U.S. patent application number 09/770075 was filed with the patent office on 2001-11-08 for novel glucosaminidase.
Invention is credited to Black, Michael Terence, Burnham, Martin Karl Russel, Foster, Simon J., Hodgson, John Edward, Knowles, David Justin Charles, Nicholas, Richard O., Pratt, Julie M., Reichard, Raymond Winfield, Rosenberg, Martin, Ward, Judith M..
Application Number | 20010039038 09/770075 |
Document ID | / |
Family ID | 25487562 |
Filed Date | 2001-11-08 |
United States Patent
Application |
20010039038 |
Kind Code |
A1 |
Black, Michael Terence ; et
al. |
November 8, 2001 |
Novel glucosaminidase
Abstract
The invention provides glucosaminidase polypeptides and DNA
(RNA) encoding glucosaminidase polypeptides and methods for
producing such polypeptides by recombinant techniques. Also
provided are methods for utilizing glucosaminidase polypeptides to
screen for antibacterial compounds.
Inventors: |
Black, Michael Terence;
(Chester Springs, PA) ; Hodgson, John Edward;
(Malvern, PA) ; Knowles, David Justin Charles;
(Boroughbridge, GB) ; Reichard, Raymond Winfield;
(Quakertown, PA) ; Nicholas, Richard O.;
(Collegeville, PA) ; Burnham, Martin Karl Russel;
(Barto, PA) ; Pratt, Julie M.; (Verona, IT)
; Rosenberg, Martin; (Royersford, PA) ; Ward,
Judith M.; (Dorking, GB) ; Foster, Simon J.;
(Sheffield, GB) |
Correspondence
Address: |
DECHERT
ATTN: ALLEN BLOOM, ESQ
4000 BELL ATLANTIC TOWER
1717 ARCH STREET
PHILADELPHIA
PA
19103
US
|
Family ID: |
25487562 |
Appl. No.: |
09/770075 |
Filed: |
January 25, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09770075 |
Jan 25, 2001 |
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08948265 |
Oct 9, 1997 |
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6228619 |
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08948265 |
Oct 9, 1997 |
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08899088 |
Jul 23, 1997 |
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5953180 |
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08948265 |
Oct 9, 1997 |
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PCT/US97/02547 |
Feb 19, 1997 |
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08948265 |
Oct 9, 1997 |
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PCT/US97/02318 |
Feb 19, 1997 |
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60011888 |
Feb 20, 1996 |
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Current U.S.
Class: |
435/69.3 ;
424/185.1; 435/200; 435/320.1; 435/325; 536/23.2 |
Current CPC
Class: |
C12Y 302/01023 20130101;
A61P 9/00 20180101; C07K 16/1275 20130101; C12N 9/78 20130101; A61P
27/02 20180101; A61P 31/04 20180101; C12Y 305/04025 20130101; A61P
1/00 20180101; C12Y 302/01096 20130101; A61P 19/02 20180101; A61K
2039/53 20130101; A61K 39/00 20130101; A61P 27/00 20180101; C07K
14/31 20130101; A61P 1/04 20180101; A61P 19/00 20180101; A61P 11/00
20180101; A61P 25/00 20180101; A61P 31/00 20180101; C12N 9/2402
20130101; A61P 13/00 20180101; A61P 17/00 20180101 |
Class at
Publication: |
435/69.3 ;
435/200; 435/325; 435/320.1; 424/185.1; 536/23.2 |
International
Class: |
A61K 039/00; C07H
021/04; C12N 009/24; C12N 005/06; C12N 015/09; C12N 015/00; C12N
015/63; C12N 015/70; C12N 015/74; C12N 005/00 |
Claims
What is claimed is:
1. An isolated polynucleotide comprising a polynucleotide sequence
selected from the group consisting of: (a) a polynucleotide having
at least a 70% identity to a polynucleotide encoding a polypeptide
comprising the amino acid sequence of SEQ ID NO:2; (b) a
polynucleotide having at least a 70% identity to a polynucleotide
encoding the same mature polypeptide expressed by the
glucosaminidase gene contained in the Staphylococcus aureus of the
deposited strain; (c) a polynucleotide encoding a polypeptide
comprising an amino acid sequence which is at least 70% identical
to the amino acid sequence of SEQ ID NO:2; (d) a polynucleotide
which is complementary to the polynucleotide of (a), (b) or (c);
and (e) a polynucleotide comprising at least 15 sequential bases of
the polynucleotide of (a), (b), (c) or (d).
2. The polynucleotide of claim 1 wherein the polynucleotide is
DNA.
3. The polynucleotide of claim 1 wherein the polynucleotide is
RNA.
4. The polynucleotide of claim 2 comprising the nucleic acid
sequence set forth in SEQ ID NO:1.
5. The polynucleotide of claim 2 comprising nucleotide 1 to 786 set
forth in SEQ ID NO:1.
6. The polynucleotide of claim 2 which encodes a polypeptide
comprising the amino acid sequence of SEQ ID NO:2.
7. A vector comprising the polynucleotide of claim 1.
8. A host cell comprising the vector of claim 7.
9. A process for producing a polypeptide comprising: expressing
from the host cell of claim 8 a polypeptide encoded by said
DNA.
10. A process for producing a glucosaminidase polypeptide or
fragment comprising culturing a host of claim 8 under conditions
sufficient for the production of said polypeptide or fragment.
11. A polypeptide comprising an amino acid sequence which is at
least 70% identical to the amino acid sequence of SEQ I) NO:2.
12. A polypeptide comprising an amino acid sequence as set forth in
SEQ ID NO:2.
13. An antibody against the polypeptide of claim 11.
14. An antagonist which inhibits the activity or expression of the
polypeptide of claim 11.
15. A method for the treatment of an individual in need of
glucosaminidase polypeptide comprising: administering to the
individual a therapeutically effective amount of the polypeptide of
claim 11.
16. A method for the treatment of an individual having need to
inhibit glucosaminidase polypeptide comprising: administering to
the individual a therapeutically effective amount of the antagonist
of claim 14.
17. A process for diagnosing a disease related to expression or
activity of the polypeptide of claim 11 in an individual
comprising: (a) determining a nucleic acid sequence encoding said
polypeptide, and/or (b) analyzing for the presence or amount of
said polypeptide in a sample derived from the individual.
18. A method for identifying compounds which interact with and
inhibit or activate an activity of the polypeptide of claim 11
comprising: contacting a composition comprising the polypeptide
with the compound to be screened under conditions to permit
interaction between the compound and the polypeptide to assess the
interaction of a compound, such interaction being associated with a
second component capable of providing a detectable signal in
response to the interaction of the polypeptide with the compound;
and determining whether the compound interacts with and activates
or inhibits an activity of the polypeptide by detecting the
presence or absence of a signal generated from the interaction of
the compound with the polypeptide.
19. A method for inducing an immunological response in a mammal
which comprises inoculating the mammal with glucosaminidase
polypeptide of claim 11, or a fragment or variant thereof, adequate
to produce antibody and/or T cell immune response to protect said
animal from disease.
20. A method of inducing immunological response in a mammal which
comprises delivering a nucleic acid vector to direct expression of
glucosaminidase polypeptide of claim 11, or fragment or a variant
thereof, for expressing said glucosaminidase polypeptide, or a
fragment or a variant thereof in vivo in order to induce an
immunological response to produce antibody and/ or T cell immune
response to protect said animal from disease.
Description
RELATED APPLICATIONS
[0001] This application is a Continuation-in-Part of U.S. patent
application Ser. No. 08/899,088 filed, Jul. 23, 1997 which claimed
benefit of U.S. Provisional Patent Application No. 60/011,888,
filed Feb. 20, 1996, PCT Application, International Application
Number PCT/US97/02547, filed Feb. 19, 1997, and PCT Application,
International Application Number PCT/US97/02318, filed Feb. 19,
1997.
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,
in these and in other regards, the invention relates to novel
polynucleotides and polypeptides of the autolysin family,
hereinafter referred to as "glucosaminidase".
