Primary Examiner: LeGuyader; John L.
Assistant Examiner: Schultz; James Douglas
Attorney, Agent or Firm: Knobbe, Martens, Olson & Bear, LLP
 

What is claimed is:

1. A method for screening a test compound for the ability to inhibit microbial proliferation, said method comprising the steps of:

(a) providing a population of microbial cells expressing an ectoenzyme or secreted enzyme, wherein said population of cells is contacted with a sublethal level of an antisense nucleic acid that is complementary to at least a portion of a nucleic acid that encodes a gene product which is required for proliferation of said population of microbial cells, to reduce the activity or amount of said gene product in said cells, to thereby produce sensitized microbial cells;

(b) determining the extent of proliferation of said sensitized cells that express said ectoenzyme or secreted ezyme by measuring the activity of said ectoenzyme or secreted enzyme;

(c) contacting said sensitized cells with a test compound and measuring the extent of proliferation of said sensitized cells in response to said test compound; and

(d) determining whether said test compound inhibits the proliferation of said sensitized cells by comparing the activity of said ectoenzyme or secreted enzyme in said sensitized cells prior to contact with the test compound with the activity of said ectoenzyme or secreted enzyme following contact with the test compound.

2. The method of claim 1, wherein said ectoenzyme or secreted enzyme is selected from the group consisting of Pseudomonas aeruginosa metalloproteinase, Moraxella (Branhamella) Catarrhalis BRO beta-lactamase, P. aeruginosa FpvA ferric pyoverdin receptor, E. coli OmpP endopeptidase, outer membrane phospho lipase A, Bacteriodes thetaiotamicron susG starch utilization protein, Haemophilus influenza? 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. aureus autolysin, hemolysin, DNase, coagulase, protein A, staphylokinase and enterotoxin.

3. The method of claim 1, wherein said ectoenzyme or secreted enzyme is a membrane-bound form of chitobiase.

4. The method of claim 1, wherein said ectoenzyme or secreted enzyme is endogenous.

5. The method of claim 1, wherein said sensitized cells contain an introduced gene encoding said ectoenzyme or secreted enzyme.

6. The method of claim 1, wherein said population of cells is from an organism selected from the group consisting of Staphylococcus aureus, Aspergillus fumigatus, Bacillus anthracis, Campylobacter jejuni, Candida albicans, Chlamydia pneumoniae, Chlamydia trachomatus, Clostridium botulinum, Cryptococcus neoformans, E. coli, Enterobacter cloacae, Enterococcus faecalis, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Mycobacterium leprae, Mycobacterium tuberculosis, Heisseria gonorrhoeae, Pseudomonas aeruginosa, Salmonella cholerasuis, Salmonella paratyphi, Salmonella typhi, Salmonella typhimurium, Staphylococcus epidermidis, Streptococcus pneumoniae, Treponema pallidum, and Yersinia pestis or any species falling within the genera of any of the above species.

7. The method of claim 1, wherein said antisense nucleic acid is transcribed from an inducible promoter.

8. The method of claim 1, further comprising the step of contacting said population of cells with a concentration of inducer which induces said antisense nucleic acid to a sublethal level.

9. The method of claim 1, wherein said sublethal level of the antisense nucleic acid is provided by contacting said population of cells with the antisense nucleic acid.

10. The method of claim 1, wherein said sublethal level of the antisense nucleic acid is provided by expressing the antisense in said population of cells.

11. The method of claim 1, wherein said gene product is a polypeptide.

12. The method of claim 1, wherein said gene product is an RNA.

13. The method of claim 1, wherein said test compound is from a combinatorial chemical library.

14. The method of claim 1, wherein said test compound is a natural product.

15. A method for screening a test compound for the ability to inhibit microbial proliferation, said method comprising the steps of:

(a) providing a first population of unsensitized microbial cells expressing an ectoenzyme or secreted enzyme, wherein said first population of cells is contacted with a sublethal level of an antisense nucleic acid that is complementary to at least a portion of a nucleic acid that encodes a gene product which is required for proliferation of said first population of microbial cells, to reduce the activity or amount of said gene product in said cells, to thereby produce sensitized microbial cells;

(b) determining the extent of proliferation of said sensitized cells that express said ectoenzyme or secreted enzyme by measuring the activity of said ectoenzyme or secreted enzyme;

(c) contacting said sensitized cells with a test compound and measuring the extent of proliferation of said sensitized cells in response to said test compound; and

(d) determining whether said test compound inhibits the proliferation of said sensitized cells by comparing the activity of said ectoenzyme or secreted enzyme in said sensitized cells prior to contact with the test compound with the activity of said ectoenzyme or secreted enzyme following contact with the test compound,

(e) providing a second population of unsensitized microbial cells, wherein said second population of unsensitized microbial cells are from the same population of microbial cells as said first population of unsensitized microbial cells, and said second population of unsensitized cells have not undergone sensitization treatment of any kind;

(f) determining the extent of proliferation for said unsensitized cells that express said ectoenzyme or secreted enzyme by measuring the activity of said ectoenzyme or secreted enzyme;

(g) contacting said unsensitized cells with a test compound and measuring the extent of proliferation of said unsensitized cells in response to said test compound; and

(h) determining whether said test compound inhibits the proliferation of said unsensitized cells by comparing the activity of said ectoenzyme or secreted enzyme in said unsensitized cells prior to contact with the test compound with the activity of said ectoenzyme or secreted enzyme following contact with the test compound;

(i) determining whether said test compound inhibits the proliferation of said sensitized cells to a greater extent than said compound inhibits the proliferation of said unsensitized cells by comparing the change in activity of said ectoenzyme or secreted enzyme in said sensitized cells following contact with the test compound with the change in activity of said ectoenzyme or secreted enzyme in said unsensitized cells following contact with the test compound.

