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WO2006063425A1 - Vecteur d'expression pour le traitement d'infections bactériennes - Google Patents

Vecteur d'expression pour le traitement d'infections bactériennes Download PDF

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Publication number
WO2006063425A1
WO2006063425A1 PCT/CA2004/002127 CA2004002127W WO2006063425A1 WO 2006063425 A1 WO2006063425 A1 WO 2006063425A1 CA 2004002127 W CA2004002127 W CA 2004002127W WO 2006063425 A1 WO2006063425 A1 WO 2006063425A1
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WIPO (PCT)
Prior art keywords
expression vector
colicin
gram
bacteriocin
nucleotide sequences
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PCT/CA2004/002127
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English (en)
Inventor
Michael E. Stiles
Marius Jacobus Van Belkum
Liru Wang
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Canbiocin Inc
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Canbiocin Inc
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Priority to PCT/CA2004/002127 priority Critical patent/WO2006063425A1/fr
Priority to CN2004800448525A priority patent/CN101111599B/zh
Priority to CA002592344A priority patent/CA2592344A1/fr
Priority to EP04802302A priority patent/EP1836305A4/fr
Publication of WO2006063425A1 publication Critical patent/WO2006063425A1/fr
Anticipated expiration legal-status Critical
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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)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/746—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K2035/11—Medicinal preparations comprising living procariotic cells

Definitions

  • the present invention relates to expression vectors that can be used for transferring at least one heterologous gene into, and expressing it in, a Gram-positive bacterium, preferably a lactic acid bacterium (LAB).
  • LAB lactic acid bacterium
  • the present invention also relates to the anti-bacterial use of the transformed host, the heterologous gene product, fermentate containing the host and/or the gene product, or combinations thereof.
  • bacteriocins antibacterial peptides or proteins
  • Table 1 An exemplary list of bacteria and their bacteriocins are shown in Table 1.
  • the classical bacteriocins are the colicins produced by Escherichia coli. Most colicins are relatively large proteinaceous compounds that are not actively secreted from the bacterial cell.
  • Microcins produced by E. coli are peptides or polypeptides that are secreted from the cell by a dedicated export pathway and are post-translationally modified (Class I microcins) or are not posttranslationally modified (Class Il microcins).
  • Posttranslational modification requires the production of enzymes that modify the ribosomally translated peptide.
  • Bacteriocins produced by LAB are normally active against other Gram- positive bacteria, especially closely-related LABs.
  • bacteriocins produced by Gram-negative bacteria are against Gram-negative target strains.
  • colicin V a bacteriocin produced by Escherichia coli , is active against a wide range of other E. coli.
  • Colicin V was the first colicin discovered from E. coli. It is a Class Il microcin that is synthesized as a 105 amino acid pre-peptide (leader + bacteriocin) that is cleaved to release the active 88 amino acid mature peptide.
  • the colicin V operon includes a structural gene, an immunity gene, and two dedicated transport genes.
  • a large number of LAB produce bacteriocins that include the lantibiotic peptides (Class I); non-lantibiotic peptides (Class II); and proteins (Class III).
  • the lantibiotics e.g., nisin produced by Lactococc ⁇ s lactis subsp.
  • lactis are post- translationally modified and have a genetic operon consisting of about 11 genes for their synthesis, immunity, modification and export from the cell.
  • the non-lantibiotic (Class II) bacteriocins are similar to colicin V in genetic complexity. These bacteriocins are produced as pre-peptides that are cleaved to form the mature peptide and exported from the cell in the same way as colicin V, e.g. camobacteriocins A and B2, leucocin A , and pediocin PA-1.
  • the non-lantibiotic divergicin A produced by Carnobacterium divergens UAL9 requires only two genes for its production and secretion from the cell.
  • Predivergicin A consists of a signal peptide and divergicin A.
  • One gene or nucleotide sequence encodes a bacteriocin.
  • the other gene encodes an immunity protein.
  • coli if it is genetically modified (GMO) to produce a bacteriocin (such as, colicin V) or another bacteriocin that is active against another target bacterium.
  • GMO genetically modified
  • bacteriocin such as, colicin V
  • another bacteriocin that is active against another target bacterium.
  • the ability to target a Gram-negative bacterium, such as E. coli, using a Gram-positive bacterium that expresses a bacteriocin effective against the Gram-negative bacterium suggests the possibility of an alternative or supplemental therapy or preventative treatment protocol against any diseases or conditions caused by the Gram-negative bacteria.
  • An example of such a condition is post-weaning diarrhea (PWD), also known as scours, which is caused by an E. coli infection in pigs.
  • PWD post-weaning diarrhea
  • scours also known as scours, which is caused by an E. coli infection in pigs.
  • the present invention provides a technology that depends on the use of LAB that are genetically-modified (GMO) to produce heterologous polypeptides, such as bacteriocin(s), that specifically target the causative agent of a disease.
  • GMO genetically-modified
  • One or many specific uses of the compositions and methods of the present invention include treating post weaning diarrhea (PWD) caused by enterotoxigenic Escherichia coli in weanling
  • This technology can be applied anywhere that Gram-positive LAB grow in a specific environment without causing harm.
