WO2003087147A1

WO2003087147A1 - Stretptococcal genes involved in osmotic and oxidative stress and in virulence

Info

Publication number: WO2003087147A1
Application number: PCT/GB2003/001638
Authority: WO
Inventors: Jeremy Stuart Brown
Original assignee: Imperial College Innovations Limited
Priority date: 2002-04-12
Filing date: 2003-04-14
Publication date: 2003-10-23
Also published as: GB0208499D0; AU2003229906A1

Abstract

According to the invention, an operon, termed the phg ABC operon, has been identified in Gram-positive bacteria, and encodes proteins required for the bacterial response to osmotic and oxidative stress. Isolated proteins and polynucleotides derived from the phg ABC operon are used in therapy, e.g. in vaccinology to treat infection by Gram-positive bacteria.

Description

STRETPTOCOCCAL GENES INVOLVED IN OSMOTIC AND OXIDATIVE STRESS AND IN VIRULENCE

Field of the Invention

This invention relates to genes and the proteins that they encode, to vaccines containing the proteins or functional fragments of the proteins, and to live attenuated bacterial vaccines lacking any or part of the genes. More particularly, the invention relates to their prophylactic and therapeutic uses and their use in diagnosis. Background to the Invention

A fundamental requirement for invasive bacterial pathogens is an ability to grow in the physiological conditions found within the host, including a temperature of 37°C, pH 7.4, a restricted availability of certain essential nutrients such as iron, and an osmoiality of around 290 mosmol kg^"1. In addition, growth in certain organs involves particular environmental stresses such as the high oxygen tension found within the lungs. Bacteria unable to grow efficiently under these conditions will not be capable of causing disease. The identification of the mechanisms by which pathogens adjust to physiological conditions may identify new therapeutic possibilities to either prevent or treat disease due to microbial pathogens.

Unlike eukaryotic cells, bacteria have to maintain the intracellular osmotic pressure greater than the extracellular pressure on the cell to prevent plasmolysis (separation of the outer membrane from the inner aspect of the bacterial cell wall) due to loss of turgor. The mechanisms by which Escherichia coli and Bacillus subtilis adjust to an increase in the osmotic potential of their environment are reasonably well defined. Initially there is a rapid influx of K⁺ ions and a slower influx of the compatible solutes (compounds which can raise intracellular osmoiality and be concentrated within the cell to high levels without affecting cellular function) proline and glycine betaine. In addition, probably to counter the increase in intracellular positive charge associated with K⁺, there is an efflux of polyamines. In addition, due to the stresses placed upon the cell wall during high osmotic stress, the structural integrity of the cell wall is likely to be important. The similarities between the Gram-negative bacteria E. coli and the Gram-positive bacteria B. subtilis in their responses to changes in the environmental osmotic potential suggest that most bacterial pathogens will use the same mechanisms. Responses to osmotic stress have been investigated for Staphylococcus aureus and Listeha monocytogenes, both of which are food- borne pathogens with a high tolerance of high salt environments, and are indeed similar to those of non-pathogenic bacteria. However, in contrast to iron uptake, there is little information on how important bacterial pathogens respond to changes in osmoiality and the importance of these responses for virulence.

Recently a 27 kb region of the S. pneumoniae genome called Pneumococcal Pathogenicity Island 1 (PPM) has been described, which has several of the features of horizontally acquired DNA and pathogenicity islands (PAIs) of Gram-negative pathogens (co-pending patent application number PCT/GB01/04749). PPI1 contains at least 18 genes with predicted protein products of greater than 100 amino acids in length.

There is still a need to provide means for the prevention and therapy of Gram-positive bacterial infections, in particular S. pneumoniae infections. Summary of the Invention

The present invention is based on the identification of an operon, referred to herein as the phg operon, that encodes proteins required for the S. pneumoniae response to osmotic and oxidative stress and for full virulence in mouse models of pneumonia and septicaemia. The operon resides within a pathogenicity island termed Pneumococcal Pathogenicity Island 1 (PPM) and contains three open reading frames termed phg A, B and C.

According to a first aspect of the invention, an isolated peptide is encoded by any of the polynucleotide sequences identified herein as SEQ ID NOS. 1 , 3 and 5, or a homoiogue thereof with at least 70% sequence similarity, or a functional fragment thereof.

According to a second aspect of the invention, as isolated polynucleotide encodes a peptide as defined above.

