragments thereof can be
constructed to conduct efficient screening of, for example, genetic
mutations, serotype, taxonomic classification or identification.
Array technology methods are well known and have general
applicability and can be used to address a variety of questions in
molecular genetics including gene expression, genetic linkage, and
genetic variability (see, for example, Chee et al., Science, 274:
610 (1996)).
[0075] Thus in another aspect, the present invention relates to a
diagnostic kit that comprises: (a) a polynucleotide of the present
invention, preferably the nucleotide sequence of SEQ ID NO: 1, or a
fragment thereof, (b) a nucleotide sequence complementary to that
of (a); (c) a polypeptide of the present invention, preferably the
polypeptide of SEQ ID NO:2 or a fragment thereof, or (d) an
antibody to a polypeptide of the present invention, preferably to
the polypeptide of SEQ ID NO:2. It will be appreciated that in any
such kit, (a), (b), (c) or (d) may comprise a substantial
component. Such a kit will be of use in diagnosing a disease or
susceptibility to a Disease, among others.
[0076] This invention also relates to the use of polynucleotides of
the present invention as diagnostic reagents. Detection of a
mutated form of a polynucleotide of the invention, preferable, SEQ
ID NO: 1, that is associated with a disease or pathogenicity will
provide a diagnostic tool that can add to, or define, a diagnosis
of a disease, a prognosis of a course of disease, a determination
of a stage of disease, or a susceptibility to a disease, that
results from under-expression, over-expression or altered
expression of the polynucleotide. Organisms, particularly
infectious organisms, carrying mutations in such polynucleotide may
be detected at the polynucleotide level by a variety of techniques,
such as those described elsewhere herein.
[0077] The differences in a polynucleotide and/or polypeptide
sequence between organisms possessing a first phenotype and
organisms possessing a different, second different phenotype can
also be determined. If a mutation is observed in some or all
organisms possessing the first phenotype but not in any organisms
possessing the second phenotype, then the mutation is likely to be
the causative agent of the first phenotype.
[0078] Cells from an organism carrying mutations or polymorphisms
(allelic variations) in a polynucleotide and/or polypeptide of the
invention may also be detected at the polynucleotide or polypeptide
level by a variety of techniques, to allow for serotyping, for
example. For example, RT-PCR can be used to detect mutations in the
RNA. It is particularly preferred to use RT-PCR in conjunction with
automated detection systems, such as, for example, GeneScan. RNA,
cDNA or genomic DNA may also be used for the same purpose, PCP As
an example, PCR primers complementary to a polynucleotide encoding
ndp polypeptide can be used to identify and analyze mutations. The
invention further provides these primers with 1, 2, 3 or 4
nucleotides removed from the 5' and/or the 3' end. These primers
may be used for, among other things, amplifing ndp DNA and/or RNA
isolated from a sample derived from an individual, such as a bodily
material. The primers may be used to amplify a polynucleotide
isolated from an infected individual, such that the polynucleotide
may then be subject to various techniques for elucidation of the
polynucleotide sequence. In this way, mutations in the
polynucleotide sequence may be detected and used to diagnose and/or
prognose the infection or its stage or course, or to serotype
and/or classify the infectious agent.
[0079] The invention further provides a process for diagnosing,
disease, preferably bacterial infections, more preferably
infections caused by Streptococcus pneumoniae, comprising
determining from a sample derived from an individual, such as a
bodily material, an increased level of expression of polynucleotide
having a sequence of Table 1 [SEQ ID NO:1]. Increased or decreased
expression of a ndp polynucleotide can be measured using any on of
the methods well known in the art for the quantitation of
polynucleotides, such as, for example, amplification, PCR, RT-PCR,
RNase protection, Northern blotting, spectrometry and other
hybridization methods.
[0080] In addition, a diagnostic assay in accordance with the
invention for detecting over-xpression of ndp polypeptide compared
to normal control tissue samples may be used to detect the presence
of an infection, for example. Assay techniques that can be used to
determine levels of a ndp polypeptide, in a sample derived from a
host, such as a bodily material, are well-known to those of skill
in the art. Such assay methods include radioirnrnunoassays,
competitive-binding assays, Western Blot analysis, antibody
sandwich assays, antibody detection and ELISA assays.
[0081] Antagonists and Agonists--Assays and Molecules
[0082] Polypeptides and polynucleotides of the invention may also
be used to assess the binding of small molecule substrates and
ligands in, for example, cells, cell-free preparations, chemical
libraries, and natural product mixtures. These substrates and
ligands may be natural substrates and ligands or may be structural
or functional mimetics. See, e.g., Coligan et al., Current
Protocols in Immunology 1(2): Chapter 5 (1991).
[0083] Polypeptides and polynucleotides of the present invention
are responsible for many biological functions, including many
disease states, in particular the Diseases herein mentioned. It is
therefore desirable to devise screening methods to identify
compounds that agonize (e.g., stimulate) or that antagonize (e.g,
inhibit) the function of the polypeptide or polynucleotide.
Accordingly, in a further aspect, the present invention provides
for a method of screening compounds to identify those that agonize
or that antagonize the function of a polypeptide or polynucleotide
of the invention, as well as related polypeptides and
polynucleotides. In general, agonists or antagonists (e.g.
inhibitors) may be employed for therapeutic and prophylactic
purposes for such Diseases as herein mentioned. Compounds may be
identified from a variety of sources, for example, cells, cell-free
preparations, chemical libraries, and natural product mixtures.
Such agonists and antagonists so-identified may be natural or
modified substrates, ligands, receptors, enzymes, etc., as the case
may be, of ndp polypeptides and polynucleotides; or may be
structural or functional mimetics thereof (see Coligan et al.,
Current Protocols in Immunology 1(2):Chapter 5 (1991)).
[0084] The screening methods may simply measure the binding of a
candidate compound to the polypeptide or polynucleotide, or to
cells or membranes bearing the polypeptide or polynucleotide, or a
fusion protein of the polypeptide by means of a label directly or
indirectly associated with the candidate compound. Alternatively,
the screening method may involve competition with a labeled
competitor. Further, these screening methods may test whether the
candidate compound results in a signal generated by activation or
inhibition of the polypeptide or polynucleotide, using detection
systems appropriate to the cells comprising the polypeptide or
polynucleotide. Inhibitors of activation are generally assayed in
the presence of a known agonist and the effect on activation by the
agonist by the presence of the candidate compound is observed.