  • animal feed such as silage
  • fermented foods and anaerobically- or vacuum-packaged foods such as raw and processed meats, vegetables and pasta products
  • animal (and human) gastrointestinal (Gl) or urogenital tracts include animal feed, such as silage; fermented foods and anaerobically- or vacuum-packaged foods, such as raw and processed meats, vegetables and pasta products.
  • some LAB strains may be probiotic (i.e., health promoting), but they may not be “targeted” against specific pathogens.
  • some LAB may be targeted by genetic modification against specific pathogens, such as E. coli.
  • the compositions and/or methods of the present invention may be preventative rather than curative. In these embodiments of the invention, the compositions and methods could be effective as a replacement for feeding sub-therapeutic levels of antibiotics as a prophylactic against Gl diseases.
  • Figure 1 is a schematic representation of pCaT.
  • Figure 2 is a schematic representation of pCV22, and illustrates the replacement of the pCaT mobilization genes (mob) with a colicin V (col V) gene.
  • Figure 3 is a schematic representation of pCB12, and illustrates the replacement of the pCaT streptomycin resistance gene and Replb gene with a
  • Carnobacterium immunity gene (cbiA).
  • FIG 4 is a schematic representation of pCB15, and illustrates the replacement in pCB12 of the cbiA gene with a brochocin C immunity gene (brcl).
  • pCB15 includes colicin V (illustrated), and pCB15s includes colicin VM (not illustrated).
  • Figure 5 provides the nucleotide and amino acid sequences of colicin V and colicin VM.
  • Figures 5A and 5C show the nucleotide and amino acid sequences of colicin V, respectively; and
  • Figures 5B and 5D show the nucleotide and amino acid sequences of colicin VM, respectively.
  • Figure 6 is a schematic representation of pCB21 , and illustrates the removal of the EcoRV restriction site from pCB15.
  • Figure 7 is a schematic representation of pCB22, and illustrates the removal of the cat gene from pCB21.
  • Figure 8 is a schematic representation of pCB23m, and of a feed-grade vector; and illustrates the change of the colicin V gene in pCB22 to a colicin VM gene
  • Figure 9 is a schematic representation of pCB19, and graphically illustrates the inclusion of a polylinker containing multiple cloning sites.
  • Figure 10 is the nucleotide sequence of the p15 promoter.
  • Figure 11 is a schematic representation of the recombinant PCR technique used to generate the DNA fragment containing the p15 promoter and colicin v gene.
  • pGKV210-p15 and pCB15 were used as templates for the first round of PCR.
  • SP signal peptide divergicin A
  • colV colicin V gene
  • p15 p15 promoter.
  • Figure 12 is a schematic representation of pCB101.
  • Figure 13 is a schematic representation of pCB103.
  • Figure 14 is a schematic representation of pCB104.
  • Figure 15 is a schematic representation of pCB110.
  • Figure 16 illustrates an expression vector pMvB of the present invention.
  • the present invention is compositions and methods for expressing a gram (-) polypeptide, such as a bacteriocin, in a Gram-positive host, such as a lactic acid bacterium.
  • the invention also includes the use of a Gram-positive host, genetically modified according to the present invention, the polypeptide produced by the genetically modified host, compositions that include the GMO bacterium and/or the polypeptide, and combinations thereof in the treatment of susceptible bacteria.
  • the present invention also includes an expression vector suitable for transforming a Gram-positive host and secreting a polypeptide effective against a Gram-negative bacterium.
  • the expression vector may be variously configured according to the choice of host, promoter, and polypeptides used.
  • the expression vectors include a signal peptide, preferably a divergicin A signal peptide, and at least one bacteriocin immunity gene.
  • the expression vector is suitable for use in a LAB host.
  • the present invention also includes compositions and methods for treating susceptible bacteria and the diseases or conditions caused by the susceptible bacteria.
  • some of the compositions and methods of the present invention are used to treat E. coli.
  • the compositions and methods are used to treat scours.
  • An embodiment of the present invention includes expression vectors for expressing a mutant colicin V bacteriocin (termed colicin VM).
  • the expression vector comprises nucleotide sequences that encode colicin VM. Exemplary nucleotide sequences include but are not limited to those shown in Seq. I. D. No. l and Seq. I. D. No. 3.
  • Exemplary amino acid sequences include but are not limited to those shown in Seq. I. D. No. 2 and Seq. I. D. No. 4.
  • promoters signal peptides, selection markers, and other conventional elements of a functional expression vector may be used to express colicin VM.
  • An exemplary embodiment of the invention comprises a pCB vector comprising a P15 or P32 promoter; a divergicin A signal peptide; nucleotide sequences encoding a colicin VM; a selection marker, including but not limited to a bacteriocin immunity gene (such as brochocin-C); and a suitable replication region or regions.
  • the expression vector includes a P32 promoter, a divergicin A signal peptide, nucleotide sequences encoding colicin VM, nucleotide sequences encoding a brochocin-C immunity gene, and the replication regions Rep1A and RepB from pCaT (see Jewell, et al.; Current Microbiology: 19:343- 346 (1989)).
