Note: Descriptions are shown in the official language in which they were submitted.
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S7REPTOCOCCUS SAG-A, A STRUCTURAL PROTEIN ASSOCIATED WITH SLS ACTIVITY
BACKGROUND TO THE INVENTION
The Streptococci are a medically important genera of
microbes known to cause several types of disease in humans. Most
strains of streptococci causing human infection belong to group A
streptococci (GAS). GAS are the cause of strep throat, scarlet
fever, impetigo, cellulitis-erysipelas, rheumatic fever, acute
glomerular nephritis, endocarditis, necrotizing fasciitis, brain
abscesses, meningitis, osteomyelitis, pharyngitis, pneumonia,
rheumatic carditis, and toxic shock. The prototype organism is
Streptococcus pyogenes.
Streptolysin S (SLS) is produced by virtually all strains of
GAS and it has a direct cytopathic effect on a broad range of cell
types (Bernheimer A.W., 1954; Freer, J.H. and J.P. Arbuthriott,
1976; Ginsburg, I. 1970). SLS is an oxygen-stable, nonimmunogenic
cytotoxin which causes a zone of beta-hemolysis observed on the
surface of blood agar. This property, used routinely in the
clinical laboratory to identify GAS, distinguishes SLS activity
from streptolysin O (SLO). SLO is an oxygen-labile, immunogenic
hemolysin which does not cause beta-hemolysis on the surface of
blood agar plates (Ginsburg, I. 1970).
The cytolytic spectrum of SLS is broad, including not only
erythrocytes of all tested eukaryotes but also lymphocytes,
polymorphonuclear leukocytes, platelets, several tissue culture
cell lines, tumor cells, bacterial protoplasts, and L forms of
bacteria as well as intracellular organelles such as mitochondria
and lysozomes (Ginsburg, I. 1970). By weight, it is one of the
most toxic agents known (Alouf, J.E. 1980; Koyama, J. and F.
Egami, 1963; and Lai, C.Y. et al, 1978).
sUI~ARY OF THS INVBNTION
The present inventors generated two SLS deficient mutants
using transposon (Tn) 9I6 mutagenesis from two clinical
Streptococcus pyogenes isolates of M1 and M18 serotypes. They
demonstrated that the non-hemolytic transconjugants were
significantly reduced in virulence in a dermonecrotic mouse model
of subcutaneous infection, despite exhibiting identical phenotypic
characteristics as their isogenic parents, including growth rates,
protease, streptolysin O, and DNAase activities and exoprotein and
M protein profiles. Further characterization of these non-
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hemolytic transconjugants revealed that each contained a single
Tn916 insertion located in the promoter region of an open reading
frame (ORF). A 390-by region of genomic DNA corresponding to the
chromosomal point of insertion of Tn916 was sequenced (see SEQ ID
NO. 12) and a novel structural gene associated with SLS activity
was identified. The gene Was designated sagA. The polypeptides
encoded by the gene are herein referred to as " SAG-A
Polypeptides" , " SAG-A" or " SAG-A Peptides" .
Studies have shown that inactivation of the sagA gene
reduces virulence of S. pyogeaes in mice, and that SAG-A plays a
direct role in tissue necrosis.
Broadly stated the present invention relates to isolated
nucleic acid molecules encoding SAG-A Polypeptides.
A further aspect of the invention provides isolated nucleic
acid molecules encoding a SAG-A Polypeptide, particularly
Streptococcus pyogenes SAG-A polypeptides, including mRNAs, DNAS,
cDNAs, genomic DNAs, PNAs, as well as antisense analogs and
biologically, diagnostically, prophylactically, clinically or
therapeutically useful variants or fragments thereof, and
compositions comprising same.
In an embodiment, the invention relates to an isolated gene
encoding SAG-A. The gene allows the production of purified SAG-A
by subcloning the gene into expression vectors under the control
of strong constitutive or inducible promoters. Since the genetic
code is degenerate, those skilled in the art will recognize that
the nucleic acid sequence in Figure 2 (SEQ ID NO: 1) is not the
only sequence which may be used to code for a peptide having the
functions of the SAG-A peptide. Changes in the nucleotide sequence
which result in production of a chemically equivalent or
chemically similar amino acid, are included within the scope of
the invention. Variants of the proteins of the invention may be
made, for example, with protein engineering techniques such as
site-directed mutagenesis which are well known in the art for
substitution of amino acids. A combination of techniques known in
the art may be used to substitute, delete, or add amino acids.
In a particular embodiment, the invention provides an
isolated nucleic acid molecule consisting of the nucleotide
sequence of SEQ ID NO: 1, 3, or 5 or a nucleotide sequence
selected from the group having at least: 40% homology, 65%
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homology, 75% homology, 85% homology, 95% homology and 98%
homology to the nucleotide sequence of SEQ ID N0: 1, 3, or 5. The
invention also includes an isolated nucleic acid molecule which
hybridizes to the above nucleic acid molecules under stringent
hybridization conditions. The nucleic acid molecule may be DNA or
RNA. The nucleic acid molecule may encode a lantibiotic or
lantibiotic fragment i.e. a polypeptide with the characteristics
of a lantibiotic. In a preferred embodiment, the nucleic acid
molecule encodes a peptide consisting of the amino acid sequence
of SEQ ID NO: 2, 4, or 6. The nucleic acid molecule may be
isolated from a group A streptococci cell.
The invention also contemplates an isolated SAG-A
polypeptide encoded by a nucleic acid molecule of the invention.
The invention also contemplates biologically, diagnostically,
prophylactically, clinically or therapeutically useful variants
thereof, including truncations, analogs, allelic or species
variations thereof, or a homolog of a polypeptide of the invention
or a truncation thereof. (Variants including truncations, analogs,
allelic or species variations, and homologs are collectively
referred to herein as " SAG-A Related Polypeptides" ). Among the
preferred embodiments of the invention are variants of SAG-A
polypeptide encoded by naturally occurring alleles of the sagA
gene.
The nucleic acid molecules of the invention may be inserted
into an appropriate vector, and the vector may contain the
necessary elements for the transcription and translation of an
inserted coding sequence. Accordingly, vectors may be constructed
which comprise a nucleic acid molecule of the invention, and where
appropriate one or more transcription and translation elements
linked to the nucleic acid molecule. Therefore, vectors are
contemplated within the scope of the invention which comprise
regulatory sequences of the invention, as well as chimeric gene
constructs wherein a regulatory sequence of the invention is
operably linked to a heterologous nucleic acid, and a
transcription termination signal.
A vector can be used to transform host cells to express a
SAG-A Polypeptide or SAG-A Related Polypeptide. Therefore, the
invention further provides host cells containing a vector of the
invention. The invention also includes a cell consisting of the
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nucleic acid molecules. In another embodiment, the invention is a
cell into which the expression vector is inserted.
The protein may be expressed by inserting a recombinant
nucleic acid molecule in a known expression system derived from
bacteria, viruses, yeast, mammals, insects, fungi or birds. The
recombinant molecule may be introduced into the cells by techniques
such as transformation, transfection and electroporation.
Retroviral vectors, adenoviral vectors, DNA virus vectors and
liposomes may be used. Suitable constructs are inserted in an
expression vector, which may also include markers for selection of
transformed cells. The construct may be inserted at a site created
by restriction enzymes. Gene expression levels may be controlled
with a transcription initiation region that regulates
transcription of the gene or gene fragment of interest in a cell
such as a prokaryotic cell or a eukaryotic cell. The
transcription initiation region may be part of the construct or
the expression vector. The transcription initiation domain or
promoter may include an RNA polymerase binding site and an mRNA
initiation site. Other regulatory regions that may be used include
an enhancer domain and a termination region. The regulatory
elements described above may be from animal, plant, yeast,
bacterial, fungal, viral, avian, insect or other sources,
including synthetically produced elements and mutated elements.
Transcription is enhanced with promoters known in the art. The
promoters may be inducible promoters and/or tissue-specific
promoters. These promoters may be selected by one skilled in the
art depending on the desired transcription initiation rate and/or
efficiency.
In one embodiment of the invention, a cell is transformed
with the gene of the invention or a fragment of the gene and
inserted in an expression vector to produce cells expressing the
SAG-A peptide. The gene or gene fragment may be either isolated
from a native source (in sense or antisense orientations),
synthesized, a mutated native or synthetic sequence, or a
combination of these.
Another embodiment of the invention relates to a method of
transforming a cell with the gene of the invention or a fragment
of the gene, inserted in an expression vector to produce a cell
expressing the SAG-A peptide. The invention also relates to a
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method of expressing the SAG-A peptides of the invention in the
cells.
Levels of gene expression may be controlled with genes that
code for anti-sense RNA inserted in the expression cassettes or
vectors described above.
The invention further broadly contemplates a recombinant
SAG-A Polypeptide, or SAG-A Related Polypeptide obtained using a
method of the invention.
The invention also includes hybrid genes and peptides, for
example where a nucleotide sequence from the gene of the invention
is combined with another nucleotide sequence to produce a fusion
polypeptide or peptide. Fusion genes and polypeptides or peptides
can also be chemically synthesized or produced using other known
techniques.
The invention further contemplates antibodies having
specificity against an epitope of a SAG-A Polypeptide, or a SAG-A
Related Polypeptide of the invention. Antibodies may be labeled
with a detectable substance and used to detect polypeptides of the
invention in biological samples, tissues, and cells.
The invention also permits the construction of nucleotide
probes that are unique to nucleic acid molecules of the invention.
Therefore, the invention also relates to a probe comprising a
sequence encoding a polypeptide of the invention, or a portion
(i.e. fragment) thereof.
DNA probes made from the sagA gene or other nucleic acid
molecules of the invention may be used to identify genes similar
to sagA. These genes could be identified using standard genetic
techniques which are well known in the art. The probes will
usually be 15 or more nucleotides in length and preferably at
Least 30 or more nucleotides. The gene fragments are capable of
hybridizing to SEQ ID NO: 1, 3, or 5 or the other sequences of the
invention under stringent hybridization conditions. A nucleic
acid molecule encoding a peptide of the invention may be isolated
from other organisms by screening a library under stringent
hybridization conditions with a labeled probe.
The nucleic acid molecules of the invention may be used for
therapeutic or prophylactic purposes, in particular genetic
immunization. Among the particularly preferred embodiments of the
invention are naturally occurring allelic variants of SAG-A and
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polypeptides encoded thereby.
The invention also provides inhibitors of SAG-A polypeptides
or SAG-A Related Polypeptides of the invention, useful as
antibacterial agents including for example antibodies of the
invention.
Still further the invention provides a method for evaluating
a test substance or compound for its ability to modulate the
activity of a SAG-A Polypeptide, or a SAG-A Related Polypeptide of
the invention. For example, a substance or compound which inhibits
or enhances the cytolytic activity of a SAG-A Polypeptide, or a
SAG-A Related Polypeptide may be evaluated.
Compounds which modulate the activity of a polypeptide of
the invention may also be identified using the methods of the
invention by comparing the pattern and level of expression of a
nucleic acid molecule or polypeptide of the invention in host
cells, in the presence, and in the absence of the compounds.
In accordance with one aspect of the invention, a
polypeptide or peptide of the invention (or the fragments of the
peptide) may be used in an assay to identify compounds that bind
the polypeptide or peptide. Methods known in the art may be used
to identify agonists and antagonists of the polypeptides or
peptides.
Methods are also contemplated that identify compounds or
substances (e. g. polypeptides) which interact with sagA regulatory
sequences (e. g. promoter sequences, enhancer sequences, negative
modulator sequences).
The substances and compounds identified using the methods of
the invention may be SagA agonists or antagonists, preferably
bacteriostatic or bactericidal agonists and antagonists.
In accordance with certain embodiments of the invention,
there are provided products, compositions, and methods for
assessing sagA expression, treating disease caused by organisms
producing streptolysin S (e. g. GAS), for example, strep throat,
scarlet fever, impetigo, cellulitis-erysipelas, rheumatic fever,
acute glomerular nephritis, endocarditis, and necrotizing
fasciitis, assaying genetic variation, and administering a SAG-A
Poiypeptide or SAG-A Related Polypeptide to an organism to raise
an immunological response against a bacteria especially a GAS.
In accordance with a further aspect of the invention, there
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are provided processes for utilizing polypeptides or nucleic acid
molecules, for in vitro purposes related to scientific research,
synthesis of DNA and manufacture of vectors.
These and other aspects, features, and advantages of the
present invention should be apparent to those skilled in the art
from the following drawings and detailed description.
BRIEF DESCRIPTION OF TH8 DRAWZNC3S
Preferred embodiments of the invention will be described in
relation to the drawings in Which:
Figure 1A is a blot of a Southern hybridization analysis of
HindIII restriction digests of genomic DNA from hemolytic wildtype
isolates and non-hemolytic transconjugants all possessing at least
one copy of Tn916, probed with tetM;
Figure 1B is a blot of a Southern hybridization analysis of
HindIII restriction digests of genomic DNA from hemolytic wildtype
isolates and non-hemolytic transconjugants all possessing at least
one copy of Tn916, probed with tetM;
Figure 2 shows the nucleotide sequence and protein
translation of sagA
Figure 3 is a blot of total RNA extracted from mutant SBNHS
(lanes 2-7) and wildtype MGAS166s (lanes 7-13) quantified,
standardized, blotted and probed using a PCR amplicon of sagA
labeled with a'~P;
Figure 4 is a graph showing comparisons of mean weight
changes of mice after infection with wild type (MGAS166s; TlBPs)
and the respective isogenic non-hemolytic mutants (SBNHS; SB30-2);
Figure 5A is a photograph of a hairless SKH1 mice 24 hours
after infection with 106 cfu of the SLS producing wildtype
MGAS166s (A);
Figure 5B is a photograph of a hairless SKH1 mice 24 hours
after infection with 106 cfu of the SLS-deficient Tn916 mutant
SBNHS;
Figure 6A is a photograph of a tissue biopsy from euthanized
mice which were infected with 106 efu of the SLS-producing
wildtype MGAS166s or the SLS-deficient Tn916 mutant SBNH5;
Figure 6B is a photograph of a tissue biopsy from euthanized
mice which were infected with 106 cfu of the SLS-deficient Tn916
mutant SBNH5; and
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_g_
Figure 7 shows the amino acid sequence of a polypeptide of
the invention with a proposed cleavage site for polypeptides of
the invention having features consistent with a lantibiotic.
DETAILBD D88CRIPTION OF TH$ INVBNTION
In accordance with the present invention there may be
employed conventional molecular biology, microbiology, and
recombinant DNA techniques within the skill of the art. Such
techniques are explained fully in the literature. See for example,
Sambrook, Fritsch, & Maniatis, Molecular Cloning: A Laboratory
Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y); DNA Cloning: A Practical Approach,
Volumes I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis
(M..J. Gait ed. 1984); Nucleic Acid Hybridization B.D. Hames &
S.J. Higgins eds. (1985); Transcription and Translation B.D. Hames
& S.J. Higgins eds (/984); Animal Cell Culture R.I. Freshney, ed.
(1986); Immobilized Cells and enzymes IRL Press, (1986); and B.
Perbal, A Practical Guide to Molecular Cloning (1984).
Nucleic Acid Molecules of the Inveatioa
As hereinbefore mentioned, the invention provides isolated
sagA nucleic acid molecules. The term "isolated" refers to a
nucleic acid (or polypeptide) removed from its natural
environment, purified or separated, or substantially free of
cellular material or culture medium when produced by recombinant
DNA techniques, or chemical reactants, or other chemicals when
chemically synthesized. Preferably, an isolated nucleic acid is at
least 60% free, more preferably at least 75% free, and most
preferably at least 90% free from other components with which they
are naturally associated. The term "nucleic acid" is intended to
include modified or unmodified DNA, RNA, including mRNAs, DNAs,
cDNAs, and genomic DNAs, or a mixed polymer, and can be either
single-stranded, double-stranded or triple-stranded. For example,
a nucleic acid sequence may be a single-stranded or 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, RNA that may be single-stranded, or more
typically, double-stranded, or triple-stranded, or a mixture of
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 DNAs or RNAs may contain one or more modified
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bases. For example, the DNAs or RNAs may have backbones modified
for stability or for other reasons. A nucleic acid sequence
includes an oligonucleotide, nucleotide, or polynucleotide.
Moreover, DNAs or RNAs comprising unusual bases, such as inosine,
or modified bases, such as tritylated bases, to name a few
examples, are nucleic acid molecules, 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 functions known to
those skilled in the art. The term 'nucleic acid molecule"
embraces such chemically, enzymatically or metabolically modified
forms of nucleic acids, as well the chemical forms of DNA and RNA
characteristic of viruses and cells, including simple and complex
cells. The term " nucleic acid molecule" and in particular DNA or
RNA, refers only to the primary and secondary structure and it
does not limit it to any particular tertiary forms.
The nucleic acid molecules Which encode for a SAG-A
polypeptide (may include only the coding sequence for the
polypeptide; the coding sequence for the polypeptide and
additional coding sequences (e. g. processing protease sequences,
transporter sequences such as sequences of translocators of the
ATP-binding cassette transporter family, immunity gene sequences,
leader or transporter sequences, propolypeptide sequences, a pre-
or pro- or prepro- protein sequences (e.g. SEQ ID NO. 4 and 6),
marker sequences]; the coding sequence for the polypeptide (and
optionally additional coding sequence) and non-coding sequences
(e. g. non-coding 5' and 3' sequences, such as transcribed, non-
translated sequences, termination signals, ribosome binding sites,
sequences that stabilize mRNA, polyadenyiation signals) of the
polypeptide. A nucleic acid molecule of the invention may
comprise a structural gene and its naturally associated sequences
that control gene expression.
Therefore, the term " nucleic acid molecule encoding a
polypeptide" encompasses a nucleic acid molecule which includes
only coding sequence for the polypeptide as well as a nucleic acid
molecule which includes additional coding and/or non-coding
sequences.
In an embodiment of the invention an isolated nucleic acid
molecule is contemplated which comprises:
(i) a nucleic acid sequence encoding a polypeptide having
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substantial sequence identity to the amino acid sequence
of SEQ. ID. NO. 2, 4 or 6;
(ii) a nucleic acid sequence having at least 95% identity
to a nucleic acid molecule encoding a polypeptide
comprising the amino acid sequence of SEQ. ID. NO. 2, 4
or 6;
(iii) a nucleic acid molecule encoding a polypeptide
comprising the amino acid sequence of SEQ. ID. N0. 2, 4
or 6;
(iv) a nucleic acid sequence complementary to (i), iii),
or (iii);
(v} a nucleic acid sequence differing from any of
(i),(ii), or (iii), in codon sequences due to the
degeneracy of the genetic code;
(vi) a nucleic acid sequence comprising at least 5
nucleotides capable of hybridizing to a nucleic acid
sequence in SEQ. ID. NO. 1, 3, or 5 or to a degenerate
form thereof;
(vii) a nucleic acid sequence encoding a truncation, an
analog, an allelic or species variation of a polypeptide
comprising the amino acid sequence shown in SEQ. ID. NO.
2, 4, or 6; or
(viii) a fragment, or allelic or species variation of (i) ,
(ii) or (iii) .
In a specific embodiment, the isolated nucleic acid molecule
comprises:
(i) a nucleic acid sequence having substantial sequence
identity or sequence similarity with a nucleic acid
sequence shown in SEQ. ID. NO. l, 3 or 5;
(ii) nucleic acid sequences complementary to (i),
preferably complementary to the full nucleic acid
sequence shown in SEQ. ID. NO. l, 3, or 5;
(iii) nucleic acid sequences differing from any of the
nucleic acid sequences of (i) or (ii) in codon
sequences due to the degeneracy of the genetic code; or
(iv) a fragment, or allelic or species variation of (i),
(ii) or (iii) .
The invention relates to a nucleic acid molecule encoding
the complementary nucleotide sequence of any of the nucleic acid
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molecules described above.The term " complementary" refers to the
natural binding of nucleic acid molecules under permissive salt
and temperature conditions by base-pairing. For example, the
sequence "A-G-T" binds to the complementary sequence " T-C-A".