16. The method of claim 15, wherein said ectoenzyme or secreted enzyme is selected from the group consisting of Pseudomonas aeruginosa metalloproteinase, Moraxella (Branhamella) Catarrhalis BRO beta-lactamase, P. aeruginosa FpvA ferric pyoverdin receptor, E. coli OmpP endopeptidase, outer membrane phospho ipase 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. aureus autolysin, hemolysin, DNase, coagulase, protein A, staphylokinase, and enterotoxin.

17. The method of claim 15, wherein said ectoenzyme or secreted enzyme is a membrane-bound form of chitobiase.

18. The method of claim 15, wherein said ectoenzyme or secreted enzyme is endogenous.

19. The method of claim 15, wherein said sensitized cells contain an introduced gene encoding said ectoenzyme or secrete enzyme.

20. The method of claim 15, wherein said population of cells is from an organism selected from the group consisting of Staphylococcus aureus, Aspergillus fumigatus, Bacillus anthracis, Campylobacter jejuni, Candida albicans, Chlamydia pneumoniae, Chlamydia trachomatus, Clostridium botulinum, Cryptococcus neoformans, E. 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 pneumoniae, Treponema pallidum, and Yersinia pestis or any species falling within the genera of any of the above species.

21. The method of claim 15, wherein said antisense nucleic acid is transcribed from an inducible promoter.

22. The method of claim 15, further comprising the step of contacting said first population of unsensitized cells with a concentration of inducer which induces said antisense nucleic acid to a sublethal level.

23. The method of claim 15, wherein said sublethal level of the antisense nucleic acid is provided by contacting said first population of unsensitized cells with the antisense nucleic acid.

24. The method of claim 15, wherein said sublethal level of the antisense nucleic aid is provided by expressing the antisense in said first population of unsensitized cells.

25. The method of claim 15, wherein said gene product is a polypeptide.

26. The method of claim 15, said gene product is an RNA.

27. The method of claim 15, wherein said test compound is from a combinatorial chemical library.

28. The method of claim 15, wherein said test compound is a natural product.

FIELD OF THE INVENTION

The present invention relates to the use of enzymes which are associated with the cell (ectoenzymes) and secreted enzymes for monitoring cellular proliferation.

BACKGROUND OF THE INVENTION

Reporter enzymes are enzymes whose activities are easily assayed when present inside cells. In order to study the regulation of a gene whose expression is regulated by various environmental and/or cellular factors or influences, 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. In addition, 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.

Since the discovery of penicillin, the use of antibiotics to treat the ravages of bacterial infections has saved millions of lives. With the advent of these "miracle drugs," for a time it was popularly believed that humanity might, once and for all, be saved from the scourge of bacterial infections. In fact, during the 1980s and early 1990s, many large pharmaceutical companies cut back or eliminated antibiotics research and development. They believed that infectious disease caused by bacteria finally had been conquered and that markets for new drugs were limited. Unfortunately, this belief was overly optimistic.

The tide is beginning to turn in favor of the bacteria as reports of drug resistant bacteria become more frequent. The United States Centers for Disease Control announced that one of the most powerful known antibiotics, vancomycin, was unable to treat an infection of the common Staphylococcus aureus (staph). This organism is commonly found in our environment and is responsible for many nosocomial infections. The import of this announcement becomes clear when one considers that vancomycin was used for years to treat infections caused by Staphylococcus species as well as other stubborn strains of bacteria. In short, bacteria are becoming resistant to our most powerful antibiotics. If this trend continues, it is conceivable that we will return to a time when what are presently considered minor bacterial infections are fatal diseases.

Over-prescription and improper prescription habits by some physicians have caused an indiscriminate increase in the availability of antibiotics to the public. The patients are also partly responsible, since they will often improperly use the drug, thereby generating yet another population of bacteria that is resistant, in whole or in part, to traditional antibiotics.

The bacterial pathogens that have haunted humanity remain, in spite of the development of modern scientific practices to deal with the diseases that they cause. Drug resistant bacteria are now an increasing threat to the health of humanity. A new generation of antibiotics is needed to once again deal with the pending health threat that bacteria present.

Discovery of New Antibiotics

As more and more bacterial strains become resistant to the panel of available antibiotics, new antibiotics are required to treat infections. In the past, practitioners of pharmacology would have to rely upon traditional methods of drug discovery to generate novel, safe and efficacious compounds for the treatment of disease. Traditional drug discovery methods involve blindly testing potential drug candidate-molecules, often selected at random, in the hope that one might prove to be an effective treatment for some disease. The process is painstaking and laborious, with no guarantee of success. Today, the average cost to discover and develop a new drug exceeds US $500 million, and the average time from laboratory to patient is 15 years. Improving this process, even incrementally, would represent a huge advance in the generation of novel antimicrobial agents.

