WO2001012842A1 - Methods and targets of antibiotic resistance - Google Patents
Methods and targets of antibiotic resistance Download PDFInfo
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- WO2001012842A1 WO2001012842A1 PCT/US2000/040676 US0040676W WO0112842A1 WO 2001012842 A1 WO2001012842 A1 WO 2001012842A1 US 0040676 W US0040676 W US 0040676W WO 0112842 A1 WO0112842 A1 WO 0112842A1
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- selective agent
- continuous culturing
- culturing
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- resistance
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/18—Testing for antimicrobial activity of a material
Definitions
- the present invention relates to methods and materials for the discovery and characterization of molecular mechanisms of drug resistance. More specifically, the present invention relates to methods and materials for the discovery and characterization of molecular mechanisms of drug resistance using directed evolution.
- Drug resistance is an extremely important aspect of the clinical use and efficacy of therapeutic compounds in the treatment of human and animal diseases. Drug resistance necessitates affirmative treatments which can be less effective and more costly. Moreover, the rapidly increasing rate of reemergence of once controlled clinical infections is seriously eroding the repertoire of effective antibiotics. For example, many common pediatric antibiotics, such as amoxicillin, have been rendered much less effective against such common infections as otitis media due to emerging resistance over the past decade. Alternative therapeutics, when available, are often much more expensive than the first line drug. Likewise, tumor cells of patients undergoing cancer treatment often become resistant to the anticancer drugs being used.
- a key in determining the cause of resistance is often found in an analysis of the drug target proteins of the pathogenic organism. Mutation of the drug target protein is responsible for much of the rapidly increasing rate of clinical antibiotic resistance. This is attributable to the large number and variety of antibiotics that target cellular proteins. Likewise, changes in the target protein structure, as a result of point mutations in the gene, results in loss of binding and efficacy of the antibiotic. This has been widely observed in clinical isolates for nearly all antibiotics targeting cellular proteins.
- the amino acid sequence of a protein determines its three- dimensional (3D) structure, which in turn determines protein function (EPST63, ANFI73).
- Shortle SHOR85
- Sauer and colleagues PAKU86, REID88a
- Caruthers and colleagues EISE85
- the 3D structure is essentially unaffected by the identity of the amino acids at some loci, while at other loci, only one or a few types of amino-acid is allowed. In most cases, the loci where wide variety is allowed have the amino acid side-chain group directed toward the solvent. Loci where limited variety is allowed frequently have the side-chain group directed toward other parts of the protein.
- substitutions of amino acids that are exposed to solvent are less likely to affect the 3D structure than are substitutions at internal loci. (See also SCHU79, p169-171 and CREI84, p239-245, 314-315).
- the secondary structure (helices, sheets, turns, loops) of a protein is determined mostly by local sequence. Certain amino acids have a propensity to appear in certain "secondary structures," they are found from time to time in other structures, and studies of pentapeptide sequences found in different proteins have shown that their conformation varies considerably from one occurrence to the next (KABS84, ARGO87). As a result, a priori design of proteins to have a particular 3D structure is difficult.
- Protein engineering is the art of manipulating the sequence of a protein in order to alter its binding characteristics.
- the factors affecting protein binding are known, (CHOT75, CHOT76, SCHU79, p98-107, and CREI84, Ch8), but designing new complementary surfaces has proved difficult.
- side groups SUTC87b
- the side groups of proteins are floppy (i.e. can move from side to side) and it is difficult to predict what conformation a new side group will take.
- the forces that bind proteins to other molecules are all relatively weak and it is difficult to predict the effects of these forces.
- TSCH87 the protein was cleaved into two fragments, a residue removed from the new end of one fragment, the substitute residue added on in its place, and the modified fragment joined with the other, original fragment.
- the mutant protein could be synthesized in its entirety (TANK77).
- Erickson et al. suggested that mixed amino acid reagents could be used to produce a family of sequence-related proteins which could then be screened by affinity chromatography (ERIC86). They envision successive rounds of mixed synthesis of variant proteins and purification by specific binding. They do not discuss how residues should be chosen for variation. Because proteins cannot be amplified, the researchers must sequence the recovered protein to learn which substitutions improve binding. The researchers must limit the level of diversity so that each variety of protein is present in sufficient quantity for the isolated fraction to be sequenced.
