WO2001021655A2 - Gene de virulence et proteine, utilisations de ces derniers - Google Patents

Gene de virulence et proteine, utilisations de ces derniers Download PDF

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Publication number
WO2001021655A2
WO2001021655A2 PCT/GB2000/003647 GB0003647W WO0121655A2 WO 2001021655 A2 WO2001021655 A2 WO 2001021655A2 GB 0003647 W GB0003647 W GB 0003647W WO 0121655 A2 WO0121655 A2 WO 0121655A2
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coli
peptide
mutants
genes
colonization
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PCT/GB2000/003647
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English (en)
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WO2001021655A3 (fr
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Christoph Tang
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Isis Innovation Limited
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Publication of WO2001021655A2 publication Critical patent/WO2001021655A2/fr
Publication of WO2001021655A3 publication Critical patent/WO2001021655A3/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
    • C07K14/245—Escherichia (G)
    • 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
    • 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
    • C07K14/29—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Richettsiales (O)
    • 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates to virulence genes and their encoded proteins. More particularly, it relates to their use in therapy and in drug discovery. Background to the Invention
  • Colonization is the first step in the disease process and is often fundamental to bacterial pathogenesis .
  • the site of colonization is the source of infection for further susceptible hosts.
  • Escherichia coli is a ubiquitous resident of the human gastrointestinal (GI) tract and is transmitted by the feco- oral route. From the GI tract, it can cause enteric, urinary, and systemic disease.
  • GI human gastrointestinal
  • Studies on E. coli colonization have identified a variety of bacterial receptors, and their corresponding ligands, that mediate E. coli attachment to, or uptake into, host intestinal cells (see, for example, Gaastra and Svennerholm, Trends Microbiol. 1996; 4:444-452 and Guerrant et al . , J . Infect. Dis. 1999; 179:5331-5337) .
  • bacterial adhesion and cell entry is only one component of colonization, which is a complex and dynamic sequence of events.
  • E. coli must survive the low pH conditions of the stomach, the high osmotic pressures in the small bowel, and other innate and acquired defence mechanisms.
  • the bacteria Once at its site of residency, the bacteria must attach to the mucosal surface and avoid mechanical expulsion by peristalsis.
  • the bacterium During its transit through, and residency within, the GI tract, the bacterium must also acquire nutrients from the surrounding microenvironment , often competing with the host microbial flora for limited resources.
  • E. coli expressing the Kl polysaccharide capsule are the major cause of Gram-negative bacteraemia and meningitis in neonates . Infants often become infected through contact with their mothers or health care workers carrying the bacterium. Furthermore, E. coli Kl isolates are among the most common cause of ascending urinary tract infection in childhood. In studies of E. coli pathogenesis , host-to-host transmission, GI tract colonisation, systemic disease, and ascending urinary tract infection have all been described. However, colonization is an aspect of E. coli Kl pathogenesis that has not been widely investigated. Summary of the Invention
  • the present invention is based on the identification of gene sequences within E. coli Kl which may code for proteins that have an important role in colonisation during infection.
  • the genes are therefore important targets for antimicrobial therapy, where disruption of the gene sequences decreases infection of the microbe.
  • a peptide or protein is encoded by any of the genes defined herein as dgc B, C, D or E from E. coli Kl , or a homologue thereof in a Gram negative bacterium, or a functional fragment thereof .
  • a peptide is encoded by any of the genes dgcA, trsE, trsC, fimH, rnr, csgE, fnr, metJ, rpoN, frdA, speA, adh, pgi , treB or emrB, or a homologue thereof in a Gram negative bacterium, or a functional fragment thereof, for therapeutic use, particularly for use in the manufacture of a medicament to treat bacterial infection.
  • polynucleotides that encode the peptides (or proteins) may also have therapeutic use.
