Classifications

• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• This invention relates to a family of bacterial polypeptides which are required for growth of both gram negative and gram positive bacteria, the genes which encode them and the use of such polypeptides and genes as tools for identifying novel broad spectrum antibiotics.
• the invention therefore provides an isolated polypeptide of the yjeQ family as defined below particularly for use in the identification of novel antibiotic agents.
• the polypeptides of the present invention are believed to be essential to the viability of a wide range of bacteria including both gram positive and gram negative bacteria.
• BLAST searches J. Mol. Biol. (1990) 215:403 -10 and Meth. Enzymol. (1996) 266: 131-141, 227-258 both incorporated herein by reference
• Such searches involve using in succession as query sequences, each of the existing yjeQ protein family member sequences to identify other full length members of the yjeQ family of proteins.
• Such family members yield high-scoring segment pairs (HSP) scores of greater than 100 in comparison to at least one member of the yjeQ family when the BLAST algorithm described in the reference above is used with a particular scoring matrix (a BLOSUM62 matrix - Proteins (1993) 17:49-61 incorporated herein by reference).
• Motif based searches may be carried out using PROSITE patterns defined for the yjeQ family members. These searches involve the representation as patterns, of the conserved sequence elements identified in the profile searches.
• HSP score of greater than or equal to 100 when compared with one of the sequences of Figure 1 when the BLAST algorithm is used with a BLOSUM62 scoring matrix ;
• the letters denote an amino acid in one letter code
• the square brackets denote a single amino acid
• the amino acids within the square brackets are alternatives
• X is any one amino acid residue
• the numbers in the curved brackets refer to the number of residues at that position.
• both of the amino acid sequences listed under iii) are present.
• the invention also provides an isolated polypeptide sequence as set out in any of Figures 2a-d.
• polypeptides are preferably recombinant and ideally purified to homogeneity.
• polypeptides according to the invention are variants, analogues and derivatives. Particularly those in which a number of amino acids have been substituted, deleted or added.
• Polypeptides which have at least 70% identity to any of the polypeptide sequences according to the invention, in particular the sequences of Figures 2a-d are encompassed within the invention.
• the identity is at least 80%, more preferably at least 90% and still more preferably at least or greater than 95%) identity for example 97%, 98% or even 99% identity to any of the sequences according to the invention, in particular the sequences of Figures 2a-d.
• Such polypeptides may also be fragments.
• a fragment is a part of a polypeptide according to the invention which retains sufficient identity of the original polypeptide to be effective for example in a screen.
• Such fragments may be fused to other amino acids or polypeptides or may be comprised within a larger polypeptide.
• Such a fragment may be comprised within a precursor polypeptide designed for expression in a host. Therefore in one aspect the term fragment means a portion or portions of a fusion polypeptide or polypeptide derived from a polypeptide according to the invention.
• Fragments also include portions of a polypeptide according to the invention characterised by structural or functional attributes of a polypeptide according to the invention. These may have similar or improved chemical or biological activity or reduced side-effect activity.
• fragments may comprise an alpha helix or alpha-helix forming region, beta sheet and beta-sheet forming region, turn and turn forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, amphipathic regions (alpha or beta), flexible regions, surface-forming regions, substrate binding regions and regions of high antigenic index.
• Fragments or portions may be used for producing the corresponding full length polypeptide by peptide synthesis.
• polypeptides according to the invention include the polypeptides of Borrellia burgdorferi, Synechocystis sp. Strain PCC6803, Haemophilus influenza, Mycobacterium tuberculosis, Neisseria gonorrhoeae, Mycoplasma genitalium,
• the present invention further provides isolated polynucleotides which encode the polypeptides as defined herein, polynucleotides complementary thereto, or polynucleotides hybridising to any of the aforesaid polynucleotides.
• Isolated polynucleotides have been removed by separation from their natural environment and those materials with which they are naturally associated.
• these polynucleotide molecules are provided in recombinant form (i.e. combined with one or more heterologous sequences).
