Genes identified as required for proliferation in Escherichia coli

• US 6,720,139 B1
• Filed: 01/27/2000
• Issued: 04/13/2004
• Est. Priority Date: 01/27/1999
• Status: Expired due to Fees

• Current Assignee: Merck & Co., Inc.
• Original Assignee: Elitra Pharmaceuticals, Inc. (Merck & Co., Inc.)
• Inventors: Forsyth, R. Allyn, Yamamoto, Robert T., Trawick, John, Froelich, Jamie M., Ohlsen, Kari L., Zyskind, Judith, Carr, Grant J., Xu, H. Howard
• Primary Examiner(s): Marschel, Ardin H.
• Application Number:
  US09/492,709
• Time in Patent Office: 1,538 Days
• Field of Search: 435/4, 435/6, 435/69.1, 514/2, 514/44, 436/501
• US Class Current: 435/6.18
• CPC Class Codes: C07K 14/245 Escherichia (G)
• CPC Class Codes: C12Q 1/18 Testing for antimicrobial activity of a material

Abstract

The sequences of nucleic acids encoding proteins required for E. coli proliferation are disclosed. The nucleic acids can be used to express proteins or portions thereof, to obtain antibodies capable of specifically binding to the expressed proteins, and to use those expressed proteins as a screen to isolate candidate molecules for rational drug discovery programs. The nucleic acids can also be used to screen for homologous genes that are required for proliferation in microorganisms other than E. coli. The nucleic acids can also be used to design expression vectors and secretion vectors. The nucleic acids of the present invention can also be used in various assay systems to screen for proliferation required genes in other organisms as well as to screen for antimicrobial agents.

Claims

1. A method of screening a candidate compound for the ability to reduce the activity or level of a gene product required for cell proliferation, said method comprising the steps of:

expressing an antisense nucleic acid against a nucleic acid encoding said gene product in a cell to reduce the activity or amount of said gene product in said cell, thereby producing a sensitized cell;

contacting said sensitized cell with a candidate compound, wherein said candidate compound is not previously known to possess the ability to reduce cell proliferation; and

determining whether said candidate compound inhibits the growth of said sensitized cell to a greater content than said candidate compound inhibits the growth of a nonsensitized cell.

2. The method of claim 1, wherein said cell is selected from the group consisting of bacterial cells, fungal cells, plant cells, and animal cells.

3. The method of claim 2, wherein said cell is an E. coli cell.

4. The method of claim 2, wherein said cell is from an organism selected from the group consisting of Staphylococcus aureus, Pseudomonas aeruginosa, Enterobacter cloacae, Helicobacter pylori, Neisseria gonorrhoeae, Enterococcus faecalis, Streptococcus pneumoniae, Haemophilus influenzae, Salmonella typhimurium, Saccharomyces cerevisiae, Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus, Klebsiella pneumoniae, Salmonella typhi, Salmonella paratyphi, Salmonella cholerasuis, Staphylococcus epidermidis, Mycobacterium tuberculosis, Mycobacterium leprae, Treponema pallidum, Bacillus anthracis, Yersinia pestis, Clostridium botulinum, Campylobacter jejuni, and Chlamydia trachomatus, Chlamydia pneumoniae or any species falling within the genera of any of the above species.

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

6. The method of claim 5, further comprising the step of contacting said cell with a concentration of inducer which induces said antisense nucleic acid to a sublethal level.

7. The method of claim 6, wherein said sub-lethal concentration of said inducer is such that growth inhibition is 8% or more.

8. The method of claim 6, wherein said inducer is isopropyl-1-thio-β

-D-galactoside.

9. The method of claim 1, wherein growth inhibition is measured by monitoring optical density of a culture growth solution.

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

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

12. The method of claim 10, wherein said gene product comprises a polypeptide having a sequence selected from the group consisting of SEQ ID NOs.:

243-357, 359-398.