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.

Nevertheless, while this approach to drug discovery is clearly the way of the future, problems remain. For example, 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.

To facilitate the identification of new drugs, automated assays which allow the effects of a large number of candidate compounds on microbial proliferation to be easily, rapidly and inexpensively evaluated are required. The present invention relates to the use of ectoenzymes and secreted enzymes in assays for measuring cellular proliferation.

SUMMARY OF THE INVENTION

One embodiment of the present 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, E. 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. aureus autolysin, hemolysin, DNase, coagulase, protein A, staphylokinase and enterotoxin. 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 sarkosyl. The method may further comprise contacting the cells with sarkosyl and NaCl. The method may further comprise contacting the cells with NaCl. In some versions of the method, 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 prokaryotic 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 maybe p-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. 0-7, Arabidopsis thaliana, Bacillus subtilis, Bombyx mori, Bos taurus, Caenorhabditis elegans, Candida albicans, Dictyostelium discoideum, Entamoeba histolytica, Felis catus, Homo sapiens, Korat cats, Lactobacillus casei, Leishmania donovani, Mus musculus, Pisum sativum, Porphyromonas gingivalis, Pseudoalteromonas sp. S9, Rattus norvegicus, Serratia marcescens, Streptomyces plicatus, Streptomyces thermoviolaceus, Sus scrofa, Trichoderma harzianum, Vibrio furnissii, Vibrio harveyi, Vibrio parahaemolyticus, and Vibrio vulnificus.

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. 0-7, Arabidopsis thaliana, Bacillus subtilis, Bombyx mori, Bos taurus, Caenorhabditis elegans, Candida albicans, Dictyostelium discoideum, Entamoeba histolytica, Felis catus, Homo sapiens, Korat cats, Lactobacillus casei, Leishmania donovani, Mus musculus, Pisum sativum, Porphyromonas gingivalis, Pseudoalteromonas sp. S9, Rattus norvegicus, Serratia marcescens, Streptomyces plicatus, Streptomyces thermoviolaceus, Sus scrofa, Trichoderma harzianum, Vibrio furnissii, Vibrio harveyi, Vibrio parahaemolyticus, and Vibrio vulnificus. 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 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 p-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. 0-7, Arabidopsis thaliana, Bacillus subtilis, Bombyx mori, Bos taurus, Caenorhabditis elegans, Candida albicans, Dictyostelium discoideum, Entamoeba histolytica, Felis catus, Homo sapiens, Korat cats, Lactobacillus casei, Leishmania donovani, Mus musculus, Pisum sativum, Porphyromonas gingivalis, Pseudoalteromonas sp. S9, Rattus norvegicus, Serratia marcescens, Streptomyces plicatus, Streptomyces thermoviolaceus, Sus scrofa, Trichoderma harzianum, Vibrio furnissii, Vibrio harveyi, Vibrio parahaemolyticus, and Vibrio vulnificus. The nucleic acid may further comprise a .lambda. 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. 0-7, Arabidopsis thaliana, Bacillus subtilis, Bombyx mori, Bos taurus, Caenorhabditis elegans, Candida albicans, Dictyostelium discoideum, Entamoeba histolytica, Felis catus, Homo sapiens, Korat cats, Lactobacillus casei, Leishmania donovani, Mus musculus, Pisum sativum, Porphyromonas gingivalis, Pseudoalteromonas sp. S9, Rattus norvegicus, Serratia marcescens, Streptomyces plicatus, Streptomyces thermoviolaceus, Sus scrofa, Trichoderma harzianum, Vibrio furnissii, Vibrio harveyi, Vibrio parahaemolyticus, and Vibrio vulnificus. 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 p-nitrophenol. 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. 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, E. 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. 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 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 Pseudomonas aeruginosa metalloproteinase, Moraxella (Branhamella) Catarrhalis BRO beta-lactamase, P. aeruginosa FpvA ferric pyoverdin receptor, E. 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. 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.

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, E. 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. aureus autolysin, hemolysin, DNase, coagulase, protein A, staphylokinase and enterotoxin. 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 fumigatus, Bacillus anthracis, Campylobacter jejuni, Candida albicans, Chlamydia pneumoniae, Chlamydia trachomatus, Clostridium botulinum, Cryptococcus neoformans, E. 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 pneumoniae, Treponema pallidunm, and Yersinia pestis or any species falling within the genera of any of the above species. 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, E. 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. aureus autolysin, hemolysin, DNase, coagulase, protein A, staphylokinase and enterotoxin. 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, E. 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. aureus autolysin, hemolysin, DNase, coagulase, protein A, staphylokinase and enterotoxin. 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, E. 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. aureus autolysin, hemolysin, DNase, coagulase, protein A, staphylokinase and enterotoxin. 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, E. 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. aureus autolysin, hemolysin, DNase, coagulase, protein A, staphylokinase and enterotoxin. 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 genomic 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, E. 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. aureus autolysin, hemolysin, DNase, coagulase, protein A, staphylokinase and enterotoxin. The ectoenzyme or secreted enzyme may bea 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.

