EP1210365A2 - Methodes et compositions destinees a restaurer la sensibilite aux antibiotiques des enterocoques resistants aux glycopeptides - Google Patents

Methodes et compositions destinees a restaurer la sensibilite aux antibiotiques des enterocoques resistants aux glycopeptides

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Publication number
EP1210365A2
EP1210365A2 EP00955471A EP00955471A EP1210365A2 EP 1210365 A2 EP1210365 A2 EP 1210365A2 EP 00955471 A EP00955471 A EP 00955471A EP 00955471 A EP00955471 A EP 00955471A EP 1210365 A2 EP1210365 A2 EP 1210365A2
Authority
EP
European Patent Office
Prior art keywords
molecule
vancomycin
sense
vana
organism
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00955471A
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German (de)
English (en)
Inventor
Roger T. Inouye
Carlos Torres-Viera
Robert Moellering
Howard Gold
George M. Eliopoulos
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Beth Israel Deaconess Medical Center Inc
Original Assignee
Beth Israel Deaconess Medical Center Inc
Beth Israel Hospital Association
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Application filed by Beth Israel Deaconess Medical Center Inc, Beth Israel Hospital Association filed Critical Beth Israel Deaconess Medical Center Inc
Publication of EP1210365A2 publication Critical patent/EP1210365A2/fr
Withdrawn legal-status Critical Current

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    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • 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
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it

Definitions

  • This invention relates to methods for reducing antibiotic resistance in vancomycin resistant bacteria.
  • Enterococci are Gram-positive cocci which, prior to DNA homology studies, were classified as Lancefield group D streptococci (Moellering, R.C. Jr., In:Mandell GL, Bennett JE and Dolin R eds. Principles and Practices of Infectious Diseases. New York:Churchhill Livingstone. 1995:1826-1835). While these organisms are known constituents of the gastrointestinal and genital tract bacterial flora, enterococci have rapidly emerged as clinically relevant pathogens especially in the nosocomial setting.
  • enterococci are the second most common cause of nosocomial infections in the United States as well as a frequent cause of nosocomial bacteremia (Eliopoulos, G.M., Infect. Dis. Clin. North. Am. 1997;11:851-65); Schaberg, et al., Am. J. Med, 1991:91(3B):72S-85S).
  • the mortality attributable to vancomycin resistant enterococcal bacteremia has been estimated to approach 25% in some studies (Edmond, et al., Clin. Infect. Dis., 1996;23:1234-1239). Vancomycin Mechanism-of-Action
  • vancomycin a glycopolypeptide antibiotic
  • beta-lactam antibiotic-resistant Gram-positive bacterial infections Fekety, et al., In: Mandell, et al. Principles and Practices of Infectious Diseases. New York:Churchhill Livingstone, 1995;346-354.
  • vancomycin While other ancillary mechanisms-of- action continue to be investigated, the major mechanism of vancomycin is the inhibition of polymerization and transpeptidation of the bacterial cell wall peptidoglycan (Ge, et al., Science 1999;284:507-11).
  • VanA Class A glycopeptide resistance
  • Enterococcus faecalis and Enterococcus faecium species, and is characterized by high-level vancomycin resistance with MICs > 64 ⁇ g/mL as well as resistance to teicoplanin, a related glycopeptide antibiotic (Eliopoulos, G.M., Infect. Dis. Clin. North. Am. 1997;11 :851-65).
  • the genotypic characterization of Class A vancomycin resistance has uncovered potential targets for gene-based anti-drug resistance determinant strategy.
  • the genetic basis for VanA phenotypic resistance is a transposon-based operon consisting of 7 genes including vanR, vanS, vanH, vanA, vanX, vanY, and vanZ (Arthur, et al., Antimicrob. agent Chemother., 1993;37:1563-1571; Bugg, et al., 5/oc/zem., 1991;30:2017-2021) ( Figure 1).
  • the products of these genes function in concert to negate the inhibitory effects of vancomycin by, in essence, allowing for an alternate biosynthetic pathway for the production of cell wall precursors which less avidly bind vancomycin.
  • vanH, -A, and -X are under the control of the vanH promoter.
  • This promoter is inducible by the binding of the phosphorylated gene product of vanR (Arthur, et al., J. Bacteriol, 1992;174:2582-2591; Holman, et al., Biochem., 1994;33:4625-4631).
  • nucleic acid binding decoys antisense nucleic acids (antisense RNA and DNA), ribozymes, and trans-dominant mutants are among the many gene therapy motifs which have been used to target the expression of key viral functions in human immunodeficiency virus, type 1 ; human papilloma virus; hepatitis viruses, and Herpesviridae infections (Chatterjee, et al., Science, 1992, 258:1485-1488; Weiss, et al., Cell. Mol. Life.
  • a cornerstone of a successful gene-based tactic is that the target nucleic acid sequence encode for pivotal, highly conserved pathogenic functions.
  • antisense nucleic acids for example, have also been specifically used to inhibit the expression of key viral or cellular functional proteins including the expression of drug resistance determinants (Gao, et al., Anticancer Res., 1998, 18:3073-3076; Inouye, et al., Antiviral Therapy, 1999, 4 (Supplement 1): 121).
  • examples of gene-based strategies in prokaryotic systems are scant (Takada-Guerrier, et al., Proc. Natl. Acad.
  • the invention overcomes the above-noted and other problems of the prior art by providing methods and related compositions for reducing antibiotic resistance in vancomycin resistant microorganisms. More particularly, the present invention provides a gene cassette comprised of the v ⁇ nH promoter and a single copy of a v ⁇ nA antisense gene in an enterococcal shuttle vector. Using this invention, we have demonstrated an ability to increase the vancomycin susceptibility in previously resistant Enterococcus f ⁇ ec ⁇ lis. According to one aspect of the invention, a method for reducing vancomycin resistance in a vancomycin-resistant organism is provided.
  • the method involves introducing into the organism at least one "anti-sense vancomycin resistance molecule" under conditions to inhibit expression of a vancomycin resistance gene.
  • inhibit expression it is meant to inhibit replication, transcription, and/or translation of a vancomycin gene since inhibition of any of these processes results in the inhibition of expression of a protein encoded by a vancomycin gene.
  • Exemplary vancomycin-resistant organisms include the Gram-positive bacteria Enterococcus faecium and Enterococcus faecalis and other bacteria to which these organisms have the potential of transferring resistance determinants, given that VanA is a transferable form of resistance and that it could be transferred to other clinically significant pathogens such as Streptococcus Pneumococcus, and Staphylococcus. (See, e.g., Brisson- Noel A., et al., J. Bacteriol, 1988, 170:1739-1745).
  • the vancomycin resistant organism is a Gram-positive bacteria and, more preferably, the organism is an Enterococcus.
  • Vancomycin resistance can take a variety of forms depending upon the nature of the gene cluster which mediates the resistance phenotype.
  • exemplary vancomycin resistant organisms of the invention may exhibit one or more of the following phenotypes: VanA resistance, VanB resistance, VanC resistance, and VanD resistance.
  • VanA resistance is mediated by a gene cluster which includes seven genes: vanR (SEQ ID NO: 18), vanS (SEQ ID NO:19), vanH (SEQ ID NO:20), vanA (SEQ ID NO:21), vanX (SEQ ID NO:22), vanY (SEQ ID NO:23), and vanZ (SEQ ID NO:24).
  • the antisense vancomycin resistance molecule is selected from the group consisting of antisense molecules which hybridize under stringent conditions to these target genes or to conserved regions of these target genes (e.g., SEQ ID NOS: 5, 6, 7, 8, 9, and 10).
