DE60037908T2 - STREPTOCOCCUS PYOGENESIS VIRULENT GENES AND PROTEINS AND THEIR USES - Google Patents

Description

Field of the invention

The The invention relates to a virulence gene and protein and its use. Specifically, it relates to a product obtained from Streptococcus pyogenes Gene and protein / polypeptide and its use in therapy and in drug screening.

Background of the invention

Group A Streptococcus (GAS) is for the majority of streptococcal diseases are responsible. A special interesting organism is S. pyogenes, which with a wide range associated with non-invasive and invasive infections is such as impetigo, pharyngitis, necrotic fasciitis, bacteremia, streptococcal-induced Toxic shock syndrome (STSS), pneumonia and rheumatic fever.

Some GAS infections can with antibiotics, including Penicillin and enrythromycin are treated. It exists, however because of the problems associated with antibiotic resistance and antibiotic allergies in patients, there is a need for more therapeutics that for treatment or prevention of GAS infections are suitable.

Summary of the invention

The The present invention is based on the discovery of a virulence gene in S. pyogenes.

According to one In a first aspect of the invention, a peptide of the invention comprises herein as SEQ ID NO: 2 designated amino acid sequence of S. pyogenes or a related virulence molecule that is at least 80% similar at the peptide level or 80% identity at the nucleotide level, in a gram positive bacterium for therapeutic or diagnostic Use.

The Peptide can be used for many therapeutic purposes Group A streptococcal infections including, use in vaccines for prophylactic use.

According to one The second aspect may be a polynucleotide having a Peptide encoded, also be suitable for therapy or diagnosis.

According to one In the third aspect, the gene encoding the peptide may be produced from weakened Microorganisms are used.

The attenuated Microorganisms become common have a mutation which indicates the expression of the present invention Gens prevents to provide a strain that lacks virulence. These microorganisms are also used in therapy and find a diagnosis.

According to one fourth aspect the peptide, the gene and the attenuated microorganisms according to the invention in the treatment or prevention a disease that is associated with infection by Streptococcus or Gram-positive bacteria is related.

Description of the invention

The The present invention is based on the discovery of a gene which encodes a peptide that has been implicated in virulence becomes.

The The peptide and the gene of the invention are accordingly for the production of Therapeutics suitable for the treatment of infections. It understands itself, that mention the therapy also preventive treatments, z. Vaccination. Furthermore, although the products of the invention are mainly used for Treatment of infections in human patients, veterinary Applications are also considered within the scope of the invention.

The present invention will be described with reference to Streptococcus pyogenes. However, all group A streptococcal strains and many other gram positive bacterial strains likely include related peptides or proteins having amino acid sequence identity or similarity to those of the present invention Have designated designated. Organisms likely to contain the peptides include, but are not limited to, the genera Lactococcus, Enterococcus, Streptococcus and Staphylococcus.

Prefers have the peptides used in the various aspects of the invention could be suitable more than 80% similarity with the peptide referred to herein. Most preferred have the peptides z. B. 95% similarity on. With respect to the polynucleotide sequences referred to herein can be related Polynucleotides used in the various aspects of the invention can be suitable more than 80% identity having the sequences indicated herein. More preferred, the polynucleotide sequences have more than e.g. B. 95% identity.

The The terms "similarity" and "identity" are in the art known. The use of the term "identity" refers to a sequence comparison, the one on perfect matches between correspondingly identical positions in the sequences, which are compared. The term "similarity" refers to a comparison between amino acid sequences and taken into account not just identical amino acids in appropriate positions, but also functionally similar amino acids in appropriate positions. Thus, there is a similarity between polypeptide sequences on a functional similarity additionally to sequence similarity out.

The Extent identity between gene sequences and the extent of identity or similarity between amino acid sequences can calculated using known methods. Relating to The present invention includes publicly available computer-based Method for determining identities and similarities the BLASTP, BLASTN and FASTA program (Atschul et al., J. Molec. Biol., 1990; 215: 403-410) the BLASTX program, available by NCBI, and the Gap program by Genetics Computer Group, Madison, WI. The dimensions of similarity and identity given herein were using the gap program with a gap penalty of 12 and a gap length penalty of 3 for the polynucleotide sequence comparisons receive.

After this characterizes the gene of the invention was, it is possible use the gene sequence to look for related genes or peptides to look in other microorganisms. This can be done by searching in existing databases, eg. EMBL or GenBank.