According to a third aspect, an attenuated microorganism comprises a mutation that disrupts expression of any of the phg ABC gene sequences or a homoiogue thereof. According to a fourth aspect of the invention, a vaccine composition comprises any of the peptide or polynucleotide sequences or attenuated microorganisms identified above, with an optional pharmaceutically acceptable diluent, carrier or adjuvant. According to a fifth aspect of the invention, a peptide of the invention is used in a screening assay for the identification of an antimicrobial drug, or in a diagnostic assay for the detection of a streptococcal microorganism.

According to a sixth aspect of the invention, there is the use of a peptide, polynucleotide or attenuated microorganism as defined above, for the manufacture of a medicament for the treatment of a condition associated with infection by S. pneumoniae or other Gram-positive bacteria.

According to a seventh aspect of the invention, an antibody is raised against any of the peptides defined above. Description of the Drawings The invention is described with reference to the accompanying drawings, wherein:

Figure 1 (A) is a schematic representation of the genetic organisation of the phg locus where the thick black line represents chromosomal DNA; the clear boxes represent phg ORFs (Sp1043 = phgA, Sp1044 = phgB, Sp1045 = phgC); the arrows represent the sites of insertion in mutant strains; (B) is a schematic representation of the transcriptional analysis of the phg locus;

Figure 2 illustrates the growth curves as measured by optical density of the phg^' and wild-type strains in (A) THY, (B) THY + 50 (open symbols) or 100 (black symbols) mM NaCI, (C) THY + 200 mM NaCI and (D) THY + 100 mM (open symbols) or 400 mM (black symbols) sucrose; the wild-type strain is represented by diamonds; phgA^' (Sp1043) by squares; phgC (Sp1045) by triangles, and PPC50 by circles (data points marked with an asterix have a p value < 0.05 when compared to the results for the wild-type strain at the same time point and with the same growth conditions); Figure 3 illustrates growth curves as measured by optical density of the phg^' and wild-type strains in (A) THY + 100 mM NaCI with (black symbols) or without (open symbols) supplemented with 0.6 M glycine betaine and (B) THY + 100 mM NaCI with (black symbols) or without (open symbols) supplemented with 0.6 M proline (the wild-type strain is represented by diamonds; phgA^' by squares; phg by triangles and PPC50 by circles); and

Figure 4 illustrates growth curves as measured by optical density of the phg^' and wild-type strains in (A) THY + 1 mM paraquat and (B) THY + 5 mM paraquat (the wild-type strain is represented by diamonds; phgA^' by squares; phgC by triangles and PPC50 by circles. Description of the Invention

In general, the techniques required to carry out the invention are those known conventionally in the art. Particular guidance is given in Sambrook et al., Molecular Cloning, A Laboratory Manual (1989), and Ausubel et al., Current Protocols in Molecular Biology (1995), John Wiley & Sons Inc.

The present invention relates to genes required for the growth of Gram- positive pathogens in vivo. The Examples below are described with reference to S. pneumoniae strain 0100993, however all pathogenic S. pneumoniae strains are expected to have the same genes.

The terms "peptide" or "peptides" are intended to include also protein or proteins.

The peptides and polynucleotides of the present invention may be suitable candidates for the production of therapeutically-effective vaccines against Gram-positive bacterial pathogens and S. pneumoniae. The term "therapeutically-effective" is intended to include the prophylactic effect of the vaccines. For example, a recombinant peptide may be used, as an antigen for direct administration to an individual. The peptide may be isolated directly from a Gram-positive bacterial pathogen or from S. pneumoniae or expressed in any suitable expression system, e.g. Escherishia coli. It is preferably administered with an adjuvant, e.g. alum.

Preferably, the peptides that may be useful for the production of vaccines have greater than 70% similarity with the peptides identified herein. More preferably, the peptides have greater than 80% sequence similarity. Most preferably the peptides have greater than 90% sequence similarity, e.g. 95% similarity. Preferably, the polynucleotide sequences that may be useful for the production of vaccines have greater than 60% identity with the polynucleotide sequences identified herein. More preferably, the polynucleotide sequences have greater than 70% sequence identity. Most preferably the polynucleotide sequences have greater than 90% sequence identity, e.g. 95% identity. "Similarity" and "identity" are known in the art. In the art, identity refers to the relatedness between polynucleotide or polypeptide sequences as determined by comparing the sequences, and particularly identical matches between nucleotides or amino acids in correspondingly identical positions in the sequences being compared. Similarity refers to the relatedness of polypeptide sequences, and takes account not only of identical amino acids in corresponding positions, but also functionally similar amino acids in corresponding positions. Thus similarity between polypeptide sequences indicates functional similarity, even if there is little apparent identity.