  • antisense molecules to these target genes are referred to as vanR antisense molecules, vanS anti-sense molecules, vanH anti-sense molecules, vanA anti-sense molecules, vanX anti-sense molecules, van Y anti-sense molecules, and vanZ anti-sense molecules, respectively.
  • the organism is a VanA type, and the anti-sense vancomycin resistance molecule hybridizes under stringent conditions to the vanA target gene (SEQ ID NO:21), or to a conserved region of the vanA gene (e.g., SEQ ID NOs: 7, and 8).
  • the organism is a VanA type, and the anti-sense vancomycin resistance molecule hybridizes under stringent conditions to the vanX target gene (SEQ ID NO:22), or to a conserved region of the vanX gene (e.g., SEQ ID NO: 10).
  • the vancomycin resistant organism can be a VanB, VanC, and/or VanD type organism and the anti-sense vancomycin resistance molecule is a nucleic acid molecule which hybridizes under stringent conditions to these target genes (SEQ ID NO:2 is the vanB gene cluster sequence; SEQ ID NO:3 is the vanC gene sequence; SEQ ID NO:4 is the vanD gene cluster sequence) or to conserved regions of these target genes (e.g., SEQ ID NOS: 1 1, 12, and 13).
  • the antisense molecules which hybridize to a conserved region of a target vancomycin resistance gene contain from about 18 to about 1500 nucleotides, more preferably from about 10 to about 30 nucleotides, and most preferably from about 20 to about 30 nucleotides.
  • the anti-sense vancomycin resistance molecules are introduced to the organism by contacting the vancomycin resistant organism with at least one cassette (typically contained in a vector) comprising one or more "anti-sense vancomycin resistance molecules" under conditions to allow the vector to enter the organism and inhibit expression of one or more vancomycin resistance genes.
  • the vector comprises an expression cassette which permits expression of the anti-sense vancomycin resistance molecules in the organism.
  • the preferred vectors are selected from the group consisting of: an enterococcal shuttle vector (e.g., see the Examples), an enterococcal bacteriophage (Merril CR, et al., Proc Natl Acad Sci USA, 1996, 93:3188-92); the nucleic acid portion of a peptide nucleic acid molecule (Good L, et al., Nat Biotechnol, 1998; 16:355-8); an enterococcal conjugative transposon or pheromone-responsive plasmid (Murray BE, Emerg Infect Dis, 1998, 4:37-47).
  • an enterococcal shuttle vector e.g., see the Examples
  • an enterococcal bacteriophage Merril CR, et al., Proc Natl Acad Sci USA, 1996, 93:3188-92
  • the nucleic acid portion of a peptide nucleic acid molecule Good L, et al., Nat Biotech
  • the cassette contains one or more copies of a vanA antisense molecule operatively coupled to a promoter, preferably, the same inducible promoter which drives expression of the vanH, vanA, and vanX resistance determinant, e.g., a F ⁇ #-responsive promoter such as the vanH promoter.
  • a ⁇ «R-responsive refers to a promoter which activates transcription in response to binding of a phosphorylated VanR protein.
  • the F ⁇ «i?-responsive promoter is a vanH promoter (P v _ m ⁇ ) or a vanR promoter (P va « «), each of which directs transcription of the genes of the vancomycin resistance operon found in several species.
  • These F «i?-responsive promoters activate transcription in response to binding of an activated VanR protein.
  • These promoters include, in addition to the VanR binding sites, all other sequences required for efficient transcriptional activation of the gene or genes located downstream of the promoters.
  • these VanR- responsive promoters include the 60 nucleotides immediately upstream (nucleotides -60 to -1) of the genes encoding a VanR protein or a VanR protein, which sequences include a VanR binding site, and other sites which contribute to efficient VanR- responsive activation of gene transcription.
  • VanR -responsive promoters can be used to effect transcription of protein coding sequences.
  • alternative Fi /rl-responsive promoters can be identified by searching databases of bacterial nucleotide sequences for sequences which have VanR binding sites in proximity to sites which contribute to efficient bacterial transcriptional activation, e.g. a consensus binding site for bacterial DNA polymerase. Such sites are well-known to one of ordinary skill in the art.
  • F ⁇ rcic-responsive promoters can also be identified by genetic screening and cloning protocols that are standard in the art, as described in Sambrook.
  • non-natural VanR promoters can be prepared by combining a VanR binding site with the other nucleotide sequences which contribute to efficient bacterial transcriptional activity.
  • Such synthetic or non-natural F ⁇ rcR-responsive promoters can be synthesized directly by chemical means, such as by use of an automated DNA synthesizer.
  • the expression cassette contains one or more copies of a different vancomycin resistance antisense molecule operatively coupled to a promoter which drives expression of the targeted antisense gene.
  • an alternative method for reducing vancomycin resistance involves enhancing expression of a VanR- ⁇ espon ⁇ ve promoter, such as a vanH promoter, in the organism to an amount sufficient to reduce vancomycin resistance in the organism, wherein the vanH promoter is not operatively coupled to a vancomycin resistance gene of the organism.
  • a vancomycin resistance gene of the organism refers to the gene in its native configuration contained within the genome of the organism, i.e., not isolated from the organism.
  • the vanH promoter is operatively coupled to an antisense vancomycin resistance molecule, such as a vanA anti-sense molecule.
  • the vanH promoter (alone or operatively coupled to an antisense vancomycin resistance molecule) is contained in a cassette.
  • the cassette is contained in a vector to facilitate transport into and out of the resistant organism.
  • the vector is an enterococcal vector and enhancing expression of the vanH promoter involves introducing the vector into the organism.
  • introducing the vector into the organism results in expression of an amount of the vanH promoter sufficient that is sufficient to bind to phosphorylated VanR and thereby reduce vancomycin resistance in the organism.
  • the VanR -responsive promoter such as a vanH promoter is co-administered into the organism together with an antisense vancomycin resistance molecule operatively coupled to a vanH promoter.
  • compositions for use in accordance with the methods of the invention are provided.
  • the compositions of the invention are isolated nucleic acids that hybridize under stringent conditions to a targeted vancomycin gene or a conserved region thereof, such as described in more detail below.
  • the isolated nucleic acid is vancomycin resistance gene sequence which has been cloned in the opposite direction (see, e.g., the Examples).
  • Exemplary target genes and conserved regions thereof include the genes which are contained in the VanA resistance gene cluster (GenBank Accession No. M97297, SEQ ID NO:l), the VanB resistance gene cluster (GenBank Accession No.
  • the anti- sense molecules of the invention have sequences which are complementary, and therefore capable of hybridizing to the target genes identified herein, as well as to conserved and/or unique regions of these genes (e.g., by using routine skill to search nucleic acid databases such as GenBank to identify regions of the vancomycin resistance genes which are conserved and/or which are unique).
  • the anti-sense molecules of the invention hybridize to regions of the target gene which encode an active site or other which encodes an active site or other functional portion of the encoded protein (e.g., the active site of the ligase encoded by the vanA gene).
  • regions of the target gene which encode an active site or other which encodes an active site or other functional portion of the encoded protein (e.g., the active site of the ligase encoded by the vanA gene).
  • vanRIM.91291 4258 to 4287 5'ggcgcggatgattatataacgaagcccttt-3' 7 vanAM91291 7719 to 7736 5 ' -cgagccggaaaaggctc-3 '
  • anti-sense molecules which contain a few nucleotide residues (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) which hybridize to either side of the above-identified conserved nucleotide regions are embraced within the meaning of the anti-sense molecules disclosed and claimed herein for use in accordance with the methods of the invention.
