WO1999049049A1 - Sag-a streptococcique, une proteine de structure a activite sls associee - Google Patents

Sag-a streptococcique, une proteine de structure a activite sls associee Download PDF

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Publication number
WO1999049049A1
WO1999049049A1 PCT/CA1999/000240 CA9900240W WO9949049A1 WO 1999049049 A1 WO1999049049 A1 WO 1999049049A1 CA 9900240 W CA9900240 W CA 9900240W WO 9949049 A1 WO9949049 A1 WO 9949049A1
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nucleic acid
polypeptide
peptide
acid molecule
sequence
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PCT/CA1999/000240
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English (en)
Inventor
Joyce De Azavedo
Darrin Bast
Sergio Borgia
Stephen Betschel
Donald Low
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Mount Sinai Hospital
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Priority to AU28232/99A priority Critical patent/AU2823299A/en
Priority to CA002290653A priority patent/CA2290653A1/fr
Publication of WO1999049049A1 publication Critical patent/WO1999049049A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • 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 is produced by virtually all strains of GAS and it has a direct cytopathic effect on a broad range of cell types (Bernhei er A.W. , 1954; Freer, J.H. and J.P. Arbuthnott, 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). SUMMARY OF THE INVENTION
  • the present inventors generated two SLS deficient mutants using transposon ( Tn) 916 mutagenesis from two clinical Streptococcus pyogenes isolates of Ml 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- hemolytic transconjugants revealed that each contained a single Tn916 insertion located in the promoter region of an open reading frame (ORF) .
  • ORF open reading frame
  • SAG-A Polypeptides SAG-A
  • SAG-A Peptides SAG-A Peptides
  • 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.
  • 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.
  • 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% homology, 75% homology, 85% homology, 95% homology and 98% homology to the nucleotide sequence of SEQ ID NO: 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.
  • 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" ) .
  • variants of SAG-A polypeptide encoded by naturally occurring alleles of the sagA gene are variants of SAG-A polypeptide encoded by naturally occurring alleles of the sagA gene.
  • 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 nucleic acid molecules.
  • 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.
  • 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 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.
  • 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.
  • nucleic acid molecules of the invention may be used for therapeutic or prophylactic purposes, in particular genetic immunization.
  • particularly preferred embodiments of the invention are naturally occurring allelic variants of SAG-A and 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.
  • 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.
  • a polypeptide or peptide of the invention 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 .
  • 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.
  • SagA agonists or antagonists preferably bacteriostatic or bactericidal agonists and antagonists.
  • 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 Polypeptide or SAG-A Related Polypeptide to an organism to raise an immunological response against a bacteria especially a GAS .
  • Figure 1A is a blot of a Southern hybridization analysis of Hindlll 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 IB is a blot of a Southern hybridization analysis of Hindlll 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 SBNH5 (lanes 2-7) and wildtype MGAS166s (lanes 7-13) quantified, standardized, blotted and probed using a PCR amplicon of sagA labeled with ⁇ 32 P;
  • Figure 4 is a graph showing comparisons of mean weight changes of mice after infection with wild type (MGAS166s; T18Ps) and the respective isogenic non-hemolytic mutants (SBNH5; SB30-2) ;
  • Figure 5A is a photograph of a hairless SKH1 mice 24 hours after infection with 10 cfu of the SLS producing wildtype MGAS166S (A) ;
  • Figure 5B is a photograph of a hairless SKH1 mice 24 hours after infection with 10 cfu of the SLS-deficient Tn916 mutant SBNH5 ;
  • Figure 6A is a photograph of a tissue biopsy from euthanized mice which were infected with 10 cfu of the SLS-producing wildtype MGAS166s or the SLS-deficient Tn916 mutant SBNH5;
  • Figure 6B is a photograph of a tissue biopsy from euthanized
  • 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.
  • 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.
  • 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.
  • 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 bases .
  • the DNAs or RNAs may have backbones modified for stability or for other reasons.
