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WO2003056008A1 - Regulation de l'expression de genes associes a la mobilite bacterienne - Google Patents

Regulation de l'expression de genes associes a la mobilite bacterienne Download PDF

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Publication number
WO2003056008A1
WO2003056008A1 PCT/JP2002/013523 JP0213523W WO03056008A1 WO 2003056008 A1 WO2003056008 A1 WO 2003056008A1 JP 0213523 W JP0213523 W JP 0213523W WO 03056008 A1 WO03056008 A1 WO 03056008A1
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expression
bacterium
motility
gene
genes
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Japanese (ja)
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Taku Oshima
Chieko Wada
Hirotada Mori
Sota Hiraga
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Japan Science and Technology Agency
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Japan Science and Technology Agency
Japan Science and Technology Corp
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    • 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
    • A61K39/02—Bacterial antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • 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
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins

Definitions

  • the present invention provides a method for mutating a bacterium, comprising controlling the expression of a group of motility-related genes in a bacterium, and in particular, mutating a bacterium by controlling the expression of a group of motility-related genes in a bacterium,
  • the present invention relates to a method for controlling invasion into cells and reducing infectivity of a bacterium, and use of the bacterium as a vaccine-producing strain.
  • E.co1i The genome sequence of E. coli (E.co1i) has already been completely decoded (Science, 277, 1453-1474, 1997), and is called "transcriptome analysis" to determine gene functions and their interactions. Comprehensive analysis of gene expression profiles has become the priority method.
  • the latest and most efficient methods in functional genomics include microarray and two-dimensional (2-D) gel electrophoresis. By using such a method, researchers have been able to add new insights into the complex micro-mechanism of cells.
  • comprehensive genome-wide expression analysis using microarrays has been conventionally performed (Nucleic Acids Res. 27, 3821-3835, 1999, J. Bacteriol.
  • Dam determines when chromosomal replication begins and regulates mismatch repair and gene transcription expression by methylating adenine residues in GATC sequences.
  • XyR is a comprehensive regulatory system that responds to oxidative stress.XyR binds the recognition sequence of the unmethylated GATC site to the regulatory region of the mom gene and suppresses operon expression. (EMBO. J. 8, 2403-2410, 1989).
  • protection analysis of the entire E. coli genome revealed that global regulators such as CRP, Fnr, and IHF prevented many GAT C sequences from methylation by Da. Nature, 360, 606-610, 1992, Nat. Biotechnol. 16, 566-571, 1998).
  • Lrp leucine-responsive regulatory protein
  • PapI PapI
  • TCA cycle enzymes and some proteins such as molecular chaperones and nucleoid proteins are strongly oxidized and lose their activity (Genes Dev. 12, 3431-3441, 1998).
  • superoxide dismutase (SodA and SodB) and chaperone protein DnaK are induced (Genes Dev. 12, 3431-3441, 1998, J. Biol. Chem. 274, 26027-26032, 1999) 0
  • the expression of the TCA cycle enzyme is down-regulated, which decreases the oxidation of proteins, and the up-regulation of superoxide dismutase and the chaperone protein D naK to protect cells from oxidative stress. It is suggested.
  • pyruvate In the presence of oxygen (O 2 ), pyruvate is converted to Acetyl-CoA by the action of a pyruvate dehydrogenase enzyme and enters the TCA cycle. Substrates are sequentially oxidized while hydrogen molecules are rearranged to NAD +. Produced by the NADH is oxidized by Yubikinon, electricity flows from NADH through the respiratory chain to O 2, the final electrical receptor (J. Biochem. 120, 1055- 1063 , 1996). O 2 is produced in such oxidation cycle (see FIG. 6) c However, when the ⁇ 2 qs not present, the electrical receptors nitrate is to assist the flow of electricity to be instead.
  • the vigorous '14 response is regulated by the Aer protein, a sensor protein produced in the redox state of cells.
  • Aer is one of the energy transducers in which the signal transduction pathway adapts to external stimuli and regulates the phosphorylation of CheA, and phosphorylation of the CheY-responsive regulae overnight. Conditioning (PNAS 94, 10541-10546, 1997, Mol. Microbiol. 28, 683-690, 1998).