Complementarity between two single-stranded molecules may be
" partial" , in which only some of the nucleic acids bind, or it
may be complete when total complementarity exists between the
single stranded molecules.
In a preferred embodiment the isolated nucleic acid
comprises a nucleic acid sequence encoding the amino acid sequence
of Streptococcus pyogenes SAG-A shown in SEQ. ID. N0. 2 or 6, or
comprises the nucleic acid sequence of Streptococcus pyogenes sagA
shown in SEQ. ID. NO. 1 or S wherein T can also be U.
The terms " sequence similarity" or " sequence identity"
refer to the relationship between two or more amino acid or
nucleic acid sequences, determined by comparing the sequences,
which relationship is generally known as " homology" . Identity in
the art also means the degree of sequence relatedness between
amino acid or nucleic acid sequences, as the case may be, as
determined by the match between strings of such sequences. Both
identity and similarity can be readily calculated (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, New York, 1987; and
Sequence Analysis Primer, Gribskov, M, and Devereux, J., eds. M.
Stockton Press, New York, 1991). While there are a number of
existing methods to measure identity and similarity between two
amino acid sequences or two nucleic acid sequences, both terms are
well known to the skilled artisan (Sequence Analysis in Molecular
Biology, von Heinje, G., Academic Press, New York, 1987; 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 for determining
identity are designed to give the largest match between the
sequences tested. Methods to determine identity are codified in
computer programs. Preferred computer program methods for
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determining 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. 215:403, 1990).
Identity or similarity may also be determined using the alignment
algorithm of Dayhoff et al [Methods in Enzymology 91: 524-545
(1983) ] .
By way of example, a nucleic acid molecule having a nucleic
acid sequence having at least, for example 95% identity to a
reference nucleic acid sequence of SEQ ID NO: 1, 3 or 5 indicates
that the nucleic acid sequence is identical to the reference
sequence except that it may include up to five point mutations per
each 100 nucleotides of the reference sequence. Therefore, to
obtain a nucleic acid molecule having at least 95% identity to a
reference sequence, up to 5% of the nucleotides in the reference
sequence must 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. Mutations of the reference sequence may occur at the 5'
or 3' terminal positions of the reference 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.
Preferably, the nucleic acids of the present invention have
substantial sequence identity using the preferred computer
programs cited herein, for example greater than 40% nucleic acid
identity; preferably greater than 50% nucleic acid identity; more
preferably greater than 65-80% sequence identity, and most
preferably at least 90% to 99% sequence identity to the sequence
shown in SEQ. ID. NO. l, 3, or 5.
Isolated nucleic acids comprising a sequence that differs
from the nucleic acid sequence shown in SEQ. ID. NO. 1, 3, or 5
due to degeneracy in the genetic code are also within the scope of
the invention. Such nucleic acids encode equivalent polypeptides
but differ in sequence from the sequence of SEQ. ID. NO. 1, 3, or
5 due to degeneracy in the genetic code. As one example, DNA
sequence mutations within sagA may result in silent mutations that
do not affect the amino acid sequence. Variations in one or more
nucleotides may exist among strains within a population due to
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natural variation. Any and all such nucleic acid variations are
within the scope of the invention. DNA sequence mutations may also
occur which lead to changes in the amino acid sequence of SAG-A
Polypeptide. These amino acid variations are also within the scope
of the present invention. In addition, strain or species
variations i.e. variations in nucleotide sequence naturally
occurring among different strains or species, are within the scope
of the invention.
Another aspect of the invention provides a nucleic acid
molecule which hybridizes under selective conditions, (e. g. high
stringency conditions), to a nucleic acid which comprises a
sequence which encodes a SAG-A Polypeptide of the invention.
Preferably the sequence encodes the amino acid sequence shown in
SEQ. ID. N0. 2 and comprises at least 5, preferably at least 10,
more preferably at least 15, and most preferably at least 20
nucleotides. In an embodiment, the nucleic acid molecule may also
consist of a sequence selected from the group consisting of 8 to
10 nucleotides of the nucleic acid molecules described above, 11
to 25 nucleotides of the nucleic acid described above and 26 to 50
nucleotides of the nucleic acid molecules described above which
hybridize to the nucleic acid molecules described above under
stringent hybridization conditions.
Selectivity of hybridization occurs with a certain degree of
specificity rather than being random. Appropriate stringency
conditions which promote DNA hybridization are known to those
skilled in the art, or can be found in Current Protocols in
Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
For example, 5.0 to 6.0 x sodium chloride/sodium citrate (SSC) or
0.5% SDS at about 45°C, followed by a wash of 2.0 x SSC at 50°C
may be employed. The stringency may be selected based on the
conditions used in the wash step. By way of example, the salt
concentration in the wash step can be selected from a high
stringency of about 0.2 x SSC at 50°C. In addition, the
temperature in the wash step can be at high stringency conditions,
at about 65°C.
It will be appreciated that the invention includes nucleic
acid molecules encoding a SAG-A Polypeptide, or a SAG-A Related
Polypeptide, including truncations of the polypeptides, allelic
and species variants, and analogs of the polypeptides as described
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herein. In particular, fragments of a nucleic acid of the
invention are contemplated that are a stretch of at least about 5,
preferably at least 10, more preferably at least 15, and most
preferably at least 20 nucleotides, more typically at least 50 to
S 200 nucleotides but less than 2 kb. It will further be appreciated
that variant forms of the nucleic acid molecules of the invention
which arise by alternative splicing of an mRNA corresponding to a
cDNA of the invention are encompassed by the invention.
In an embodiment of the invention, peptide fragments of the
proteins of the invention are provided which retain activity
similar to SAG-A and the other peptides of the invention. The
invention also includes peptide fragments of the proteins of the
invention which can be used as a research tool to characterize the
protein or its activity. Such peptides preferably consist of at
least 5 amino acids. In preferred embodiments, they may consist of
6 to 10, 11 to 15, 16 to 25 or 26 to 50 amino acids of the
proteins of the invention.
An isolated nucleic acid molecule of the invention which
comprises DNA can be isolated by preparing a labeled nucleic acid
probe based on all or part of the nucleic acid sequence shown in
SEQ. ID. NO. 1, 3, or 5. The labeled nucleic acid probe is used
to screen an appropriate DNA library (e.g. a cDNA or genomic DNA
library). For example, a cDNA library can be used to isolate a
cDNA encoding a SAG-A Polypeptide, or a SAG-A Related Polypeptide,
by screening the library with the labeled probe using standard
techniques. Alternatively, a genomic DNA library can be similarly
screened to isolate a genomic clone encompassing a sagA gene.
Nucleic acids isolated by screening of a cDNA or genomic DNA
library can be sequenced by standard techniques.
An isolated nucleic acid molecule of the invention that is
DNA can also be isolated by selectively amplifying a nucleic acid
of the invention. N Amplifying" or ~~ amplification " refers to
the production of additional copies of a nucleic acid sequence and
is generally carried out using polymerase chain reaction (PCR)
technologies well known in the art (Dieffenbach, C. W. and G. S.
Dveksler (1995) PCR Primer, a Laboratory Manual, Cold Spring
Harbor Press, Plainview, N.Y.). In particular, it is possible to
design synthetic oligonucleotide primers from the nucleotide
sequence shown in SEQ. ID. NO. 1, 3, or 5 (e.g. SEQ. ID. Nos. 5-
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14) for use in PCR. A nucleic acid can be amplified from cDNA or
genomic DNA using these oligonucleotide primers and standard PCR
amplification techniques. The nucleic acid so amplified can be
cloned into an appropriate vector and characterized by DNA
sequence analysis. cDNA may be prepared from mRNA, by isolating
total cellular mRNA by a variety of techniques, for example, by
using the guanidinium-thiocyanate extraction procedure of Chirgwin
et al., Biochemistry, 18, 5294-5299 (1979). cDNA is then
synthesized from the mRNA using reverse transcriptase (for
example, Moloney MLV reverse transcriptase available from
Gibco/BRL, Bethesda, MD, or AMV reverse transcriptase available
from Seikagaku America, Inc., St. Petersburg, FL).
An isolated nucleic acid molecule of the invention which is
RNA can be isolated by cloning a cDNA encoding a SAG-A
Polypeptide, or a SAG-A Related Polypeptide into an appropriate
vector which allows for transcription of the cDNA to produce an
RNA molecule which encodes a SAG-A Polypeptide, or a SAG-A Related
Polypeptide. For example, a cDNA can be cloned downstream of a
bacteriophage promoter, (e.g. a T7 promoter) in a vector, eDNA can
be transcribed in vitro with T7 polymerise, and the resultant RNA
can be isolated by conventional techniques.
Nucleic acid molecules of the invention may be chemically
synthesized using standard techniques. Methods of chemically
synthesizing polydeoxynucleotides are known, including but not
limited to solid-phase synthesis which, like peptide synthesis,
has been fully automated in commercially available DNA
synthesizers (See e.g., Itakura et al. U.S. Patent No. 4,598,049;
Caruthers et al. U.S. Patent No. 4,458,066; and Itakura U.S.
Patent Nos. 4,401,796 and 4,373,071).
Determination of whether a particular nucleic acid molecule
is a sagA gene or encodes a SAG-A Polypeptide, or a SAG-A Related
Polypeptide can be accomplished by expressing the cDNA in an
appropriate host cell by standard techniques, and testing the
expressed polypeptide in the methods described herein. A sagA
cDNA or cDNA encoding a SAG-A Polypeptide, or a SAG-A Related
Polypeptide can be sequenced by standard techniques, such as
dideoxynucleotide chain termination or Maxim-Gilbert chemical
sequencing, to determine the nucleic acid sequence and the
predicted amino acid sequence of the encoded polypeptide.
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The initiation codon and untranslated sequences of a nucleic
acid molecule of the invention may be determined using computer
software designed for the purpose, such as PC/Gene
(IntelliGenetics Inc., Calif.). The transcription regulatory
sequences of a nucleic acid molecule of the invention and/or
encoding a SAG-A Polypeptide, or a SAG-A Related Polypeptide may
be identified by using a nucleic acid molecule of the invention to
probe a genomic DNA clone library. Regulatory elements can be
identified using standard techniques. The function of the
elements can be confirmed by using these elements to express a
reporter gene such as the lacZ gene which is operatively linked to
the elements. These constructs may be introduced into cultured
cells using conventional procedures.
In an embodiment of the invention a nucleic acid molecule is
provided comprising a regulatory sequence of sagA as shown in SEQ.
ID. N0. 7.
The invention contemplates nucleic acid molecules comprising
all or a portion of a nucleic acid molecule of the invention
comprising a regulatory sequence of a sagA contained in appropriate
vectors. The vectors may contain heterologous nucleic acid
sequences. ~~ Heterologous nucleic acid" refers to a nucleic acid
not naturally located in the cell. Preferably, the heterologous
nucleic acid includes a nucleic acid foreign to the cell.
In accordance with another aspect of the invention, the
nucleic acid molecules isolated using the methods described herein
are mutant sagA genes. For example, the mutant genes may be
isolated from strains either known or proposed to have altered
cytolytic activity. Mutant genes and mutant gene products may be
used in therapeutic and diagnostic methods described herein. For
example, a cDNA of a mutant sagA gene may be isolated using PCR as
described herein, and the DNA sequence of the mutant gene may be
compared to the normal gene to ascertain the mutations)
responsible for the loss or alteration of function of the mutant
gene product. A genomic library can also be constructed using DNA
from a strain known to carry a mutant gene, or a cDNA library can
be constructed using RNA from strains suspected of expressing the
mutant allele. A nucleic acid encoding a normal sagA gene or any
suitable fragment thereof, may then be labeled and used as a probe
to identify the corresponding mutant genes in such libraries.
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Clones containing mutant sequences can be purified and subjected to
sequence analysis. In addition, an expression library can be
constructed using cDNA from RNA isolated from a strain known or
suspected to express a mutant sagA gene. Gene products from
putatively mutant strains may be expressed and screened, for
example using antibodies specific for a SAG-A Polypeptide, or a
SAG-A Related Polypeptide as described herein. Library clones
identified using the antibodies can be purified and subjected to
sequence analysis.
Antisense molecules and ribozymes are contemplated within the
scope of the invention. They may be prepared by any method known in
the art for the synthesis of nucleic acid molecules. These include
techniques for chemically synthesizing oligonucleotides such as
solid phase phosphoramidite chemical synthesis. Alternatively, RNA
molecules may be generated by in vitro and in vivo transcription of
DNA sequences encoding SAG-A Polypeptide. Such DNA sequences may be
incorporated into a wide variety of vectors with suitable RNA
polymerase promoters such as T7 or SP6. Alternatively, these cDNA
constructs that synthesize antisense RNA constitutively or
inducibly can be introduced into cell lines, and cells. RNA
molecules may be modified to increase intracellular stability and
half-life. Possible modifications include, but are not limited to,
the addition of flanking sequences at the 5~ and/or 3~ ends of the
molecule or the use of phosphorothioate or 2' 0-methyl rather than
phosphodiesterase linkages within the backbone of the molecule.
This concept is inherent in the production of PNAs and can be
extended in all of these molecules by the inclusion of
nontraditional bases such as inosine, queosine, and wybutosine, as
well as acetyl-, methyl-, thio-, and similarly modified forms of
adenine, cytidine, guanine, thymine, and uridine which are not as
easily recognized by endogenous endonucleases.
Polvpe~tides of the Invention
The term " polypeptide" used herein generally refers to any
protein or peptide comprising two or more amino acids joined to
each other by peptide bonds or modified peptide bonds. The term
refers to both short chains (i.e. peptides, oligopeptides and
oligomers) and to longer chains (i.e. proteins). Polypeptides may
contain amino acids other than the 20 gene encoded amino acids.
Polypeptides include those modified by natural processes (e. g.
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processing and other post-translational modifications) and by
chemical modification techniques. The same type of modification
may be present in the same or varying degree at several sites in a
given polypeptide and a polypeptide may contain many
modifications. Modifications may occur in the peptide backbone,
the amino acid side-chains, and the amino or carboxyl termini.
Examples of modifications include acetylation; acylation; ADP-
ribosylation; amidation; covalent attachment of flavin, a heme
moiety, a nucleotide or nucleotide derivative, a lipid or lipid
derivative, or phosphotidylinositol; cross-linking; cyclization;
disulfide bond formation; demethylation, formation of covalent
cross-links; glycosylation; hydroxylation; iodination;
methylation; myristoylation; oxidation; proteoytic processing;
phosphorylation;, racemization; lipid attachment; sulfation,
gamma-carboxylation of glutamic acid residues; and hydroxylation.
fBy way of example see Proteins-Structure and Molecular Properties
2"° Ed., T.E. Creighton, W.H. Freeman and Company, New York (1993),
and Wold, F., Posttranslational Protein Modifications:
Perspectives and Prospects, pages 1-12 in Posttranslational
Covalent Modification Of Proteins, B.C. Johnson, Ed. Academic
Press, New (1983); Seifer et al., Meth. Enzymol 182:626 (1990);
and Rattan et al., Protein Synthesis: Posttranslational
Modificatios and Aging, Ann. N.Y. Acad. Sci. 663:48 (1992)]. The
polypeptides may be branched or cyclic, with or without branching.
The polypeptides of the invention include the polypeptide
comprising the sequence of SEQ. ID. NO. 2, 4, or 6. In addition
to the amino acid sequences of SEQ. ID. N0.2, 4, or 6 the
polypeptides of the present invention include truncations of the
polypeptides of the invention, and analogs, and homologs of the
polypeptides and truncations thereof as described herein.
Truncated polypeptides may comprise peptides having an amino
acid sequence of at least five consecutive amino acids in SEQ.ID.
NO. 2, 4, or 6 where no amino acid sequence of five or more, six
or more, seven or more, or eight or more, consecutive amino acids
present in the fragment is present in a polypeptide other than a
SAG-A Polypeptide. In an embodiment of the invention the fragment
is a stretch of amino acid residues of at least 12 to 30
contiguous amino acids from particular sequences such as the
sequences shown in SEQ.ID. NO. 2, 4 or 6.
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The truncated polypeptides may have an amino group (-NH2), a
hydrophobic group (for example, carbobenzoxyl, dansyl, or T-
butyloxycarbonyl), an acetyl group, a 9-fluorenylmethoxy-carbonyl
(PMOC) group, or a macromolecule including but not limited to
lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates
at the amino terminal end. The truncated polypeptides may have a
carboxyl group, an amido group, a T-butyloxycarbonyl group, or a
macromolecule including but not limited to lipid-fatty acid
conjugates, polyethylene glycol, or carbohydrates at the carboxy
terminal end.
A truncated polypeptide or fragment 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, of a single
larger polypeptide.
In a preferred embodiment, the truncated polypeptides or
fragments are biologically active and mediate activities of SAG-A.
The fragments may have similar activity or an improved activity,
or a decreased undesirable activity. The fragments may be
immunogenic in an animal and preferably are not immunoreactive
with antibodies that are immunoreactive to polypeptides other than
SAG-A. Particularly preferred fragments are those that confer a
function essential for viability of GAS, or for initiation,
maintaining or causing disease in an individual, particularly a
human.
Cyclic polypeptides of the invention are also part of the
present invention. Cyclization may allow the polypeptide to assume
a more favorable conformation. Cyclization may be achieved using
techniques known in the art. For example, disulfide bonds may be
formed between two appropriately spaced components having free
sulfhydryl groups, or an amide bond may be formed between an amino
group of one component and a carboxyl group of another component.
Cyclization may also be achieved using an azobenzene-containing
amino acid as described by Ulysse, L., et al., J. Am. Chem. Soc.
1995, 117, 8466-8467. The side chains of Tyr and Asn may be
linked to form cyclic peptides. The components that form the bonds
may be side chains of amino acids, non-amino acid components or a
combination of the two.
It may be desirable to produce a cyclic polypeptide that is
more flexible. A more flexible peptide may be prepared by
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introducing cysteines at the right and left position of the
peptide and forming a disulphide bridge between the two cysteines.
The two cysteines are arranged so as not to deform the beta-sheet
and turn. The peptide is more flexible as a result of the length
of the disulfide linkage and the smaller number of hydrogen bonds
in the beta-sheet portion. The relative flexibility of a cyclic
peptide can be determined by molecular dynamics simulations.
Mimetics of polypeptides of the invention are also
contemplated. Mimetics may be designed based on information
i0 obtained by systematic replacement of L-amino acids by D-amino
acids, replacement of side chains with groups having different
electronic properties, and by systematic replacement of peptide
bonds with amide bond replacements. Local conformational
constraints can also be introduced to determine conformational
requirements for activity of a candidate peptide mimetic. The
mimetics may include isosteric amide bonds, or D-amino acids to
stabilize or promote reverse turn conformations and to help
stabilize the molecule. Cyclic amino acid analogues may be used to
constrain amino acid residues to particular conformational states.
Peptoids may also be used which are oligomers of N-substituted
amino acids and can be used as motifs for the generation of
chemically diverse libraries of novel molecules.
Peptides having one or more D-amino acids are contemplated
within the invention. Also contemplated are peptides where one or
more amino acids are acetylated at the N-terminus. Those skilled
in the art recognize that a variety of techniques are available
for constructing peptide mimetics with the same or similar desired
biological activity as the corresponding peptide compound of the
invention but with more favorable activity than the peptide with
respect to solubility, stability, and/or susceptibility to
hydrolysis and proteolysis. See for example, Morgan and Gainor,
Ann. Rep. Med. Chem., 24:243-252 (1989?. Mimetics of a
lantibivtic, nisin A, prepared by substitution, deletion and
insertion of amino acids in the lantibiotic are taught in U.S.
Patent No. 5,594,103 (De Vos et aI.). Examples of other peptide
mimetics are described in U.S. Patent No. 5,643,873. Mimetics of
the proteins of the invention may also be made according to other
techniques known in the art. For example, by treating a protein of
the invention with an agent that chemically alters a side group by
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converting a hydrogen group to another group such as a hydroxy or
amino group.