Levels of identity between genes (polynucleotides) and levels of identity and similarity between proteins can be calculated using known methods. In relation to the present invention, publicly available computer based methods for determining identity and similarity between polypeptide sequences and identity between polynucleotide sequences include but are not limited to those of the GCG package (Genetics Computer Group, (1991 ), Program Manual for the GCG Package, Version 7, April 1991 , 575 Science Drive, Madison, Wisconsin, USA 53711), BLASTP, BLASTN, and FASTA (Atschul, S.F. etal., J. Molec. Biol.215: 403-410 (1990)). The BLASTX program is available from NCBI and other sources. The Smith Waterman algorithm may also be used to determine identity. The parameters for polypeptide sequence comparison include the following: Algorithm: Needleman and Wunsch (J. Mol Biol. 48: 443-453 (1970)).

Comparison matrix: BLOSSUM62 (Hentikoff and Hentikoff, PNAS 89: 10915- 10919 (1992)). Gap penalty: 12 Gap length penalty: 4 These parameters are the default parameters of the "Gap" program from Genetics Computer Group, Madison Wl. The parameters for polynucleotide sequence comparison include the following: Algorithm: Needleman and Wunsch (J. Mol Biol. 48: 443-453 (1970)). Comparison matrix: matches = +10, mismatch = 0 Gap penalty: 50 Gap length penalty: 3 available as the "Gap" program from Genetics Computer Group, Madison Wl. These parameters are the default parameters for nucleic acid comparisons.

In one embodiment, the peptide comprises any of the amino acid sequences identified herein as SEQ ID NOS. 2, 4 or 6. However, the peptide may be a mutant peptide in comparison to wild-type peptide, a fragment of the peptide or a chimeric peptide comprising different fragments or peptides, provided an effective immune response is generated.

The term "functional fragment" is used herein to mean a fragment of a peptide sequence disclosed herein, which retains the ability to raise an immune response, i.e. to act as an antigen. The functional fragment will retain the ability to bind to an antibody raised against the native peptide/protein. Alternatively, or in addition, a functional fragment is a peptide fragment that retains a biological function described herein for the native peptide/protein. Preferably, the peptide fragments are at least 20 amino acids in size, more preferably, at least 30 amino acids and most preferably, at least 50 amino acids in size. An alternative therapeutic approach is to use a live attenuated Gram- positive bacterium, e.g. S. pneumoniae, as a vaccine. This may be produced by deleting or disrupting the expression of a phg ABC gene or a homoiogue thereof. Preferably, the attenuated strain comprises additional virulence gene mutations, i.e. the strain is a double mutant. The double mutant may be prepared by disrupting a combination of phgABC genes. Other mutations will be apparent to the skilled person.

The mutated microorganisms of the invention may be prepared by known techniques, e.g. by deletion mutagenesis or insertional inactivationofapfrgABC gene. The gene does not necessarily have to be mutated, provided that the expression of its product is in some way disrupted. For example, a mutation may be made upstream of the gene, or to a gene regulatory system. The preparation of mutant microorganisms having a deletion mutation are shown in WO-A-96/17951 , which is incorporated herein by reference. In one suitable technique, a suicide plasmid comprising a mutated gene and a selective marker is introduced into a microorganism by conjugation. The wild-type gene is replaced with the mutated gene via homologous recombination, and the mutated microorganism is identified using the selective marker.

The attenuated microorganisms may be used as carriers of heterologous antigens or therapeutic proteins/polynucleotides. For example, a DNA vector expressing an antigen may be inserted into the attenuated strain for delivery to a patient. Conventional techniques may be used to carry out this embodiment, including those disclosed in European patent publication No. EP-A-0184086 and US 5877159.