Inventive peptides or proteins can be purified and isolated by methods known in the art. In particular, since the gene sequence has been determined, it will be possible recombinant techniques for expression of the gene in a suitable Host to use. Related molecules can be identified and can be suitable for therapy. For example, the peptides may be used as antigenic determinants used in a vaccine to trigger an immune response. she can also in the production of antibodies to passive immunization or for diagnostic applications are used. Suitable antibodies include monoclonal antibodies or fragments thereof, including single-chain Fv fragments. Methods for producing antibodies are a specialist obvious.

It is also understood that the invention additionally to related molecules from other microorganisms, includes modifications to the made in the present case designated peptides and polynucleotides which do not significantly affect biological function. It is obvious to a person skilled in the art that the degeneration of the genetic codes polynucleotides with minor base changes over the in this case specified, but nevertheless the same Encode peptides. complementary Polynucleotides are also included in the invention. conservative Exchanges at the amino acid level are also provided, d. H. different acidic or basic amino acids can be replaced without significant loss of function.

The preparation of vaccines based on attenuated microorganisms is known to those skilled in the art. Vaccine compositions may, as necessary or desired, be treated with suitable carriers or excipients, e.g. Alum, to provide effective immunization against infections. The preparation of vaccine compositions will be apparent to one skilled in the art. The attenuated microorganisms can be produced with a mutation which prevents the expression of any of the presently designated genes. The person skilled in the art knows of methods for suppressing the expression of certain genes. Techniques that may be used include insertion inactivation or gene deletion techniques. Attenuated microorganisms according to the invention may also comprise additional mutations in other genes, for example in a second gene referred to herein, or in another gene required for the growth of the microorganism, e.g. B. an aro mutation. Attenuated microorganisms may also be used as delivery systems for the administration of heterologous antigens, therapeutic proteins or nucleic acids (DNA or RNA). In this embodiment, the attenuated microorganisms are used to administer a heterologous antigen, protein or nucleic acid in vivo at a particular site. Introduction of a heterologous antigen, peptide or nucleic acid into the attenuated microorganism can be accomplished by conventional techniques, including the use of recombinant constructs, e.g. Vectors comprising polynucleotides expressing the heterologous antigen or therapeutic protein, and further comprising suitable promoter sequences. Alternatively, the gene encoding the heterologous antigen or protein can be incorporated into the genome of the organism, and the endogenous promoters can be used to regulate expression.

More generally, and as is well known to those skilled in the art, may be a suitable one Amount of a drug of the invention for therapeutic use selected as well as suitable carriers or excipients and routes of administration. These factors would be known according to Criteria such as the type / severity of the disease to be treated, the Type and / or health of the subject, etc., selected or be determined.

at another embodiment can the products of the invention in screening assays for identification of potential antimicrobial medicines or for detection a virulence can be used. Routine screening tests are the Known and knowledgeable using the products of the invention appropriately be adjusted. For example, the products of the invention as the goal for a potential drug may be used with the drug is able to inactivate or bind the target, which indicates its potential antimicrobial activity.

The Various products of the invention may also be used in veterinary Applications are used.

The The following is a brief overview of that experimental procedures used to identify the virulence gene. Virulence genes in S. pyogenes were tagged using the Signature Mutagenesis (STM) for the screening of a S. pyogenes mutant library for attenuated mutants (Hensel et al., 1995. Science 269 (5222): 400-3).

mutants were transfected by Tn917 transposon insertion using a Plasmid vector generated. additionally to a fragment of Tn917, the vector included a spectinomycin resistance gene, a chloramphenicol resistance gene (CAT gene) with a synthetic Promoter, a Gram-negative replication origin (rop-ori) and a cloning site for the STM tags. After ligation of the tags into the vector, a E. coli transformation performed, and 96 plasmids that hybridized with the starting tags were selected.

Of the S. pyogenes strain B514-SM (type M50) was labeled with each of the 96-day Plasmids were transformed, and transformants were determined by resistance across from Spectinomycin and chloramphenicol selected. For each of the transformed S. pyogenes strains were 20 mutants generated by Tn917 transposon insertion to a Create mutant bank.

The Mutant library was either in a mouse invasive invasive model skin lesions (Schrager et al., J. Clinical Investigation 1996; 98: 1954-1958) or a mouse infection model for Pharyngeal colonization (Husmann et al., Infection and Immunity, 1997; 65 (4): 935-944) searched.