  • a nucleic acid sequence includes an oligonucleotide, nucleotide, or polynucleotide.
  • 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.
  • 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.
  • 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.
  • a nucleic acid molecule of the invention may comprise a structural gene and its naturally associated sequences that control gene expression.
  • 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 .
  • nucleic acid sequence encoding a polypeptide having substantial sequence identity to the amino acid sequence of SEQ. ID. NO. 2, 4 or 6;
  • the isolated nucleic acid molecule comprises :
  • the invention relates to a nucleic acid molecule encoding the complementary nucleotide sequence of any of the nucleic acid molecules described above.
  • complementary refers to the natural binding of nucleic acid molecules under permissive salt and temperature conditions by base-pairing.
  • sequence "A-G-T” binds to the complementary sequence M T-C-A” .
  • Complementarity between two single-stranded molecules may be
  • the isolated nucleic acid comprises a nucleic acid sequence encoding the amino acid sequence of Streptococcus pyogenes SAG-A shown in SEQ. ID. NO. 2 or 6, or comprises the nucleic acid sequence of Streptococcus pyogenes sagA shown in SEQ. ID. NO. 1 or 5 wherein T can also be U.
  • sequence similarity refers 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.
  • 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 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)] .
  • 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.
  • 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. 1, 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.
  • 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 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.
  • strain or species variations i.e. variations in nucleotide sequence naturally occurring among different strains or species, are within the scope of the invention.
  • 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.
  • sequence encodes the amino acid sequence shown in SEQ. ID. NO. 2 and comprises at least 5, preferably at least 10, more preferably at least 15, and most preferably at least 20 nucleotides.
  • 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.
  • 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 herein.
  • 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 200 nucleotides but less than 2 kb.
  • 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.
  • 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) .
  • 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.
  • 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.
  • 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.).
  • PCR polymerase chain reaction
  • 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
  • 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.
  • a cDNA can be cloned downstream of a bacteriophage promoter, (e.g. a T7 promoter) in a vector, cDNA can be transcribed in vi tro with T7 polymerase, and the resultant RNA can be isolated by conventional techniques.
  • a bacteriophage promoter e.g. a T7 promoter
  • 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 Maxam-Gilbert chemical sequencing, to determine the nucleic acid sequence and the predicted amino acid sequence of the encoded polypeptide.
  • 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.
  • nucleic acid molecule comprising a regulatory sequence of sagA as shown in SEQ. ID. NO. 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.
  • the heterologous nucleic acid includes a nucleic acid foreign to the cell .
  • the nucleic acid molecules isolated using the methods described herein are mutant sagA genes.
  • 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.
  • 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 mutation(s) 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.
  • Clones containing mutant sequences can be purified and subjected to sequence analysis.
  • 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.
  • RNA molecules 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.
  • RNA molecules may be generated by in vi tro 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.
  • 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.
  • flanking sequences at the 5 ' and/or 3 ' ends of the molecule 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 ' O-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 .
  • Polypeptides of the Invention are not limited to, the addition of flanking sequences at the 5 ' and/or 3 ' ends of the molecule or the use of phosphorothio
  • 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. 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.
  • 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.
  • 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.
  • 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 .
  • 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.
  • 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 a ido 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.
  • 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.
  • a more flexible peptide may be prepared by 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 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.
  • 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 lantibiotic, 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 al . ) . 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 converting a hydrogen group to another group such as a hydroxy or amino group.
  • 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. conserveed 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.
  • 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 .
  • 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.
  • 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 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.
  • 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. NO.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 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.
  • 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.
  • 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) .
  • 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. These techniques are known to those skilled in the art.
  • 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 sequence 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.
  • marker genes are genes encoding a polypeptide such as G418 and hygromycin which confer resistance to certain drugs, ⁇ - 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.
  • 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 pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST) , maltose E binding polypeptide, or polypeptide A, respectively, to the recombinant polypeptide.
  • GST glutathione S-transferase
  • 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.