  • cassettes containing the isolated nucleic acids of the invention, as well as vectors containing such nucleic acids and/or cassettes also are provided.
  • the cassettes further comprise a vancomycin- inducible promoter (e.g., a F ⁇ raR-responsive promoter such as a vanH promoter) operatively coupled to one or more isolated nucleic acid molecules of the invention.
  • a vancomycin- inducible promoter e.g., a F ⁇ raR-responsive promoter such as a vanH promoter
  • isolated vancomycin resistant organisms containing any of the foregoing isolated nucleic acids, cassettes and/or vectors also are provided.
  • SEQ ID NO:l The nucleic acid encoding the VanA resistance gene cluster of Enterococcus faecium. GenBank accession number M97297.
  • SEQ ID NO:2 The nucleic acid encoding the VanB resistance gene cluster of Enterococcus faecalis. GenBank accession number U35369.
  • SEQ ID NO:3 The nucleic acid encoding the VanC resistance gene cluster of Enterococcus casseliflavus. GenBank accession number L29638.
  • SEQ ID NO:4 The nucleic acid encoding the VanD resistance gene cluster of
  • SEQ ID NO: 5 A conserved nucleic acid region of the vanS gene of the VanA gene cluster.
  • SEQ ID NO:6 A conserved nucleic acid region of the vanR gene of the VanA gene cluster.
  • SEQ ID NO: 7 A conserved nucleic acid region of the vanA gene of the VanA gene cluster (nucleotides 7719 to 7736).
  • SEQ ID NO:8 A conserved nucleic acid region of the vanA gene of the VanA gene cluster (nucleotides 7339 to 7358).
  • SEQ ID NO:9 A conserved nucleic acid region of the vanH gene of the VanA gene cluster.
  • SEQ ID NO: 10 A conserved nucleic acid region of the vanX gene of the VanA gene cluster.
  • SEQ ID NO: 11 ⁇ A conserved nucleic acid region of the vanB gene cluster (nucleotides 5708 to 5725).
  • SEQ ID NO: 12 A conserved nucleic acid region of the vanB gene cluster (nucleotides 5328 to 5347).
  • SEQ ID NO: 13 A conserved nucleic acid region of the vanD gene cluster.
  • SEQ ID NO: 14 A 5' -PCR primer oligonucleotide sequence for the vanH promoter, used in conjunction with the primer of SEQ ID NO: 15.
  • SEQ ID NO: 15 A 3' -PCR primer oligonucleotide sequence for the vanH promoter, used in conjunction with the primer of SEQ ID NO: 14.
  • SEQ ID NO: 16 A 5' -PCR primer oligonucleotide sequence for the vanA gene, used in conjunction with the primer of SEQ ID NO: 17.
  • SEQ ID NO: 17 A 3' -PCR primer oligonucleotide sequence for the vanA gene, used in conjunction with the primer of SEQ ID NO: 16.
  • SEQ ID NO: 18 The nucleotide sequence of the vanR gene of the VanA gene cluster (SEQ ID NO: 1).
  • SEQ ID NO: 19 The nucleotide sequence of the vanS gene of the VanA gene cluster (SEQ ID NO: 1).
  • SEQ ID NO:20 The nucleotide sequence of the vanH gene of the VanA gene cluster (SEQ ID NO: 1).
  • SEQ ID NO:21 The nucleotide sequence of the vanA gene of the VanA gene cluster
  • SEQ ID NO:22 The nucleotide sequence of the vanX gene of the VanA gene cluster (SEQ ID NO: 1).
  • SEQ ID NO:23 The nucleotide sequence of the vanY gene of the VanA gene cluster (SEQ ID NO: 1).
  • SEQ ID NO:24 The nucleotide sequence of the vanZ gene of the VanA gene cluster (SEQ ID NO: 1).
  • SEQ ID NO:25 A 3' -PCR primer oligonucleotide sequence for the vanA gene, used in conjunction with the primer of SEQ ID NO: 16.
  • SEQ ID NO:26 The nucleotide sequence of the vanRB gene of the VanB gene cluster (SEQ ID NO:2).
  • SEQ ID NO:27 The nucleotide sequence of the vanSB gene of the VanB gene cluster (SEQ ID NO:2).
  • SEQ ID NO:28 The nucleotide sequence of the vanYB gene of the VanB gene cluster (SEQ ID NO:2).
  • SEQ ID NO:29 The nucleotide sequence of the vanHB gene of the VanB gene cluster (SEQ ID NO:2).
  • SEQ ID NO:30 The nucleotide sequence of the vanB gene of the VanB gene cluster
  • SEQ ID NO:31 The nucleotide sequence of the vanXB gene of the VanB gene cluster (SEQ ID NO:2).
  • SEQ ID NO:32 The nucleotide sequence of the van W gene of the VanB gene cluster (SEQ ID NO:2).
  • SEQ ID NO:33 The nucleotide sequence of the vanC-2 gene of the VanC gene cluster (SEQ ID NO:3).
  • SEQ ID NO:34 The nucleotide sequence of the vanRD gene of the VanD gene cluster (SEQ ID NO:4).
  • SEQ ID NO:35 The nucleotide sequence of the vanSD gene of the VanD gene cluster (SEQ ID NO:4).
  • SEQ ID NO:36 The nucleotide sequence of the vanYD gene of the VanD gene cluster (SEQ ID NO:4).
  • SEQ ID NO:37 The nucleotide sequence of the vanHD gene of the VanD gene cluster (SEQ ID NO:4).
  • SEQ ID NO:38 The nucleotide sequence of the vanD gene of the VanD gene cluster (SEQ ID NO:4).
  • SEQ ID NO:39 The nucleotide sequence of the vanXD gene of the VanD gene cluster (SEQ ID NO:4).
  • FIG. 1 A schematic showing the organization of genes in the VanA vancomycin resistance operon.
  • FIG. 2 A shows the parent vector, pAM401
  • Fig. 2B shows the restriction sites for the varnH promoter insertion into pAM401
  • Fig. 2C shows the restriction sites for the vanA antisense insertion into v ⁇ nHpromoter/pAM401 construct.
  • Figure 3 A schematic showing the proposed nucleic acid binding decoy mechanism with the introduction of a shuttle vector carrying the vanH promoter alone.
  • Figure 4 A schematic of the proposed mechanism-of-action of the pAM401-v ⁇ nH promoter-v ⁇ « ⁇ antisense recombinant shuttle vector.
  • vancomycin has been the mainstay of treatment for beta-lactam antibiotic- resistant enterococci
  • the increasing prevalence of vancomycin-resistant enterococci has prompted a continued search for new therapeutic approaches.
  • gene transfer has been used to define molecular pathogenesis as well as applied towards therapeutic ends.
  • the elucidation of the genetic basis for vancomycin resistance has uncovered potential targets for a unique anti-drug resistance gene-based strategy.
  • the preferred embodiments of the present invention consist of a gene cassette comprised of the enterococcal vanH promoter and a single copy of a vanA antisense gene in the shuttle vector, pAM401. Using this invention, we have demonstrated the ability to increase the vancomycin susceptibility of a vancomycin-resistant Enterococcus faecalis by up to 32-fold.
  • a method for reducing vancomycin resistance in a vancomycin-resistant organism involves introducing into the organism at least one "anti-sense vancomycin resistance molecule" under conditions to inhibit expression of a vancomycin resistance gene.