  • An object of the present invention is to provide a method for mutating a bacterium by controlling the expression of a motility-related gene group of a bacterium, and in particular, to mutating a bacterium by slowly controlling the expression of a motility-related gene group of a bacterium
  • Dam In order to elucidate the biological function of the bacterial DNA methylase, Dam, we used microarray and proteome methods to study various changes in environmental conditions throughout the growth cycle. Was analyzed for gene expression in E. coli. As a result, they found that a number of genes, including energy and nucleotide metabolism, cellular processes, and translation-related genes, were up-regulated and down-regulated by Dam. In the E.
  • coli dam gene damaged DNA methylase gene
  • the expression of genes involved in chemotaxis and oxygen motility the expression of enzymes involved in anaerobic respiration, and the expression of flagella-related genes Since the expression is greatly changed and the expression of the gene is deeply involved in the motility of the bacterium, the global control of the expression of the motility-related genes of the bacterium is controlled by the bacterial host cells.
  • the present inventors have found that invasiveness can be controlled, and have made the present invention.
  • the present invention controls the expression of a group of motility-related genes in a bacterium to mutate the bacterium and controls the invasion of the bacterium into a host cell, thereby deficient or reducing the infectivity of the bacterium It consists of producing bacteria.
  • the bacterium of the present invention which has been mutated to reduce infectivity, can be used as a vaccine-producing strain.
  • the present invention provides a method for mutating a bacterium characterized by controlling the expression of a group of motility-related genes in a bacterium (Claim 1).
  • the method for mutating a bacterium according to claim 1 wherein the methylase gene is controlled by mutating the gene (claim 2), and the DNA methylase gene of the bacterium is a Dam DNA methylase gene.
  • the method for mutating a bacterium according to claim 2 wherein the control of the expression of a group of motility-related genes in the bacterium reduces the methylation of adenine in the GATC sequence of the DNA of the group of motility-related genes.
  • the method for mutating a bacterium according to claim 3 (claim 4).
  • the present invention also relates to any one of claims 1 to 4, wherein the control of the expression of the motility-related genes in the bacterium is the control of the expression of a gene encoding a TCA cycle enzyme.
  • the method of mutating a bacterium according to claim 5 or controlling the expression of a motility-related gene group of the bacterium is controlling the expression of a gene encoding an enzyme involved in anaerobic respiration.
  • the method for mutating a bacterium according to any one of claims 1 to 4 (Claim 6), and the control of the expression of a group of motility-related genes of the bacterium is the control of the expression of a chemotaxis-related gene.
  • the method for mutating a bacterium according to any one of claims 1 to 4 (Claim 7) and the control of expression of a motility-related gene group of the bacterium is the control of expression of a flagella-related gene.
  • the present invention provides a bacterial mutant strain (claim 9) characterized by being mutated by the method for mutating a bacterium according to any one of claims 1 to 8, and a bacterium which is Escherichia coli.
  • the bacterial mutant according to claim 9 (claim 10) or a bacterial mutation controls bacterial host cell invasion by controlling the expression of a group of motility-related genes. 10.
  • a method for producing a vaccine comprising using the bacterial mutant according to claim 9 or 10 (claim 11) or the bacterial mutant according to any one of claims 9 to 11 as a vaccine-producing strain.
  • a vaccine (claim 13) produced by the method (claim 12) and the production method according to claim 12.
  • FIG. 1 is a diagram showing the appearance of the frequency distribution of GATC class in the upstream region of the gene over the entire E. coli genome in the experimental example of the present invention.
  • FIG. 2 is a view showing a state of cell growth under different aerobic conditions in an experimental example of the present invention.
  • FIG. 3 is a photograph showing a clustergram of an expression profile in a 400.27 Escherichia coli gene, that is, an effect of the Adam mutation in an experimental example of the present invention.
  • FIG. 4 is a photograph showing the results of analysis of proteins in wild-type and ⁇ dam mutant strains in the logarithmic phase or stationary phase using the FHRR2-DPAGE (proteome) in the experimental examples of the present invention. It is.
  • FIG. 5 shows the results of microarray analysis (italic letters) and 2-D PAGE analysis (ordinary letters) in the experimental examples of the present invention, showing the expression of TCA cycle enzymes in the ⁇ dam strain.
  • FIG. 6 relates to the present invention and shows the metabolizable energy and the production of ⁇ 2 in E. coli. It is the schematic which shows a mode of the oxidation process in raw.
  • FIG. 7 is a schematic diagram relating to the present invention and schematically illustrating the relationship between bacterial chemotaxis, mobility, nitrate reduction, and host invasion.
  • the present invention comprises a method of mutating a bacterium by controlling the expression of a motility-related gene group of a bacterium. And to reduce or reduce the infectivity of the bacterium so that it can be used as a vaccine-producing strain.