  • the expression vector and the host transformed by the expression vector are food or feed-grade. In the most preferred embodiments of the invention, the host and the expression vector do not contain a gene or nucleotide sequence that encodes or confers antibiotic resistance.
  • Another embodiment of the present invention includes a host cell transformed by an expression vector of the present invention.
  • the compositions and methods include CB4, a Lactobacillus reuteri host transformed with expression vector pCB15s that contains nucleotide sequences encoding colicin VM bacteriocin.
  • host lactic acid bacteria are capable of expressing or secreting one or more polypeptides, including one or more bactehocins, and include an expression vector as described herein that permit the secretion of one or more bacteriocins.
  • the expression vector may be introduced into the host bacterium by conjugation, transformation, protoplast fusion, or other gene or nucleotide transfer method.
  • Another embodiment of the present invention includes an expression vector and methods of use thereof wherein the vector includes a bacteriocin immunity gene selected from the group consisting of , but not limited to, brochocin-C and camobacteriocin A.
  • Another embodiment of the present invention includes an animal feed comprising a host bacterium transformed with an expression vector of the present invention, a bacterium produced by a transformed host of the present invention, or
  • Another embodiment of the present invention includes a probiotic composition
  • a probiotic composition comprising a host bacterium transformed with an expression vector of the present invention, a bacteriocin produced by a transformed host of the present invention, or combinations thereof.
  • Another embodiment of the present invention includes a method of treating bacterial infections in animals or humans using a composition comprising a host bacteria transformed with an expression vector of the present invention, a bacteriocin produced by a transformed host of the present invention, or combinations thereof.
  • Another embodiment of the present invention includes compositions and methods for treating any E. coli susceptible to a bacteriocin expressed in accordance with the present invention.
  • Preferred embodiments of the invention include treating E. coli and/or the diseases and conditions caused by E. coli.
  • the most preferred embodiments of the invention include treating post-weaning diarrhea or scours, and/or promoting weight gain or preventing weight loss, in pigs.
  • An expression vector of the present invention may be derived from LAB, in particular LAB of the genus Lactobacillus.
  • the plasmids according to the invention can advantageously be stably transferred into lactic acid bacteria that belong to the genera Carnobacterium, Leuconostoc, Lactobacillus, Pediococcus, or Enterococcus, among others.
  • the invention also relates to a plasmid or host transformed with the plasmid, as previously defined, the plasmid comprising the nucleotide sequence SEQ ID No. 1 , or Seq. LD. No.
  • the invention also relates to a plasmid or host transformed with the plasmid, as previously defined, the plasmid expressing the amino acid sequence comprising Seq. ID No. 2, or Seq. LD. No. 4, or a sequence which differs from this sequence by the insertion, deletion or mutation of one to several amino acids, and which retains the ability to replicate.
  • the invention also relates to an expression vector as shown in Figures 2-4, 6-9, and 12-15, the vector comprising the nucleotide sequence or sequences as shown, or a sequence which differs from this sequence by the insertion, deletion or mutation of one or several base pairs and which retains the ability of the plasmid to replicate stably in suitable bacterial host cells, e.g., LAB.
  • the invention also relates to bacterial host cells that comprise an expression vector according to the invention.
  • Exemplary expression vectors of the present invention include but are not limited to pJKM37, pCV22, pCB12, pCB15, pCB15s, pCB21 , pCB22, pCB23M, pCB19, pGKV210, pGKV210-P15, pCB101 , pCB103, pCB104, pCB110, and pCB111.
  • Exemplary hosts transformed by at least one of these expression vectors include but are not limited to Carnobacterium maltaromaticum UAL26, Lactobacillus reuteri CB4, two other strains of Lactobacillus reuteri and one strain of Lactobacillus johnsonii.
  • the plasmids according to the present invention constitute outstanding tools for cloning and expressing heterologous nucleotide sequences in host LAB.
  • the plasmids according to the invention can be used for expressing heterologous proteins, such as bacteriocins, and proteins for resistance to these bacteriocins, also termed immunity proteins.
  • any suitable host bacterium may be used.
  • the host bacterium is a Gram-positive bacterium.
  • the host bacterium is a lactic acid bacterium (LAB).
  • Exemplary suitable host include, but are not limited to, those shown in Table 1 and in the Examples. The choice of a suitable host is well within the skill of one skilled in the art.
  • the host is L. reuteri. In the most preferred embodiments of the invention, the host is CB4, a Lactobacillus reuteri strain.
  • any promoter suitable for use with expressing a bacteriocin gene may be used.
  • any promoter may be employed that is compatible with the host strain in which the secretion system of the present invention is used. Suitable promoters and the choice of a particular promoter are apparent to one skilled in the art. Suitable exemplary promoters include but are not limited to P15 and P32. See for example U.S. Patent 5,939,317, incorporated herein by reference.
  • the expression vector includes a P15 promoter, operatively associated with the bacteriocin gene of interest.
  • a promoter having nucleotide sequences corresponding to Seq. ID No. 5 may be used (see Figure 10).
  • any signal peptide suitable for use with expressing a bacteriocin gene may be used.
  • Suitable signal peptides include, but are not limited to, a signal peptide of divergicin A.