Suitable heterologous antigens will be apparent to the skilled person, and include any bacterial, viral or fungal antigens and allergens, e.g. tumour- associated antigens. For example, suitable viral antigens include: hepatitis A, B and C antigens, herpes simplex virus HSV, human papilloma virus HPV, respiratory syncytial virus RSV, (human and bovine), rotavirus, norwalk, HIV, and varicella zooster virus (shingles and chickenpox). Suitable bacterial antigens include those from: ETEC, Shigella, Campylobacter, Helicobacter, Vibrio cholera, EPEC, EAEC, Staphylococcus aureus toxin, Chlamydia, Mycobacterium tuberculosis, Plasmodium falciparum, Malaria and Pseudomonas spp.

The heterologous antigen may be expressed in the host cell utilising a eukaryotic DNA expression cassette, delivered by the mutant. Alternatively, the heterologous antigen may be expressed by the mutant bacterium utilising a prokaryotic expression cassette. The microorganism may alternatively be used to deliver a therapeutic heterologous peptide or polynucleotide to a host cell. For example, cytokines are suitable therapeutic peptides (proteins), which may be delivered by the microorganisms for the treatment of patients infected with hepatitis. The delivery of a polynucleotide is desirable for gene therapy, for example, anti-sense nucleotides, such as anti-sense RNA, or catalytic RNA, such as ribozymes or interfering RNA (RNAi). Methods for preparing microorganisms with the heterologous antigens etc, will be apparent to the skilled person and are disclosed in Pasetti et al., Clin. Immunol, 1999; 92(1): 76-89, and Sharma et al., Infect. Immun., 2001 May; 69(5): 2928-2934, both of which are incorporated herein by reference. The polynucleotide that encodes the heterologous product may be provided on a recombinant construct that contains the regulatory apparatus necessary for expression, e.g. promoter, enhancers etc. For example, a prokaryotic or eukaryotic expression cassette may be incorporated into the microorganism, e.g. as a multi-copy plasmid. Alternatively, the heterologous polynucleotide may be targeted to a gene endogenous to the microorganism, including the gene that naturally comprises a polynucleotide of the invention, so that the heterologous polynucleotide becomes incorporated into the genome of the microorganism, and uses endogenous or cloned regulatory apparatus for its expression. The peptide (or fragments thereof) of the present invention may also be used in the production of monoclonal and polyclonal antibodies for use in passive immunisation. Methods for the production of antibodies are well known in the art. Antibody fragments and humanised antibodies are intended to be encompassed by the term "antibody". In a further embodiment of the invention, the peptide or corresponding polynucleotide may be used as a target for screening potentially useful compounds, especially antimicrobials. Suitable compounds may be selected for their ability to bind to the peptide or. polynucleotide to exert their effects. Suitable compounds may be selected for their ability to affect (i.e. prevent or reduce) the expression of Gram-positive pathogenicity island genes required for in vivo growth, for example the phg genes, thereby reducing or altering the ability of the bacterium to survive in vivo or in a particular environment. Assays for screening for suitable compounds and which make use of the peptide or polynucleotides of the invention will be apparent to those skilled in the art. The assays may be performed in vitro by contacting the target compound with the peptide/polynucleotide of the invention, and measuring the extent to which the compound interacts with, e.g. binds to, the peptide/polynucleotide, or the level of expression from the polynucleotide, compared to a control. Those compounds that interact can then be selected for further testing, e.g. against the wild-type microorganism.

Although the peptides, polynucleotides, attenuated mutants and antibodies raised against the peptides and attenuated mutants are described for use in the diagnosis or treatment of individuals, veterinary uses are also considered to be within the scope of the present invention.

In a further embodiment, a promoter sequence associated with the phg genes identified herein may be used to regulate expression of heterologous genes. This may be achieved either by incorporating the promoter in a vector system, e.g. a conventional gene expression vector. Alternatively, the heterologous gene may be inserted into the bacterial chromosome such that the promoter regulates expression. Sequences incorporating the promoter will be apparent to the skilled person based on the knowledge provided herein on the phg ABC genes, and is within the sequence identified herein as SEQ ID NO. 13.

The promoter may be of use due to the potential for in vivo expression under different osmotic conditions.

To formulate the vaccine compositions, the mutant microorganisms may be present in a composition together with any suitable excipient. For example, the compositions may comprise any suitable adjuvant. Alternatively, the microorganisms may be produced to express an adjuvant endogenously.

Suitable formulations will be apparent to the skilled person. The formulations may be developed for any suitable means of administration. Preferred administration is via the oral, mucosal (e.g. nasal) or systemic routes. The polynucleotides representing the phg A, B and C genes are identified in the accompanying sequence listing as SEQ ID NOS. 1 , 3 and 5, respectively.