  • 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.
  • the polypeptides of the invention may be expressed in bacterial cells such as streptococci, staphylococci, enterococci, E. coli , streptomyces, lactic acid bacteria, and Bacillus substilis, 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, 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.
  • a method for preparing a SAG-A Polypeptide, or SAG-A Related Polypeptide utilizing the purified and isolated nucleic acid molecules of the invention.
  • a method for preparing a SAG-A Polypeptide, or a SAG-A Related Polypeptide comprising:
  • 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 LI 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 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.
  • 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 nd 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.
  • 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.
  • 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 . Lantibiotics constitute a unique class of ribosomally- synthesized, antimicrobial peptides produced by gram positive bacteria.
  • 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 Helico acter pylori associated peptic ulcer disease in humans (Blackburn and Projan 1994) .
  • nisin 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).
  • lantibiotics also have numerous commercial applications.
  • 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
  • U.S. Patent Nos. 5,512,269 and 5,683,675 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.
  • 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.
  • 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.
  • 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) . 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, epidermin, gallidermin, lacticin, streptoccin, salivaricin A, mutacin, lactocin S, carnocin, or cytolysin LI or L2, to a propeptide part of the protein (e.g. SEQ ID NO. 6) .
  • 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, Pep5, subtilin, epilancin, epidermin, gallidermin, lacticin, streptoccin, salivaricin A, mutacin, lactocin S, carnocin, or cytolysin LI 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 .
  • the invention relates to methods for identifying substances that affect a polypeptide having characteristics of a lantibiotic .
  • 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 known 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 method for producing a lantibiotic by inserting an expression vector in a cell and expressing the peptide.
  • a polypeptide of the invention (e.g. SEQ ID NO 2, 4, or 6) 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.
  • the invention can employ intact monoclonal or polyclonal 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 F v molecule (Ladner et al, U.S. Pat. No.
  • a chimeric antibody for example, an antibody which contains the binding specificity of a murine antibody, but in which the remaining portions are of human 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.
  • 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.
  • 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) . Diagnostic Methods
  • 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 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 .
  • 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 3 P, 3 H, 14 C or the like.
  • detectable substances 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 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.
  • nucleic acids e.g. recombinant DNA molecules, cloned genes
  • 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 assays of biological samples to detect abnormalities involving sagA structure, including point mutations, insertions, deletions, and chromosomal rearrangements.
  • 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 SI protection or a chemical cleavage method
  • 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.
  • primers derived from SEQ ID NO: 1 or 5 may be used to amplify nucleic acids isolated from an infected individual and the amplified nucleic acids may be subjected to various techniques for elucidation of the DNA sequence .
  • mutations may be detected and used to diagnose infection and to serotype and/or classify the infectious agent.
  • a method for diagnosing disease 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:l 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.
  • 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.
  • immunoassays may also be used to assess or monitor the efficacy of particular therapies .
  • the antibodies of the invention may also be used in vi tro 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 (e.g.
  • 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.
  • 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.
  • detectable substances include, but are not limited to, the following: radioisotopes (e.g., 3 H, 14 C, 5 S, 125 I, 131 I) , fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), luminescent labels such as luminol; enzymatic labels (e.g., horseradish peroxidase, ⁇ -galactosidase, luciferase, alkaline ph ⁇ sphatase, 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) .
  • a secondary reporter
  • the antibody or sample may be immobilized on a carrier or solid support which is capable of immobilizing cells, antibodies, etc.
  • 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 second antibody, having specificity for the antibody reactive against a polypeptide of the invention.
  • the second antibody may be goat anti-rabbit gamma-globulin labeled with a detectable substance as described herein.
  • 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 .
  • the methods described herein are designed to identify substances or compounds that modulate the activity of a SAG-A
  • 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.
  • the invention also provides methods for identifying substances which interact with a SAG-A Polypeptide or SAG-A 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.
  • agonist refers to a molecule that increases the amount of, or prolongs the duration of, the activity of the polypeptide.