Constitutively active polypeptide and/or constitutively expressed
polypeptides and polynucleotides may be employed in screening
methods for inverse agonists, in the absence of an agonist or
antagonist, by testing whether the candidate compound results in
inhibition of activation of the polypeptide or polynucleotide, as
the case may be. Further, the screening methods may simply comprise
the steps of mixing a candidate compound with a solution comprising
a polypeptide or polynucleotide of the present invention, to form a
mixture, measuring ndp polypeptide and/or polynucleotide activity
in the mixture, and comparing the ndp polypeptide and/or
polynucleotide activity of the mixture to a standard. Fusion
proteins, such as those made from Fc portion and ndp polypeptide,
as herein described, can also be used for high-throughput screening
assays to identify antagonists of the polypeptide of the present
invention, as well as of phylogenetically and and/or functionally
related polypeptides (see D. Bennett et al., J Mol Recognition,
8:52-58 (1995); and K. Johanson et al., J Biol Chem,
270(16):9459-9471 (1995)).
[0085] The polynucleotides, polypeptides and antibodies that bind
to and/or interact with a polypeptide of the present invention may
also be used to configure screening methods for detecting the
effect of added compounds on the production of mRNA and/or
polypeptide in cells. For example, an ELISA assay may be
constructed for measuring secreted or cell associated levels of
polypeptide using monoclonal and polyclonal antibodies by standard
methods known in the art. This can be used to discover agents that
may inhibit or enhance the production of polypeptide (also called
antagonist or agonist, respectively) from suitably manipulated
cells or tissues.
[0086] The invention also provides a method of screening compounds
to identify those that enhance (agonist) or block (antagonist) the
action of ndp polypeptides or polynucleotides, particularly those
compounds that are bacteristatic and/or bactericidal. The method of
screening may involve high-throughput techniques. For example, to
screen for agonists or antagonists, a synthetic reaction mix, a
cellular compartment, such as a membrane, cell envelope or cell
wall, or a preparation of any thereof, comprising ndp polypeptide
and a labeled substrate or ligand of such polypeptide is incubated
in the absence or the presence of a candidate molecule that may be
a ndp agonist or antagonist. The ability of the candidate molecule
to agonize or antagonize the ndp polypeptide is reflected in
decreased binding of the labeled ligand or decreased production of
product from such substrate. Molecules that bind gratuitously,
i.e., without inducing the effects of ndp polypeptide are most
likely to be good antagonists. Molecules that bind well and, as the
case may be, increase the rate of product production from
substrate, increase signal transduction, or increase chemical
channel activity are agonists. Detection of the rate or level of,
as the case may be, production of product from substrate, signal
transduction, or chemical channel activity may be enhanced by using
a reporter system. Reporter systems that may be useful in this
regard include but are not limited to colorimetric, labeled
substrate converted into product, a reporter gene that is
responsive to changes in ndp polynucleotide or polypeptide
activity, and binding assays known in the art.
[0087] Polypeptides of the invention may be used to identify
membrane bound or soluble receptors, if any, for such polypeptide,
through standard receptor binding techniques known in the art.
These techniques include, but are not limited to, ligand binding
and crosslinking assays in which the polypeptide is labeled with a
radioactive isotope (for instance, .sup.125I), chemically modified
(for instance, biotinylated), or fused to a peptide sequence
suitable for detection or purification, and incubated with a source
of the putative receptor (e.g, cells, cell membranes, cell
supernatants, tissue extracts, bodily materials). Other methods
include biophysical techniques such as surface plasmon resonance
and spectroscopy. These screening methods may also be used to
identify agonists and antagonists of the polypeptide that compete
with the binding of the polypeptide to its receptor(s), if any.
Standard methods for conducting such assays are well understood in
the art.
[0088] The fluorescence polarization value for a
fluorescently-tagged molecule depends on the rotational correlation
time or tumbling rate. Protein complexes, such as formed by ndp
polypeptide associating with another ndp polypeptide or other
polypeptide, labeled to comprise a fluorescently-labeled molecule
will have higher polarization values than a fluorescently labeled
monomeric protein. It is preferred that this method be used to
characterize small molecules that disrupt polypeptide
complexes.
[0089] Fluorescence energy transfer may also be used characterize
small molecules that interfere with the formation of ndp
polypeptide dimers, trimers, tetramers or higher order structures,
or structures formed by ndp polypeptide bound to another
polypeptide. Ndp polypeptide can be labeled with both a donor and
acceptor fluorophore. Upon mixing of the two labeled species and
excitation of the donor fluorophore, fluorescence energy transfer
can be detected by observing fluorescence of the acceptor.
Compounds that block dimerization will inhibit fluorescence energy
transfer.
[0090] Surface plasmon resonance can be used to monitor the effect
of small molecules on ndp polypeptide self-association as well as
an association of ndp polypeptide and another polypeptide or small
molecule. Ndp polypeptide can be coupled to a sensor chip at low
site density such that covalently bound molecules will be
monomeric. Solution protein can then passed over the ndp
polypeptide-coated surface and specific binding can be detected in
real-time by monitoring the change in resonance angle caused by a
change in local refractive index. This technique can be used to
characterize the effect of small molecules on kinetic rates and
equilibrium binding constants for ndp polypeptide self-association
as well as an association of ndp polypeptide and another
polypeptide or small molecule.
[0091] A scintillation proximity assay may be used to characterize
the interaction between an association of ndp polypeptide with
another ndp polypeptide or a different polypeptide. Ndp polypeptide
can be coupled to a scintillation-filled bead. Addition of
radio-labeled ndp polypeptide results in binding where the
radioactive source molecule is in close proximity to the
scintillation fluid. Thus, signal is emitted upon ndp polypeptide
binding and compounds that prevent ndp polypeptide self-association
or an association of ndp polypeptide and another polypeptide or
small molecule will diminish signal.
