WO2003027140A2 - Genes, proteines, et utilisation - Google Patents

Genes, proteines, et utilisation Download PDF

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Publication number
WO2003027140A2
WO2003027140A2 PCT/GB2002/004333 GB0204333W WO03027140A2 WO 2003027140 A2 WO2003027140 A2 WO 2003027140A2 GB 0204333 W GB0204333 W GB 0204333W WO 03027140 A2 WO03027140 A2 WO 03027140A2
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Prior art keywords
protein
putative
genes
gene
microorganism
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PCT/GB2002/004333
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English (en)
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WO2003027140A3 (fr
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Gordon Dougan
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Microscience Limited
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Priority to AU2002334081A priority Critical patent/AU2002334081A1/en
Publication of WO2003027140A2 publication Critical patent/WO2003027140A2/fr
Publication of WO2003027140A3 publication Critical patent/WO2003027140A3/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
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/255—Salmonella (G)
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
    • 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 genes and proteins, and their use. More particularly, it relates to genes and proteins/peptides obtained from enteric microorganisms, and their use in therapy and in screening for drugs .
  • Background of the Invention relates to genes and proteins/peptides obtained from enteric microorganisms, and their use in therapy and in screening for drugs .
  • Salmonella typhimurium is one of the major causes of food poisoning, resulting in gastro-enteritis. The source of most infections is ingestion of contaminated water or food, such as poultry, eggs and diary products. Salmonella typhimurium is also able to cause typhoid in mice and to colonise the alimentary tracts of poultry. While infection of adult poultry leads to limited excretion of Salmonella in the faeces, infection of newly hatched chicks, which have a relatively simple gut flora, results in rapid multiplication and extensive excretion. This can lead to a rapid spread of a Salmonella strain through a flock as the housing and the water and feeding systems become contaminated. There is also a risk of contamination of the carcasses at slaughter, with entry of Salmonella typhimurium into human food.
  • Salmonella typhimurium is a member of the genera Enterobacter.
  • the Enterobacteria provide an example of genetic adaptation and have evolved genes that allow them to survive in diverse conditions found outside of their hosts. They will survive in water and soil and will resist large temperature fluctuations. In addition to these genes for environmental survival, the Enterobacteria harbor many genes that contribute directly to colonization of the intestine. These genes may be regarded as a core set of genes needed for the commensal lifestyle.
  • Pathogenic strains of ⁇ . coli and most Salmonella enterica have acquired additional genes that can enhance virulence for the host, increasing their pathogenic potential. For example, S.
  • enterica have acquired sets of genes that facilitate the invasion of tissues [Galan, 1989 1 ; Galan, 2000 2 ] and the ability to survive and grow within mammalian cells, including immune cells such as macrophages [Fields, 1986 3 ; Garcia-del Portillo, 1995 4 ] .
  • This enhanced ability to target immune cells makes S. enterica potentially highly immunogenic.
  • S. enterica have also evolved into, a large number of different serovars that exhibit distinct characteristics, such as host restriction and the ability to induce different disease syndromes, depending on the particular host/pathogen combination.
  • Host-restricted bacteria may be regarded as mutant forms of their more promiscuous parents that have lost the ability to grow in some hosts but have become better adapted to a specific host.
  • S. enterica serovar Typhimurium S. typhimurium
  • S. enterica serovar Typhi S. typhi isolates are limited in their pathogenic potential to humans. Further, they cause systemic infection leading to typhoid [Neidhardt, 1996 5 ] .
  • Salmonella serovars to cause different types of disease in the same target species or become host-adapted is not well understood but is almost certainly due to the different gene repertoires of the two serovars . In a sense the bacteria have become adapted to different environmental niches within the host.
  • the present invention is based on the discovery of genes in Salmonella typhimurium, the products of which are implicated in virulence and colonisation.
  • an isolated peptide or gene of the invention listed in Table 1, from S . typhimurium or a homologue thereof in a Gram- negative bacterium, or a functional fragment thereof, is used for therapy or diagnosis.
  • peptides have many therapeutic uses for treating Salmonella infections, including use in vaccines for prophylactic application.
  • a polynucleotide encoding a peptide defined above is also useful for therapy or diagnosis.
  • the genes that encode the peptides may be utilised to prepare attenuated microorganisms.
  • the attenuated microorganisms will usually have a mutation that disrupts the expression of one or more of the genes, to provide a strain that lacks virulence. These microorganisms will also have use in therapy and diagnosis.
  • the peptides, genes and attenuated microorganisms according to the invention may be used in the treatment or prevention of a condition associated with infection by Salmonella or Gram-negative bacteria.
  • a method for the identification of a gene, the product of which is required for the colonisation of the intestine by an enteric pathogen, the method comprising comparing the genome of the enteric pathogen with the genome of a related, non-pathogenic strain, and identifying those genes that are functional in the pathogen but which are inactive in the non-pathogen.
