WO2002010442A1 - Utilisation d'ectoenzymes et d'enzymes secretees pour suivre la proliferation cellulaire - Google Patents

Utilisation d'ectoenzymes et d'enzymes secretees pour suivre la proliferation cellulaire Download PDF

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Publication number
WO2002010442A1
WO2002010442A1 PCT/US2000/021049 US0021049W WO0210442A1 WO 2002010442 A1 WO2002010442 A1 WO 2002010442A1 US 0021049 W US0021049 W US 0021049W WO 0210442 A1 WO0210442 A1 WO 0210442A1
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Prior art keywords
ectoenzyme
cells
activity
chitobiase
secreted enzyme
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PCT/US2000/021049
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English (en)
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Judith Zyskind
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Elitra Pharmaceuticals, Inc.
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Priority to PCT/US2000/021049 priority Critical patent/WO2002010442A1/fr
Priority to AU2000268924A priority patent/AU2000268924A1/en
Publication of WO2002010442A1 publication Critical patent/WO2002010442A1/fr

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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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • 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
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • 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
    • 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
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
    • G01N2333/918—Carboxylic ester hydrolases (3.1.1)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)

Definitions

  • the present invention relates to the use of enzymes which are associated with the cell (ectoenzymes) and secreted enzymes for monitoring cellular proliferation.
  • Reporter enzymes are enzymes whose activities are easily assayed when present inside cells.
  • a gene encoding a reporter enzyme may be fused to the coding region or to the regulatory region of the regulated gene.
  • Reporter genes may be used to determine whether a sequence contains a promoter or other cis-acting element which directs transcription, such as an enhancer.
  • reporter genes may be used to identify regulatory sites in promoters or other cis-acting elements and to determine the effects of mutating these regulatory sites on the level of gene expression directed by the promoters or other cis-acting elements.
  • Reporter genes may also be used to detect successful transformation, to monitor gene expression under various conditions, to assess the subcellular location of an expressed protein and to identify drugs such as antibiotics.
  • Newly emerging practices in drug discovery utilize a number of biochemical techniques to provide for directed approaches to creating new drugs, rather than discovering them at random. For example, gene sequences and proteins encoded thereby that are required for the proliferation of a microorganism make excellent targets since exposure of bacteria to compounds active against these targets would result in the inactivation of the microorganism. Once a target is identified, biochemical analysis of that target can be used to discover or to design molecules that interact with and alter the functions of the target. Use of physical and computational techniques to analyze structural and biochemical properties of targets in order to derive compounds that interact with such targets is called rational drug design and offers great potential. Thus, emerging drug discovery practices use molecular modeling techniques, combinatorial chemistry approaches, and other means to produce and screen and/or design large numbers of candidate compounds.
  • the initial step of identifying molecular targets for investigation can be an extremely time consuming task. It may also be difficult to design molecules that interact with the target by using computer modeling techniques. Furthermore, in cases where the function of the target is not known or is poorly understood, it may be difficult to design assays to detect molecules that interact with and alter the functions of the target. To improve the rate of novel drug discovery and development, methods of identifying important molecular targets in pathogenic microorganisms and methods for identifying molecules that interact with and alter the functions of such molecular targets are urgently required.
  • the present invention relates to the use of ectoenzymes and secreted enzymes in assays for measuring cellular proliferation.
  • Summary of the Invention is a method for measuring cellular proliferation in a sample comprising obtaining a sample of cells which express an ectoenzyme or a secreted enzyme, determining the level of activity of the ectoenzyme or secreted enzyme in the sample and correlating the level of activity of the ectoenzyme or secreted enzyme with the extent of cellular proliferation.
  • the step of determining the level of activity of the ectoenzyme or secreted enzyme may comprise contacting the cells with an agent which yields a detectable product when acted upon by the ectoenzyme or secreted enzyme and determining the level of the detectable product in the sample.
  • the ectoenzyme or secreted enzyme may be selected from the group consisting of Pseudomonas aeruginosa metalloproteinase, Moraxella (Branhamella) catarrhalis BRO beta-lactamase, P.
  • aeruginosa FpvA ferric pyoverdin receptor £ coli OmpP endopeptidase, outer membrane phospholipase A
  • Bacteriodes thetaiotamicron susG starch utilization protein Hae ophilus influenzae phosphomonoesterase, streptococcal protein Sir, streptococcal C5a peptidase, Lactococcus lactis serine protease NisP, proteinase PrtB, proteinase PrtH, proteinase PrtP, proteinase ScpA, S. pneumoniae beta-N-acetylglucosaminidase, S.
  • pneumoniae neuraminidase Streptococcus sobrinus dextranase, Streptococcus suis muramidase, Streptococcus mutans exo-beta-D-fructosidase, Staphylococcus aureus murine hydrolase, staphylococcal lipases, lysostaphin, endo-beta-N-acetylglucosaminidase, sulfhydryl protease, staphylococcal esterase, S. aureus nuclease, S. aureus fatty acid modifying enzyme, chitinase, S.
  • the determining step may comprise determining the level of activity of a secreted enzyme by contacting the growth medium of the cells with an agent which yields a detectable product when acted upon by the secreted enzyme and determining the level of the detectable product in the sample.
  • the determining step may comprise determining the level of activity of a secreted enzyme by contacting a supernatant with an agent which yields a detectable product when acted upon by the secreted enzyme and determining the level of the detectable product in the sample, wherein the supernatant comprises growth media from which the cells have been removed.
  • the ectoenzyme or secreted enzyme may comprise a membrane-bound form of chitobiase.
  • the method may further comprise introducing a gene encoding the membrane-bound form of chitobiase into the cells prior to obtaining the sample of cells.
  • the method may further comprise contacting the cells with sarkos ⁇ l.
  • the method may further comprise contacting the cells with sarkosyl and NaCl.
  • the method may further comprise contacting the cells with NaCl.
  • the cells are intact.
  • the ectoenzyme or secreted enzyme may be expressed transiently.
  • the ectoenzyme or secreted enzyme may be expressed stably.
  • the ectoenzyme or secreted enzyme may be expressed from a plasmid.
  • the ectoenzyme or secreted enzyme may be endogenous.
  • the ectoenzyme or secreted enzyme may be exogenous.
  • the ectoenzyme or secreted enzyme may be expressed from an inducible promoter.
  • the determining step may comprise determining the level of activity of an ectoenzyme.
  • the method may further comprise preparing a membrane fraction comprising the ectoenzyme.
  • the ectoenzyme or secreted enzyme may be expressed from a gene encoding the ectoenzyme or secreted enzyme which has been introduced into the genomes of the cells.
  • the cells may be selected from the group consisting of prokar ⁇ otic cells and eukaryotic cells.
  • the step of determining the level of activity of the ectoenzyme or secreted enzyme may be selected from the group consisting of measuring the amount of a chemiluminescent product produced from a substrate, measuring the amount of a fluorescent product produced from a substrate, measuring the amount of light absorbed by a product produced from a substrate and measuring a decrease in the amount of a detectable substrate.
  • the product may be 7-nitrophenol.
  • Another embodiment of the present invention is a method for determining the level of membrane-bound chitobiase gene activity in intact cells, comprising the steps of introducing a nucleic acid encoding the membrane-bound chitobiase into a cell population and contacting the cells with a chitobiase substrate.
  • Another embodiment of the present invention is a gene construct comprising a heterologous promoter operably linked to a nucleic acid encoding a membrane-bound form of chitobiase.
  • the portion of the nucleic acid encoding a membrane-bound form of chitobiase comprises a signal sequence from a gene other than the chitobiase gene.
  • Another embodiment of the present invention is a cell into which a gene encoding a membrane-bound form of chitobiase has been introduced.
  • the portion of the nucleic acid encoding the membrane-bound chitobiase signal sequence may be heterologous.
  • the gene encoding membrane-bound chitobiase may be introduced into the genome of the cell.
  • Another embodiment of the present is a method for characterizing a promoter comprising providing a construct comprising the promoter operably linked to a nucleic acid encoding a membrane-bound form of chitobiase, introducing the construct into host cells, and identifying sequences in the promoter which regulate transcription levels.
  • the nucleic acid encoding a membrane-bound form of chitobiase encodes a membrane-bound form of chitobiase may be obtained from an organism selected from the group consisting of Alteromonas sp.
  • the method of identifying sequences which are involved in regulating transcription may comprise mutagenizing the promoter.
  • the method of identifying sequences which are involved in transcription may comprise constructing deletions in the promoter.
  • Another embodiment of the present invention is a method for identifying a regulatory element capable of modulating transcription within a test nucleic acid sequence, comprising providing a construct comprising the test nucleic acid sequence operably linked to a nucleic acid encoding a membrane-bound form of chitobiase; introducing the construct into host cells and determining the level of chitobiase activity.
  • the nucleic acid encoding a membrane-bound form of chitobiase may encode a membrane-bound form of chitobiase obtained from an organism selected from the group consisting of Alteromonas sp.
  • the construct may be introduced transiently.
  • The may also be introduced stably.
  • the host cells may be selected from the group consisting of prokaryotic cells and eukaryotic cells.
  • the method may further comprise the step of preparing membrane fractions of the cells.
  • the step of determining the level of membrane-bound chitobiase activity may be selected from the group consisting of measuring the amount of a chemiluminescent product prod uced from a substrate, measuring the amount of a fluorescent product produced from a substrate, measuring the amount of light absorbed by a product produced from a substrate and measuring a decrease in the amount of a detectable substrate.
  • the product may be /7-nitrophenol.
  • the test nucleic acid sequence may comprise a portion of genomic DNA.
  • the step of determining the level of membrane-bound chitobiase activity may comprise determining the level of membrane-bound chitobiase activity after exposing the host cells to a desired set of environmental conditions.
  • the step of determining the level of membrane-bound chitobiase activity may comprise determining the level of membrane- bound chitobiase activity after contacting the host cells with a compound to be tested for its influence on the level of transcription from the regulatory element.
  • Another embodiment of the present invention is a method of detecting successful transformation, comprising the steps of introducing a nucleic acid encoding a membrane-bound form of chitobiase into host cells and detecting membrane-bound chitobiase expression in the host cells.
  • the nucleic acid may encode a membrane-bound form of chitobiase obtained from an organism selected from the group consisting of Alteromonas sp.
  • the nucleic acid may further comprise a ⁇ site-specific recombination sequence.
  • Another embodiment of the present invention is a method for monitoring the activity of a promoter comprising providing a construct comprising the promoter operably linked to a nucleic acid encoding a membrane-bound form of chitobiase, introducing the construct into host cells, and determining the level of membrane-bound chitobiase activity.
  • the nucleic acid encoding a membrane-bound form of chitobiase may encode a membrane-bound form of chitobiase obtained from an organism selected from the group consisting of Alteromonas sp.
  • the reporter gene construct may be introduced transiently.
  • the reporter gene construct may be introduced stably.
  • the reporter gene may be incorporated into the genome of the host cells.
  • the host cells may be selected from the group consisting of prokaryotic cells and eukaryotic cells.
  • the method may further comprise the step of preparing membrane fractions of the host cells.
  • the step of determining the level of membrane-bound chitobiase activity may be selected from the group consisting of measuring the amount of a chemiluminescent product produced from a substrate, determining the level of chitobiase activity comprises measuring the amount of a fluorescent product produced from a substrate, measuring the amount of light absorbed by a product produced from a substrate and measuring a decrease in the amount of a detectable substrate.
  • the product may be y ⁇ -nitrophenol.
  • the step of determining the level of membrane-bound chitobiase activity may comprise determining the level of membrane-bound hitobiase activity after exposing the host cells to a desired set of environmental conditions.
  • the step of determining the level of membrane-bound chitobiase activity may comprise determining the level of membrane-bound chitobiase activity after contacting the host cells with a compound to be tested for its influence on the level of transcription from the regulatory element.
  • the compound may comprise a compound to be tested for activity as a drug.
  • Another embodiment of the present invention is a method for determining whether a test protein is associated with the outer membrane, comprising the steps of: fractionating a cell population and assaying the fractions for membrane-bound chitobiase activity and test protein activity, wherein if the test protein and membrane- bound chitobiase are found in the same fraction, the test protein is a membrane protein.
  • the test protein may be an antibiotic target.
  • Another embodiment of the present invention is a method of determining whether a test compound inhibits cellular proliferation comprising contacting a first population of cells expressing an ectoenzyme or a secreted enzyme with the test compound and comparing the activity of the ectoenzyme or the secreted enzyme in the first population of cells with the activity of the ectoenzyme or the secreted enzyme in a second population of cells expressing the ectoenzyme or the secreted enzyme, wherein the second population of cells was not contacted with the test compound and wherein if the level of activity of the ectoenzyme or the secreted enzyme in the first population of cells is significantly less than the level of activity of the ectoenzyme or the secreted enzyme in the second population of cells, then the test compound inhibits cellular proliferation.
  • the ectoenzyme or secreted enzyme may be selected from the group consisting of Pseudomonas aeruginosa metalloproteinase, Moraxella (Branhamella) Catarrhalis BRO beta- lactamase, P.
  • aeruginosa FpvA ferric pyoverdin receptor £ coli OmpP endopeptidase, outer membrane phospholipase A
  • Bacteriodes thetaiotamicron susG starch utilization protein Haemophilus influenzae phosphomonoesterase, streptococcal protein Sir, streptococcal C5a peptidase, Lactococcus lactis serine protease NisP, proteinase PrtB, proteinase PrtH, proteinase PrtP, proteinase ScpA, S. pneumoniae beta-N-acetylglucosaminidase, S.
  • pneumoniae neuraminidase Streptococcus sobrinus dextranase, Streptococcus suis muramidase, Streptococcus mutans exo-beta- D-fructosidase, Staphylococcus aureus murine hydrolase, staphylococcal lipases, lysostaphin, endo-beta-N- acetylglucosa inidase, sulfhydryl protease, staphylococcal esterase, S. aureus nuclease, S. aureus fatty acid modifying enzyme, chitinase, S.
  • aureus autolysin hemolysin, DNase, coagulase, protein A, staphylokinase and enterotoxin.
  • the ectoenzyme or secreted enzyme may comprise a membrane-bound form of chitobiase.
  • the ectoenzyme or secreted enzyme may be endogenous.
  • the method may further comprise introducing a gene encoding the ectoenzyme or secreted enzyme into the cells prior to comparing the activity of the ectoenzyme or secreted enzyme in the first population of cells with the activity of the ectoenzyme or secreted enzyme in a second population of cells.
  • Another embodiment of the present invention is a method for identifying a compound which inhibits cellular proliferation comprising contacting a first population of cells expressing an ectoenzyme or secreted enzyme with the compound wherein the first population of cells has been sensitized by reducing the level or activity of a gene product required for proliferation and determining whether the compound inhibits cellular proliferation by detecting the activity of the ectoenzyme or secreted enzyme.
  • the method may further comprise contacting a second population of cells expressing an ectoenzyme or secreted enzyme with the compound wherein the second population of cells has not been sensitized and comparing the activity of the ectoenzyme or secreted enzyme in the first population of cells with the activity of the ectoenzyme or secreted enzyme in the second population of cells, wherein the compound inhibits cellular proliferation if the level of activity of the ectoenzyme or secreted enzyme in the first population of cells is significantly less than the level of activity of the ectoenzyme or secreted enzyme in the second population of cells.