  • reducing vancomycin resistance refers to enhancing the susceptibility of a vancomycin resistant organism to vancomycin to a statistically significant extent.
  • the methods of the invention have been used to increase the vancomycin susceptibility of a vancomycin-resistant Enterococcus faecalis by at least about 16-fold and up to about 32-fold compared to organisms which have not been so treated.
  • the methods involve inhibiting expression of a vancomycin resistance gene.
  • inhibit expression refers to inhibiting (i.e., reducing to a detectable extent) replication, transcription, and/or translation of a vancomycin gene since inhibition of any of these processes results in the inhibition of expression of a protein encoded by a vancomycin gene.
  • Exemplary vancomycin-resistant organisms include the Gram-positive bacteria Enterococcus faecium and Enterococcus faecalis and other bacteria to which these organisms have the potential of transferring resistance determinants, given that VanA is a transferable form of resistance and that it could be transferred to other clinically significant pathogens such as Streptococcus species Pneumococcus, and Staphylococcus species.
  • VanA is a transferable form of resistance and that it could be transferred to other clinically significant pathogens
  • Streptococcus species Pneumococcus and Staphylococcus species.
  • Brisson-Noel A. Arthur, M. Courvalin P. "Evidence for natural gene transfer from Gram-positive cocci to Escherichia coli? J. Bacteriol 170:1739-1745, 1988).
  • the vancomycin resistant organism is a Gram-positive bacteria and, more preferably, the organism is an Enterococcus.
  • Vancomycin resistance can take a variety of forms depending upon the nature of the gene(s) which mediates the resistance phenotype.
  • exemplary vancomycin resistant organisms of the invention may exhibit one or more of the following phenotypes: VanA resistance, VanB resistance, VanC resistance, and VanD resistance.
  • VanA resistance is mediated by a gene cluster (SEQ ID NO:l) which includes seven genes: vanR (SEQ ID NO: 18), vanS (SEQ ID NO: 19), v ⁇ «H(SEQ ID NO:20), vanA (SEQ ID NO:21), vanX (SEQ ID NO:22), vanY (SEQ ID NO:23), and vanZ (SEQ ID NO:24), as described in GenBank Accession No.
  • VanB resistance is mediated by a gene cluster which includes seven genes: vanRB (SEQ ID NO:26), vanSB (SEQ ID NO:27), vanYB (SEQ ID NO:28), vanHB (SEQ ID NO:29), vanB (SEQ ID NO:30), vanXB (SEQ ID NO:31), and vanW (SEQ ID NO:32), as described in GenBank Accession No. U35369 (SEQ ID NO:2); VanC resistance is mediated by a vanC-2 gene (SEQ ID NO:33), as described in GenBank Accession No.
  • VanD resistance is mediated by a gene cluster which includes at least six genes: vanRD (SEQ ID NO:34), vanSD (SEQ ID NO:35), vanYD (SEQ ID NO:36), vanHD (SEQ ID NO:37), vanD (SEQ ID NO:38), and vanXD (SEQ ID NO:39), as described in GenBank Accession No. AF130997 (SEQ ID NO:4).
  • vanRD SEQ ID NO:34
  • vanSD SEQ ID NO:35
  • vanYD SEQ ID NO:36
  • vanHD SEQ ID NO:37
  • vanD SEQ ID NO:38
  • vanXD SEQ ID NO:39
  • the antisense vancomycin resistance molecule is selected from the group consisting of antisense molecules which hybridize under stringent conditions to these target genes or to conserved, unique, or functionally important regions of these target genes as described above.
  • antisense molecules to these target genes are referred to as vanA anti- sense molecules, vanR antisense molecules, vanS anti-sense molecules, vanH anti-sense molecules, vanX anti-sense molecules, vanY anti-sense molecules, and vanZ anti-sense molecules, respectively.
  • the organism carries a VanA phenotype and the anti-sense vancomycin resistance molecule hybridizes under physiological conditions to the vanA target gene or to a conserved region of the vanA gene.
  • the vancomycin-resistant organism can be a VanB, VanC, and/or VanD resistant organism and the anti-sense vancomycin resistance molecule is selected which hybridizes under stringent conditions to these target genes (SEQ ID NO:2 is the VanB gene cluster sequence; SEQ ID NO:3 is the VanC gene sequence; SEQ ID NO:4 is the VanD gene cluster sequence) or to conserved regions of these target genes.
  • the antisense molecules are isolated molecules which hybridize to a conserved region of a target vancomycin resistance gene contain from about 18 to about 1500 nucleotides, more preferably from about 10 to about 30 nucleotides, and most preferably, from about 20 to about 30 nucleotides.
  • nucleic acid molecules described herein preferably are isolated.
  • isolated means: (i) amplified in vitro by, for example, polymerase chain reaction (PCR); (ii) recombinantly produced by cloning; (iii) purified, as by cleavage and gel separation; or (iv) synthesized by, for example, chemical synthesis.
  • An isolated nucleic acid is one which is readily manipulable by recombinant DNA techniques well known in the art.
  • nucleotide sequence contained in a vector in which 5' and 3' restriction sites are known or for which polymerase chain reaction (PCR) primer sequences have been disclosed is considered isolated but a nucleic acid sequence existing in its native state in its natural host is not.
  • An isolated nucleic acid may be substantially purified, but need not be.
  • a nucleic acid that is isolated within a cloning or expression vector is not pure in that it may comprise only a tiny percentage of the material in the cell in which it resides.
  • Such a nucleic acid is isolated, however, as the term is used herein because it is readily manipulable by standard techniques known to those of ordinary skill in the art.
  • An isolated nucleic acid as used herein is not a naturally occurring chromosome.
  • antisense oligonucleotide or “antisense” describes an oligonucleotide that is oligoribonucleotide, oligodeoxyribonucleotide, modified oligoribonucleotide, or modified oligodeoxyribonucleotide which hybridizes under physiological conditions to DNA comprising a particular gene or to a messenger RNA (mRNA) transcript of that gene and, thereby, inhibits the transcription of that gene and/or the translation of that mRNA.
  • mRNA messenger RNA
  • the antisense molecules are designed so as to interfere with transcription or translation of a target gene upon hybridization with the target gene or transcript.
  • the exact length of the antisense oligonucleotide and its degree of complementarity with its target will depend upon the specific target selected, including the sequence of the target and the particular bases which comprise that sequence. It is preferred that the antisense oligonucleotide be constructed and arranged so as to bind selectively with the target under the physiological conditions of the target organism, i.e., to hybridize substantially more to the target sequence than to any other sequence in the target cell under physiological conditions.
  • antisense oligonucleotides should comprise at least 10 and, more preferably, at least 15 consecutive bases which are complementary to the target, although in certain cases modified oligonucleotides as short as 7 bases in length have been used successfully as antisense oligonucleotides. (Wagner et al., Nature BiotechnoL 14:840-844, 1996).
  • the antisense oligonucleotides comprise a complementary sequence of 20-30 bases.
  • oligonucleotides may be chosen which are antisense to any region of the gene or mRNA transcripts, in preferred embodiments the antisense oligonuleotides correspond to N-terminal or 5' upstream sites such as translation initiation, transcription initiation or promoter sites.
  • 3 '-untranslated regions may be targeted. Targeting to mRNA splicing sites has also been used in the art but may be less preferred if alternative mRNA splicing occurs.