  • Bacterial motility-related genes include genes involved in chemotaxis such as chemotaxis and oxygen motility, genes encoding TCA cycle enzymes, genes involved in anaerobic respiration, and flagella-related genes. And so on.
  • Chemotaxis-related genes include aer, tar, tap, cheA, cheY, motA, motB, and genes encoding TCA cycle enzymes include gltA, lpdA, acnBicdA. , SdhA, and mdh; genes for enzymes involved in anaerobic respiration; nitrate reductase, etc .; and flagella-related genes, f1a, mo related to flagella formation and movement. t, and che and the like.
  • the expression of these motility-related genes in bacteria can be controlled by means such as deletion or mutation of the base sequence of these motility-related genes, or antisense. In order to globally control the expression of a group of sex-related genes, for example, a DNA methylase gene such as dam can be mutated and effectively controlled.
  • the motility-related genes there is a consensus sequence GATC that is methylated by Dam methylase, and DNA methylase such as Dam is present. Since the methylation of these sequences by the element is deeply involved in the expression of the motility-related gene, for example, the DNA in the motility-related gene is mutated by mutating a DNA methylase gene such as dam. By reducing sequence methylation, the expression of these motility-related genes can be controlled globally.
  • an appropriate method such as deletion of the gene or isomerization method such as partial deletion, substitution, or insertion of the base sequence of the gene can be employed.
  • the invasiveness of the bacterium to the host cell can be controlled. It can be used as a vaccine production strain.
  • gene products in a number of functional clusters were more prevalent in the ⁇ dam strain than in the wild type (Tables 1 and 2, and Figure 4).
  • Such products include energy metabolism (atpG, frdA, gapA, lpdA, mdh, tkt;), translation (fusA, glnS, infC, ppiB, tufA), cell envelope (omp F, yea F, unc D), amino acid metabolism (gly A), nucleotide metabolism (adk, deo C, gua A, gua B)> stress response (dna K, fkp A, gro EL, ppi B, tig) ) Includes the genes involved.
  • the oxidative stress responsive gene sod A, the nucleoid protein hns and h1 pA gene products, and the Opp A, RbsB, Asg2, IpyR, and YrbC proteins are also constant. Significantly increased in the ⁇ dam strain during the period.
  • IpyR P a Inorganic pyrophosphatase Central intermediate metabolism PRS 1.4 nd 1
  • Microarray analysis indicates that the expression of most genes involved in the TCA cycle is more frequently expressed in adam mutants than in wild-type under aerobic conditions, and especially in the stationary phase. It became clear (Fig. 5).
  • genes include g 1 t A (synthetic citrate) lpd A (Lipore Middehydrogenase 'subunit E 3), acn B (aconitic acid hydrahydrogenase 2), icd A (isoquenate dehydrogenase), sdh A (succinate dehydrogenase) , And mdh (lignoic acid dehydrogenase).
  • This enzyme is mainly responsible for its dismutase activity in aerobic culture (J. Biochem. 120, 1055-1063, 1996).
  • the synthesis of Sod A and Sod B is induced in the late stationary phase under aerobic conditions and prevents the oxidation of protein (J. Biol. Chem. 274, 26027-26032, 1999).
  • wild-type ! In the dagger comparison, since sod A and sod B are more frequently expressed in the ⁇ dam mutant in both the logarithmic phase and the stationary phase, Dam It is thought that the cells are protected from oxidative damage according to the level of growth rate.
  • Flagellar, motility, and chemotactic genes cluster in four distinct regions on the chromosome: regions I (24 minutes), II (41 minutes), IIIa and IIIb (43 minutes) (Macnab, RM., In Eschericia coli and Salmonella: celluar and Molecular Biology, F. Neidhardt, Ed. (American Society for Microbiology, Washington, DC, ed. 2, 1996), pp l23- 145). Expression of most genes located specifically in region II was significantly less frequent in the ⁇ dam strain than in the wild type (Table 3B).
  • nitrates Leda reductase encoding na r G and nar Z is this TogaTomo you are related to bacterial infectious (Mol. Microbiol. 35, 1017-1025, 2000, Microbiology 145, 3035-3045, 1999).
  • the results of the present invention show that under low aerobic conditions, the expression levels of certain chemotaxis-related genes (region 11 in Table 3B) and nitrate reduction-related genes (Table 3A) are reduced to the wild type. In comparison, it is extremely low in the ⁇ dam mutant.