  • the ectoenzyme or secreted enzyme may be selected from the group consisting of Pseudomo ⁇ as aeruginosa metalloproteinase, Moraxella (Branhamella) Catarrha/is BRO beta-lactamase, P.
  • aeruginosa FpvA ferric pyoverdin receptor £ coli OmpP endopeptidase, outer membrane phospholipase A
  • Bacteriodes thetaiotamicron susG starch utilization protein Haemophilus influenzae phospho onoesterase, streptococcal protein Sir, streptococcal C5a peptidase, Lactococcus lactis serine protease NisP, proteinase PrtB, proteinase PrtH, proteinase PrtP, proteinase ScpA, S. pneumoniae beta-N- acetylglucosaminidase, S.
  • pneumoniae neuraminidase Streptococcus sobrinus dextranase, Streptococcus suis muramidase, Streptococcus mutans exo-beta-D-fructosidase, Staphylococcus aureus murine hydrolase, staphylococcal lipases, lysostaphin, endo-beta-N-acetylglucosaminidase, sulfhydryl protease, staphylococcal esterase, S. aureus nuclease, S. aureus fatty acid modifying enzyme, chitinase, S.
  • the ectoenzyme or secreted enzyme may comprise a membrane-bound form of chitobiase.
  • the ectoenzyme or secreted enzyme may be endogenous.
  • Another embodiment of the present invention is a compound identified using the method of the preceding paragraph.
  • Another embodiment of the present invention is a method for identifying a compound which reduces the activity or level of a gene product required for proliferation of a microorganism wherein the activity or expression of the gene product is inhibited by an antisense nucleic acid, the method comprising the steps of (a) expressing a sublethal level of an antisense nucleic acid complementary to a nucleic acid encoding the gene product in a first population of cells expressing an ectoenzyme or secreted enzyme to reduce the activity or amount of the gene product in the cells, thereby producing sensitized cells (b) contacting the sensitized cells with a compound and (c) determining whether the compound alters cellular proliferation by measuring the level of activity of the ectoenzyme or secreted enzyme.
  • the method may further comprise the steps of (d) contacting a second population of cells expressing an ectoenzyme or secreted enzyme with the compound and (e) comparing the activity of the ectoenzyme or secreted enzyme in the first population of cells with the activity of the ectoenzyme or secreted enzyme in the second population of cells, wherein the compound inhibits cellular proliferation if the level or activity of the ectoenzyme or secreted enzyme in the first population of cells is significantly less than the level or activity of the ectoenzyme or secreted enzyme in the second population of cells.
  • the ectoenzyme or secreted enzyme may be selected from the group consisting of Pseudomonas aeruginosa metalloproteinase, Moraxella (Branhamella) Catarrhalis BRO beta-lactamase, P.
  • aeruginosa FpvA ferric pyoverdin receptor £ coli OmpP endopeptidase, outer membrane phospholipase A
  • Bacteriodes thetaiotamicron susG starch utilization protein Haemophilus influenzae phosphomonoesterase, streptococcal protein Sir, streptococcal C5a peptidase, Lactococcus lactis serine protease NisP, proteinase PrtB, proteinase PrtH, proteinase PrtP, proteinase ScpA, S. pneumoniae beta-N-acetylglucosaminidase, S.
  • pneumoniae neuraminidase Streptococcus sobrinus dextranase, Streptococcus suis muramidase, Streptococcus mutans exo-beta-D-fructosidase, Staphylococcus aureus murine hydrolase, staphylococcal lipases, lysostaphin, endo-beta-N-acetylglucosaminidase, sulfhydryl protease, staphylococcal esterase, S. aureus nuclease, S. aureus fatty acid modifying enzyme, chitinase, S.
  • the ectoenzyme or secreted enzyme may be a membrane-bound form of chitobiase.
  • the ectoenzyme or secreted enzyme amy be endogenous.
  • the sensitized cell may contain an introduced gene encoding the ectoenzyme or secreted enzyme.
  • the first population of cells may be from an organism selected from the group consisting of Staphylococcus aureus, Aspergillus fu igatus, Bacillus anthracis, Campylobacter j ' ejuni, Candida albicans, Chlamydia pneumoniae, Chlamydia trachomatus, Clostridium botulinum, Cryptococcus neoformans, £ coli, Enterobacter cloacae, Enterococcus faecalis, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Mycobacterium leprae, Mycobacterium tuberculosis, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Salmonella cholerasuis, Salmonella paratyphi, Salmonella typhi, Salmonella typhimurium, Staphylococcus epidermidis, Streptococcus pneumonia
  • the antisense nucleic acid may be transcribed from an inducible promoter.
  • the method may further comprise the step of contacting the first population of cells with a concentration of inducer which induces the antisense nucleic acid to a sublethal level.
  • the gene product may be a polypeptide.
  • the gene product may be an RNA. Another embodiment of the present invention is a compound identified using the method of the preceding paragraph.
  • Another embodiment of the present invention is a method for screening a test compound for activity against a gene or gene product that is essential for microbial proliferation, comprising providing a cell containing a gene encoding a gene product that is essential for microbial proliferation, wherein the cell further produces an ectoenzyme or secreted enzyme sensitizing the cell by reducing the activity or level of expression of the gene product contacting the sensitized cell with a test compound and determining whether the test compound alters cellular proliferation by mesuring the level of ectoenzyme or secreted enzyme activity.
  • the sensitizing step may comprise contacting the cell with an antisense polynucleotide that inhibits production of the gene product.
  • the ectoenzyme or secreted enzyme activity may be detected by detecting the action of the ectoenzyme or secreted enzyme on a substrate.
  • the ectoenzyme or secreted enzyme may be selected from the group consisting of Pseudomonas aeruginosa metalloproteinase, Moraxella (Branhamella) Catarrhalis BRO beta-lactamase, P.
  • aeruginosa FpvA ferric pyoverdin receptor £ coli OmpP endopeptidase, outer membrane phospholipase A
  • Bacteriodes thetaiotamicron susG starch utilization protein Haemophilus influenzae phosphomonoesterase, streptococcal protein Sir, streptococcal C5a peptidase, Lactococcus lactis serine protease NisP, proteinase PrtB, proteinase PrtH, proteinase PrtP, proteinase ScpA, S. pneumoniae beta-N-acetylglucosaminidase, S.
  • pneumoniae neuraminidase Streptococcus sobrinus dextranase, Streptococcus suis muramidase, Streptococcus mutans exo-beta-D-fructosidase, Staphylococcus aureus murine hydrolase, staphylococcal lipases, lysostaphin, endo-beta-N-acetylglucosaminidase, sulfhydryl protease, staphylococcal esterase, S. aureus nuclease, S. aureus fatty acid modifying enzyme, chitinase, S.
  • the ectoenzyme or secreted enzyme may be a membrane-bound form of chitobiase.
  • the sensitizing step may comprise contacting the cell with an agent which reduces the activity or level of a gene product required for proliferation or growth of a microorganism.
  • the agent may be a peptide or polypeptide.
  • the cell may contain a mutation which reduces the activity or level of the gene product required for proliferation of the cell.
  • the ectoenzyme or secreted enzyme may be endogenous.
  • Another embodiment of the present invention is a compound identified using the method of the preceding paragraph.
  • Another embodiment of the present invention is a method for identifying the biological pathway in which a proliferation-required gene or its gene product lies, wherein the gene or gene product comprises a gene or gene product whose activity or expression is inhibited by an antisense nucleic acid, the method comprising (a) expressing a sublethal level of an antisense nucleic acid which inhibits the activity or expression of the proliferation-required gene or gene product in a first population of cells expressing an ectoenzyme or secreted enzyme (b) contacting the first population of cells with a compound known to inhibit growth or proliferation of a microorganism, wherein the biological pathway on which the compound acts is known and (c) determining whether the compound alters cellular proliferation by measuring the level of activity of the ectoenzyme or secreted enzyme.
  • the method may further comprise (d) contacting a second population of cells expressing an ectoenzyme or secreted enzyme with the compound and (e) determining whether the first population of cells has a significantly greater sensitivity to the compound than the second population of cells by comparing the activity of the ectoenzyme or secreted enzyme expressed by the first and second population of cells.
  • the ectoenzyme or secreted enzyme may be selected from the group consisting of Pseudomonas aeruginosa metalloproteinase, Bacteriodes thetaiotamicron susG starch utilization protein, Haemophilus influMoraxella (Branhamella) Catarrhalis BRO beta-lactamase, P.
  • aeruginosa FpvA ferric pyoverdin receptor £ coli OmpP endopeptidase, outer membrane phospholipase A, enzae phosphomonoesterase, streptococcal protein Sir, streptococcal C5a peptidase, Lactococcus lactis serine protease NisP, proteinase PrtB, proteinase PrtH, proteinase PrtP, proteinase ScpA, S. pneumoniae beta-N-acetylglucosaminidase, S.
  • pneumoniae neuraminidase Streptococcus sobrinus dextranase, Streptococcus suis muramidase, Streptococcus mutans exo-beta-D-fructosidase, Staphylococcus aureus murine hydrolase, staphylococcal lipases, lysostaphin, endo-beta-N-acetylglucosaminidase, sulfhydryl protease, staphylococcal esterase, S. aureus nuclease, S. aureus fatty acid modifying enzyme, chitinase, S.
  • the ectoenzyme or secreted enzyme may be a membrane-bound form of chitobiase.
  • the ectoenzyme or secreted enzyme may be endogenous.
  • Another embodiment of the present invention is a method for determining the biological pathway on which a test compound acts comprising (a) expressing a sublethal level of an antisense nucleic acid complementary to a proliferation-required nucleic acid in a first population of cells expressing an ectoenzyme or secreted enzyme, wherein the activity or expression of the proliferation-required nucleic acid is inhibited by the antisense nucleic acid and wherein the biological pathway in which the proliferation-required nucleic acid or a protein encoded by the proliferation-required polypeptide lies is known (b) contacting the first population of cells with the test compound and (c) determining whether the compound alters cellular proliferation by measuring the level of activity of the ectoenzyme or secreted enzyme.
  • the method may further comprise (d) contacting a second population of cells with the test compound and (e) determining whether the first population of cells has a significantly greater sensitivity to the test compound that the second population of cells by comparing the activity of the ectoenzyme or secreted enzyme expressed by the cell populations.
  • the ectoenzyme or secreted enzyme may be selected from the group consisting of Pseudomonas aeruginosa metalloproteinase, Moraxella (Branhamella) Catarrhalis BRO beta-lactamase, P.
  • aeruginosa FpvA ferric pyoverdin receptor £ coli OmpP endopeptidase, outer membrane phospholipase A
  • Bacteriodes thetaiotamicron susG starch utilization protein Haemophilus influenzae phosphomonoesterase, streptococcal protein Sir, streptococcal C5a peptidase, Lactococcus lactis serine protease NisP, proteinase PrtB, proteinase PrtH, proteinase PrtP, proteinase ScpA, S. pneumoniae beta-N-acetylglucosaminidase, S.
  • pneumoniae neuraminidase Streptococcus sobrinus dextranase, Streptococcus suis muramidase, Streptococcus mutans exo-beta-D-fructosidase, Staphylococcus aureus murine hydrolase, staphylococcal lipases, lysostaphin, endo-beta-N-acetylglucosaminidase, sulfhydryl protease, staphylococcal esterase, S. aureus nuclease, S. aureus fatty acid modifying enzyme, chitinase, S.
  • the ectoenzyme or secreted enzyme may be a membrane-bound form of chitobiase.
  • the ectoenzyme or secreted enzyme may be endogenous.
  • the method may further comprise (f) expressing a sublethal level of a second antisense nucleic acid complementary to a second proliferation- required nucleic acid in a third population of cells, wherein the second proliferation-required nucleic acid is in a different biological pathway than the proliferation-required nucleic acid in step (a) and (g) determining whether the third cell does not have a significantly greater sensitivity to the test compound than a cell which does not express the sublethal level of the second antisense nucleic acid, wherein the test compound is specific for the biological pathway against which the antisense nucleic acid of step (a) acts if the third cell does not have significantly greater sensitivity to the test compound.
  • Another embodiment of the present invention is a method for manufacturing an antibiotic comprising the steps of screening one or more candidate compounds to identify a compound that reduces the activity or level of a gene product required for proliferation, wherein the effect of the compound on proliferation is determined by measuring the activity of an ectoenzyme or secreted enzyme expressed by the cell and manufacturing the compound so identified.
  • the ectoenzyme or secreted enzyme may be selected from the group consisting of Pseudomonas aeruginosa metalloproteinase, Moraxella (Branhamella) Catarrhalis BRO beta-lactamase, P.
  • aeruginosa FpvA ferric pyoverdin receptor £ coli OmpP endopeptidase, outer membrane phospholipase A
  • Bacteriodes thetaiotamicron susG starch utilization protein Haemophilus influenzae phosphomonoesterase, streptococcal protein Sir, streptococcal C5a peptidase, Lactococcus lactis serine protease NisP, proteinase PrtB, proteinase PrtH, proteinase PrtP, proteinase ScpA, S. pneumoniae beta-N-acetylglucosaminidase, S.
  • pneumoniae neuraminidase Streptococcus sobrinus dextranase, Streptococcus suis muramidase, Streptococcus mutans exo-beta-D-fructosidase, Staphylococcus aureus murine hydrolase, staphylococcal lipases, lysostaphin, endo-beta-N-acetylglucosaminidase, sulfhydryl protease, staphylococcal esterase, S. aureus nuclease, S. aureus fatty acid modifying enzyme, chitinase, S.
  • the ectoenzyme or secreted enzyme may be a membrane-bound form of chitobiase.
  • the gene product may comprise a gene product whose activity or expression is inhibited by an antisense nucleic acid.
  • the ectoenzyme or secreted enzyme may be endogenous.
  • Another embodiment of the present invention is a method for identifying nucleic acids which inhibit cellular proliferation, comprising the steps of transcribing a first level of a nucleic acid in a first population of cells expressing a gene encoding an ectoenzyme or secreted enzyme and comparing the activity of the ectoenzyme or secreted enzyme in the first population of cells to the activity of the ectoenzyme or secreted enzyme in a second population of cells expressing the ectoenzyme or secreted enzyme, wherein the second population of cells transcribes the nucleic acid at a lower level than the first population of cells, or does not transcribe the nucleic acid, wherein the nucleic acid inhibits proliferation if the activity of the ectoenzyme or secreted enzyme is significantly less in the first population of cells than in the second population of cells.
  • the nucleic acid may be a random gen ⁇ mic fragment.
  • the nucleic acid may be an antisense nucleic acid.
  • the nucleic acid may be a sense nucleic acid which encodes a peptide or polypeptide.
  • the peptide or polypeptide may comprise a peptide or polypeptide that is normally expressed in the cell.
  • the nucleic acid may encode an RNA comprising an RNA that is normally expressed inside the cell.
  • the ectoenzyme or secreted enzyme may be selected from the group consisting of Pseudomonas aeruginosa metalloproteinase, Moraxella (Branhamella) Catarrhalis BRO beta-lactamase, P.