  • the antisense is targeted, preferably, to sites in which mRNA secondary structure is not expected (see, e.g., Sainio et al., Cell Mol. Neurobiol. 1994, 14(5):439-457) and at which proteins are not expected to bind.
  • the listed sequences may include cDNA sequences, one of ordinary skill in the art may easily derive the genomic DNA corresponding to the cDNA of a vancomycin resistance gene.
  • the present invention also provides for antisense oligonucleotides which are complementary to the genomic DNA corresponding to nucleic acids encoding vancomycin resistance proteins.
  • antisense to allelic or homologous cDNAs and genomic DNAs are enabled without undue experimentation.
  • Exemplary U.S. patents which describe and claim antisense molecules for reducing gene expression include U.S. Patent Nos. 5,734,039; 5,783,683; 5,859,229; 5,858,987; 5,919,677; and 5,916,807; the entire contents of which patents are incorporated in their entirety herein by reference.
  • the antisense oligonucleotides of the invention may be composed of "natural" deoxyribonucleotides, ribonucleotides, or any combination thereof. That is, the 5' end of one native nucleotide and the 3' end of another native nucleotide may be covalently linked, as in natural systems, via a phosphodiester internucleoside linkage.
  • These oligonucleotides may be prepared by art recognized methods which may be carried out manually or by an automated synthesizer. They also may be produced recombinantly by vectors.
  • the antisense oligonucleotides of the invention also may include "modified" oligonucleotides. That is, the oligonucleotides may be modified in a number of ways which do not prevent them from hybridizing to their target but which enhance their stability or targeting or which otherwise enhance their therapeutic effectiveness.
  • modified oligonucleotide as used herein describes an oligonucleotide in which (1) at least two of its oligonucleotides are covalently linked via a synthetic internucleoside linkage (i.e., a linkage rather than a phosphodiester linkage between the 5' end of one oligonucleotide and the 3' end of another nucleotide) and/or (2) a chemical group not normally associated with nucleic acids has been covalently attached to the oligonucleotide.
  • a synthetic internucleoside linkage i.e., a linkage rather than a phosphodiester linkage between the 5' end of one oligonucleotide and the 3' end of another nucleotide
  • a chemical group not normally associated with nucleic acids has been covalently attached to the oligonucleotide.
  • Preferred synthetic internucleoside linkages are phosphorothioates, alkylphosphonates, phosphorodithioates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, carbonates, phosphate triesters, acetamidates, carboxymethyl esters and peptides.
  • modified oligonucleotide also encompasses oligonucleotides with a covalently modified base and/or sugar.
  • modified oligonucleotides include oligonucleotides having backbone sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3' position and other than a phosphate group at the 5' position.
  • modified oligonucleotides may include a 2'-O- alkylated ribose group.
  • modified oligonucleotides may include sugars such as arabinose instead of ribose.
  • the present invention contemplates pharmaceutical preparations containing modified antisense molecules that are complementary to and hybridizable with, under physiological conditions, nucleic acids encoding vancomycin resistance polypeptides, together with acceptable carriers to deliver these molecules into the target organism.
  • compositions of the invention may be administered as part of a pharmaceutical composition to a mammal (e.g., humans, domestic animals, such as dogs, cats, livestock, such as horses, sheep, cows, pigs) hosting a vancomycin resistant organism.
  • a pharmaceutical composition may include the antisense oligonucleotides in combination with any standard physiologically and/or pharmaceutically effective amount of the antisense oligonucleotides in a unit of weight or volume suitable for administration to a patient.
  • pharmaceutically acceptable means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients.
  • physiologically acceptable refers to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism.
  • a biological system such as a cell, cell culture, tissue, or organism.
  • the characteristics of the carrier will depend on the rout of administration.
  • Physiologically and pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials which are well known in the art, as further described below.
  • compositions of the invention also may be introduced into vancomycin resistant organisms which is ex vivo, i.e., not contained within a mammal.
  • the applications of such compositions include both treatment of vancomycin-resistant enterococci or other clinically significant pathogen infections and colonization including, for example: (1) ex vivo eradication of vancomycin-resistant enterococci from frequently colonized settings (e.g., intensive care units, hemodialysis units, chronic care facilities); (2) in vivo clearance of vancomycin-resistant enterococci from colonized gastrointestinal or genitourinary tracts of human and animal subjects; and (3) primary or adjuvant therapy for vancomycin-resistant enterococcal infections.
  • antisense oligonucleotides e.g., a synthetic antisense DNA strand
  • the genes which code for antisense RNA e.g., by conjugation, transformation, or transduction with bacteriophage.
  • the antisense motif and other anti-resistance determinant genetic elements of the invention e.g., nucleic acid binding decoys, transdominant mutants, suicide genes, ribozymes etc.
  • a "vector" may be any of a number of nucleic acids into which a desired sequence may be inserted by restriction and ligation for transport between different genetic environments or for expression in a host organism.
  • Vectors are typically composed of DNA although RNA vectors are also available.
  • Vectors include, but are not limited to, plasmids, phagemids and virus genomes.
  • a cloning vector is one which is able to replicate autonomously or integrated in the genome or host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence by be ligated such that the new recombinant vector retains its ability to replicate in the host cell.
  • replication of the desired sequence may occur many times as the plasmid increases in copy number within the host bacterium or just a single time per host before the host reproduces by mitosis. In the case of phage, replication may occur actively during a lytic phase or passively during a lysogenic phase.
  • An expression vector is one into which a desired DNA sequence may be inserted by restriction and ligation such that it is operably joined to regulatory sequences and may be expressed as an RNA transcript. Vectors may further contain one or more marker sequences suitable for use in the identification of cells which have or have not been transformed or transfected with the vector.
  • Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes which encode enzymes whose activities are detectable by standard assays known in the art (e.g., ⁇ -galactosidase, luciferase or alkaline phosphatase), and genes which visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques (e.g., green fluorescent protein).
  • Preferred vectors are those capable of autonomous replication and expression of the structural gene products present in the DNA segments to which they are operably joined.
  • a coding sequence and regulatory sequences are said to be "operably” joined when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences.
  • two DNA sequences are said to be operably joined if induction of a promoter in the 5' regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein.
  • a promoter region would be operably joined to a coding sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript might be translated into the desired protein or polypeptide.
  • regulatory sequences needed for gene expression may vary between species or cell types, but shall in general include, as necessary, 5' non-transcribed and 5' non-translated sequences involved with the initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, and the like.
  • 5' non-transcribed regulatory sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined gene.
  • Regulatory sequences may also include enhancer sequences or upstream activator sequences as desired.
  • the vectors of the invention may optionally include 5' leader or signal sequences. The choice and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art.
  • RNA heterologous DNA
  • RNA heterologous DNA
  • That heterologous DNA (RNA) is placed under operable control of transcriptional elements to permit the expression of the heterologous DNA in the host bacterium.
  • the vancomycin resistance operons of a targeted organism include, e.g., the naturally occurring operon of Enterococcus faecium, or such operons which are substantially identical thereto, e.g., homologs of the vancomycin resistance operon of Enterococcus faecium from other species, functionally equivalent variant of the vancomycin resistance operon containing variants of the genes which constitute the naturally occurring operon.
  • Such variants may be sequence variants, e.g., containing conservative substitutions of amino acids and the like as defined herein, or may be different genes which have the same or a similar function as one of the genes found in the naturally-occurring vancomycin operon.
  • the ddlB gene of E for example, the ddlB gene of E.
  • a prefened vancomycin resistance operon of a targeted organism typically includes a v ⁇ wHgene, a ddlB gene and a vanX gene.