  • 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 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 polypeptide 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 non-complexed polypeptides may be isolated by conventional isolation techniques, for example, salting out, chromatography, electrophoresis, gel filtration, fractionation, absorption, polyacrylamide gel electrophoresis, agglutination, or combinations thereof.
  • 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.
  • the method may comprise contacting a polypeptide of nucleic acid molecule of the invention with a compound to be screened under conditions to permit binding to or other interaction between the compound and the polypeptide or 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 inhibitor) 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.
  • 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 .
  • 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 .
  • 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 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
  • the marked impairment in the virulence of two clinically relevant S. pyogenes strains by transposon insertion in the sagA 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.
  • 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
  • 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.
  • 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 polypeptides.
  • 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.
  • 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, 5,147,643, 5,085,862 and 5,073,494.
  • 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 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” 1:83-92, CRC Press, Inc. Boca Raton, Fla., 1987).
  • 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).
  • 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.
  • 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) .
  • streptococcal disease such as necrotizing fasciitis (NF) and streptococcal toxic-shock syndrome (STSS) .
  • 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 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.
  • 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 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 .
  • 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 1 , Fab, Dab, as well as engineered antibodies such as recombinant antibodies, humanized antibodies, bispecific 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.
  • growth factors 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 ⁇ (transforming growth factor beta) , and pleitotropin.
  • 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).
  • 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 .
  • 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.
  • 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.
  • 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 periods 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)).
  • 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 th ed, (1990, Mack Publishing Company) and subsequent editions) .
  • 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.
  • compositions used to treat patients having diseases, disorders or abnormal physical states could include SAG- A or another peptide of the invention and an acceptable vehicle or excipient.
  • 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 .
  • 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, creams, ointments, powders, emulsions, gelatin capsules, capsules, suppositories, gels or membranes.
  • compositions of the invention may also be conjugated to transport 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.
  • 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 .
  • cells may be transformed with sequences complementary to nucleic acid molecules encoding a SAG-A Polypeptide or SAG-A Related Polypeptide.
  • 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 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 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.
  • 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.
  • the antisense molecules are oligonucleotides derived from the transcription initiation site (e.g. between positions -10 and +10 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
  • 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
  • Ribozyme action involves sequence- specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
  • hammerhead motif ribozyme molecules may be engineered that can specifically and efficiently catalyze endonucleolytic cleavage of 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, GUU, 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 ribonuclease protection assays.
  • the activity of the substances, compounds, antibodies, polypeptides, nucleic acid molecules, and compositions of the invention may be confirmed in in vi tro cell systems or in animal experimental model systems (e.g. the dermonecretic mouse model described herein) .
  • 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 .
  • GG lantibiotic double glycine motif cleavage site
  • the two fragments generated would be of similar size and amino acid composition as those generated by the proteolytic cleavage of other lantibiotics.
  • 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.
  • cysteine residues in the amino half of the SAG-A pro-peptide and the serine residues in its carb ⁇ xy terminus is characteristic of the type-A group of lantibiotics (Jung 1991) .
  • posttranslational 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.
  • Examples 2-12 describe in detail the studies leading to the identification of the sagA gene and its role in virulence.
  • Example 2 Generation of non-hemolytic transconjugants.
  • E. faecalis CG110 at a frequency of 10 "4 for both recipients.
  • Transconjugants maintained the non-hemolytic phenotype after subculture on selective media.
  • Tn916 excision assays were conducted on non-hemolytic transconjugants derived from T18Ps 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 "8 and 10 '7 for SBNH5 and SB30-2, respectively. Because of the low frequency of Tn916 excision, it was necessary to screen for hemolytic revertants on a confluent mat of bacteria.
  • Example 3 Genetic characterization of the non-hemolytic transconjugants and hemolytic revertants.