  • aeruginosa FpvA ferric pyoverdin receptor £ coli OmpP endopeptidase, outer membrane phospholipase A
  • Bacteriodes thetaiotamicron susG starch utilization protein Haemophilus influenzae phosphomonoesterase, streptococcal protein Sir, streptococcal C5a peptidase, Lactococcus lactis serine protease NisP, proteinase PrtB, proteinase PrtH, proteinase PrtP, proteinase ScpA, S. pneumoniae beta-N- acetylglucosaminidase, S. pneumoniae neuraminidase.
  • Streptococcus sobrinus dextranase Streptococcus suis muramidase, Streptococcus mutans exo-beta-D-fructosidase, Staphylococcus aureus murine hydrolase, staphylococcal lipases, lysostaphin, endo-beta-N-acetylglucosaminidase, sulfhydryl protease, staphylococcal esterase, S. aureus nuclease, S. aureus fatty acid modifying enzyme, chitinase, S.
  • the ectoenzyme or secreted enzyme may be a membrane-bound form of chitobiase.
  • the ectoenzyme or secreted enzyme may be endogenous.
  • the nucleic acid may be transcribed from an inducible promoter.
  • the transcribed nucleic acid may be a recombinant nucleic acid that has been introduced into the first and second populations of cells.
  • FIG. 1 is a diagram of plasmid pJFK4 comprising the wild type chitobiase (chb) gene encoding the membrane-bound form of chitobiase, and the £ coli chromosomal attB site. Restriction sites shown are unique with the exception of Sac ⁇ of which there are two sites created by the construction of the plasmid, and Not ⁇ , of which there are two sites flanking the P15A origin.
  • chb wild type chitobiase
  • FIG. 1 is a schematic diagram showing the integration of the wild type chb gene into the £ coli chromosome by site-specific recombination between attB and attP.
  • Figure 3 is a graph showing that cells carrying the integrated wild type chitobiase gene can be detected with greater sensitivity than turbidity measurements.
  • Control cells pLEX5BA
  • cells containing the integrated chb gene DJKGC4 were stopped for growth, diluted to 0.2 OD 6D0 serially diluted and a fluorescent chitobiase substrate was added.
  • Relative fluorescence units RRU were charted after a 2 hour incubation at room temperature. Fluorescence was clearly detectable above background for cultures calculated to have an 0D 600 of 0.0016 and 0.00032, below detectable limits of common-use spectrophotometers.
  • Figure 4 is a graph comparing the sensitivity of measurement of the growth of £ coli in a 1536-well microplate by turbidity at OD 600 and chitobiase activity was determined by measuring release of p- ⁇ itrophenol from the substrate PNAG by monitoring 0D 415 .
  • Figure 5 is a graph showing the measurement of £ coli growth in a 1536-well microplate using a chitobiase assay in £ coli strain DJ GC4 which contain a chromosomally integrated, constitutively expressed chitobiase gene.
  • the parental chitobiase negative £ co/ strain of DJKGC4 is MG1655.
  • Figure 6 is an £ coli dose response curve to gentamicin.
  • FIG. 7 is a graph showing that sarkosyl, sodium chloride and the combination of sarkosyl and sodium chloride increase the sensitivity of detection of cells by the chitobiase assay.
  • Figure 8 is a graph showing sensitive detection of cell growth using the chitobiase assay.
  • MG1655 £ coli cells transfected with the pJFK4 plasmid were grown in LB medium to an OD 600 of 0.2-0.3.
  • Cells were diluted into M9 media (M9 salts supplemented with 0.4% glucose, 0.02 mg/ml uracil, 0.005 mg/ml each of thymine and thiamine, 1 M MgS0 4 and 0.1 mM CaCI 2 ), with or without 1 mM PNAG to a final OD 600 of 0.002.
  • FIG. 9 is an IPTG dose response curve in £ coli transformed with an IPTG-inducible plasmid containing either an antisense clone to the £ coli ribosomal protein rplW which is essential for cell proliferation, or an antisense clone to the elaD gene which is not essential for proliferation.
  • Figure 10A is a tetracycline dose response curve in £ coli transfected with an IPTG-inducible plasmid containing antisense to rplW ' m the presence of 0.20 or 50 ⁇ M IPTG.
  • Figure 10B is a tetracycline dose response curve in £ coli transfected with an IPTG-inducible plasmid containing antisense to elaD in the presence of 0, 20 or 50 ⁇ M IPTG.
  • Figure 11 is a graph showing the fold increase in tetracycline sensitivity of £ co// transfected with antisense clones to essential ribosomal proteins L23 (t&rplW) and L7/L12 and L10 (hSrplLrplA Antisense clones to genes known not to be involved in protein synthesis (atpB/E, visC, elaD, yohH] are much less sensitive to tetracycline.
  • the present invention relates to the use of ectoenzymes or secreted enzymes to measure microbial proliferation.
  • ectoenzyme any enzyme which is associated with a cell either covalently or non- covalently such that its active site is available to compounds which are on the exterior of the cell.
  • these ectoenzymes are membrane-bound proteins.
  • Some ectoenzymes are attached to the cell wall (Navarre et al., MicrobioL Mol. Biol. Rev. 63:174-229, 1999).
  • the ectoenzyme is linked to the bacterial cell wall through another molecule, such as the protein encoded by the srtA gene of Gram-positive bacteria (Mazmanian et al., Science 285:760-763, 1999).
  • Secreted enzymes also can be converted into ectoenzymes which are anchored to the cell wall by addition of an appropriate sequence at their C-terminus.
  • the C-terminal 35 residues of protein A comprising an LPXTG (SEQ ID NO: 1) sequence motif, hydrophobic domain and charged tail (Navarre et al., supra.) may be linked to the C-terminus of the secreted protein to link the secreted protein to the cell wall.
  • chitobiase is used as an exemplary ectoenzyme herein, it will be appreciated by one of ordinary skill in the art that other ectoenzymes are also suitable for use in the present invention, including, but not limited to, Pseudomonas aeruginosa metalloproteinase (Fricke et al., Biochim. Biophys. Acta. 1454:236-250, 1999), Moraxella (Branhamella) catarrhalis BRO beta-lactamase (Bootsma et al., J. Bacteriol 181:5090-5093, 1999), P.
  • the ectoenzyme may be an endogenous ectoenzyme or an exogenous ectoenzyme introduced using genetic engineering methods. It will also be appreciated that ectoenzymes other than chitobiase may be substituted for chitobiase in each of the embodiments discussed below.
  • the enzyme is a bacterial ectoenzyme.
  • the ectoenzyme is a membrane-bound form of chitobiase. The - membrane-bound form of chitobiase may be the native form of chitobiase or may be generated, for example, via genetic engineering or microbial selection techniques.
  • Chitobiase normally has its own signal peptide which directs it to the cell membrane.
  • DNA encoding the native signal sequence of chitobiase may be exchanged for DNA encoding a heterologous signal peptide.
  • Those in the art will further appreciate that almost any enzyme could be expressed as an ectoenzyme by addition of appropriate signal sequences to ensure its secretion and the appropriate anchoring sequence such as a membrane anchor or cell wall attachment signal to ensure that at least a portion of the enzyme extends into the extracellular milieu.
  • signal sequences, membrane anchors and cell wall attachment signals are familiar to those skilled in the art.
  • secreted enzymes may also be used in each of the embodiments discussed below.
  • Secreted enzymes are enzymes which are secreted into the medium or environment in which the cells are growing.
  • the secreted enzyme may be an endogenous enzyme or an exogenous enzyme introduced using genetic engineering methods.
  • Secreted enzymes suitable for use in the methods described below include, but are not limited to, Streptococcus mutans exo-beta-D-fructosidase (Igarashi, MicrobioL Immunol. 36:643-647, 1992), Staphylococcus aureus murein hydrolase (Groicher et al., J.
  • aureus nucleases A and B (Suciu et al., Mol. MicrobioL 21:181-195, 1996), S. aureus fatty acid modifying enzyme (FAME) (Chamberlain et al., J. Med. MicrobioL 44:125-129, 1996); bacterial chitinases (Hayashi et al., Biosci. Biotechnol. Biochem. 59:1981-1982, 1995); S. aureus autolysin (Proc. Natl. Acad. Sci. U.S.A.
  • the secreted enzymes may be naturally-occurring.
  • an enzyme which is not naturally secreted can be made into a secreted protein by insertion into a secretion vector adjacent to a signal sequence which will direct its secretion.
  • Secretion vectors are used routinely in the art to generate a secreted form of a desired protein.
  • the signal sequence fused to a coding region of a protein of interest will function regardless of the coding region to which it is fused.
  • Secretion vectors are described by Murphy et al. (Protein Expr. Purif. 4:349-357, 1993; Sivaprasadarao et al., Biochem. J. 296:209-215, 1993).
  • secretion vectors are commercially available.
  • Invitrogen Carlsbad, CA
  • pBAD/glll kit which is designed to express recombinant proteins in £ coli.
  • the leader peptide from the bacteriophage fd gene III protein (gill) directs the secretion of the polypeptide encoded by any adjacent sequence into the periplasmic space.
  • pSecTag2 and pSecTag2/Hygro are secretion vectors for use in mammalian host cells in which a mouse secretion signal directs secretion of the polypeptide encoded by any adjacent sequence.
  • Ectoenzymes may be used for monitoring proliferation of bacterial cells, plant cells, mammalian cells and other cell types.
  • a genetic construct comprising a nucleic acid encoding an ectoenzyme or a non-secreted enzyme adjacent a signal sequence is introduced into a population of cells, and the number of cells in the population is determined by measurement of ectoenzyme or secreted enzyme activity using substrates which result in a detectable product, such as a colored product, fluorescent product or luminescent product.
  • the proliferation of cells which have been contacted with a compound is compared to the proliferation of cells which were not contacted with the compound to determine whether the compound affects proliferation of the cells.
  • the cells are sensitized as discussed below.
  • the ectoenzyme is chitobiase.
  • Chitobiase is one of two enzymes that hydrolyze chitin, an abundant insoluble polysaccharide, to its monomeric unit, /IZ-acetylglucosamine (GlcNac). Chitobiase is known to be present in a number of organisms.
  • the chitobiase enzyme is known to be present in various genera including Arabidopsis, Bacillus, Bombyx, Bos, Caenorhabditis, Candida, Dictyostelium, Entamoeba, Felis, Homo, Korat, Lactobacillus, Leishmania, Mus, Pisum, Porphyromonas, Pseudoalteromonas, Rattus, Serratia, Streptomyces, Sus, Trichoderma, and Vibrio.
  • Specific examples of organisms known to contain chitobiase include Alteromonas sp.
  • chitobiase is the marine bacterium, Vibrio harveyi.
  • Escherichia coli cells harboring a plasmid carrying the chb gene from Vibrio harveyi were reported to produce the enzyme, which was found to be associated with the outer membrane of the bacterial cells (Jannatipour et al., J. Bacteriol. 169:3785-3791, 1987; Soto-Gil et al., J. Biol Chem. 264:14778-14782, 1989).
  • an advantage of the assays for measuring the activity of ectoenzymes, such as membrane-bound chitobiase, or of secreted enzymes is that the cells need not be permeabilized prior to the assay.
  • the ectoenzyme or secreted enzyme substrate may be added directly to intact cells.
  • the substrate may also be added to the medium in which the cells are growing or to a supernatant obtained by removing the cells from the growth medium.
  • the assay may be a "homogeneous assay" in which washing steps are not required. Standard per eabilization techniques such as sonication, freeze-thaw, treatment with organic compounds and detergent lysis are time-consuming and can inhibit enzyme activity.
  • the absence of cell lysis and washing procedures significantly increases the sensitivity of the assay.
  • assays performed in the absence of detergent are easier to automate such as for high throughput screening, and assays performed in intact cells allow real time determination of cell number in growing cultures which are difficult to perform in permeabilized cells.
  • the membrane-bound chitobiase assay described herein is extremely sensitive, facilitating miniaturization and automation of the assay because large numbers of cells are not required.
  • the present invention also relates to various protein expression vectors that can be used to express membrane-bound chitobiase.
  • the structure of a construct encoding an ectoenzyme, such as a membrane-bound form of chitobiase, or a secreted enzyme, will vary according to its purposes.
  • constructs are prepared according to standard techniques of molecular biology well known in the art.
  • the vector may integrate the gene encoding the ectoenzyme into the host's genome or may be extrachromosomal, such as a plasmid.
  • Extrachromosomal constructs can contain an origin of replication with activity in the host cell of interest. This feature provides the ability to replicate within the host cell in which it has been introduced.
  • the construct may contain sequences that will facilitate incorporation.
  • Constructs may also contain a promoter for expressing the gene encoding an ectoenzyme, a multiple cloning site, and a selectable marker.
  • the promoter may be a heterologous promoter from a gene other than the ectoenzyme gene or may be the natural promoter from the gene encoding the ectoenzyme.
  • Constructs for use in eukaryotic cells may also contain a polyA site adjacent to the gene encoding the ectoenzyme or secreted enzyme.
  • integration sequences that can be included in a construct encoding the ectoenzyme or secreted enzyme is the ⁇ attP site. This site permits a single copy of the gene encoding the ectoenzyme or secreted enzyme to be incorporated into a host bacterial genome.
  • Integration-promoting sequences with utility in mammalian cells include the long terminal repeats found in retroviral genomes. These sequences promote viral genome integration in a host genome and have been used extensively by those of skill in the art to promote the integration of exogenous sequences in mammalian host cells.
  • the gene encoding the ectoenzyme such as membrane-bound chitobiase, or secreted enzyme
  • a constitutive promoter for obtaining constant gene expression.
  • the gene encoding the ectoenzyme, such as membrane-bound chitobiase, or secreted enzyme is operably linked to an inducible promoter for providing variable levels of expression.
  • the gene encoding the ectoenzyme, such as membrane-bound chitobiase, or secreted enzyme is operably linked to a tissue-specific promoter for obtaining gene expression in particular cell and tissue types. Such promoters are well known in the art.
  • kits One aspect of this embodiment includes a construct encoding an ectoenzyme such as membrane-bound chitobiase, or a secreted enzyme.
  • the construct also contains a multiple cloning site containing a variety of restriction endonuclease cutting sites that facilitate the introduction of exogenous DNA into the construct.
  • the kit embodiment of the present invention also includes those components necessary to assay for ectoenzyme activity or secreted enzyme activity produced by the gene construct.
  • the kit will include a supply of a suitable chitobiase substrate whose metabolism into product by the enzyme can be assayed.
  • constructs encoding an ectoenzyme such as a membrane-bound form of chitobiase, or a secreted enzyme, may be introduced into prokaryotic or eukaryotic cells.
  • ectoenzymes such as membrane-bound chitobiase, or a secreted enzyme
  • the constructs may be introduced into bacteria using calcium chloride transformation, electroporation, or viral vectors such as the filamentous phages.
  • the constructs encoding an ectoenzyme may be introduced into eukaryotic cells, including yeast, mammalian, plant, and insect cells.
  • the sequence encoding an ectoenzyme, such as membrane-bound chitobiase, or a secreted enzyme may be inserted into a yeast artificial chromosome, a yeast plasmid, a bovine papilloma virus vector or other extrachromosomal vector, a retroviral vector, a Ti-plasmid, or a baculovirus vector.
  • yeast artificial chromosome a yeast plasmid
  • bovine papilloma virus vector or other extrachromosomal vector a retroviral vector
  • Ti-plasmid or a baculovirus vector.