BACKGROUND OF THE INVENTION
[0003] It is particularly preferred to employ Staphylococcal genes
and gene products as targets for the development of antibiotics.
The Staphylococci make up a medically important genera of microbes.
They are known to produce two types of disease, invasive and
toxigenic. Invasive infections are characterized generally by
abscesss formation effecting both skin surfaces and deep tissues.
Staphylococcus aureus is the second leading cause of bacteremia in
cancer patients. Osteomyelitis, septic arthritis, septic
thrombophlebitis and acute bacterial endocarditis are also
relatively common. There are at least three clinical conditions
resulting from the toxigenic properties of Staphylococci. The
manifestation of these diseases result from the actions of
exotoxins as opposed to tissue invasion and bacteremia. These
conditions include: Staphylococcal food poisoning, scalded skin
syndrome and toxic shock syndrome.
[0004] The frequency of Staphylococcus aureus infections has risen
dramatically in the past 20 years. This has been attributed to the
emergence of multiply antibiotic resistant strains and an
increasing population of people with weakened immune systems. It is
no longer uncommon to isolate Staphylococcus aureus strains which
are resistant to some or all of the standard antibiotics. This has
created a demand for both new anti-microbial agents and diagnostic
tests for this organism.
[0005] Autolysin enzymes are able to disrupt the peptidoglycan
chains of bacterial cell walls. It is thought they are employed by
bacteria in a carefully regulated manner in essential cell
processes such as cell wall growth and septation. Inhibition of
their action or disruption of their controlled function represents
a probable antibacterial target.
[0006] Clearly, there is a need for factors, such as the novel
compounds of the invention, that have a present benefit of being
useful to screen compounds for antibiotic activity. Such factors
are also useful to determine their role in pathogenesis of
infection, dysfunction and disease. There is also a need for
identification and characterization of such factors and their
antagonists and agonists which can play a role in preventing,
ameliorating or correcting infections, dysfunctions or
diseases.
[0007] The polypeptides of the invention have amino acid sequence
homology to a known sp.vertline.P52081.vertline.ATL_STAAU AUTOLYSIN
PRECURSOR protein (autolysin AtlE; GENBANK: locus SEU71377,
accession U71377).
SUMMARY OF THE INVENTION
[0008] It is an object of the invention to provide polypeptides
that have been identified as novel glucosaminidase polypeptides by
homology between the amino acid sequence set out in Table 1 [SEQ ID
NO: 2] and a known amino acid sequence or sequences of other
proteins such as sp.vertline.P52081.vertline.ATL_STAAU AUTOLYSIN
PRECURSOR protein.
[0009] It is a further object of the invention to provide
polynucleotides that encode glucosaminidase polypeptides,
particularly polynucleotides that encode the polypeptide herein
designated glucosaminidase.
[0010] In a particularly preferred embodiment of the invention, the
polynucleotide comprises a region encoding glucosaminidase
polypeptides comprising the sequence set out in Table 1 [SEQ ID
NO:1] which includes a full length gene, or a variant thereof.
[0011] In another particularly preferred embodiment of the
invention, there is a novel glucosaminidase protein from
Staphylococcus aureus comprising the amino acid sequence of Table 1
[SEQ ID NO:2], or a variant thereof.
[0012] In accordance with another aspect of the invention, there is
provided an isolated nucleic acid molecule encoding a mature
polypeptide expressible by the Staphylococcus aureus WCUH 29 strain
contained in the deposited strain.
[0013] As a further aspect of the invention, there are provided
isolated nucleic acid molecules encoding glucosaminidase,
particularly Staphylococcus aureus glucosaminidase, including
mRNAs, cDNAs, genomic DNAs. Further embodiments of the invention
include biologically, diagnostically, prophylactically, clinically
or therapeutically useful variants thereof, and compositions
comprising the same.
[0014] In accordance with another aspect of the invention, there is
provided the use of a polynucleotide of the invention for
therapeutic or prophylactic purposes, in particular genetic
immunization. Among the particularly preferred embodiments of the
invention are naturally occurring allelic variants of
glucosaminidase and polypeptides encoded thereby.
[0015] As another aspect of the invention, there are provided novel
polypeptides of Staphylococcus aureus referred to herein as
glucosaminidase as well as biologically, diagnostically,
prophylactically, clinically or therapeutically useful variants
thereof, and compositions comprising the same.
[0016] Among the particularly preferred embodiments of the
invention are variants of glucosaminidase polypeptide encoded by
naturally occurring alleles of the glucosaminidase gene.
[0017] In a preferred embodiment of the invention, there are
provided methods for producing the aforementioned glucosaminidase
polypeptides.
[0018] In accordance with yet another aspect of the invention,
there are provided inhibitors to such polypeptides, useful as
antibacterial agents, including, for example, antibodies.
[0019] In accordance with certain preferred embodiments of the
invention, there are provided products, compositions and methods
for assessing glucosaminidase expression, treating disease, for
example, disease, such as, infections of the upper respiratory
tract (e.g., otitis media, bacterial tracheitis, acute
epiglottitis, thyroiditis), lower respiratory (e.g., empyema, lung
abscesss), cardiac (e.g., infective endocarditis), gastrointestinal
(e.g., secretory diarrhoea, splenic abscess, retroperitoneal
abscess), CNS (e.g., cerebral abscess), eye (e.g., blepharitis,
conjunctivitis, keratitis, endophthalmitis, preseptal and orbital
cellulitis, darcryocystitis), kidney and urinary tract (e.g.,
epididymitis, intrarenal and perinephric abscess, toxic shock
syndrome), skin (e.g., impetigo, folliculitis, cutaneous abscesses,
cellulitis, wound infection, bacterial myositis) bone and joint
(e.g., septic arthritis, osteomyelitis), assaying genetic
variation, and administering a glucosaminidase polypeptide or
polynucleotide to an organism to raise an immunological response
against a bacteria, especially a Staphylococcus aureus
bacteria.
[0020] In accordance with certain preferred embodiments of this and
other aspects of the invention, there are provided polynucleotides
that hybridize to glucosaminidase polynucleotide sequences,
particularly under stringent conditions.
[0021] In certain preferred embodiments of the invention, there are
provided antibodies against glucosaminidase polypeptides.
[0022] In other embodiments of the invention, there are provided
methods for identifying compounds which bind to or otherwise
interact with and inhibit or activate an activity of a polypeptide
or polynucleotide of the invention comprising: contacting a
polypeptide or polynucleotide of the invention with a compound to
be screened under conditions to permit binding to or other
interaction between the compound and the polypeptide or
polynucleotide to assess the binding to or other interaction with
the compound, such binding or interaction being associated with a
second component capable of providing a detectable signal in
response to the binding or interaction of the polypeptide or
polynucleotide with the compound; and determining whether the
compound binds to or otherwise interacts with and activates or
inhibits an activity of the polypeptide or polynucleotide by
detecting the presence or absence of a signal generated from the
binding or interaction of the compound with the polypeptide or
polynucleotide.
[0023] In accordance with yet another aspect of the invention,
there are provided glucosaminidase agonists and antagonists,
preferably bacteriostatic or bacteriocidal agonists and
antagonists.
[0024] In a further aspect of the invention, there are provided
compositions comprising a glucosaminidase polynucleotide or a
glucosaminidase polypeptide for administration to a cell or to a
multicellular organism.
[0025] 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.
GLOSSARY
[0026] The following definitions are provided to facilitate
understanding of certain terms used frequently herein.
[0027] "Host cell" is a cell which has been transformed or
transfected, or is capable of transformation or transfection by an
exogenous polynucleotide sequence.
[0028] "Identity," as known in the art, is a relationship between
two or more polypeptide sequences or two or more polynucleotide
sequences, 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" and "similarity" 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). Preferred
methods to determine identity are designed to give the largest
match between the sequences tested. Methods to determine identity
and similarity are codified in publicly available computer
programs. Preferred computer program methods to determine identity
and similarity 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 (Atschul, S.