[0092] In other embodiments of the invention there are provided
methods for identifying compounds that bind to or otherwise
interact with and inhibit or activate an activity or expression of
a polypeptide and/or polynucleotide of the invention comprising:
contacting a polypeptide and/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
and/or polynucleotide to assess the binding to or other interaction
with the compound, such binding or interaction preferably being
associated with a second component capable of providing a
detectable signal in response to the binding or interaction of the
polypeptide and/or polynucleotide with the compound; and
determining whether the compound binds to or otherwise interacts
with and activates or inhibits an activity or expression of the
polypeptide and/or polynucleotide by detecting the presence or
absence of a signal generated from the binding or interaction of
the compound with the polypeptide and/or polynucleotide.
[0093] Another example of an assay for ndp agonists is a
competitive assay that combines ndp and a potential agonist with
ndp-binding molecules, recombinant ndp binding molecules, natural
substrates or ligands, or substrate or ligand mimetics, under
appropriate conditions for a competitive inhibition assay. Ndp can
be labeled, such as by radioactivity or a colorimetric compound,
such that the number of ndp molecules bound to a binding molecule
or converted to product can be determined accurately to assess the
effectiveness of the potential antagonist.
[0094] It will be readily appreciated by the skilled artisan that a
polypeptide and/or polynucleotide of the present invention may also
be used in a method for the structure-based design of an agonist or
antagonist of the polypeptide and/or polynucleotide, by: (a)
determining in the first instance the three-dimensional structure
of the polypeptide and/or polynucleotide, or complexes thereof, (b)
deducing the three-dimensional structure for the likely reactive
site(s), binding site(s) or motif(s) of an agonist or antagonist;
(c) synthesizing candidate compounds that are predicted to bind to
or react with the deduced binding site(s), reactive site(s), and/or
motif(s); and (d) testing whether the candidate compounds are
indeed agonists or antagonists.
[0095] It will be further appreciated that this will normally be an
iterative process, and this iterative process may be performed
using automated and computer-controlled steps.
[0096] In a further aspect, the present invention provides methods
of treating abnormal conditions such as, for instance, a Disease,
related to either an excess of, an under-expression of, an elevated
activity of, or a decreased activity of ndp polypeptide and/or
polynucleotide.
[0097] If the expression and/or activity of the polypeptide and/or
polynucleotide is in excess, several approaches are available. One
approach comprises administering to an individual in need thereof
an inhibitor compound (antagonist) as herein described, optionally
in combination with a pharmaceutically acceptable carrier, in an
amount effective to inhibit the function and/or expression of the
polypeptide and/or polynucleotide, such as, for example, by
blocking the binding of ligands, substrates, receptors, enzymes,
etc., or by inhibiting a second signal, and thereby alleviating the
abnormal condition. In another approach, soluble forms of the
polypeptides still capable of binding the ligand, substrate,
enzymes, receptors, etc. in competition with endogenous polypeptide
and/or polynucleotide may be administered. Typical examples of such
competitors include fragments of the ndp polypeptide and/or
polypeptide.
[0098] In still another approach, expression of the gene encoding
endogenous ndp polypeptide can be inhibited using expression
blocking techniques. This blocking may be targeted against any step
in gene expression, but is preferably targeted against
transcription and/or translation. An examples of a known technique
of this sort involve the use of antisense sequences, either
internally generated or separately administered (see, for example,
O'Connor, J Neurochem (1991) 56:560 in Oligodeoxynucleotides as
Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton,
Fla. (1988)). Alternatively, oligonucleotides that form triple
helices with the gene can be supplied (see, for example, Lee et
al., Nucleic Acids Res (1979) 6:3073; Cooney et al., Science (1988)
241:456; Dervanetal., Science (1991)251:1360). These oligomers can
be administered per se or the relevant oligomers can be expressed
in vivo.
[0099] Each of the polynucleotide sequences provided herein may be
used in the discovery and development of antibacterial compounds.
The encoded protein, upon expression, can be used as a target for
the screening of antibacterial drugs. Additionally, the
polynucleotide sequences encoding the amino terminal regions of the
encoded protein or Shine-Delgarno or other translation facilitating
sequences of the respective mRNA can be used to construct antisense
sequences to control the expression of the coding sequence of
interest.
[0100] The invention also provides the use of the polypeptide,
polynucleotide, agonist or antagonist of the invention to interfere
with the initial physical interaction between a pathogen or
pathogens and a eukaryotic, preferably mammalian, host responsible
for sequelae of infection. In particular, the molecules of the
invention may be used: in the prevention of adhesion of bacteria,
in particular gram positive and/or gram negative bacteria, to
eukaryotic, preferably mammalian, extracellular matrix proteins on
in-dwelling devices or to extracellular matrix proteins in wounds;
to block bacterial adhesion between eukaryotic, preferably
mammalian, extracellular matrix proteins and bacterial ndp proteins
that mediate tissue damage and/or; to block the normal progression
of pathogenesis in infections initiated other than by the
implantation of in-dwelling devices or by other surgical
techniques.
[0101] In accordance with yet another aspect of the invention,
there are provided ndp agonists and antagonists, preferably
bacteristatic or bactericidal agonists and antagonists.
[0102] The antagonists and agonists of the invention may be
employed, for instance, to prevent, inhibit and/or treat
diseases.
[0103] Helicobacter pylori (herein "H. pylori") bacteria infect the
stomachs of over one-third of the world's population causing
stomach cancer, ulcers, and gastritis (International Agency for
Research on Cancer (1994) Schistosomes, Liver Flukes and
Helicobacter Pylori (International Agency for Research on Cancer,
Lyon, France, http://www.uicc.ch/ecp/ecp2904.htrn). Moreover, the
International Agency for Research on Cancer recently recognized a
cause-and-effect relationship between H. pylori and gastric
adenocarcinoma, classifying the bacterium as a Group I (definite)
carcinogen. Preferred antimicrobial compounds of the invention
(agonists and antagonists of ndp polypeptides and/or
polynucleotides) found using screens provided by the invention, or
known in the art, particularly narrow-spectrum antibiotics, should
be useful in the treatment of H. pylori infection. Such treatment
should decrease the advent of H. pylori-induced cancers, such as
gastrointestinal carcinoma. Such treatment should also prevent,
inhibit and/or cure gastric ulcers and gastritis.