  • the peptides (or proteins) may be used as antigens to raise an immune response. Alternatively, they may be used to produce an attenuated microorganism that can be used in a vaccine. For example, an attenuated microorganism may be produced by a deletion in one or more of the genes encoding the peptides (or proteins) . Description of the Invention
  • the present invention is based on discoveries made using signature tagged utagenesis (STM) (Hensel et al . , Science, 1995; 269:400-402) to identify genes required by E. coli Kl for GI colonization.
  • STM signature tagged utagenesis
  • a gene as identified herein, or its encoded product may be used in various ways in antimicrobial therapy.
  • the product may be used as a vaccine, to elicit an immune response.
  • the product may be used to generate a monoclonal or polyclonal antibody, which may have therapeutic or diagnostic use. Techniques for generating antibodies are known to those skilled in the art.
  • antibody is used herein to refer to whole antibodies, single chain antibodies and antibody fragments, e.g. Fv or Fab fragments.
  • a microorganism which is attenuated by inactivation of a gene identified herein may also be used as a vaccine.
  • the gene may be inactivated by any suitable technique, including a deletion or insertion mutation. It may be desirable to include further attenuating mutations in one or more other genes, to provide a greater degree of certainty that reversion to the wild-type strain cannot occur.
  • An attenuated microorganism of the invention may also be used as a carrier of another antigen or therapeutic agent. In this way, the microorganisms are useful as delivery vehicles.
  • a gene or polynucleotide of the invention may also be a useful therapeutic or diagnostic agent. For example, the gene, or functional fragment thereof, may be used in gene therapy. Alternatively, the polynucleotides may be used as probes in a diagnostic test to identify possible pathogenic microorganisms.
  • a nucleotide sequence identified herein may have homology to sequences in other microorganisms. Homology refers to either sequence similarity or identity. Typically, the degree of homology will be greater than 20%, preferably greater than 50%, more preferably greater than 80% and most preferably greater than 90%. Determining the level of homology may be carried out using conventional sequence comparison programmes, for example, the BASTN and BLASTX programmes. The homology is determined over at least 20 nucleotides, preferably 50 nucleotides and most preferably the full sequence identified herein. Similar homology in respect of an amino acid sequence may also be achieved.
  • the present invention also relates to functional fragments of the peptides and genes.
  • the term "functional fragment” is used to refer to a subunit of a sequence identified herein, which retains either functional activity, i.e. its biological role, or therapeutic activity, i.e. its ability to elicit an immune response.
  • the invention includes, of course, mutated versions of the genes or encoded products, which have reduced or no biological activity. The mutants are intended to produce an attenuated microorgansm.
  • a nucleotide fragment may be 20 nucleotides, preferably 50 nucleotides and more preferably at least 100 nucleotides, in length.
  • An amino acid fragment may be from 10 amino acids, preferably 20 amino acids, more preferably 40 amino acids and most preferably at least 50 amino acids, in length.
  • the E. coli isolate used in this study expresses the 018 serotype lipopolysaccharide .
  • This serotype is responsible for a significant proportion of all cases of E. coli bacteraemia, and 018 strains have a propensity to cause invasive infections in experimental models.
  • E. coli CC118- ⁇ pir was transformed with pUTmini-Tn5Km2 containing signature tags (de Lorenzo et al , J. Bacteriol . 1990; 172:6568-72; Hensel et al , supra) .
  • a total of 288 individual transformants were screened to identify a subset of 96 plasmids harboring tags that gave consistent and specific signals. Colony blots of the transformants were probed, with all tags labelled with ( 32 P]-dCTP. Transformants that failed to give a hybridization signal were discarded. The remainder were then analyzed for the presence of cross-hybridizing tags by probing the blots with tags amplified from subsets of the transformants .
  • 96 uniquely tagged pUTmini-Tn5Km2 derivatives were selected for the construction of the mutant library. These plasmids were transformed separately into the donor strain, S117-Xpir, and used in independent matings with the E. coli Kl recipient, RS228nal R . A total of 2,304 mutants were arrayed into 24 pools of 96 mutants each. To determine the diversity of insertion sites of mini-Tn5 in RS228nal R , DNA from 30 ex-conjugates from three different matings was subjected to Southern analysis.