  • the expression vector includes a divergicin A signal peptide, operatively associated with the bacteriocin gene of interest.
  • a divergicin A signal peptide having nucleotide sequences corresponding to those disclosed in U.S. Patent 6,403,082 (Stiles et a/.), incorporated herein by reference, may be used.
  • any bacteriocin gene may be used. See, for example, Table 1. Suitable bacteriocin genes include but are not limited to colicin V, colicin Y101 , colicin VM, leucocin A, and brochocin-C.
  • the expression vector includes a nucleotide sequence or gene encoding one of more of the above bacteriocins.
  • the expression vector comprises nucleotide sequences or a gene encoding colicin VM. Exemplary nucleotide sequences for a bacteriocin are well known to those skilled in the art. See, for example, U.S. Patent 6,403,082 (Stiles, et al.).
  • the compositions and methods include a host and/or an expression vector that comprises nucleotide sequences or a gene that encodes a mutated colicin V that contains the following nucleotide sequence: gtggctggaggtgtggctggaggt (Seq. I. D. No. 1). See Figure 5B.
  • the compositions and methods include a host and/or an expression vector that comprises nucleotide sequences or a gene that encodes a mutated colicin V that contains the nucleotide sequences shown in Figure 5B (Seq., LD. No. 3).
  • compositions and methods include a host and/or an expression vector that encodes the following colicin VM amino acid sequence: VAGGVAGG (Seq. LD. No. 2).
  • the compositions and methods include a host and/or an expression vector that encodes A colicin VM amino acid sequence corresponding to (Seq. LD. No. 4). See Figure 5D.
  • any selection marker suitable for use with expressing a bacteriocin gene may be used. Suitable selection markers include but are not limited to immunity genes for carnobacteriocin A, piscicolin 126, and brochocin-C; and antibiotic resistance genes, e.g., chloramphenicol, erythromycin, and streptomycin.
  • the expression vector includes a bacteriocin immunity gene, preferably a brochocin C immunity gene, operatively associated with the bacteriocin gene of interest.
  • Exemplary nucleotide sequences for an immunity gene are well known to those skilled in the art. See, for example, U.S.
  • Patent 6,403,082 (Stiles, et al.), incorporated herein by reference. As noted above, it may be highly desirable to produce and use a feed-grade vector and host; such vectors and host lack functional antibiotic resistance genes and, in accordance with the present invention, include nucleotide sequences or genes that encode bacteriocin immunity.
  • the invention also includes a method of treating a bacterial infection or a method of treating an animal (including a human) by administering or contacting the bacteria or animal with one or more of the following compositions: a composition comprising one or more hosts transformed by an expression vector of the present invention; a composition comprising one or more bacteriocins produced by a transformed host; one or more bacteriocins produced naturally or by GMO (see, for example Table 1); or combinations thereof.
  • any of the compositions of the present invention may be used to treat an E. coli disease or condition, including but not limited to scours. In some embodiments of the invention, any of the compositions of the present invention may be used to promote weight gain in the subject animal. In some embodiments of the present invention, any of the compositions of the present invention may be used to treat or affect indigenous microflora in the treated subject.
  • An embodiment of the present invention includes expression vector pMvB, comprising a suitable promoter, e.g., P15; a signal peptide encoding DNA, e.g., divergicin A signal peptide; a gene encoding a polypeptide, e.g., encoding a bacteriocin, including but not limited to colicin V; a selection marker, including but not limited to a bacteriocin immunity gene, e.g., brochocin C; and a suitable replication region or regions, e.g., pCaT (a commercially available plasmid).
  • a suitable promoter e.g., P15
  • a signal peptide encoding DNA e.g., divergicin A signal peptide
  • a gene encoding a polypeptide e.g., encoding a bacteriocin, including but not limited to colicin V
  • a selection marker including but not limited to a bac
  • sequences from a pCaT plasmid that is not required and/or unwanted are deleted to result in a fragment of pCaT that may be used as a replicon.
  • sequences from a pCaT plasmid that is not required and/or unwanted are deleted to result in a fragment of pCaT that may be used as a replicon.
  • several additions are made to the pCaT replicon, including but not limited to any desired genes (such as bacteriocin and immunity genes), promoters (such as P15) and expression signals.
  • a replication sequence (or replication sequences) suitable for use in a lactic acid bacteria host may be used. Suitable replication sequences include but are not limited to the replication region(s) of pCaT.
  • the replication sequences include a pCaT segment derived from L. plantarum.
  • gene refers to a DNA sequence, including but not limited to a DNA sequence that can be transcribed into mRNA which can be translated into polypeptide chains, transcribed into rRNA or tRNA or serve as recognition sites for enzymes and other proteins involved in DNA replication, transcription and regulation.
  • genes include, but are not limited to, structural genes, immunity genes and secretory (transport) genes.
  • the term "vector” as used herein refers to any DNA material capable of transferring genetic material into a bacterial host organism.
  • the vector may be linear or circular in topology and includes but is not limited to plasmids, food grade plasmids or bacteriophages.
  • the vector may include amplification genes, enhancers or selection markers and may or may not be integrated into the genome of the host organism.
  • secretion vector or "expression vector” refers to a vector designed to provide secretion of a polypeptide such as a protein from the host organism.