[0104] All publications and references, including but not limited
to patents and patent applications, cited in this specification are
herein incorporated by reference in their entirety as if each
individual publication or reference were specifically and
individually indicated to be incorporated by reference herein as
being fully set forth. Any patent application to which this
application claims priority is also incorporated by reference
herein in its entirety in the manner described above for
publications and references.
GLOSSARY
[0105] The following definitions are provided to facilitate
understanding of certain terms used frequently herein.
[0106] "Bodily material(s) means any material derived from an
individual or from an organism infecting, infesting or inhabiting
an individual, including but not limited to, cells, tissues and
waste, such as, bone, blood, serum, cerebrospinal fluid, semen,
saliva, muscle, cartilage, organ tissue, skin, urine, stool or
autopsy materials.
[0107] "Disease(s)" means any disease caused by or related to
infection by a bacteria, 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.
[0108] "Host cell(s)" is a cell that has been introduced (e.g.,
transformed or transfected) or is capable of introduction (e.g.,
transformation or transfection) by an exogenous polynucleotide
sequence.
[0109] "Identity," as known in the art, is a relationship between
two or more polypeptide sequences or two or more polynucleotide
sequences, as the case may be, as determined by comparing the
sequences. In the art, "identity" also means the degree of sequence
relatedness between polypeptide or polynucleotide sequences, as the
case may be, as determined by the match between strings of such
sequences. "Identity" can be readily calculated by known methods,
including but not limited to those described in (Computational
Molecular Biology, Lesk, A. M., ed., Oxford University Press, New
York, 1988; Biocomputing: Informatics and Genome Projects, Smith,
D. W., ed., Academic Press, New York, 1993; Computer Analysis of
sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds.,
Humana Press, New Jersey, 1994; Sequence Analysis in Molecular
Biology, von Heinje, G., Academic Press, 1987; and Sequence
Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton
Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J.
Applied Math., 48: 1073 (1988). Methods to determine identity are
designed to give the largest match between the sequences tested.
Moreover, methods to determine identity are codified in publicly
available computer programs. Computer program methods to determine
identity between two sequences include, but are not limited to, the
GCG program package (Devereux, J., et al., Nucleic Acids Research
12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S. F. et
al., J. Molec. Biol. 215: 403-410 (1990). The BLAST X program is
publicly available from NCBI and other sources (BLAST Manual,
Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul,
S., et al., J. Mol. Biol. 215: 403-410 (1990). The well known Smith
Waterman algorithm may also be used to determine identity.
[0110] Parameters for polypeptide sequence comparison include the
following: Algorithm: Needleman and Wunsch, J. Mol Biol. 48:
443-453 (1970)
[0111] Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff,
Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992)
[0112] Gap Penalty: 12
[0113] Gap Length Penalty: 4
[0114] A program useful with these parameters is publicly available
as the "gap" program from Genetics Computer Group, Madison Wis. The
aforementioned parameters are the default parameters for peptide
comparisons (along with no penalty for end gaps).
[0115] Parameters for polynucleotide comparison include the
following: Algorithm: Needleman and Wunsch, J. Mol Biol. 48:
443-453 (1970)
[0116] Comparison matrix: matches=+10, mismatch=0
[0117] Gap Penalty: 50
[0118] Gap Length Penalty: 3
[0119] Available as: The "gap" program from Genetics Computer
Group, Madison Wis. These are the default parameters for nucleic
acid comparisons.
[0120] A preferred meaning for "identity" for polynucleotides and
polypeptides, as the case may be, are provided in (1) and (2)
below.
[0121] (1) Polynucleotide embodiments further include an isolated
polynucleotide comprising a polynucleotide sequence having at least
a 95, 97, 99, 99.5 or 100% identity to the reference sequence of
SEQ ID NO:1, wherein said polynucleotide sequence may be identical
to the reference sequence of SEQ ID NO:1 or may include up to a
certain integer number of nucleotide alterations as compared to the
reference sequence, wherein said alterations are selected from the
group consisting of at least one nucleotide deletion, substitution,
including transition and transversion, or insertion, and wherein
said alterations may occur at the 5' or 3' terminal positions of
the reference nucleotide sequence or anywhere between those
terminal positions, interspersed either individually among the
nucleotides in the reference sequence or in one or more contiguous
groups within the reference sequence, and wherein said number of
nucleotide alterations is determined by multiplying the total
number of nucleotides in SEQ ID NO:1 by the integer defining the
percent identity divided by 100 and then subtracting that product
from said total number of nucleotides in SEQ ID NO:1, or:
n.sub.n<x.sub.n-(x.sub.n.multidot.y),
[0122] wherein n.sub.n is the number of nucleotide alterations,
x.sub.n is the total number of nucleotides in SEQ ID NO:1, y is
0.95 for 95%, 0.97 for 97%, 0.99 for 99%, 0.995 for 99.5% or 1.00
for 100%, and .multidot. is the symbol for tile multiplication
operator, and wherein any non-integer product of x.sub.n and y is
rounded down to the nearest integer prior to subtracting it from
x.sub.n. Alterations of a polynucleotide sequence encoding the
polypeptide of SEQ ID NO:2 may create nonsense, missense or
frameshift mutations in this coding sequence and thereby alter the
polypeptide encoded by the polynucleotide following such
alterations.
[0123] (2) Polypeptide embodiments further include an isolated
polypeptide comprising a polypeptide having at least a 95, 97 or
100% identity to a polypeptide reference sequence of SEQ ID NO:2,
wherein said polypeptide sequence may be identical to the reference
sequence of SEQ ID NO:2 or may include up to a certain integer
number of amino acid alterations as compared to the reference
sequence, wherein said alterations are selected from the group
consisting of at least one amino acid deletion, substitution,
including conservative and non-conservative substitution, or
insertion, and wherein said alterations may occur at the amino- or
carboxy-terminal positions of the reference polypeptide sequence or
anywhere between those terminal positions, interspersed either
individually among the amino acids in the reference sequence or in
one or more contiguous groups within the reference sequence, and
wherein said number of amino acid alterations is determined by
multiplying the total number of amino acids in SEQ ID NO:2 by the
integer defining the percent identity divided by 100 and then
subtracting that product from said total number of amino acids in
SEQ ID NO:2, or:
n.sub.a<x.sub.a-(x.sub.a.multidot.y),
[0124] wherein n.sub.a is the number of amino acid alterations,
x.sub.a is the total number of amino acids in SEQ ID NO:2, y is
0.95 for 95%, 0.97 for 97% or 1.00 for 100%, and .multidot. is the
symbol for the multiplication operator, and wherein any non-integer
product of x.sub.a and y is rounded down to the nearest integer
prior to subtracting it from x.sub.a.