  • auxotrophic mutants were replica plated onto complete and minimal media. Five mutants (approximately 1 % failed to grow on minimal media alone, and these were randomly distributed in the STM pools.
  • a total of 2,140 mutants were screened for mutants defective in GI colonization. Pairs of animals received 5 x 10 8 cfu, containing 47 to 95 mutants intragastrically, and bacteria were recovered from the descending colon 48 hr later. In the initial screen, 37 mutants (1.7%) were present in the inoculum but failed to be recovered from the large bowel of infected rats. All these mutants were re- assessed in animals. Therefore, three pools were assembled containing the mutants identified in the first screen, and the pools re-tested in the infant rat model. Of the 37 original mutants, 19 were consistently attenuated in subsequent examination; all these mutants were prototrophic .
  • the insertion site of mini-Tn5 in all 19 colonization defective mutants was identified. Genomic DNA flanking the insertion sites was amplified, the PCR products cloned, and the nucleotide sequence of the insert determined. To confirm that the DNA sequence flanking the insertion site had been correctly amplified by arbitrary PCR, Southern analysis was performed on eight mutants. Blots wore prepared containing genomic DNA from the mutants and RS228nal R digested with Clal or Pstl , and probed with the corresponding arbitrary PCR product . In each instance the hybridization pattern obtained for the mutant differed from that of the wild-type strain, confirming that sequences flanking the transposon insertion site of mini-Tn5 had been isolated. DNA and protein database searches were undertaken using flanking sequences.
  • mutants Of the 19 colonization defective mutants, 12 have transposon insertion sites in genes with homologues in the published whole genome sequence of E. coli K12 (Blattner et al . , Science, 1997; 277:1453-1474), and no gene or protein with significant similarity ( ⁇ 10 10 ) was found in database searches for four insertion sites.
  • the mutants fall into five categories that contain transposon insertions in genes encoding: i) cell surface structures, ii) transcriptional regulators, iii) enzymes in metabolic pathways, iv) proteins with membrane transport functions, and v) proteins of unknown function.
  • Type-1 pili are filamentous surface organelles that bear an adhesin, encoded by fimH, that mediates attachment of bacteria to mannosylated host receptors. FimH is required for adhesion to the bladder mucosal surface via uroplakins (Wu et al . , PNAS, 1996; 93:9630-35 and Mulvey et al . , Science, 1998; 282:1494- 1497) , and for the pathogenesis of lower urinary tract infections.
  • Curli are cell surface structures that are encoded by two divergently transcribed operons, csgBA and csgDEFG (Hammar et al . , Mol. Microbiol., 1995; 18:661-670).
  • the genes in the latter operon are required for the expression of curli, though their specific functions are not known except for csgD, a transcriptional activator for the csgBA operon.
  • the gene mutation was found to be within csgE.
  • the first is a mutant with an insertion in fnr (fumarate nitrate reduction) , was colonization defective and had a markedly reduced growth rate in static culture.
  • the fnr product modulates the expression of over 70 genes whose products are involved in anaerobic metabolism (Stewart, J. Bacteriol . , 1982; 151:1320-1325 and Jones and Gunsalus, J. Bacteriol., 1987; 169:3340-49).
  • the identified gene sequence is shown as SEQ ID NO . 5.
  • the second was mutated in metJ, which is a repressor of the methionine synthesis pathway.
  • the third mutant, rpoN encodes the alternative sigma factor ⁇ 54 that has pleiotropic functions in the cell .
  • the identified gene sequences are shown as SEQ ID NOS . 6 and 7, respectively.
  • Transposon insertions were also found in genes with general metabolic functions, with several expressed under anaerobic conditions.
  • FrdA (SEQ ID NO. 8) encodes a subunit of fumarate reductase (Lohmeier et al . , Can. J. Biochem., 1981; 59:158-164) which is part of a pathway used as a terminal electron acceptor during anaerobic respiration.
  • the enzyme arginine decarboxylase (encoded by speA) catalyses the first step in the degradation of L- arginine to succinate, and is a component an acid survival system in E. coli that protects the stationary-phase cells in acidic environments (Castanie-Comet et al . , J. Bacteriol., 1999; 181:3525-3535).
  • This gene sequence is identified as SEQ ID NO. 9.