• Polynucleotide molecules which hybridise to polynucleotides encoding substances of the present invention, or to complementary polynucleotides thereto, preferably do so under stringent hybridisation conditions.
• stringent hybridisation conditions which is sometimes used is where attempted hybridisation is carried out at a temperature of from about 35°C to about 65°C using a salt solution which is about 0.9 molar.
• the invention also provides polynucleotide variants, analogues, derivatives and fragments which encode polypeptides according to the invention.
• Polynucleotides are included which preferably have at least 70% identity over their entire length to a polynucleotide encoding a polypeptide according to the invention, most preferably those set out in Figures 2A-D. More preferred are those sequences which have at least 80%) identity over their entire length to a polynucleotide encoding a polypeptide according to the invention. Even more preferred are polynucleotides which demonstrate at least 90% for example 95%, 97%, 98% or 99% identity over their entire length to a polynucleotide encoding a polypeptide according to the invention.
• Polynucleotide molecules of the present invention may be used as probes for other members of the gene family or in anti-sense therapy to block or to reduce the expression of one or more of the polypeptides of the invention. Since these substances are believed to be essential to the bacteria expressing them, blocking or reducing their expression can provide an effective way of treating bacterial mediated diseases or disorders. Polynucleotides may also be used directly in screening and in generating whole cell screens by expression of a polypeptide of the inventions.
• the polynucleotides may be joined to other polynucleotides such as to form fusions or to regulatory elements for expression.
• Isolated polynucleotides alone or joined to other polynucleotides can be in introduced into a vector which itself will contain other elements of DNA or RNA for expression in a host cells.
• the invention therefore comprises a vector containing a polynucleotide generally operatively linked to appropriate expression control sequences.
• Vectors for use in the invention include plasmid vectors, phage vectors and DNA or RNA viral vectors. These vectors may include gene sequences which render them inducible under certain conditions such as manipulation of the environmental conditions under which the host cells are maintained for example by temperature alteration or nutrient additives. Regulatory sequences include for example a promoter to direct mRNA transcription. Such promoters include for example E. coli. lac, trp, tac and araB AD as well as the SV40 early and late promoters Such systems and sequences would be well known to those skilled in the art.
• Host cells expressing a polynucleotide of the present invention can be generated by any of the traditional routes such as transfection or electroporation see for example Davis et al, Basic Methods in Molecular Biology, (1986) and Sambrook et al Molecular Cloning: A Laboratory Manual, 2 nd Edition., Cold Spring Harbor Lab. Press, Cold Spring Harbor, N.Y. (1989).
• This invention also provides a method for identification of molecules such as antagonists, that bind to the polypeptide or a polynucleotide encoding a polypeptide of the present invention.
• Biochemical assays for inhibition of polypeptide activity with purified polypeptides or bacterial extracts can be more sensitive than whole cell killing assays and provide direct evidence for a compound's mode of action.
• this approach requires that the target polypeptide is known and the activity of the polypeptide be amenable to in vitro assays. Nor does it address other factors, such as membrane permeability or compound stability, which can limit a compounds effectiveness as an antibiotic.
• Whole cell screening of compounds for killing activity will identify molecules which kill cells at the concentrations tested, but provide no information on the mode of action of the compound and may not have the sensitivity needed to detect less potent compounds.
• Bacterial strains which contain surrogate markers whose activity is linked to that of the target gene or which have been engineered to over-express or under- express the target polypeptide can be used for selective whole-cell screens.
• the invention further provides a host cell comprising a vector as defined herein and a reporter gene encoding a reporter molecule whose activity is linked to that of the polypeptide encoded by the vector.
• a reporter gene encoding a reporter molecule whose activity is linked to that of the polypeptide encoded by the vector. Examples of such systems include a transcriptional fusion of the E. coli lacZ gene to vanH promoter in a B. subtilis strain expressing VanS and R as a reporter for inhibition of cell wall biosynthesis (J. Bacteriol.