- DILL87 protein surgery
- Reidhaar-Olson and Sauer have used synthetic degenerate oligo-nts to vary simultaneously two or three residues through all twenty amino acids. See also Vershon et al. (VERS86a; VERS86b). Reidhaar-Olson and Sauer do not discuss the limits on how many residues could be varied at once nor do they mention the problem of unequal abundance of DNA encoding different amino acids. They looked for proteins that either had wild-type dimerization or that did not dimehze. They did not seek proteins having novel binding properties and did not find any. This approach is likewise limited by the number of colonies that can be examined (ROBE86).
- Ferenci and collaborators have published a series of papers on the chromatographic isolation of mutants of the maltose-transport protein LamB of E. coli (FERE82a, FERE82b, FERE83, FERE84, CLUN84, HEIN87 and papers cited therein).
- the mutants were either spontaneous or induced with nonspecific chemical mutagens.
- Levels of mutagenesis were picked to provide single point mutations or single insertions of two residues. No multiple mutations were sought or found.
- Ferenci also taught that there was no need to clone the structural gene, or to know the protein structure, active site, or sequence. Ferenci did not limit the mutations to particular loci or particular substitutions. Ferenci does not suggest that surface residues should be preferentially varied. In consequence, Ferenci's selection system is much less efficient than that disclosed herein. A number of researchers have directed unmutated foreign antigenic epitopes to the surface of bacteria or phage, fused to a native bacterial or phage surface protein, and demonstrated that the epitopes were recognized by antibodies. Thus, Charbit, et al. (CHAR86) genetically inserted the C3 epitope of the VP1 coat protein of poliovirus into the LamB outer membrane protein of E.
- CHAR86 Charbit, et al.
- Charbit, et al. (CHAR87) likewise produced chimeras of LamB and the A (or B) epitopes of the preS2 region of hepatitis B virus.
- a chimeric LacZ/OmpB protein has been expressed in E. coli and is, depending on the fusion, directed to either the outer membrane or the periplasm (SILH77).
- a chimeric LacZ/OmpA surface protein has also been expressed and displayed on the surface of E. coli cells (Weinstock et al., WEIN83). Others have expressed and displayed on the surface of a cell chimeras of other bacterial surface proteins, such as E. coli type 1 fimbriae (Hedegaard and Klemm (HEDE89)) and Bacterioides nodusus type 1 fimbriae (Jennings et al., JENN89). In none of the recited cases was the inserted genetic material mutagenized.
- Dulbecco suggests a procedure for incorporating a foreign antigenic epitope into a viral surface protein so that the expressed chimeric protein is displayed on the surface of the virus in a manner such that the foreign epitope is accessible to antibody.
- Smith reported inserting a nonfunctional segment of the EcoRI endonuclease gene into gene III of bacte ophage f1 , "in phase". The gene III protein is a minor coat protein necessary for infectivity. Smith demonstrated that the recombinant phage were adsorbed by immobilized antibody raised against the EcoRI endonuclease, and could be eluted with acid. De la Cruz et al.
- DELA88 have expressed a fragment of the repeat region of the circumsporozoite protein from Piasmodium faiciparum on the surface of M13 as an insert in the gene III protein. They showed that the recombinant phage were both antigenic and immunogenic in rabbits, and that such recombinant phage could be used for B epitope mapping. The researchers suggest that similar recombinant phage could be used for T epitope mapping and for vaccine development.
- McCafferty et al. (MCCA90) expressed a fusion of an Fv fragment of an antibody to the N-terminal of the pill protein. The Fv fragment was not mutated.
- Parmley and Smith suggested that an epitope library that exhibits all possible hexapeptides could be constructed and used to isolate epitopes that bind to antibodies.
- the authors did not suggest that it was desirable to balance the representation of different amino acids.
- the insert should encode a complete domain of the exogenous protein. Epitopes are considered to be unstructured peptides as opposed to structured proteins.
- a method of evolving and selecting cells resistant to a selective agent by inducing directed evolution in continuous culture while applying both a mutagenic and selective agent to the cells to determine the cells having resistance. This also provides a method of generating mutant drug targets useful for screening for effective compounds.
- Figure 1A is a generalized schematic diagram of a chemostat of the present invention.
- Figure 1 B is a schematic diagram of a specific chemostat of the present invention.
- Figure 2 is a sequence alignment of the quinolone resistance determining region from resistant mutants generated with the present invention compared to the wild type.
- the present invention is directed to a method for evolving and/or selecting for microorganisms from a susceptible culture or sample, that are resistant to one or more therapeutic agents.