The polypeptides of the invention may also include analogs,
and/or truncations thereof as described herein, which may include,
but are not limited to the polypeptides, containing one or more
amino acid substitutions, insertions, and/or deletions. Amino
acid substitutions may be of a conserved or non-conserved nature.
Conserved amino acid substitutions involve replacing one or more
amino acids with amino acids of similar charge, size, and/or
hydrophobicity characteristics. When only conserved substitutions
are made the resulting analog should be functionally equivalent to
the native polypeptide. Non-conserved substitutions involve
replacing one or more amino acids with one or more amino acids
which possess dissimilar charge, size, and/or hydrophobicity
characteristics. For example, a hydrophobic residue such as
methionine can be substituted for another hydrophobic residue such
as alanine. An alanine residue may be substituted with a more
hydrophobic residue such as leucine, valine or isoleucine. An
aromatic residue such as phenylalanine may be substituted for
tyrosine. An acidic, negatively charged amino acid such as
aspartic acid may be substituted for glutamic acid. A positively
charged amino acid such as lysine may be substituted for another
positively charged amino acid such as arginine.
One or more amino acid insertions may be introduced into a
polypeptide of the invention. Amino acid insertions may consist of
single amino acid residues or sequential amino acids ranging from
about 2 to 15 amino acids in length.
Deletions may consist of the removal of one or more amino
acids, or discrete portions from the polypeptide sequence. The
deleted amino acids may or may not be contiguous. The lower limit
length of the resulting analog with a deletion mutation is about
10 amino acids, preferably 20 amino acids.
An allelic variant at the polypeptide level differs from
another polypeptide by only one, or at most, a few amino acid
substitutions. A species variation of a polypeptide of the
invention is an allelic variation which is naturally occurring
among different species. The polypeptides of the invention also
include homologs and/or truncations thereof as described herein.
Such homologs include polypeptides whose amino acid sequences are
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comprised of the amino acid sequences of regions from other
species that hybridize under selective hybridization conditions
(see discussion of selective and in particular stringent
hybridization conditions herein) with a probe used to obtain a
polypeptide of the invention. These homologs will generally have
the same regions which are characteristic of a polypeptide of the
invention. It is anticipated that a polypeptide comprising an
amino acid sequence which is at least 20% identical, preferably at
least 40% identical, more preferably at least 60% identical, and
most preferably at least 80%-95% identical with an amino acid
sequence of SEQ. ID. N0.2, 4, or 6 will be a homolog. A percent
amino acid sequence similarity or identity is calculated using the
methods described herein, preferably the computer programs
described herein.
The invention also contemplates isoforms of the polypeptides
of the invention. An isoform contains the same number and kinds
of amino acids as the polypeptide of the invention, but the
isoform has a different molecular structure. The isoforms
contemplated by the present invention are those having the same
properties as a polypeptide of the invention as described herein.
The present invention also includes polypeptides of the
invention conjugated with a selected polypeptide (see description
of targeting agents below), or a marker polypeptide (see below) to
produce fusion polypeptides. Additionally, immunogenic portions of
a polypeptide of the invention are within the scope of the
invention.
Antigenically, epitopically, or immunologically equivalent
variants of a SAG-A polypeptide form a particular aspect of this
invention. Antigenically equivalent variants encompass a
polypeptide or its equivalent which will be recognized by certain
antibodies which when raised to the polypeptide of the invention,
interfere with the activity of a polypeptide of the invention. An
immunologically equivalent derivative encompasses a peptide or
equivalent which when used in a suitable formulation to raise
antibodies in a vertebrate, produces antibodies which interfere
with the activity of a polypeptide of the invention.
A polypeptide of the invention may be prepared using
recombinant DNA methods. Accordingly, the nucleic acid molecules
of the present invention having a sequence which encodes a
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polypeptide of the invention may be incorporated in a known manner
into an appropriate expression vector which ensures good
expression of the polypeptide. Possible expression vectors
include but are not limited to chromosomal, episomal, and virus-
derived vectors. For example, the vectors may be derived from
bacterial plasmids, from bacteriophage, from transposons, from
yeast episomes, from insertions elements, from yeast chromosomal
elements, from viruses such as baculoviruses, papova virus, 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.
Generally, any system or vector suitable to maintain, produce or
express a nucleic acid of the invention and/or to express a
polypeptide of the invention in a selected host cell may be used.
The invention therefore contemplates a vector of the
invention containing a nucleic acid molecule of the invention, and
optionally the necessary regulatory sequences for the
transcription and translation of the inserted
polypeptide-sequence. Suitable regulatory sequences may be
derived from a variety of sources, including bacterial, fungal,
plant, viral, avian, mammalian, or insect genes, or other sources
(For example, see the regulatory sequences described in Goeddel,
Gene Expression Technology: Methods in Enzymology 185, Academic
Press, San Diego, CA (1990). Selection of appropriate regulatory
sequences is dependent on the host cell chosen as discussed below,
and may be readily accomplished by one of ordinary skill in the
art. The necessary regulatory sequences may be supplied by a
native polypeptide and/or its flanking regions.
~ In an embodiment of the invention, a recombinant nucleic
acid molecule for a SAG-A peptide is provided that contains
suitable transcriptional or translational regulatory elements.
Suitable regulatory elements are derived from a variety of
sources, and they may be readily selected by one with ordinary
skill in the art. For example, if one were to upregulate the
expression of the gene, one could insert the sense sequence and
the appropriate promoter into the vehicle. If one Were to
downregulate the expression of the gene, one could insert the
antisense sequence and the appropriate promoter into the vehicle.
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These techniques are known to those skilled in the art.
Examples of regulatory elements include a transcriptional
promoter and enhancer or RNA polymerase binding sequence, a
ribosomal binding sequence, including a translation initiation
signal. Additionally, depending on the vector employed, other
genetic elements, such as selectable markers, may be incorporated
into the recombinant molecule.
The invention further provides a vector comprising a DNA
nucleic acid molecule of the invention cloned into the vector in
an antisense orientation. That is, the DNA molecule is linked to
a regulatory seguence in a manner which allows for expression, by
transcription of the DNA molecule, of an RNA molecule which is
antisense to a nucleic acid sequence of a nucleic acid molecule of
the invention. Regulatory sequences linked to the antisense
nucleic acid can be chosen which direct the continuous expression
of the antisense RNA molecule in a variety of cell types, for
instance a viral promoter and/or enhancer, or regulatory sequences
can be chosen which direct tissue or cell type specific expression
of antisense RNA.
The expression vector of the invention may also contain a
marker gene which facilitates the selection of host cells
transformed or transfected with a vector of the invention.
Examples of marker genes are genes encoding a polypeptide such as
6418 and hygromycin which confer resistance to certain drugs, ~i-
galactosidase, chloramphenicol acetyltransferase, firefly
luciferase, or an immunoglobulin or portion thereof such as the Fc
portion of an immunoglobulin preferably IgG. The markers can be
introduced on a separate vector from the nucleic acid of interest.
The vectors may also contain genes which encode a fusion
moiety which provides increased expression of the recombinant
polypeptide; increased solubility of the recombinant polypeptide;
and aid in the purification of the target recombinant polypeptide
by acting as a ligand in affinity purification. For example, a
proteolytic cleavage site may be added to the target recombinant
polypeptide to allow separation of the recombinant polypeptide
from the fusion moiety subsequent to purification of the fusion
polypeptide. Typical fusion expression vectors include pGEX
(Amrad Corp., Melbourne, Australia), pMAL (New England Biolabs,
Beverly, MA) and pRITS (Pharmacia, Piscataway, NJ) which fuse
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glutathione S-transferase (GST), maltose E binding polypeptide, or
polypeptide A, respectively, to the recombinant polypeptide.
Appropriate secretion signals may also be incorporated into the
expressed polypeptide to facilitate secretion of the translated
polypeptide.
The vectors may be introduced into host cells to produce a
transformed or transfected host cell. The terms "transfected" and
"transfection" encompass the introduction of nucleic acid (e.g. a
vector) into a cell by one of many standard techniques. A cell is
~~ transformed" by a nucleic acid when the transfected nucleic
acid effects a phenotypic change. Prokaryotic cells can be
transformed with nucleic acid by, for example, electroporation or
calcium-chloride mediated transformation. Nucleic acid can be
introduced into mammalian cells via conventional techniques such
as calcium phosphate or calcium chloride co-precipitation, DEAE-
dextran-mediated transfection, lipofectin, transvection, cationic
lipid-mediated transfection, scrape loading, transduction,
ballistic introduction, infection. electroporation or
microinjection. Suitable methods for transforming and
transfecting host cells can be found in Sambrook et al. (Molecular
Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor
Laboratory press (1989)), and other laboratory textbooks.
Suitable host cells include a wide variety of prokaryotic
and eukaryotic host cells. For example, the polypeptides of the
invention may be expressed in bacterial cells such as
streptococci, staphylococci, enterococci, E. coli, streptomyces,
lactic acid bacteria, and Bacillus swbstilis, fungal cells such as
yeast cells and Aspergillus cells, insect cells such as Drosophila
S2 and Spodoptera Sf9; animal cells such as CHO, COS, HeLa, C127,
3T3, BHK, 293, and plant cells. Other suitable host cells can be
found in Goeddel, Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, CA (199 1).
A host cell may also be chosen which modulates the
expression of an inserted nucleic acid sequence, or modifies (e. g.
glycosylation or phosphorylation) and processes (e.g. cleaves) the
polypeptide in a desired fashion. Host systems or cell lines may
be selected which have specific and characteristic mechanisms for
post-translational processing and modification of polypeptides.
For long-term high-yield stable expression of the polypeptide,
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cell lines and host systems which stably express the gene product
may. be engineered.
Polypeptides of the invention can be recovered and purified
from recombinant host cells 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, lectin
chromatography, and in particular high performance liquid
chromatography. If a polypeptide is denatured during isolation and
purification well known refolding techniques may be used to
regenerate the active conformation.
In accordance with one aspect of the invention a method is
provided for preparing a SAG-A Polypeptide, or SAG-A Related
Polypeptide utilizing the purified and isolated nucleic acid
molecules of the invention. In particular, a method for preparing
a SAG-A Polypeptide, or a SAG-A Related Polypeptide is provided
comprising:
(a) transferring a vector of the invention comprising a
nucleic acid sequence encoding a SAG-A Polypeptide, or a SAG-A
Related Polypeptide, into a host cell;
(b) selecting transformed host cells from untransformed
host cells;
(c) culturing a selected transformed host cell under
conditions which allow expression of the SAG-A Polypeptide, or
a SAG-A Related Polypeptide; and
(d) isolating the SAG-A Polypeptide, or SAG-A Related
Polypeptide.
Host cells may also comprise genes encoding accessory
proteins including but not limited to processing proteases (e. g.
see SEQ ID NO. 8 and 9), translocators of the ATP-binding cassette
transporter family (e. g. see SEQ. ID. NO. 10 and 11), regulatory
proteins, and dedicated producer self-protection mechanisms. These
genes may be those naturally associated with SAG-A or associated
with other proteins including nisin, Pep5, subtilin, epilancin,
epidermin, gallidermin, lacticin, streptoccin, salivaricin A,
mutacin, lactocin S, carnocin, or cytolysin L1 or L2 (see Sahl et
al Eur. J. Biochem. 230:827, 1995). The genes encoding the
accessory proteins may be introduced into the host cell as part of
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the vector comprising a nucleic acid molecule of the invention or
they may be on a separate vector.
Host cells and in particular cell lines produced using the
methods described herein may be particularly useful in screening
and evaluating substances or compounds that modulate the activity
of a polypeptide of the invention.
The polypeptides of the invention may also be prepared by
chemical synthesis using techniques well known in the chemistry of
polypeptides such as solid phase synthesis or synthesis in
homogenous solution ( See for example, Merrifield, 1964, J. Am.
Chem. Assoc. 85:2149-2154, Houbenweyl, 1987, Methods of Organic
Chemistry, ed. E. Wansch, Vol. 15 I and II, Thieme,
Stuttgartsee, J. M. Stewart, and J.D. Young, Solid Phase Peptide
Synthesis, 2"d Ed., Pierce Chemical Co., Rockford III. (1984) and
G. Barany and R.B. Merrifield, The Peptides: Analysis Synthesis,
Biology editors E. Gross and J. Meienhofer Vol. 2 Academic Press,
New York, 1980, pp. 3-254 for solid phase synthesis techniques;
and M Bodansky, Principles of Peptide Synthesis, Springer-Verlag,
Berlin 1984, and E. Gross and J. Meienhofer, Eds., The Peptides:
Analysis, Synthesis, Biologu, supra, Vol 1, for classical solution
synthesis.)
N-terminal or C-terminal fusion or chimeric polypeptides
comprising a polypeptide of the invention conjugated with other
molecules, such as polypeptides (e. g. markers or targeting agents)
may be prepared by fusing, through recombinant techniques, the
N-terminal or C-terminal of a polypeptide of the invention, and
the sequence of a selected polypeptide or marker polypeptide with
a desired biological function. The resultant fusion polypeptides
contain a polypeptide of the invention fused to the selected
polypeptide or marker polypeptide as described herein.
Polv~efltidas With Lantibiotic Characteristics
The amino acid sequence of the SAG-A polypeptide shown in
SEQ. ID. NO. 1 exhibits close similarities with the class of
bacterial peptides known as lantibiotics (Borgia 1997). Sequence
characterization information for sagA is described in Example 1.
Several features characteristic of this class of molecules are
described in Example 1 and known in the art. The similarity of
many of these features with SAG-A shows that it is related to the
lantibiotic class of molecules.
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Lantibiotics constitute a unique class of ribosomally-
synthesized, antimicrobial peptides produced by gram positive
bacteria. Their unique structural properties result from the
presence of intra-molecular rings formed by thioether bonds of the
post-translationally modified amino acids lanthionine (Lan) and 3-
methyllanthionine (MeLan) (Nes and Tagg 1996).
Lantibiotics are synthesized on the ribosome as a prepeptide
or precursor which undergoes several post-translational
modifications and removal of leader sequences. The modifications
may include dehydration of specific hydroxyl amino acids to form
dehydroamino acids, addition of neighbouring sulfhydryl groups to
form thioethers and in specific cases other modifications such as
introduction of D-alanine residues from L-serine, formation of
lysino-alanine bridges, formation of novel N-terminal blocking
groups and oxidative decarboxylation of a C-terminal cysteine.
The first identified lantibiotic, nisin, produced by certain
strains of Lactococcus lactis subsp. lactis, is the most widely
used lantibiotic in the industrial sector (Delves-Broughton et al.
1996). Following its first successful application as a
preservative in processed cheese products, it has since been used
in numerous other foods and beverages, including beer, wine and
low pH foods such as salad dressings. It is used in natural cheese
production and as an adjunct in food processing (Delves-Broughton
et al. 1996). It is also used in the treatment and prophylaxis of
Helicobacter pylori associated peptic ulcer disease in humans
(Blackburn and Projan 1994). Since nisin has also been
demonstrated to be particularly bactericidal towards both
Staphylococcus and Streptococcus species, it is used as an
effective therapeutic agent in the treatment of bovine mastitis
(Delves-Broughton et al. 1996).
Other lantibiotics also have numerous commercial
applications. For example, the lantibiotic, mersacidin, produced
by Bacillus subtilis HIL Y-85,54728 may be an alternative
therapeutic agent for the treatment of staphylococcal infections
since it is active in vivo against methicillin-resistant
Staphylococcus aureus (MRSA) strains (Chatterjee et al. 1992).
U.S. Patent No. 5,667,991 (Koller et aI.) teaches a recombinant
gene encoding mersacidin. U.S. Patent No. 5,112,806 (De Vos et
al.) discloses pharmaceutical compositions containing mersacidin.
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Several patents have been filed on the use of lantibiotics
in other therapeutic combinations. U.S. Patent No. 5,458,876
(Monticello) discloses a composition for lysing Listeria
monocytogenes containing lysozyme and either of the lantibiotics
nisin and subtilin. U.S. Patent Nos. 5,512,269 and 5,683,675
(Molina y Vedia, et a~.) teach a method of facilitating the
clearance of retained pulmonary secretions in a subject by
administering lantibiotics topically to the lungs. U.S. Patent No.
5,043,176 discloses a synergistic antimicrobial composition
consisting of an antimicrobial polypeptide, a buffering component
and a hypothiocyanate component. U.S. Patent No. 5,670,138
discloses a lantibiotic mouth care product. A more comprehensive
review of additional lantibiotics and their applications is found
in Ray and Daeschel 1992, Klaenhammer 1993 and De Vuyst and
Vandamme 1994.
Despite the widespread potential applications of
lantibiotics, they are individually distinct in their bactericidal
activity. This limitation creates a need for novel lantibiotics
which can be used in the food and pharmaceutical industries.
The invention provides a novel peptide with features
consistent with or characteristic of a lantibiotic, encoded by a
gene of the invention. The invention also includes an isolated
peptide produced from nucleic acid molecules described herein,
including an isolated peptide produced from an expression vector.
In a preferred embodiment, the isolated peptide consists of the
amino acid sequence in SEQ ID NO: 2 or an isolated peptide having
at least 40% homology, 65% homology, 75% homology, 85% homology,
95% homology and 98% homology to the peptide of SEQ ID NO: 2. The
peptide is preferably a lantibiotic. The peptide can be isolated
from a group A streptococci cell. The invention also includes an
isolated peptide consisting of at least 5 amino acids, 6 to 15
amino acids or 15 to 30 amino acids of the peptides described
above.
The invention also contemplates a precursor of a polypeptide
of the invention which when expressed in bacteria is converted
after translation to the protein streptolysin A. In particular,
the invention contemplates a prepeptide or precursor protein (SEQ
ID NO 2) having a propeptide part of the polypeptide (e.g. SEQ ID
NO. 6) fused to one or more leader sequences (e. g. SEQ~ID No.4).
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Some or all of the leader sequences may be removed (e.g. SEQ ID
NO. 4) to provide a propeptide which is modified during
biosynthesis to form a polypeptide having features consistent with
a mature lantibiotic. See Figure 7 showing the proposed Gly-Gly
cleavage site.
The invention provides a gene leader fragment encoding a
peptide leader sequence which induces post-translational
modification of amino acids selected from the group consisting of
Cys, Ser, Thr, and mixtures thereof, the fragment comprising the
sequence of SEQ ID NO. 3. A polypeptide sequence is also provided
which when attached as a leader to a protein precursor which
undergoes post-translational modification, assists in inducing the
modification, comprising a polypeptide having the biological
function of the amino acid sequence of SEQ ID NO. 4.
The invention also contemplates modifications of the
prepeptides produced by coupling leader sequences from other
lantibiotics including nisin, Pep5, subtilin, epilancin,
epidermis, gallidermin, lacticin, streptoccin, salivaricin A,
mutacin, lactocin S, carnocin, or cytolysis L1 or L2, to a
propeptide part of the protein (e. g. SEQ ID N0. 6). In addition,
leader sequences of a polypeptide of the invention having a
structure consistent with a lantibiotic can be coupled to
propeptides of other lantibiotics including nisin, Peps, subtilin,
epilancin, epidermis, gallidermin, lacticin, streptoccin,
salivaricin A, mutacin, lactocin S, carnocin, or cytolysis L1 or
L2 (See Sahl et al Eur. J. Biochem. 230:827, 1995).
Polypeptides of the invention with features characteristic
of a lantibiotic may be produced by inserting an expression vector
containing a nucleic acid of the invention in a cell and
expressing the peptide.
Still further the invention relates to methods for
identifying substances that affect a polypeptide having
characteristics of a lantibiotic. Such substances may be
identified by determining if a test substance affects the
conversion of a precursor of a polypeptide of the invention to the
mature protein. The precursor or mature protein may be assayed
using 3snown methods to determine the affect of the substance.
The invention also relates to food products, pharmaceutical
compositions or vaccines containing these peptides, and to a
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method for producing a lantibiotic by inserting an expression
vector in a cell and expressing the peptide.