  • baculovirus vector A variety of such vectors are known to those skilled in the art.
  • the vectors may be introduced into any of the yeast, mammalian, plant, and insect cells familiar to those skilled in the art
  • transfections can be transient or stable.
  • suitable transfer protocols include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection, and viral transfection.
  • the enzymatic activity of the enzyme is measured.
  • the assays are performed on intact cells expressing the ectoenzyme on the cell surface or secreting the secreted enzyme into the medium.
  • Ectoenzyme assays may also be performed on cell membrane fractions produced by methods well known in the art.
  • cellular chitobiase activity can be measured quantitatively by following the hydrolysis of chitobiase substrates.
  • substrates with utility in chitobiase activity assays include N,N'- diacetylchitobiase (chitobiase), -nitrophenyl-V-acetyl- ⁇ -D-glucosaminide (PNAG)(Sigma Chemical, St.
  • Products produced by the hydrolysis of the chitobiase substrates are monitored using various means familiar to those skilled in the art.
  • various optical means are known to those skilled in the art.
  • One such optical means may comprise detection of chemiluminescent or fluorescent products released from a substrate, measuring the amount of light absorbed by a product produced from a substrate, or measuring a decrease in the amount of a detectable substrate.
  • /7-nitrophenol is released from the substrate and measured colorimetrically at 400 nm.
  • fluorescence excitation and emission of the fluorescent substrate MNAG is measured at 360 nm and 425 nm, respectively.
  • Other monitoring methods well known in the art can be used to quantitate signals produced in the chitobiase assay. These may include use of radioactive substrates or substrates having radio frequency tags.
  • blue/white colony indicator plates are used to monitor enzyme activity.
  • the membrane-bound chitobiase gene construct of the invention can also be used for measuring cell number.
  • cells are transfected with an expression vector encoding membrane- bound chitobiase and the level of chitobiase activity is assayed on intact cells or cell membrane preparations. The higher the chitobiase activity, the greater the number of cells in the sample.
  • a standard curve may be constructed using known numbers of cells transfected with the gene encoding membrane-bound chitobiase.
  • relative measurements of chitobiase activity may be used to compare cellular proliferation in multiple samples. Cell number is determined using a chitobiase assay as described herein.
  • each cell in the cell population is similar.
  • each cell may contain an identical number of genes encoding chitobiase in its genome.
  • the cells may contain a single copy of a gene encoding chitobiase in its genome.
  • the cells may each contain a similar or identical number of multicopy plasmids encoding chitobiase.
  • the chitobiase assay is performed on cells which have not been lysed or permeabilized.
  • the substrate is placed in contact with cells expressing membrane-bound chitobiase and chitobiase activity is measured without performing washing steps.
  • cells expressing an ectoenzyme such as membrane-bound chitobiase, or a secreted enzyme
  • a test-cell population such as a microbial, plant, fungal or animal cell population, which expresses the ectoenzyme or secreted enzyme, is grown in the presence of a candidate compound.
  • the candidate compound may be a compound produced using combinatorial chemical syntheses.
  • a control cell population such as a microbial, plant, fungal, or animal cell population, which expresses the ectoenzyme, such as membrane-bound chitobiase, is grown in the absence of the candidate compound.
  • Assays are performed on the test-cell population and the control population to determine the level of ectoenzyme in each population. If the level of ectoenzyme or secreted enzyme activity in the test-cell population is significantly less than the level in the control population, the candidate compound inhibits proliferation and may be used as a drug to inhibit cellular proliferation.
  • a difference of at least 2, at least 10, at least 20, at least 50, at least 100 or more than 100 fold in the level of ectoenzyme or secreted enzyme activity in the test cell population relative to the control cell population may constitute a significant difference for the purposes of determining whether the compound inhibits proliferation.
  • the ability of the cell-based assays to identify compounds which inhibit proliferation is enhanced by increasing the sensitivity of cells expressing an ectoenzyme such as membrane-bound chitobiase, or a secreted enzyme, to potential inhibitors of the target of interest.
  • an ectoenzyme such as membrane-bound chitobiase, or a secreted enzyme
  • the target cells are sensitized by reducing expression or activity of a proliferation-required gene to the point where the presence or absence of its function becomes the rate determining step for cellular proliferation.
  • Bacterial, fungal, plant, or animal cells can all be used with the present method.
  • target molecules are proteins such as enzymes, receptors and the like.
  • target molecules may also include other molecules such as DNAs, lipids, carbohydrates and RNAs including messenger RNAs, ribosomal RNAs, tRNAs and the like.
  • a number of highly sensitive cell-based assay methods are available to those of skill in the art to detect binding and interaction of test compounds with specific target molecules. However, these methods are generally not highly effective when the test compound binds to or otherwise interacts with its target molecule with moderate or low affinity.
  • the target molecule may not be readily accessible to a test compound in solution, such as when the target molecule is located inside the cell or within a cellular compartment such as the periplasm of a bacterial cell.
  • current cell- based assay methods are limited in that they are not effective in identifying or characterizing compounds that interact with their targets with moderate to low affinity or compounds that interact with targets that are not readily accessible.
  • the effectiveness of the cell-based assays may be further augmented by employing an ectoenzyme or a secreted enzyme.
  • the cell-based assay methods using cells expressing an ectoenzyme have substantial advantages over current cell-based assays when used in a context in which the level or activity of at least one proliferation-required gene product (the target molecule) has been specifically reduced to the point where the presence or absence of its function becomes a rate-determining step for cellular proliferation.
  • Bacterial, fungal, plant, or animal cells can all be used with the present method. Such sensitized cells become much more sensitive to compounds that are active against the affected target molecule.
  • cell-based assays using cells expressing an ectoenzyme are capable of detecting compounds exhibiting low or moderate potency against the target molecule of interest because such compounds are substantially more potent on sensitized cells than on non-sensitized cells.
  • the effect may be such that a test compound may be two to several times more potent, at least 10 times more potent, at least 20 times more potent, at least 50 times more potent, or even at least 100 times more potent when tested on the sensitized cells as compared to the non-sensitized cells.
  • sensitized cells of the current invention which express an ectoenzyme or secreted enzyme provides a solution to the above problem in two ways.
  • desired compounds acting at a target of interest whether a new target or a previously known but poorly exploited target, can now be detected above the "noise" of compounds acting at the "old” targets due to the specific and substantial increase in potency of such desired compounds when tested on the sensitized cells of the current invention.
  • the methods used to sensitize cells to compounds acting at a target of interest may also sensitize these cells to compounds acting at other target molecules within the same biological pathway.
  • an antisense molecule to a gene encoding a ribosomal protein is expected to sensitize the cell to compounds acting at that ribosomal protein and may also sensitize the cells to compounds acting at any of the ribosomal components (proteins or rRNA) or even to compounds acting at any target which is part of the protein synthesis pathway.
  • an important advantage of the present invention is the ability to reveal new targets and pathways that were previously not readily accessible to drug discovery methods.
  • Sensitized cells of the present invention are prepared by reducing the activity or level of a target molecule.
  • the target molecule may be a gene product, such as an RNA or polypeptide produced from the proliferation-required nucleic acids described herein.
  • the target may be a gene product such as an RNA or polypeptide which is produced from a sequence within the same operon as the proliferation-required nucleic acids described herein.
  • the target may be an RNA or polypeptide in the same biological pathway as the proliferation-required nucleic acids described herein.
  • biological pathways include, but are not limited to, enzymatic, biochemical and metabolic pathways as well as pathways involved in the production of cellular structures such the cell wall.
  • the sensitized cells contain a gene encoding a membrane-bound form of chitobiase.
  • the gene encoding an ectoenzyme, such as a membrane-bound form of chitobiase, or a secreted enzyme may be on a chromosome or in an extrachromosomal vector.
  • This information is used to design subsequent directed libraries containing compounds with enhanced activity against the target molecule. After one or several iterations of this process, compounds with substantially increased activity against the target molecule are identified and may be further developed as drugs. This process is facilitated by use of the sensitized cells expressing an ectoenzyme, such as a membrane-bound form of chitobiase, or a secreted enzyme, since compounds acting at the selected targets exhibit increased potency in such cell-based assays, thus; more compounds can now be characterized providing more useful information than would be obtained otherwise.
  • an ectoenzyme such as a membrane-bound form of chitobiase, or a secreted enzyme
  • cell-based assays of the present invention identify or characterize compounds that previously would not have been readily identified or characterized including compounds that act at targets that previously were not readily exploited using cell-based assays.
  • the process of evolving potent drug leads from initial hit compounds is also substantially improved by the cell-based assays of the present invention because, for the same number of test compounds, more structure-function relationship information is likely to be revealed.
  • the method of sensitizing a cell entails selecting a suitable gene or operon.
  • a suitable gene or operon is one whose expression is required for the proliferation of the cell to be sensitized.
  • the next step is to introduce an antisense RNA capable of hybridizing to the suitable gene or operon or to the RNA encoded by the suitable gene or operon into the cells to be sensitized.
  • Introduction of the antisense RNA can be in the form of an expression vector in which antisense RNA is produced under the control of an inducible promoter.
  • the amount of antisense RNA produced is regulated by varying the inducer concentration to which the cell is exposed and thereby varying the activity of the promoter driving transcription of the antisense RNA.
  • cells are sensitized by exposing them to an inducer concentration that results in a sub-lethal level of antisense RNA expression.
  • sensitized cells expressing an ectoenzyme or secreted enzyme are contacted with compounds to be tested for the ability to inhibit proliferation.
  • compounds to be tested for the ability to inhibit proliferation Preferably, a large number of compounds are tested for the ability to inhibit proliferation.
  • the test compounds may be generated using combinatorial chemistry or may be a library of naturally occuring compounds. The ability of the test compounds to inhibit proliferation is determined by measuring the level of activity of the ectoenzyme or secreted enzyme.
  • Those compounds which result in reduced levels of ectoenzyme or secreted enzyme activity are then tested for their specificity for the proliferation-required gene product whose level or activity was reduced in the sensitized cell by comparing the level of ectoenzyme or secreted enzyme activity in sensitized cells contacted with the compound to the level of ectoenzyme or secreted enzyme activity in unsensitized cells contacted with the compound.
  • the compound is acting on the proliferation-required gene product whose level or activity was reduced in the sensitized cells or a gene product which lies in the same biological pathway as the proliferation-required gene product whose level or activity was reduced in the sensitized cells.
  • a large number of compounds is initially screened to identify those compounds that inhibit proliferation and subsequently the inhibitory compounds are screened to identify those which act on the gene product whose level or activity was reduced in the sensitized cells or a gene product in the same biological pathway as the gene product whose level or activity was reduced.
  • both sensitized and unsensitized cells are initially contacted with a large number of compounds and those compounds which act on a gene product whose level or activity was reduced in sensitized cells or a gene product in the same biological pathway as the proliferation-required gene product whose level or activity was reduced are identified by comparing the effects of the test compound on the sensitized and unsensitized cells as described above.
  • a single screening step is performed to identify those compounds which act on the gene product whose level or activity was reduced or a gene product in the same biological pathway as the gene product whose level or activity was reduced.
  • EXAMPLE 1 Construction of chitobiase integration plasmid pJFK4 (Fig. 1; SEQ ID NO: 4) was constructed by ligating a Sac ⁇ digested PCR product containing the wild type (WT) chitobiase promoter and additional 5' open reading frame (ORF) sequence into the Sac ⁇ site of a variant of pJMF4 (BioTechniques 25:1030, 1998) which contains a 146 base pair (bp) Asel-Sa ⁇ deletion, removing the promoter. Proper orientation of the Sac ⁇ fragment was determined by both restriction digest and chitobiase assay.
  • pRSG192 J. Biol. Chem.
  • the electroporated cells were incubated at 42°C with shaking for 30 min, then moved to 37°C for 1 hour, followed by selection on LB agar plates containing 25 ⁇ g/ml chloramphenicol at 42°C. Transformants were screened both for chitobiase activity and loss of kanamycin resistance, and therefore loss of pLDR ⁇ .
  • Chitobiase activity is located on the surface of cells which express the gene encoding the native, membrane- bound protein. Accordingly, chitobiase assays may be performed on intact cells, lysed cells or cell membrane fractions. Membrane fractions may be prepared using well known techniques.
  • the cells were diluted to an OD eoo of 0.2 in M9-DB, then serially diluted five-fold in duplicate in a 96 well white microtiter plate (black plates are also suitable) to a final 0D 600 of 0.000064, lOO ⁇ l each.
  • the plate was then read in an LJL Analyst spectrofluorimeter using an excitation wavelength of 360 nm and an emission wavelength of 425 nm. Readings were performed every 5 minutes for 2 hours.
  • chitobiase activity is assayed colorimetrically by the release of //-nitrophenol at 400 nm from the substrate p-nitrophenyl-N-acetyl- ⁇ -D-glucopyranoside (PNAG), and turbidity is measured at 550 nm.
  • PNAG p-nitrophenyl-N-acetyl- ⁇ -D-glucopyranoside
  • turbidity is measured at 550 nm.
  • p- Nitrophenol release is measured immediately at 400 nm with a molar absorptivity of 1.8 x 10 3 liters mol '1 cm '1 . Units are calculated after subtracting the light scattering factor (1.5 x 0D 550 ) from 0D 4 ⁇ 0 of the sample. The normalizing factor of 1.5 was determined previously by measuring the light scattering ratio of bacteria at OD 400 and OD 550 .
  • One unit of chitobiase activity is the amount of enzyme that catalyzes the formation of 1 pmol of / -nitrophenol per min at 28°C.
  • Miller units of ⁇ -galactosidase described in Miller, J. H., A Short Course in Bacterial Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1992)
  • the units are normalized to 1 ml of culture at 0D 450 - 1.
  • Constructs encoding ectoenzymes other than chitobiase may also be used to measure cellular proliferation in the methods described herein.
  • the activities of the ectoenzymes H. influenzae outer membrane phosphomonoesterase e, SusG and neuraminidase may be measured as described in Examples 4-6 below.
  • Constructs encoding secreted enzymes may also be used to measure cellular proliferation in the methods described herein, For example, the activities of the secreted enzymes ⁇ -N-acetylglucosaminidase, fatty acid modifying enzyme and Staphylococcus esterase may be measured as described in Examples 7-9 below.
  • the activity of secreted enzymes may be measured by contacting a culture of cells expressing the secreted enzyme with a substrate which yields a detectable product when acted upon by the secreted enzyme.
  • a substrate which yields a detectable product when acted upon by the secreted enzyme.
  • medium or supernatants obtained from cultures of cells expressing the secreted enzyme may be contacted with a substrate which yields a detectable product when acted upon by the secreted enzyme.
  • Enzyme activity is determined using the discontinuous colorimetric assay described by Reilly et al. (Protein Expression and Purification 17:401-409, 1999.
  • the 0.2-ml standard assay mixture contains 0.2 M sodium acetate, pH. 5.5, 0.1 mM CuS0 , 1.0 mM p-nitrophenylphosphate (pNPP), and varying amounts of H. influenzae.
  • the mixtures are incubated at 37°C for 15 min with constant agitation.
  • the reaction is stopped by addition of 100 ⁇ l 0.5 M glycine, pH 10.0.