[0125] "Individual(s)" means a multicellular eukalyote, including,
but not limited to a metazoan, a mammal, an ovid, a bovid, a
simian, a primate, and a human.
[0126] "Isolated" means altered "by the hand of man" from its
natural state, i.e., if it occurs in nature, it has been changed or
removed from its original environment, or both. For example, a
polynucleotide or a polypeptide naturally present in a living
organism is not "isolated," but the same polynucleotide or
polypeptide separated from the coexisting materials of its natural
state is "isolated", as the term is employed herein. Moreover, a
polynucleotide or polypeptide that is introduced into an organism
by transformation, genetic manipulation or by any other recombinant
method is "isolated" even if it is still present in said organism,
which organism may be living or non-living.
[0127] "Organism(s)" means a (i) prokaryote, including but not
limited to, a member of the genus Streptococcus, Staphylococcus,
Bordetella, Corynebacterium, Mycobacterium, Neisseria, Haemophilus,
Actinomycetes, Streptomycetes, Nocardia, Enterobacter, Yersinia,
Fancisella, Pasturella, Morarella, Acinetobacter, Erysipelothrix,
Branhamella, Actinobacillus, Streptobacillus, Listeria,
Calymmatobacterium, Brucella, Bacillus, Clostridium, Treponema,
Escherichia, Salmonella, Kleibsiella, Vibrio, Proteus, Erwinia,
Borrelia, Leptospira, Spirillum, Campylobacter, Shigella,
Legionella, Pseudomonas, Aeromonas, Rickettsia, Chlamydia, Borrelia
and Mycoplasma, and further including, but not limited to, a member
of the species or group, Group A Streptococcus, Group B
Streptococcus, Group C Streptococcus, Group D Streptococcus, Group
G Streptococcus, Streptococcus pneumoniae, Streptococcus pyogenes,
Streptococcus agalactiae, Streptococcus faecalis, Streptococcus
faecium, Streptococcus durans, Neisseria gonorrheae, Neisseria
meningitidis, Staphylococcus aureus, Staphylococcus epidermidis,
Corynebacterium diptheriae, Gardnerella vaginalis, Mycobacterium
tuberculosis, Mycobacterium bovis, Mycobacterium ulcerans,
Mycobacterium leprae, Actinomyctes israelii, Listeria
monocytogenes, Bordetella pertusis, Bordatella parapertusis,
Bordetella bronchiseptica, Escherichia coli, Shigella dysenteriae,
Haemophilus influenzae, Haemophilus aegyptius, Haemophilus
parainfluenzae, Haemophilus ducreyi, Bordetella, Salmonella typhi,
Citrobacter freundii, Proteus mirabilis, Proteus vulgaris, Yersinia
pestis, Kleibsiella pneumoniae, Serratia marcessens, Serratia
liquefaciens, Vibrio cholera, Shigella dysenterii, Shigella
flexneri, Pseudomonas aeruginosa, Franscisella tularensis, Brucella
abortis, Bacillus anthracis, Bacillus cereus, Clostridium
perfringens, Clostridium tetani, Clostridium botulinum, Treponema
pallidum, Rickettsia rickettsii and Chlamydia trachomitis, (ii) an
archaeon, including but not limited to Archaebacter, and (iii) a
unicellular or filamentous eukaryote, including but not limited to,
a protozoan, a fungus, a member of the genus Saccharomyces,
Kluveromyces, or Candida, and a member of the species Saccharomyces
ceriviseae, Kluveromyces lactis, or Candida albicans.
[0128] "Polynucleotide(s)" generally refers to any
polyribonucleotide or polydeoxyribonucleotide, that may be
unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide(s)"
include, without limitation, single- and double-stranded DNA, DNA
that is a mixture of single- and double-stranded regions or
single-, double- and triple-stranded regions, single- and
double-stranded RNA, and RNA that is mixture of single- and
double-stranded regions, hybrid molecules comprising DNA and RNA
that may be single-stranded or, more typically, double-standed, or
triple-stranded regions, or a mixture of single- and double-sranded
regions. In addition, "polynucleotide" as used herein refers to
triple-stranded regions comprising RNA or DNA or both RNA and DNA.
The strands in such regions may be from the same molecule or from
different molecules. The regions may include all of one or more of
the molecules, but more typically involve only a region of some of
the molecules. One of the molecules of a triple-helical region
often is an oligonucleotide. As used herein, the term
"polynucleotide(s)" also includes DNAs or RNAs as described above
that comprise one or more modified bases. Thus, DNAs or RNAs with
backbones modified for stability or for other reasons are
"polynucleotide(s)" as that term is intended herein. Moreover, DNAs
or RNAs comprising unusual bases, such as inosine, or modified
bases, such as tritylated bases, to name just two examples, are
polynucleotides as the term is used herein. It will be appreciated
that a great variety of modifications have been made to DNA and RNA
that serve many useful purposes known to those of skill in the art.
The term "polynucleotide(s)" as it is employed herein embraces such
chemically, emmmatically or metabolically modified forms of
polynucleotides, as well as the chemical forms of DNA and RNA
characteristic of viruses and cells, including, for example, simple
and complex cells. "Polynucleotide(s)" also embraces short
polynucleotides often referred to as oligonucleotide(s).
[0129] "Polypeptide(s)" refers to any peptide or protein comprising
two or more amino acids joined to each other by peptide bonds or
modified peptide bonds. "Polypeptide(s)" refers to both short
chains, commonly referred to as peptides, oligopeptides and
oligomers and to longer chains generally referred to as proteins.