  • signal peptide as used herein refers to amino-terminal amino acid residues that, when attached to a target polypeptide, permits the export of the target
  • DOC;1 ⁇ 1 5 polypeptide from the cell and cleavage of the signal peptide.
  • the signal peptide accesses the general protein secretion pathway.
  • An example of a signal peptide is the Divergicin A signal peptide described in U.S. Patent 6,403,082, incorporated herein by reference. Other signal peptides can be used and are known to those skilled in the art.
  • feed or food-grade refer to the origin of the DNA material and its constituents. Food-grade indicates that a regulatory agency would consider the substance as coming from a food source and therefore suitable for inclusion in food or food products, typically those intended for human or animal consumption.
  • Organisms that are food-grade can be added directly to food without concern for pathogenicity.
  • Food or feed grade as used herein also refers to the quality of a substance, specifically whether it is free of elements or the like that might be undesirable.
  • a food or feed grade expression vector or a food or feed grade bacterium of the present invention is free of or lacks an antibiotic resistance gene, or is free of or lacks an expressible or functional antibiotic resistance gene.
  • the food or feed grade compositions of the present invention may be used in or comprise silage, foods, feeds, diary products, meat, vegetables, or pasta.
  • bacteriocin refers to polypeptides and the like produced by the bacteria that inhibit one or more bacterial species. This includes, but is not limited to, polypeptides that are derived from specific strains of bacteria, proteins that are derived from other types of organisms, or proteins developed through genetic engineering. The bacteriocin can be bacteriostatic or bactericidal.
  • immuno gene refers to a gene that produces a protein that protects the host organism against the bacteriocin that it produces. An immunity gene may also be used as a selection marker.
  • susceptible bacterium refers to a species or strain of bacteria that is inhibited by the presence of one or more bacteriocins in its environment.
  • microcins produced by gram-negative bacteria:
  • Klebsiella pneumoniae RYC492 microcin E492 (same as 107)
  • E. coli microcin V (same as 101 , colicin is "old" name)
  • Escherichia coli DH5 ⁇ cells were grown in Luria Broth (LB) medium (Difco Laboratories Inc.) at 37°C; Carnobacterium maltaromaticum UAL26 was grown in APT (All Purpose Tween) medium (Difco) at 25 0 C; and Lactobacillus reuteri CB4 was grown in Lactobacilli MRS medium (MRS; Difco) at 37°C. Bacteriocin production was tested as described previously (van Belkum and Stiles, 1995). Colicin V production was tested using E. coli (DH5 ⁇ ) as the indicator organism grown on APT medium supplemented with 1.5% (wt/vol) agar for solid plating.
  • LB Luria Broth
  • Carnobacterium maltaromaticum UAL26 was grown in APT (All Purpose Tween) medium (Difco) at 25 0 C
  • Lactobacillus reuteri CB4 was grown in Lactobacilli
  • UAL26 and CB4 For transformation of UAL26 and CB4, cells were grown in APT or MRS medium supplemented with 2% (wt/vol) glycine, respectively. Exponentially growing cells were harvested and washed twice with ice-cold water and twice with ice-cold electroporation buffer (0.5 M sucrose, 10% glycerol, 1 mM MgC ⁇ , 5 mM potassium phosphate buffer [pH6] and concentrated 100-fold in the same buffer. Cells were divided into 50 ⁇ l portions and stored at -7O 0 C.
  • electroporation buffer 0.5 M sucrose, 10% glycerol, 1 mM MgC ⁇ , 5 mM potassium phosphate buffer [pH6]
  • Electroporation was done as described by van Belkum and Stiles (1995) with the following modification for CB4: cells were incubated at 44 0 C for 20 min and chilled on ice for an additional 10 min prior to the addition of DNA. Electroporation was done in a Gene-Pulser instrument (Bio-Rad). One pulse of 25 ⁇ F, 200 ⁇ , 2.5 kV was used for UAL26 and one pulse of 25 ⁇ F, 800 ⁇ , 1.0 kV for CB4.
  • FIG. 1 shows a schematic representation of plasmid pCaT from Lactobacillus plantarum caTC2R (Jewell and Collins-Thompson, 1989).
  • the pCaT plasmid was reported to contain the genetic information for chloramphenicol resistance (cat gene).
  • the inventors have fully sequenced and partially characterized the plasmid.
  • the plasmid has been transformed into various Carnobacterium spp., L. plantarum NC8 and L case/ ATCC 393, demonstrating chloramphenicol resistance in these strains (Ahn et al., 1992).
  • the pCaT plasmid contains 8951 base pairs.
  • pCaT transposase
  • the inventors have used pCaT as a cloning vector for genes related to the production of proteins such as, but not limited to, bacteriocins produced by Gram-positive bacteria.
  • the P32 promoter was isolated from Lactococcus lactis subsp. lactis (van der Vossen et al., 1987) and this promoter been used to express colicin V gene in pJKM37 (McCormick et al., 1999). Plasmid pJKM37 contains P32 promoter, divergicin A signal peptide, and colicin V gene (colV). A 28-mer oligonucleotide, (5' - CCC GCATGC TGA ATT CGG TCC TCG GGA T - 3') (Seq. I. D. No.