Antibodies
A polypeptide of the invention (e.g. SEQ ID NO 2, 4, or 6)
S can be used to prepare antibodies specific for the polypeptides.
Antibodies can be prepared which bind a distinct epitope in an
unconserved region of the polypeptide. An unconserved region of
the polypeptide is one that does not have substantial sequence
homology to other polypeptides. A region from a conserved region
such as a well-characterized sequence can also be used to prepare
an antibody to a conserved region of a polypeptide of the
invention. Antibodies having specificity for a polypeptide of the
invention may also be raised from fusion polypeptides created by
expressing fusion polypeptides in host cells as described herein.
IS The invention can employ intact monoclonal or polycional
antibodies; chimeric, single chain antibodies (see U.S. Pat No.
4,946,778), simianized antibodies, humanized antibodies (Jones, P.
et al., Nature 321:522, 1986 or Tempest et al., Biotechnology
9:266, 1991), immunologically active fragments (e.g. a Fab or
(Fab)2 fragment), an antibody heavy chain, and antibody light
chain, a genetically engineered single chain Fv molecule (Ladner
et al, U.S. Pat. No. 4,946,778), or a chimeric antibody, for
example, an antibody which contains the binding specificity of a
marine antibody, but in which the remaining portions are of human
2S origin. Antibodies including monoclonal and polyclonal
antibodies, fragments and chimeras, etc. may be prepared using
methods known to those skilled in the art.
The antibodies of the invention may be used to isolate or to
identify clones expressing a polypeptide of the invention or to
purify the polypeptides using affinity chromatography. The
antibodies of the invention may also be used in diagnostic and
therapeutic applications as described herein.
Ac~lications of the Nuclaic Acid Molecules, Polvoectida , and
Antibodies of the Invention
3S It would be apparent to one skilled in the art that the
nucleic acid molecules and polypeptides of the invention may be
employed as research reagents and materials for the discovery of
treatments of, and diagnostics for disease, particularly human
disease, as further discussed herein.
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The nucleic acid molecules, SAG-A Polypeptide, or SAG-A
Related Polypeptide, and antibodies of the invention may be used
in the diagnosis of disease. For example, they may have utility in
the diagnosis of the stage of infection and the type of infection.
Eukaryotes (herein also " individuals" ), particularly mammals,
and especially humans infected with an organism comprising a
nucleic acid or polypeptide of the invention may be monitored or
diagnosed by detecting and/or localizing the nucleic acids and
polypeptides of the invention.
The applications of the present invention also include
methods for the identification of substances or compounds that
modulate the biological activity of a polypeptide of the invention
(See below). The substances and compounds, as well as
polypeptides, nucleic acids, and antibodies of the invention, etc.
may be used for the treatment of diseases. (See below).
Diaanoetic Methods
A variety of methods can be employed for the diagnostic and
prognostic evaluation of diseases. Such methods may, for example,
utilize nucleic acid molecules of the invention, and fragments
thereof, and antibodies directed against polypeptides of the
invention, including peptide fragments.
The methods described herein for detecting nucleic acid
molecules and polypeptides can be used in the diagnosis of
infectious diseases especially caused by GAS by detecting
polypeptides and nucleic acid molecules of the invention.
The nucleic acid molecules and polypeptides of the invention
are markers for group A streptococci and accordingly the
antibodies and probes described herein may also be used to
characterize a species or strain of GAS.
The methods described herein may be performed by utilizing
pre-packaged diagnostic kits comprising at least one specific
nucleic acid or antibody described herein, which may be
conveniently used, e.g., in clinical settings, to screen and
diagnose patients and to screen and identify those individuals
having a particular type or stage of infection.
Nucleic acid-based detection techniques and peptide
detection techniques are described below. The samples that may be
analyzed using the methods of the invention include those which
are known or suspected to contain sagA or a polypeptide of the
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invention. The methods may be performed on biological samples
including but not limited to cells, lysates of cells which have
been incubated in cell culture, DNA (in solutions or bound to a
solid support such as for Southern analysis), RNA (in solution or
bound to a solid support such as for northern analysis), an
extract from cells or a tissue, and biological fluids such as
serum, urine, blood, and CSF. The samples may be derived from a
patient or a culture.
Methods for Detectinc Nucleic Acid Molecules of th~ Invention
The nucleic acid molecules of the invention allow those
skilled in the art to construct nucleotide probes for use in the
detection of nucleic acid sequences of the invention in biological
materials. Suitable probes include nucleic acid molecules based
on nucleic acid sequences encoding at least 5 sequential amino
acids from regions of the SAG-A Polypeptide, or a SAG-A Related
Polypeptide (see SEQ. ID. No. 1 or 3), preferably they comprise 15
to 30 nucleotides.
A nucleotide probe may be labeled with a detectable
substance such as a radioactive label that provides for an
adequate signal and has sufficient half-life such as 32p, 3g~ 14C
or the like. Other detectable substances that may be used include
antigens that are recognized by a specific labeled antibody,
fluorescent compounds, enzymes, antibodies specific for a labeled
antigen, and luminescent compounds. An appropriate label may be
selected having regard to the rate of hybridization and binding of
the probe to the nucleotide to be detected and the amount of
nucleotide available for hybridization. Labeled probes may be
hybridized to nucleic acids on solid supports such as
nitrocellulose filters or nylon membranes as generally described
in Sambrook et al, 1989, Molecular Cloning, A Laboratory Manual
(2nd ed.). The nucleic acid probes may be used to detect sagA
genes, preferably in human biological samples. The nucleotide
probes may also be useful for example in the diagnosis or
prognosis of infections particularly those caused by GAS, and in
monitoring the progression of these conditions, or monitoring a
therapeutic treatment.
The probe may be used in hybridization techniques to detect
a sagA gene. The technique generally involves contacting and
incubating nucleic acids (e. g. recombinant DNA molecules, cloned
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genes) obtained from a sample from a patient or other cellular
source with a probe of the present invention under conditions
favourable for the specific annealing of the probes to
complementary sequences in the nucleic acids. After incubation,
the non-annealed nucleic acids are removed, and the presence of
nucleic acids that have hybridized to the probe if any are
detected.
The detection of nucleic acid molecules of the invention may
involve the amplification of specific gene sequences using an
amplification method such as PCR, followed by the analysis of the
amplified molecules using techniques known to those skilled in the
art. Suitable primers can be routinely designed by one skilled in
the art.
Genomic DNA may be used in hybridization or amplification
i5 assays of biological samples to detect abnormalities involving
sagA structure, including point mutations, insertions, deletions,
and chromosomal rearrangements. For example, direct sequencing,
single stranded conformational polymorphism analyses, heteroduplex
analysis, denaturing gradient gel electrophoresis, chemical
mismatch cleavage, and oligonucleotide hybridization may be
utilized.
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 sagA nucleic acid sequences.
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. Nuclease
protection assays (e. g. RNase and S1 protection or a chemical
cleavage method) may be used to detect sequence changes at
specific locations.
Mutations or polymorphisms in a nucleic acid molecule of the
invention may be detected by a variety of known techniques to
allow for example, for serotyping. RT-PCR preferably in
conjunction with automated detection systems (e.g. GeneScan) can
be used. For example, primers derived from SEQ ID NO: 1 or 5 may
be used to amplify nucleic acids isolated from an infected
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individual and the amplified nucleic acids may be subjected to
various techniques for elucidation of the DNA sequence. Using this
method, mutations may be detected and used to diagnose infection
and to serotype and/or classify the infectious agent.
In an embodiment of the invention a method is provided for
diagnosing disease, preferably bacterial infections, more
preferably infections caused by GAS, comprising determining from a
sample derived from an individual an increased level of expression
of a nucleic acid molecule of the invention, in particular a
nucleic acid molecule of SEQ ID NO:1 or 5. Increased or decreased
expression of sagA nucleic acids may be measured using any of the
methods well known in the art for the quantification of nucleic
acids such as for example, amplification, PCR, RT-PCR, RNase
production, Northern blotting, and other hydridization methods.
t5 Methods for Detectinc Polvceptides
Antibodies specifically reactive with a SAG-A Polypeptide, a
SAG-A Related Polypeptide, or derivatives, such as enzyme
conjugates or labeled derivatives, may be used to detect SAG-A
Polypeptides or SAG-A Related Polypeptides in various biological
materials. They may be used as diagnostic or prognostic reagents
and they may be used to detect increased or decreased levels of
SAG-A Polypeptides or SAG-A Related Polypeptides, expression, or
abnormalities in the structure of the polypeptides. A diagnostic
assay may be used to detect the presence of an infection by
detecting increased levels of SAG-A polypeptide to a control.
Immunoassays as well as other techniques such as Western Blot
analysis can be used to determine levels of a polypeptide of the
invention.
In vitro immunoassays may also be used to assess or monitor
the efficacy of particular therapies. The antibodies of the
invention may also be used in vitro to determine the level of SAG-
A Polypeptide or SAG-A Related Polypeptide expression in cells
genetically engineered to produce a SAG-A Polypeptide, or SAG-A
Related Polypeptide.
Antibodies of. the invention may be used in any known
immunoassays that rely on the binding interaction between an
antigenic determinant of a polypeptide of the invention, and the
antibodies. Examples of such assays are radioimmunoassays, enzyme
immunoassays te.g. ELISA), immunofluorescence, competitive binding
*rB
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assays, immunoprecipitation, latex agglutination,
hemagglutination, and histochemical tests. The antibodies may also
be used in Western Blot analysis. The antibodies may be used to
detect and quantify polypeptides of the invention in a sample in
order to determine their role in particular cellular events or
pathological states, and to diagnose and treat such pathological
states.
Cytochemical techniques known in the art for localizing
antigens using light and electron microscopy may be used to detect
a polypeptide of the invention. Generally, an antibody of the
invention may be labeled with a detectable substance and a
polypeptide may be detected based upon the presence of the
detectable substance. Various methods of labeling antibodies are
known in the art and may be used. Examples of detectable
IS substances include, but are not limited to, the following:
radioisotopes (e.g. , 3 Fi, 1'C, 'sS, msl, 1'lI) , fluorescent labels
(e. g., FITC, rhodamine, lanthanide phosphors), luminescent labels
such as luminol; enzymatic labels (e. g., horseradish peroxidase,
~i-galactosidase, luciferase, alkaline phosphatase,
acetylcholinesterase), biotinyl groups (which can be detected by
marked avidin e.g., streptavidin containing a fluorescent marker
or enzymatic activity that can be detected by optical or
calorimetric methods), predetermined polypeptide epitopes
recognized by a secondary reporter (e. g., leucine zipper pair
sequences, binding sites for secondary antibodies, metal binding
domains, epitope tags). In some embodiments, labels are attached
via spacer arms of various lengths to reduce potential steric
hindrance. Antibodies may also be coupled to electron dense
substances, such as ferritin or colloidal gold, which are readily
visualised by electron microscopy.
The antibody or sample may be immobilized on a carrier or
solid support which is capable of immobilizing cells, antibodies,
etc. For example, the carrier or support may be nitrocellulose, or
glass, polyacrylamides, gabbros, and magnetite. The support
material may have any possible configuration including spherical
(e.g. bead), cylindrical (e.g. inside surface of a test tube or
well, or the external surface of a rod), or flat (e. g. sheet, test
strip). Indirect methods may also be employed in which the primary
antigen-antibody reaction is amplified by the introduction of a
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second antibody, having specificity for the antibody reactive
against a polypeptide of the invention. By way of example, if the
antibody having specificity against a polypeptide of the invention
is a rabbit IgG antibody, the second antibody may be goat
anti-rabbit gamma-globulin labeled with a detectable substance as
described herein.
Where a radioactive label is used as a detectable substance,
a polypeptide of the invention may be localized by
radioautography. The results of radioautography may be
quantitated by determining the density of particles in the
radioautographs by various optical methods, or by counting the
grains.
Methods for Identifvina or 8valuatina Substanoea/Comaounds
The methods described herein are designed to identify
substances or compounds that modulate the activity of a SAG-A
Polypeptide or SAG-A Related Polypeptide. " Modulate" refers to a
change or an alteration in the biological activity of a
polypeptide of the invention. Modulation may be an increase or a
decrease in activity, a change in characteristics, or any other
change in the biological, functional, or immunological properties
of the polypeptide.
Substances and compounds identified using the methods of
the invention include but are not limited to peptides such as
soluble peptides including Ig-tailed fusion peptides, members of
random peptide libraries and combinatorial chemistry-derived
molecular libraries made of D- and/or L-configuration amino acids,
phosphopeptides (including members of random or partially
degenerate, directed phosphopeptide libraries), antibodies [e. g.
polyclonal, monoclonal, humanized, anti-idiotypic, chimeric,
single chain antibodies, fragments, (e.g. Fab, F(ab)2, and Fab
expression library fragments, and epitope-binding fragments
thereof)], and small organic or inorganic molecules. A substance
or compound may be an endogenous physiological compound or it may
be a natural or synthetic compound.
Substances which modulate a SAG-A Polypeptide or SAG-A
Related Polypeptide can be identified based on their ability to
interact with a SAG-A Polypeptide or SAG-A Related Polypeptide.
Therefore, the invention also provides methods for identifying
substances which interact with a SAG-A Polypeptide or SAG-A
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Related Polypeptide. Substances identified using the methods of
the invention may be isolated, cloned and sequenced using
conventional techniques. A substance that interacts with a
polypeptide of the invention may be an agonist or antagonist of
the biological or immunological activity of a polypeptide of the
invention.
The term "agonist", refers to a molecule that increases the
amount of, or prolongs the duration of, the activity of the
polypeptide. The term °antagonist" refers to a molecule which
decreases the biological or immunological activity of the
polypeptide. Agonists and antagonists may include proteins,
nucleic acids, carbohydrates, or any other molecules that interact
with a polypeptide of the invention.
Substances which can interact with a polypeptide of the
1S invention may be identified by reacting the polypeptide with a
test substance which potentially interacts with the polypeptide,
under conditions Which permit the interaction, and removing and/or
detecting complexes of the polypeptides and substance. Substance-
polypeptide complexes, free substance, non-complexed polypeptide,
or activated golypeptide may be assayed. Conditions which permit
the formation of complexes may be selected having regard to
factors such as the nature and amounts of the substance and the
polypeptide.
Substance-polypeptide complexes, free substances or
2S non-complexed poiypeptides may be isolated by conventional
isolation techniques, for example, salting out, chromatography,
electrophoresis, gel filtration, fractionation, absorption,
polyacrylamide gel electrophoresis, agglutination, or combinations
thereof. To facilitate the assay of the components, antibody
against the polypeptide or the substance, or labelled polypeptide,
or a labelled substance may be utilized. The antibodies,
polypeptides, or substances may be labelled with a detectable
substance as described above. A polypeptide, or the substance used
in the method of the invention may be insolubilized.
In an embodiment 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 nucleic acid molecule of the invention. The method may comprise
contacting a polypeptide of nucleic acid molecule of the invention
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with a compound to be screened under conditions to permit binding
to or other interaction between the compound and the polypeptide
or nucleic acid molecule 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 nucleic acid molecule with the compound, and
determining whether the compound binds to or otherwise interacts
with and activates or inhibits an activity of the polypeptide or
nucleic acid molecule by detecting the presence or absence of a
signal generated from the binding or interaction of the compound
with the polypeptide or nucleic acid molecule.
The invention also contemplates a method for evaluating a
compound for its ability to modulate the activity of a polypeptide
of the invention, by assaying for an agonist or antagonist (i.e.
enhancer or inhibitor3 of the interaction of the polypeptide with
a substance that binds or otherwise interacts with the
polypeptide. The basic method for evaluating if a compound is an
agonist or antagonist of the interaction of a polypeptide of the
invention and a substance that interacts with the polypeptide, is
to prepare a reaction mixture containing the polypeptide and the
substance under conditions which permit the formation of
substance- polypeptide complexes, in the presence of a test
compound. The test compound may be initially added to the mixture,
or may be added subsequent to the addition of the polypeptide and
substance. Control reaction mixtures without the test compound or
with a placebo are also prepared. The formation of complexes is
detected and the formation of complexes in the control reaction
but not in the reaction mixture indicates that the test compound
interferes with the interaction of the polypeptide and substance.
The reactions may be carried out in the liquid phase or the
polypeptide, substance, or test compound may be immobilized as
described herein.
The reagents suitable for applying the methods of the
invention to evaluate compounds that modulate a polypeptide of the
invention may be packaged into convenient kits providing the
necessary materials packaged into suitable containers. The kits
may also include suitable supports useful in performing the
methods of the invention.
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The nucleic acid 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 nucleic acid
sequences encoding the amino terminal regions of the encoded
protein or the translation facilitating sequences of the
respective mRNA can be used to construct antisense sequences to
control the expression of the coding sequence of interest.
Polypeptides of the invention that have characteristics of a
lantibiotic may be used to design drugs. Since lantibiotics are
gene-encoded peptides as opposed to peptide antibiotics
synthesized by multi-enzyme complexes, site-directed mutagenesis
can be used in the construction of modified SAG-A peptides. One
skilled in the art is familiar with techniques to substitute amino
acids for certain residues of SAG-A to optimize chemical and
physical properties such as enhanced bactericidal action and
stability. Techniques for the genetic engineering of ~~ new drugs"
are used to engineer SAG-A as has been done with the lantibiotic
subtilin (Liu and Hansen 1992).
Vaccines
The marked impairment in the virulence of two clinically
relevant S. pyogenes strains by transposon insertion in the sag~1
promoter region shows that SAG-A plays an important role in GAS
pathogenesis. Therefore, antibodies directed against the SAG-A
peptide may provide protection against streptococcal infections
and the peptide may be used in a human vaccine. The invention
includes the antibodies, fragments of the antibodies and the
hybridoma, which secretes the monoclonal antibodies.
Accordingly broadly stated, the invention contemplates a
vaccine comprising an immunogenic polypeptide of the invention.
The polypeptides provided by the invention can be used to
vaccinate a subject for protection from a particular disease,
infection, or condition caused by an organism producing a SAG-A
polypeptide, particularly a GAS infection. A SAG-A Polypeptide or
SAG-A Related Polypeptide (e.g, a fragment or variant), can be
used to inoculate a host organism such that the host produces an
active immune response (e. g. an antibody and/or T cell immune
response) to the presence of the polypeptide which can later
protect the host from infection by an organism producing the
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polypeptide. One skilled in the art will appreciate that an immune
response especially a cell-mediated immune response to a
polypeptide of the invention can provide later protection from
reinfection or from infection from a closely related strain.
Immunization can be achieved through artificial vaccination
(Kuby, J. Immunology W.H. Freeman and Co. New York, 1992). This
immunization may be achieved by administering to individuals the
polypeptide either alone or with a pharmaceutically acceptable
carrier.
Immunogenic amounts of a polypeptide of the invention can be
determined using standard procedures. Briefly, various
concentrations of the polypeptide are prepared, administered to
individuals, and the immunogenic response (e.g. production of
antibodies or cell mediated immunity) to each concentration is
determined. Procedures for monitoring the immunogenic response of
individuals after inoculation with the polypeptide are well known.
For example, samples can be assayed using ELISA to detect the
presence of specific antibodies, or lymphocytes, or cytokine
production can be monitored. The specificity of a putative
immunogenic antigen of a polypeptide can be determined by testing
sera, other fluids or lymphocytes from the inoculated individual
for cross-reactivity with any closely related poiypeptides.
The amount of the polypeptide administered will depend on
the individual, the condition of the individual, the size of the
individual etc. but will be at least an immunogenic amount. The
polypeptide can be formulated with adjuvants and with additional
compounds including cytokines, with a pharmaceutically acceptable
carrier.
Techniques for preparing or using vaccines are known in the
art. To prepare the vaccine, the peptide, or a fragment of the
peptide, may be mixed with other antigens, a vehicle or an
excipient. Examples of peptide vaccines are found in U.S. Patent
Nos. 5,679,352, 5,194,254 and 4,950,480. Techniques for preparing
vaccines involving site directed mutagenesis are described in U.S.