  • Alcohol dehydrogenase, encoded by adh (SEQ ID NO. 10), is involved in fermentation, reducing acetyl CoA to ethanol, thereby generating two molecules of NAD + to act as electron acceptors under anaerobic conditions (Gupta and Clark, J.
  • the di-saccharide trehalose is phosphorylated and taken up by the treB gene (SEQ ID NO. 12) product which is part of the phosphotransferase system (Boos et al., J. Bacteriol., 1990; 172:3450-61).
  • EmrB (SEQ ID NO. 13) is a component of the ErmAB efflux pump, one of a family of proteins known as membrane-fusion-proteins that includes AcrAB (Lomovskaya and Lewis, J. Bacteriol., 1992; 177:2328- 34; and Miller and Sulavik, Mol. Microbiol., 1996 21:44- 448) .
  • This gene was not identified in E. coli K12, but is present in Rickettsia prowazekii .
  • a mutant was also found to have a disruption in a gene corresponding to the rnr gene (SEQ ID NO. 14) in Shigella (Cheng et al . , J. Biol . Chem. , 1998; 273:14077-14080).
  • the mutant has a general growth defect which may be responsible for its attenuation.
  • dgc defective in GI colonisation
  • A, B, C, D and E genes of currently unknown function that were designated dgc (defective in GI colonisation) A, B, C, D and E, and are shown as SEQ ID NOS 15 to 19, respectively.
  • dgcA a homologue was present in the whole genome sequence of E. coli K12 (Blattner et al . , supra) , while for the remainder, no related sequences were identified through database searches.
  • the colonization potential of the dgcB strain was reduced compared with the wild-type isolate, but the strain grows equally well in vitro, compared to the wild-type.
  • dgc genes are present in other enteric pa thogens
  • low stringency Southern analysis was performed. Blots containing DNA from a range of bacterial pathogens (UPEC, EPEC, EHEC, ETEC, enteroaggregative E. coli , Shigella flexneri , Salmonella typhi , Salmonella typhimuiium, Yetsinia enteracoli tica and Yersinia pseudotuberculosis) were probed with fragments of the dgc genes under conditions that allow sequences of 60% identity to be detected.
  • UPEC UPEC
  • EPEC EHEC
  • ETEC enteroaggregative E. coli
  • Shigella flexneri Salmonella typhi
  • Salmonella typhimuiium Salmonella typhimuiium
  • Yetsinia enteracoli tica and Yersinia pseudotuberculosis were probed with fragments of the dgc genes under conditions that allow sequences of 60% identity to be detected
  • the mutant strains were subjected to SDS-PAGE analysis.
  • the trsE and trsC strains had altered glycoforms on LPS analysis compared with the wild-type and another colonization defective mutant (pgi ) ⁇
  • the core portion of the LPS of both the trsC and trsE mutants was affected.
  • the trsC mutant had lower molecular weight core glycoforms, while the trsE mutant structures were of similar size but of altered relative abundance in comparison with the wild- type.
  • mAb monoclonal antibody
  • PIBS phosphate buffered saline
  • Wild-type bacteria RS228nal R
  • PIBS phosphate buffered saline
  • the bacteria are predominantly in the lumen of the large bowel in association with faecal matter, and are also present in small numbers in close proximity to the mucosal surface of the bowel. No staining was seen in sections from inoculated animals when incubation with the primary antibody was omitted.
  • the dgcB mutant was detected in a similar distribution to the parental strain but at lower density at all locations. Although the levels of the dgcC mutant are greatly reduced in the small intestine, significant numbers are found in the descending colon. The dgcD mutant appears to have a specific defect for lower GI colonization; this strain colonizes the small intestine as efficiently as the wild- type but is absent entirely from the colon. The results are shown in Table 1.
  • mutant (nal R , kan R ) and wild-type bacteria (nal R ) were grown to mid log in LB broth, equal amounts of bacteria (10 7 each in 100 ml PBS) were mixed then administered intragastrically to animals. After 48 hr, animals were euthanised and bacteria recovered by plating dilutions of homogenized large bowel to media containing nal alone. To determine the proportion of wild- type to mutant bacteria in samples, 200 colonies were replica plated to media with or without kan. The colonization potential of each mutant was analyzed in two or more animals, and the results given as an average (Table 2) . The competitive index (CI) was calculated as the proportion of mutant to wild-type bacteria recovered from animals divided by the proportion of mutant to wild-type in the inoculum. A competitive index of less than 1 indicates that the mutant is avirulent.
  • Ec refers to E. coli ; Ye to Yersinia enterocoli tica ; and Rp to Rickettsia prowazekii .