  • the Tn916 probe used spanned the only Hindlll restriction site within Tn916. Cleavage at this site divides the transposon into two fragments of approximately 6 kb and 12 kb (Clewell et al. 1993) . After Hindlll digestion, each copy of Tn916 which has integrated into the chromosome of the recipient strains yields two bands that hybridize with the probe. Two Hindlll fragments, approximately 14 and 7.8 kb, from each of the non-hemolytic mutants derived from MGAS166s hybridized with the tetM probe.
  • Tn916 is capable of precise excision (Gawron-Burke and Clewell 1984) .
  • Two revertants were selected for further analysis, NH5rev 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.
  • Example 4 Analysis of Tn916 insertion site.
  • a genomic library of MGAS166s was generated using the low copy number plasmid, pACYC184. 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.
  • 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 .
  • sagA was not found in the Oklahoma GAS genomic sequence data base.
  • PCR products were generated using primers based on the known 3.8 kb sequence 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 bp downstream of the -35 element and 6 bp upstream of the -10 TATA box.
  • 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 bp which was in keeping with the expected size of an mRNA product from sagA.
  • No detectable transcript was observed from RNA isolated from SBNH5 at any time point . Probing the same membranes with the 16s rRNA probe did not yield any differences between RNA from MGAS166s and SBNH5.
  • 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 Ml parent strains respectively. No difference in M-protein quantity was seen between MGAS166s and SBNH5 by Western blotting using a monoclonal antibody to the constant region of Ml 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 SBNH5 at all points measured.
  • Example 10 Reduced virulence of SLS deficient transconjugants. Reproducible, non-lethal lesions were generated following injection of 10 6 CFU MGAS166s and 10 7 CFU T18Ps subcutaneously into mice. The difference in inoculum size, needed to produce the same virulence profile, is likely due to inherent differences in virulence between Ml and M18 serotypes of GAS.
  • 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,
  • mice which received 10 CFU of SBNH5 demonstrated a mean weight gain of +1.15 + 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 + 0.29 g seen in the uninfected
  • mice injected with 10 CFU of the wildtype hemolytic T18Ps exhibited a significant mean weight loss
  • 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 mm 2 . No necrotic lesions were observed in animals infected with SBNH5, 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, T18Ps 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 2 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 mm 2 .
  • biopsies of tissue from animals which had been infected with MGAS166s differed histologically from SBNH5 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 SBNH5 did not show evidence of acute inflammation and no 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.
  • 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.
  • 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.
  • MBP maltose-binding protein
  • 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, epidermin and Pep5 (Saris et al . 1996).
  • Example 14 Antibodies directed to SAG-A
  • the MBP-SAG-A fusion protein is used to raise antibodies in rabbits .
  • Monoclonal and polyclonal 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.
  • vi tro hemolysis inhibition studies are performed to characterize SAG-A specific antibodies that abrogate SLS activity.
  • Monoclonal and polyclonal antibodies are prepared according to other techniques known in the art .
  • 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, 5,225,331 and 5,124,147.
  • Bacterial strains and culture conditions 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 ⁇ g/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) .
  • LB Luria Bertani
  • Difco Luria Bertani
  • M-typing and quantitation 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 constant region of the Ml protein (kindly performed by Vincent
  • Southern hybridization analysis A probe specific for the tetM gene of Tn916 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.
  • ECL enhanced chemiluminescence
  • Genomic DNA was isolated from GAS as previously described (O'Connor and Cleary 1983) .
  • DNA was digested with tfindlll (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 with Hindlll, ligated into the Hindlll site of pACYC184 (New England Biolabs, Mississauga, ON) and transformed into E. coli DH5 ⁇ MCR 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 .
  • RNA was extracted using Trizol (Gibco BRL) according to manufacturers directions . RNA was isolated from bacteria at mid-log phase (O.D. 550 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 ⁇ 32 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 . Excision of Tn916. Phenotypic revertants were produced in a manner similar to that described by Nida and Cleary (1983) .