Polypeptides may comprise amino acids other than the 20 gene
encoded amino acids. "Polypeptide(s)" include those modified either
by natural processes, such as processing and other
post-translational modifications, but also by chemical modification
techniques. Such modifications are well described in basic texts
and in more detailed monographs, as well as in a voluminous
research literature, and they are well known to those of skill in
the art. It will be appreciated that the same type of modification
may be present in the same or varying degree at several sites in a
given polypeptide. Also, a given polypeptide may comprise many
types of modifications. Modifications can occur anywhere in a
polypeptide, including the peptide backbone, the amino acid
side-chains, and the amino or carboxyl termim. Modifications
include, for example, acetylation, acylation, ADP-ribosylation,
amidation, covalent attachment of flavin, covalent attachment of a
heme moiety, covalent attachment of a nucleotide or nucleotide
derivative, covalent attachment of a lipid or lipid derivative,
covalent attachment of phosphotidylinositol, cross-inking,
cyclization, disulfide bond formation, demethylation, formation of
covalent cross-links, formation of cysteine, formation of
pyroglutamate, formylation, gamma-carboxylation, GPI anchor
formation, hydroxylation, iodination, methylation, myristoylation,
oxidation, proteolytic processing, phosphorylation, prenylation,
racemization, glycosylation, lipid attachment, sulfation,
gamma-carboxylation of glutamic acid residues, hydroxylation and
ADP-ribosylation, selenoylation, sulfation, transfer-RNA mediated
addition of amino acids to proteins, such as arginylation, and
ubiquitination. See, for instance, PROTEINS--STRUCTURE AND
MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and
Company, New York (1993) and Wold, F., Posttr lational Protein
Modifications: Perspectives and Prospects, pgs. 1-12 in
POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson,
Ed., Academic Press, New York (1983); Seifter et al., Meth.
Enzymol. 182:626-646 (1990) and Rattan et al., Protein Synthesis:
Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci.
663: 48-62 (1992). Polypeptides may be branched or cyclic, with or
without branching. Cyclic, branched and branched circular
polypeptides may result from post-translational natural processes
and may be made by entirely synthetic methods, as well.
[0130] "Recombinant expression system(s)" refers to expression
systems or portions thereof or polynucleotides of the invention
introduced or transformed into a host cell or host cell lysate for
the production of the polynucleotides and polypeptides of the
invention.
[0131] "Variant(s)" as the term is used herein, is a polynucleotide
or polypeptide that differs from a reference polynucleotide or
polypeptide respectively, but retains essential properties. A
typical variant of a polynucleotide differs in nucleotide sequence
from another, reference polynucleotide. Changes in the nucleotide
sequence of the variant may or may not alter the amino acid
sequence of a polypeptide encoded by the reference polynucleotide.
Nucleotide changes may result in amino acid substitutions,
additions, deletions, fusion proteins and truncations in the
polypeptide encoded by the reference sequence, as discussed below.
A typical variant of a polypeptide differs in amino acid sequence
from another, reference polypeptide. Generally, differences are
limited so that the sequences of the reference polypeptide and the
variant are closely similar overall and, in many regions,
identical. A variant and reference polypeptide may differ in amino
acid sequence by one or more substitutions, additions, deletions in
any combination. A substituted or inserted amino acid residue may
or may not be one encoded by the genetic code. The present
invention also includes include variants of each of the
polypeptides of the invention, that is polypeptides that vary from
the referents by conservative amino acid substitutions, whereby a
residue is substituted by another with like characteristics.
Typical such substitutions are among Ala, Val, Leu and Ile; among
Ser and Thr; among the acidic residues Asp and Glu; among Asn and
Ghn; and among the basic residues Lys and Arg; or aromatic residues
Phe and Tyr. Particularly preferred are variants in which several,
5-10, 1-5, 1-3, 1-2 or 1 amino acids are substituted, deleted, or
added in any combination. A variant of a polynucleotide or
polypeptide may be a naturally occurring such as an allelic
variant, or it may be a variant that is not known to occur
naturally. Non-naturally occurring variants of polynucleotides and
polypeptides may be made by mutagenesis techniques, by direct
synthesis, and by other recombinant methods known to skilled
artisans.
EXAMPLES
[0132] The examples below are carried out using standard
techniques, that are well known and routine to those of skill in
the art, except where otherwise described in detail. The examples
are illustrative, but do not limit the invention.
Example 1
Strain selection, Library Production and Sequencing
[0133] The polynucleotide having a DNA sequence given in Table 1
[SEQ ID NO:1] was obtained from a library of clones of chromosomal
DNA of Streptococcus pneumoniae in E. coli. The sequencing data
from two or more clones comprising overlapping Streptococcus
pneumoniae DNAs was used to construct the contiguous DNA sequence
in SEQ ID NO:1. Libraries may be prepared by routine methods, for
example: Methods 1 and 2 below.
[0134] Total cellular DNA is isolated from Streptococcus pneumoniae
0100993 according to standard procedures and size-fractionated by
either of two methods.
[0135] Method 1
[0136] Total cellular DNA is mechanically sheared by passage
through a needle in order to size-fractionate according to standard
procedures. DNA fragments of up to 11 kbp in size are rendered
blunt by treatment with exonuclease and DNA polymerase, and EcoRI
linkers added. Fragments are ligated into the vector Lambda ZapII
that has been cut with EcoRI, the library packaged by standard
procedures and E. coli infected with the packaged library. The
library is amplified by standard procedures.
[0137] Method 2
[0138] Total cellular DNA is partially hydrolyzed with a one or a
combination of restriction enzymes appropriate to generate a series
of fragments for cloning into library vectors (e.g., RsaI, PalI,
AluI, Bshl235I), and such fragments are size-fractionated according
to standard procedures. EcoRI linkers are ligated to the DNA and
the fragments then ligated into the vector Lambda ZapII that have
been cut with EcoRI, the library packaged by standard procedures,
and E. coli infected with the packaged library. The library is
amplified by standard procedures.
Example 2
ndp Characterization
[0139] The determination of expression during infection of a gene
from Streptococcus pneumoniae
[0140] Excised lungs from a 48 hour respiratory tract infection of
Streptococcus pneumoniae 0100993 in the mouse is efficiently
disrupted and processed in the presence of chaotropic agents and
RNAase inhibitor to provide a mixture of animal and bacterial RNA.