F. et al., J. Molec. Biol. 215: 403410 (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: 403410 (1990). As an illustration,
by a polynucleotide having a nucleotide sequence having at least,
for example, 95% "identity" to a reference nucleotide sequence of
SEQ ID NO:1 it is intended that the nucleotide sequence of the
polynucleotide is identical to the reference sequence except that
the polynucleotide sequence may include up to five point mutations
per each 100 nucleotides of the reference nucleotide sequence of
SEQ ID NO: 1. In other words, to obtain a polynucleotide having a
nucleotide sequence at least 95% identical to a reference
nucleotide sequence, up to 5% of the nucleotides in the reference
sequence may be deleted or substituted with another nucleotide, or
a number of nucleotides up to 5% of the total nucleotides in the
reference sequence may be inserted into the reference sequence.
These mutations of the reference sequence may occur at the 5 or 3
terminal positions of the reference nucleotide sequence or anywhere
between those terminal positions, interspersed either individually
among nucleotides in the reference sequence or in one or more
contiguous groups within the reference sequence. Analogously, by a
polypeptide having an amino acid sequence having at least, for
example, 95% identity to a reference amino acid sequence of SEQ ID
NO:2 is intended that the amino acid sequence of the polypeptide is
identical to the reference sequence except that the polypeptide
sequence may include up to five amino acid alterations per each 100
amino acids of the reference amino acid of SEQ ID NO: 2. In other
words, to obtain a polypeptide having an amino acid sequence at
least 95% identical to a reference amino acid sequence, up to 5% of
the amino acid residues in the reference sequence may be deleted or
substituted with another amino acid, or a number of amino acids up
to 5% of the total amino acid residues in the reference sequence
may be inserted into the reference sequence. These alterations of
the reference sequence may occur at the amino or carboxy terminal
positions of the reference amino acid sequence or anywhere between
those terminal positions, interspersed either individually among
residues in the reference sequence or in one or more contiguous
groups within the reference sequence.
[0029] "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.
[0030] "Polynucleotide(s)" generally refers to any
polyribonucleotide or polydeoxribonucleotide, which 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-stranded, or
triple-stranded regions, or a mixture of single- and
double-stranded 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 contain 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, enzymatically 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).
[0031] "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 contain 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 contain 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 termini. 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-linking, cyclization,
disulfide bond formation, demethylation, formation of covalent
cross-links, formation of cysteine, formation of pyroglutamate,
formylation, gamma-carboxylation, glycosylation, 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., Posttranslational 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.
[0032] "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, fusions 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. 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.
DESCRIPTION OF THE INVENTION
[0033] The invention relates to novel glucosaminidase polypeptides
and polynucleotides as described in greater detail below. In
particular, the invention relates to polypeptides and
polynucleotides of a novel glucosaminidase of Staphylococcus
aureus, which is related by amino acid sequence homology to
sp.vertline.P52081.vertline.ATL_STAAU AUTOLYSIN PRECURSOR
polypeptide. The invention relates especially to glucosaminidase
having the nucleotide and amino acid sequences set out in Table 1
[SEQ ID NO: 1] and Table 1 [SEQ ID NO: 2] respectively, and to the
glucosaminidase nucleotide sequences of the DNA in the deposited
strain and amino acid sequences encoded thereby.
1TABLE 1 glucosaminidase Polynucleotide and Polypeptide Sequences
(A) Sequences from Staphylococcus aureus glucosaminidase
polynucleotide sequence [SEQ ID NO:1]. 5'-1 GGTTTTGTCT ATATGAAGAA
GAATTTCAAG TTACGCATTT CAACGCTACT 51 ATTGATAGTT ATTTTAGTTG
TTTTTGCTGT ATTACTCATC GTGAATGAAA 101 CTAAATTGTT TAAAAATGAT
GTGAATTACT CTTTTGATGA GGCTGTTTCA 151 ATGCAACAAG GGAAAGGTAT
TGTACAGACA AAAGAAGAGG ATGGTAAATT 201 TGTTGAAGCA AATAATAATG
AAATTGCTAA AGCAATGACT ATTTCAGATA 251 AAGACAATGA TATGAAGTAT
ATGGATATCA CAGAAAAAGT GCCAATGTCG 301 GAATCTGAAG TTAACCAATT
GCTAAAAGGT AAGGGGATTT TAGAAAATCG 351 AGGGAAAGTT TTTCTAGAAG
CTCAAGAAAA ATATGAGGTT AATGTCATTT 401 ATCTTGTTAG CCATGCATTA
GTAGAAACAG GTAACGGCAA ATCAGAATTA 451 GCAAAAGGCA TTAAAGATGG
GAAAAAACGC TATTACAACT TTTTTGGTAT 501 AGGAGCATTC GATAGTAGTG
CTGTTCGTAG TGGGAAAAGT TATGCTGAAA 551 AGGAACAATG GACATCACCA
GATAAGGCGA TTATTGGTGG TGCAAAGTTC 601 ATTCGTAATG AATATTTTGA
AAACAATCAA CTGAATTTAT ATCAAATGCG 651 ATGGAATCCA GAAAATCCTG
CGCAACATCA ATATGCGAGT GACATTCGCT 701 GGGCAGATAA AATTGCCAAA
TTAATGGATA AATCCTATAA GCAGTTTGGT 751 ATAAAGAAAG ATGATATTAG
ACAAACATAT TATAAATAA -3' (B) glucosaminidase polypeptide sequence
deduced from the polynucleotide sequence in this table [SEQ ID
NO:2]. NH.sub.2-1 GFVYMKKNFK LRISTLLLIV ILVVFAVLLI VNETKLFKND
VNYSFDEAVS 51 MQQGKGIVQT KEEDGKFVEA NNNEIAKAMT ISHKDNDMKY
MDITEKVPMS 101 ESEVNQLLKG KGILENRGKV FLEAQEKYEV NVIYLVSHAL
VETGNGKSEL 151 AKGIKDGKKR YYNFFGIGAF DSSAVRSGKS YAEKEQWTSF
DKAIIGGAKF 201 IRNEYFENNQ LNLYQMRWNP ENPAQHQYAS DIRWADKIAK
LMDKSYKQFG 251 IKKDDIRQTY YK-COOH (C) Polynucleotide sequence
embodiments [SEQ ID NO:1]. X-(R.sub.1).sub.n-1 GGTTTTGTCT
ATATGAAGAA GAATTTCAAG TTACGCATTT CAACGCTACT 51 ATTGATAGTT
ATTTTAGTTG TTTTTGCTGT ATTACTCATC GTGAATGAAA 101 CTAAATTGTT
TAAAAATGAT GTGAATTACT CTTTTGATGA GGCTGTTTCA 151 ATGCAACAAG
GGAAAGGTAT TGTACAGACA AAAGAAGAGG ATGGTAAATT 201 TGTTGAAGCA
AATAATAATG AAATTGCTAA AGCAATGACT ATTTCACATA 251 AAGACAATGA
TATGAAGTAT ATGGATATCA CAGAAAAAGT GCCAATGTCG 301 GAATCTGAAG
TTAACCAATT GCTAAAAGGT AAGGGGATTT TAGAAAATCG 351 AGGGAAAGTT
TTTCTAGAAG CTCAAGAAAA ATATGAGGTT AATGTCATTT 401 ATCTTGTTAG
CCATGCATTA GTAGAAACAG GTAACGGCAA ATCAGAATTA 451 GCAAAAGGCA
TTAAAGATGG GAAAAAACGC TATTACAACT TTTTTGGTAT 501 AGGAGCATTC
GATAGTAGTG CTGTTCGTAG TGGGAAAAGT TATGCTGAAA 551 AGGAACAATG
GACATCACCA GATAAGGCGA TTATTGGTGG TGCAAAGTTC 601 ATTCGTAATG