  • 10 CFU determined by an optical density at 550 nm (O.D.5 50 ) of 1.0-1.2, of a late log phase culture of non-hemolytic transconjugants were inoculated into 50 ml of non-selective Todd- Hewitt broth. After overnight incubation, 10 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 subcultured on non-selective media to isolate the hemolytic revertants . Tetracycline resistance was determined by growth on Columbia blood agar plates containing tetracycline (5 ⁇ g/ml) . Growth rate analysis.
  • SLS activity was also measured during early, mid and late log phase using the above method. Overnight broth cultures of MGAS166s and SBNH5 were subcultured in Todd-Hewitt broth and samples withdrawn hourly for 8 hours and immediately frozen at -
  • 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 10% SDS-polyacrylamide gel and stained with Coomassie brilliant blue R.
  • 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. Quantitation of hyaluronic acid. Bacteria were grown in 150 ml of Todd-Hewitt broth to an O.D. 5S0 of 0.6 - 0.8. Mutants were grown in the presence of tetracycline.
  • Virulence of GAS strains was determined using a dermonecrotic mouse model as previously described (Bunce et al . 1992) .
  • a 100 ⁇ l 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 ⁇ l 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 for 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.
  • 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
  • mice infected with sagB, D and G produced lesions similar to NZ131. Mutants for sagH 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 NZ131.
  • T18Ps ..- -. thoroughly r __ s _,. prepare+ See text M18,St , Tc ,SLS Spontaneous Str derivative of T18P
  • Complete lysis was determined by lysing 750 ⁇ L of 5% washed sheep erythrocytes in hypotonic saline and adding to an equal volume of sterile THB.
  • Results are zone diameters surrounding inoculum after overnight anaerobic incubation of assay plates at 37 C. Measurements are mean +/- standard deviation of three experiments.
  • FIG. 1 Southern hybridization analysis of Hindlll restriction digests of genomic DNA probed tetM.
  • A Hemolytic wildtype T18P (Lane 1) does not hybridize with the tetM probe.
  • the Tn916 donor strain CG110 contains several copies of Tn916.
  • B Hemolytic wildtype MGAS166s (Lane 1) does not hybridize with the tetM probe.
  • the non- hemolytic transconjugants SBNHl, SBNH3 , SBNH4 , SBNH5 , SBNH6 , SBNH7, and SBNH8 (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 (B) .
  • Figure 2. The nucleotide sequence and protein translation of sagA.
  • a 390 bp region of genomic DNA from MGAS166s is represented corresponding to the chromosomal point of insertion of Tn916 (V) .
  • 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 pep5 (from Staphylococcus epidermidis) matching 44% and 40% similarity respectively, and 22% and 20% identity respectively.
  • 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 SBNH5 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 strain is devoid of any transcripts from sagA while the wildtype contains sagA transcripts at all time points.
  • FIG. 4 Comparisons of mean weight change are shown 24 hours after infection with wild type (MGAS166s; T18Ps) and the respective 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 .
  • MGAS166s A or the SLS-deficient Tn916 mutant SBNH5 (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 SBNH5.
  • Figure 6. Tissue biopsies from euthanized mice which were c infected with 10 cfu of either the SLS-producing wildtype MGAS166s (A) or the SLS-deficient Tn916 mutant SBNH5 (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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Abstract

La présente invention concerne des molécules d'acides nucléiques qui codent pour sagA, des molécules d'acides nucléiques homologues, aussi bien que des peptides codés par les molécules d'acides nucléiques.