  • the present invention is based on the discovery of genes encoding peptides which are implicated in virulence and/or colonisation.
  • the peptides/genes of the invention were identified by comparing the genome of the pathogenic S . typhimurium microorganism with that from the less pathogenic S. typhi strain. The comparison was intended to identify genes in S . typhi that were inactive, but for which there was an active corresponding gene in S. typhimurium. The invention therefore made use of the genome data available for S . typhi at www.sanger.ac.uk/Projects/S_typhi, and that for
  • S . typhimurium at http://genome.wustl.edu/gsc/Projects/ S . typhimurium.
  • the genes identified for S. typhi may be termed pseudogenes, as they are inactive. However, they can be used to identify their active counterparts in other organisms based on homology/similarity searching, e.g. in S . typhimuri urn .
  • the pseudogenes identified in S . typhi are listed in Table 1. Although the pseudogenes themselves may have a therapeutic application, it is expected that the active counterparts (i.e. those in S . typhimurium) will be of particular therapeutic importance, as they can be disrupted to produce less pathogenic microorganisms. It should be understood that references to therapy also include preventative treatments, e.g. vaccination. Furthermore, while the products of the invention are intended primarily for treatment of infections in human patients, veterinary applications are also considered to be within the scope of the invention.
  • the present invention is described with reference to Salmonella typhimurium .
  • Salmonella strains and many other Gram-negative bacterial strains are likely to include related peptides or proteins having amino acid sequence identity or similarity to those identified herein.
  • Organisms likely to contain the peptides include, but are not limited to the genera Enterobacter , Klebsiella, Shigella and Yersinia .
  • the peptides that may be useful in the various aspects of the invention have greater than a 70% similarity with the peptides identified herein. More preferably, the peptides have greater than 80% sequence similarity. Most preferably, the peptides have greater than
  • polynucleotide sequences that encode the peptides may have greater than 70% identity with the nucleotide sequences. More preferably, the polynucleotide sequences have greater- than 80% 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. The use of the term “identity” refers to a sequence comparison based on identical matches between correspondingly identical positions in the sequences being compared.
  • similarity refers to a comparison between amino acid sequences, and takes into account not only identical amino acids in corresponding positions, but also functionally similar amino acids in corresponding positions. Thus similarity between polypeptide sequences indicates functional similarity, in addition to sequence similarity.
  • Levels of identity between gene sequences and levels of identity or similarity between amino acid sequences can be calculated using known methods.
  • publicly available computer based methods for determining identity and similarity include the BLASTP, BLASTN and FASTA (Atschul et al . , J. Molec . Biol . , 1990; 215:403-410), the BLASTX program available from NCBI, and the Gap program from Genetics Computer Group, Madison WI .
  • the levels of similarity and identity referred to above, are based on the use of the Gap program, with a Gap penalty of 12 and a Gap length penalty of 4 for determining the amino acid sequence comparisons, and a Gap penalty of 50 and a Gap length penalty of 3 for the polynucleotide sequence comparisons.
  • gene sequence it is possible to use the gene sequence to search for related genes or peptides in other microorganisms. This may be carried out by searching in existing databases, e.g. EMBL or GenBank.
  • Peptides or proteins according to the invention may be purified and isolated by methods known in the art. In particular, having identified the gene sequence, it will be possible to use recombinant techniques to express the genes in a suitable host. Active fragments and related molecules can be identified and may be useful in therapy. For example, the peptides or their active fragments may be used as antigenic determinants in a vaccine, to elicit an immune response. They may also be used in the preparation of antibodies, for passive immunisation, or diagnostic applications. Suitable antibodies include monoclonal antibodies, or fragments thereof, including single chain Fv fragments. Methods for the preparation of antibodies will be apparent to those skilled in the art .
  • Active fragments of the peptides are those that retain a biological function.
  • the fragment when used to elicit an immune response, the fragment will be of sufficient size, such that antibodies generated from the fragment will discriminate between that peptide and other peptides of the bacterial microorganism.
  • the fragment will be at least 30 nucleotides (10 amino acids) in size, preferably 60 nucleotides (20 amino acids) and most preferably greater than 90 nucleotides (30 amino acids) in size.
  • the invention encompasses modifications made to the peptides and polynucleotides identified herein which do not significantly alter the biological function. It will be apparent to the skilled person that the degeneracy of the genetic code can result in polynucleotides with minor base changes from those specified herein, but which nevertheless encode the same peptides. Complementary polynucleotides are also within the invention. Conservative replacements at the amino acid level are also envisaged, i.e. different acidic or basic amino acids may be substituted without substantial loss of function.