AATATTTTGA AAACAATCAA CTGAATTTAT ATCAAATGCG 651 ATGGAATCCA
GAAAATCCTG CGCAACATCA ATATGCGAGT GACATTCGCT 701 GGGCAGATAA
AATTGCCAAA TTAATGGATA AATCCTATAA GCAGTTTGGT 751 ATAAAGAAAG
ATGATATTAG ACAAACATAT TATAAATAA -(R.sub.2).sub.n-Y (D) Polypeptide
sequence embodiments [SEQ ID NO:2]. X-(R.sub.1).sub.n-1 GFVYMKKNFK
LRISTLLLIV ILVVFAVLLI VNETKLFKND VNYSFDEAVS 51 MQQGKGIVQT
KEEDGKFVEA NNNEIAKAMT ISHKDNDMKY MDITEKVPMS 101 ESEVNQLLKG
KGILENRGKV FLEAQEKYEV NVIYLVSHAL VETGNGKSEL 151 AKGIKDGKKR
YYNFFGIGAF DSSAVRSGKS YAEKEQWTSP DKAIIGGAKF 201 IRNEYFENNQ
LNLYQMRWNP ENPAQHQYAS DIRWADKIAK LMDKSYKQFG 251 IKKDDIRQTY
YK-(R.sub.2).sub.n-Y (E) Sequences from Staphylococcus aureus
glucosaminidase polynucleotide ORF sequence [SEQ ID NO:3]. 5'-1
GGTTTTGTCT ATATGAAGAA GAATTTCAAG TTACGCATTT CAACGCTACT 51
ATTGATAGTT ATTTTAGTTG TTTTTGCTGT ATTACTCATC GTGAATGAAA 101
CTAAATTGTT TAAAAATGAT GTGAATTACT CTTTTGATGA GGCTGTTTCA 151
ATGCAACAAG GGAAAGGTAT TGTACAGACA AAAGAAGAGG ATGGTAAATT 201
TGTTGAAGCA AATAATAATG AAATTGCTAA AGCAATGACT ATTTCACATA 251
AAGACAATGA TATGAAGTAT ATGGATATCA CAGAAAAAGT GCCAATGTCG 301
GAATCTGAAG TTAACCAATT GCTAAAAGGT AAGGGGATTT TAGAAAATCG 351
AGGGAAAGTT TTTCTAGAAG CTCAAGAAAA ATATGAGGTT AATGTCATTT 401
ATCTTGTTAG CCATGCATTA GTAGAAACAG GTAACGGCAA ATCAGAATTA 451
GCAAAAGGCA TTAAAGATGG GAAAAAACGC TATTACAACT TTTTTGGTAT 501
AGGAGCATTC GATAGTAGTG CTGTTCGTAG TGGGAAAAGT TATGCTGAAA 551
AGGAACAATG GACATCACCA GATAAGGCGA TTATTGGTGG TGCAAAGTTC 601
ATTCGTAATG AATATTTTGA AAACAATCAA CTGAATTTAT ATCAAATGCG 651
ATGGAATCCA GAAAATCCTG CGCAACATCA ATATGCGAGT GACATTCGCT 701
GGGCAGATAA AATTGCCAAA TTAATGGATA AATCCTATAA GCAGTTTGGT 751
ATAAAGAAAG ATGATATTAG ACAAACATAT TATAAA-3' (F) glucosaminidase
polypeptide sequence deduced from the polynucleotide ORF sequence
in this table [SEQ ID NO:4]. NH.sub.2-1 GFVYMKKNFK LRISTLLLIV
ILVVFAVLLI VNETKLFKND VNYSFDEAVS 51 MQQGKGIVQT KEEDGKFVEA
NNNEIAKAMT ISHKDNDMKY MDITEKVPMS 101 ESEVNQLLKG KGILENRGKV
FLEAQEKYEV NVIYLVSHAL VETGNGKSEL 151 AKGIKDGKKR YYNFFGIGAF
DSSAVRSGKS YAEKEQWTSP DKAIIGGAKF 201 IRNEYFENNQ LNLYQMRWNP
ENPAQHQYAS DIRWADKIAK LMDKSYKQFG 251 IKKDDIRQTY YK-COOH
[0034] Deposited Materials
[0035] A deposit containing a Staphylococcus aureus WCUH 29 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 Sep. 11, 1995 and assigned NCIMB
Deposit No. 40771, and is referred to as Staphylococcus aureus
WCUH29 on deposit. The Staphylococcus aureus strain deposit is
referred to herein as "the deposited strain" or as "the DNA of the
deposited strain."
[0036] The deposited strain contains the full length
glucosaminidase gene. The sequence of the polynucleotides contained
in the deposited strain, as well as the amino acid sequence of the
polypeptide encoded thereby, are controlling in the event of any
conflict with any description of sequences herein.
[0037] 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 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.
[0038] A license may be required to make, use or sell the deposited
strain, and compounds derived therefrom, and no such license is
hereby granted.
[0039] Polypeptides
[0040] The polypeptides of the invention include the polypeptide of
Table 1 [SEQ ID NO:2] (in particular the mature polypeptide) as
well as polypeptides and fragments, particularly those which have
the biological activity of glucosaminidase, and also those which
have at least 70% identity to a polypeptide of Table 1 [SEQ ID
NOS:2 and 4] or the relevant portion, preferably at least 80%
identity to a polypeptide of Table 1 [SEQ ID NOS:2 and 4], and more
preferably at least 90% similarity (more preferably at least 90%
identity) to a polypeptide of Table 1 [SEQ ID NOS:2 and 4] and
still more preferably at least 95% similarity (still more
preferably at least 95% identity) to a polypeptide of Table 1 [SEQ
ID NOS:2 and 4] and also include portions of such polypeptides with
such portion of the polypeptide generally containing at least 30
amino acids and more preferably at least 50 amino acids.
[0041] The invention also includes polypeptides of the formula set
forth in Table 1 (D) [SEQ ID NO:2] wherein, at the amino terminus,
X is hydrogen, and at the carboxyl terminus, Y is hydrogen or a
metal, R.sub.1 and R.sub.2 is any amino acid residue, and n is an
integer between 1 and 1000. Any stretch of amino acid residues
denoted by either R group, where R is greater than 1, may be either
a heteropolymer or a homopolymer, preferably a heteropolymer.
[0042] A fragment is a variant polypeptide having an amino acid
sequence that entirely is the same as part but not all of the amino
acid sequence of the aforementioned polypeptides. As with
glucosaminidase 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, a single
larger polypeptide.
[0043] Preferred fragments include, for example, truncation
polypeptides having a portion of an amino acid sequence of Table 1
[SEQ ID NOS:2 and 4], or of variants thereof, such as a continuous
series of residues that includes the amino terminus, or a
continuous series of residues that includes the carboxyl terminus.
Degradation forms of the polypeptides of the invention in a host
cell, particularly a Staphylococcus aureus, 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.
[0044] Also preferred are biologically active fragments which are
those fragments that mediate activities of glucosaminidase,
including those with a similar activity or an improved activity, or
with a decreased undesirable activity. Also included are those
fragments that are antigenic or immunogenic in an animal,
especially in a human. Particularly preferred are fragments
comprising receptors or domains of enzymes that confer a function
essential for viability of Staphylococcus aureus or the ability to
initiate, or maintain cause disease in an individual, particularly
a human.
[0045] Variants that are 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.
[0046] Polynucleotides
[0047] Another aspect of the invention relates to isolated
polynucleotides, including the full length gene, that encode the
glucosaminidase polypeptide having a deduced amino acid sequence of
Table 1 [SEQ ID NOS:2 and 4] and polynucleotides closely related
thereto and variants thereof.
[0048] Using the information provided herein, such as a
polynucleotide sequence set out in Table 1 [SEQ ID NOS: 1 and 3], a
polynucleotide of the invention encoding glucosaminidase
polypeptide may be obtained using standard cloning and screening
methods, such as those for cloning and sequencing chromosomal DNA
fragments from bacteria using Staphylococcus aureus WCUH 29 cells
as starting material, followed by obtaining a full length clone.