  • Dam methylase is present in a limited bacterial species, the gamma subclass, of proteobacterial steel, including E. coli and Salmonella typhimurium. Dam methylation has been implicated in the regulation of metabolism, infectivity, replication, and mismatch repair in E. coli (Marinus, MG. In Eschericia coli and Salmonella: celluar and Molecular Biology, F. Neidhardt, Ed. (American Society for Microbiology, Washington, DC, ed. 2, 1996), pp782-791, J. Bacteriol. 182, 463-468, 2000, Science, 284, 967-970, 1999, Proc. Natl. Acad. Sci. USA, 96, 11578-11583, 1999).
  • DNA methylation by Dam is essential for the phylogeny of sister chromosome segregation. Se QA and Muk FEB also play important roles in sister chromosome localization, and it is interesting that Dam, Seq A, and Muk FEB are only present in E. coli and related bacteria ( Genes Cells 5, 327-341, 2000).
  • DNA methylase in eubacterium has been found in limited numbers, DNA methylation is a universal regulator of gene expression. This mechanism is found in eukaryotes as well as bacteria.
  • CpG methylase plays an important role in mammalian gene expression, and the expression of such methylase itself depends on tumor type and cell age (Pro Natl. Acad. Sci. USA. 96 , 8681-8686, 1999).
  • methylation by dam plays an important role in adapting cell energy levels to meet various requirements under different growth periods and various oxygen supply conditions. It is suggested that Under aerobic conditions, the Adam mutant apparently failed to adapt its energy synthesis level to growth conditions and partially induced oxidative stress. In addition, the ⁇ dam mutant under reduced aerobic conditions showed reduced motility and expression of the electroreceptor chemotaxis gene compared to the wild type, and the ddam mutant under reduced aerobic conditions Also suggests that it cannot adapt its energy synthesis. Such growth-dependent regulation of gene expression by Dam methylation plays an important role in that Dam is a typical methylation and adapts cell activity in response to environmental changes to achieve survival. Observed in E. coli and related bacteria.
  • the bacterial strains are KK46 (YK1100: trpC994l) (Mol.Gen. Genet. 250, 241-251, 1996) and KK333 (dam-16: : kam) (Gene 73, 531-535, 1988).
  • KK335 is a dam-deficient mutant (Adam) derived from KK46 (Hiraga, unpablished data). All cultures were performed in LB (Luria Bertani) medium. The aerobic culture conditions were a rotation of 170 rpm in a 37-liter flask containing 200 ml of liquid medium. Low aerobic culture conditions were the same as aerobic conditions except that a 300 ml flask was used. And
  • RNA from cell suspensions was prepared according to a modified version of the hot phenol method (J. Biol. Chem. 260, 3063-3070, 1985). After culturing the spores, the cells are harvested by centrifugation (12,000 X g) for 2 minutes and the cells are reconstituted in 0.5 ml of solution A (0.5% SDS, 20 mM sodium acetate). After suspending with thorium, 10 mM EDTA), the mixture was mixed with 0.5 ml of acidic phenol (pH 5.5), which had been warmed to 60, by pipetting. The mixture was allowed to stand at 60 for 5 minutes, and then centrifuged at 1200 rpm for 3 minutes to obtain a precipitate.
  • the array consists of 40 27 genes from E. coli and has been cloned previously in our laboratory (details on the website: http: @ ecoli.aist-nara.ac.jp).
  • the human / 3 actin gene was used as a negative control.
  • Slide to sports The amplified E. coli gene was amplified by PCR using the following vector-specific primers.
  • E.c01i microarrays are currently available from Takara Shuzo (Otsu, Japan).
  • c DNA labeling and microarray hybridization were performed according to the M guide (http://cmgm.stanford.edu/ Drown / mguiae / index.html, Science 278, 680-686, 1997).
  • Fluorescently labeled cDNA probes were prepared with the kit supplied by XL (Life Science) and 4 nmo 1 of Cy3—dUTP or Cy5—dUDP (Amersham Pharmacia) The cDNA probe was purified by Centri-sep (Princeton Separations), phenol-chloroform extraction and ethanol precipitation. c After the DNA probe was dried, it was dissolved in 9/1 water.
  • the intensity of the negative control spot consisting of the human / 3-actin gene and determined the mean and standard deviation.
  • the data from the E.co1i gene hybridization were classified into three groups.
  • the signal intensities of Cy3 and Cy5 in group 1 were greater than the +1 SD of the negative control.