• surrogate markers for the activity of the gene can be identified using at least two approaches. Two dimensional electrophoresis coupled with mass spectrometry analysis of isolated polypeptides, proteome mapping, has been used to identify specific polypeptides which increase in abundance in response to polypeptide or RNA synthesis inhibitors (Microbial & Comparative Genomics (1996) 1 :375). Tightly regulated promoters used to demonstrate that the E. coli and B. subtilis conserved, essential polypeptides are essential can also be used to reduce the concentrations of these polypeptides.
• proteome maps generated from bacteria depleted of the conserved essential genes can be used to detect polypeptides which change in abundance as compared to wild-type bacteria. Transcriptional or translational fusions to these polypeptides can be used as reporter molecules to screen for antagonists of members of the conserved essential gene family.
• transposons or other mobile genetic elements containing reporter genes can be used to search for reporter molecules. Such an approach has been used to identify vancomycin responsive genes in S. aureus (Antibiot. (Tokyo) (1991) 44:210-217).
• bacteria in which conserved essential genes are controlled by tightly regulated promoters can be used to screen for transposon carrying strains in which expression of promoterless reporter genes is induced upon depletion of the polypeptides.
• Standard broth or plate assays can be used in many different formats. Such assays will detect molecules which antagonise the response which couples the activity of the conserved, target polypeptide to the reporter molecule. Thus, the compounds identified may act directly upon the target polypeptide or on another stage in the pathway which leads to activation of the reporter.
• Screens for inhibitors of the target which do not require the use of surrogate markers may be designed by manipulating expression levels of the target polypeptide.
• quinolone resistant strains of E. coli have been made by over-expression of gyrA (FEMS Microbiol. Lett. (1997) 154:271-276)
• over-expression of alanine racemase has been shown to increase resistance to cycloserine in M. smegmatis
• multicopy plasmids carrying murZ have been shown to increase phosphomycin resistance in both E. coli (J. Bacteriol. (1992) 174:5748-5752) and . calcoaceticus (FEMS Microbiol.
• strains more sensitive to antibiotics may be made by reducing expression levels of the polypeptide targeted by the antibiotic.
• Over or under-expression of members of the conserved, essential gene family may be used to screen for antibiotics which act either directly on gene or gene product or indirectly on the pathway which it is involved.
• an assay for antagonists is a competitive assay that combines the polypeptide of the present invention and a potential antagonist with membrane-bound binding molecules, recombinant binding molecules, natural substrates or ligands, or substrate or ligand mimetics, under appropriate conditions for a competitive inhibition assay.
• the polypeptide can be labelled, such as by radioactivity or a colorimetric compound, such that the number of polypeptide molecules bound to a binding molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist.
• the present invention therefore provides a method of assaying compounds for activity against bacteria comprising:
• the present invention also provides a method of assaying compounds for activity against bacteria comprising:
• the present invention further provides a method of screening for an antibiotic which method comprises:
• the present invention also provides a method of assaying compounds for activity against bacteria comprising:
• Potential antagonists include small organic molecules, ions which interact specifically with a polypeptide or polynucleotide for example a substrate, cell membrane component, receptor a fragment thereof or a peptide.
• Such molecules may include antibodies, antibody-derived reagents or chimaeric molecules.
• Potential antagonists also may be small organic molecules, a peptide, a polypeptide such as a closely related protein or antibody that binds to the same sites on a binding molecule without inducing functional activity of the polypeptide of the invention.
• the antibodies may be monoclonal or polyclonal. Techniques for producing monoclonal and polyclonal antibodies which bind to a particular polypeptide are now well developed in the art. They are discussed in standard immunology textbooks, for example in Roitt et al (Immunology, Churchill Livingston, 2nd Edition (1989)).
• the present invention covers variants thereof which are capable of binding to an epitope present or a substance of the present invention.
• the variants may be antibody fragments or synthetic constructs. Examples of antibody fragments and synthetic constructs are given by Dougall et al in Tibtech 12 372-379 (September 1994).
• Antibody fragments include Fab and Fv fragments.