For example, to obtain a polynucleotide sequence of the invention,
such as a sequence given in Table 1 [SEQ ID NOS: 1 and 3],
typically a library of clones of chromosomal DNA of Staphylococcus
aureus WCUH 29 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
conditions. By sequencing the individual clones thus identified
with sequencing primers designed from the original sequence it is
then possible to extend the sequence in both directions to
determine the full gene sequence. Conveniently, such sequencing is
performed 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).
Illustrative of the invention, the polynucleotide set out in Table
1 [SEQ ID NO: 1] was discovered in a DNA library derived from
Staphylococcus aureus WCUH 29.
[0049] The DNA sequence set out in Table 1 [SEQ ID NOS: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 in the art. The
polynucleotide of SEQ ID NO: 1, between nucleotide number 1 through
number 786 encodes the polypeptide of SEQ ID NO:2. The stop codon
begins at nucleotide number 787 of SEQ ID NO: 1.
[0050] The glucosaminidase polypeptide of the invention is
structurally related to other proteins of the autolysin family, as
shown by the results of sequencing the DNA encoding glucosaminidase
of the deposited strain. The protein exhibits greatest homology to
sp.vertline.P52081.vert- line.ATL_STAAU AUTOLYSIN PRECURSOR protein
among known proteins (see also, autolysin AtIE (GENBANK: locus
SEU71377, accession U71377). The glucosaminidase polypeptide of
Table 1 [SEQ ID NO:2] has about 45% identity over its entire length
and about 70% similarity over its entire length with the amino acid
sequence of sp.vertline.P52081.vertline.ATL_ST- AAU AUTOLYSIN
PRECURSOR polypeptide.
[0051] The invention provides a polynucleotide sequence identical
over its entire length to the coding sequence in Table 1 [SEQ ID
NO: 1]. Also provided by the invention is the coding sequence for
the mature polypeptide or a fragment thereof, by itself as well as
the coding sequence for the mature polypeptide or a fragment in
reading frame with other coding sequence, such as those encoding a
leader or secretory sequence, a pre-, or pro- or prepro- protein
sequence. The polynucleotide may also contain non-coding sequences,
including for example, but not limited to non-coding 5' and 3'
sequences, such as the transcribed, non-translated sequences,
termination signals, ribosome binding sites, sequences that
stabilize mRNA, introns, polyadenylation signals, and additional
coding sequence which encode additional amino acids. For example, a
marker sequence that facilitates purification of the 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 tag
(Wilson et al., Cell 37: 767 (1984). 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.
[0052] A preferred embodiment of the invention is a polynucleotide
of comprising nucleotide 1 to 786 or 789 set forth in SEQ ID NO: 1
of Table 1 which encode the glucosaminidase polypeptide.
[0053] The invention also includes polynucleotides of the formula
set forth in Table 1 (C)[SEQ ID NO: 1] wherein, at the 5' end of
the molecule, X is hydrogen, and at the 3' end of the molecule, Y
is hydrogen or a metal, R.sub.1 and R.sub.2 is any nucleic acid
residue, and n is an integer between 1 and 1000. Any stretch of
nucleic acid residues denoted by either R group, where R is greater
than 1, may be either a heteropolymer or a homopolymer, preferably
a heteropolymer.
[0054] 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
Staphylococcus aureus glucosaminidase having the 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,
interrupted by integrated phage or an insertion sequence or
editing) together with additional regions, that also may contain
coding and/or non-coding sequences.
[0055] The invention further relates to variants of the
polynucleotides described herein that encode for variants of the
polypeptide having the deduced amino acid sequence of Table 1 [SEQ
ID NO:2]. Variants that are fragments of the polynucleotides of the
invention may be used to synthesize full-length polynucleotides of
the invention.
[0056] Further particularly preferred embodiments are
polynucleotides encoding glucosaminidase variants, that have the
amino acid sequence of glucosaminidase 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, deleted or added, in any
combination. Especially preferred among these are silent
substitutions, additions and deletions, that do not alter the
properties and activities of glucosaminidase.
[0057] Further preferred embodiments of the invention are
polynucleotides that are at least 70% identical over their entire
length to a polynucleotide encoding glucosaminidase polypeptide
having an amino acid sequence set out in Table I [SEQ ID NOS:2 and
4], and polynucleotides that are complementary to such
polynucleotides. Alternatively, most highly preferred are
polynucleotides that comprise a region that is at least 80%
identical over its entire length to a polynucleotide encoding
glucosaminidase polypeptide of the deposited strain and
polynucleotides complementary thereto. In this regard,
polynucleotides at least 90% identical over their entire length to
the same are particularly preferred, and among these particularly
preferred polynucleotides, those with 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% being the more preferred.
[0058] Preferred embodiments are polynucleotides that encode
polypeptides that retain substantially the same biological function
or activity as the mature polypeptide encoded by the DNA of Table 1
[SEQ ID NO: 1].
[0059] The invention further relates to polynucleotides that
hybridize to the herein above-described sequences. In this regard,
the invention especially relates to polynucleotides that hybridize
under stringent conditions to the herein above-described
polynucleotides. As herein used, the terms "stringent conditions"
and "stringent hybridization conditions" mean hybridization will
occur only if there is at least 95% and preferably at least 97%
identity between the sequences. An 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 nM sodium phosphate (pH7.6),
5.times.Denhardt's solution, 10% dextran sulfate, and 20
micrograms/ml 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.
[0060] The invention also provides a polynucleotide consisting
essentially of a polynucleotide sequence obtainable by screening an
appropriate library containing the complete gene for a
polynucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO:3
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 DNA sequence. Fragments
useful for obtaining such a polynucleotide include, for example,
probes and primers described elsewhere herein.
[0061] As discussed additionally herein regarding polynucleotide
assays of the invention, for instance, polynucleotides of the
invention as discussed above, may be used as a hybridization probe
for RNA, cDNA and genomic DNA to isolate full-length cDNAs and
genomic clones encoding glucosaminidase and to isolate cDNA and
genomic clones of other genes that have a high sequence similarity
to the glucosaminidase gene. Such probes generally will comprise at
least 15 bases. Preferably, such probes will have at least 30 bases
and may have at least 50 bases. Particularly preferred probes will
have at least 30 bases and will have 50 bases or less.
[0062] For example, the coding region of the glucosaminidase gene
may be isolated by screening using the DNA sequence provided in 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.
[0063] 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 disease,
particularly human disease, as further discussed herein relating to
polynucleotide assays.
[0064] Polynucleotides of the invention that are oligonucleotides
derived from the sequences of SEQ ID NOS: 1 and/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.
[0065] The invention also provides polynucleotides that may encode
a polypeptide that is the mature protein plus additional amino or
carboxyl-terminal amino acids, or amino acids interior to the
mature polypeptide (when the 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 things. As generally is the case in vivo, the additional
amino acids may be processed away from the mature protein by
cellular enzymes.
[0066] A precursor protein, having the 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.
[0067] 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, which is a precursor to a
proprotein, having a leader sequence and one or more prosequences,
which generally are removed during processing steps that produce
active and mature forms of the polypeptide.
[0068] Vectors, Host Cells, Expression
[0069] 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.
[0070] For recombinant production, 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.
[0071] Representative examples of appropriate hosts include
bacterial cells, such as streptococci, staphylococci, enterococci
E. coli, streptomyces and Bacillus subtilis cells; fungal cells,
such as yeast cells and Aspergillus cells; insect cells such as
Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO,
COS, HeLa, C127, 3T3, BHK, 293 and Bowes melanoma cells; and plant
cells.
[0072] 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, e.g.,
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 and retroviruses, and vectors
derived from combinations thereof, such as those derived from
plasmid and bacteriophage genetic elements, such as cosmids and
phagemids. The expression system constructs may contain 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).
[0073] For secretion of the 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.
[0074] 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.
[0075] Diagnostic Assays
[0076] This invention is also related to the use of the
glucosaminidase polynucleotides of the invention for use as
diagnostic reagents. Detection of glucosaminidase in a eukaryote,
particularly a mammal, and especially a human, will provide a
diagnostic method for diagnosis of a disease. Eukaryotes (herein
also "individual(s)"), particularly mammals, and especially humans,
infected with an organism comprising the glucosaminidase gene may
be detected at the nucleic acid level by a variety of
techniques.