  • the concentration of p-nitrophenol produced is measured with a microplate reader at 410 nm using an extinction coefficient of 18.3 ⁇ 0.2 mM ⁇ cm '1 .
  • One unit of enzyme activity is defined as the amount of activity required to convert 1 nmol substrate to product per hour at 37°C.
  • S. pneumoniae neuraminidase assay S. pneumoniae neuraminidase assay is performed as described by Camara et al. (Infect. Immun. 62:3688-
  • S. pneumoniae cells or membrane preparations are mixed with an equal volume of 0.35% (w/v) of the fluorogenic substrate 2'-(4-methylumbelliferyl)- ⁇ -D-N-acetylneuraminic acid (MUAN) (Sigma).
  • MUAN fluorogenic substrate 2'-(4-methylumbelliferyl)- ⁇ -D-N-acetylneuraminic acid
  • the reaction mixture is incubated for 5 min at 37°C, and the reaction is stopped by the addition of 2 ml of 50 mM sodium carbonate buffer, pH 9.6. Fluorescence resulting from the release of 4-methylumbelliferone from MUAN is detected by using a Perkin- Elmer LS2B fluorimeter at an excitation wavelength of 366 nm and an emission wavelength of 446 nm.
  • the reactants are desalted, and hydrolysis is monitored by Dionex HPAEC (Dionex BioLC system) using a CarboPac PA-1 column eluted at 1 ml/min with 150 mM NaOH, 30 mM NaOAC, and the reaction products were detected using triple-pulsed amperometric detection with the following pulse potentials and durations: E--0.01 V (t,- 120 ms), E 2 -0.6 V (t 2 - 120 ms), and E 3 -0.93 V (t 3 - 130 ms).
  • the extent of hydrolysis is calculated from empirically derived response factors for substrate and reaction products, and the data are plotted using a weighted nonlinear regression analysis (Multifit 2.0, Day Computing, Cambridge, UK).
  • FAME Staphylococcus aureus fattv acid modifying enzyme
  • Lipids are extracted from the solution with 200 ⁇ l of ethyl ethe ⁇ methanol (6:1, EE:M). The lower phase is discarded and the upper organic phase is dried in a stream of nitrogen. The dried lipids are suspended in 100 ⁇ l of hexane:ethyl ethe ⁇ glacial acetic acid (73:25:2; H:EE:AA). The cholesterol ester is separated from the radiolabeled cholesterol with silica gel columns and a solvent system used for TLC to separate cholesterol esters from fatty acids and cholesterol. Slurries of silica gel (average particle size 40 ⁇ M, VWR Scientific, St.
  • H:EE:AA 0.6 g columns (dry weight; 5.3 cm x 0.5 cm) in 23 cm Pasteur pipettes plugged with siliconized glass wool.
  • the suspended samples are placed on the column and the cholesterol esters are eluted in 2 ml of H:EE:AA.
  • the eluant is collected in liquid scintillation vials and 10 ml of scintillation fluid is added. Radioactivity (cpm) of the samples is measured in a liquid scintillation counter as a direct measure of FAME activity (esterification of cholesterol with oleic acid).
  • Staphylococcus esterase assay The Staphylococcus esterase assay is performed as described by Talon et al. (Int. J. Food MicrobioL 36:207- 214, 1997). Esterase activities of cell-free extracts (CFE) or extracellular concentrates (EC) are determined using p- nitrophenyl esters (PN) of acetic, butyric, caproic, caprylic, capric and lauric acids (Sigma). The PN substrates are prepared in acetone at a concentration of 10 mM, then diluted in 0.1 M phosphate buffer, pH 7.0, to a final concentration of 0.16 mM.
  • CFE cell-free extracts
  • EC extracellular concentrates
  • PN substrates are prepared in acetone at a concentration of 10 mM, then diluted in 0.1 M phosphate buffer, pH 7.0, to a final concentration of 0.16 mM.
  • Esterase activities are measured in microplates using the incubator of a Bioscreen C (Labsyste , Finland).
  • the assay mixture contains 340 ⁇ l of PN substrate at pH 7.0 and 10 ⁇ l of CFE or EC.
  • the samples are incubated at 25°C with shaking for 2 hours.
  • the release of p-nitrophenol is measured directly by its absorption at 405 nm.
  • the standard curve is produced using p-nitrophenol.
  • Esterase activity is expressed as n ol of p-nitrophenol/min/mg protein.
  • enzymes which are not naturally secreted may be converted into secreted enzymes by fusing them to signal sequences in secretion vectors which direct their secretion.
  • the secreted enzymes may be used in the methods for measuring cellular proliferation described herein.
  • Secretion vectors include a promoter capable of directing gene expression in the host cell of interest.
  • promoters include the Rous Sarcoma Virus (RSV) promoter, the SV40 promoter, the human cytomegalovirus (CMV) promoter and other promoters well known in the art.
  • a signal sequence which directs protein secretion out of the cell is operably linked to a promoter such that the mRNA transcribed from the promoter directs translation of the signal peptide.
  • the host cell may be any cell which recognizes the signal peptide encoded by the signal sequence.
  • the secretion vector contains cloning sites for inserting genes encoding the proteins which are to be secreted.
  • the cloning sites facilitate the cloning of the insert gene in frame with the signal sequence such that a fusion protein in which the signal peptide is fused to the protein encoded by the inserted gene is expressed from the mRNA transcribed from the promoter.
  • the signal peptide directs the extracellular secretion of the fusion protein.
  • nucleic acid backbones suitable for use as secretion vectors are known to those skilled in the art, including retroviral vectors, SV40 vectors, bovine papillomavirus vectors, yeast integrating plasmids, yeast episomal plasmids, yeast artificial chromosomes, human artificial chromosomes, P element vectors, baculovirus vectors, or bacterial plasmids capable of being transiently introduced into the host.
  • secretion vectors are also commercially available from sources such as Invitrogen (Carlsbad, CA).
  • the secretion vector pBAD/glll may be used.
  • the secretion vector may also contain a polyA signal located downstream of the inserted gene.
  • the gene encoding the protein for which secretion is desired is inserted into the secretion vector using well known methods.
  • Suitable genes include, but are not limited to, those encoding chloramphenicol acetyltransferase, firefly luciferase, ⁇ -glucuronidase, green fluorescent protein, thermostable DNA polymerase, hypoxanthine-guanine phosphoribosyltransferase, bovine growth hormone, proteinase K, ricin A chain, hirudin/proteinase inhibitor and human interferon- ⁇ .
  • the secretion vector is then introduced into the host cell using methods including, but not limited to, calcium phosphate precipitation, DEAE-dextran, electroporation, liposome-mediated transfection, viral particles or as naked DNA.
  • the protein is expressed by the cells and secreted into the culture medium.
  • EXAMPLE 11 Use of single-copy chitobiase gene system to follow cell growth Measurement of cell growth based on light-scattering (turbidity) has limitations in sensitivity and dynamic range, which preclude its effective use with currently available 1536 well plates and plate readers. To overcome this limitation, a bacterial strain was constructed that contains a constitutively expressed chitobiase gene in its chromosome. The expressed chitobiase enzyme localizes to the outer cell membrane of the bacteria, permitting assay of enzyme activity without lysis of the cell, and allows continuous measurement of cell growth. Chitobiase activity is easily monitored by adding substrates which will generate colorimetric or fluorescent products, for example, PNAG and MNAG.
  • the increased sensitivity and dynamic range of the chitobiase activity-based cell growth assay allows bacterial cell growth to be effectively monitored in high density microplates (1536-well or higher), and presents a method for scaling to ultra high throughput screening (UHTS) of chemical entities and natural products.
  • UHTS ultra high throughput screening
  • the substrate PNAG is cleaved by chitobiase to generate the colorimetric product, p-nitrophenol, which absorbs light at 405-415 nm.
  • the results (Fig. 4) indicate that the chitobiase assay is significantly more sensitive that the turbidity assay because of the relatively higher OD observed at each time point.
  • £ coli cells of strain DJKGC4 containing a chromosomally integrated, constitutively expressed chitobiase gene were added to an appropriate growth medium containing 100 ⁇ M MNAG, and the resulting cell suspension was dispensed into the wells of 1536-well microplates as described above.
  • EXAMPLE 12 Chitobiase assay sensitivity is increased by addition of sarkosyl Assays were performed to determine whether sarkosyl, NaCl or a combination of both could increase the sensitivity of chitobiase detection in whole cells. Either control cells or cells containing the integrated chitobiase gene were suspended in M9 salts to a final density equivalent to an OD 600 of 0.0018. MNAG was added either in Tris HCI (pH 8.0) or Tris HCI containing sarkosyl, NaCl or both, to cells such that the final concentration of each was 100 mM Tris, 0.5% sarkosyl and 0.5 M NaCl.
  • Chitobiase activity was measured after a 2 hour incubation at room temperature. Addition of sarkosyl and NaCl, either alone or in combination, increased chitobiase activity (Fig. 7). Addition of sarkosyl alone worked best, increasing chitobiase activity about three fold.
  • Detection of chitobiase activity does not require disruption of the cell membranes and both the PNAG substrate and PNP product are innocuous to cell growth. This allows sensitive detection of cell proliferation in real time.
  • Log phase cells grown in LB were subbed to an OD 600 of 0.002 in 200 ⁇ l M9 media in a 96 well microtiter plate in the presence of 1 mM PNAG. Cells were read every 5 minutes at 0D 405 and 0D 6QQ . OD 6Q0 detects turbidity only, whereas 0D 4Q5 detects a combination of turbidity and PNP formation.
  • Fig. 8 The comparison between chitobiase assay and turbidity for detection of cell growth in shown in Fig. 8.
  • chitobiase assay cells can be detected over noise only after 4 hours growth, at which time the 0D 405 detection of PNP product is 12 fold higher. After 8 hours, the OD 405 detection of the PNP product is nearly 16 fold greater than the OD 6 ⁇ o turbidity measurement.
  • cells were grown in M9 plus 0.4% glucose. Because there is a putative cAMP catabolite activator protein binding site found in the chitobiase promoter, gene expression could be subject to glucose repression. Accordingly, in some embodiments, growth in the presence of a different carbon source (e.g. galactose) may be used to increase the sensitivity of the assay.
  • a different carbon source e.g. galactose
  • chitobiase activity may appear to be decreased because of the lowered substrate concentration.
  • activity may therefore be increased at higher time points by titration of the substrate for optimal concentration or, alternatively, switching to a fluorogenic substrate, for example MNAG. Similar methods may be performed using other ectoenzymes or secreted enzymes
  • ectoenzymes such as membrane-bound chitobiase, or a secreted enzyme, is used to measure cellular proliferation in methods for identifying genes required for proliferation, such as those described in copending application Serial No. 09/164,415.
  • a proliferation-required gene is one where, in the absence of a gene transcript and/or gene product, growth or viability of the microorganism is reduced or eliminated. These proliferation- required genes can be used as targets for the generation of new antimicrobial agents.
  • Cell proliferation assays to identify genes required for proliferation may be performed as follows. Nucleic acids to be evaluated for the ability to inhibit proliferation are cloned into an expression vector next to an inducible promoter. In some embodiments, the nucleic acids to be evaluated for the ability to inhibit proliferation are fragments of genomic DNA. In some embodiments, the fragments are random fragments of genomic DNA. Random fragments may be generated by digestion with restriction endonucleases, partial digestion with DNase I, physical shear or other methods familiar to those skilled in the art. Nucleic acid fragments obtained by partial or total restriction digestion or by shearing can be size selected by agarose gel electrophoresis or sucrose gradients, if desired. Nucleic acids to be evaluated for the ability to inhibit proliferation can also be obtained by chemical synthesis, from a cDNA library, or by other means known in the art.
  • the vector is then introduced into cells expressing an ectoenzyme, such as membrane-bound chitobiase, or secreted enzyme, and the growth of induced cells is compared to uninduced cells by measuring the activity of the ectoenzyme or secreted enzyme.
  • the vector is introduced into a cell containing a gene encoding membrane-bound chitobiase.
  • the gene encoding the ectoenzyme, such as membrane-bound chitobiase, or a secreted enzyme may be integrated into the genome of the cell or present on an extrachromosomal vector. Expression of the nucleic acids to be evaluated for the ability to inhibit proliferation in the cell is then activated.
  • Cell number is then determined using a chitobiase assay as described herein or an appropriate assay for the ectoenzyme or secreted enzyme expressed by the cell, to determine the effect of expression of the nucleic acids being evaluated on cell proliferation.
  • the expression vectors that, upon activation and expression, negatively impact the growth of the host cells are identified, isolated and purified for further study of the inserts contained therein.
  • the inserts which inhibit cell growth may be sequenced to determine whether they are antisense inserts (i.e., whether the DNA strand being transcribed from the promoter in the expression vector is noncoding) or whether the insert encodes a polypeptide or portion thereof.
  • Random genomic fragments were cloned into an inducible expression vector and assayed for growth inhibition activity.
  • the expression vector pLEX5BA contains: I) a Bujard Promoter that has binding sites for lac repressor centered at -22 and + 11 relative to the start of transcription, II) a multiple cloning site downstream of the promoter, and III) an rrnBtH2 transcriptional terminator after the multiple cloning site. Expression of fragments cloned downstream of the Bujard promoter can be induced with IPTG.
  • £ coli chromosomal DNA was digested with either Pst ⁇ and Hi ⁇ dlW or EcoR ⁇ and BamHI and ligated into vector pLEX5BA cut with the same enzymes (Diederich et al., Biotechniques 16:916-923, 1994). The double digestions were chosen to give fragments with a median length of 2-3 kb (Churchill et al., Nucl. Acids Res. 18:589-597, 1990). The ligation mix was transformed into £ coli DH5 and transformants were selected on plates containing ampicillin. Colonies that grew on ampicillin were subsequently replica plated by physical transfer to a second ampicillin plate containing the inducing agent IPTG at a concentration of 100 ⁇ M. Colonies that did not grow in the presence of IPTG were chosen for further characterization.
  • genomic fragments may be generated by fully digesting genomic DNA with the restriction enzyme, Sau3r ⁇ .
  • random genomic fragments may be generated by partially digesting genomic DNA with DNase I or mechanically shearing genomic DNA , and "blunt-ending" the resulting fragments by incubating with T4 DNA polymerase. Random genomic fragments between 200 and 800 base pairs in length, or any other desired length, may be selected by gel purification. The size-selected genomic fragments are added to a linearized and dephosphorylated vector at a molar ratio of 0.1 to 1, and ligated to form a shotgun library. The ligated products are transformed into host cells and plasmids are purified therefrom.
  • Example 15 describes the examination of a library of random genomic fragments obtained by performing digests with Pst ⁇ and Hind ⁇ or EcoRV and BamHI cloned into IPTG-inducible expression vectors. Upon activation or induction, the expression vectors produced an RNA molecule corresponding to the subcloned random genomic fragments. In those instances where the random genomic fragments were in an antisense orientation with respect to the promoter, the transcript produced was complementary to at least a portion of an mRNA encoding a proliferation- required gene product, such that they interacted with the sense mRNA to decrease its translation efficiency or its level, thereby decreasing production of the protein encoded by the sense mRNA.
  • the sense mRNA encoded a protein required for proliferation In cases where the sense mRNA encoded a protein required for proliferation, bacterial cells containing an activated expression vector failed to grow or grew at a substantially reduced rate.