- This agent is typically an antibiotic or anticancer compound.
- the method relies upon the principles of directed evolution and is specifically implemented through continuous culture in the presence of both selective agent (antibiotic) and mutagen.
- a "chemostat” as used herein is meant to include any apparatus which properly controls the environment such that bacterial culture is maintained in a continuous state of cell division. Any chemostat can be used that is appropriate for the experimental conditions at hand as are known to those of skill in the art.
- a generalized schematic is shown in Figure 1A (Dykhuizen, 1993), and can be of simple, ready-made and/or of custom design.
- a proprietary design for a very versatile, variable volume chemostat is shown in Figure 1 B.
- the term "selective agent” as used herein is meant to include any chemotherapeutics or any other compound that can act to permit phenotypic or genotypic differentiation between mutant and wild type cells. Additional examples would include industrial chemicals that can also be used as nutrients by microorganisms.
- Mutagens used in the present invention include Ethyl Methanesulfonate (EMS), 4-Nitroquinoline-1 -Oxide (NQO), and N- Methyl-N'-Nitro-N-Nitrosoguanidine (MNNG), but can include any mutagenic agent, physical or chemical, without departing from the disclosure of the present invention.
- EMS Ethyl Methanesulfonate
- NQO 4-Nitroquinoline-1 -Oxide
- MNNG N- Methyl-N'-Nitro-N-Nitrosoguanidine
- UV irradiation of a chemostatic culture specially constructed of UV transparent glass can be implemented.
- the design of the chemostat shown in Figure 1 B is especially amenable to this application due to the columnar shape and relatively light transparent culture vessel with the chemostat.
- any apparatus that can provide for continuous culture including chemostats, nutristats, and fermentors
- the drug sensitive microorganism is cultured in a chemostat via continuous culture.
- the selective agent e.g. antibiotic
- mutagenic agents are added to the continuous culture at a concentration predetermined by a survivability vs. dose curve. The optimum concentration of mutagen produces the maximum number of non-lethal mutations.
- the optimum concentration of mutagen is determined empirically by determining a survivability vs. dose curve. This concentration must be determined empirically for each pure strain or mixed culture used. In general, for stepwise mutagenesis and selection, the optimum concentration for many bacteria, including several strains of E. coli, is approximately 0.5-3% (v/v or w/w) for 20 to 120 minutes.
- the mutagen can be infused slowly and continuously into the continuous culture, with adjustments, if necessary, in reduced medium influx to compensate for decreasing numbers of cells in culture as a result of lethal mutations This is also determined empirically from the survivability vs dose curve
- a typical evolution/discovery chemostat consists of a 100 to 1000 ml culture grown for several generations to establish stability and equilibrium Subsequently, mutagen is added to the chemostatic culture, as described in the preceding paragraph, to the predetermined concentration of mutagenic agent Subsequently, antibiotic selection is commenced at a predetermined concentration (either step-wise or constant infusion) Typically, and as in the case of antibiotic used for selection, the selective agent concentration is titered to give a differential growth rate between wild type and resistant mutants
- the use of a material that mimics a physiological attachment or colonization site in the chemostat can effect the rate of evolution and molecular mode of resistance
- the inclusion of a relatively small amount of glass or polystyrene beads, or biological matrix increases the rate of mutant evolution and selection in the nalixidic acid/E coli ATCC 11775 model system described above
- the present invention is also useful for discovery and characterization of virulence mechanisms and determinants, and can have important applications in the area of biofilms as relevant to virulence.
- GENERAL METHODS General methods in molecular biology: Standard molecular biology techniques known in the art and not specifically described were generally followed as in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York (1989), and in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989) and in Perbal, A Practical Guide to Molecular Cloning, John Wiley & Sons, New York (1988), and in Watson et al., Recombinant DNA, Scientific American Books, New York and in Birren et al (eds) Genome Analysis: A Laboratory Manual Series, Vols.
- PCR Polymerase chain reaction
- the present invention has been used to evolve quinolone resistant mutants from the antibiotic sensitive E. coli reference strain ATCC 11775 evolved with EMS mutagen in the presence of nalidixic acid as the selective agent, and to correlate the level of resistance with specific point mutations occurring within the quinolone resistance determining region (QRDR) of the gyrase A gene (gyrA).
- QRDR quinolone resistance determining region
- gyrA gyrase A gene
- the mutagenic reaction was terminated by immediately washing and diluting the bacterial cells in suitable buffer or growth medium.