[0077] Nucleic acids for diagnosis may be obtained from an infected
individual's cells and tissues, such as bone, blood, muscle,
cartilage, and skin. Genomic DNA may be used directly for detection
or may be amplified enzymatically by using PCR or other
amplification technique prior to analysis. RNA or cDNA may also be
used in the same ways. Using amplification, characterization of the
species and strain of prokaryote present in an individual, may be
made by an analysis of the genotype of the prokaryote gene.
Deletions and insertions can be detected by a change in size of the
amplified product in comparison to the genotype of a reference
sequence. Point mutations can be identified by hybridizing
amplified DNA to labeled glucosaminidase polynucleotide sequences.
Perfectly matched sequences can be distinguished from mismatched
duplexes by RNase digestion or by differences in melting
temperatures. DNA sequence differences may also be detected by
alterations in the electrophoretic mobility of the DNA fragments in
gels, with or without denaturing agents, or by direct DNA
sequencing. See, e.g., Myers et al., Science, 230: 1242 (1985).
Sequence changes at specific locations also may be revealed by
nuclease protection assays, such as RNase and S1 protection or a
chemical cleavage method. See, e.g., Cotton et al., Proc. Natl.
Acad. Sci., USA, 85:4397-4401 (1985).
[0078] Cells carrying mutations or polymorphisms in the gene of the
invention may also be detected at the DNA level by a variety of
techniques, to allow for serotyping, for example. For example,
RT-PCR can be used to detect mutations. It is particularly
preferred to used RT-PCR in conjunction with automated detection
systems, such as, for example, GeneScan. RNA or cDNA may also be
used for the same purpose, PCR or RT-PCR. As an example, PCR
primers complementary to a nucleic acid encoding glucosaminidase
can be used to identify and analyze mutations. Examples of
representative primers are shown below in Table 2.
2TABLE 2 Primers for amplification of glucosaminidase
polynucleotides SEQ ID NO PRIMER SEQUENCE 5 5'-G TTAACCAATT
GCTAAAAGG-3' 6 5'-AAACTGCTTATAGGATTTATC-3- '
[0079] 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, amplifying glucosaminidase DNA
isolated from a sample derived from an individual. The primers may
be used to amplify the gene isolated from an infected individual
such that the gene may then be subject to various techniques for
elucidation of the DNA sequence. In this way, mutations in the DNA
sequence may be detected and used to diagnose infection and to
serotype and/or classify the infectious agent.
[0080] The invention further provides a process for diagnosing,
disease, preferably bacterial infections, more preferably
infections by Staphylococcus aureus, and most preferably disease,
such as, infections of the upper respiratory tract (e.g., otitis
media, bacterial tracheitis, acute epiglottitis, thyroiditis),
lower respiratory (e.g., empyema, lung abscess), cardiac (e.g.,
infective endocarditis), gastrointestinal (e.g., secretory
diarrhoea, splenic abscess, retroperitoneal abscess), CNS (e.g.,
cerebral abscess), eye (e.g., blepharitis, conjunctivitis,
keratitis, endophthalmitis, preseptal and orbital cellulitis,
darcryocystitis), kidney and urinary tract (e.g., epididymitis,
intrarenal and perinephric abscess, toxic shock syndrome), skin
(e.g., impetigo, folliculitis, cutaneous abscesses, cellulitis,
wound infection, bacterial myositis) bone and joint (e.g., septic
arthritis, osteomyelitis), comprising determining from a sample
derived from an individual a increased level of expression of
polynucleotide having the sequence of Table 1 [SEQ ID NO: 1].
Increased or decreased expression of glucosaminidase polynucleotide
can be measured using any on of the methods well known in the art
for the quantation of polynucleotides, such as, for example,
amplification, PCR, RT-PCR, RNase protection, Northern blotting and
other hybridization methods.
[0081] In addition, a diagnostic assay in accordance with the
invention for detecting over-expression of glucosaminidase protein
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 glucosaminidase protein, in a sample
derived from a host are well-known to those of skill in the art.
Such assay methods include radioimmunoassays, competitive-binding
assays, Western Blot analysis and ELISA assays.
[0082] Antibodies
[0083] The polypeptides of the invention or variants thereof, or
cells expressing them can be used as an immunogen to produce
antibodies immunospecific for such polypeptides. "Antibodies" as
used herein includes monoclonal and polyclonal antibodies,
chimeric, single chain, simianized antibodies and humanized
antibodies, as well as Fab fragments, including the products of an
Fab immunolglobulin expression library.
[0084] Antibodies generated against the polypeptides of the
invention can be obtained by administering the polypeptides or
epitope-bearing fragments, analogues or cells to an animal,
preferably a nonhuman, using routine protocols. For preparation of
monoclonal antibodies, any technique known in the art that provides
antibodies produced by continuous cell line cultures can be used.
Examples include various techniques, such as those in Kohler, G.
and Milstein, C., Nature 256: 495-497 (1975); Kozbor et al.,
Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONAL
ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).
[0085] Techniques for the production of single chain antibodies
(U.S. Pat. No. 4,946,778) can be adapted to produce single chain
antibodies to polypeptides of this invention. Also, transgenic
mice, or other organisms such as other mammals, may be used to
express humanized antibodies.
[0086] Alternatively phage display technology may be utilized to
select antibody genes with binding activities towards the
polypeptide either from repertoires of PCR amplified v-genes of
lymphocytes from humans screened for possessing
anti-glucosaminidase or from naive libraries (McCafferty, J. et
al., (1990), Nature 348, 552-554; Marks, J. et al., (1992)
Biotechnology 10, 779-783). The affinity of these antibodies can
also be improved by chain shuffling (Clackson, T. et al., (1991)
Nature 352, 624-628).
[0087] If two antigen binding domains are present each domain may
be directed against a different epitope--termed `bispecific`
antibodies.
[0088] The above-described antibodies may be employed to isolate or
to identify clones expressing the polypeptides to purify the
polypeptides by affinity chromatography.
[0089] Thus, among others, antibodies against
glucosaminidase-polypeptide may be employed to treat infections,
particularly bacterial infections and especially disease, such as,
infections of the upper respiratory tract (e.g., otitis media,
bacterial tracheitis, acute epiglottitis, thyroiditis), lower
respiratory (e.g., empyema, lung abscess), cardiac (e.g., infective
endocarditis), gastrointestinal (e.g., secretory diarrhoea, splenic
abscess, retroperitoneal abscess), CNS (e.g., cerebral abscess),
eye (e.g., blepharitis, conjunctivitis, keratitis, endophthalmitis,
preseptal and orbital cellulitis, darcryocystitis), kidney and
urinary tract (e.g., epididymitis, intrarenal and perinephric
abscess, toxic shock syndrome), skin (e.g., impetigo, folliculitis,
cutaneous abscesses, cellulitis, wound infection, bacterial
myositis) bone and joint (e.g., septic arthritis,
osteomyelitis).
[0090] Polypeptide variants include antigenically, epitopically or
immunologically equivalent variants that form a particular aspect
of this invention. The term "antigenically equivalent derivative"
as used herein encompasses a polypeptide or its equivalent which
will be specifically recognized by certain antibodies which, when
raised to the protein or polypeptide according to the invention,
interfere with the immediate physical interaction between pathogen
and mammalian host. The term "immunologically equivalent
derivative" as used herein encompasses a peptide or its equivalent
which when used in a suitable formulation to raise antibodies in a
vertebrate, the antibodies act to interfere with the immediate
physical interaction between pathogen and mammalian host.
[0091] The polypeptide, such as an antigenically or immunologically
equivalent derivative or a fusion protein thereof is used as an
antigen to immunize a mouse or other animal such as a rat or
chicken. The fusion protein may provide stability to the
polypeptide. The antigen may be associated, for example by
conjugation, with an immunogenic carrier protein for example bovine
serum albumin (BSA) or keyhole limpet haemocyanin (KLH).