Definitions

As used herein, the term "proliferation" means an increase in the number of cells with time. By "inhibition of proliferation" is meant that growth, replication or viability of the microorganism is reduced or eliminated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of plasmid pJFK4 comprising the wild type chitobiase (chb) gene encoding the membrane-bound form of chitobiase, and the E. coli chromosomal attB site. Restriction sites shown are unique with the exception of SacI of which there are two sites created by the construction of the plasmid, and NotI, of which there are two sites flanking the P15A origin. Closed arrows represent genes and gene orientation, the open box represents the origin of replication (ori) and the small arrow represents the transcription start site.

FIG. 2 is a schematic diagram showing the integration of the wild type chb gene into the E. coli chromosome by site-specific recombination between attB and attP.

FIG. 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) or cells containing the integrated chb gene (DJKGC4) were stopped for growth, diluted to 0.2 OD.sub.600 serially diluted and a fluorescent chitobiase substrate was added. Relative fluorescence units (RFU) were charted after a 2 hour incubation at room temperature. Fluorescence was clearly detectable above background for cultures calculated to have an OD.sub.600 of 0.0016 and 0.00032, below detectable limits of common-use spectrophotometers.

FIG. 4 is a graph comparing the sensitivity of measurement of the growth of E. coli in a 1536-well microplateby turbidity at OD.sub.600 and chitobiase activity was determined by measuring release of p-nitrophenol from the substrate PNAG by monitoring OD.sub.415.

FIG. 5 is a graph showing the measurement of E. coli growth ina 1536-well micro plate using a chitobiase assay in E. coli strain DJKGC4 which contain a chromosomally integrated, constitutively expressed chitobiase gene. The parental chitobiase negative E. coli strain of DJKGC4 is MG1655. LB=Luria-Bertani broth; S=chitobiase substrate 4-methylumbelliferyl N-acetyl .beta.-D-glucosaminide (MNAG).

FIG. 6 is an E. coli dose response curve to gentamicin. E. coli strain DJKGC4 was inubated with various concentrations of gentamicin for 5 hours in a 1536 well plate in the presence of 100 .mu.M 4-methylumbelliferyl N-acetyl .beta.-D-glucosaminide (MNAG). After removal of background fluorescence, the inhibition of cell growth produced by each gentamicin concentration was calculated by comparison to the fluorescence generated by control cells growing in the absence of 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. Control E. coli cells (pLEX5BA) or E. coli containing the integrated chitobiase gene (DJKGC4) were grown in LB medium stopped for growth with kanamycin, pelleted and resuspended in M9 dilution buffer (M9-DB). Cells were serially diluted and 100 .mu.l of "Tris MNAG" buffer, supplemented with NaCl, sarkosyl, both, or neither, was added to each well such that final concentrations were: 100 mM Tris-Cl (pH 8.0), 50 .mu.M MNAG, 0.5 M NaCl, 0.5% sarkosyl. Data for cells corresponding to the turbidimetric measurement of OD.sub.600 =0.0018 is plotted after 2 hours of incubation with MNAG.

FIG. 8 is a graph showing sensitive detection of cell growth using the chitobiase assay. MG1655 E. coli cells transfected with the pJFK4 plasmid were grown in LB medium to an OD.sub.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 mM MgSO.sub.4 and 0.1 mM CaCl.sub.2), with or without 1 mM PNAG to a final OD.sub.600 of 0.002. Two hundred .mu.l of each were aliquoted into a 96 well microtiter plate and OD.sub.405 and OD.sub.600 were read in a Spectramax plate reader (Molecular Devices) in 5 minute intervals for 14 hours. Duplicate samples were plotted. OD.sub.405 detects both PNP product and turbidity, whereas OD.sub.600 detects only turbidity.

FIG. 9 is an IPTG dose response curve in E. coli transformed with an IPTG-inducible plasmid containing either an antisense clone to the E. coli ribosomal protein rplW which is essential for cell proliferation, or an antisense clone to the elaD gene which is not essential for proliferation.

FIG. 10A is a tetracycline dose response curve in E. coli transfected with an IPTG-inducible plasmid containing antisense to rplW in the presence of 0.20 or 50 .mu.M IPTG.

FIG. 10B is a tetracycline dose response curve in E. coli transfected with an IPTG-inducible plasmid containing antisense to elaD in the presence of 0, 20 or 50 .mu.M IPTG.

FIG. 11 is a graph showing the fold increase in tetracycline sensitivity of E. coli transfected with antisense clones to essential ribosomal proteins L23 (ASrplW) and L7/L12 and L10 (ASrplLrplJ). Antisense clones to genes known not to be involved in protein synthesis (atpB/E, visC, elaD, yohH) are much less sensitive to tetracycline.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to the use of ectoenzymes or secreted enzymes to measure microbial proliferation. By the term "ectoenzyme" is meant 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. In a preferred embodiment, 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). In another embodiment, 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. For example, 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.