Patent Nos. 5,714,372, 5,543,302, 5,433,945, 5,358,868, 5,332,583,
5,244,657, 5,221,618, 5,14?,643, 5,085,862 and 5,073,494. It will
be appreciated that a SAG-A Polypeptide or SAG-A Related
Polypeptide may be chemically treated (e. g. glutaraldehyde) before
it is used as a vaccine. Chemical treatment may substantially
*rB
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decrease or destroy the biological activity of the polypeptide.
The pharmaceutically acceptable carrier or adjuvant employed
in a vaccine of the present invention can be selected by standard
criteria (Arnon, R. (ed.) " Synthetic Vaccines" I:83-92, CRC
Press, Inc. Boca Raton, Fla., 1987). By ~ pharmaceutically
acceptable" is meant material that is not biologically or
otherwise undesirable that is, the material may be administered to
an individual along with the selected compound without causing any
undesirable biological effects or interacting in an undesirable
manner with any of the other components of the pharmaceutical
compositions in which it is contained. The carrier or adjuvant may
depend on the method of administration and the particular
individual.
Methods of administration can be oral, sublingual, mucosal,
inhaled, absorbed, or by injection. Actual methods of preparing
appropriate dosage forms are known or will be apparent to those
skilled in the art. (See for example, Remington's Pharmaceutical
Sciences (Martin E.W. (ed) latest edition Mack Publishing Co.
Easton, Pa}.
Parenteral administration if used is generally characterized
by injection. Injectables can be prepared in conventional forms,
either as liquid solutions or suspensions, solid forms suitable
for solution or suspension in liquid prior to injection or as
emulsions. A more recently revised approach for parenteral
administration involves use of a slow release or sustained release
system, such that a constant level of dosage is maintained (see
for example U.S. Pat. No. 3,710,795}.
It is also contemplated that immunization can be achieved by
a genetic immunization approach. A nucleic acid molecule of the
invention may be used in genetic immunization employing a suitable
delivery system. Examples of such systems include 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 1986, 83: 9551),
encapsulation of DNA in various forms of liposomes (Kaneda et al.,
Science 243:375, 1989); particle bombardment (Tang et al. Nature,
1992, 356:152, Eisenbraun et al, DNA Cell Biol 1993, 12:791); and
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in vivo infection using cloned retroviral vectors (Seeger et al,
PNAS 81:5849, 1984).
In an embodiment of the invention, a peptide of the
invention is used as a human vaccine for preventing streptococcal
disease, such as necrotizing fasciitis (NF) and streptococcal
toxic-shock syndrome (STSS).
Comvositions and Treatmeats
The polypeptides, nucleic acid molecules, substances or
compounds identified by the methods described herein, antibodies,
and antisense nucleic acid molecules of the invention may be used
for modulating the activity of a polypeptide or nucleic acid
molecule of the invention. The polypeptides etc. may have
particular application in the treatment of diseases. Inhibitors
or antagonists of a polypeptide of the invention having cytolytic
IS activity may be used to treat disorders including diseases caused
by streptococcal infections such as endocarditis, cellulitis,
brain abscesses, glomerulonephritis, pneumonia, meningitis,
osteomyelitis, pharyngitis, rheumatic fever, pneumonia, strep
throat, scarlet fever, impetigo, necrotizing fasciitis, rheumatic
carditis, and toxic shock.
Inhibitors and antagonists of a polypeptide of the invention
are particularly useful in reducing tissue necrosis caused by an
organism producing a polypeptide of the invention. Therefore, in a
preferred embodiment the inhibitors or antagonists are used to
treat necrotizing fasciitis.
A polypeptide of the invention which has characteristics of
a lantibiotic (e.g. a SAG-A peptide) may be useful in both the
pharmaceutical and food industries. It may exhibit antibacterial
activity against a wide variety of gram-negative and gram-positive
bacteria and it may be used as a food preservative, an
antibacterial agent for medical use, a preservative for
construction materials and/or paints, an antibacterial agent for
horticultural use, a preservative for livestock feed, a
preservative for fish feed, and the like, and it may be used as an
antibacterial agent in a wide variety of fields.
The methods of preparing food preservative agents, including
lantibiotics, and their use are well known in the art. For
examples, see U.S. Patent Nos. 5,646,014, 5,453,420, 5,397,499,
5,260,271, 5,213,833, 5,026,856, 4,961,945, 4,728,376, 4,670,288,
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4,538,002, 4,410,547, and 3,936,359. The applications for such
polypeptides (e.g. SAG-A peptide) include some of the same uses of
other lantibiotics known in the art. Some of the preferred
advantages of the SAG-A peptide are due to its unique stability
and solubility as compared with other lantibiotics when exposed to
different food environments, features which are important when
using SAG-A as a biopreservative. SAG-A may be used with a variety
of solid, semi-solid and liquid food products. Also, the distinct
antimicrobial activity of SAG-A against multidrug-resistant
bacteria may be tested and characterized using techniques well
known in the art.
Polypeptides of the invention having cytolytic activity may
be used to lyse microbial and eukaryotic cells. Accordingly, the
invention provides a method for lysing microbial and eukaryotic
cells comprising contacting the cells with a polypeptide of the
invention having cytolytic activity in an amount effective to lyse
the cells. The cells include gram positive and gram negative
procaryotic microorganisms (e.g. bacteria, fungi, viruses, or
protozoans), neoplastic cells including lymphomas, leukemias, or
carcinomas, or eukaryotic cells infected With an intracellular
pathogenic microorganism. Cytolytic polypeptides of the invention
may therefore be used to treat plants and animals against
microbial infections, including bacterial, yeast, fungal, viral
and protozoan infections and they may be used in the treatment of
cancer. They may function synergistically with conventional
therapeutic agents such as antibiotics and anti-cancer treatments,
and they may be used as adjuvants.
Cytolytic polypeptides of the invention may be used to
selectively lyse cells. Cells may be selectively lysed using a
chimeric toxin comprising a cytolytic polypeptide of the invention
operatively linked to a targeting agent. The polypeptide may be
linked to the targeting agent via peptide linkages. The chimeric
toxins allow therapeutic targeting of the toxic action of a
cytolytic polypeptide of the invention to target cells such as
tumor cells.
The targeting agent may be an any immunologic binding agent
such as IgG, IgM, IgA, IgE, F(ab~)2, a univalent fragment such as
Fab~, Fab, Dab, as well as engineered antibodies such as
recombinant antibodies, humanized antibodies, bispecific
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antibodies, and the like. Monoclonal antibodies that bind
specifically to carcinoma-associated antigens including
glycoproteins, glycolipids, and mucins may be employed in the
chimeric toxins of the invention (See Fink et al. Prog. Clin.
Pathol. 9:121-33, 1984; U.S. Pat. No. 4,737,579 describing
monoclonal antibodies to non-small cell lung carcinomas; U.S. Pat.
No. 4,753,894 describing monoclonal antibodies to human breast
cancer; U.S. Pat. No. 4,579,827 describing monoclonal antibodies
to human gastrointestinal cancer; U.S. Pat. No. 4,713,352
describing monoclonal antibodies to human renal carcinoma; U.S.
Pat. No. 4, 612,282 describing monoclonal antibody B72.3
recognizing a tumor-associated mucin antigen; U.S. Pat. No.
4,708,930 describing monoclonal antibody KC-4; Young et al J. Exp
Med 150:1008, 1979, Kneip et aI J. Immunol 131(3):1591, 1983,
Rosen et al Cancer Research 44:2052, 1984, Varki et al Cancer
Research 44:681, 1984, and U.S. Pat. Nos. 4, 507,391 and 4,579,827
describes monoclonal antibodies specific for glycolipid antigens
associated with tumor cells).
Alternatively, growth factors, rather than antibodies, may
be utilized as the reagents to target therapeutic agents to target
cells. Any growth factor may be used for such a targeting
purpose, so long as it binds to a target cell, generally by
binding to a growth factor receptor present on the surface of such
a cell. Suitable growth factors for targeting include, but are not
limited to, VEGF/VPF (vascular endothelial growth factor/vascular
permeability factor), FGF (which, as used herein, refers to the
fibroblast growth factor family of proteins), TFG(3 (transforming
growth factor beta), and pleitotropin. Preferably, the growth
factor receptor to which the targeting growth factor binds should
be present at a higher concentration on the surface of target
cells (i.e. disease cells such as tumor cells) than on non-target
cells (i.e. normal cells). Most preferably, the growth factor
receptor to which the targeting growth factor binds should,
further, be present at a higher concentration on the surface of
target cells than on non-target cells.
A chimeric toxin of the invention may be produced using
either standard recombinant DNA techniques or standard synthetic
chemistry techniques, both of which are well known to those
skilled in the art.
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The polypeptides, substances, antibodies, and compounds of
the invention may be formulated into pharmaceutical compositions
for administration to subjects in a biologically compatible form
suitable for administration in vivo. By ~~biologically compatible
form suitable for administration in vivo" is meant a form of the
substance to be administered in which any toxic effects are
outweighed by the therapeutic effects. The substances may be
administered to living organisms including humans, and animals.
Administration of a therapeutically active amount of the
pharmaceutical compositions of the present invention is defined as
an amount effective, at dosages and for periods of time necessary
to achieve the desired result. For example, a therapeutically
active amount of a substance may vary according to factors such as
the disease state, age, sex, and weight of the individual, and the
ability of antibody to elicit a desired response in the
individual. Dosage regima may be adjusted to provide the optimum
therapeutic response. For example, several divided doses may be
administered daily or the dose may be proportionally reduced as
indicated by the exigencies of the therapeutic situation. Dosages
to be administered depend on individual patient condition,
indication of the drug, physical and chemical stability of the
drug, toxicity, the desired effect and on the chosen route of
administration (Robert Rakel, ed., Conn's Current Therapy (1995,
W.B. Saunders Company, USA)).
The compositions described herein can be prepared by per se
known methods for the preparation of pharmaceutically acceptable
compositions which can be administered to subjects, such that an
effective quantity of the active substance is combined in a
mixture with a pharmaceutically acceptable vehicle. Suitable
vehicles are described, for example, in Remington~s Pharmaceutical
Sciences (Remington's Pharmaceutical Sciences 18'h ed, (1990, Mack
Publishing Company) and subsequent editions). On this basis, the
compositions include, albeit not exclusively, solutions of the
substances or compounds in association With one or more
pharmaceutically acceptable vehicles or diluents, and contained in
buffered solutions with a suitable pH and iso-osmotic with the
physiological fluids.
Pharmaceutical compositions used to treat patients having
diseases, disorders or abnormal physical states could include SAG-
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A or another peptide of the invention and an acceptable vehicle or
excipient. Examples of vehicles include saline and D5W (5%
dextrose and water). Excipients include additives such as a
buffer, solubilizer, suspending agent, emulsifying agent,
viscosity controlling agent, flavor, lactose filler, antioxidant,
preservative or dye. There are preferred excipients for
stabilizing peptides for parenteral and other administration. The
excipients include serum albumin, glutamic or aspartic acid,
phospholipids and fatty acids. The protein may be formulated in
solid or semisolid form, for example pills, tablets, dreams,
ointments, powders, emulsions, gelatin capsules, capsules,
suppositories, gels or membranes.
Routes of administration include oral, topical, rental,
parenteral (injectable), local, inhalant and epidural
IS administration. The compositions of the invention may also be
conjugated to transgort molecules to facilitate transport of the
molecules. The methods for the preparation of pharmaceutically
acceptable compositions which can be administered to patients are
known in the art.
The polypeptides etc. and compositions of the invention may
be used alone, or in combination with another pharmaceutically
active agent.
The invention also contemplates an antibody that
specifically binds the therapeutically active ingredient used in a
treatment or composition of the invention. The antibody may be
used to measure the amount of the therapeutic molecule in a sample
taken from a patient for purposes of monitoring the course of
therapy.
The nucleic acid molecules encoding a polypeptide of the
invention or any fragment thereof. or antisense sequences may be
used for therapeutic purposes. Antisense to a nucleic acid
molecule encoding a polypeptide of the invention may be used in
situations to block the synthesis of the polypeptide. In
particular, cells may be transformed with sequences complementary
to nucleic avid molecules encoding a SAG-A Polypegtide or SAG-A
Related Polypeptide. Thus, antisense sequences may be used to
modulate activity or to achieve regulation of gene function. This
technology is well known in the art, and sense or antisense
oligomers or larger fragments, can be designed from various
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locations along the coding or regulatory regions of sequences
encoding a polypeptide of the invention.
Expression vectors may be derived from retroviruses,
adenoviruses, herpes or vaccinia viruses or from various bacterial
S plasmids for delivery of nucleic acid sequences to the target
cells. Vectors that express antisense nucleic acid sequences of
SAG-A Polypeptides can be constructed using techniques well known
to those skilled in the art (see for example, Sambrook et al.).
Genes encoding a SAG-A Polypeptide can be turned off by
transforming cells with expression vectors that express high
levels of a nucleic acid molecule or fragment thereof which
encodes a polypeptide of the invention. Such constructs may be
used to introduce untranslatable sense or antisense sequences into
a cell. Even if they do not integrate into the DNA, the vectors
may continue to transcribe RNA molecules until all copies are
disabled by endogenous nucleases. Transient expression may last
for extended periods of time (e.g a month or more) with a non-
replicating vector, or if appropriate replication elements are
part of the vector system.
Modification of gene expression may be achieved by designing
antisense molecules, DNA, RNA, or PNA, to the control regions of a
sagA gene i.e. the promoters, and enhancers. Preferably the
antisense molecules are oligonucleotides derived from the
transcription initiation site (e.g. between positions -10 and +1o
from the start site). Inhibition can also be achieved by using
triple-helix base-pairing techniques. Triple helix pairing causes
inhibition of the ability of the double helix to open sufficiently
for the binding of polymerases, transcription factors, or
regulatory molecules (see Gee J.E. et al (1994) In: Huber, B.E.
and B.I. Carr, Molecular and Immunologic Approaches, Futura
Publishing Co., Mt. Kisco, N.Y.). An antisense molecule may also
be designed to block translation of mRNA by inhibiting binding of
the transcript to the ribosomes.
Ribozymes, enzymatic RNA molecules, may be used to catalyze
the specific cleavage of RNA. Ribozyme action involves sequence-
specific hybridization of the ribozyme molecule to complementary
target RNA, followed by endonucleolytic cleavage. For example,
hammerhead motif ribozyme molecules may be engineered that can
specifically and efficiently catalyze endonucleolytic cleavage of
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sequences encoding a polypeptide of the invention.
Specific ribosome cleavage sites within any RNA target may
be initially identified by scanning the target molecule for
ribozyme cleavage sites which include the following sequences:
GUA, GW, and GUC. Short RNA sequences of between 15 and 20
ribonucleotides corresponding to the region of the cleavage site
of the target gene may be evaluated for secondary structural
features which may render the oligonucleotide inoperable. The
suitability of candidate targets may be evaluated by testing
accessibility to hybridization with complementary oligonucleotides
using ribonuciease protection assays.
The activity of the substances, compounds, antibodies,
polypeptides, nucleic acid molecules, and compositions of the
invention may be confirmed in in vitro cell systems or in animal
experimental model systems (e. g. the dermonecretic mouse model
described herein).
The following non-limiting examples are illustrative of the
present invention:
BXAMPLBS
Facample 1. Characterization of sagA.
The sagA gene has features that are fundamental for encoding
a functional transcript, specifically, consensus promoter elements
upstream of an ATG start codon (Fig. 2). Also, Northern blot
analyses have revealed that while sagA is transcriptionally active
in the wild-type parent strains, mRNA transcripts are not produced
in the non-hemolytic transconjugants. Based on sequence analysis,
sagA encodes a 53 amino acid peptide containing a long string of
cysteine residues spanning seven out of nine consecutive residues.
Its estimated translational size is consistent with the findings
of Lai and colleagues (1978) that suggest SLS is a low molecular
weight protein. Therefore, sagA is the proposed structural gene
for SLS. The absence of features characteristic of a DNA binding
regulator or processing enzyme shows that the gene is not encoding
for a regulatory element of SLS. The sagA sequence is unique as it
lacks homology with all known regulatory or structural
determinants.
The amino acid sequence analysis of the sagA translational
product revealed that a characteristic lantibiotic double glycine
motif cleavage site (GG) is present 22 and 23 residues from the
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amino terminus, with a corresponding proline residue at position
21 also common to many lantibiotics (Sahl et al. 1995). As
peptide cleavage has been shown to play a fundamental role in the
biosynthesis of all lantibiotics, the presence of such a site in
the SAG-A peptide shows a significant similarity between SAG-A and
other lantibiotics. Moreover, if the SAG-A peptide were cleaved at
this site, the two fragments generated would be of similar size
and amino acid composition as those generated by the proteolytic
cleavage of other lantibiotics. In particular, the unusually high
percentage composition of cysteine, serine and threonine residues
in the C-terminal fragment of SAG-A is characteristic of the
lantibiotic pro-peptide or active fragment and shows that this
domain of the SAG-A peptide represents the active
lantibiotic/hemolysin. A distinct separation of the cysteine
residues in the amino half of the 5AG-A pro-peptide and the serine
residues in its carboxy terminus is characteristic of the type-A
group of lantibiotics (Jung 1991). In addition, the post-
translational modification of cysteine residues may account for
the lack of cysteine content in previously reported amino acid
compositions of SLS (Alouf and Geoffroy 1988; Koyama 1963), as
free cysteines are never found in lantibiotics. Also, the presence
of intra-molecular rings formed by the thioether amino acids,
lanthionine and 3-methyllanthionine, derived from the N-terminal
cysteine residues in the putative pro-peptide of SAG-A may account
for the previous unsuccessful attempts by Alouf (1988) to sequence
SLS by Edman degradation. Finally, in terms of function, the
association of SAG-A with hemolysis parallels the membrane and
pore-forming ability of several lantibiotics of the type-A
category. In particular, Brock and Davie (1963) showed that the
hemolytic activity of the lantibiotic cytolysin LL/LS, produced by
Enterococcus faecalis, correlates with its bacteriocin action.
Interestingly, studies of the enterococcal cytolysin were first
initiated because of its potential role as a virulence factor in
infectious disease.
8RP$RI>IO;NTS LBADINO TO THB IDENTIFICATION OF T8B sagA Q8N8
Examples 2-12 describe in detail the studies leading to the
identification of the sagA gene and its role in virulence.
8xamole 2 - Generation of non-hamolvtic tranaconiuQanta.
In this investigation, SLS-deficient Tn916 mutants were
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generated from two strains of GAS. Each of the wild-types were
associated with severe streptococcal disease in humans (Musser et
al. 1993; Schlievert et al. 1977). The mutants were compared to
wild- type using a murine model of subcutaneous streptococcal
infection, and a gene associated with SLS production designated
sagA, for streptolysin S-associated gene, was identified. Non-
hemolytic, tetracycline resistant transconjugants resulted from
mating GAS strains MGAS166s and TlBPs with the Tn916 donor strain
E. faecalis CG110 at a frequency of 10-' fox both recipients.
Transconjugants maintained the non-hemolytic phenotype after
subculture on selective media. When Tn916 excision assays were
conducted on non-hemolytic transconjugants derived from TlBPs and
MGAS166s, the wildtype, beta-hemolytic phenotype was restored and
detected as a zone of beta-hemolysis within a confluent mat of
non-hemolytic bacteria. The frequency of excision of Tn916 was in
the order of 10-° and 10-' for SBNfiS and SH30-2, respectively.
Because of the low frequency of Tn916 excision, it was necessary
to screen fox hemolytic revertants on a confluent mat of bacteria.
Bxam~le 3 - aeaetic characterizatioa of the aoa-hamolvtic
transconiuaaats and hemolvtic revertaate.