  • the signal intensity of either Cy3 or Cy5 in group 2 was greater than the intensity value of 1000.
  • Group 3 Cy 3 and Cy 5 signals were lower than the +1 SD of the negative control.
  • the present inventor has standardized all the spots in Group 1 by using the average value of the ratio of all the spots in Group 1 (Cy5 to Cy3).
  • the average of the ratios (Cy5ZCy3) was defined as 1.
  • E. coli was cultured under the same aerobic conditions used in microarray analysis. The cells of 400 m1 were converted to 1 in Fig. 2 (log phase, OD600). And 2 (stationary phase, about 8 hours later). Approximately 1.0-1.2 g of cells are wetted with 1.5 ml of buffer 1 (100 mM CH 3 CO ⁇ NH 4 , 15 mM (CH 3 C ⁇ 0) 2 Mg, 20 mM T ris_HCl (H7.6), 6 mM) 3-mercaptoethanol, 0.5 mM phenylmethylsulfonylfluoride (PMS F)), sonicate, 900 xg Centrifuged for 10 minutes.
  • buffer 1 100 mM CH 3 CO ⁇ NH 4 , 15 mM (CH 3 C ⁇ 0) 2 Mg, 20 mM T ris_HCl (H7.6), 6 mM
  • PMS F phenylmethylsulfonylfluoride
  • the supernatant (sup) was further centrifuged at 100 OOOX g for 20 minutes.
  • the precipitate (Ppt) was suspended in 2 ml of buffer 1 and centrifuged at l OOOOX g for 20 minutes.
  • the resulting precipitate was suspended in 0.5-0.7 ml of buffer-11. This fraction was defined as "CD”.
  • the supernatant obtained after centrifugation at 10,000 X g for 20 minutes was further centrifuged at 1 OOOOOX g for 180 minutes. This supernatant was defined as "RPS”, and the precipitate was suspended in 0.5 ml of buffer-11 and centrifuged at 17 OOOX g for 10 minutes. This supernatant was defined as "CR”.
  • PRS, CD were suspended respectively CR Furakusho in to a solution of 6 7% acetic acid containing 3 3 mM M g C l 2 , and centrifuged for 10 minutes at 1 0 0 0 0 X g. The precipitate was suspended in the same buffer, and the elution procedure was repeated. These two supernatants were combined and desalted with Sephadex G-25 (Medium). This sample was then lyophilized.
  • Freeze-dried protein (about 1 to 2 mgZ gel) is described (J. Biochem. 100, 1583-1594, 1986, http://www.osaka-med.ac.jp/ ⁇ yhide/rfhr_2-d_page.htm) ), And analyzed by RF HR 2 -DP AG E.
  • the volume of glacial acetic acid in the sample charging buffer (50X) was 7.4 mL instead of 74 mL, and a 2 mm thick gel was used in the modified solution.
  • the protein spot increased in the Adam strain was compared with the wild strain, and confirmed by the gene protein index for RHFR2-DPAGE (http: ⁇ ecoli.aist-nara.ac.jp /). Spots not included in the gene protein index were confirmed by peptide sequence or MALDI-TO FMS. That is, the protein is plotted on a PVDF membrane by RF HR2-DPAGE, and the protein is digested with a proteinase (endoproteinase LysC) using Shimadzu PPSQ-23 peptide sequencer or the protein spot is digested with proteinase (endoproteinase LysC). The N-terminal amino acid sequence was determined by MALDI-TO FMS analysis (Voyager).
  • FIG. 1 GAT C in the upstream region of the gene throughout the E. coli genome It is a figure showing a situation of a frequency distribution of a raster. The number of GAT C sequences existing upstream of the 500p region of each gene was calculated for all open reading frames (ORF) assumed in the genome. The frequency of ORFs with different numbers of GATC sequences was plotted.
  • FIG. 1 Diagram of cell growth under different aerobic conditions. Growth of aerobic (A) and hypoaerobic (B) cultures of YL110 (wild-type, solid circle) and KK335 (Adam, open square) At an optical density (OD) of 600 nm. In the first (log phase) and the second (stationary phase) cells were harvested, RNA or protein was extracted and analyzed by microarray and 2-D PAGE, respectively.
  • A aerobic
  • B hypoaerobic cultures of YL110 (wild-type, solid circle) and KK335 (Adam, open square)
  • OD optical density
  • FIG. 3 Cluster graph of the expression profile of the E. coli gene, that is, the effect of the ⁇ dam mutation.