Although 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, the disclosure of which is incorporated by reference in its entirety), Moraxella (Branhamella) catarrhalis BRO beta-lactamase (Bootsma et al., J. Bacteriol 181:5090-5093, 1999, the disclosure of which is incorporated by reference in its entirety), P. aeuriginosa FpvA ferric pyoverdin receptor (Schalk et al., Biochemistry 38:9357-9365, 1999, the disclosure of which is incorporated by reference in its entirety), E. coli OmpP endopeptidase (Striebel et al., Eur. J. Biochem. 262:832-839, 1999, the disclosure of which is incorporated by reference in its entirety), outer-membrane phospholipase A (OMPLA) of Gram-negative bacteria (Dekker, Mol. Microbiol. 35:711-717, 2000, the disclosure of which is incorporated by reference in its entirety), OmpT endopeptidase of Gram-negative bacteria (Stathopoulos, Membr. Cell Biol. 1:1-8, 1998, the disclosure of which is incorporated by reference in its entirety), Bacteroides thetaiotamicron susG starch utilization protein (Shipman et al., J. Bacteriol. 181:7206-7211, 1999, the disclosure of which is incorporated by reference in its entirety), Haemophilus influenzae phosphomonoesterase (Reilly et al., Protein Expr. Purif. 17:401-409, 1999; Reilly et al., J. Bacteriol. 181:6797-6805, 1999, the disclosure of which is incorporated by reference in its entirety), streptococcal protein Sir (Stenberg et al., J. Biol. Chem. 269:13458-13464, 1994, the disclosure of which is incorporated by reference in its entirety), C5a peptidase genes in group A and B streptococci (Chmouryguina et al., Infect. Immun. 64:2387-2390, 1996, the disclosure of which is incorporated by reference in its entirety), Lactococcus lactis nisin serine protease NisP (van der Meer et al., J. Bacteriol. 175:2578-2588, 1993, the disclosure of which is incorporated by reference in its entirety), proteinases PrtB, PrtH, PrtP, ScpA (Siezen, Multi-domain, cell-envelope proteinases of lactic acid bacteria. Antonie van Leeuwenhoek, 76:139-155, 1999, the disclosure of which is incorporated by reference in its entirety); Streptococcus mutans dextranase (Igarashi, Microbiol. Immunol. 36:969-976, 1992, the disclosure of which is incorporated by reference in its entirety), Streptococcus pneumoniae beta-N-acetylglucosaminidase (Clarke et al., J. Biol. Chem. 270:8805-8814, 1995, the disclosure of which is incorporated by reference in its entirety), S. penumoniae neuraminidase (Infect. Immun. 62:3688-3695, 1995, the disclosure of which is incorporated by reference in its entirety), Streptococcus sobrinus dextranase (J. Bacteriol. 176:3839-3850. 1994, the disclosure of which is incorporated by reference in its entirety) and Streptococcus suis muramidase (Infect. Immun. 60:2361-2367, the disclosure of which is incorporated by reference in its entirety). Methods for measuring the activity of these ectoenzymes are described in these references, the disclosures of which are incorporated herein by reference in their entireties.

Accordingly, 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. In some embodiments, the enzyme is a bacterial ectoenzyme. In other embodiments, 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. However, in another embodiment, 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. Such signal sequences, membrane anchors and cell wall attachment signals are familiar to those skilled in the art.

In addition to ectoenzymes, 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, the disclosure of which is incorporated by reference in its entirety); Staphylococcus aureus murein hydrolase (Groicher et al., J. Bacteriol 182:1794-1801, 2000, the disclosure of which is incorporated by reference in its entirety); Staphylococcal lipases (Gotz et al., Chem. Phys. Lipids 93:15-25, 1998, the disclosure of which is incorporated by reference in its entirety); staphylolytic glycylglycine (lysostaphin), endo-beta-N-acetylglucosaminidase (hexosaminidase) and sulfhydryl protease (Bunn et al., FEMS Microbiol. Lett. 165:123-127, 1998, the disclosure of which is incorporated by reference in its entirety); staphylococci ester hydrolyzing enzymes (Talon et al., Int. J. food Microbiol. 36:207-214, 1997, the disclosure of which is incorporated by reference in its entirety); S. aureus nucleases A and B (Suciu et al., Mol. Microbiol. 21:181-195, 1996, the disclosure of which is incorporated by reference in its entirety), S. aureus fatty acid modifying enzyme (FAME) (Chamberlain et al., J. Med. Microbiol. 44:125-129, 1996, the disclosure of which is incorporated by reference in its entirety); bacterial chitinases (Hayashi et al., Biosci. Biotechnol. Biochem. 59:1981-1982, 1995, the disclosure of which is incorporated by reference in its entirety); S. aureus autolysin (Proc. Natl. Acad. Sci. U.S.A. 92:285-289, 1995, the disclosure of which is incorporated by reference in its entirety) and alpha- and beta-hemolysins, DNase, coagulase, protein A, proteases, lipase, staphylokinase and enterotoxin A of S. aureus (Giraudo et al., Can. J. Microbiol. 40:677-681, 1994, the disclosure of which is incorporated by reference in its entirety). Methods for measuring the activity of these secreted enzymes are described in these references, the disclosures of which are incorporated herein by reference in their entireties.