  • molecules that comprise a polynucleotide which hybridizes under stringent hybridization conditions to a portion of a polynucleotide of the invention are molecules that comprise a polynucleotide which hybridizes under stringent hybridization conditions to a portion of a polynucleotide of the invention.
  • stringent hybridization conditions is intended overnight incubation at 42°C in a solution comprising: 50% formamide, 5x SSC (150 nM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt ' s solution, 10% dextran sulfate, and 20 ⁇ g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0. Ix SSC at about 65°C.
  • a polynucleotide which hybridizes to a "portion" of a polynucleotide is intended a polynucleotide (either DNA or RNA) hybridization to at least about 15 nucleotides bases, and more preferably at least about 20 nucleotides bases, still more preferably at least about 30 nucleotides bases, and even more preferably about 30-70 (e.g., 50) nucleotides bases of the reference polynucleotide.
  • Vaccine compositions can be formulated with suitable carriers or adjuvants, e.g. alum, as necessary or desired, to provide effective immunisation against infection.
  • suitable carriers or adjuvants e.g. alum
  • the preparation of vaccine formulations will be apparent to the skilled person.
  • the attenuated microorganisms may be prepared with a mutation that disrupts the expression of any of the genes identified herein. The skilled person will be aware of methods for disrupting expression of particular genes. Techniques that may be used include insertional 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 identified herein or in a separate gene required for growth of the microorganism, e.g. an aro mutation or, with regard to Salmonella, in a gene located in the SPI2 region identified in WO-A-96/17951.
  • Attenuated microorganisms may also be used as carrier systems for the delivery of heterologous antigens, therapeutic proteins or nucleic acids (DNA or RNA) .
  • the attenuated microorganisms are used to deliver a heterologous antigen, protein or nucleic acid to a particular site in vivo .
  • Introduction of a heterologous antigen, peptide or nucleic acid into an attenuated microorganism can be carried out by conventional techniques, including the use of recombinant constructs, e.g. vectors, which comprise polynucleotides that express the heterologous antigen or therapeutic protein, and also include suitable promoter sequences.
  • the gene that encodes the heterologous antigen or protein may be incorporated into the genome of the organism and the endogenous promoters used to control expression.
  • a suitable amount of an active component of the invention can be selected, for therapeutic use, as can suitable carriers or excipients, and routes of administration. These factors would be chosen or determined according to known criteria such as the nature/severity of the condition to be treated, the type and/or health of the subject etc.
  • the products of the invention may be used in screening assays for the identification of potential antimicrobial drugs or for the detection for virulence. Routine screening assays are known to those skilled in the art, and can be adapted using the products of the invention in the appropriate way.
  • the products of the invention may be used as the target for a potential drug, with the ability of the drug to inactivate or bind to the target indicating its potential antimicrobial activity.
  • the various products of the invention may also be used in veterinary applications, e.g. for the immunisation of poultry.
  • the pseudogenes identified in S. typhi are shown in Table 1.
  • the genes are identified by a STY number, which is the annotation of the genome sequence that identifies individual genes on the genome, which is deposited at NCBI as Accession No. NC_003198.
  • the full gene and protein sequence can be obtained from NCBI (see www.ncbi.nlm.nih.gov).
  • the corresponding genes for S . typhimurium can be obtained by searching the WUSTL database, using the gene sequences obtained from the NCBI database. The skilled person will appreciate that a sequence of, say, 30 nucleotides from a S. typhi gene sequence can be used to identify the corresponding gene in S . typhimurium.
  • the genome and proteome sequences of S. typhi and S. typhimurium or E. coli were compared in parallel to identify deletions and insertions using the Artemis Comparison Tool (ACT) (see www.sanger.ac.uk/Software/ACT/).
  • ACT Artemis Comparison Tool
  • Pseudogenes had one or more mutations that would ablate expression, and were identified by direct comparison with S. typhimurium; each of the inactivating mutations was subsequently checked against the original sequencing data.
  • pseudogenes 124/204
  • Many of' these genes might be described as "cryptic' (i.e. capable of reverting to full function) , but in the absence of any experimental information about reversion, they are all described as pseudogenes .
  • DNA coding for five of these genes (priC, ushA, fepE, and fliB) was sequenced from several independent S. typhi and in each case the sequence was identical.
  • Frameshifts due to changes in the length of homopolymeric tracts account for 45 pseudogenes; this is a mechanism of variation previously shown to occur in E.
  • pseudogenes are the remnants of insertion sequence (IS) transposases, integrases and genes. of bacteriophage origin.
  • Examples of this latter group include components of seven of the twelve chaperone/usher fimbrial operon [Townsend, 2001 11 ] , the gene responsible for flagellar methylation, fliB [Burnens, 1997 12 ] ; genes within or associated with previously described Salmonella pathogenicity islands (SPI) (for example the sensor kinase ttrS associated with SPI2 [Hensel , 1999 13 ] ; cigR marT and misL from SPI3 [Blanc-Potard, 1999 14 ) .
  • SPI Salmonella pathogenicity islands
  • SPI2 STY1735 ttrS Sensor kinase TtrS protein
  • SPI2 STY1739 - Putative ribokinase
  • SopE2 a Salmonella secreted protein which is highly homologous to SopE and involved in bacterial invasion of epithelial cells. J Bacteriol 182, 2341-4. (2000).