  • the PCR product containing P32 promoter and colicin V gene (coN) fused to divergicin A signal peptide was digested with Sph ⁇ .
  • the digested PCR product was cloned into pCaT by replacing the 2.1 kb Sph ⁇ fragment of pCaT containing the mobilization genes.
  • the resulting plasmid, pCV22 ( Figure 2), was transformed into a plasmidless host, Carnobacterium maltaromatic ⁇ m UAL26. These transformed cells inhibited the growth of the colicin V sensitive indicator strain E. coli DH5 ⁇ .
  • Immunity genes for bacteriocins were introduced into pCV22 as genetic selection markers. Two different functional polynucleotide seguences encoding bacteriocin immunity proteins were selected for this procedure: camobacteriocin A immunity gene and brochocin-C immunity gene (Franz et al., 2000; McCormick et al., 1998). In plasmid pCF08 the mid-sequence encoding camobacteriocin A immunity was cloned behind the P32 promoter (functional) (Franz et al., 2000). A 28-mer oligonucleotide (5' - TAT ATG ATC AGG TCC TCG GGA TAT GATA- 3') (Seq.
  • A40-mer oligonucleotide (5' - ATA TAT CGA TAG GAA GTA TGA TCAATG GTAAAAACT ATA C - 3') (Seq. I. D. No. 10) containing a C/al restriction site (underlined) was added to the 5' end of the brochocin-C immunity gene in pJKM61 (McCormick et al., 1998) and a 35- mer oligonucleotide (5' - ATA TCT GCA GAT ATC TAG TTA GAG AAT ATA ATC CA - 3') (Seq. I. D. No.
  • Lactobacillus reuteri CB4 Isolation and selection of Lactobacillus reuteri CB4 for use as a host to develop a targeted probiotic organism
  • the gastrointestinal tract (GIT) of two healthy pigs was obtained from a small, provincially inspected meat packing plant at time of slaughter.
  • the GIT was excised, sealed at the anterior and posterior ends and transported to the Animal Science laboratory at the University of Alberta Research Station (Edmonton, Canada).
  • the GIT was flushed with tap water to remove the intestinal contents and segments were excised from the pars esophagea, ileum, jejunum, cecum and colon.
  • the internal surface of the excised segments was scraped with a sterile microscope slide to remove the surface of the epithelial layer.
  • the scrapings were washed into a dilution bottle, plated onto Difco Lactobacilli MRS agar (MRS) and incubated anaerobically at 37°C for 18 to 24 hours. A total of 18 morphologically distinct colonies was randomly selected and checked for Gram-positive, catalase negative, rod-shaped characteristics and inoculated into MRS broth for storage. These strains were checked for bacteriological purity and tested for transformability with pCB15. Only Lactobacillus spp. that could be transformed were selected for further study. The isolate CB4 was able to be transformed, and was confirmed to be Lactobacillus reuteri by 16S rDNA analysis (Willson et al., 1990). L. reuteri CB4 was chosen as a strain of interest based on the stability of the transformed plasmid.
  • Electroporation of pCB15 isolated from C. maltaromaticum UAL26 into L. reuteri CB4 resulted in a low transformation rate.
  • a L. reuteri CB4 transformant was isolated that contained a plasmid denoted pCB15s, that was stable in the host strain and produced a bacteriocin that inhibited growth of colicin V-sensitive indicator organisms such as E. coli DH5 ⁇ .
  • the plasmid pCB15s from L reuteri CB4 was isolated from this transformant and electroporated back into plasmidless C. maltaromaticum UAL26. When pCB15s that was re-isolated from these C.
  • the colicin VM consists of 92 amino acids instead of the 88 amino acids that constitute colicin V ( Figure 5). Both C. maltaromaticum UAL26 and L reuteri CBA transformants containing pCB15s inhibited E. coli DH5 ⁇ indicating that colicin VM retains antibacterial activity against E. coli.
  • the host strains for use in this technology will be harmless or beneficial (probiotic) microorganisms that are commonly associated with the Gl tract of the target animal.
  • Post-weaning diarrhea (PWD) that causes morbidity or mortality of pigs. is an example of a Gl disease that can be prevented using this technology.
  • the efficacy of the transformed host strain, Lactobacillus reuteri CB4 containing pCB15s, producing colicin VM (colVM) to target enterotoxigenic Escherichia coli (ETEC) that cause post-weaning diarrhea (PWD) in pigs was determined.
  • the organism was tested in an established pig infection model. Efficacy of the preventative treatment is measured by reduction of PWD and normal weight gain of the weanling pigs.
  • piglets Twenty 17-day-old weaned piglets were divided into two groups of 10 pigs. Group 1 was untreated and Group 2 was treated by administration of approximately 1 x 10 9 L reuteri CB4, containing pCB15s in the drinking water from Day 1 to Day 9 of the experiment. On Day 7 both groups were challenged with approximately 5 x 10 8 of an ETEC-F4 strain (known to cause PWD), administered by oesophageal tube. In the model the presence of F4 receptor-positive animals (those specifically susceptible to colonization by the ETEC-F4 strain) were selected for separate analysis. Health of the experimental animals was monitored and on Day 10 they pigs were euthanized for necropsy.