The secreted enzymes may be naturally-occurring. Alternatively, 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, the disclosure of which is incorporated by reference in its entirety). A number of secretion vectors are commercially available. For example, Invitrogen (Carlsbad, Calif.) sells secretion vectors for use in a variety of host cells. One such vector is the pBAD/gIII kit which is designed to express recombinant proteins in E. coli. In this vector, the leader peptide from the bacteriophage fd gene III protein (gIII) directs the secretion of the polypeptide encoded by any adjacent sequence into the periplasmic space. pSecTag2 and pSecTag2/Hygro (Invitrogen) 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, particularly membrane-bound chitobiase, or secreted enzymes, 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. In some embodiments of the present invention, 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. In some embodiments, the cells are sensitized as discussed below. In one embodiment, the ectoenzyme is chitobiase.

Chitobiase is one of two enzymes that hydrolyze chitin, an abundant insoluble polysaccharide, to its monomeric unit, N-acetylglucosamine (GlcNac). Chitobiase is known to be present in a number of organisms. For example, 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. 0-7, Arabidopsis thaliana, Bacillus subtilis, Bombyx mori, Bos taurus, Caenorhabditis elegans, Candida albicans, Dictyostelium discoideum, Entamoeba histolytica, Felis catus, Homo sapiens, Korat cats, Lactobacillus casei, Leishmania donovani, Mus musculus, Pisum sativum, Porphyromonas gingivalis, Pseudoalteromonas sp. S9, Rattus norvegicus, Serratia marcescens, Streptomyces plicatus, Streptomyces thermoviolaceus, Sus scrofa, Trichoderma harzianum, Vibrio furnissii, Vibrio harveyi, Vibrio parahaemolyticus, and Vibrio vulnificus.

One source of 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; both of which are incorporated herein by reference in their entireties). Replacement of the first 22 amino acids of prechitobiase with the first 21 amino acids of lacZ from pUC19 resulted in a soluble chimeric protein with chitobiase activity which remained in the cytoplasm. This soluble chitobiase has been used as a reporter enzyme in toluene-solubilized cells (Jannatipour et al., supra.). The complete nucleotide and amino acid sequences of membrane-bound chitobiase from V. harveyi is shown in SEQ ID NOS: 2 (endogenous chitobiase promoter and coding sequence) and SEQ ID NO: 3 (amino acid sequence of membrane-bound chitobiase). However, it will be appreciated that chitobiase from other organisms may also be used.

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. In addition, in the case of secreted enzymes, 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. Thus, the assay may be a "homogeneous assay" in which washing steps are not required. Standard permeabilization 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. In addition, 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. In particular, 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. The constructs are prepared according to standard techniques of molecular biology well known in the art. When the construct is a vector, 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. When integration of the gene encoding the ectoenzyme is a desired result, 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.

One example of integration sequences that can be included in a construct encoding the ectoenzyme or secreted enzyme is the .lambda. 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.

In some preferred embodiments, the gene encoding the ectoenzyme, such as membrane-bound chitobiase, or secreted enzyme, is operably linked to a constitutive promoter for obtaining constant gene expression. In other embodiments, 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. In further embodiments, 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.

Another embodiment of the present invention is a kit. One aspect of this embodiment includes a construct encoding an ectoenzyme such as membrane-bound chitobiase, or a secreted enzyme. In some embodiments, 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. For example, in one embodiment where the ectoenzyme is membrane-bound chitobiase, the kit will include a supply of a suitable chitobiase substrate whose metabolism into product by the enzyme can be assayed.

The constructs encoding an ectoenzyme, such as a membrane-bound form of chitobiase, or a secreted enzyme, may be introduced into prokaryotic or eukaryotic cells. A variety of methods are available to introduce constructs encoding ectoenzymes, such as membrane-bound chitobiase, or a secreted enzyme, into prokaryotic cells. For example, the constructs may be introduced into bacteria using calcium chloride transformation, electroporation, or viral vectors such as the filamentous phages. These and other protocols for introducing nucleic acids into prokaryotes are well known in the art.

Alternatively, the constructs encoding an ectoenzyme, such as the membrane-bound form of chitobiase, or a secreted enzyme, may be introduced into eukaryotic cells, including yeast, mammalian, plant, and insect cells. For example, 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. 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.

The introduction of the construct encoding an ectoenzyme, such as chitobiase, or a secreted enzyme, into mammalian cells can likewise utilize a number of transfection protocols well known to those of skill in the art. As discussed above, transfections can be transient or stable. Examples of suitable transfer protocols include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection, and viral transfection. These and other eukaryotic transformation protocols are well known in the art.

Following introduction of the construct encoding an ectoenzyme, such as chitobiase, or a secreted enzyme into the host cell of interest, the enzymatic activity of the enzyme is measured. Preferably, 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.

Where the ectoenzyme is chitobiase, cellular chitobiase activity can be measured quantitatively by following the hydrolysis of chitobiase substrates. Examples of substrates with utility in chitobiase activity assays include N,N'-diacetylchitobiase (chitobiase), p-nitrophenyl-N-acetyl-.beta.-D-glucosaminide (PNAG)(Sigma Chemical, St. Louis, Mo.), 4-methylumbelliferyl-N-acetyl-.beta.-D-glucosaminide dihydrate (MNAG) (Fluka), 5-bromo-4-chloro-3-indolyl-N-acetyl-.beta.-D-glucosaminide (X-Gluc)(Sigma Chemical, St. Louis, Mo.). Other substrates are also contemplated for use in the assays of the present invention.