  • the sense mRNA encodes a peptide or polypeptide which is not normally produced by the cell, but is produced from an open reading frame which encodes an aptamer, defined herein as a peptide which inhibits cellular proliferation by interfering with a protein required for proliferation.
  • EXAMPLE 15 Inhibition of Bacterial Proliferation after IPTG induction
  • growth curves were carried out by back diluting cultures 1:200 into fresh media with or without 1 mM IPTG and measuring the 0D 450 every 30 minutes (min).
  • 10 2 , 10 3 , 10 4 , 10 5 , 10°, 10 7 and 10 8 fold dilutions of overnight cultures were prepared. Aliquots of from 0.5 to 3 ⁇ l of these dilutions were spotted on selective agar plates with or without 1 mM IPTG. After overnight incubation, the plates were compared to assess the sensitivity of the clones to IPTG.
  • the gene to which the inserted nucleic acid sequence corresponds, or a gene within the operon containing the inserted nucleic acid may be required for proliferation in £ coli.
  • liquid endpoint scoring protocol may be used.
  • liquid growth characteristics of non- induced and induced cells are used to score sensitive clones according to a "% inhibition.”
  • growth conditions e.g., temperature, media and dilutions
  • type of inducer e.g., xylose and IPTG
  • concentration of inducer will vary depending on the organism to be assayed.
  • Two 96-well “hit” plates containing stationary phase cells and two 96-well Nunc plates filled with 90 ⁇ L LB broth and Sm100 are brought to room temperature.
  • the cells are diluted 1:10 into fresh media plates using Biomek 2000 (Beckman Instruments). Innoculated plates are placed on a LabNet Shaker30 (E&K Scientific) at setting 500-600 for 2-3 hours. After 2 hours, the cell density is checked by reading the OD 600 and subtracting the background. If the cells are not between 0D 600 1.0-1.2 after subtracting background, they are returned to the shaker and readings are continued as needed (e.g., every 30 min) until cells reach the correct density.
  • Two 96 well plates containing log phase cells are removed from a plate shaker and the wells diluted 1:10 into duplicate wells of a 384 well plate.
  • One of the duplicate wells contains media and the other contains media plus inducer (for example LB broth with 1mM IPTG).
  • the 384-well plate is placed on the LabNet Shaker 30 at 37°C at setting 500- 600 for 2 hours. The cell density is checked as described above.
  • the 384-well plate is removed from the plate shaker and carried to a SpectraMax Plus 384 plate reader.
  • SoftMax Pro 3.3.1 software is opened.
  • the "untitled” assay is closed. From the “Assays” file menu, "Basic Protocols” is chosen, followed by "Endpoint Assay.”
  • the assay is saved and named according to the date and plates being read to an appropriate folder.
  • the "Setup” button in the assay window is selected and the following parameters are changed: Wavelength is set to 600 nm and Plate Type is 384 Standard (there is no need to change any other parameters).
  • the OK button is selected and the assay is saved again. The bottom of the plate is wiped, the reader drawer is opened and the plate is placed in the holder (A1 in the top left corner), and the lid is removed. The reader drawer is closed, and the "Read” button in the assay window is selected.
  • the vector controls are extracted out to establish the background cut-off for each plate as follows.
  • Vector controls are arrayed into 4 wells in the 96-well hit plates: A1, D7, E6, H12.
  • the % inhibition for each of the 4 vector controls from each input plate is calculated.
  • An average (-AVERAGE(range))
  • Vector% inhibition from the 4 vector samples is obtained.
  • the standard deviation (-STDEV(range)) from the % inhibition of all 4 vector samples is calculated.
  • the 2x StDev cut-off « (2*STDEV)+AVERAGE is calculated. The cut-off should fall between
  • Clones are sorted from each plate by decreasing % inhibition, ensuring that the well coordinates of each clone is next to the % inhibition.
  • the cut-off on the sorted data is set to separate sensitive clones from non-sensitive clones by creating a border. Characterization of Isolated Clones Negatively Affecting £ coli Proliferation
  • nucleic Acid Fragments with Detrimental Effects of £ coli Proliferation The nucleotide sequences for the exogenous identified sequences of Example 15 were determined using plasmid DNA isolated using QIAPREP (Qiagen, Valencia, CA) and methods supplied by the manufacturer. Primers flanking the pol ⁇ linker in pLEX5BA were used for sequencing the inserts. Sequence identification numbers (SEQ ID NOs) for the identified inserts are listed in Table I of copending Application Serial No. 09/492,709, and discussed below.
  • EXAMPLE 17 Comparison Of Isolated Seguences to Known Sequences
  • the nucleic acid sequences of the subcloned fragments obtained from the expression vectors discussed in Examples 15 and 16 above were compared to known £ coli sequences in GenBank using BLAST version 1.4 or version 2.0.6 using the following default parameters: Filtering off, cost to open a gap -5, cost to extend a gap -2, penalty for a mismatch in the blast portion of run— 3, reward for a match in the blast portion of run- 1, expectation value (e)» 10.0, word size- 11, number of one-line descriptions - 100, number of alignments to show (B)- 100.
  • BLAST is described in Altschul, J Mol Biol. 215:403-10 (1990).
  • Expression vectors were found to contain nucleic acid sequences in both the sense and antisense orientations. The presence of known genes, open reading frames, and ribosome binding sites was determined by comparison to public databases holding genetic information and various computer programs such as the Genetics Computer Group programs FRAMES and CODONPREFERENCE. Clones were designated as "antisense” if the cloned fragment was oriented to the promoter such that the RNA transcript produced was complementary to the expressed mRNA from a chromosomal locus. Clones were designated as "sense” if they coded for an RNA fragment that was identical to a portion of a wild type mRNA from a chromosomal locus. Alternative databases may also be used to determine whether a clone is "sense” or "antisense.” For example, the PathoSeqTM database available from Incyte Genomics.or the TIGR databases may also be used.
  • the nucleic acids which inhibit proliferation may be used in cell-based assay methods using cells expressing an ectoenzyme or secreted enzyme of the present invention.
  • cell-based assays using sensitized cells in which the level or activity of at least one proliferation-required gene product has been specifically reduced to the point where its presence or absence becomes rate-determining for cellular proliferation have significant advantages.
  • the identified antisense nucleic acids which inhibit bacterial proliferation as measured by determining the activity of the ectoenzyme or secreted enzyme, for example, by using the chitobiase assay described herein, are used to inhibit the production of a proliferation-required protein.
  • Expression vectors producing antisense RNA complementary to identified genes required for proliferation are used to limit the concentration of a proliferation-required protein without completely inhibiting growth.
  • a growth inhibition dose curve of inducer is calculated by plotting various doses of inducer against the corresponding growth inhibition caused by the antisense expression and measured using a chitobiase assay. From this curve, various percentages of antisense induced growth inhibition, from 1 to 99% can be determined. Any suitable inducer may be used, including IPTG. For example, the highest concentration of the inducer that does not reduce the growth rate significantly can be estimated from the curve.
  • Cellular proliferation can be monitored by measuring ectoenzyme or secreted enzyme activity as described herein.
  • the concentration of inducer that reduces growth by 25% can be predicted from the curve.
  • a concentration of inducer that reduces growth by 50% can be calculated.
  • a concentration of inducer that reduces growth by at least 10%, 25%, 50%, 60%, 75%, 90%, 95% or more than 95% can be predicted from the curve. Additional parameters such as colony forming units (cfu) can be used to measure cellular viability.
  • Cells to be assayed are exposed to one of the above-determined concentrations of inducer.
  • the presence of the inducer at this sub-lethal concentration reduces the amount of the proliferation required gene product to the lowest amount in the cell that will support growth.
  • Cells grown in the presence of this concentration of inducer are therefore specifically more sensitive to inhibitors of the proliferation-required protein or RNA of interest or to inhibitors of proteins or RNAs in the same biological pathway as the proliferation-required protein or RNA of interest but not to inhibitors of proteins or RNAs in a different biological pathway.
  • the sub-lethal concentration of inducer may be any concentration consistent with the intended use of the assay to identify candidate compounds to which the cells are more sensitive.
  • the sub-lethal concentration of the inducer may be such that growth inhibition is at least about 5%, at least about 8%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60% at least about 75%, at least about 90%, at least about 95% or more.
  • Cells which are pre-sensitized using the preceding method are more sensitive to inhibitors of the target protein or RNA because these cells contain less target protein or RNA to inhibit than wild-type cells.
  • the sensitized cells may be used to identify compounds which inhibit proliferation using the methods described herein.
  • the following examples describe methods for conducting cell based assays in which ectoenzymes or secreted enzymes are used to measure cellular proliferation.
  • EXAMPLE 18 Cell based assay to determine the effect of antisense expression on cell sensitivity £ coli clones expressing antisense nucleic acids complementary to the mRNA encoding the proliferation- required ribosomal proteins L7/L12, L10 and L23 were used to test the effect of antisense expression on cell sensitivity to the antibiotics known to bind to these proteins.
  • expression vectors containing antisense to either the genes encoding L7/L12 and L10 or L23 were introduced into separate £ coli cell populations along with an expression vector encoding the membrane-bound form of chitobiase. Vector introduction is a technique well known to those of ordinary skill in the art.
  • the expression vectors contain IPTG inducible promoters that drive the expression of the antisense sequence in the presence of the inducer. Suitable expression vectors are also well known in the art.
  • the cell populations were exposed to a range of IPTG concentrations in liquid medium to obtain the growth inhibitory dose curve for each clone (Fig. 9).
  • seed cultures were grown to a particular turbidity that is measured by the optical density (OD) of the growth solution.
  • the OD of the solution is directly related to the number of bacterial cells contained therein.
  • IPTG concentration of IPTG that inhibits cell growth to 20% (IC 20 ) and 50% (IC 50 ) as compared to the 0 mM IPTG control (0% growth inhibition) was then calculated from the curve.
  • concentrations of IPTG produced an amount of antisense RNA that reduced the expression levels of L7/L12, L10 and L23 to a degree such that growth was inhibited by 20% and 50%, respectively.
  • Cells were pretreated with the selected concentration of IPTG and then used to test the sensitivity of cell populations to tetracycline, erythromycin and other protein synthesis inhibitors.
  • An example of a tetracycline dose response curve is shown in Figures 10A and 10B for the rplW and elaD genes, respectively. Cells were grown to log phase and then diluted into media alone or media containing IPTG at concentrations which give 20% and 50% growth inhibition as determined by IPTG dose response curves.
  • the cells were diluted to a final OD 600 of 0.002 into 96 well plates containing (1) +/• IPTG at the same concentrations used for the 2.5 hour pre-incubation; and (2) serial two-fold dilutions of tetracycline such that the final concentrations of tetracycline range from 1 ⁇ g/ml to 15.6 ng/ml and 0 ⁇ g/ml.
  • the 96 well plates were incubated at 37°C and the OD 6 ⁇ o was read by a plate reader every 5 minutes for up to 15 hours. Tetracycline dose response curves were determined for each IPTG concentration and the no IPTG control.
  • tetracycline IC 50 s (the concentration of tetracycline that further inhibits growth by 50%) were determined from the dose response curves (Figs. 10A-B).
  • Cells with reduced levels of the ribosomal protein L23 (rplW gene product) showed increased sensitivity to the ribosomal inhibitory antibiotic tetracycline (Fig. 10A) as opposed to cells with reduced levels of elaD gene product, which is not a ribosomal protein and is not in the protein synthesis pathway (Fig. 10B).
  • Figure 11 shows a summary bar chart in which the ratios of tetracycline IC 50 s determined in the presence of IPTG which gives 50% growth inhibition versus tetracycline IC SD s determined without IPTG (fold increase in tetracycline sensitivity) were plotted.
  • Cells with reduced levels of either L7/L12 (rplL and rplJ gene products) or L23 (rp/W Qene product) showed increased sensitivity to tetracycline (Fig. 11).
  • sensitization was measured by optical density rather than ectoenzyme or secreted enzyme activity in the example above, it will be appreciated that cells expressing an ectoenzyme, such as a membrane-bound form of chitobiase, or a secreted enzyme, may be used in similar assays in which sensitization is measured by determining ectoenzyme activity.
  • an ectoenzyme such as a membrane-bound form of chitobiase, or a secreted enzyme
  • the level or activity of a proliferation required gene product is reduced in a cell expressing an ectoenzyme, such as a membrane-bound form of chitobiase, or a secreted enzyme, using both a mutation in the proliferation-required gene that reduces the activity of the proliferation-required gene and an antisense nucleic acid complementary to the proliferation-required sequence.
  • the mutation in the proliferation-required gene is a condition mutation, such as a temperature sensitive mutation. Growing the cells at an intermediate temperature between the permissive and restrictive temperatures of the temperature sensitive mutant produces cells with reduced activity of the proliferation-required gene product.
  • the antisense RNA complementary to the proliferation-required sequence further reduces the activity of the proliferation required gene product by reducing the amount of the gene product.
  • Drugs that may not have been found using either the temperature sensitive mutation or the antisense nucleic acid alone may be identified by determining whether cells in which expression of the antisense nucleic acid has been induced and which are grown at a temperature between the permissive temperature and the restrictive temperature are substantially more sensitive to a test compound than cells in which expression of the antisense nucleic acid has not been induced and which are grown at a permissive temperature.
  • Cell sensitivity to a test compound may be determined by performing a chitobiase assay as described herein.
  • compounds identified using either the antisense nucleic acid alone or the temperature sensitive mutation alone may exhibit a different sensitivity profile when used in cells combining the two approaches, and that sensitivity profile may indicate a more specific action of the drug in inhibiting one or more activities of the gene product.
  • Temperature sensitive mutations may be located at different sites within the gene and correspond to different domains of the protein.
  • the dnaB gene of Escherichia coli encodes the replication fork DNA helicase. DnaB has several domains, including domains for oligomerization, ATP hydrolysis, DNA binding, interaction with primase, interaction with DnaC, and interaction with DnaA (Biswas, E.E. and Biswas, S.B. 1999.
  • growth inhibition of cells containing a limiting amount of that proliferation-required gene product can be assayed. Growth inhibition can be measured by directly comparing the amount of growth, measured by ectoenzyme or secreted enzyme activity, between an experimental sample and a control sample.
  • the effect of compounds on cellular proliferation may be tested entirely in liquid phase using microtiter plates as described below.
  • Liquid phase screening may be performed in microtiter plates containing 96, 384, 1536 or more wells per microtiter plate to screen multiple plates and thousands to millions of compounds per day.
  • the improved sensitivity of the methods of the present invention are particularly useful as the size of the wells in the microtater plates decreases, such as in the 1536 well plates.
  • Automated and semi-automated equipment may be used for addition of reagents (for example cells and compounds) and determination of cell density.
  • the cell based assay described above may also be used to identify the biological pathway in which a proliferation-required nucleic acid or its gene product lies.
  • cells containing a gene encoding an ectoenzyme, such as a membrane-bound form of chitobiase, or a secreted enzyme which express a sub-lethal level of antisense to a target proliferation-required nucleic acid and control cells in which expression of the antisense has not been induced are contacted with a panel of antibiotics known to act in various pathways.
  • Cell sensitivity to a test compound may be determined by performing an ectoenzyme assay, such as a chitobiase assay, or a secreted enzyme assay, as described herein.