- the different dilutions of the cells were plated on agar medium to determine the surviving fractions of the cells, which were compared with the non-treated bacterial cell used as control for 100% survival.
- the selected concentration of mutagens and the reaction period for E. coli ATCC 11775 resulting in an acceptable range of survival rates with reasonable mutational frequencies are shown in Table I.
- Proprietary microcarrier spinner flask chemostats were used for continuous culture. Two hundred ml of LB or TS broth containing antifoam A (50 ppm concentration) was autoclaved in the chemostat flask with associated tubes and connectors attached to them. The chemostats were set up, by placing them on a multi-magnetic stirrer, and by connecting them to a constant water supply for providing constant temperature, to a vacuum pump through the waste-disposal reservoir, and to the medium reservoir through a controllable peristaltic pump.
- the starter culture of bacteria was inoculated into the flask through a sample port by using a 3.0-ml sterile syringe followed by washing the port with an aliquot of sterile broth.
- the culture was allowed to grow in batch mode with constant stirring until it reached log to late log phase (usually 3.0 to 4.5 hours).
- continuous culture mode was established by feeding the flask with a continuous supply of fresh medium from the reservoir. The flow rate was maintained so that the culture medium could be replaced with the fresh medium during the time period when the culture attains its log phase.
- OD ⁇ was monitored at regular intervals by withdrawing the culture through the sample port with a sterile syringe. The culture was, thus, allowed to maintain several generations at its log phase.
- Mutagen EMS (2% w/v) or MNNG (10 ⁇ g/ml) was added to the continuous culture through one of the infusion ports for a specific period, during which the chemostat was operated in batch mode (30 minutes for EMS and 10 minutes for MNNG).
- the continuous culture was resumed at a slightly higher flow rate to ensure washing of the mutagen from the chemostat before selection of the culture with the target antibiotic or inhibitor.
- a low dose or sub-lethal concentrations of mutagen e.g., 0.1-0.5 ⁇ g MNNG/ml
- Antibiotic was also directly incorporated into the medium reservoir for the selection of mutants from the mutagenized culture after several generations, starting with the sub- lethal concentrations of antibiotic and then with the gradual increase of the antibiotic concentration, depending on the culture growth. At 8-12 hour intervals a small aliquot of the culture was withdrawn from the chemostat, OD 6 ⁇ was measured, and an aliquot of the culture was spread on an agar medium containing a gradient of the selective antibiotic (0-30 ⁇ g nalidixic acid/ml).
- Antibiotic-resistant clones were selected at varying concentrations along the gradient and further analyzed by a standard antimicrobial disk- diffusion test (antibiogram) for extent of resistance phenotypes (whether partially or completely resistant). Genomic DNA from the selected mutant was prepared and used as template in a PCR designed to amplify the target resistance gene. The PCR product was sequenced directly and analyzed to detect the expected point mutations within the resistance-determining region (QRDR) (Table II). Sequences were routinely aligned and compared with the wild-type (non-mutated) gene or gene region ( Figure 2).
- E. coli strain 11775 was also subjected to mutagenesis by EMS in a chemostat continuous culture system and allowed to grow for seven days for over 400 generations. Culture was selected for NA R mutants on NA- containing gradient plate every 12 hours E. coli NA R clones, thus obtained, from different time-points were analyzed for their phenotype and genetic mutation, especially in the gyrA QRDR. Pont mutations in the QRDR of several selected NA R clones from different time-points (generations) are shown in Table III.
- This clinical strain was subjected to mutagenesis with MNNG in the continuous culture system for at least eight days under continuous mutagen and antibiotic infusion.
- ESBL resistant mutants were selected with CTX in the range of 25-50 ⁇ g/ml.
- CTX R mutants were obtained and analyzed for their muiti-drug- resistance phenotypes (Table IV).
- This example again demonstrates the utility of the present invention in providing the methods and materials to discover and characterize antibiotic resistance prior to clinical emergence.
- the use of a material that mimics a physiological attachment or colonization site in the chemostat can effect the rate of evolution and molecular mode of resistance.
- the inclusion of a relatively small amount of glass or polystyrene beads, or biological matrix increases the rate of mutant evolution and selection in the nalixidic acid/E. coli ATCC 11775 model system described above.
- the present invention is also useful for discovery and characterization of virulence mechanisms and determinants, and can have important applications in the area of biofilms as relevant to virulence.