The optimal conditions for disruption and processing to give stable
preparations and high yields of bacterial RNA are followed by the
use of hybridisation to a radiolabelled oligonucleotide specific to
Streptococcus pneumoniae 16S RNA on Northern blots. The RNAase
free, DNAase free, DNA and protein free preparations of RNA
obtained are suitable for Reverse Transcription PCR (RT-PCR) using
unique primer pairs designed from the sequence of each gene of
Streptococcus pneumoniae 0100993. Using this procedure it was
possible to demonstrate that NDP is transcibed during
infection.
[0141] a) Isolation of tissue infected with Streptococcus
pneumoniae 0100993 from a mouse animal model of infection
(lungs)
[0142] Streptococcus pneumoniae 0100993 is grown either on TSA/5%
horse blood plates or in AGCH medium overnight, 37.degree. C., 5%
CO.sub.2. Bacteria are then collected and resuspended in
phosphate-buffered saline to an A.sub.600 of approximately 0.4.
Mice are anaesthetized with isofluorane and 50 ml of bacterial
suspension (approximately 2.times.10.sup.5 bacteria) is
administered intranasally using a pipetman. Mice are allowed to
recover and have food and water ad libitum. After 48 hours, the
mice are euthanized by carbon dioxide overdose, and lungs are
aseptically removed and snap-frozen in liquid nitrogen.
[0143] b) Isolation of Streptococcus pneumoniae 0100993 RNA from
infected tissue samples
[0144] Infected tissue samples, in 2-ml cryo-strorage tubes, are
removed from -80.degree. C. storage into a dry ice ethanol bath. In
a microbiological safety cabinet the samples are disrupted up to
eight at a time while the remaining samples are kept frozen in the
dry ice ethanol bath. To disrupt the bacteria within the tissue
sample, 50-100 mg of the tissue is transfered to a FastRNA tube
containing a silica/ceramic matrix (BIO101). Immediately, 1 ml of
extraction reagents (FastRNA reagents, BIO101) are added to give a
sample to reagent volume ratio of approximately 1 to 20. The tubes
are shaken in a reciprocating shaker (FastPrep FP120, BIO101) at
6000 rpm for 20-120 sec. The crude RNA preparation is extracted
with chloroform/isoamyl alcohol, and precipitated with
DEPC-treated/Isopropanol Precipitation Solution (BIO101). RNA
preparations are stored in this isopropanol solution at -80.degree.
C. if necessary. The RNA is pelleted (12,00 g for 10 min.), washed
with 75% ethanol (v/v in DEPC-treated water), air-dried for 5-10
min, and resuspended in 0.1 ml of DEPC-treated water, followed by
5-10 minutes at 55.degree. C. Finally, after at least 1 minute on
ice, 200 units of Rnasin (Promega) is added.
[0145] RNA preparations are stored at -80.degree. C. for up to one
month. For longer term storage the RNA precipitate can be stored at
the wash stage of the protocol in 75% ethanol for at least one year
at -20.degree. C.
[0146] Quality of the RNA isolated is assessed by running samples
on 1% agarose gels. 1.times.TBE gels stained with ethidium bromide
are used to visualise total RNA yields. To demonstrate the
isolation of bacterial RNA from the infected tissue 1.times.MOPS,
2.2M formaldehyde gels are run and vacuum blotted to Hybond-N
(Amersham). The blot is then hybridised with a .sup.32P-labelled
oligonucletide probe, of sequence 5'AACTGAGACTGGCTTTAAGAGATTA 3'
[SEQ ID NO: 3], specific to 16S rRNA of Streptococcus pneumoniae.
The size of the hybridising band is compared to that of control RNA
isolated from in vitro grown Streptococcus pneumoniae 0100993 in
the Northern blot. Correct sized bacterial 16S rRNA bands can be
detected in total RNA samples which show degradation of the
mammalian RNA when visualised on TBE gels.
[0147] c) The removal of DNA from Streptococcus pneumoniae-derived
RNA
[0148] DNA was removed from 50 microgram samples of RNA by a 30
minute treatment at 37.degree. C. with 20 units of RNAase-free
DNAaseI (GenHunter) in the buffer supplied in a final volume of 57
microliters.
[0149] The DNAase was inactivated and removed by treatment with
TRIzol LS Reagent (Gibco BRL, Life Technologies) according to the
manufacturers protocol. DNAase treated RNA was resuspended in 100
microlitres of DEPC treated water with the addition of Rnasin as
described before.
[0150] d) The preparation of cDNA from RNA samples derived from
infected tissue
[0151] 3 microgram samples of DNAase treated RNA are reverse
transcribed using a SuperScript Preamplification System for First
Strand cDNA Synthesis kit (Gibco BRL, Life Technologies) according
to the manufacturers instructions. 150 nanogram of random hexamers
is used to prime each reaction. Controls without the addition of
SuperScriptII reverse transcriptase are also run. Both +/-RT
samples are treated with RNaseH before proceeding to the PCR
reaction
[0152] e) The use of PCR to determine the presence of a bacterial
cDNA species
[0153] PCR reactions are set up on ice in 0.2 ml tubes by adding
the following components: 43 microlitres PCR Master Mix (Advanced
Biotechnologies Ltd.); 1 microlitre PCR primers (optimally 18-25
basepairs in length and designed to possess similar annealing
temperatures), each primer at 10 mM initial concentration; and 5
microlitres cDNA.
[0154] PCR reactions are run on a Perkin Elmer GeneAmp PCR System
9600 as follows: 2 minutes at 94.degree. C., then 50 cycles of 30
seconds each at 94.degree. C., 50.degree. C. and 72.degree. C.
followed by 7 minutes at 72 .degree. C. and then a hold temperature
of 20.degree. C. (the number of cycles is optimally 30-50 to
determine the appearance or lack of a PCR product and optimally
8-30 cycles if an estimation of the starting quantity of cDNA from
the RT reaction is to be made); 10 microlitre aliquots are then run
out on 1% 1.times.TBE gels stained with ethidium bromide, with PCR
product, if present, sizes estimated by comparison to a 100 bp DNA
Ladder (Gibco BRL, Life Technologies). Alternatively if the PCR
products are conveniently labelled by the use of a labelled PCR
primer (e.g. labelled at the 5' end with a dye) a suitable aliquot
of the PCR product is run out on a polyacrylamide sequencing gel
and its presence and quantity detected using a suitable gel
scanning system (e.g. ABI Prism.TM. 377 Sequencer using
GeneScan.TM. software as supplied by Perkin Elmer).