The encoded products are identified as SEQ ID NOS. 2, 4 and 6, respectively. The phg ABC genes were identified using the experimental procedure detailed in co-pending international application number PCT/G B01/04749, the content of which is hereby incorporated by reference.

Example 1

Identification of the phg ABC operon The phg ABC operon was identified by using isogenic mutant strains of

S. pneumoniae containing gene disruptions within the PPI1 region (co-pending international patent application number PCT/GB01 /04749) and investigating the loss of virulence using mixed infections with the wild-type strain.

5 Construction of mutant strains:

Plasmids, primers and S. pneumoniae strains constructed and used for this work are described in Table 1 and reference is also made to Lau etai, Mol.

Micro. Biol., 2001 May; 40(3): 555-571.

Table 1

Name Description / sequence

Strains

100993 capsular serotype 3 clinical isolate phgA^' 0100993 containing an insertion mphgA (Spl043): cm^r phgC^' 0100993 containing an insertion inphgC (Spl045): cm^r

PPC50 0100993 containing an insertion downstream of Sp 1045 : cm^r

Plasmids pID701 disruption vector for S. pneumoniae derived from pENP3 : amp^r cm^r pPC33 pID701 with ORF3.1 / ORF3.2 PCR product ligated into the Xbal site: amp^r cm^r pPC49 pID701 with ORF5.6 / ORF5.7 PCR product ligated into the Xbal site: amp^r cm¹ pPC50 pJD701 with ORF5.8 / ORF5.9 PCR product ligated into the Xbal site: amp^r cm'

Primers (5' to 3')

ORF3.1 GCT CTA GAGTAAATT ACC AAGTGAGG (SEQ IDNO.7)

ORF3.2 CGC TCT AGATCA CCT GTATAG GGT CG (SEQ IDNO.8)

ORF5.6 GCT CTAGAACCC TAG TTC TGGTGGC (SEQ IDNO.9)

ORF5.7 CGC TCT AGATTGTGAATC GCC TCA GGC (SEQ IDNO.10)

ORF5.8 GCT CTA GAT CGGTTC TCT GCC TGA GGC (SEQ IDNO.11) ORF5.9 CGC TCT AGATTT GCAAGGAAT GTC CGT TG (SEQ ID NO.12)

To construct Sp1043lphgA and Sp1045lphgC disruption vectors, internal portions of the genes (bp 22 to 495 for Sp1043, and bp 12 to 428 for Sp1045) were amplified by PCR (primers ORF3.1/3.2 and ORF5.6/ORF5.7 respectively) and ligated into the suicide vector plD701 to make pPC33 and pPC49 respectively (both cm resistant). Plasmid insert identities were confirmed by DNA sequencing, and S. pneumoniae mutant strains containing disrupted copies of Sp1043 and Sp1045 were constructed by insertion-duplication mutagenesis with pPC33 and pPC49 according to a previously described transformation protocol utilising Competence Stimulating Peptide 1 (Lau era/., supra). A strain containing an insertion 83 bp 3' to Sp1045, termed PPC50, was constructed by transformation of S. pneumoniaewWh pPC50 (plD701 containing a length of DNA homologous to the terminal portion of Sp1045 and 83 bp 3' to the stop codon (amplified by PCR with primers ORF5.8/5.9). Mutant constructs were confirmed by PCR. All mutations were stable after two 8 h growth cycles (each representing approximately 10 rounds of cell division) in THY broth without antibiotic selection.

The mutant strain containing a disruption in the gene Sp1043 (TIGR genome assignation, previously identified as ORF3) had a Cl of 0.007 +/- 0.004 (SD) (n = 4) when compared to the wild-type strain in a mouse model of pneumonia, suggesting that this strain has a severe defect in vivo growth. Hence Sp1043 was investigated further to identify why its disruption resulted in loss of virulence. The protein encoded by Sp1043 has a predicted length of 252 amino acid residues, and is likely to be transcribed as an operon with two adjacent ORFs, Sp1044 (predicted length 284 amino acid residues) and Sp1045 (predicted length 294 amino acid residues) (Figure 1 A). To confirm that these three genes are cotranscribed, the transcript structure of this region was analysed using RT- PCR. PCR with primers which span the junctions of Sp1043/Sp1044 and Sp1044/Sp1045, amplified identical products when either DNA or cDNA made from total RNA was used as the target (Figure 1 B). However, PCR carried out using primers designed to amplify the region spanning from Sp1042 to Sp1043 or from an internal portion of Sp1045 to 209 bp 3' to its stop codon failed to amplify products from cDNA, although products were amplified from DNA. Hence, Sp1043-45 are transcribed as a single transcript, transcription of which starts with Sp1043 and terminates after Sp1045. The results of sequence similarity searches with BLAST for each of the derived amino acid sequences of Sp1043, Sp1044 and Sp1045 are shown in Table 2.