PCT/CA1999/000240 1998-03-20 1999-03-18 Sag-a streptococcique, une proteine de structure a activite sls associee WO1999049049A1 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002057315A2 (fr) * 2000-10-10 2002-07-25 University Of Tennessee Research Corporation Vaccins anti-streptocoques à base de streptolysine s
US7160547B2 (en) * 2000-10-10 2007-01-09 University Of Tennessee Research Corporation Streptococcal streptolysin S vaccines
WO2009092790A1 (fr) * 2008-01-24 2009-07-30 Teagasc, The Agriculture And Food Development Authority Cytotoxine de listeria monocytogenes, la listériolysine s
WO2009081274A3 (fr) * 2007-12-21 2009-08-13 Novartis Ag Formes mutantes de la streptolysine o
WO2016172476A1 (fr) * 2015-04-24 2016-10-27 The Rockefeller University Microorganismes modifiés exprimant un antigène saga en tant qu'agents anti-infectieux, probiotiques, et composants alimentaires
EP3219721A1 (fr) * 2016-03-16 2017-09-20 Institut Pasteur Listériolysine s ou peptides apparentés en tant qu'agents antibactériens
WO2020172406A1 (fr) * 2019-02-20 2020-08-27 The Rockefeller University Micro-organismes modifiés exprimant des saga et compositions associées pour l'immunomodulation contre une infection et l'immunothérapie anticancéreuse

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BETSCHEL S. D. ET AL.: "Reduced virulence of group A streptococcal Tn916 mutants that do not produce streptolysin S.", INFECTION AND IMMUNITY, vol. 66, no. 4, April 1998 (1998-04-01), pages 1671 - 1679, XP002113084 *
BORGIA S. M. ET AL.: "Cloning of a chromosomal region responsible for streptolysin S production in Streptococcus pyogenes.", ADV. EXP. MED. BIOL., vol. 418, 1997, pages 733 - 736, XP002113083 *
DATABASE EMBL NUCLEOTIDE SEQU 1 January 1900 (1900-01-01), XP002113085, Database accession no. AF067649 *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002057315A3 (fr) * 2000-10-10 2003-06-19 Univ Tennessee Res Corp Vaccins anti-streptocoques à base de streptolysine s
US7160547B2 (en) * 2000-10-10 2007-01-09 University Of Tennessee Research Corporation Streptococcal streptolysin S vaccines
WO2002057315A2 (fr) * 2000-10-10 2002-07-25 University Of Tennessee Research Corporation Vaccins anti-streptocoques à base de streptolysine s
US8409589B2 (en) 2007-12-21 2013-04-02 Novartis Ag Mutant forms of streptolysin O
WO2009081274A3 (fr) * 2007-12-21 2009-08-13 Novartis Ag Formes mutantes de la streptolysine o
US7731978B2 (en) 2007-12-21 2010-06-08 Novartis Ag Mutant forms of streptolysin O
US8039005B2 (en) 2007-12-21 2011-10-18 Novartis Ag Mutant forms of streptolysin O
EP2537857A3 (fr) * 2007-12-21 2013-01-02 Novartis AG Formes mutantes de streptolysine O
WO2009092790A1 (fr) * 2008-01-24 2009-07-30 Teagasc, The Agriculture And Food Development Authority Cytotoxine de listeria monocytogenes, la listériolysine s
WO2016172476A1 (fr) * 2015-04-24 2016-10-27 The Rockefeller University Microorganismes modifiés exprimant un antigène saga en tant qu'agents anti-infectieux, probiotiques, et composants alimentaires
US10723771B2 (en) 2015-04-24 2020-07-28 The Rockefeller University Modified microorganisms expressing SagA as anti-infective agents, probiotics and food components
EP3219721A1 (fr) * 2016-03-16 2017-09-20 Institut Pasteur Listériolysine s ou peptides apparentés en tant qu'agents antibactériens
WO2017158108A1 (fr) * 2016-03-16 2017-09-21 Institut Pasteur Listériolysine s ou peptides apparentés en tant qu'agents antibactériens
WO2020172406A1 (fr) * 2019-02-20 2020-08-27 The Rockefeller University Micro-organismes modifiés exprimant des saga et compositions associées pour l'immunomodulation contre une infection et l'immunothérapie anticancéreuse
EP3927358A4 (fr) * 2019-02-20 2022-11-09 The Rockefeller University Micro-organismes modifiés exprimant des saga et compositions associées pour l'immunomodulation contre une infection et l'immunothérapie anticancéreuse

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