  • the VanA protein product has two activities: a D-Ala-D-hydroxybutyrate depsipeptide ligase activity (Bugg et al., Biochemistry 30:2017-2021, 1991). VanA shares 28% amino acid identity with an E. coli enzyme, DdlB, which is a D-Ala-D-Ala dipeptide ligase. Two point mutants of DdlB recently have been reported that exhibit depsipeptide ligase activity (S150A and Y126F; Shi & Walsh, Biochemistry 34:2768-2776, 1995; Park et al., Biochemistry, 1996, in press). Thus, these mutants appear to be functional homologs of VanA.
  • Other functional homologs include, for example, genes encoding a VanA or DdlB protein that are present in other vancomycin operons, including such genes present in other species which encode vancomycin resistance.
  • other vancomycin resistant strains of bacteria i.e., not Enterococci which have a VanA operon
  • Non-VanA vancomycin resistance operons such as the VanB vancomycin resistance operon, contain functionally equivalent VanA homologs.
  • Other functional homologs, either natural or non-natural, are also embraced by the invention.
  • the anti-sense vancomycin resistance molecules are introduced to the organism by contacting the vancomycin resistant organism with at least one cassette, preferably contained in a vector, which cassette comprises one or more "anti-sense vancomycin resistance molecules" operably coupled to a promoter (e.g., a VanR response promoter).
  • a promoter e.g., a VanR response promoter
  • the cassette is contacted with the organism under conditions which allow the cassette and/or vector to enter the organism and inhibit expression of one or more vancomycin resistance genes.
  • the vector comprises an expression cassette which permits expression of the anti-sense vancomycin resistance molecules in the organism.
  • the prefened vectors are selected from the group consisting of: an enterococcal shuttle vector (e.g., see the Examples), an enterococcal bacteriophage (Menil CR, Biswas B, Carlton R, Jensen NC, Creed GJ, Zullo S, Adhya S, "Long-Circulating Bacteriophage as Antibacterial Agents.” Proc Natl Acad Sci USA, 1996; 93:3188-92); the nucleic acid portion of a peptide nucleic acid molecule (Good L, Nielsen PE, "Antisense Inhibition of Gene Expression in Bacteria by PNA Targeting To mRNA," Nat Biotechnol 1998; 16:355-8); an enterococcal conjugative transposon or pheromone-responsive plasmid (Murray BE, "Diversity Among Multidrug-Resistant Enterococci,” Emerg Infect Dis 1998; 4:37-47).
  • an enterococcal shuttle vector e
  • the cassette contains one or more copies of a vanA antisense molecule, e.g., in tandem, operatively coupled to a promoter, preferably, the same inducible promoter which drives expression of the vanA resistance determinant, e.g., a F ⁇ «i?-responsive promoter such as the vanH promoter.
  • a F ⁇ «i?-responsive refers to a promoter which activates transcription in response to binding of an activated VanR protein.
  • These promoters include, in addition to the VanR binding site, all other sequences required for efficient transcriptional activation of the gene or genes located downstream of the promoters.
  • other embodiments can be prepared in which the expression cassette contains one or more copies of a different vancomycin antisense molecule operatively coupled to a promoter which drives expression of the targeted antisense gene.
  • an alternative method for reducing vancomycin resistance involves enhancing expression of a VanR -responsive promoter (e.g., a vanH promoter) in the organism to an amount sufficient to reduce vancomycin resistance in the organism, wherein the vanH promoter is not operatively coupled to a vancomycin resistance gene of the organism.
  • a Vancomycin resistance gene of the organism refers to the gene in its native configuration contained within the genome of the organism, i.e., not isolated from the organism or attached to nucleic acid which is not contained within the genome of the orgamsm.
  • the Fi ft-responsive promoter is operatively coupled to an antisense vancomycin resistance molecule, such as a vanA anti-sense molecule. More preferably, the VanR -responsive promoter (alone or operatively coupled to an antisense vancomycin resistance molecule) is contained in a cassette. Typically, the cassette is contained in a vector to facilitate transport into and out of the resistant organism. In a particularly prefened embodiment, the vector is an enterococcal vector and enhancing expression of the VanR- ⁇ esponsive promoter involves introducing the vector into the organism.
  • An exemplary cassette, vector and process for introducing the cassette into a vancomycin resistant organism and representative experimental evidence showing the efficacy of the claimed methods for reducing antibiotic resistance in a vancomycin resistant organism are described in the Examples.
  • VanR-responsive promoter e.g., a vanH promoter
  • compositions for use in accordance with the methods of the invention are provided.
  • the compositions of the invention are isolated nucleic acids that hybridize under stringent conditions to a targeted vancomycin gene or a conserved region thereof, such as described in more detail below.
  • the isolated nucleic acid is vancomycin resistance gene sequence which has been cloned in the opposite direction (see, e.g., the Examples).
  • Exemplary target genes and conserved regions thereof include the genes which are contained in the vanA resistance gene cluster (GenBank Accession No. M97297, SEQ ID NO:l), the vanB resistance gene cluster (GenBank Accession No.
  • the anti- sense molecules of the invention have sequences which are complementary, and therefore capable of hybridizing to the target genes identified herein, as well as to conserved and/or unique regions of these genes (e.g., by using routine skill to search nucleic acid databases such as GenBank to identify regions of the vancomycin resistance genes which are conserved and/or which are unique).
  • the anti-sense molecules of the invention hybridize to regions of the target gene which encode an active site or other which encodes an active site or other functional portion of the encoded protein (e.g., the active site of the ligase encoded by the vanA gene).
  • regions of the target gene which encode an active site or other which encodes an active site or other functional portion of the encoded protein (e.g., the active site of the ligase encoded by the vanA gene).
  • Applicants have identified the following nucleotide regions of representative target genes to which the anti-sense molecules can be designed to hybridize (i.e., the anti-sense molecules have complementary nucleotide sequences to the target genes or the selected regions).
  • anti-sense molecules which contain a few nucleotide residues (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) which hybridize to either side of the above-identified conserved nucleotide regions are embraced within the meaning of the anti-sense molecules disclosed and claimed herein for use in accordance with the methods of the invention.
  • stringent conditions refers to parameters with which the art is familiar. More specifically, stringent conditions, as used herein, refers to hybridization at 65°C in hybridization buffer (3.5 x SSC, 0.02% Ficoll, 0.02% polyvinyl pynolidone, 0.02% Bovine Serum Albumin, 2.5mM NaH 2 PO 4 (pH7), 0.5% SDS, 2mM EDTA).
  • SSC is 0.15M sodium chloride/0.15M sodium citrate, pH7; SDS is sodium dodecyl sulphate; and EDTA is ethylenediaminetetracetic acid.
  • the membrane upon which the DNA is transfened is washed at 2 x SSC at room temperature and then at 0.1 x SSC/0.1 x SDS at 65°C.
  • the cassettes further comprise a vancomycin- inducible promoter (e.g., a VanR-responsive promoter such as a vanH promoter) operatively coupled to one or more isolated nucleic acid molecules of the invention.
  • a vancomycin- inducible promoter e.g., a VanR-responsive promoter such as a vanH promoter
  • isolated vancomycin resistant organisms containing any of the foregoing isolated nucleic acids, cassettes and/or vectors also are provided.
  • Co-administering refers to administering simultaneously two or more compounds (constructs) of the invention (e.g., the ⁇ «y -responsive promoter, such as a vanH promoter, and an antisense vancomycin resistance molecule operatively coupled to a vanH promoter), as an admixture in a single composition, or sequentially, close enough in time so that the compounds may exert an additive or even synergistic effect, i.e., on reducing vancomycin resistance.