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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Abstract

Selon l'invention, la comparaison de gènes inactifs dans un micro-organisme non pathogène, mais actifs dans un micro-organisme pathogène associé, a permis d'identifier des gènes pouvant être employés en thérapie ou diagnostic. Ainsi, des peptides codés par une gène de S. typhimurium, correspondant aux gènes regroupés dans le tableau (1), peuvent être employés en thérapie ou diagnostic.
PCT/GB2002/004333 2001-09-26 2002-09-26 Genes, proteines, et utilisation WO2003027140A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2002334081A AU2002334081A1 (en) 2001-09-26 2002-09-26 Identification of s. typhimurium genes by software-based genome comparison

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GBGB0123170.3A GB0123170D0 (en) 2001-09-26 2001-09-26 Genes,proteins,and their use
GB0123170.3 2001-09-26

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Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
DAIGLE FRANCE ET AL: "Identification of Salmonella typhi genes expressed within macrophages by selective capture of transcribed sequences (SCOTS)." MOLECULAR MICROBIOLOGY, vol. 41, no. 5, September 2001 (2001-09), pages 1211-1222, XP002226474 ISSN: 0950-382X *
DATABASE EMBL [Online] 1 November 1999 (1999-11-01) "Fimbrial usher, BCFC, Salmonella typhimurium" retrieved from EBI Database accession no. Q9WW45 XP002227016 *
DATABASE EMBL [Online] 3 June 1999 (1999-06-03) "S. typhimurium bovine colonization factor operon, completet sequence." retrieved from EBI Database accession no. AF130422 XP002226591 *
ELTZE S M ET AL: "Salmonella Pathogenicity Island-2 (SPI-2) mutants of Salmonella typhi and S. typhimurium as live vaccine vectors." ABSTRACTS OF THE GENERAL MEETING OF THE AMERICAN SOCIETY FOR, vol. 101, 2001, page 339 XP009003750 101st General Meeting of the American Society for Microbiology;Orlando, FL, USA; May 20-24, 2001, http://www.asmusa.org/mtgsrc/generalmeetin g.htm 2001 ISSN: 1060-2011 *
MCCLELLAND MICHAEL ET AL: "Complete genome sequence of Salmonella enterica serovar Typhimurium LT2." NATURE (LONDON), vol. 413, no. 6858, 2001, pages 852-856, XP002224529 ISSN: 0028-0836 *
SYDENHAM MARK ET AL: "Salmonella enterica serovar Typhimurium surA mutants are attenuated and effective live oral vaccines." INFECTION AND IMMUNITY., vol. 68, no. 3, March 2000 (2000-03), pages 1109-1115, XP002226473 ISSN: 0019-9567 *

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AU2002334081A1 (en) 2003-04-07
GB0123170D0 (en) 2001-11-21

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