Table 2 gene % identity/ length of designation organism similarity amino acids compared

So 1043 ig Acidiihiobacillus ferrooxidans 36/48 112

Nc0270 Vibrio cholerae 28/42 182

Spl044 ig Streptococcus mitis* 91/95 244 ig Streptococcus gordonii 85/93 254 ig Desulfitobacterium hafniense 50/64 253 ig Fibrobacter succinogened 45/56 264 ig Prevotella intermedia 40/53 257 ig Magnetospirillum magnetotacticum 41/58 216

Lmo0067 Listeria monocytogenes 24/43 286

Spl045 ig S. mitis* 98/98 293 ig Enterococcus faecalis 39/60 293 ig Streptococcus equi 38/63 292

Lmo0774 L. monocytogenes 34/57 300 ig Staphylococcus epidermis 34/54 299

Savl898 Staphylococcus aureus 33/53 295 yerQ Bacillus subtilis 32/52 297 ig Clostridia difficile 29/50 293

Spy0814 Streptococcus pyogenes 26/49 302

* adjacent ORFS in the incomplete S. mitis genome ig = incomplete genome

The predicted protein product of Sp1043 has only relatively low levels of similarity over a proportion of its length to two ORFs from bacterial species unrelated to S. pneumoniae, whereas the predicted protein product of Sp1044 has almost identical homologues in Streptococcus mitis and Streptococcus gordonii, species which are closely related to S. pneumoniae, as well as moderate degrees of similarity to proteins from a wide variety of other bacterial species. An ORF in the S. mitis genome also has very high levels of identity at the amino acid level to Sp1045, and forms a probable operon with the S. mitis homoiogue of Sp1044. Strikingly, Sp1045 has similarity to many proteins present in the available genome sequences of numerous Gram-positive (but not Gram-negative) bacteria, forming a previously undescribed protein family. The derived amino acid sequence of Sp1045 and related proteins from other Gram- positive bacteria have high levels of sequence similarity to the consensus diacyl glycerol (DAG) kinase domain of eukaryotic organisms (48% over 123 amino acids for Sp1045) suggesting this protein family may function as kinases. Example 2

In order to investigate the function of proteins encoded by the Sp1043- Sp1045 operon, S. pneumoniae strains containing disruptions of the genes were constructed by insertional mutagenesis of the capsular serotype 3 wild-type strain 010093 as described above. All mutant strains had normal growth in the complete medium THY. Strains carrying mutations in Sp1043 and Sp1045 had reduced growth rates in THY medium containing 100 mM NaCI or 200 mM sucrose (calculated osmoiality of around 300 mM, similar to that of extracellular fluid and blood) compared to the wild-type strain and the strain containing a mutation downstream of Sp1045 (Figure 2). Supplementation of high osmotic medium with 0.6 M of the osmoprotectants betaine or proline improved growth of both the wild-type and the Sp1043 mutant, showing that the impaired growth in high osmotic medium of the mutant strain was not due to a defect in betaine or proline acquisition (Figure 3). The Sp1043 and Sp1045 mutant strains also had growth defects compared to the wild-type strain in THY supplemented with 1 mM paraquat, indicating that this strain has increased sensitivity to oxidative stress (Figure 4). The number of Sp1043 mutant strain cfu present in human blood after 4 hours incubation was 66% of the cfu of the wild-type strain (Sp1043 mutant strain 6.9 x 10⁷ cfu ml^"1, wild-type strain 1.05 x 10⁸ cfu ml^"1).