  • the results of the assay may be confirmed by contacting a panel of cells containing a gene encoding an ectoenzyme, such as a membrane-bound form of chitobiase, or a secreted enzyme, which express antisense nucleic acids to many different proliferation-required genes including the target proliferation-required gene with the panel of antibiotics.
  • an ectoenzyme such as a membrane-bound form of chitobiase, or a secreted enzyme, which express antisense nucleic acids to many different proliferation-required genes including the target proliferation-required gene with the panel of antibiotics.
  • the antibiotic is acting specifically, heightened sensitivity to the antibiotic as measured by performing an ectoenzyme or secreted enzyme assay as described herein, will be observed only in the cells expressing antisense to a target proliferation-required gene (or cells expressing antisense to other proliferation-required genes in the same pathway as the target proliferation-required gene) but will not be observed generally in all cells expressing antisense to proliferation-required genes.
  • the above method may be used to determine the pathway on which a test compound, such as a test antibiotic, acts.
  • the sensitivity of the panel of cells to the test compound is determined by performing an ectoenzyme or secreted enzyme assay on cells in which expression of the antisense has been induced and on control cells in which expression of the antisense has not been induced.
  • test compound acts on the pathway on which an antisense nucleic acid acts
  • cells in which expression of the antisense has been induced will be more sensitive to the compound, as determined by performing an ectoenzyme or secreted enzyme assay, than cells in which expression of the antisense has not been induced.
  • control cells expressing an ectoenzyme, such as a membrane-bound form of chitobiase, or a secreted enzyme in which expression of antisense to proliferation-required genes in other pathways has been induced will not exhibit heightened sensitivity to the compound. In this way, the pathway on which the test compound acts may be determined.
  • frozen stocks of host bacteria containing a gene encoding a secreted enzyme or an ectoenzyme, such as a membrane-bound form of chitobiase, as well as the desired construct in which antisense expression is under the control of an inducible promoter, such as the IPTG inducible lac promoter, are prepared using standard microbiological techniques. For example, a single clone of the microorganism can be isolated by streaking out a sample of the original stock onto an agar plate containing nutrients for cell growth.
  • the antisense construct is on a plasmid which includes a selectable marker gene, such as an antibiotic resistance gene.
  • the agar also contains an antibiotic or other compounds appropriate for selecting for the presence of the plasmid containing the antisense construct.
  • an isolated colony is picked from the plate with a sterile needle and transferred to an appropriate liquid growth media containing the antibiotic required for maintenance of the plasmid.
  • the cells are incubated at 30°C to 37°C with vigorous shaking for 4 to 6 hours to yield a culture in exponential growth.
  • Sterile glycerol is added to 15% (volume to volume) and 10O ⁇ L to 500 ⁇ L aliquots are distributed into sterile cryotubes, snap frozen in liquid nitrogen, and stored at -80°C for future assays.
  • a stock vial is removed from the freezer, rapidly thawed (37°C water bath) and a loop of culture is streaked out on an agar plate containing nutrients for cell growth and an antibiotic to which the plasmid or vector comprising the antisense construct confers resistance.
  • a loop of culture is streaked out on an agar plate containing nutrients for cell growth and an antibiotic to which the plasmid or vector comprising the antisense construct confers resistance.
  • ten randomly chosen, isolated colonies are transferred from the plate (sterile inoculum loop) to a sterile tube containing 5 mL of LB medium containing the antibiotic to which the plasmid or vector comprising the antisense construct confers resistance.
  • the optical density of the suspension is measured at 600 nm (0D ⁇ 00 ) and if necessary an aliquot of the suspension is diluted into a second tube of 5 mL, sterile, LB medium plus antibiotic to achieve an 0D 600 ⁇ 0.02 absorbance units.
  • the culture is then incubated at 37° C for 1-2 hrs with shaking until the OD 600 reaches OD 0.2 - 0.3. At this point the cells are ready to be used in the assay.
  • Two-fold dilution series of antibiotics of known mechanism of action are generated in the culture media selected for further assay development that has been supplemented with the antibiotic used to maintain the construct.
  • a panel of test antibiotics known to act on different pathways is tested side by side with three to four wells being used to evaluate the effect of a test antibiotic on cell growth at each concentration.
  • Equal volumes of test antibiotic and cells are added to the wells of a microtiter plate and mixed. Cells are prepared as described above using the media selected for assay development supplemented with the antibiotic required to maintain the antisense construct and are diluted 1:100 in identical media immediately prior to addition to the microtiter plate wells. For a control, cells are also added to several wells that lack antibiotic, but contain the solvent used to dissolve the antibiotics.
  • ectoenzyme assay such as a chitobiase assay, or a secreted enzyme assay, as described herein.
  • the percent inhibition of growth produced by each concentration of antibiotic is calculated by comparing the rates of logarithmic growth against that exhibited by cells growing in media without antibiotic. Growth rates are determined by performing a secreted ezyme assay or an ectoenzyme assay, such as a chitobiase assay, as described herein.
  • a plot of percent inhibition against log.antibiotic concentration] allows extrapolation of an IC 50 value for each antibiotic.
  • the culture media selected for use in the assay is supplemented with inducer at concentrations shown to inhibit cell growth by 50% and 80% as described above, as well as the antibiotic used to maintain the construct.
  • Two fold dilution series of the panel of test antibiotics used above are generated in each of these media.
  • Several antibiotics are tested side by side in each medium with three to four wells being used to evaluate the effects of an antibiotic on cell growth at each concentration.
  • Equal volumes of test antibiotic and cells are added to the wells of a microtiter plate and mixed. Cells are prepared as described above using the media selected for use in the assay supplemented with the antibiotic required to maintain the antisense construct.
  • the cells are diluted 1:100 into two 50 mL aliquots of identical media containing concentrations of inducer that have been shown to inhibit cell growth by 50% and 80 % respectively and incubated at 37°C with shaking for 2.5 hours.
  • the cultures are adjusted to an appropriate 0D 600 (typically 0.002) by dilution into warm (37°C) sterile media supplemented with identical concentrations of the inducer and antibiotic used to maintain the antisense construct.
  • 0D 600 typically 0.002
  • cells are also added to several wells that contain solvent used to dissolve test antibiotics but which contain no antibiotic.
  • ectoenzyme assays such as chitobiase assays or secreted enzyme assays
  • the percent inhibition of growth produced by each concentration of antibiotic is calculated by comparing the rates of logarithmic growth against that exhibited by cells growing in media without antibiotic. Growth rates are measured by performing ectoenzyme assays, such as chitobiase assays, or secreted enzyme assays, as described herein.
  • a plot of percent inhibition against log[antibiotic concentration] allows extrapolation of an IC 5D value for each antibiotic.
  • the cell based assay may also be used to determine the pathway against which a test antibiotic acts.
  • the pathways against which each member of a panel of antisense nucleic acids acts are identified as described above.
  • a panel of cells expressing an ectoenzyme, such as a membrane-bound form of chitobiase, or a secreted enzyme, each containing an inducible vector which transcribes an antisense nucleic acid complementary to a gene in a known proliferation-required pathway is contacted with a test antibiotic for which it is desired to determine the pathway on which it acts under inducing and non-inducing conditions.
  • test antibiotic acts against the pathway for which heightened sensitivity was observed.
  • Cell sensitivity to the test antibiotic is determined by performing a chitobiase assay as described herein.
  • the concentration of inducer used to induce antisense expression and/or the growth conditions used for the assay may further increase the selectivity and/or magnitude of the antibiotic sensitization exhibited.
  • concentration of inducer used to induce antisense expression and/or the growth conditions used for the assay for example incubation temperature and media components
  • the growth conditions used for the assay for example incubation temperature and media components
  • sensitized cells expressing antisense complementary to a nucleic acid required for proliferation may be used to determine the biological pathway in which the proliferation-required nucleic acid lies.
  • EXAMPLE 20 Identification of the Biological Pathway in which a Proliferation-Required Gene Lies
  • the effectiveness of the above assays was validated using proliferation-required genes from £ coli which were identified using procedures similar to those described above.
  • Antibiotics of various chemical classes and modes of action were purchased from Sigma Chemicals (St. Louis, MO). Stock solutions were prepared by dissolving each antibiotic in an appropriate aqueous solution based on information provided by the manufacturer. The final working solution of each antibiotic contained no more than 0.2% (w/v) of any organic solvent.
  • each antibiotic was serially diluted two or three fold in growth medium supplemented with the appropriate antibiotic for maintenance of the anti-sense construct. At least ten dilutions were prepared for each antibiotic. 25 ⁇ L aliquots of each dilution were transferred to discrete wells of a 384-well microplate (the assay plate) using a multi-channel pipette. Quadruplicate wells were used for each dilution of an antibiotic under each treatment condition (plus and minus inducer).
  • Each assay plate contained twenty wells for cell growth controls (growth media replacing antibiotic), ten wells for each treatment (plus and minus inducer, in this example IPTG). Assay plates were usually divided into the two treatments: half the plate containing induced cells and an appropriate concentrations of inducer (in this example IPTG) to maintain the state of induction, the other half containing non-induced cells in the absence of IPTG. Cells for the assay were prepared as follows.
  • Bacterial ceils containing a construct, from which expression of antisense nucleic acid complementary to rplL and rplJ, which encode proliferation-required 50S ribosomal subunit proteins, is inducible in the presence of IPTG were grown into exponential growth (OD 600 0.2 to 0.3) and then diluted 1:100 into fresh media containing either 400 ⁇ M or 0 ⁇ M inducer (IPTG). These cultures were incubated at 37° C for 2.5 hr. After a 2.5 hr incubation, induced and non-induced cells were respectively diluted into an assay medium at a final OD 600 value of 0.0004. The medium contained an appropriate concentration of the antibiotic for the maintenance of the antisense construct.
  • the medium used to dilute induced cells was supplemented with 800 ⁇ M IPTG so that addition to the assay plate would result in a final IPTG concentration of 400 ⁇ M.
  • Induced and non-induced cell suspensions were dispensed (25 ⁇ l/well) into the appropriate wells of the assay plate as discussed previously. The plate was then loaded into a plate reader, incubated at constant temperature, and cell growth was monitored in each well by the measurement of light scattering at 595 nm. Growth was monitored every 5 minutes until the cell culture attained a stationary growth phase. For each concentration of antibiotic, a percentage inhibition of growth was calculated at the time point corresponding to mid-exponential growth for the associated control wells (no antibiotic, plus or minus IPTG).
  • sensitization was measured by optical density rather than ectoenzyme activity in the example above, it will be appreciated that cells expressing an ectoenzyme, such as a membrane-bound form of chitobiase, or a secreted enzyme, may be used in similar assays in which sensitization is measured by determining the ectoenzyme or secreted enzyme activity.
  • Assays utilizing antisense constructs to essential genes in cells expressing an ectoenzyme, such as a membrane-bound form of chitobiase, or a secreted enzyme can be used to identify compounds that interfere with the activity of those gene products. Such assays could be used to identify drug leads, for example antibiotics.
  • Panels of cells expressing an ectoenzyme such as a membrane-bound form of chitobiase, or a secreted enzyme, which express different antisense nucleic acids, can be used to characterize the point of intervention of a compound affecting an essential biochemical pathway including antibiotics with no known mechanism of action.
  • an ectoenzyme such as a membrane-bound form of chitobiase, or a secreted enzyme, which express different antisense nucleic acids
  • Assays utilizing antisense constructs to essential genes in cells expressing an ectoenzyme, such as a membrane-bound form of chitobiase, or a secreted enzyme, can be used to identify compounds that specifically interfere with the activity of multiple targets in a pathway. Such constructs can be used to simultaneously screen a sample against multiple targets in one pathway in one reaction (Combinatorial HTS). Furthermore, as discussed above, panels of antisense construct-containing cells expressing an ectoenzyme, such as a membrane-bound form of chitobiase, or a secreted enzyme, may be used to characterize the point of intervention of any compound affecting an essential biological pathway including antibiotics with no known mechanism of action.
  • an ectoenzyme such as a membrane-bound form of chitobiase, or a secreted enzyme
  • Another embodiment of the present invention is a method for determining the pathway against which a test antibiotic compound is active, in which the activity of target proteins or nucleic acids involved in proliferation-required pathways is reduced by contacting cells expressing an ectoenzyme, such as a membrane-bound form of chitobiase, or a secreted enzyme, with a sublethal concentration of a known antibiotic which acts against the target protein or nucleic acid.
  • an ectoenzyme such as a membrane-bound form of chitobiase, or a secreted enzyme
  • the target protein or nucleic acid corresponds to a proliferation-required nucleic acid identified using the methods described above.
  • the method is similar to those described above for determining which pathway a test antibiotic acts against, except that rather than reducing the activity or level of a proliferation-required gene product using a sublethal level of antisense to a proliferation-required nucleic acid, the activity or level of the proliferation-required gene product is reduced using a sublethal level of a known antibiotic which acts against the proliferation required gene product.
  • Mecillinam (Amdinocillin) binds to and inactivates the penicillin binding protein 2 (PBP2, product of the mrdA in £ coli).
  • PBP2 penicillin binding protein 2
  • This antibiotic interacts with other antibiotics that inhibit PBP2 as well as antibiotics that inhibit other penicillin binding proteins such as PBP3 [(Gutmann, L Do Vincent, S., Billot-Klein, D., Acar, J.F., Mrena, E., and Williamson, R.
  • the proton pump inhibitor, Omeprazole, and the antibiotic, Amoxicillin, two synergistic compounds acting together, can cure Helicobacter pylori infection [( Gabryelewicz, A., Laszewicz, W., Dzieniszewski, J., Ciok, J., Marlicz, K., Bielecki, D., Popiela, T., Legutko, J., Knapik, Z., Poniewierka, E. (1997) Multicenter evaluation of dual-therapy (omeprazole and amoxicillin) for Helicobacter /jy/ ⁇ /7-associated duodenal and gastric ulcer (two years of the observation). J. Physiol. Pharmacol. 48 Suppl 4:93-105)].
  • the growth inhibition from the sublethal concentration of the known antibiotic may be at least about 5%, at least about 8%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, or at least about 75%, at least about 90%, at least about 95% or more.
  • Cells expressing an ectoenzyme such as a membrane-bound form of chitobiase, or a secreted enzyme, are contacted with a combination of each member of a panel of known antibiotics at a sublethal level and varying concentrations of the test antibiotic. As a control, the cells are contacted with varying concentrations of the test antibiotic alone.
  • the IC 50 of the test antibiotic in the presence and absence of the known antibiotic is determined by measuring the activity of the ectoenzyme. If the IC 50 s in the presence and absence of the known drug are substantially similar, then the test drug and the known drug act on different pathways. If the IC 50 s are substantially different, then the test drug and the known drug act on the same pathway.
  • Another embodiment of the present invention is a method for identifying a candidate compound for use as an antibiotic in which the activity of target proteins or nucleic acids involved in proliferation-required pathways is reduced by contacting cells expressing an ectoenzyme, such as a membrane-bound form of chitobiase, or a secreted enzyme, with a sublethal concentration of a known antibiotic which acts against the target protein or nucleic acid.
  • the target protein or nucleic acid is a target protein or nucleic acid corresponding to a proliferation- required nucleic acid identified using the methods described above.