The Tn916 probe used spanned the only HindIII restriction
site within Ta916. Cleavage at this site divides the transposon
into two fragments of approximately 6 kb and 12 kb (Clewell et
al. 1993). After HindIII digestion, each copy of Tn916 which has
integrated into the chromosome of the recipient strains yields two
bands that hybridize with the probe. Two HindIII fragments,
approximately 14 and 7.8 kb, from each of the non-hemolytic
mutants derived from MGAS166s hybridized with the tetM probe. For
each non-hemolytic mutant derived from TlBPs, HindIII fragments of
14 and 6.5 kb hybridized with the tetM probe. This pattern was
seen even in those non-hemolytic transconjugants which possessed
more than a single insertion (Fig. 1). Two non-hemolytic
transconjugants derived from Tl8Ps, designated SB30-2 and one from
MGAS166s, designated SBNHS, were chosen for further study.
Excision of Tn916 from SB30-2 and SBNHS was permitted by
growth in the absence of tetracycline and confirmed by detecting
tetracycline susceptible, hemolytic revertants. Restoration of the
wild phenotype is consistent with previous reports that Tn916 is
capable of precise excision (Gawron-Burke and Clewell 1984). Two
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revertants were selected for further analysis, NHSrev and 30-2rev,
derived from SBNH5 and SB30-2 respectively. Neither revertant
hybridized with the tetM specific probe and excision of Tn916 was
precise as it resulted in restoration of the hemolytic phenotype.
Bxam~la 4 - Analysis of Tn9I6 insertion site.
In order to identify the wild-type region into which Tn916
inserted, a genomic library of MGAS166s was generated using the
low copy number plasmid, pACYCl84. Clones containing the wild-type
region corresponding to the insertion site of Tn916 were
identified using a 2.2 kb PI-PCR product which was generated using
Tn916 derived outward reading primers. Three clones were
identified containing a 3.8 kb fragment which hybridized with the
Tn916 flanking region probe. A single clone, SL-1, was chosen for
further analysis. Confirmation that the 3.8 kb insert in pSL-1
corresponded to the region interrupted by Tn916 insertion in the
wildtype was done by probing HindIII digested genomic DNA from
both MGAS166s and SBNHS with the 3.8 kb insert. A single band at
3.8 kb was detected in MGAS166s while two bands, at approximately
14 and 7.8 kb were detected in SBNHS.
The entire 3.8 kb insert in pSL-1 was sequenced in both
directions yielding a fragment of exactly 3,732 bp. Analysis of
the sequence using the Wisconsin GCG computer program identified
several putative ORF~s. However, only a single ORF, designated
sagA, demonstrated nearly all of the conserved elements of a
functional transcript (Fig. 2). A consensus Shine-Dalgarno
(AGGAGG) sequence is located exactly 10 by upstream of the ATG
start codon. Approximately 150 by upstream of this site is the -
l0 promoter (TATAAT), and 167 by upstream of this lies the -35
promoter region sequence of TTTACA. The sagA ORF appears to code
for a peptide of 53 amino acids which is devoid of a signal
sequence. It is also interesting to note the unusual presence of
several cysteine residues near the amino terminal; seven
cysteines, five consecutive, followed by two tyrosines, followed
by two more cysteine residues. Analysis of the sequence of sagA
using FASTA and BLAST searches failed to detect significant
homology with other known sequences. Furthermore, the sequence of
sagA was not found in the Oklahoma GAS genomic sequence data base.
To determine the exact insertion site of Tn916, PCR products
were generated using primers based on the known 3.8 kb sequence
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coupled with outward reading primers from Tn916. PCR products were
sequenced and allowed precise determination of the Tn916 insertion
point Which was midway within the putative promoter region of
sagA, 11 by downstream of the -35 element and 6 by upstream of the
-10 TATA box.
Sequence analysis of PCR products of the genomic DNA
flanking Tn916 derived from SB30-2 confirmed that Tn9I6 insertion
was in exactly the same locus. Furthermore, Tn916 was oriented in
the same direction as it was in SBNHS.
Sxaamle 5 - Transcription of aavA.
To show that sagA was transcribed, RNA was isolated from
MGAS166s and SBNH5 and probed with DNA corresponding only to sagA
(Fig. 3). A transcript was detected in RNA isolated from MGAS166s
which gave a maximal signal at 4-6 hours post mid-log phase. The
transcription product corresponded to a size of approximately 400
by which was in keeping with the expected size of an mRNA product
from sagA. No detectable transcript was observed from RNA
isolated from SBNHS at any time point. Probing the same membranes
with the 16s rRNA probe did not yield any differences between RNA
from MGAS166s and SBNHS.
Example 6 - M-tvninc of mutants.
M-typing of non-hemolytic transconjugants confirmed that M-
protein was produced and both SB30-2 and SBNH5 had the same M-
protein phenotype as their M18 and M1 parent strains respectively.
No difference in M-protein quantity was seen between MGAS166s and
SBNHS by Western blotting using a monoclonal antibody to the
constant region of M1 protein.
Example 7 - Hemolytic activity.
The non-hemolytic mutants SBNH5 and SB30-2 showed no beta-
hemolysis on blood agar indicating that SLS activity had been
ablated. Hemolysis profiles were identical to ATCC27762 which does
not produce SLS but does produce SLO (Bernheimer 1954). An assay
specific for SLO conducted under reducing conditions showed
continued SLO production in all strains of GAS tested. Hemolysis
was detected in the presence of the SLS inhibitor trypan blue but
not in the presence of both trypan blue and the SLO inhibitor
cholesterol (Table 2). SLS production peaked at late log phase for
MGAS166s whereas there Was no detectable SLS activity for SBNHS at
all points measured. These results confirm that SLO was not
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affected by the insertion of Tn9I6 and the absence of beta-
hemolysis was attributable to the loss of SLS activity, a profile
similar to the SLS-deficient Tn9I6 insertion mutants of Nida and
Cleary (1983).
BxamDle 8 - Growth rate comaarisoas.
To determine if the mutation conferred by Tn9~6 insertion
had affected growth in addition to ablating SLS activity, the
growth rates of the mutants were compared to their parent strains.
There was no difference between the growth rate of either SB30-2
or SBNHS when each strain was compared to its respective parent
strain.
8xampla 9 - Protein and hvaluronic acid capsule vroduction.
There was no difference in production of cell-associated and
extracellular proteins, resolved by SDS-PAGE, between the non-
hemolytic mutants and their parent strains suggesting that the
Tn916 had no gross pleiotropic effect. In addition, hyaluronic
acid production was measured and strains were tested for DNase and
caseinase activity. Both non-hemolytic insertion mutants retained
their respective wild types in all three assays (Table 3).
8xxsamla 10 - Reduced virulence of SLS deficient transconiuaants.
Reproducible, non-lethal lesions were generated following
injection of 106 CFU MGAS166s and 10' CFU TlBPs subcutaneously into
mice. The difference in inoculum size, needed to produce the same
virulence profile, is likely due to inherent differences in
virulence between M1 and Mle serotypes of GAS.
The non-hemolytic transconjugants, SB30-2 and SBNAS, showed
markedly reduced virulence compared to their wild-type
counterparts. Mice infected with 106 CFU of the wildtype strain
MGAS166s exhibited a mean weight loss, -1.16 ~ 0.42 g, compared to
mice Which received either SBNH5 or sterile cytodex alone (p<0.05,
Fisher's PSLD). Mice which received 106 CFU of SBNH5 demonstrated
a mean weight gain of +1.15 t 0.2 g in the first 24 hours after
injection. This change in weight was not significantly different
from the mean weight gain of +1.44 t 0.29 g seen in the uninfected
controls (Fig. 4). Similarly, mice injected with lob CFU of the
wildtype hemolytic TlBPs exhibited a significant mean weight loss,
-0.66 t 0.28 g, in the first 24 hours after infection when
compared to the weight gain observed in mice which received the
i
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same infective dose of the non-hemolytic SB30-2, +0.54 ~ 0.13 g
(p<0.05 Fisher's PS7~D) .
None of the nine mice which received the non-hemolytic
transconjugant SBNHS developed a necrotic lesion, while 7 of the 9
mice (78%) which received the wildtype MGAS166s developed necrotic
lesions (p=0.0007, Fisher s Exact test). Similarly, of the nine
mice which received SB30-2, only one mouse (11%) developed a
necrotic lesion compared to a of the 9 mice (89%) which developed
necrotic lesion when injected with the wildtype Tl8Ps (p=0.001,
Fisher s Exact test). Data for the M1 and M18 strain were similar
to each other in two separate experiments.
Two phenotypic revertants, 30-2rev and NHSrev, from which
Ta916 had excised, derived from SB30-2 and SBNH5 respectively were
compared to the wild types, TlBPs and MGAS166s. The number of
necrotic lesions and weight changes were not significantly
different from that produced by the wild type in each case.
Sxamole 11 - Gross and histoloaical characterization of infected
tissue.
In mice which were infected with MGAS166s, initial
examination of the lesions revealed indurated zones surrounded by
edema. The indurated zones subsequently progressed, yielding
centralized ulceration and necrosis which did not penetrate the
underlying musculature (Fig. 5). MGAS166s produced a maximum mean
necrotic lesion size of 90.4 mmz. No necrotic lesions were observed
in animals infected with SBNHS, though some animals did develop
slight localized edema within 24 hours of infection similar to the
mice which received sterile cytodex. Animals infected with the M18
strains, TlBPs and SB30-2, showed a similar pattern when comparing
the wild-type with the non-hemolytic mutant. The maximum mean
necrotic lesion area was 31 mm~ in animals infected with T18P. For
the single animals which were infected with SB30-2 and developed
lesions in two separate experiments, the maximum area was 10 mmz.
Twenty four hours post infection, biopsies of tissue from
animals which had been infected with MGA5166s, differed
histologically from SBNFiS or sterile cytodex inoculated animals.
Sections of tissue from mice which received MGAS166s demonstrated
evidence of profuse acute inflammation with dense infiltration of
neutrophils and tissue necrosis. Biopsies obtained from mice which
received SBNHS did not show evidence of acute inflammation and no
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tissue damage was evident (Fig. 6). Gram staining of the sections
revealed Gram positive cocci distributed throughout the tissue
obtained from mice infected with MGAS166s, while tissue from mice
which received SBNH5 failed to demonstrate any bacteria in all
fields scanned. Examination of hematoxylin and eosin stained or
Gram stained tissue sections from mice which received SBNFi5, did
not show an appreciable difference compared with tissue from mice
which received a sterile cytodex injection.
Example 12 - Culturing of lesions.
To determine if the phenotype of the infecting strains had
remained the same as the injected organisms, lesions were cultured
from animals which had received either MGAS166s or SBNH5 after 1
and 5 days. As there were no necrotic lesions on mice infected
with SBNH5, the erythematous injection site, comparable in size to
the lesion on the mice which received sterile cytodex, was excised
for culturing. All lesions from animals which received MGAS166s
grew tetracycline susceptible, hemolytic GAS. However, no
organisms grew from tissue cultured at either 1 or 5 days from
mice which had received SBNH5. In two separate experiments, one
out of 9 mice infected with SB30-2 developed necrotic lesions
from which hemolytic, tetracycline susceptible, GAS were cultured.
Animals infected with SB30-2 received an inoculum of 10~ CFU,
probably sufficient to permit the emergence of revertants from
which Tn9I6 had excised. Growth of the revertants may explain the
production of the small necrotic lesion in animals infected with
the non-hemolytic SB30-2 in two separate experiments.
Sxamnle 13. - Expression of the Sag-A Peptide
A translational fusion between the C-terminal portion of the
Escherichia coli produced maltose-binding protein (MBP) gene and
the sagA sequence is constructed to allow the expression and
subsequent purification of large quantities of the SAG-A peptide.
Expression systems that have all the components required for
the correct post-translational modifications of the precursors and
are known in the art are examined for the expression of the SAG-A
peptide. Expression systems have been described for nisin,
subtilin, epidermis and Peps (Saris et al. 1996).
Example 14. - Antibodies directed to SACi-A
Subsequent to its purification, the MBP-SAG-A fusion protein
is used to raise antibodies in rabbits. Monoclonal and polyclonal
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antibodies are prepared according to established techniques
(Harlow E & Lane D (1988). Antibodies: a laboratory manual. Cold
Spring Harbor Laboratory Press. New York). The protective role of
anti-SAG-A antibodies was also identified in an animal model of
infection. In addition, in vitro hemolysis inhibition studies are
performed to characterize SAG-A specific antibodies that abrogate
BLS activity.
Monoclonal and polyclonal antibodies are prepared according
to other techniques known in the art. For examples of methods of
the preparation and uses of monoclonal antibodies, see U.S. Patent
Nos. 5,688,681, 5,688,657, 5,683,693, 5,667,781, 5,665,356,
5,591,628, 5,510,241, 5,503,987, 5,501,988, 5,500,345 and
5,496,705. Examples of the preparation and uses of polyclonal
antibodies are disclosed in U.S. Patent Nos. 5,512,282, 4,828,985,
IS 5,225,331 and 5,124,147.
MATERIALS AND MSTFFODS USED 80R SXAMPhBS 1-12
Bacterial strains and culture conditions. Strains used in this
investigation are listed in Table 1. Gram positive bacteria were
grown in Todd-Hewitt broth (Oxoid, Basingstoke, England) or on
Columbia agar (Oxoid) plates containing 5% defibrinated sheep
blood (WOOdlyn Laboratories, Guelph, ON). When antibiotic
selection was required, 2000 ug/ml streptomycin (Sigma
Laboratories, St. Louis, MO) and 5 ~g/ml tetracycline (Sigma) were
added to the appropriate media. Escherichia coli were propagated
using Luria Bertani (LB) broth (Difco). When appropriate, 25
~g/ml tetracycline and/or 50 ug/ml chloramphenicol were added to
the media. Strains T18P, MGAS166, and CG110 were kindly provided
by Drs. Patrick Schlievert, (University of Minnesota, Minneapolis,
MN), James Musser (Baylor College of Medicine, Houston, TX) and
Don Clewell (University of Michigan, Ann Arbor, MI) respectively.
Escherichia coli strain RN6851 (pRN6680) contains a 2.2 kb tetM
gene from Tn551 cloned into pBS-bluescript and was provided by Dr.
Barry Krieswirth (New York Public Health Research Institute, New
York, NY).
M-typing and quantatation. Serotyping of recipient and non-
hemolytic transconjugants was conducted by the National Reference
Center for Streptococci (Edmonton, AB) in a blinded fashion
according to standard techniques (Griffith 1934). M protein was
quantitated by Western blot using monoclonal antibody to the
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constant region of the M1 protein (kindly performed by Vincent
Fischetti, Rockefeller University) using published methods
(Fischetti et aI. 1985).
Generation of transconjugants. Strains MGAS166 and T18P were made
resistant to streptomycin by plating each strain on Columbia blood
agar containing streptomycin and selecting a colony which became
spontaneously resistant to streptomycin. Tn9I6 was mobilized from
Enterococcus faecalis CG110 to MGAS166s and TlBPs by a variation
of a method described by Nida et al. (1983). Cells of recipient
and donor were added to a non-selective Columbia blood agar plate
in a ratio of 1:1, which corresponded to 107 CFU of each strain,
and the entire plate was cross-streaked using a sterile loop.
After overnight incubation at 37°C in 5% C02 the bacterial mat was
replica-plated using Acutran sterile replicators (Schleicher and
Schuell, Keene, NH) to selective media containing tetracycline and
streptomycin. Non-hemolytic transconjugants were chosen which
were devoid of a beta-hemolytic phenotype and were passaged at
least l0 times on selective media to ensure stability of the
mutant phenotype. Lancefield grouping was conducted on the non-
hemolytic transconjugants (Prolab, Richmondhill, ON) as outlined
by the manufacturer.
Southern hybridization aaalysis. A probe specific for the tetM
gene of Tn9I6 was used to identify the transposon insertion in the
transconjugants. The tetM determinant was amplified from pRN6680
by the polymerase chain reaction (PCR) using T3/T7 universal
primers (Stratagene Cloning Systems, LaJolla, CA) and parameters
recommended by the manufacturer. The PCR product was confirmed by
its size on a 0.7% agarose gel and was purified from the gel using
the Qiaex II Gel Extraction Kit (Qiagen, Chatsworth, CA). The
purified product was labeled using the enhanced chemiluminescence
(ECL) direct labeling system (Amersham, oakville, ON) as outlined
by the manufacturer. Genomic DNA was isolated from GAS as
previously described (O'Connor and Cleary 1983). DNA was digested
with HindIII (Boehringer-Mannheim, Laval, PQ), subjected to 0.7%
agarose gel electrophoresis, transferred to Hybond N+ nylon
membranes (Amersham) and probed with the enhanced
chemiluminescence labeled tetM specific probe as indicated by the
manufacturer.
Cloning and sequencing. Genomic DNA from MGAS166s was digested
i
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with HindIII, ligated into the HindIII site of pACYC184 (New
England Biolabs, Mississauga, ON) and transformed into E. coli
DHSaMCR high efficiency competent cells (Gibco BRL, Burlington,
ON} using standard techniques (Gilman 1997). Plasmid DNA from
transformants was isolated by alkaline lysis (Maniatis et al.
1989) and dot blotted by vacuum suction onto Hybond N+ membranes.
In order to identify transformants harboring the sequence
disrupted by Tn916 in SBNHS, a probe based on the sequence
flanking the transposon in SBNH5 was generated by partial-inverse
PCR (PI-PCR) as follows (Pang and Knedt 1997). Genomic DNA from
SBNHS was digested with HindIII, self-ligated and used as a
template with outward reading primers based on the ends of Tn916.
The resulting amplicon consequently consisted of the sequence
flanking Tn916 and was used as a probe for identifying
transformants from MGAS166s harbouring the sequence associated
with SLS production. Sequencing was done commercially (Mobix,
Inc., Hamilton, ON) using an automated sequencer (Applied
Biosystems, Oakville, ON) according to manufacturer s guidelines.
Analysis of sequence data was done using the Wisconsin GCG
sequence analysis software as well as the FASTA algorithm and
BLAST search engines of the National Biotechnology Institute.
Northern Analysis. Total RNA was extracted using Trizol (Gibco
BRL) according to manufacturers directions. RNA was isolated from
bacteria at mid-log phase (O.D.sso=0.6 - 0.8) and then every two
hours thereafter for a maximum of ten hours. Total RNA was
standardized spectrophotometrically and resolved using 1.9%
formaldehyde/agarose gels. RNA electrophoresis and Northern blot
transfer were performed using standard techniques (Gilman 1997).
DNA probes were labeled with a"PdCTP using Ready-to-Go DNA
labeling beads (Pharmacia Biotech, PQ) according to the
manufacturer's instructions. Integrity of the RNA was checked
simultaneously by probing all samples with a conserved 16S rRNA
sequence.
Bxcieion of Tn916. Phenotypic revertants were produced in a
manner similar to that described by Nida and Cleary (1983).
Briefly, 106 CFU, determined by an optical density at 550 nm
(O.D.55p) of 1.0-1.2, of a late log phase culture of non-hemolytic
transconjugants were inoculated into 50 ml of non-selective Todd-
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Hewitt broth. After overnight incubation, 108 CFU were plated
onto a single non-selective blood agar plate. Following overnight
incubation, zones of hemolysis were identified within the
bacterial mat, and colonies within the hemolytic zones were
S subcultured on non-selective media to isolate the hemolytic
revertants. Tetracycline resistance was determined by growth on
Columbia blood agar plates containing tetracycline (5 Ng/ml}.
Growth rate analysis. To determine the growth rate of wildtype and
mutant GAS, lOml of THB was inoculated with a single colony.
Mutants were grown in the presence of tetracycline. After
overnight growth 106 CFU were used to inoculate 50 ml of Todd-
Hewitt broth. O.D.55o readings using a Beckman spectrophotometer
(Beckman Instruments Inc., Fullerton, CA) were taken at the time
of inoculation and every hour subsequently for a period of 12
hours. Actual CFU at each time point were confirmed by serial
dilutions and plating on Columbia blood agar.