• CDR peptides include CDR peptides. These are synthetic peptides comprising antigen binding determinants. Peptide mimetics may also be used. These molecules are usually conformationally restricted organic rings which mimic the structure of a CDR loop and which include antigen-interactive side chains. Synthetic constructs include chimaeric molecules. Thus, for example, humanised antibodies or derivatives thereof are within the scope of the present invention. An example of a humanised antibody is an antibody having human framework regions, but a rodent or other non-human hypervariable regions. Synthetic constructs also include molecules comprising a covalently linked moiety which provides the molecule with some desirable property in addition to antigen binding. For example the moiety may be a label (e.g. a fluorescent or radioactive label) or a pharmaceutically active agent.
• a label e.g. a fluorescent or radioactive label
• antisense molecules see Okano, J. Neurochem. 56:560 (1 91); Oligodeoxynucleotides As Antisense Inhibitors Of Gene Expression, CRC Press, Boca Raton, FL (1988), for a description of these molecules).
• the invention provides the use of the polypeptide, polynucleotide or antagonist of the invention to interfere with the initial physical interaction between a pathogen and mammalian host responsible for sequelae of infection.
• the invention further includes molecules which block the function of the polypeptides according to the invention or a polynucleotide encoding the same, identifiable by any of the above described methods.
• An antagonist of the invention may be provided in pharmaceutical compositions which may include a carrier. They may be provided in unit dosage form. Such agents and pharmaceutical compositions are within the scope of the present invention. In order to prepare such pharmaceutical compositions the inhibitors will normally be provided in substantially pure form. They can then be combined with a carrier under sterile conditions.
• the present invention also provides a method of treatment which comprises administering to a patient an effective amount of an antagonist of the expression or function of a polypeptide as defined herein.
• the present invention further provides the use of an antagonist of a polypeptide as defined herein or a polynucleotide encoding the same for the manufacture of a medicament for the treatment of a bacterial infection.
• Figure 1 shows the multiple sequence alignment of the yjeQ family members which may be used for BLAST-based identification of yjeQ family members.
• Figures 2a-d show position-dependant scoring matrices for profile-based identification of yjeQ family members.
• Figure 2a shows examples of motif 1 in yjeQ family members.
• Figure 2b shows examples of motif 2 in yjeQ family members.
• Figure 2c shows examples of motif 3 in yjeQ family members.
• Figure 2d shows examples of motif 4 in yjeQ family members.
• Figure 3 shows the PROSITE patterns which may be used to identify yjeQ family members.
• Figure 4 shows the outline cloning strategy for a plasmid used for gene disruption in E. coli.
• the black box represents the adapter sequence.
• Figure 5 is a diagram of the vector used to create conditional mutants in B. subtilis.
• Seq ID No. 2 and Seq ID No. 3 are respectively elements of a single sequence and are separated from each other by 20-80 Xaa residues where each Xaa is any one amino acid.
• Example 1 Identification of conserved bacterial open reading frames.
• the predicted open reading frames obtained from the complete E. coli genomic sequence were compared in a serial manner to the predicted open reading frames of the H. influenzae (Science (1995) 270:397-403), M. genatilum (Science (1995) 270:397-403), Synechocystis (Nuc. Acids Res. (1998) 26: 63-67) and B. subtilis (Nature (1997) 390:249-256) complete genome sequences using the BLAST algorithm (J. Mol. Biol. (1990) 215:403-10).
• a disruption plasmid was constructed using DNA containing an in-frame deletion of the gene of interest plus -900 base pairs of 5' and 3' flanking DNA for homologous recombination.
• the plasmid was cloned into the gene-replacement vector pKO3 as follows: Two separate PCR reactions were used to amplify fragments of approximately 900 base pairs of 5' and 3' sequence flanking the gene of interest. Chromosomal DNA from E .coli strain MG1655 was used as the template.
• Primers 2 and 3 carry a 5' extension of a 33 bp adapter sequence
• the 2 PCR products were purified using High PureTMPCR Product Purification Kit (Boehringer Mannheim Inc., Mannheim, GE).