Comparison of the virulence of Sp1043 and Sp1045 mutant strains to the wild-type strains using mixed infections and the competitive index (Cl) demonstrated that the mutant strains were outcompeted by the wild-type strains in mouse models of pneumonia (Cl < 0.05 for bacteria recovered from the lungs after 48 hours) and septicaemia (Cl < 0.1 for bacteria recovered from the spleen after 24 hours). The results are shown in Table 3, where IN relates to the mouse model of pneumoniae, and IP relates to the mouse model of systemic infection. A Cl substantially < 1.0 indicates loss of full virulence.

Table 3

Strain mouse strain timepoint route CI (SD) n (hours) wild-type v. phgA^' wild-type CD1 24 IP 0.10 (0.03) 5 wild-type v. phgC wild-type CD1 24 π 0.16 (0.11) 5 wild-type v. PPC50 wild-type GDI 24 IP 1.24 (0.36) 5 wild-type v. phgA^' wild-type CD1 6 ΓP 0.20 (0.10) 4 wild-type v. phgA^' wild-type C57B/6 24 IP 0.3 (0.10)¹ 4 wild-type v. phgA^' gp91phox'- C57B/6 24 IP 0.35 (0.16)¹ 5 wild-type v. phgA^' wild-type CD1 48+ IN 0.007 (0.004) 4 wild-type v. phgC^' wild-type CD1 48+ IN 0.03 (0.014) 5 wild-type v. PPC50^" wild-type GDI 48+ IN 0.73 (0.22) 4 wild-type v. phgA^' gp91phox'^' C57B/6 48+ IN 0.011 (0*) 3

These results show that proteins encoded by the operon have a role in the S. pneumoniae response to osmotic and oxidative stress and during infection. For this reason, the genes were renamed Pneumococcal Hyperosmotic Growth (phg) A (Sp1043), B (Sp1044) and C (Sp1045).

Claims

I . An isolated peptide encoded by any of the polynucleotide sequences identified herein as SEQ ID NOS. 1 , 3 or 5, or a homoiogue thereof with at least 70% sequence similarity, or a functional fragment thereof.

2. A peptide according to claim 1 , comprising at least 30 amino acids.

3. A peptide according to claim 1 or claim 2, comprising at least 50 amino acids.

4. A peptide according to any preceding claim comprising any of the amino acid sequences identified herein as SEQ ID NOS. 2, 4 or 6.

5. A peptide according to any preceding claim, for therapeutic or diagnostic use.

6. An isolated polynucleotide encoding a peptide according to any of claims 1 to 4.

7. A polynucleotide according to claim 6, having at least 40 nucleotides.

8. A polynucleotide according to claim 7, having at least 80 nucleotides.

9. A polynucleotide according to any of claims 6 to 8, for therapeutic or diagnostic use.

10. An attenuated Gram-positive microorganism comprising a mutation that disrupts expression of any of the polynucleotide sequences identified herein as SEQ ID NO. 1, 3 or 5.

I I. A microorganism according to claim 10, wherein the polynucleotide is any of the phg A, B or C genes.

12. A microorganism according to claim 10 or claim 11 , wherein the mutation is a deletion mutation.

13. A microorganism according to any of claims 10 to 12, comprising a further attenuating mutation in a second gene.

14. A microorganism according to claim 13, wherein the further mutation is an auxotrophic mutation.

15. A microorganism according to any of claims 10 to 14, genetically modified to express a heterologous antigen.

16. A construct comprising a promoter naturally associated with a phg A, B or C gene, and a heterologous gene.

17. A vaccine comprising a microorganism according to any of claims 10 to 14 in a pharmaceutically acceptable diluent, excipient or adjuvant.

18. A vaccine comprising a peptide according to claim 1 , or a polynucleotide according to claim 6.

19. A vaccine comprising at least two peptides according to claim 1.

20. Use of a product according to any of claims 1 to 15, in a screening assay for the identification of an antimicrobial compound.

21. Use of a product according to any of claims 1 to 15, in a diagnostic assay for the detection of a streptococcal microorganism.

22. Use of a product according to any of claims 1 to 15, for the manufacture of a medicament for the treatment or prevention of a condition associated with infection by S. pneumoniae or other Gram-positive bacteria.

23. Use according to claim 22, wherein the treatment is veterinary treatment.

24. An antibody, raised against any of the products of claims 1 to 5.

25. An antibody according to claim 24, for therapeutic or diagnostic use.

26. Use of an antibody according to claim 24, in the manufacture of a medicament for the treatment or prevention of an infection by S. pneumoniae or other Gram-positive bacteria.