  • the ⁇ «y -responsive promoter such as a vanH promoter, and an antisense vancomycin resistance molecule operatively coupled to a vanH promoter
  • Plasmids The parent shuttle plasmid used in the test vector constructs was pAM401 (American
  • This plasmid is a high copy shuttle vector containing both Gram-negative bacillary (Eschericia coli) and enterococcal (Enterococcus faecalis) elements necessary for replication in these two bacterial types ( Figure 2). To aid in selection of appropriately transformed clones, this plasmid also contains tetracycline and chloramphenicol resistance genes.
  • the cloning vector, pAMPl (Gibco BRL, Rockville, MD), was also employed for the cloning of polymerase chain reaction-amplified fragments. Construction of Recombinant Enterococcal Shuttle Vectors
  • vanA was digested out of pAMPl-vanA antisense with Xho I and Sal I and cloned into the Sal I site in pAM401- vanHP in the anti-coding direction.
  • VanA phenotype clinical isolates obtained from E. Cercenada (Hospital General Gregorio Maran ⁇ n, Madrid, Spain).
  • A1221 is a VanA strain of Enterococcus faecium resulting from the transconjugation with a VanA strain of Enterococcus faecalis (A312) obtained from F. Tenover (Centers for Disease Control, Atlanta, GA).
  • These strains were identified as Enterococcus faecalis or faecium by the use of API-Rapid Strep Strips (bioMeriux Vitex, Inc., Hazelwood, MO).
  • the presence of the vanA genotype was confirmed by DNA probe analysis as previously described (Eliopoulos, et al., Antimicrob. Agents Chemother, 1998, 42:1088-92).
  • Vancomycin susceptibilities were determined by the National Committee for Clinical Laboratory Standards agar dilution method (National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard M7-A4. Wayne, PA: NCCCLS, 1997).
  • Commercially prepared competent DH5-alpha Eschericia coli (Gibco BRL, Rockville, MD) were also used in the cloning and sub-cloning of the vectors via a standard transformation protocol (Sambrook, et al., ln:Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. 1989; 1.74). Antibiotics, Culture Media, Cloning Reagents
  • Vancomycin and other antimicrobial agents were purchased from Sigma (St. Louis, MO). Restriction and modifying enzymes were obtained from Promega (Madison, WI) and New England BioLabs, Inc. (Beverly, MA). Eschericia coli were grown in Luria-Bertani medium and enterococci were grown on Mueller-Hinton or Blood-agar medium. Plasmid preparations were performed using Promega Wizard DNA Purification systems (Madison, WI).
  • vanH Promoter and vanA Antisense Construction An approximate 450 base-pair fragment containing the vanH promoter - previously described to be necessary for expression of vanH, -A, and -X - was amplified using genomic DNA from a known strain of VanA strain Enterococcus faecium (A 1221) as a template (Arthur, et al., J. Bacter., 1992, 174:2582-2591). 5' and 3' primers were synthesized by Gibco BRL (Rockville, MD). The primer sequences for the respective 5' and 3' vanH promoter primers as follows:
  • vanHP vanH promoter
  • the vanA gene was amplified using the following primer pair and subcloning the product into pAMPl to create a plasmid designated pAM? ⁇ -vanA antisense: 5'-CUA CUA CUA CUA CTC GAG GCT TAT CAC CCC TTT AAC GC-3' (SEQ ID NO: 16) 5'-CAU CAU CAU GGA GAC AGG AGC ATG AAT AG-3' (SEQ ID NO:17)
  • the polymerase chain reaction with these primers consisted of 30 cycles of 94° C,
  • Vancomycin susceptibilities were determined using the standard National Committee for Clinical Laboratory Standards (NCCLS) agar dilution protocol (National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard M7-A4. Wayne, PA: NCCCLS, 1997).
  • the test antibiotic in this case, vancomycin
  • NCCLS National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved
  • a single colony of A407 with the pAM40 ⁇ -vanHP-vanA antisense construct was grown in brain-heart infusion (BHI) liquid media with sub-inhibitory concentrations of vancomycin (1 ⁇ g/ml) and chloramphenicol (10 ⁇ g/ml).
  • Bacterial RNA was prepared using the Qiagen RNeasy ® protocol for the isolation of total RNA (Qiagen Inc. Valencia, CA) modified to inco ⁇ orate a step of treatment with RNAse free DNAse applied directly on the QIAamp ® column (both Qiagen Inc. Valencia, CA). Then a modified TitanTM One tube RT- PCR protocol (Roche molecular biochemicals, Indianapolis, IN) was followed.
  • Each reaction mix contained template RNA (5 ⁇ g), enzyme (either Titan enzyme mix, reverse and forward PCR primers and buffer components recommended for optimal enzyme activity.
  • the forward (5'- CUA CUA CUA CUA CUA CTC GAG GCT TAT CAC CCC TTT AAC GC -3' -SEQ ID NO: 16) and the reverse primer (5'-CGA ATA CCG CAA GCG ACA G-3' -SEQ ID NO:25) were designed to amplify a 1.1 kb bacterial RNA sequence.
  • the RT reaction was performed at 45°C for 60 min, followed by PCR in a Perkin Elmer Model 9600 Thermal Cycler with the following thermal profile: Initial denaturation: 95°C for 3 min then 35 cycles of denaturation (93°C, 15 s), annealing (55°C, 30 s), elongation (68°C, 70 s) and a final extension step (72°C, 1 min). Amplification products were analyzed by gel electrophoresis. Results Changes in Vancomycin Phenotypic Susceptibility
  • vancomycin susceptibility of a vanA Enterococcus faecalis strain, A407 was assessed after electroporation with either pAM401; pAM401-v ⁇ «HP; or pAM401-v ⁇ HE- vanA antisense. While the vancomycin minimum inhibitory concentration (MIC) remained at 128 ⁇ g/ml in A407 containing the pAM401 shuttle vector alone, the introduction of pAM401 with the vanH promoter decreased the vancomycin MIC to 16 - 32 ⁇ g/ml. The vancomycin MIC was further decreased in response to the pAM401 containing both the vanH promoter and the vanA antisense, typically in the 8 ⁇ g/ml range. VanH promoter effect on vancomycin resistance
  • the pVanR binding domain within the vanH promoter has previously been characterized and consists of an approximate 80 bp region that is considered to have the capacity to bind multiple p-VanR molecules ( ⁇ olman, et al., Biochemistry, 1994, 33:4625- 31). Therefore, it was reasoned that the introduction of an exogenous vanH promoter cloned into a recombinant enterococcal shuttle vector could increase the vancomycin susceptibility of a target VanA enterococcal isolate through the binding and sequestration of pVanR from the native vanH promoter.
  • pAM401 enterococcal shuttle vectors with or without the vanH promoter were constructed and electroporated into a VanA strain of E. faecalis (A407).
  • the successful transfer of the vectors by electroporation was confirmed through the purification of shuttle vector plasmids from the transformants followed by restriction digest analysis as well as by dideoxy-sequencing.
  • the retention of the resistance determinant gene cluster was confirmed by the polymerase chain reaction (PCR) amplification of relevant genes.
  • the vancomycin MIC of A407 enterococci transformed with the shuttle vector containing the vanH promoter demonstrated a four- fold reduction in the MIC from 256 ⁇ g/mL to 64 ⁇ g/mL.