  • Gene expression profiles are shown in the columns. Growth and aerobic conditions are shown in the left column (1: log phase under aerobic conditions, 2: stationary phase under aerobic conditions, 3: log under aerobic conditions) Phase 4: stationary phase under low aerobic conditions).
  • Phase 4 stationary phase under low aerobic conditions.
  • the relative expression level of each gene is the result of two experiments. Estimated from Shows expression profiles of several genes involved in chemotaxis, mobility, anaerobic respiration, SOS response, and TCA cycle.
  • FIG. 4 Analysis of protein in wild-type and ⁇ dam mutant strains in log phase or stationary phase by FRHR2-DPAGE (proteome). It is a figure which shows the result.
  • Samples of wild-type and ⁇ dam cells grown in L-broth and under aerobic conditions were fractionated and subjected to 2-D electrophoresis according to the method described in “Materials and Methods”. analyzed.
  • PRS post ribosome supernatant: post ribosomal supernatant;
  • the 2-D patterns of (crude: crude debris and CR (crude ribosome) fractions are shown in (A), (B), and (C), respectively.
  • FIG. 1 Microarray analysis (italic letters) and 2-DP AGE analysis (ordinary letters) show that TCA cycle enzymes are more highly expressed in the A dam strain than in the wild type. .
  • the expression rate (AdamZ wild type) of each gene by the microarray method is shown in parentheses.
  • Figure 6. is a schematic diagram showing the state of oxidation processes in the production of metabolic energy and ⁇ 2 in E. coli.
  • the products of the TCA cycle and the nuo operon are part of the aerobic respiratory mechanism.
  • Pyruvate produced by glycolysis of glucose is a substrate that initiates the TCA cycle, and NADH is produced by oxidizing NAD.
  • N ADZN ADH cycle circulates, to produce 0 2 bound to Q 8.
  • the 0 2 is converted to H 2 0 2 by S od A and S od B peroxide Jisumu evening Ichize.
  • FIG. 7 Schematic representation of the relationship between chemotaxis, mobility, nitrate reduction, and host entry. Under aerobic conditions, the amount of oxides as final receptors in the respiratory chain was low. Under such conditions, in E. coli cells, nitrate may be the ultimate receptor instead of oxide. Low aerobic conditions Or, under anaerobic conditions, Aer senses a deficiency in energy production and induces motility and electroreceptive motility. Genes related to motility, mobility, flagella and nitrate reduction play important roles in maintaining cell survival. The chemotaxis-sensing proteins Tar and Ta belonged to similar chemotaxis processes and induced chemotaxis. These genes also play important roles in invading host cells. Industrial applicability
  • the present invention provides a bacterium which is mutated by controlling the expression of a group of motility-related genes of the bacterium to thereby mutate the bacterium and controls the invasiveness of the bacterium into a host cell, thereby producing a bacterium having a reduced or reduced infectivity of the bacterium.
  • the bacterium of the present invention which has been mutated to reduce infectivity, can be used as a vaccine-producing strain. In other words, it is suggested that reduction of the infectivity of the mutant bacterial strain to the host is a global effect of the genes that control these motility, and the genes that control the motility of the bacteria are found in many bacteria.
  • bacterial infectivity can be deleted or reduced by a method of collectively mutating this group of genes, since they are concentrated on the genome.
  • this method since this method is not restricted to a specific bacterial species, it can produce an effective vaccine-producing strain for a wide range of bacterial species.

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Abstract

L'invention concerne un procédé permettant de provoquer la mutation d'une bactérie et ainsi de réguler l'invasion de cette bactérie dans des cellules hôtes et de diminuer le pouvoir infectieux de ladite bactérie. L'invention se rapporte également à l'utilisation de cette bactérie en tant que souche productrice de vaccin. L'expression des gènes associés à la mobilité bactérienne est régulée par mutation d'un gène d'ADN méthylase, etc., ce qui entraîne la mutation d'une bactérie. Par conséquent, l'invasion de la bactérie dans des cellules hôtes est régulée, ce qui permet d'obtenir une bactérie présentant un pouvoir infectieux réduit ou nul. La bactérie mutante présentant un pouvoir infectieux réduit peut servir de souche productrice de vaccin.
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Citations (5)

* Cited by examiner, † Cited by third party
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WO2000045840A1 (fr) * 1999-02-02 2000-08-10 The Regents Of The University Of California Compositions et methodes de traitement et de prevention d'infections bacteriennes pathogenes basees sur le role essentiel de la methylation de l'adn dans la virulence bacterienne
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