13. The method of claim 1, wherein said candidate compound is a natural product.

14. A method of screening a candidate compound for the ability to inhibit proliferation of a microorganism said method comprising:

(a) identifying a gene or gene product required for proliferation in a first microorganism;

(b) identifying a homolog of said gene or gene product in a second microorganism;

(c) identifying an inhibitory nucleic acid sequence which inhibits the activity of said homolog in said second microorganism;

(d) contacting said second microorganism with a proliferation-inhibiting amount of said inhibitory nucleic acid, thus sensitizing said second microorganism,

(e) contacting the sensitized microorganism of step (d) with a candidate compound; and

(f) determining whether said candidate compound inhibits proliferation of said sensitized microorganism to a greater extent than said candidate compound inhibits proliferation of a nonsensitized microorganism.

15. The method of claim 14, wherein said step of identifying a gene involved in proliferation in a first microorganism comprises:

introducing a nucleic acid comprising a random genomic fragment from said first microorganism operably linked to a promoter wherein said random genomic fragment is in the antisense orientation; and

comparing the proliferation of said first microorganism transcribing a first level of said random genomic fragment to the proliferation of said first microorganism transcribing a lower level of said random genomic fragment, wherein a difference in proliferation indicates that said random genomic fragment comprises a gene involved in proliferation.

16. The method of claim 15, wherein said step of identifying a homolog of said gene is a second microorganism comprises identifying a homologous nucleic acid or a nucleic acid encoding a homologous polypeptide in a database using an algorithm selected from the group consisting of BLASTN version 2.0 with the default parameters and FASTA version 3.0t78 algorithm with the default parameters.

17. The method of claim 15, wherein said step of identifying a homolog of said gene in a second microorganism comprises identifying a homologous nucleic acid or a nucleic acid encoding a homologous polypeptide by identifying nucleic acids which hybridize to said first gene.

18. The method of claim 15, wherein the step of identifying a homolog of said gene in a second microorganism comprises expressing a nucleic acid which inhibits the proliferation of said first microorganism in said second microorganism.

19. The method of claim 15, wherein said inhibitory nucleic acid is an antisense nucleic acid.

20. The method of claim 15, wherein said inhibitory nucleic acid comprises an antisense nucleic acid to a portion of said homolog.

21. The method of claim 15, wherein said inhibitory nucleic acid comprises an antisense nucleic acid to a portion of the operon encoding said homolog.

22. The method of claim 15, wherein the step or contacting the second microorganism with a proliferation-inhibiting amount of said nucleic acid sequence comprises directly contacting said second microorganism with said nucleic acid.

23. The method of claim 15, wherein the step of contacting the second microorganism with a proliferation-inhibiting amount of said nucleic acid sequence comprises expressing all antisense nucleic acid to said homolog in said second microorganism.

24. The method of claim 14, wherein said candidate compound is a compound not previously known to possess the ability to inhibit cellular proliferation.

25. The method of claim 14, wherein said candidate compound is a natural product.

26. A method of screening a candidate compound for the ability to inhibit proliferation said method comprising:

(a) identifying an inhibitory nucleic acid sequence which inhibits the activity of a gene or gene product required for proliferation in a first microorganism;

(b) contacting a second microorganism with a proliferation-inhibiting amount of said inhibitory nucleic acid, thus sensitizing said second microorganism;

(c) contacting the proliferation-inhibited microorganism of step (b) with a candidate compound; and

(d) determining whether said candidate compound inhibits proliferation of said sensitized second microorganism to a greater extent than said candidate compound inhibits proliferation of a nonsensitized second microorganism.

27. The method of claim 26, wherein said inhibitory nucleic acid is an antisense nucleic acid which inhibits the proliferation of said first microorganism.

28. The method of claim 26, wherein said inhibitory nucleic acid comprises a portion of an antisense nucleic acid which inhibits the proliferation of said first microorganism.