Hemolysis assays. To confirm that SLO was still being produced by
the non-hemolytic mutants, assays similar to those previously
described by Smyth and Duncan were employed (Smyth and Duncan
1978). Late-log phase cultures (O.D.550 = 1.0-1.2) of GAS were
centrifuged to pellet bacteria. Culture supernatants (750 ~1) were
reduced by adding L-cysteine to a final concentration of 20 mM and
incubating at ambient temperature for 10 min. An equal volume of
a 5% solution of sheep erythrocytes, washed three times in 0.15 M
sodium phosphate buffer, pH 6.8 (PBS), and resuspended in the same
buffer, was added to culture supernatants and samples were
incubated at 37°C for 60 min. After centrifugation, the optical
density of the supernatant Was read at 540 nm to determine the
release of hemoglobin. An equivalent amount of lysed erythrocytes
suspended in sterile Todd-Hewitt broth was used as a control to
represent 100% hemolysis and sample values were recorded as a
fraction of this value. Trypan blue (Sigma), at a final
concentration of 13 ~g/ml, and cholesterol (Sigma), at a final
concentration of 0.5 mg/ml were used as inhibitors of SLS and SLO
respectively. ATCC 21547 (SLO+, SLS+) and 27762 (SLO+, SLS-) were
used as control strains (Table 1).
SLS activity Was also measured during early, mid and late
log phase using the above method, overnight broth cultures of
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MGAS166s and SBNHS were subcultured in Todd-Hewitt broth and
samples withdrawn hourly for a hours and immediately frozen at -
70°C. Samples were thawed and bacteria pelleted by
centrifugation. Serial dilutions of culture supernatant were added
to PBS-washed 5% rabbit erythrocytes and incubated at 37°C for 60
min. Cells were removed by centrifugation and the O.D. determined
as above.
Preparation of ~xtracellular and call associated proteins.
Bacteria were grown in 200 ml of Todd-Hewitt broth and samples
were collected at either mid-log phase (O.D.SSO=0~6) or late-log
phase (O.D.SSO=1.0-1.2). Bacteria were pelleted by centrifugation
at 10,000 g for 15 min at 4°C. Ammonium sulfate was added to the
culture supernatants gradually with constant shaking at 4°C until
solution reached e0% saturation. After mixing gently overnight at
4°C, tubes were centrifuged at 10,000 rpm and supernatant was
discarded. Ammonium sulfate precipitate was dissolved in 2 ml of
0.01 M ammonium bicarbonate (pH 7.0) and dialyzed against the same
solution using Slide-A-Lyzer dialysis cassettes (Pierce Chemical
Co., IL) overnight at 4°C. Dialysate samples were boiled for 5
min in SDS-PAGE loading buffer (Lamemelli 1970), resolved using a
l0% SDS-polyacrylamide gel and stained with Coomassie brilliant
blue R.
For analysis of cellular proteins, bacteria were grown in 10
ml of Todd-Hewitt broth and bacteria were pelleted when they
reached mid-log or late-log phase. Supernatants were discarded
and the pellets Were resuspended in 15 ~tl of 0.1% Triton X
(Sigma) and 25 mM PBS (pH 7.2), and vortexed briefly. After
incubating cells at 37°C for 30 min, 15 ~1 of SDS-PAGE loading
buffer was added and samples were resolved as described above.
Production of caseinas~ and DNaea. Caseinase activity was
determined by the method of Wheeler et al. (Wheeler et al. 1991).
DNase production was determined using commercial media (Difco,
Detroit, MI). In both assays, an equivalent inoculum of late-log
phase organisms was spotted onto assay plates. Plates were
incubated anaerobically overnight and zones of opacity or clearing
were measured to determine caseinase or DNase activity
respectively. SBNH5 and SB30-2 were tested with and without 5
ug/ml of tetracycline in the media.
Quantitatioa of hyaluronic acid. Bacteria were grown in 150 ml of
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Todd-Hewitt broth to an O.D.sso of 0.6 - 0.8. Mutants were grown
in the presence of tetracycline. Aliquots of the cultures were
removed, serially diluted and subcultured to confirm the exact
number of CFU. The bacterial pellet was harvested by
centrifugation and washed once with sterile distilled water. The
pellet was resuspended in 1.5 ml of water and an equal volume of
chloroform was added, mixed vigorously and incubated at room
temperature for 1 hour. The mixture was centrifuged to separate
the aqueous phase from the chloroform. The aqueous phase was used
in the carbazole method of uronic acid quantitation as described
by Knutson et aI (Knutson and Jeanes 1969). Human umbilical
hyaluronic acid (Sigma) was used as a standard.
Dermoaecrotic mouse modal. Virulence of GAS strains was
determined using a dermonecrotic mouse model as previously
described (Bunce et al. 1992). Organisms were grown to mid-log
phase (O.D,55p = 0.6 - 0.8) in Todd-Hewitt broth with appropriate
antibiotic selection. A 100 ~1 volume of mid-log phase organisms
was mixed with an equal volume of sterilized cytodex beads (Sigma)
suspended in PBS at a concentration of 20 ~g/mL. The 200 gel
cytodex/bacterial suspension was injected subcutaneously in the
right flank of hairless, 4 week-old, male, crl:SKH1(hrhr)Br mice
(Charles River, Wilmington, MA) weighing 15-20 g using a 1 ml
tuberculin syringe. Nine animals were injected for each strain
examined. Viable counts were performed on all cultures to confirm
the exact number of CFU injected. Animals were weighed
immediately prior to inoculation and every 24 hours subsequently
fox a total of 5 days. The length and width of the lesions were
measured daily by an observer blinded to the identity of the
infecting strain. The wound area (A) was determined by A=(LxW)/2
where L is the longest axis and W is the shortest axis.
Culturing of necrotic laeioas and histopathology. To determine
the phenotypes of the organisms in the lesions, a single animal
was randomly chosen from each group 24 and 120 hours after
infection and euthanized. The wounds were excised from euthanized
animals and divided equally. One half of each lesion was cultured
and the remainder was used for the preparation of histological
specimens. Tissue for culture was suspended in 1 ml of sterile
PBS and then ground in a sterile tissue homogenizer. Aliquots of
the PBS/tissue homogenate were serially diluted and inoculated on
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either selective or non-selective Columbia blood agar plates and
scored for beta-hemolysis. Histologic sections were prepared by
immersion in 10% buffered formalin and embedded in paraffin.
Sections were stained with hematoxylin and eosin or tissue. Gram
stain (Brown-Benn stain) and examined by light microscopy by a
pathologist blinded to the source of the biopsies.
Statistics. Statistical analysis was conducted as described
previously (Bunce et al. 1992). Group means for weight loss and
lesion size were compared among groups by using analysis of
variance (ANOVA). Post hoc tests were done using Fisher's
protected least significant difference (Fisher s PSLD). P values
reported, refer to the ANOVA tests. Significant differences
between pairs of groups were reported if P < 0.05. Fisher's
Exact test was used to compare counts of abscesses and
dermonecrotic lesions.
Example 15
Using Tn917 mutagenesis followed by chromosome walking
steps, eight genes located immediately downstream of sagA have
been shown to be important for SLS production. Furthermore, the
inactivation of each of these genes with the vector pVE6007 has
lead to the generation of non-hemolytic mutants. The virulence of
six of these mutants was examined by using a dermonecrotic mouse
model as previously described (see above). The results are shown
in Table 4. In contrast to mice infected with the wild-type
strain, NZ131, those infected with sagA,E and F produced no
necrotic lesion. However, due to the excision of the plasmid from
the chromosome and hence reversion to their hemolytic phenotype,
those mice infected with sagB,D and G produced lesions similar to
NZ131. Mutants for sagX and sagl have not been tested. From these
data, it can be concluded that sagA, E and F play an important
role in the virulence of N2131.
The present invention has been described in terms of
particular embodiments found or proposed by the present inventors
to comprise preferred modes for the practice of the invention. It
will be appreciated by those of skill in the art that, in light of
the present disclosure, numerous modifications and changes can be
made in the particular embodiments exemplified without departing
from the intended scope of the invention. All such modifications
are intended to be included within the scope of the appended
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claims.
All articles, patents and other documents described in this
application are incorporated by reference in their entirety to the
same extent as if each individual publication, patent or document
was specifically and individually indicated to be incorporated by
reference in its entirety. They are also incorporated to the
extent that they supplement, explain, provide a background for, or
teach methodology, techniques and/or compositions employed herein.
i
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TABLg 1. and relevantproperties
Bacterial
strains
Strain Relevant Reference Comments
Phenotype
S.pyogenes
T18P MlB,St',Tc',SLS"Schilvert Isolate associated with
1977 rheumatic fever
MGAS166 Ml,St',Tc',SLS"Musser Invasive clinical isolate
1993
TlBPS MlB,Str, Tcs,SLS+See text Spontaneous Strr derivative
of T18P
MGAS166s Ml,Str,Tcs, See text Spontaneous Str derivative
SLS+
of MGAS166
SB30-2 MlB,Str,Tcr, See text Nonhemolytic derivative
SLS of
TlBPs
30-2rev MlB,Str,Tcs,SLS+See text Hemolytic derivative
of
SB30-2
SBNHSs Ml,Str, Tcr, See text non-hemolytic derivative
SLS of
MGAS166s
NHSrev Ml,Str, Tcs, See text Hemolytic derivative
SLS+ of
SBNHS
SLS+, SLO+ ~ Hemolytic control strain
ATCC21S47
SLS , SLO+ Ginsburg non-hemolytic control
strain
ATCC27762 1970
E. faecalis
CG110 Sts, Tcr Gawron Tn9Z6 donor strain
1982
E.coli
RN6851 Tc' NR Contains pRN6680
DHSaMCR mcrA,~80dlacAZMlSGibcoBRL Library efficiency competent
cells
SL-1 Tc, Cmr See Text Contains pACYC184 with
3.8kb
insert
Str, streptomycin Sts, streptomycin
resistant; sensitive;
Tcs,
tetracyclinesensitive; tetracycline
Tcr, resistant;
Cmr,
chloramphenicol resistant;
SLS, streptolysin
S; NR, no
reference.
i
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TABLB 2. Streptolysin O activity of wildtype and mutant
streptococci
r;ssay Fraction of complete lysisa exhibited by bacterial
Contents
strains
Tl8Ps SB30-2 MGAS166s SBNHS ATCC2772 ATCC2154
7
Supernatant 0.57 0.48 0.56 0.45 0.62 0.78
Supernatant 0.64 0.50 0.48 0.39 0.58 0.69
with Trypan
blush
Supernatant 0.05 0.09 0.10 0.07 0.11 0.04
with trypan
blue and
cholesterolc
a Complete lysis was determined by lysing 750 ~tL of 5% washed
sheep erythrocytes in hypotonic saline and adding to an equal
volume of sterile THB.
b Concentration of trypan blue was 13 ~g/mL
c Concentration of cholesterol was 0.5 mg/mL
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TABLS 3. Phenotypic comparisons between hemolytic and non-hemolytic
GAS
Strain Assay
Caseinasea Dnasea Hyaluronic acid
MGAS166s 12.8 +/- 1.3 mm 15.1 +/- 0.9 mm 2 fg / cfu
SBNHS 12.2 +/- l.lmm 16.3 +/- 1.3 mm 3.1 fg / cfu
TlBPs 0 mm 16.1 +/- 1.6 mm 68 fg / cfu
SB30-2 0 mm 17.4 +/- 2.0 mm 54 fg / cfu
aResults are zone diameters surrounding inoculum after overnight
anaerobic incubation of assay plates at 37°C. Measurements are
mean +/- standard deviation of three experiments.
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N
N
_ a.G o m
Q C O
Z Z ~ f~
aZE moo! 0
NON
NJ
t
N
O ~ i tlt ~ O
C
0 ~ O J ~ ~
p
p
c~ o J~ r C 0 N
D. 7, O C
J ~ C r- Z v
C C
V
r
p
O . . . . 1 . ,
N .
P7
J
V V
N
O O O ~ O
O + M ~ N ~
W
t t
O (f
CG st n ~ c~ 0~
O O ~ ~ O O ~ m
m
O O ~ ~ O O ~
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'~
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~'9
er
N
O ~ ",
O
(~ Q ~ O O O ~ O O
~ N
o E
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0.-~ 0 0 0 0 0 0 0
0
~N
+ + + ~. ~ +
v v v ~. ~. v 'r e~ t_
tp ~ N " v ~ W N
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ii o; n ~ a; ~! ~E
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C
C~ N M ~ O ~ ~- Gf
7. ~ ~,j~~O ,.,u~N~j ,~ tl NO
~ '1'1* ~ * * II * tf~
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~ ~ 0f ~O O v ~ O
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N N N ~ N N N m
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N 0f tO N N C~ 1~ m
N - ~ ~ ~ N
?. i1 ~ C Wi ~ p ~
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a
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DETAILED DESCRIPTION OF THE DRAPTINGS
Figure 1. Southern hybridization analysis of HindIII restriction
digests of genomic DNA probed tetM. (A) Hemolytic wildtype T18P
(Lane 1) does not hybridize with the tetM probe. Non-hemolytic
transconjugants SB1-4, SB2-9, SB30-2, SB1-9, SB5-9, SB6-9, SB7-9,
SBl-1, and SB8-2 (Lanes 2-20) all contain at least one copy of
Tn916 and hybridize with the tetM probe. Isolates in lanes 2, 7
and 9 possess more than one insertion of Tn916. All lanes possess
two bands hybridizing with the tetM probe corresponding to
approximately 6.5 kb and l4kb. The Tn916 donor strain CG110 (Lane
11) contains several copies of Tn916. (B) Hemolytic wildtype
MGAS166s (Lane 1) does not hybridize with the tetM probe. The non-
hemolytic transconjugants SBNH1, SBNH3, SBNH4, SBNH5, SBNH6, SBNH7,
and SBNHB (Lanes 2-7) all possess at least one copy of Tn916.
Isolates in lanes 3, 4, 7, and 8 possess more than a single
insertion of Tn916. Isolates in all lanes possess two bands of a
similar size of approximately 14 kb and 7.5 kb. The migration of
molecular size standards (1 kb ladder) is indicated (in kilobases)
on the left for both (A) and (H).
Figure 2. The nucleotide sequence and protein translation of
sagA. A 390 by region of genomic DNA from MGAS166s is represented
corresponding to the chromosomal point of insertion of Tn916 (D).
The conserved elements of the sagA ORF are indicated and the
putative 53 amino acid translation product is represented. S.D.
indicates the Shine-Dalgarno consensus sequence. (The highest
degree of homology was observed with epidermin and peps (from
Staphylococcus epidermidis) matching 44% and 40% similarity
respectively, and 22~ and 20% identity respectively. Calculations
of homology are done using the FASTA (Pearson W.R. and Lipman
D.J., 1988, PNAS 85: 2444-2448) and BLAST (Altschul S.F. and
Lipman D.J., 1990 PNAS 87: 5509-5513) algorithms.)
Figure 3. Total RNA extracted from mutant SBNHS (lanes 2-7) and
wildtype MGAS166s (lanes 7-13) was quantified, standardized,
blotted and probed using a PCR amplicon of sagA labeled with a'~P.
Lane 1 is a 0.16-1.77 kb RNA standard, lane 2 is SBNH5 RNA
harvested at mid-log phase, lanes 3-7 are SBNHS RNA at 2, 4, 6, 8
and 10 hours post mid-log phase respectively. Lane 8 is MGAS166s
RNA harvested at mid log phase, lanes 9-13 are MGAS166s RNA at 2,
4, 6, 8 and 10 hours post mid-log phase respectively. The mutant
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strain is devoid of any transcripts from sagA while the wildtype
contains sagA transcripts at all time points.
Figure 4. Comparisons of mean weight change are shown 24 hours
after infection with wild type (MGAS166s; Tl8Ps) and the respective
S isogenic non hemolytic mutants (SBNH5; SB30-2). Animals infected
with non hemolytic mutants of each wild type gained weight in
contrast to the marked weight loss caused by infection with the
parent strains.
Figure 5. Photographs of hairless SKH1 mice 24 hours after
infection with 106 cfu of either the SLS producing wildtype
MGAS166s (A) or the SLS-deficient Tn916 mutant SBNHS (B). A well
demarcated zone in induration with centralized necrosis is depicted
on the right flank of a mouse infected with MGAS166s. No necrosis
was seen in mice infected with SBNHS.
Figure 6. Tissue biopsies from euthanized mice which were
infected with 106 cfu of either the SLS-producing wildtype
MGAS166s (A) or the SLS-deficient Tn9I6 mutant SBNHS (B). The
tissue section in (A) demonstrates acute inflammation with edema
and tissue necrosis. The tissue depicted in (B) does not show
evidence of necrosis and the inflammation is markedly reduced when
compared with (A). Tissue samples were stained With hemotoxylin
and eosin and final magnification is approximately x25.
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RBFgRSNCEB
Alouf, J.E. and Geoffroy, C. (1988). Meth. In Enzym. 165:59-64.
Alouf, J.E. 1980, Pharmacol. Ther. 11:661-717.
Berraheimer, A. W. 1954. Streptococcal Infections, p. 19. Columbia
University Press, New York. N.Y.
Blackburn, P. and Projan, S.J. (1994). United States Patent No:
5,304,540. Applied Microbiology Inc., New York, USA.
Borgia, S.M., Betschel, S.D., Low, D.E. and de Azavedo, J.C.S.
(1997). Advances in Experimental Medicine and Biology. 418:733-
748.
Brock, T.D. and Davie, J.M. (1963). J. Bact. 86:708-712.
Bunce, C., Wheeler, L., Reed, G., Musser, J., and Barg, N. (1992)
Infect. Immun. 60:2636-2640.
Chatterjee, S., Chatterjee, D.K., Jani, R.H., Blumbach, J.,
IS Ganguli, B.N., Klesel, N., Limbert, M. and Seibert, G. (1992).
J. Antibiot. 45:839-845.
Clewell, D. B. and Flannagan, S.E. (1993) The conjugative
transposons of gram-positive bacteria. p. 369-393. Plenum
Press, New York, N.Y.
Da Vuyst, L. and Vandamme, E.J. (eds) (1994}. Bacteriocins of
lactic acid bacteria, Chapman and Hall, London.
Delves-Broughton, J., Blackburn, P., Evans, R.J. and Hugenholtz,
J. (1996). Antonie van Leeuwenhoek 69:193-202.
Fischetti, V. A., Jones, K.F. and Scott, J.R. (1985) J. Exp. Med.
161:1384-1401.
freer, J.H. and J.P. Arbuthnott, 1976. Biochemical and
Morphological alterations of membranes by bacterial toxins, p.
170-193. In A.W. Bernheimer (ed.) Mechanisms in bacterial
toxicology. John Wiley and Sons, New York, N.Y.
Gawron-BUrke, C. and Clewell, D.B. (1982) Nature (London).
300:281-284.
Gaarron-Burke, C. and Clewell, D.B. (1984) J. Bacteriol. 159:214-
221.
Gilmaa, M. (1997) Current protocols in molecular biology, p.
4.1.1-4.1.23. In F. M. Ausubell, R. Brent, R. E. Kingston, R.
D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (ed.),
John Wiley and Sons, New York, N.Y.
Ginsburg, I. (1970) Streptolysin S. p. 99-171. In T. C. Montie,
S. Kadis, and S. J. Ajl (ed.), Microbial Toxins, Academic
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Press, New York, N. Y.
Jung, G. (1991). Agnew. Chem. Int. Ea. Eng. 30:1051-1192.
Klasnhammsr, T.R. (1993). FEMS Microbiol. Rev. 12:39-86.
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Maniatis, T., Fritsch, E. and Sambrook, J. (1989) Small scale
plasmid extraction, p. 1.21-1.31. In T. Maniatis, E. Fritsch,
and J. Sambrook (ed.), Experiments in Molecular Genetics, Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
Mussar, J., Kanjilal, S., Musher, D. et al. (1993) J. Infect. Dis.
167:337-346.
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504.