- An important aspect of the utility of this invention is its ability to predict antibiotic resistance prior to clinical emergence.
- Prior to the development of the present invention there had only been a single case where the molecular mechanism of resistance was predicted in vitro prior to this characterization from a clinical isolate (Arlet, G., et al., 1993).
- the present invention provides validated drug intervention targets, useful for screening in the development of next generation compounds. These targets can be either the actual mutant protein target or the resistant strain itself.
- Validated targets differ from putative or hypothetical targets in that they are proven to confer the observed resistance, and therefore, serve as useful intervention targets for future therapeutic development.
- Validated targets are provided through the practice of the present invention because directed evolution, mutation, and selection are conducted in vivo, and in the relevant biological host. This differs greatly from any form of in vitro mutagenesis or even evolution conducted in a non-relevant host (non-clinical), and is likely the reason that predicting the molecular mechanism of clinical resistance a prior has only been accomplished once.
- NA nalidixic acid-resistant
- NQO NX-N6 Completely NA K Ser-83 (TCG)- Leu (TTG) Ser-83 — ⁇ Leu MNNG NX-M3 Partially NA R Asp-87 (GAC) Gly (GGC) Asp-8 — ⁇ Gly NX-M5 Partially NA R Asp-87 (GAC) • Asn (AAC) Asp-87 — ⁇ Asn NX-M6 Partially NA R Asp-87 (GAC) Gly (GGC) Asp-87 — ⁇ Gly
- CTX B Mutants (8 analyzed) CTX R , CAZ R , FOX R , ATM R TEM-1— ⁇ TEM-3
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Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
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US10/069,490 US6875582B1 (en) | 1999-08-19 | 2000-08-18 | Methods and targets of antibiotic resistance |
AU78828/00A AU7882800A (en) | 1999-08-19 | 2000-08-18 | Methods and targets of antibiotic resistance |
EP00968994A EP1210450A4 (en) | 1999-08-19 | 2000-08-18 | Methods and targets of antibiotic resistance |
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US14976199P | 1999-08-19 | 1999-08-19 | |
US60/149,761 | 1999-08-19 |
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WO2001012842A1 true WO2001012842A1 (en) | 2001-02-22 |
WO2001012842A9 WO2001012842A9 (en) | 2002-08-01 |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
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US6875582B1 (en) * | 1999-08-19 | 2005-04-05 | Omniscience Pharmaceuticals, Inc. | Methods and targets of antibiotic resistance |
WO2012074476A1 (en) * | 2010-11-29 | 2012-06-07 | Rational Enzyme Mining Rem Ab | Rational enzyme mining |
Citations (1)
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US5766842A (en) * | 1994-09-16 | 1998-06-16 | Sepracor, Inc. | In vitro method for predicting the evolutionary response of a protein to a drug targeted thereagainst |
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US4528773A (en) * | 1983-09-08 | 1985-07-16 | University Of Tennessee Research Corporation | Method for producing genes conferring resistance to herbicides, growth regulators or other chemical agents in vascular plants |
US5652098A (en) * | 1993-03-12 | 1997-07-29 | The United States Of America As Represented By The United States Department Of Energy | Method for rapid isolation of sensitive mutants |
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2000
- 2000-08-18 EP EP00968994A patent/EP1210450A4/en not_active Withdrawn
- 2000-08-18 WO PCT/US2000/040676 patent/WO2001012842A1/en active Application Filing
- 2000-08-18 AU AU78828/00A patent/AU7882800A/en not_active Abandoned
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US5766842A (en) * | 1994-09-16 | 1998-06-16 | Sepracor, Inc. | In vitro method for predicting the evolutionary response of a protein to a drug targeted thereagainst |
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6875582B1 (en) * | 1999-08-19 | 2005-04-05 | Omniscience Pharmaceuticals, Inc. | Methods and targets of antibiotic resistance |
WO2012074476A1 (en) * | 2010-11-29 | 2012-06-07 | Rational Enzyme Mining Rem Ab | Rational enzyme mining |
US9365887B2 (en) | 2010-11-29 | 2016-06-14 | Rational Enzyme Mining Rem Ab | Rational enzyme mining |
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Publication number | Publication date |
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EP1210450A4 (en) | 2003-07-23 |
WO2001012842A9 (en) | 2002-08-01 |
AU7882800A (en) | 2001-03-13 |
EP1210450A1 (en) | 2002-06-05 |
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