  • the method is similar to those described above for identifying candidate compounds for use as antibiotics except that rather than reducing the activity or level of a proliferation-required gene product using a sublethal level of antisense to a proliferation-required nucleic acid, the activity or level of the proliferation-required gene product is reduced using a sublethal level of a known antibiotic which acts against the proliferation required gene product.
  • the growth inhibition from the sublethal concentration of the known antibiotic may be at least about 5%, at least about 8%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, or at least about 75%, at least about 90%, at least about 95% or more.
  • test compounds of interest In order to characterize test compounds of interest, cells expressing an ectoenzyme, such as a membrane- bound form of chitobiase, or a secreted enzyme, are contacted with a panel of known antibiotics at a sublethal level and one or more concentrations of the test compound. As a control, the cells are contacted with the same concentrations of the test compound alone.
  • the IC 50 of the test compound in the presence and absence of the known antibiotic is determined by measuring ectoenzyme activity, such as chitobiase activity. If the IC 50 of the test compound is substantially different in the presence versus the absence of the known drug then the test compound is a good candidate for use as an antibiotic or for use as a structural lead to design an antibiotic.
  • ectoenzyme such as a membrane- bound form of chitobiase, or a secreted enzyme
  • antibiotics which may be used in each of the above methods are provided in Table II below. However, it will be appreciated that other antibiotics may also be used.
  • Streptovaricin an acyclic ansamycin inhibits RNA polymerase rpoB Actinomycin D+EDTA Intercalates between 2 successive G-C pairs, rpoB, p/dA inhibits RNA synthesis
  • Sulfonamides Sulfanilamide blocks synthesis of dihydrofolate,dihydro- folP, gpt, pabA, pabB, pteroate synthesis, folP pabC
  • Trimethoprim Inhibits dihydrofolate reductase, folA folA, thyA
  • Psicofuranine Adenosine glycoside antibiotic, target is GMP guaA,B synthetase
  • Triclosan Inhibits fatty acid synthesis fabl(envM)
  • Diazoborines Isoniazid, heterocyclic, contains boron, inhibit fatty acid fabl(envM)
  • Phenylpropanoids Binds to ribosomal peptidyl transfer center
  • Chloramphenicol preventing peptide translocation/ binds to S6, rrn, cm/A, mar A, ompF,
  • Macrolides (type 1 polyketides) Binding to 50 S ribosomal subunit, 23S rRNA,
  • Tiamulin Inhibits protein synthesis rplC, rplD
  • Negamycin Inhibits termination process of protein prfB synthesis
  • Nitrofurantoin Inhibits protein synthesis, nitroreductases nfnA.B convert nitrofurantoin to highly reactive electrophilic intermediates which attack bacterial ribosomal proteins non-specificall ⁇
  • Viomycin rrmA (23S rRNA methyltransferase; mutant has slow growth rate, slow chain elongation rate, and viomycin resistance)
  • Thiopeptides Binds to L11-23S RNA complex
  • Penicillin, Ampicillin transpeptidases, endopeptidases, and Methicillin, glycosidases (PBPs), of the 12 PBPs only 2 are ampC, ampD, ampE, essential: mrdA (PBP2) and ftsKpbpB, PBP3) envZ, galU, hip A, hipQ, ompC, ompF, ompR, ptsl, rfa, tolD,
  • Cephalosporins, tolE Mecillinam (amdinocillin) Binds to and inactivates PBP2 (mrdA) tonB Inactivates PBP3 (ftsl) alaS, argS, crp, cyaA, envB, mrdA,B,
  • Cyclic lipopeptide Daptomycin Disrupts multiple aspects of membrane function, including peptidoglycan synthesis, lipoteichoic acid synthesis, and the bacterial membrane potential
  • Globomycin Inhibits signal peptidase II (cleaves Ipp, dnaE prolipoproteins subsequent to lipid modification, IspA
  • reporter genes encoding an ectoenzyme may also be used as reporters.
  • Reporter genes and reporter gene constructs play a number of important roles in a variety of molecular biology techniques. For example, reporter genes may be used to determine whether a sequence contains a promoter or other y-acting element which directs transcription, such as an enhancer.
  • reporter genes may be used to identify regulatory sites in promoters or other ⁇ s-acting elements and to determine the effects of mutating these regulatory sites on the level of gene expression directed by the promoters or other c/s-acting elements. Reporter genes may also be used to detect successful transformation.
  • reporter genes may be used to monitor gene expression under various conditions and to identify drugs. Given the utility of reporter gene constructs, it is not surprising that a number of reporter gene constructs and different reporter genes are available for use by those of skill in the art.
  • the cytoplasmic reporter enzymes chloramphenicol acetyltransferase (CAT), firefly luciferase, ⁇ -glucuronidase (GUS), green fluorescent protein (GFP), and ⁇ -galactosidase have been used extensively.
  • CAT chloramphenicol acetyltransferase
  • GUS ⁇ -glucuronidase
  • GFP green fluorescent protein
  • ⁇ -galactosidase have been used extensively.
  • such reporters all have individual shortcomings that may limit or preclude their usage under some conditions. For example, high levels of GFP are toxic to the cell.
  • reporter enzymes are not expressed equally in all cell types nor are they equally stable when expressed in all cell types.
  • the cytoplasmic enzyme ⁇ -galactosidase is widely used as a reporter gene in various microbiological and molecular biological studies. This enzyme is used in both in vitro and in vivo assays. The wide acceptance of this reporter system results, in part, because it is non-isotopic and extremely flexible. It is used in a number of assay formats and has an extremely broad linear range. Nevertheless, because ⁇ -galactosidase is present in the cytoplasm of various host cells such as Escherichia coli, deletion of the lacZ gene, the source of the enzyme, is often required prior to its use in a host cell system. In addition, cells must be lysed or solubilized prior to assaying the reporter enzyme.
  • One embodiment of the present invention is an alternative enzyme for use as a reporter, particularly one that is a secreted enzyme or an ectoenzyme.
  • a secreted enzyme or an ectoenzyme reporter enzyme will obviate the need to lyse the cells prior determining its activity.
  • membrane-bound chitobiase as a reporter is that genes encoding chitobiase are missing from many bacteria, including £ coli, some fungi, and some eukaryotic cells. Thus, it is not necessary to engineer many host cells to lack endogenous enzyme activity as is the case with the commonly used reporter ⁇ - galactosidase.
  • the present invention particularly contemplates the use of expressed membrane-bound chitobiase as a reporter enzyme.
  • the present invention also contemplates the generation of fusion proteins comprising a fusion polypeptide joined in frame to chitobiase.
  • the fusion polypeptide comprises a polypeptide other than chitobiase, such as a heterologous protein.
  • the heterologous polypeptide may comprise a polypeptide having a biological activity (such as an enzymatic or other activity besides activity as an immunogen) or the heterologous polypeptide may not have a biological activity.
  • the fusion reporter gene construct contains a sequence encoding the fusion polypeptide genetically fused in frame with a sequence encoding chitobiase.
  • a nucleic acid prospectively containing a promoter is inserted upstream of a nucleic acid encoding the membrane- bound form of chitobiase as described above.
  • the nucleic acid prospectively containing a promoter may be inserted into a restriction site in a sequence containing a plurality of restriction sites, such as a polylinker, which is located upstream of the nucleic acid encoding chitobiase.
  • the test sequence may comprise any nucleic acid to be tested for promoter activity.
  • the test sequence may comprise a genomic DNA sequence.
  • the genomic DNA sequence may be a randomly generated DNA fragment, such as a fragment generated using shotgun cloning techniques, a restriction fragment, or any other sequence.
  • the vectors containing the test sequence upstream of the nucleic acid encoding membrane-bound chitobiase are introduced into an appropriate host cell.
  • the level of chitobiase activity is assayed and compared to the level obtained from a control vector which lacks an insert in the cloning site.
  • the presence of an elevated expression level in cell containing the vector containing the insert with respect to the level in cells containing the control vector without the insert indicates the presence of a promoter in the insert.
  • the activity of the promoter in the test sequence may be assayed after exposure of the host cells to conditions which may influence the level of transcription from the promoter.
  • the environment of the host cells may be altered to determine whether the transcription level is influenced by environmental factors, including factors such as temperature, pH, nutrients, or availability of oxygen.
  • chitobiase levels are assayed under a variety of environmental conditions to determine the effects of the environmental conditions on transcription levels from the promoter.
  • the activity of the promoters may be examined in the presence or absence of compounds to be tested for regulatory activity.
  • the activity of the promoters may be tested by determining the levels of chitobiase produced in the presence or absence of compounds to be tested for activity as drugs.
  • Promoter sequences within the test sequences may be further defined by constructing nested deletions in the test sequences using conventional techniques such as Exonuclease III digestion.
  • the resulting deletion fragments can be inserted into the promoter reporter vector to determine whether the deletion has reduced or obliterated promoter activity as determined by measuring chitobiase activity in cells containing the deletion vectors.
  • the boundaries of the promoters may be defined.
  • potential individual regulatory sites within the promoter may be identified using techniques such as site directed mutagenesis or linker scanning to obliterate potential transcription factor binding sites within the promoter individually or in combination.
  • the effects of these mutations on transcription levels may be determined by inserting the mutations into the cloning sites in the promoter reporter vectors and measuring the levels of chitobiase produced from the mutated promoters.
  • the activity of known promoters may also be monitored by operably linking them to a nucleic acid encoding a membrane-bound form of chitobiase.
  • the activity of the promoters may be analyzed under various environmental conditions as described above.
  • the activity of the promoters may be analyzed in the presence or absence of compounds to be tested for the ability to affect transcription from the promoters.
  • the compounds may be tested for activity as drugs.
  • mutations may be introduced into promoters which are linked to the reporter enzyme.
  • the mutations may be screened to determine whether they increase the ability of the promoter to direct transcription in a cell or organism of interest.
  • the constructs encoding a membrane-bound form of chitobiase may be used in systems for identifying compounds that modulate cell surface protein-mediated activity or compounds which modulate the activities of intracellular signaling systems.
  • Techniques for using reporter genes to identify compounds which modulate cell surface protein-mediated activity have been described in U.S. Patent Nos. 5,401,629 and 5,436,128. Briefly, in such methods, a construct comprising a promoter operably linked to a nucleic acid encoding a reporter enzyme is introduced into cells which express the cell surface protein and cells which do not express the cell surface protein. Each of the cells are contacted with test compounds and the effects of these compounds on transcription levels is measured by determining the level of activity of the reporter enzyme. The level of expression of the reporter gene in cells expressing the cell surface protein is compared to the level in cells which do not express the cell surface protein to identify compounds that modulate cell surface protein activity.
  • the chitobiase reporter constructs may be used to identify compounds which influence the activity of intracellular signaling pathways, such as cAMP-based or phosphorylation-based pathways.
  • a promoter which is activated via such pathways is operably linked to a nucleic acid encoding a membrane-bound form of chitobiase.
  • the cells are contacted with test compounds. Those compounds which activate the pathway to which the promoter responds will produce an enhanced level of chitobiase activity in the cells as compared to the level of chitobiase activity in control cells which have not been contacted with the test compound.
  • a vector comprising a sequence encoding a membrane-bound form of chitobiase operably linked to a sequence capable of directing transcription of the chitobiase gene is introduced into a host cell.
  • the host cells are contacted with a chitobiase substrate and those host cells which contain chitobiase activity are identified as cells which were successfully transformed or transfected.
  • a portion or replica of a colony may be lysed or permeabilized prior and the lysate or permeabilized cells may be contacted with the chitobiase substrate.
  • membrane-bound chitobiase is used as a marker for the outer membrane in cell fractionation studies. If a protein X co-purifies or co-segregates with chitobiase activity, then protein X is in the outer membrane. This is especially useful in studies of £ coli or other bacterial species where it is not known which enzymes are in the outer membrane. To determine the location of an enzyme, cells are fractionated into cytoplasmic, inner membrane, outer membrane and periplasmic fractions using well known methods. Activities of enzymes associated with a particular cell compartment are included to show the extent of purity of the fractions. Chitobiase is used as a marker for the outer membrane is such a study.
  • Membrane-bound chitobiase may also be used to identify outer membrane proteins which are desirable drug targets, particularly targets for antibiotics. Outer membrane proteins which are essential for cell growth are particularly attractive antibiotic targets because the antibiotic does not have to pass through the membrane to arrive at its target. Genes essential for cell proliferation are identified as described in U.S. Patent Application No.
  • One method to determine if an essential protein is an outer membrane protein comprises fractionating cells, performing sucrose density gradient ultracentrifugation on the fractionated cells, and the fractions containing the chitobiase activity are assayed for the protein of interest.

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Abstract

L'invention porte sur des procédés de mesure de la prolifération cellulaire à l'aide d'ectoenzymes telles que la chitobiase (N,N'-diacétylchitobiase) liée à la membrane, et d'acides nucléiques nécessaires auxdits procédés.
PCT/US2000/021049 2000-08-02 2000-08-02 Utilisation d'ectoenzymes et d'enzymes secretees pour suivre la proliferation cellulaire WO2002010442A1 (fr)

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PCT/US2000/021049 WO2002010442A1 (fr) 2000-08-02 2000-08-02 Utilisation d'ectoenzymes et d'enzymes secretees pour suivre la proliferation cellulaire
AU2000268924A AU2000268924A1 (en) 2000-08-02 2000-08-02 Use of ectoenzymes and secreted enzymes to monitor cellular proliferation

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10857243B2 (en) 2014-04-09 2020-12-08 Brandeis University Enzymatically responsive magnetic particles and their use

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4259442A (en) * 1977-10-04 1981-03-31 Laboratoire De Recherche Api S.A.R.L. Process of rapid identification of bacteria of the genus Streptococcus
EP0174477A1 (fr) * 1984-08-14 1986-03-19 MERCK PATENT GmbH Moyen et procédé pour déterminer les substances ayant une activité antimicrobienne

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4259442A (en) * 1977-10-04 1981-03-31 Laboratoire De Recherche Api S.A.R.L. Process of rapid identification of bacteria of the genus Streptococcus
EP0174477A1 (fr) * 1984-08-14 1986-03-19 MERCK PATENT GmbH Moyen et procédé pour déterminer les substances ayant une activité antimicrobienne

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DOERN G V ET AL: "ANTIMICROBIAL SUSCEPTIBILITY TESTING OF HAEMOPHILUS-INFLUENZAE BRANHAMELLA-CATARRHALIS AND NEISSERIA-GONORRHOEAE", ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, vol. 32, no. 12, 1988, pages 1747 - 1753, XP002940594, ISSN: 0066-4804 *
KALABAT D Y ET AL: "Chitobiase, a new reporter enzyme", BIOTECHNIQUES,US,EATON PUBLISHING, NATICK, vol. 25, no. 6, December 1998 (1998-12-01), pages 1030 - 1035, XP002106689, ISSN: 0736-6205 *
OLSSON-LILJEQUIST BARBRO ET AL: "Antimicrobial susceptibility testing in Sweden: III. Methodology for susceptibility testing.", SCANDINAVIAN JOURNAL OF INFECTIOUS DISEASES SUPPLEMENTUM, no. 105, 1997, pages 13 - 23, XP002940595, ISSN: 0300-8878 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10857243B2 (en) 2014-04-09 2020-12-08 Brandeis University Enzymatically responsive magnetic particles and their use

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