In the skin model, five CD-1 mice were each inoculated intracutaneously with 1 x 10 ⁸ cells in a volume of 50 μl representing the collection of 96 different and easily distinguishable mutants. Forty-eight hours after inoculation, samples were taken and bacteria were collected. The skin lesions were macerated in 2xBHI medium and bacteria were released from the lesions by treatment in a Stomacher homogenizer for 10 minutes. The released bacteria were plated and at least 10,000 colonies were obtained from at least 3 mice. From these samples, the DNA was isolated and used to amplify the tag-labeled DNA present in the recovered bacteria. The DNA isolated from each recovered pool was used as a hybridization probe to reveal those mutants in each pool that could not be recovered from the animals and thus were attenuated in this animal infection model.

In the throat colonization model, 2x10 ⁸ cells were inoculated intranasally into six C57BL / 6 mice and sampled after 48 hours. As with the skin model, bacteria that had a trans poson-Tn917 insertion within a virulence gene, were not obtained from mice inoculated with a mixed mutant population, and were thus likely to be attenuated.

Additional experiments were performed on mutants identified by the STM screen to determine the competitive index (CI). Single mutants were tested in mixed infections with the wild-type strain in the skin lesion infection model (Chiang, SL and Mekalanos, JJ Molecular Microbiology 1998; 27 (4): 797-805). As with the baseline screen, groups of four CD1 mice were inoculated with equal numbers of both wild-type and mutant cells, in a total of 1 x 10 ⁸ cells. Bacteria recovered after 48 hours were plated on selection media allowing to distinguish between wild type and mutant colonies. The ratio of mutants to wild type bacteria observed in the inoculum compared to the ratio in recovered bacteria is the competitive index (ratio of mutants to wildtype in inoculum divided by the ratio of mutants obtained from the animal model to wildtype bacteria obtained from the animal model).

The the following example explains The invention.

example 1

It a mutant was identified with a nucleotide sequence that 99% sequence identity with the S. pyogenes nucleotide sequence, present as SEQ ID NO: 1 is designated. The predicted amino acid sequence is shown as SEQ ID NO: 2.

The amino acid sequence shows at the amino acid level 37% identity with the CitX protein from E. coli (accession number: SW: P77563).

CitX is required for citrate fermentation.

This shows that the disrupted gene is at least partially associated with the CitX gene from E. coli is identical. The genes involved in citrate fermentation are involved in E. coli and K. pneumonia together on a single locus located on the chromosome. Accordingly, the attenuation of S. pyogenes mutants may be the result of a failure of their CitX gene or downstream genes to express. This gene has not yet had any function in the Attributed virulence.

in view of the similarity the S. pyogenes genes with those present in E. coli the skilled artisan recognize that similar Sequences in other streptococci and Gram-positive bacteria the virulence could be involved.

CitX mutants were re a weakening tested for virulence. It has been shown that the mutated microorganisms with a competitive index (CI) of 0.025 (CitX).

Claims (16)

A peptide for therapeutic or diagnostic use comprising the amino acid sequence of S. pyogenes referred to herein as SEQ ID NO: 2, or a related virulence molecule having a peptide-level sequence similarity of at least 80% or a nucleotide sequence identity of at least 80% in a Gram-positive bacterium. The peptide of claim 1, wherein the sequence similarity or identity at least 90%. Polynucleotide for therapeutic or diagnostic Use that for a peptide according to claim 1 or claim 2 encoded. attenuated Microorganism that has a mutation that reduces expression the defined in claim 3 nucleotide sequence cancels. The microorganism of claim 4, wherein the mutation an insertion inactivation or a gene deletion. A microorganism according to claim 4 or claim 5, wherein the microorganism is a group A Streptococcus. Microorganism according to one of claims 4 to 6 for therapeutic or diagnostic use. Microorganism according to one of claims 4 to 7, which has a mutation in another nucleotide sequence. Microorganism according to one of claims 4 to 8 containing a heterologous antigen, therapeutic peptide or nucleic acid. A vaccine comprising a peptide according to claim 1 or 2. Vaccine, which is a microorganism according to one of the claims 4 to 9. Use of a product according to any one of claims 1 to 9 for the manufacture of a medicament for use in the treatment or prevention a disease associated with infection by Streptococcus or Gram-positive bacteria is related. Use according to claim 12, wherein the disease is a pneumonia. Use according to claim 12 or claim 13 to veterinary Treatment. Antibody, which is directed against a peptide according to claim 1 or claim 2. Use of a product according to any one of claims 1 to 9 in a screening test to identify an antimicrobial Active ingredient.