  • control A407 enterococci transformed with the pAM401 vector alone maintained the baseline (MIC of 256 ⁇ g/mL) resistance phenotype.
  • the pVanR binding domain portion of the vanH promoter was amplified and cloned into pAM401 (pAM401-p ⁇ «i?-BD+).
  • pAM401-p ⁇ «i?-BD+ a shuttle vector containing a mutant pVanR binding domain-deficient vanH promoter
  • pAM401 shuttle vectors were then created which contained a gene cassette consisting of the vanH promoter and downstream vanA antisense gene (pAM401- vanHP-vanA antisense), a configuration in which antisense expression would thus be upregulated in parallel that of the native VanA operon in the presence of vancomycin.
  • a control vector that expressed vanH promoter-driven vanA sense transcripts was also cloned (pAM401-v ⁇ «HP-vanA sense) and was electroporated into respective A407 VanA E. faecalis.
  • the expression of the vanH promoter-vcm,4 coding and antisense messenger RNA were confirmed by reverse transcriptase PCR (RT-PCR). In A407 E.
  • the vancomycin MIC was reduced to a susceptible range, from 256 ⁇ g/mL to 2 ⁇ g/mL.
  • the MIC of A407 transformed with pAM401- vanHP-vanA sense remained at the baseline level of 256 ⁇ g/mL.
  • a gene cassette targeting a key antibiotic resistance determinant of the clinically relevant Gram-positive bacterium, Enterococcus has been constructed and consists of the enterococcal v ⁇ «H-promoter driving the expression of a vanA antisense gene introduced in an enterococcal shuttle vector.
  • the target gene, vanA is a highly conserved component of a gene cluster that confers high-level resistance to vancomycin, a pivotal antibiotic used to treat infections caused by Enterococcus resistant to beta-lactam antibiotics.
  • the vanH promoter employed in this construct is the same inducible enterococcal promoter which drives expression of the vanA resistance determinant expression (Figure 3).
  • the enterococcal transcriptional factor, phosphorylated VanR which induces the vanH promoter (Arthur, et al., J. Bacter., 1992, 174:2582-2591), is at the same time, sequestrated from the native vanH promoter, but also allows for induction of the an -vanA antisense in parallel with the expression of the vanHAX.
  • this gene cassette inhibits vancomycin resistance both by an inducible antisense mechanism as well as by functioning as a transcriptional factor binding decoy ( Figure 4).
  • recombinant shuttle vectors containing the vanH promoter or the pVanR binding domain effected a partial restoration of vancomycin susceptibility, while full restoration of vancomycin susceptibility resulted with the introduction of a vector containing both vanH promoter and vanA antisense gene. More specifically, the introduction of a shuttle vector containing the vanH promoter alone into a vancomycin-resistant, vanA -containing Enterococcus faecalis resulted in up to a 16-fold reduction of the minimum inhibitory concentration for vancomycin while a shuttle vector containing both vanH promoter and vanA antisense increased vancomycin susceptibility even further (approximately 32-fold).
  • Recombinant shuttle vectors which target other genes in the vanA operon such as vanX, as well as polycistronic vectors which contain genetic elements designed to interfere with multiple VanA operon functions (e.g. vanA, vanH, and vanX), can be constructed using routine experimentation and no more than ordinary skill in the art.
  • an operon analogous to that associated with the VanA phenotype also forms the genetic basis for class B (VanB) vancomycin resistance
  • analogous compositions against Class B (VanB) as well as other classes of vancomycin resistance operons and genes can be developed as described above.
  • a vanX antisense strategy analogous to the vanA antisense strategy was also tested, resulting in lowering vancomycin MICs to the 2 ⁇ g/ml range.
  • compositions optimally include gene delivery systems such as bacteriophage, highly efficient transconjugative plasmids, and peptide-nucleic acids.
  • VanA vancomycin resistance operon vanR represents a response regulator which, after phosphorylation, activates the vanH promoter which results in activation of vanH, vanA, and vanX transcription;
  • vanS a signal sensor, is responsible for the inducibility of the operon by glycopeptide antibiotics;
  • the vanH gene product is a dehydrogenase that generates lactate from pyruvate;
  • vanA codes for a ligase which preferentially synthesizes D-ala-D-lac;
  • vanX codes for a dipeptidase which degrades the native D-ala-D-ala produced by the wildtype ligase;
  • vanY is a carboxypeptidase which removes terminal alanines;
  • vanZ is responsible for increased resistance to teicoplanin.
  • FIG. 1 Maps of the shuttle vectors and relevant cloning sites.
  • A The parent vector, pAM401. This vector is composed of both Enterococcus faecalis (shaded half on right) and Eschericia coli (bold portion on left) components. The cat region is the chloramphenicol acetyl-transferase gene. The tet region is the tetracycline resistance gene.
  • B The vanH promoter insertion.
  • C The vanA antisense insertion.
  • Figure 3 The proposed nucleic acid binding decoy mechanism by which the observed vancomycin minimum inhibitory concentrations are reduced with the introduction of the pAM401 shuttle vector with the vanH promoter alone.
  • Figure 4 A schematic of the proposed mechanism-of-action of the pAM401-v ⁇ nH promoter-van ⁇ antisense recombinant shuttle vector.

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  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne des méthodes et des compositions destinées à réduire la résistance à la vancomycine dans un organisme résistant à la vancomycine. Ces méthodes consistent à fournir à l'organisme une molécule isolée d'acide nucléique qui s'hybride à un gène-cible vancomycine et/ou qui agit comme leurre promoteur de réponse VanR.
EP00955471A 1999-08-17 2000-08-11 Methodes et compositions destinees a restaurer la sensibilite aux antibiotiques des enterocoques resistants aux glycopeptides Withdrawn EP1210365A2 (fr)

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AU2002243751A1 (en) 2001-01-31 2002-08-12 Mayo Foundation For Medical Education And Research Detection of herpes simplex virus
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US7365176B2 (en) 2002-09-26 2008-04-29 Mayo Foundation For Medical Education And Research Detection of Epstein-Barr virus
US7074599B2 (en) 2002-09-27 2006-07-11 Mayo Foundation For Medical Education And Research Detection of mecA-containing Staphylococcus spp.
WO2005017202A2 (fr) * 2003-05-13 2005-02-24 Gen-Probe Incorporated Procede et trousse permettant l'identification de micro-organismes resistants aux antibiotiques
US7427475B2 (en) 2003-11-18 2008-09-23 Mayo Foundation For Medical Education And Research Detection of group B streptococcus
GB0906130D0 (en) * 2008-10-03 2009-05-20 Procrata Biosystems Ltd Transcription factor decoys
US8865158B2 (en) * 2012-05-22 2014-10-21 Ramot At Tel-Aviv University Ltd. Bacteriophages for reducing toxicity of bacteria
US10227661B2 (en) * 2014-11-21 2019-03-12 GeneWeave Biosciences, Inc. Sequence-specific detection and phenotype determination
CN106884038A (zh) * 2015-12-16 2017-06-23 北京大学人民医院 一种检测革兰氏阳性细菌耐药基因的基因芯片试剂盒
CN106636426A (zh) * 2017-01-13 2017-05-10 重庆威斯腾生物医药科技有限责任公司 一种糖肽类抗生素耐药基因检测试剂盒
CN114891908A (zh) * 2022-06-25 2022-08-12 上海交通大学医学院附属瑞金医院 用于预测屎肠球菌对抗生素药敏表型的特征基因组合、试剂盒及测序方法

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