Alternatively a multiple antigenic peptide comprising multiple
copies of the protein or polypeptide, or an antigenically or
immunologically equivalent polypeptide thereof may be sufficiently
antigenic to improve immunogenicity so as to obviate the use of a
carrier.
[0092] Preferably, the antibody or variant thereof is modified to
make it less immunogenic in the individual. For example, if the
individual is human the antibody may most preferably be
"humanized"; where the complimentarily determining region(s) of the
hybridoma-derived antibody has been transplanted into a human
monoclonal antibody, for example as described in Jones, P. et al.
(1986), Nature 321, 522-525 or Tempest et al., (1991) Biotechnology
9, 266-273.
[0093] The use of a polynucleotide of the invention in genetic
immunization will preferably employ a suitable delivery method such
as direct injection of plasmid DNA into muscles (Wolff et al., Hum
Mol Genet 1992, 1:363, Manthorpe et al., Hum. Gene Ther. 1963:4,
419), delivery of DNA complexed with specific protein carriers (Wu
et al., J Biol Chem. 1989: 264, 16985), coprecipitation of DNA with
calcium phosphate (Benvenisty & Reshef, PNAS USA, 1986:83,
9551), encapsulation of DNA in various forms of liposomes (Kaneda
et al., Science 1989:243, 375), particle bombardment (Tang et al.,
Nature 1992, 356:152, Eisenbraun et al., DNA Cell Biol 1993,
12:791) and in vivo infection using cloned retroviral vectors
(Seeger et al., PNAS USA 1984:81,5849).
[0094] Antagonists and Agonists--Assays and Molecules
[0095] Polypeptides 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).
[0096] The invention also provides a method of screening compounds
to identify those which enhance (agonist) or block (antagonist) the
action of glucosaminidase polypeptides or polynucleotides,
particularly those compounds that are bacteriostatic and/or
bacteriocidal. The method of screening may involve high-throughput
techniques. For example, to screen for agonists or antagoists, a
synthetic reaction mix, a cellular compartment, such as a membrane,
cell envelope or cell wall, or a preparation of any thereof,
comprising glucosaminidase 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 glucosaminidase
agonist or antagonist. The ability of the candidate molecule to
agonize or antagonize the glucosaminidase 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 glucosaminidase polypeptide
are most likely to be good antagonists. Molecules that bind well
and increase the rate of product production from substrate are
agonists. Detection of the rate or level of production of product
from substrate 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 glucosaminidase
polynucleotide or polypeptide activity, and binding assays known in
the art.
[0097] Another example of an assay for glucosaminidase antagonists
is a competitive assay that combines glucosaminidase and a
potential antagonist with glucosaminidase-binding molecules,
recombinant glucosaminidase binding molecules, natural substrates
or ligands, or substrate or ligand mimetics, under appropriate
conditions for a competitive inhibition assay. The glucosaminidase
molecule can be labeled, such as by radioactivity or a calorimetric
compound, such that the number of glucosaminidase molecules bound
to a binding molecule or converted to product can be determined
accurately to assess the effectiveness of the potential
antagonist.
[0098] Potential antagonists include small organic molecules,
peptides, polypeptides and antibodies that bind to a polynucleotide
or polypeptide of the invention and thereby inhibit or extinguish
its activity. Potential antagonists also may be small organic
molecules, a peptide, a polypeptide such as a closely related
protein or antibody that binds the same sites on a binding
molecule, such as a binding molecule, without inducing
glucosaminidase-induced activities, thereby preventing the action
of glucosaminidase by excluding glucosaminidase from binding.
[0099] Potential antagonists include a small molecule that binds to
and occupies the binding site of the polypeptide thereby preventing
binding to cellular binding molecules, such that normal biological
activity is prevented. Examples of small molecules include but are
not limited to small organic molecules, peptides or peptide-like
molecules. Other potential antagonists include antisense molecules
(see Okano, J. Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS
ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton,
Fla. (1988), for a description of these molecules). Preferred
potential antagonists include compounds related to and variants of
glucosaminidase.
[0100] Each of the DNA 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 DNA 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.
[0101] The invention also provides the use of the polypeptide,
polynucleotide or inhibitor of the invention to interfere with the
initial physical interaction between a pathogen and 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 bacteria, to mammalian
extracellular matrix proteins on in-dwelling devices or to
extracellular matrix proteins in wounds; to block glucosaminidase
protein-mediated mammalian cell invasion by, for example,
initiating phosphorylation of mammalian tyrosine kinases
(Rosenshine et al., Infect. Immun. 60:2211 (1992); to block
bacterial adhesion between mammalian extracellular matrix proteins
and bacterial glucosaminidase proteins that mediate tissue damage
and; to block the normal progression of pathogenesis in infections
initiated other than by the implantation of in-dwelling devices or
by other surgical techniques.
[0102] The antagonists and agonists of the invention may be
employed, for instance, to inhibit and treat disease, such as,
infections of the upper respiratory tract (e.g., otitis media,
bacterial tracheitis, acute epiglottitis, thyroiditis), lower
respiratory (e.g., empyema, lung abscess), cardiac (e.g., infective
endocarditis), gastrointestinal (e.g., secretory diarrhoea, splenic
abscess, retroperitoneal abscess), CNS (e.g., cerebral abscess),
eye (e.g., blepharitis, conjunctivitis, keratitis, endophthalmitis,
preseptal and orbital cellulitis, darcryocystitis), kidney and
urinary tract (e.g., epididymitis, intrarenal and perinephric
abscess, toxic shock syndrome), skin (e.g., impetigo, folliculitis,
cutaneous abscesses, cellulitis, wound infection, bacterial
myositis) bone and joint (e.g., septic arthritis,
osteomyelitis).
[0103] Vaccines
[0104] Another aspect of the invention relates to a method for
inducing an immunological response in an individual, particularly a
mammal which comprises inoculating the individual with
glucosaminidase, or a fragment or variant thereof, adequate to
produce antibody and/ or T cell immune response to protect said
individual from infection, particularly bacterial infection and
most particularly Staphylococcus aureus infection. Also provided
are methods whereby such immunological response slows bacterial
replication. Yet another aspect of the invention relates to a
method of inducing immunological response in an individual which
comprises delivering to such individual a nucleic acid vector to
direct expression of glucosaminidase, or a fragment or a variant
thereof, for expressing glucosaminidase, or a fragment or a variant
thereof in vivo in order to induce an immunological response, such
as, to produce antibody and/ or T cell immune response, including,
for example, cytokine-producing T cells or cytotoxic T cells, to
protect said individual from disease, whether that disease is
already established within the individual or not. One way of
administering the gene is by accelerating it into the desired cells
as a coating on particles or otherwise.
[0105] Such nucleic acid vector may comprise DNA, RNA, a modified
nucleic acid, or a DNA/RNA hybrid.
[0106] A further aspect of the invention relates to an
immunological composition which, when introduced into an individual
capable or having induced within it an immunological response,
induces an immunological response in such individual to a
glucosaminidase or protein coded therefrom, wherein the composition
comprises a recombinant glucosaminidase or protein coded therefrom
comprising DNA which codes for and expresses an antigen of said
glucosaminidase or protein coded therefrom. The immunological
response may be used therapeutically or prophylactically and may
take the form of antibody immunity or cellular immunity such as
that arising from CTL or CD4+ T cells.
[0107] A glucosaminidase polypeptide or a fragment thereof may be
fused with co-protein which may not by itself produce antibodies,
but is capable of stabilizing the first protein and producing a
fused protein which will have immunogenic and protective
properties. Thus fused recombinant protein, preferably further
comprises an antigenic co-protein, such as lipoprotein D from
Hemophilus influenzae, Glutathione-S-transferase (GST) or
beta-galactosidase, relatively large co-proteins which solubilize
the protein and facilitate production and purification thereof.
Moreover, the co-protein may act as an adjuvant in the sense of
providing a generalized stimulation of the immune system. The
co-protein may be attached to either the amino or carboxy terminus
of the first protein.