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microbial origin, CRC Press, Boca Raton FL.
Sahl, H.-G., Jack, R.W. and Bierman, G. (1995). FEES. 230:827-853.
Saris, P.E.J. Immonen, T,., Reis, M., and Sahl, H.-G. (1996)
Arttonie van Leewenhoek 69:151-159.
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Immun. 16:673-679.
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Todd, J.K. (1991)JAMAa66:533-537.
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SEQUENCE LISTING
1) GENERAL INFORMATION:
(i) APPLICANTS: DE AZAVEDO, Joyce
BAST, Darrin
BORGIA, Sergio
BETSCHEL, Stephen
LOW, Donald
(ii) TITLE OF INVENTION: Streptococcus Streptolysin S Nucleic
Acid Molecule
(iii) NUMBER OF SEQUENCES: 2
(2) INFORMATION FOR SEQ ID NO: l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 390 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE: DNA sequence for sagA gene
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
AGGGTTTACA TATTAATCAT TTTTTACTAT AATAAAAGTG 50
ATAAGAACTA
GATAGTTGTT GTGTTACAAC AGTACAATTG AGCTAGCCTT 100
GTCCTTGTTG
TGTTAACTTT ATTTTTAAAA TAAGGTTAAA AATAAACGAC 150
TCGCGTTCTT
ATCAGTTACT TATTAGATAA GGAGGTAAAC CTTATGTTAA 200
AATTTACTTC
AAATATTTTA GCTACTAGTG TAGCTGAAAC AACTCAAGTT 250
GCTCCTGGAG
GCTGCTGTTG CTGCTGTACT ACTTGTTGCT TCTCAATTGC 300
TACTGGAAGT
GGTAATTCTC AAGGTGGTAG CGGAAGTTAT ACGCCAGGTA 350
AATAATCTAT
TTAGCATCTC TATGTGGTAG TGATATTAAG GTAATGAGTT 390
SUBSTITUTE SHEET (RULE 26)
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2I9
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 53 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE: Protein sequence for SAG-A
(xi) SEQUENCE DESCRIPTION: SEQ ID
N0:2:
M L K F T S N I L A
1 5 10
T S V A E T T Q V A
15 20
P G G C C C C C T T
25 30
C C F S I A T G S G
35 40
N S Q G G S G S y T
45 50
P G K
53
SEQUENCE DESCRIPTION: SEQ ID N0:3
Leader na sequence
CTTATGTTAA AATTTACTTC AAATATTTTA TAGCTGAAAC
GCTACTAGTG AACTCAAGTT
GCTCCTG GAG
SEQUENCE DESCRIPTION: SEQ ID N0:4
Leader amino acid sequence
M L K F T S N I L A T
S V A E T T Q V A P G G
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SEQUENCE DESCRIPTION: SEQ ID N0:5
propeptide na sequence
GCTGCTGTTG CTGCTGTACT ACTTGTTGCT TCTCAATTGC TACTGGAAGT
GGTAATTCTC AAGGTGGTAG CGGAAGTTAT ACGCCAGGTA AATAATCTAT TTAGCATCTC
TATGTGGTAG TGATATTAAG GTAATGAGTT
SEQUENCE DESCRIPTION: SEQ ID N0:6
propeptide amino acid sequence
C C C C C T T C C F S
I A T G S G N S Q G G
S G S Y T P G K
SEQUENCE DESCRIPTION: SEQ ID N0:7
regulatory na sequence
AGGGTTTACA TATTAATCAT TTTTTACTAT AATAAAAGTG ATAAGAACTA 50
GATAGTTGTT GTGTTACAAC AGTACAATTG AGCTAGCCTT GTCCTTGTTG 100
TGTTAACTTT ATTTTTAAAA TAAGGTTAAA AATAAACGAC TCGCGTTCTT 150
ATCAGTTACT TATTAGATAA GGAGGTAAAC CTT
SEQUENCE DESCRIPTION: SEQ ID N0:8
processing protein na
tgt cattttttac aaaggaacaa caacctaaag
721 agaattgtcc gccaataact gttgaaaaag caaggcaatt gtttgaattt
aatacaaacc
781 acttgtcctt atcggattac catcatcaaa cggtgctaaa aacgtcaaag
cagctagttg
841 ctcaacattt aatgcctaat gcaaccgata atcttagtca acattttttg
atgaactata
901 aagctaataa taattattta ggcttccaag ctagtattgt cgactttttt
acagattctg
961 ccgttgctaa tttttcaagt agttacgttt atgaaagtca ggaaaagata
attcgtttac
SUBSTITUTE SHEET (RULE 28)
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1021 caaaacctac caagatatca actgctctgt cgacatgtat tataaaacga
agaagtcatc
1081 gtcaattttc agatagacaa atgcctcttc aagatttatc aaacattctt
tattatgcat
1141 gtggtgttag ttcacaagca tcaattagag atggagcatc agataagatt
acactcagaa
1201 actgtgcttc aggtggaggt ttatacccta ttcatttagt tttttatgct
agaaacatca
1261 gtaaattaat agatggtttc tatgaatatc taccctatca gcatgcacta
aggtgttatc
1321 ggcatagctc tgaggaaaac gttagagatt ttgcggaata cggtgctatt
aatgctgaaa
1381 attgtaatat tattattatt tatgtctacc attacatgaa aaatacacgt
aaatatggga
1441 atcaggcgac tgcctatgct tttattgaat caggagaaat agcccagaat
attcaattga
1501 ctgcaactgc cttaacttat ggaagtattg atattggtgg ttataataag
gaatatctcc
1561 aagaattatt agatttagat gggctaggag agcatgtgat tcacatgaca
ctcgtaggaa
1621 ctaaggagtc tcaatga
SEQUENCE DESCRIPTION: SEQ ID N0:9
processing protein amino acid sequence
MSFFTKEQQPKENCPPITVEKARQLFEFNTNHLSLSDYHHQTVL
KTSRQLVAQHLMPNATDNLSQHFLMNYKANNNYLGFQASIVDFFTDSAVANFSSSYVY
ESQEKIIRLPKPTKISTALSTCIIKRRSHRQFSDRQMPLQDLSNILYYACGVSSQASI
RDGASDKITLRNCASGGGLYPIHLVFYARNISKLIDGFYEYLPYQHALRCYRHSSEEN
VRDFAEYGAINAENCNIIIIYVYHYMKNTRKYGNQATAYAFIESGEIAQNIQLTATAL
TYGSIDIGGYNKEYLQELLDLDGLGEHVIHMTLVGTKESQ
SUBSTITUTE SHEET (RULE 26)
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SEQUENCE DESCRIPTION: SEQ ID NOS:10 AND 11
Transporter na sequence and amino acid sequence
x912 atqagttttqtaeaattaacaaacgttgtcaagtcctacaaaaac
M S F Y Q L T N V V K S Y K N
:95~ ggcaagaaagetgtcaatgacgcctcettgtctactgaagcaggt
G K K A V N 0 V S L S I E A G
5002 aatatttatggtttgttaggsccaaatggtgccggtaagtccaec
N I Y G L L G P N G A G K S T
5017 ctgattaatettstcttaggcttgatccctttgagttccqgcaaa
L I N L I L G L I P L S S G K
5092 attaetgttttagggcsatcccaaaagactattcgaasaacsagt
I T V L G Q S Q K T I R K I S
5137 tcgcagataggttatgttccteasgacattgccgtttatecagac
S Q I G Y V P Q D I A V y p p
5182 etaactgcttacgaaaatgcagaaciatttgggtcactttatqge
L t A Y E N V E L F G S L Y G
5227 ttssagggagctcagcttaaaaaaeaagttctassaagtttagas
L K G A Q L K K Q V L K S L E
5172 ettgtqgggccacactcccaagctsagcagtttccsagtcsaccc
F V G L H S Q A K Q F P S Q f
5317 tcagqaggsatgaagagacggttasatategcttgcgcqetagtt
S G G N K R R L N I A C A L V
5362 catteacccaaattsatcatttttqsegaaecgactgtagqgact
H S P K L I I f D E P T V G I
5147 gatectcaatcaegtaatcatattttagagtcgattcgtttgcts
D P Q S R N N I L E S I R L L
5152 aseaaagaaggcgetacagttatttatacgacecactstatgqss
N K E G A T V I Y T T N Y PI E
5197 gsagtaqaggctetttgtgatcatstttttattatggatcatggt
E V E A L C D Y I F I M D N G
551? caagttattgaagasggacctaastttgaaetggsasaaegttae
Q V I E E G P K F E L E K R Y
5587 gttgesaatctagcaaaccagaccattgtsaetctaacagsctea
V A N L J1 N Q I I V T L T D S
5632 eqteatttggaaetggeagataagcctgsctggtctttgatsqas
R H L E L A D K P D YI S L I E
5677 gatqgagaaaascteatgttgaagsttgatastagtgatatgaca
D G E K L N L K I D N S D !1 1
5722 ccagttgttcsteagcteaescaggecaststtacttttagcgsg
S V V H Q L T Q A N I T F S E
5767 attagacataaccatttgaatttagasgasattttcttacactta
I R N N H L N L E E I F L N L
5812 acsgqtasgaagttscgagatcag 5A35
T G K K L R D
SEQUENCE DESCRIPTION: SEQ ID N0:12
Sequence of the originally cloned 3.8 kb insert.
SU6ST'ITUTE SHEET (RULE 26)
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1 ACCGTCCAGG TGAAGGTGTT GAAGCACATA AAGAAGCTGC TAAAGCTAAT
51 CTTGAAAAAG TAGCTAAAGA AACTAAAGCT CTTATTTCAG GAGACCGTTA
101 CTTGAGCGAA ACTGAAAAAG CAGTCCAAAA ACAAGCTGTT GAGCAAGCTC
151 TTGCGAAAGC ACTTGGTCAA GTTGAGGCTG CTAAGACAGT TGAAGCTGTT
201 AAGTTGGCAG AAAACCTTGG TACTGTAGCT ATCCGTTCAG CATATGTTGC
251 TGGTTTAGCT AAAGATACTG ATCAAGCAAC AGCTGCTCNT TAACAAGCGA
301 AACAAGCTGC TATTGAAGCT CTTAAACANG CTGCGGCAGA AACACTTGCT
351 AAGATTACAA CTGATGCTAN ATTGACTGAA GCTCAARA.AG CTGAACAATC
401 AGAAAATGTN TCNNTAGCGC TTAAGACGGC TATTGCGACT GTTCGTTCAG
451 CACAATCTAT TGCGTCTGTG AAAGAAGCAA AAGATAAAGG TATTACTGCT
501 ATCCGTGCAG CCTATGTGCC TAATAAGGCA GTCGCAAAAT CATCGTCNGC
S51 GAACCATCTT CCAANATCAG GTGANGCAAA CTCNATTGTT CTNGTTGGCT
601 TAGGAGTTAT GTCTCTTCTN TTAGGTATGG TGCTTTATAG CCCNGAAAAA
651 AGAAAGTAAA GACTAAGAAC CTGGCTTTAT CCAAATTGCT TTCTACTATA
701 TTTTCAAACC AAGGTGATTT TGAAATCACC TTGGTTTTTT GGATCGTAAG
751 GGCCCAAGAA CGGAGTGTAT TGAAAACATT TCAAAGTTCA ATTGAAAAGG
801 CAACAGTTTT CTGTACAGAA TTTATAAGGC GTTTTTTGTT CAAACGATGA
851 CTCATTTAGT TATGGGTGTT TGATAATTAA GATTACTGTG AAGGTGATGG
901 TAGTTCCACC AGTGGACTTC CTCCTTGGTT TTAAGGGTTA ATTGTTCTAG
951 TGAATAAAAG GTTTCTTGGT CGCGCAGTAG GGATCAAGCG AGCAGATAAA
1001 ATGCCTATTT TAAATCAGTT CGAGCGAATT TTCGTGCTAC AAGCTCAAAA
1051 GAGGCTACTA AAGTATTAGA ATAGTTTATC TCTGAGTGAA GTNCTCGTTA
1101 TAAGAAGATG ATGTTATCGT TGAAACGACC AAAAAATCTC CTAACTTTTA
1151 CTAATTTCTA CATGAAATCT AGTCCAGTAT TTACTCTACA ACCTTGATTG
1201 ATTCGCTTAA TAAAGACATT TCTACTTGAT TGTTTACACA TAGTTATTGA
1251 TAGAATCTAT TATAAAATTC AGTATATCAG ATCATAATTG TTTCTATATT
1301 AATCAATTAT TATTTTTCTT GTCATTTTTG ATAATATTAA AAAGAAAGGG
-35 -10
1351 TTTACATATT AATCATTTTT TACTATAATA AAAGTGATAA GAACTAGATA
SUBSTITUTE SHEET (RULE 26)
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1401 GTTGTTGTGT TACAACAGTA CAATTGAGCT AGCCTTGTCC TTGTTGTGTT
1451 AACTTTATTT TTAAAATAAG GTTAAAAATA AACGACTCGC GTTCTTATCA
S.D. sagA (1529-1691)
1501 GTTACTTATT AGATAAGGAG GTAAACCTTA TGTTAAAATT TACTTCAAAT
1551 ATTTTAGCTA CTAGTGTAGC TGAAACAACT CAAGTTGCTC CTGGAGGCTG
1601 CTGTTGCTGC TGTACTACTT GTTGCTTCTC AATTGCTACT GGAAGTGGTA
1651 ATTCTCAAGG TGGTAGCGGA AGTTATACGC GGGTAAATA ATCTATTTAG
1701 CATCTCTATG TGGTAGTGAT ATTAAGGTAA TGAGTTTGCT AGCAACTAAG
1751 TTATCTTTGG TAACTTCTGA TTATAGATGA ATGGCATAGA AGTGTTAGAA
1801 AACATGAGAC AAAAGTTATC TTTCTTGATG CTAACGTCTC AGGTAATTAG
1851 CAGGTACTAG ATAGTACCTG CTAATTACTA TATGTTTAGT AAAATGAGAT
saga (1914-2866)
1901 AGGAAAGGTA GTTATGTGT TTTTTACAAA GGAACAACAA CCTAAAGAGA
1951 ATTGTCCGCC AATAACTGTT GAAAAAGCAA GGCAATTGTT TGAATTTAAT
2001 ACAAACGCT TGTCCTTATC GGATTACGT GTCAAACGG TGCTAAAAAC
2051 GTCAAAGGG CTAGTTGCTC AAGTTTAAT GCCTAATGG ACCGATAATC
2101 TTAGTCAAG TTTTTTGATG AACTATAAAG CTAATAATAA TTATTTAGGC
2151 TTCCAAGCTA GTATTGTCGA CTTTTTTAG GATTCCGCCG TTGCTAATTT
2201 TTCAAGTAGT TACGTTTATG AAAGTGGGA AAAGATAATT CGTTTACCG
2251 AAACCTACG AGATATCAAC TGCTCTGTCG AGTGTATTA TAAAACGAAG
2301 AAGTGTCGT CAATTTTGG ATAGAGAAT GCCTCTTCAA GATTTATCAA
2351 AGTTCTTTA TTATGGTGT GGTGTTAGTT GCAAGGTC AATTAGAGAT
2401 GGAGGTCAG ATAAGATTAC ACTGGAAAC TGTGCTTGG GTGGAGGTTT
2451 ATACCCTATT GTTTAGTTT TTTATGCTAG AAAGTGGT AAATTAATAG
2501 ATGGTTTCTA TGAATATCTA CCCTATGGC ATGGCTAAG GTGTTATCGG
2551 GTAGCTCTG AGGAAAACGT TAGAGATTTT GCGGAATACG GTGCTATTAA
2601 TGCTGAAAAT TGTAATATTA TTATTATTTA TGTCTACGT TAGTGAAAA
2651 ATACAACGTA AATATGGGAA TGGGCGACT GCCTATGCTT TTATTGAATC
2701 AGGAGAAATA GCCCAGAATA TTCAATTGAC TGCAACTGCC TTAACTTATG
2751 GAAGTATTGA TATTGGTGGT TATAATAAGG AATATCTCG AGAATTATTA
2801 GATTTAGATG GGCTAGGAGA GGTGTGATT GGTGAGC TCGTAGGAAC
SUBSTfTUTE SHEET (RULE 26~
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2851 TAAGGAGTCT CAATGAAATA TCAACTTAAT AGTAATGTTC GTGTCGTGAC
2901 ATTTCAAGAT ACATTTTGTT TTAGAAAAGG AATCTGGGAC TTTAATGAAG
2951 CCATTCTAGA TTTGACACAA GAACCTCAAA ATTTAAAGAT AGTTTATCAA
3001 GAAATAGNGC CACAGTTGGT TAAGGGAGTA GCCATTGATA CTGAAACTTA
3051 TGAAAATACT TTAGAACCTG AGACGTTCGC TAATTTAATG GAAGTCATTT
3101 CAGGCTTGTA TTACAATGAT ATGCTGATGC TTGAGGATGA CTACAATCTT
3151 GAAGAAAATG TCATGAAAAT TTTAATGGGT AATTTCCGTT TTATGGCGCA
3201 AGCAGGTCAT ATGGTAAGCA ATGATCCTGT TTTGTTTATC AGTGATTCGA
3251 CTTATGTTAA TGAATCTGCT GAGTTGCTAG CTGAACAACT GCATTTACAA
3301 TTGCAAGTGG CTAGTGATGA CTTAAAAATG TTAATTCCGG CAGACAGATG
3351 TTAGTTTGGC GTTTAGGATG CTTTGGGAGC ATCGCCGTNA ACATGAATTG
3401 TTTATCAGAT GCCTTGTGCG ATTACCAAAG TATTATTATT TGTCAAGAAC
3451 GCTTGAATAT TATGATGTTG CGACACTTGA ATGAAGTTAG CGTAGCTATG
3501 AAAAAACAAT TAGTGATTGG TTTTGTAGAT GGACCATTCT TGCACACTTG
3551 TACTTTGAAT CCTCCCCATA GTGCTGACTT TGATAGCTTA GAAAGAAGAG
3601 TTTTGGCACG TTTACAAGAC TCAACTCTCT ATCAACATTT TGCTAATCAG
3651 GTTTTACCAG CCACACAAGA TGTGAGCCAG GCCTATTTAC CATTGTTAAA
3701 TGTTTTAATG AATTTAGTCG TTACGCAAGC TT
SUBSTITUTE SHEET (RULE 26)
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FIGURE 2
-35 p
-10
1 AGGGTTTACA TATTAATCAT TTTTTACTAT AATAAAAGTG ATAAGAACTA
51 GATAGTTGTT GTGTTACAAC AGTACAATTG AGCTAGCCTT GTCCTTGTTG
101 TGTTAACTTT ATTTTTAAAA TAAGGTTAAA AATAAACGAC TCGCGTTCTT
S.D.
Start
151 ATCAGTTACT TATTAGATAA GGAGGTAAAC CTTATGTTAA AATTTACTTC
M L K F T S
201 AAATATTTTA GCTACTAGTG TAGCTGAAAC AACTCAAGTT GCTCCTGGAG
N I L A T S V A E
T T Q V A P G G
251 GCTGCTGTTG CTGCTGTACT ACTTGTTGCT TCTCAATTGC TACTGGAAGT
C C C C C T T C C
F S I A T G S
Stop
301 GGTAATTCTC AAGGTGGTAG CGGAAGTTAT ACGCCAGGTA AATAATCTAT
G N S Q G G S G S
Y T P G K
351 TTAGCATCTC TATGTGGTAG TGATATTAAG GTAATGAGTT
(The highest degree of homology was observed with epidermin and
peps (from Staphylococcus epidermidis) matching 44% and 40%
similarity respectively, and 22% and 20% identity respectively.
Calculations of homology are done using the FASTA (Pearson W.R.
and Lipman D.J., 1988, PNAS 85: 2444-2448) and BLAST (Altschul
S.F. and Lipman D.J., 1990 PNAS
87: 5509-5513) algorithms.)
SUBSTITUTE SHEET (RULE 26)
*rB