Products produced by the hydrolysis of the chitobiase substrates are monitored using various means familiar to those skilled in the art. For example, 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. In one embodiment, p-nitrophenol is released from the substrate and measured colorimetrically at 400 nm. In another embodiment, 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. In another embodiment, blue/white colony indicator plates are used to monitor enzyme activity.

In a preferred embodiment, the membrane-bound chitobiase gene construct of the invention can also be used for measuring cell number. In this embodiment, 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. If desired, a standard curve may be constructed using known numbers of cells transfected with the gene encoding membrane-bound chitobiase. Alternatively, 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. In a preferred embodiment, the level of chitobiase activity in each cell in the cell population is similar. For example, each cell may contain an identical number of genes encoding chitobiase in its genome. In some embodiments, the cells may contain a single copy of a gene encoding chitobiase in its genome. Alternatively, the cells may each contain a similar or identical number of multicopy plasmids encoding chitobiase. In some embodiments, the chitobiase assay is performed on cells which have not been lysed or permeabilized. In other embodiments, the substrate is placed in contact with cells expressing membrane-bound chitobiase and chitobiase activity is measured without performing washing steps.

In another embodiment, cells expressing an ectoenzyme, such as membrane-bound chitobiase, or a secreted enzyme, are used in methods for identifying compounds which inhibit cellular proliferation. 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. In some embodiments, 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.

In another embodiment, 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. As discussed below, 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.

Current cell-based assays used to identify or to characterize compounds for drug discovery and development frequently depend on detecting the ability of a test compound to modulate the activity of a target molecule located within a cell or located on the surface of a cell. Most often such target molecules are proteins such as enzymes, receptors and the like. However, 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. In addition, 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. Thus, 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.

For antibiotic screening using cell based assays, inhibition of growth of bacterial or fungal cells is commonly detected using turbidity or light scattering measurements, This is a relatively insensitive method because of the large number of cells required for detection. The activity of cytoplasmic enzymes such as .beta.-galactosidase can also be used to measure cell growth, but this method requires that the cells be lysed or otherwise made permeable to the substrate. The advantage of using an enzyme over green fluorescent protein or bioluminescence (PCT WO99/14311, incorporated herein by reference) is that the catalytic activity of an enzyme produces a much greater and amplified signal for detection.

The cell-based assay methods using cells expressing an ectoenzyme, such as a membrane-bound form of chitobiase, or a secreted enzyme, 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. Thus, cell-based assays using cells expressing an ectoenzyme, such as a membrane-bound form of chitobiase, or a secreted enzyme, 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.

Due in part to the increased appearance of antibiotic resistance in pathogenic microorganisms and to the significant side-effects associated with some currently used antibiotics, novel antibiotics acting at new targets are highly sought after in the art. Yet, another limitation in the current art related to cell-based assays is the problem of repeatedly identifying hits against the same kinds of target molecules in the same limited set of biological pathways. This may occur when compounds acting at such new targets are discarded, ignored or fail to be detected because compounds acting at the "old" targets are encountered more frequently and are more potent than compounds acting at the new targets. As a result, the majority of antibiotics in use currently interact with a relatively small number of target molecules within an even more limited set of biological pathways.

The use of sensitized cells of the current invention which express an ectoenzyme or secreted enzyme provides a solution to the above problem in two ways. First, 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. Second, 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. For example, expression of 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. Thus 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. Alternatively, 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. In addition, the target may be an RNA or polypeptide in the same biological pathway as the proliferation-required nucleic acids described herein. Such 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. In addition, 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.

Current methods employed in the arts of medicinal and combinatorial chemistries are able to make use of structure-activity relationship information derived from testing compounds in various biological assays including direct binding assays and cell-based assays. Occasionally compounds are directly identified in such assays that are sufficiently potent to be developed as drugs. More often, initial hit compounds exhibit moderate or low potency. Once a hit compound is identified with low or moderate potency, directed libraries of compounds are synthesized and tested in order to identify more potent leads. Generally these directed libraries are combinatorial chemical libraries consisting of compounds with structures related to the hit compound but containing systematic variations including additions, subtractions and substitutions of various structural features. When tested for activity against the target molecule, structural features are identified that either alone or in combination with other features enhance or reduce activity. 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.

Thus, it is now possible using cell-based assays of the present invention to 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. Thus, cells are sensitized by exposing them to an inducer concentration that results in a sub-lethal level of antisense RNA expression.

In some embodiments of the cell-based assays described herein, sensitized cells expressing an ectoenzyme or secreted enzyme are contacted with compounds to be tested for the ability to inhibit proliferation. Preferably, a large number of compounds are tested for the ability to inhibit proliferation. For example, 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. If the level of enzyme activity in sensitized cells is significantly lower than the level of activity in unsensitized cells, 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. Thus, in this method, 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.

Alternatively, a large number of compounds can be intially screened for the ability to inhibit the proliferation of unsensitized cells and those compounds which inhibit proliferation can be further screened by comparing their effect on sensitized and unsensitized cells as described above.