[0108] Provided by this invention are compositions, particularly
vaccine compositions, and methods comprising the polypeptides or
polynucleotides of the invention and immunostimulatory DNA
sequences, such as those described in Sato, Y. et al. Science 273:
352 (1996).
[0109] Also, provided by this invention are methods using the
described polynucleotide or particular fragments thereof which have
been shown to encode non-variable regions of bacterial cell surface
proteins in DNA constructs used in such genetic immunization
experiments in animal models of infection with Staphylococcus
aureus will be particularly useful for identifying protein epitopes
able to provoke a prophylactic or therapeutic immune response. It
is believed that this approach will allow for the subsequent
preparation of monoclonal antibodies of particular value from the
requisite organ of the animal successfully resisting or clearing
infection for the development of prophylactic agents or therapeutic
treatments of bacterial infection, particularly Staphylococcus
aureus infection, in mammals, particularly humans.
[0110] The polypeptide may be used as an antigen for vaccination of
a host to produce specific antibodies which protect against
invasion of bacteria, for example by blocking adherence of bacteria
to damaged tissue. Examples of tissue damage include wounds in skin
or connective tissue caused, e.g., by mechanical, chemical or
thermal damage or by implantation of indwelling devices, or wounds
in the mucous membranes, such as the mouth, mammary glands, urethra
or vagina.
[0111] The invention also includes a vaccine formulation which
comprises an immunogenic recombinant protein of the invention
together with a suitable carrier. Since the protein may be broken
down in the stomach, it is preferably administered parenterally,
including, for example, administration that is subcutaneous,
intramuscular, intravenous, or intradermal. Formulations suitable
for parenteral administration include aqueous and non-aqueous
sterile injection solutions which may contain anti-oxidants,
buffers, bacteriostats and solutes which render the formulation
insotonic with the bodily fluid, preferably the blood, of the
individual; and aqueous and non-aqueous sterile suspensions which
may include suspending agents or thickening agents. The
formulations may be presented in unit-dose or multi-dose
containers, for example, sealed ampules and vials and may be stored
in a freeze-dried condition requiring only the addition of the
sterile liquid carrier immediately prior to use. The vaccine
formulation may also include adjuvant systems for enhancing the
immunogenicity of the formulation, such as oil-in water systems and
other systems known in the art. The dosage will depend on the
specific activity of the vaccine and can be readily determined by
routine experimentation.
[0112] While the invention has been described with reference to
certain glucosaminidase protein, it is to be understood that this
covers fragments of the naturally occurring protein and similar
proteins with additions, deletions or substitutions which do not
substantially affect the immunogenic properties of the recombinant
protein.
[0113] Compositions, Kits and Administration
[0114] The invention also relates to compositions comprising the
polynucleotide or the polypeptides discussed above or their
agonists or antagonists. The polypeptides of the invention may be
employed in combination with a non-sterile or sterile carrier or
carriers for use with cells, tissues or organisms, such as a
pharmaceutical carrier suitable for administration to a subject.
Such compositions comprise, for instance, a media additive or a
therapeutically effective amount of a polypeptide of the invention
and a pharmaceutically acceptable carrier or excipient. Such
carriers may include, but are not limited to, saline, buffered
saline, dextrose, water, glycerol, ethanol and combinations
thereof. The formulation should suit the mode of administration.
The invention further relates to diagnostic and pharmaceutical
packs and kits comprising one or more containers filled with one or
more of the ingredients of the aforementioned compositions of the
invention.
[0115] Polypeptides and other compounds of the invention may be
employed alone or in conjunction with other compounds, such as
therapeutic compounds.
[0116] The pharmaceutical compositions may be administered in any
effective, convenient manner including, for instance,
administration by topical, oral, anal, vaginal, intravenous,
intraperitoneal, intramuscular, subcutaneous, intranasal or
intradermal routes among others.
[0117] In therapy or as a prophylactic, the active agent may be
administered to an individual as an injectable composition, for
example as a sterile aqueous dispersion, preferably isotonic.
[0118] Alternatively the composition may be formulated for topical
application for example in the form of ointments, creams, lotions,
eye ointments, eye drops, ear drops, mouthwash, impregnated
dressings and sutures and aerosols, and may contain appropriate
conventional additives, including, for example, preservatives,
solvents to assist drug penetration, and emollients in ointments
and creams. Such topical formulations may also contain compatible
conventional carriers, for example cream or ointment bases, and
ethanol or oleyl alcohol for lotions. Such carriers may constitute
from about 1% to about 98% by weight of the formulation; more
usually they will constitute up to about 80% by weight of the
formulation.
[0119] For administration to mammals, and particularly humans, it
is expected that the daily dosage level of the active agent will be
from 0.01 mg/kg to 10 mg/kg, typically around 1 mg/kg. The
physician in any event will determine the actual dosage which will
be most suitable for an individual and will vary with the age,
weight and response of the particular individual. The above dosages
are exemplary of the average case. There can, of course, be
individual instances where higher or lower dosage ranges are
merited, and such are within the scope of this invention.
[0120] In-dwelling devices include surgical implants, prosthetic
devices and catheters, i.e., devices that are introduced to the
body of an individual and remain in position for an extended time.
Such devices include, for example, artificial joints, heart valves,
pacemakers, vascular grafts, vascular catheters, cerebrospinal
fluid shunts, urinary catheters, continuous ambulatory peritoneal
dialysis (CAPD) catheters.
[0121] The composition of the invention may be administered by
injection to achieve a systemic effect against relevant bacteria
shortly before insertion of an in-dwelling device. Treatment may be
continued after surgery during the in-body time of the device. In
addition, the composition could also be used to broaden
perioperative cover for any surgical technique to prevent bacterial
wound infections, especially Staphylococcus aureus wound
infections.
[0122] Many orthopaedic surgeons consider that humans with
prosthetic joints should be considered for antibiotic prophylaxis
before dental treatment that could produce a bacteremia. Late deep
infection is a serious complication sometimes leading to loss of
the prosthetic joint and is accompanied by significant morbidity
and mortality. It may therefore be possible to extend the use of
the active agent as a replacement for prophylactic antibiotics in
this situation.
[0123] In addition to the therapy described above, the compositions
of this invention may be used generally as a wound treatment agent
to prevent adhesion of bacteria to matrix proteins exposed in wound
tissue and for prophylactic use in dental treatment as an
alternative to, or in conjunction with, antibiotic prophylaxis.
[0124] Alternatively, the composition of the invention may be used
to bathe an indwelling device immediately before insertion. The
active agent will preferably be present at a concentration of 1
.mu.g/ml to 10 mg/ml for bathing of wounds or indwelling
devices.
[0125] A vaccine composition is conveniently in injectable form.
Conventional adjuvants may be employed to enhance the immune
response. A suitable unit dose for vaccination is 0.5-5
microgram/kg of antigen, and such dose is preferably administered
1-3 times and with an interval of 1-3 weeks. With the indicated
dose range, no adverse toxicological effects will be observed with
the compounds of the invention which would preclude their
administration to suitable individuals.
[0126] Each reference disclosed herein is incorporated by reference
herein in its entirety. Any patent application to which this
application claims priority is also incorporated by reference
herein in its entirety.
EXAMPLES
[0127] The examples below are carried out using standard
techniques, which 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
[0128] Strain selection, Library Production and Sequencing
[0129] The polynucleotide having the DNA sequence given in SEQ ID
NO: 1 was obtained from a library of clones of chromosomal DNA of
Staphylococcus aureus in E. coli. The sequencing data from two or
more clones containing overlapping Staphylococcus aureus DNAs was
used to construct the contiguous DNA sequence in SEQ ID NO: 1.
Libraries may be prepared by routine methods, for example:
[0130] Methods 1 and 2 below.
[0131] Total cellular DNA is isolated from Staphylococcus aureus
WCUH 29 according to standard procedures and size-fractionated by
either of two methods.
[0132] Method 1
[0133] 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.
[0134] Method 2
[0135] 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.
Sequence CWU 1
1
* * * * *