• the 2 PCR products are assembled in a second PCR reaction to give a single product .
• preparative agarose gel electrophoresis and purification using JetsorbTMGel Extraction Kit (Genomed Inc.) the final product was cloned into pKO3 using standard techniques.
• This clone is referred to as the disruption plasmid. All PCR reactions described in this section were performed with PWOTM DNA Polymerase (Boehringer Mannheim Inc., Mannheim, GE). In the final product the gene of interest was deleted from the start to the stop codon and replaced by the 33 bp adapter sequence [e.g. 5'-
• the disruption vector pKO3 (A.J.Link et al., J. Bacteriol. 179:6228-6237,1997) is a derivative of pMAK700 (C.A.Hamilton et al., J. Bacteriol. 171 :4617-4622). It features the repA (Ts) replication origin derived from pSClOl [permissive at 30°C but inactive at 42 to 44°C], the cat gene encoding chloramphenicol resistance and the sacB gene for counter selection against vector sequences in the presence of 5% sucrose.
• chromosomal integrates (cointegrates produced by a single homologous recombination event) of the plasmid were isolated by selecting clones on chloramphenicol at 44°C. Following 2-times purification under the same conditions, the cointegrates are grown at 30°C in the presence of 5% sucrose to force resolution of the cointegrate and elimination of the plasmid from the cell. At this step, a preliminary assignment if a given gene is essential or non-essential for growth of E. coli in complex media was made.
• the genotype of the chloramphenicol-sensitive clones obtained following cointegration and resolution of the disruption plasmid was determined by colony-PCR using primers cl and c2 (see Fig.4).
• the second recombination event can result in either a wild-type or a mutant genotype.
• the testing of 20 independent clones showed routinely that a ⁇ 1 :1 distribution of wild-type versus mutant genotype in case of a non-essential gene. Recovery of only wild-type genotype in 50 independent clones was considered as preliminary evidence for a gene's essentiality.
• a vector, pRDC15 was designed, which allows a copy of a putative essential gene to be placed in ectopic position on the chromosome under the control of a tightly regulated promoter.
• the plasmid is a derivative of pKO3.
• pRDC15 carries a DNA fragment consisting of the araC gene, the arabinose promoter, a cloning site [BamHl-Nhel-Sfll-Xhol-Sphl-Sfil] and the polB gene.
• the wild-type copy of a putative essential gene was amplified by PCR and cloned into the vector pRDC15 using restriction sites Nhel and Xhol.
• the resulting construct was used for gene replacement in a manner identical to the disruption plasmids described above.
• the araC and polB genes of pRDC15 represent the homologous DNA for recombination at the araCBADpolB locus of the E. coli chromosome.
• the araBAD genes in the E. coli chromosome are replaced by the wild-type copy of the gene of interest, which is now under the control of the arabinose promoter.
• This merodiploid strain is then used to construct an in frame deletion of the wild-type target gene using the disruption plasmid described above in the presence of 0.2% arabinose.
• the deletion mutant can be obtained since a wild-type copy is expressed in trans from the arabinose locus.
• the resulting strain is a conditional mutant as expression of the target gene is now dependent on the presence of arabinose.
• the inability of such a strain to grow in the absence of arabinose can provide final proof that a given gene is essential for growth of E. coli.
• growth of a mutant in yjeQ was not dependant upon arabinose.
• Example 3 yloQ is an essential gene in Bacillus subtilis.
• An integrative plasmid allowing the expression of genes under the control of a xylose inducible promoter was constructed as follows: A DNA fragment carrying the repressor gene xylR and the xylA promoter was PCR amplified from B. subtilis genomic DNA with the following primers:
• pxyl-4 5 '-atcgctcgagAGATGCACCTTCTATACCCG-3 '
• pxyI-7 5'-atcgaagcttAGCGATCCTACACAATCATG-3'
• the primers were designed such that they introduced a unique Ec ⁇ RI site at the 5' end of the PCR product and a unique BamHl site at the 3' end of the product.