[0155] RT/PCR controls may include +/-reverse transcriptase
reactions, 16S rRNA primers or DNA specific primer pairs designed
to produce PCR products from non-transcribed Streptococcus
pneumoniae 0100993 genomic sequences.
[0156] To test the efficiency of the primer pairs they are used in
DNA PCR with Streptococcus pneumoniae 0100993 total DNA. PCR
reactions are set up and run as described above using approx. 1
microgram of DNA in place of the cDNA.
[0157] Primer pairs which fail to give the predicted sized product
in either DNA PCR or RTIPCR are PCR failures and as such are
uninformative. Of those which give the correct size product with
DNA PCR two classes are distinguished in RT/PCR: 1. Genes which are
not transcribed in vivo reproducibly fail to give a product in
RT/PCR; and 2.Genes which are transcribed in vivo reproducibly give
the correct size product in RT/PCR and show a stronger signal in
the +RT samples than the signal (if at all present) in -RT
controls
Example 3
Demonstration of gene essentiality to bacterial viability
[0158] An allelic replacement cassette was generated using PCR
technology. The cassette consisted of a pair of 500 bp chromosomal
DNA fragments flanking an erythromycin resistance gene. The
chromosomal DNA sequences are the 500 bp preceding and following
the DNA sequence encoding the NDP contained in Seq. ID NO. 1
[0159] The allelic replacement cassette was introduced into S.
pneumoniae R6 by transformation. Competent cells were prepared
according to published protocols. DNA was introduced into the cells
by incubation of ng quantities of allelic replacement cassette with
10.sup.6 cells at 30.degree. C. for 30 minutes. The cells were
transferred to 37.degree. C. for 90 minutes to allow expression of
the erythromycin resistance gene. Cells were plated in agar
containing lug erythromycin per ml. Following incubation at
37.degree. C. for 36 hours, colonies are picked and grown overnight
in Todd-Hewitt broth supplemented with 0.5% yeast extract.
Typically 1000 transformants containing the appropriate allelic
replacement are obtained. If no transformants are obtained in three
separate transformation experiments as was the case for this gene,
then the gene is considered as being essential in vitro.
Sequence CWU 1
1
3 1 708 DNA Streptococcus pneumoniae 1 atgatttatg caggaattct
tgccggtgga actggcacac gcatggggat cagtaacttg 60 ccaaaacaat
ttttagagct aggtgatcga cctattttga ttcatacaat tgaaaaattt 120
gtcttggagc caagtattga aaaaattgta gttggtgttc atggagactg ggtttctcat
180 gcagaagatc ttgtagataa atatcttcct ctttataagg aacgtatcat
cattacaaag 240 ggtggtgctg accgcaatac aagtattaag aaaatcattg
aagccattga tgcttatcgt 300 ccgcttactc cagaggatat cgttgttacc
cacgattctg ttcgtccatt tattacactt 360 cgcatgattc aggacaatat
ccaacttgcc caaaatcatg acgcagtgga cacagtggta 420 gaagcggttg
atactatcgt tgaaagtacc aatggtcaat ttattacaga tattccaaat 480
cgtgctcacc tttatcaagg acaaacacct caaacattcc gttgcaagga cttcatggac
540 ctttatggat ctctttctga tgaagagaag gaaatcttga cagatgcatg
taaaatcttt 600 gtgatcaaag gaaaagatgt ggccttggcc aaaggtgaat
actcaaatct gaagattaca 660 accgtaacag atttgaagat tgcaaaaagt
atgattgaga aagactag 708 2 235 PRT Streptococcus pneumoniae 2 Met
Ile Tyr Ala Gly Ile Leu Ala Gly Gly Thr Gly Thr Arg Met Gly 1 5 10
15 Ile Ser Asn Leu Pro Lys Gln Phe Leu Glu Leu Gly Asp Arg Pro Ile
20 25 30 Leu Ile His Thr Ile Glu Lys Phe Val Leu Glu Pro Ser Ile
Glu Lys 35 40 45 Ile Val Val Gly Val His Gly Asp Trp Val Ser His
Ala Glu Asp Leu 50 55 60 Val Asp Lys Tyr Leu Pro Leu Tyr Lys Glu
Arg Ile Ile Ile Thr Lys 65 70 75 80 Gly Gly Ala Asp Arg Asn Thr Ser
Ile Lys Lys Ile Ile Glu Ala Ile 85 90 95 Asp Ala Tyr Arg Pro Leu
Thr Pro Glu Asp Ile Val Val Thr His Asp 100 105 110 Ser Val Arg Pro
Phe Ile Thr Leu Arg Met Ile Gln Asp Asn Ile Gln 115 120 125 Leu Ala
Gln Asn His Asp Ala Val Asp Thr Val Val Glu Ala Val Asp 130 135 140
Thr Ile Val Glu Ser Thr Asn Gly Gln Phe Ile Thr Asp Ile Pro Asn 145
150 155 160 Arg Ala His Leu Tyr Gln Gly Gln Thr Pro Gln Thr Phe Arg
Cys Lys 165 170 175 Asp Phe Met Asp Leu Tyr Gly Ser Leu Ser Asp Glu
Glu Lys Glu Ile 180 185 190 Leu Thr Asp Ala Cys Lys Ile Phe Val Ile
Lys Gly Lys Asp Val Ala 195 200 205 Leu Ala Lys Gly Glu Tyr Ser Asn
Leu Lys Ile Thr Thr Val Thr Asp 210 215 220 Leu Lys Ile Ala Lys Ser
Met Ile Glu Lys Asp 225 230 235 3 25 DNA Streptococcus pneumoniae 3
aactgagact ggctttaaga gatta 25
* * * * *
References