In another embodiment, rather than first contacting sensitized cells with test compounds to identify those compounds which inhibit proliferation and subsequently testing the inhibitory compounds on both sensitized and unsensitized cells, 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. Thus, in this method, 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 SacI digested PCR product containing the wild type (WT) chitobiase promoter and additional 5' open reading frame (ORE) sequence into the SacI site of a variant of pJMF4 (BioTechniques 25:1030, 1998) which contains a 146 base pair (bp) AseI-SalI deletion, removing the promoter. Proper orientation of the SacI fragment was determined by both restriction digest and chitobiase assay. pRSG192 (J. Biol. Chem. 264:14778, 1989) was used as a template for polymerase chain reaction (PCR) amplification using primers 5'-CAAGGTTATCAGCCAGTGAG-3' (SEQ ID NO: 5) and 5'-CCTCTAGAGTCGACCTGCAGGCATTAATGCATGCG-3' (SEQ ID NO: 6) to amplify the 609 bp product. The variant of pJMF4 was produced by AseI-SalI digestion, blunt end formation using Klenow polymerase, gel isolation of the 5524 bp fragment and re-circularization using T4 DNA ligase. This variant is missing the lac promoter which is present in pJMF4.

EXAMPLE 2

Integration of the Membrane-bound Chitobiase Gene Into the E. coli Chromosome

The WT chitobiase gene in pJFK4, prepared as described in Example 1, was integrated into the attB site in the E. coli chromosome (FIG. 2). and transduced to a wild type strain (MG1655) as described previously (BioTechniques, supra.). Briefly, an E. coli strain containing a plasmid (pLDR8) which expresses integrase from the .lambda. P.sub.R promoter and contains the .lambda. cl.sub.857 repressor gene, a kanamycin-resistance gene and a temperature-sensitive origin of replication. The electroporated cells were incubated at 42.degree. C. with shaking for 30 min, then moved to 37.degree. C. for 1 hour, followed by selection on LB agar plates containing 25 .mu.g/ml chloramphenicol at 42.degree. C. Transformants were screened both for chitobiase activity and loss of kanamycin resistance, and therefore loss of pLDR8.

Transduction with PI bacteriophage (Zyskind et al., Recombinant DNA Laboratory Manual, 1992) was used to construct an integrated chitobiase gene in a wild type E. coli background and to confirm the chromosomal location of the integration. Co-transduction of the chloramphenicol acetyltransferase (cat) gene (carried by the integration), chitobiase activity and galK (linked to attB) indicated that all three genes were linked on the chromosome of the DJKGC4 strain.

EXAMPLE 3

Chitobiase Assay

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.

Overnight cultures of DJKGC4 and MG1655 containing pLEX5BA (vector control) (OD.sub.600 =4-6) were grown in LB supplemented with either 25 .mu.g/ml chloramphenicol or 200 .mu.g/ml carbenicillin, respectively. Cultures were pelleted by centrifugation and resuspended in M9-DB comprising M9 salts supplemented with 180 mM potassium phosphate (pH 7.7) and 100 .mu.g/ml kanamycin. The kanamycin was added to prevent additional cell growth. The cells were diluted to an OD.sub.600 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 OD.sub.600 of 0.000064, 100 .mu.l each. The fluorogenic chitobiase substrate 4-methylumbelliferyl-N-acetyl-.beta.-D-glucosamine dihydrate (MNAG, fluka) (100 .mu.l of 100 .mu.M MNAG), diluted in M9-DB, was added to the wells for a final concentration of 50 .mu.M. 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.

The results are shown in FIG. 3 and demonstrate that chitobiase activity can be detected with simple addition of substrate to whole cells. Two hours after addition of MNAG, cells resuspended to density equivalent to an OD.sub.600 of 0.00032 can be detected over background. By turbidity, accurate readings below 0.005 are difficult to attain. Thus, simple addition of MNAG to the integrated chitobiase strain results in at least 15-fold greater sensitivity of detection.

In another embodiment, chitobiase activity is assayed colorimetrically by the release of p-nitrophenol at 400 nm from the substrate p-nitrophenyl-N-acetyl-.beta.-D-glucopyranoside (PNAG), and turbidity is measured at 550 nm. p-Nitrophenol release is measured immediately at 400 nm with a molar absorptivity of 1.8.times.10.sup.3 liters mol.sup.- cm.sup.-1. Units are calculated after subtracting the light scattering factor (1.5.times.OD.sub.550) from OD.sub.400 of the sample. The normalizing factor of 1.5 was determined previously by measuring the light scattering ratio of bacteria at OD.sub.400 and OD.sub.550. One unit of chitobiase activity is the amount of enzyme that catalyzes the formation of 1 pmol of p-nitrophenol per min at 28.degree. C. For comparison to Miller units of .beta.-galactosidase (described in Miller, J. H., A Short Course in Bacterial Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1992)), the units are normalized to 1 ml of culture at OD.sub.450 =1.

Constructs encoding ectoenzymes other than chitobiase may also be used to measure cellular proliferation in the methods described herein. For example, the activities of the ectoenzymes H. influenzae outer membrane phosphomonoesterase e, SusG an