• the PCR fragment was then cloned as an EcoRI-BamHI fragment into the B. subtilis integrative vector pDG648 to yield pRDC9 ( Figure 5).
• a DNA fragment containing approximately 100 bp sequence from the 5' region of yloQ was amplified by PCR from B. subtilis genomic DNA.
• the PCR primers were designed such that the resulting PCR product contains unique restrictions site at both the 5' and 3 'ends of the PCR product. Subsequently, the PCR product was cloned into pRDC9. 3C - Construction of a conditional mutant.
• the disruption plasmid was inserted into B. subtilis strain JH642. Chromosomal integration of the plasmid via single-reciprocal Campbell-like recombination at the yloQ locus into the chromosome was driven by selection on LB plates containing erythromycin (1 ⁇ g/ml), lincomycin (25 ⁇ g/ml) and 10 mM xylose. The resulting strain is a conditional mutant in which expression of yloQ is dependent on the presence of xylose into the growth medium.
• pRDC19 is a derivative of pDG1664 (Gene (1996) 180:57-61) a plasmid for ectopic integration at the thrC locus.
• pDG1664 Gene (1996) 180:57-61) a plasmid for ectopic integration at the thrC locus.
• a PCR fragment containing xylR-P lA generated with synthetic primers such that it was flanked with an EcoRI site 5' of xylR and Hindlll, Sail, Sphl and BamRl sites dowstream of ? xylA at its 3' extremity was digested with EcoRI and BamHl and subcloned into pDG1664.
• the yloQ gene was cloned into pRDC19 and integrated into the thrC locus.
• yloQ was then replaced by a spectinomycin cassette engineered to enable non-polar insertions.
• upstream and downstream regions of yloQ were PCR amplified, digested with appropriate enzymes and linked by ligation with a spectinomycin resistance cassette derived from pJL74 (J. Bacteriol. (1995) 177:166- 175).
• This spec r cassette was a PCR product amplified such that it did not include any transcription termination sequence was cloned in the same transcriptional orientation as the gene to be replaced.
• the resulting ligation product was cloned into pJHIOl and gene replacement was obtained by introducing the spec 1 cassette into the B.
• subtilis chromosome by a double homologous recombination event. Chromosomal DNA was then prepared from the resulting Em r and Spc r strain and used to transform B. subtilis at saturating levels of DNA. If yloQ is essential, then all transformants selected on spectinomycin should also be Em r since the strain should only able to grow if the copy of the gene which is linked to Em r at thrC is cotransformed by congression. Twenty out of 20 transformants selected on Spc were also Em r . Demonstrating that yloQ is indeed essential in B. subtilis.
• Each of the four motifs are shown as they exist in each of the family members and are explicitly described as position-dependent scoring matrices, or profiles. Together these profiles can be used by the motif alignment and search tool, MAST, described in the same reference, to search databases for yjeQ family members, which are positively identified when p-values of less than 1 x 10 "16 are obtained. Where p-values are based on a random sequence model that assumes each position in a random sequence is generated according to the average letter frequencies of all sequences in the peptide non-redundant database (ftp://ncbi.nlm.nih.gov/blast/db/) on September 22, 1996.
• Tables 1 to 4 show the position dependent scoring used to define the yjeQ family. Values in the position-dependent scoring matrix are calculated by taking the log (base 2) of the ratio p/f at each position in the motif where p is the probability of a particular letter at that position in the motif, and f is the average frequency of that letter in the training set. Columns correspond to 1 letter amino acid codes and rows correspond to the position in the motif.
• PROSITE patterns using the conventions outlined in PROSITE: A dictionary of protein sites and patterns (http://www.expasy.ch/sprot/prosite.html) and Bairoch A., Bucher P., Hofmann K. The PROSITE datatase, its status in 1995. Nucleic Acids Res. 24:189- 196(1995). YjeQ family members are positively identified when exact matches to any one of the two prosite patterns pattern 1 or pattern 2 as set out in Figure 3 are found in the protein sequence.

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