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

On a identifié dans des entérobactéries des séquences de gènes qui peuvent jouer un rôle dans la colonisation lors d'une infection. Les gènes et leurs produits codés sont par conséquent des cibles appropriées pour la thérapie antibactérienne.
PCT/GB2000/003647 1999-09-22 2000-09-22 Gene de virulence et proteine, utilisations de ces derniers WO2001021655A2 (fr)

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EP99307495 1999-09-22

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008108805A2 (fr) * 2006-07-19 2008-09-12 North Carolina State University Entérobactérie atténuée déficitaire en fnr
EP2053058A1 (fr) * 2007-10-24 2009-04-29 Imperial Innovations Limited Atténuation de virulence bactérienne

Citations (3)

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Publication number Priority date Publication date Assignee Title
WO1996000233A1 (fr) * 1994-06-24 1996-01-04 Children's Hospital And Medical Center Adhesine epithelial d'escherichia coli o157:h7
WO1996038171A1 (fr) * 1995-06-02 1996-12-05 Department Of The Army, Us Government Procede visant a dresser des anticorps contre l'e. coli de la famille cs4cfa/1
US5698416A (en) * 1995-06-02 1997-12-16 The United States Of America As Represented By The Secretary Of The Army Methods for production of antigens under control of temperature-regulated promotors in enteric bacteria

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WO1996000233A1 (fr) * 1994-06-24 1996-01-04 Children's Hospital And Medical Center Adhesine epithelial d'escherichia coli o157:h7
WO1996038171A1 (fr) * 1995-06-02 1996-12-05 Department Of The Army, Us Government Procede visant a dresser des anticorps contre l'e. coli de la famille cs4cfa/1
US5698416A (en) * 1995-06-02 1997-12-16 The United States Of America As Represented By The Secretary Of The Army Methods for production of antigens under control of temperature-regulated promotors in enteric bacteria

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ADAMS L.M. ET AL.: "Identification and characterization of a K88- and CS31A-like operon of a rabbit enteropathogenic Escherichia coli strain which encodes fimbriae involved in the colonization of rabbit intestine" INFECTION AND IMMUNITY., vol. 65, December 1997 (1997-12), pages 5222-5230, XP000905644 AMERICAN SOCIETY FOR MICROBIOLOGY. WASHINGTON., US ISSN: 0019-9567 *
HICKS S ET AL.: "Role of intimin and bundle-forming pili in enteropathogenic Escherichia coli adhesion to pediatric intestinal tissue in vitro" INFECTION AND IMMUNITY., vol. 66, April 1998 (1998-04), pages 1570-1578, XP000906845 AMERICAN SOCIETY FOR MICROBIOLOGY. WASHINGTON., US ISSN: 0019-9567 *
MCKEE M L ET AL: "ENTEROHEMORRHAGIC ESCHERICHIA COLI O157:HM REQUIRES INTIMIN TO COLONIZE THE GNOTOBIOTIC PIG INTESTINE AND TO ADHERE TO HEP-2 CELLS" INFECTION AND IMMUNITY,US,AMERICAN SOCIETY FOR MICROBIOLOGY. WASHINGTON, vol. 63, no. 9, 1 September 1995 (1995-09-01), pages 3739-3744, XP002040902 ISSN: 0019-9567 *
SWEENEY N.J. ET AL.: "Escherichia coli F-18 and E.coli k-12 eda mutants do not colonize the streptomycin-treated mouse large intestine" INFECTION AND IMMUNITY., vol. 64, 1996, pages 3504-3511, XP000905643 AMERICAN SOCIETY FOR MICROBIOLOGY. WASHINGTON., US ISSN: 0019-9567 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008108805A2 (fr) * 2006-07-19 2008-09-12 North Carolina State University Entérobactérie atténuée déficitaire en fnr
WO2008108805A3 (fr) * 2006-07-19 2008-12-31 Univ North Carolina State Entérobactérie atténuée déficitaire en fnr
US8101168B2 (en) 2006-07-19 2012-01-24 North Carolina State University Attenuated FNR deficient enterobacteria
US8435506B2 (en) 2006-07-19 2013-05-07 North Carolina State University Attenuated FNR deficient enterobacteria
EP2053058A1 (fr) * 2007-10-24 2009-04-29 Imperial Innovations Limited Atténuation de virulence bactérienne

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WO2001021655A3 (fr) 2001-10-11

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