  • ETEC-F4 strain known to cause PWD
  • test organism The effect of the test organism was measured by analysis of weight gain, the diarrhea score, consistency of the intestinal contents and colonization of the ileum by the challenge strain at the day of necropsy.
  • a feed-grade vector is a plasmid that lacks or contains a truncated antibiotic resistance genes and uses an alternate selection system, such as a bacteriocin immunity gene, for animal feed applications.
  • pCB15 a derivative of pCB15, named pCB21 ( Figure 6) was made that has unique EcoRV and BstEW restriction sites in the cat gene.
  • an EcoFN restriction site located immediately downstream of the brochocin-C immunity gene of pCB15 was removed by the following procedure: a 40-mer oligonucleotide (5' - ATA TAT CGA TAG GAA GTA TGA TCA ATG GTAAAAACTATA C - 3') (Seq. I. D. No. 14) described in Example 2, and a 27-mer oligonucleotide (5' - ATA TCT GCA GTC TAG TTA GAG AATATA- 3') (Seq. I. D. No.
  • UAL26 transformants were selected by plating on APT plates containing 20% heat-treated (100 0 C for 5 min) spent supernatant from a culture of Brochothrix campestris ATCC 43754 grown in APT medium.
  • the resulting UAL26 transformants contained plasmid pCB22 ( Figure 7) and were sensitive to chloramphenicol and produced colicin V.
  • maltaromaticum UAL26 containing pCB23M inhibited E. coli DH5 ⁇ was immune to brochocin-C and sensitive to chloramphenicol.
  • Plasmid pCB23M was isolated from C. maltaromaticum UAL26 and transferred by electroporation into L. reuteri CB4 using 4000 AU/ml of brochocin-C as selection agent.
  • Transformants of CB4 containing pCB23M were sensitive to chloramphenicol, immune to brochocin-C and inhibited growth of the indicator organism E. coli DH5 ⁇ . This result showed that we obtained a strain of L reuteri CB4 that inhibited E. coli using a feed-grade plasmid.
  • a cloning vector pCB19 based on pCaT was constructed by introducing a multiple cloning site that can be used to clone DNA fragments of interest.
  • A4.6-kb Sph ⁇ -Pst ⁇ DNA fragment from pCaT that contains the open reading frames that could encode proteins involved in horizontal transfer of plasmids as well as the streptomycin resistance gene was replaced by a polylinker (5'-GCATGC GAATTC GAG CTC GGT ACC CGG GGATCC TCC TGC AG-3') (Seq. I. D. No. 16) that contains multiple cloning sites (Figure 9).
  • the resulting 4.3-kb plasmid, pCB19 can be selected when transformed into lactic acid bacteria using the chloramphenicol resistance gene (cat).
  • This plasmid has been transformed into lactic acid bacteria such as C. maltaromaticum and L. reuteri.
  • Other selection markers including, but not limited to, bacteriocin immunity genes can be cloned into the multiple cloning sites of pCB19.
  • the inventors have demonstrated that genes encoding proteins such as bacteriocins can be cloned into the multiple cloning sites of pCB19 resulting in export of the recombinant proteins by the lactic acid bacteria.
  • a promoter from the chromosomal DNA of C. maltaromaticum LV17 was cloned. Chromosomal DNA was isolated by the inventors from C. maltaromaticum LV17, digested completely with the restriction enzyme Mbo ⁇ and cloned into the promoter screening vector pGKV210 (van der Vossen et al., 1985). The ligation mixture was transferred by electroporation into C maltaromaticum UAL26 and transformants were selected on APT agar plates containing 20 ⁇ g of chloramphenicol per ml.
  • pGKV210-P15 One such transformant obtained, designated as pGKV210-P15, grew on APT plates with chloramphenicol concentration as high as 45 to 50 ⁇ g/ml.
  • the promoter in pGKV210 that was isolated from C. maltaromaticum LV17 was labeled P15.
  • a pair of primers MP11 forward primer 5' GAATTCGAGCTCGCCCGG 3' (Seq. I. D. No. 17) containing a EcoR ⁇ restriction site (underlined) and reverse primer 5' CTGCAGGTCGACTCTAGAG 3' (Seq. I. D. No. 18), were used to amplify the insert containing the P15 promoter from pGKV210-P15.
  • the sequence of the fragment containing the P15 promoter was determined and showed to contain 276 nucleotides ( Figure 10).
  • the two PCR products from above were used as templates and the primers MP11 forward and C were used for recombinant PCR to amplify the fragment containing the DNA from both PCR products.
  • the resulting PCR product contains P15 promoter, in front of DNA encoding colicin V fused to the signal peptide of divergicin A.
  • Plasmid pCB101 was transferred by electroporation into C. maltaromaticum UAL26.
  • the strain containing pCB101 inhibited the growth of colicin V indicator strain E. coli DH5 ⁇ .