29. The method of claim 26, wherein said inhibitory nucleic acid comprises an antisense molecule against the entire coding region of the gene involved in proliferation of the first microorganism.

30. The method of claim 26, wherein said inhibitory nucleic acid comprises an antisense nucleic acid to a portion of the operon encoding the gene involved in proliferation of the first microorganism.

31. The method of claim 26, wherein said candidate compound is a compound not previously known to possess the ability to inhibit cellular proliferation.

32. The method of claim 26, wherein said candidate compound is a natural product.

33. A method of screening a candidate compound for activity against a biological pathway required for proliferation, said method comprising:

sensitizing a cell by expressing an antisense nucleic acid against a nucleic acid encoding a gene product required for proliferation in a cell to reduce the activity or amount of said gene product;

contacting the sensitized cell with a candidate compound, wherein said candidate compound is not previously known to possess the ability to reduce proliferation; and

determining whether said candidate compound inhibits the growth of said sensitized cell to a greater extent than said candidate compound inhibits the growth of an nonsensitized cell.

34. The method of claim 33, wherein said cell is selected from the group consisting of bacterial cells, fungal cells, plant cells, and animal cells.

35. The method of claim 34, wherein said cell is an E. coli cell.

36. The method of claim 33, wherein said cell is from an organism selected from the group consisting of Staphylococcus aureus, Pseudomonas aeruginosa, Enterobacter cloacae, Helicobacter pylori, Neisseria gonorrhoeae, Enterococcus faecalis, Streptococcus pneumoniae, Haemophilus influenzae, Salmonella typhimurium, Saccharomyces cerevisiae, Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus, Klebsiella pneumoniae, Salmonella typhi, Salmonella paratyphi, Salmonella cholerasuis, Staphylococcus epidermidis, Mycobacterium tuberculosis, Mycobacterium leprae, Treponema pallidum, Bacillus anthracis, Yersinia pestis, Clostridium botulinum, Campylobacter jejuni, and Chlamydia trachomatus, Chlamydia pneumoniae or any species falling within the genera of any of the above species.

37. The method of claim 33, wherein said antisense nucleic acid is transcribed from an inducible promoter.

38. The method of claim 37, further comprising contacting the cell with an agent which induces expression of said antisense nucleic acid from said inducible promoter, wherein said antisense nucleic acid is expressed at a sublethal level.

39. The method of claim 38, wherein said sublethal level of said antisense nucleic acid inhibits proliferation by 8% or more.

40. The method of claim 38, wherein said agent is isopropyl-1-thio-β

-D-galactoside (IPTG).

41. The method of claim 39, wherein inhibition of proliferation is measured by monitoring the optical density of a liquid culture.

42. The method of claim 33, wherein said gene product comprises a polypeptide having a sequence selected from the group consisting of SEQ ID NOs:

243-357, 359-398.

43. The method of claim 33, wherein said candidate compound is a natural product.

44. A method of screening a candidate compound for the ability to inhibit cellular proliferation, said method comprising:

contacting a cell with an agent which reduces the activity or level of a gene product required for proliferation of said cell;

contacting said cell with a candidate compound, wherein said candidate compound is not previously known to possess the ability to reduce cell proliferation; and

determining whether said candidate compound reduces proliferation to a greater extent than said candidate compound reduces proliferation of a cell which has not been contacted with said agent.

45. The method of claim 44, wherein said agent which reduces the activity or level of a gene product required for proliferation of said cell comprises an antisense nucleic acid to a gene or operon required for proliferation.

46. The method of claim 44, wherein said agent which reduces the activity or level of a gene product required for proliferation of said cell comprises an antibiotic.

47. The method of claim 44, wherein said cell contains a temperature sensitive mutation which reduces the activity or level of said gene product required for proliferation of said cell.

48. The method of claim 47, wherein said agent is an antisense nucleic acid directed to a gene or operon required for proliferation.

49. The method of claim 44, wherein said candidate compound is a natural product.