  • Primer (5' GTAACTCTAGAAGGAAGTATGATCAATGGTA 3') (Seq. I. D. No. 23) containing a Xba ⁇ site (underlined) and primer (5' TATCTG CAGTCTAGTTAG AG AATAT AATCCA 3') (Seq. I. D. No. 24) containing a Pst ⁇ site (underlined) were used to amplify the brochocin-C immunity gene using pCB15 DNA as the template.
  • the PCR product was inserted into the appropriate sites of pCB101 , giving the plasmid pCB103 ( Figure 13).
  • pCB103 was transformed into C. maltaromaticum UAL26, the strain containing the plasmid inhibited the growth of E. coli DH5 ⁇ and was resistant to chloramphenicol.
  • plasmid pCB103 was digested with the unique restriction enzyme sites EcoRV and BsO, which are located within the cat gene, to remove most of the cat gene. The linear fragment was blunted by DNA polymerase I, self-ligated and transformed into C. maltaromaticum UAL26.
  • the resulting feed-grade plasmid pCB104 contains the DNA encoding the signal peptide of divergicin A, fused to colicin V, and brochocin-C immunity, under control of the P15 promoter ( Figure 14).
  • C. maltaromaticum UAL26 containing pCB104 was selected on APT agar plates containing 80 AU of brochocin-C per ml.
  • C. maltaromaticum UAL26 containing plasmids pCB101 , pCB103 and pCB104 all produced a bacteriocin at a similar level that inhibited the growth of E.coli DH5 ⁇ .
  • C. maltoromaticum UAL26 containing pCB104 showed resistance to brochocin-C, but sensitivity to chloramphenicol.
  • promoter P15 to express the production of colicin VM in a feed-grade vector
  • the resulting plasmid pCB110 is feed-grade vector containing P15 promoter and signal peptide of divergicin A fused to co/VM ( Figure 15).
  • a feed-grade plasmid, designated as pCB111 was constructed by replace the P32 promoter in pCB23M with P15 promoter.
  • Plasmid pCB111 is similar to pCB23M except it has the P15 promoter instead of P32 promoter.
  • maltatomaticum UAL26 containing pCB110 or pCB111 shows activity against E. coli, sensitivity to chloramphenicol, and resistance to brochocin C.
  • Plasmid pCB110 and pCB111 were transformed into L reuteri CB4.
  • Strain L. reuteri CB4 containing pCB110 or pCB111 inhibited the growth of E. coli, was sensitive to chloramphenicol and resistant to brochocin C.
  • Colicin V can be produced by lactic acid bacteria. Lett. Appl. Microbiol. 29: 37-41. Sambrook, J., Fritsch, E. R, and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual, 2 nd edn. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.

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Abstract

La présente invention concerne des préparations et des méthodes pour la synthèse de polypeptides antibactériens, ainsi que l'utilisation de ces préparations et méthodes dans le traitement de maladies et d'états pathologiques causés par une infection bactérienne. Plus spécifiquement, lesdites préparations et méthodes incluent le traitement d'une bactérie Gram-négative à l'aide d'un hôte Gram-positif produisant un polypeptide actif contre ladite bactérie Gram-négative.
PCT/CA2004/002127 2004-12-14 2004-12-14 Vecteur d'expression pour le traitement d'infections bactériennes Ceased WO2006063425A1 (fr)

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WO2009011940A3 (fr) * 2007-04-06 2009-09-11 University Of Maryland, Baltimore Système de sélection de plasmide de la microcine h47
CN103289944A (zh) * 2012-03-01 2013-09-11 深圳市圣西马生物技术有限公司 一种分泌抗菌肽的乳酸链球菌基因工程制备方法与应用
CN110791509A (zh) * 2019-11-18 2020-02-14 深圳市圣西马生物技术有限公司 片球菌素优化表达序列、表达载体及其制作方法和菌株
CN119242550A (zh) * 2024-12-06 2025-01-03 吉林农业大学 表达etec抗原的新功能型乳酸菌及其制备方法和应用

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US6403082B1 (en) 1996-09-05 2002-06-11 Michael E. Stiles Bacteriocins, transport and vector system and method of use thereof

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WO1999002555A1 (fr) * 1997-07-09 1999-01-21 The Governors Of The University Of Alberta Nouvelles bacteriocines, leur transport et systeme de vecteurs et procede d'utilisation correspondants

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US6403082B1 (en) 1996-09-05 2002-06-11 Michael E. Stiles Bacteriocins, transport and vector system and method of use thereof

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009011940A3 (fr) * 2007-04-06 2009-09-11 University Of Maryland, Baltimore Système de sélection de plasmide de la microcine h47
CN103289944A (zh) * 2012-03-01 2013-09-11 深圳市圣西马生物技术有限公司 一种分泌抗菌肽的乳酸链球菌基因工程制备方法与应用
CN110791509A (zh) * 2019-11-18 2020-02-14 深圳市圣西马生物技术有限公司 片球菌素优化表达序列、表达载体及其制作方法和菌株
CN110791509B (zh) * 2019-11-18 2023-09-08 深圳市圣西马生物技术有限公司 片球菌素优化表达序列、表达载体及其制作方法和菌株
CN119242550A (zh) * 2024-12-06 2025-01-03 吉林农业大学 表达etec抗原的新功能型乳酸菌及其制备方法和应用

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