Teichoic acid enzymes and assays

• US 6,428,971 B1
• Filed: 05/05/1997
• Issued: 08/06/2002
• Est. Priority Date: 05/07/1996
• Status: Expired due to Fees

• Current Assignee: Pharmacia & Upjohn Company (Pfizer Inc.)
• Original Assignee: Pharmacia & Upjohn Company (Pfizer Inc.)
• Inventors: Morin, Sara E., Swaney, Steven M., Shinabarger, Dean L.
• Primary Examiner(s): Slobodyansky, Elizabeth
• Application Number:
  US08/851,435
• Time in Patent Office: 1,919 Days
• Field of Search: 435/193, 435/194, 435/195, 435/196, 435/4, 435/15, 514/1, 514/23, 558/70, 558/156
• US Class Current: 435/15
• CPC Class Codes: C12N 9/1288 Transferases for other substituted phosphate groups (2.7.8)
• CPC Class Codes: C12N 9/503 derived from viruses
• CPC Class Codes: C12Q 1/48 involving transferase
• CPC Class Codes: C12Y 207/08012 CDP-glycerol glycerophosphotransferase (2.7.8.12)
• CPC Class Codes: G01N 2500/00 Screening for compounds of potential therapeutic value

Abstract

This invention discloses a novel substrate and assay for the TAP enzyme. In addition novel DNA, proteins and peptides from genes and proteins associated with bacterial teichoic acid biosynthetic pathways, specifically the rodC gene and proteins and variations thereof are disclosed.

Claims

1. A process for determining teichoic acid polymerase activity in a sample, the method comprising:

combining CDP-glycerol, water, a lipoteichoic acid substrate from a bacterium selected from the group consisting of Staphylococcus aureus, Enterococcus faecalis, and Bacillus subtilis, and the sample to form a mixture;

incubating the mixture under conditions effective for a teichoic acid polymerase in the sample, to cause the incorporation of at least one glycerol-3-phosphate moiety into the lipoteichoic acid substrate to form a lipoteichoic acid product having at least one additional glycerol-3-phosphate moiety incorporated therein, wherein the teichoic acid polymerase is encoded by DNA having at least about 70% homology with SEQ ID NO;

1; and

determining the presence or absence of the lipoteichoic acid product.

2. The process of claim 1 wherein determining the presence or absence of the lipoteichoic acid product comprises measuring the amount of the lipoteichoic acid product formed.

3. The process of claim 1 wherein determining the presence or absence of the lipoteichoic acid product comprises adding an acid to the mixture after incubation to precipitate the lipoteichoic acid product.

4. The process of claim 1 wherein the CDP-glycerol is CDP[³H]glycerol.

5. The process of claim 4 wherein determining the presence or absence of the lipoteichoic acid product comprises:

adding streptavidin SPA beads comprising a lectin to the mixture after incubation to bind the lipoteichoic acid product; and

measuring the amount of lipoteichoic acid product bound to the SPA beads.

6. The process of claim 1 wherein the teichoic acid polymerase comprises SEQ ID No:

2.

7. The process of claim 1 wherein the teichoic acid polymerase comprises the first 20 N-terminal amino residues of SEQ ID No:

2.

8. The process of claim 1 wherein the lipoteichoic acid substrate is pretreated to remove alanine bound to the lipoteichoic acid as an ester and free alanine associated with lipoteichoic acid in solution before the lipoteichoic acid substrate is combined with the other ingredients.

27. The process of claim 1 wherein the teichoic acid polymerase is encoded by DNA represented by SEQ ID NO:

1.

28. The process of claim 1 wherein the amino acid sequence of the teichoic acid polymerase has at least about 70% homology with SEQ ID NO:

2.

29. The process of claim 1 wherein the amino acid sequence of the teichoic acid polymerase comprises an amino acid sequence represented by SEQ ID NO:

2.

9. A process for determining the presence or absence of lipoteichoic acid from a bacterium selected from the group consisting of Staphylococcus aureus, Enterococcus faecalis, and Bacillus subtilis in a sample, the method comprising:

combining CDP-glycerol, water, a teichoic acid polymerase encoded by DNA having at least about 70% homology with SEQ ID NO;

1, and the sample to form a mixture;

incubating the mixture under conditions effective for the teichoic acid polymerase to cause the incorporation of at least one glycerol-3-phosphate moiety into a lipoteichoic acid present in the sample to form a lipoteichoic acid product having at least one additional glycerol-3-phosphate moiety incorporated therein; and

determining the presence or absence of the lipoteichoic acid product.

10. The process of claim 9 wherein determining the presence or absence of the lipoteichoic acid product comprises measuring the amount of the lipoteichoic acid product formed.

11. The process of claim 9 wherein determining the presence or absence of the lipoteichoic acid product comprises adding an acid to the mixture after incubation to precipitate the lipoteichoic acid product.

12. The process of claim 9 wherein the CDP-glycerol is CDP[³H]glycerol.

13. The process of claim 12 wherein determining the presence or absence of the lipoteichoic acid product comprises:

adding streptavidin SPA beads comprising a lectin to the mixture after incubation to bind the lipoteichoic acid product; and

measuring the amount of lipoteichoic acid product bound to the SPA beads.

14. The process of claim 9 wherein the teichoic acid polymerase is expressed from a cloned cell.

15. The process of claim 14 wherein the teichoic acid polymerase comprises the first 20 N-terminal amino residues of SEQ ID No:

2.

16. The process of claim 9 wherein the teichoic acid polymerase is a soluble enzyme.

30. The process of claim 9 wherein the teichoic acid polymerase is encoded by DNA represented by SEQ ID NO:

1.

31. The process of claim 9 wherein the amino acid sequence of the teichoic acid polymerase has at least about 70% homology with SEQ ID NO:

2.

32. The process of claim 9 wherein the amino acid sequence of the teichoic acid polymerase comprises an amino acid sequence represented by SEQ ID NO:

2.

17. A process for screening teichoic acid polymerase inhibitors, the method comprising:

combining CDP-glycerol, water, a lipoteichoic acid substrate from a bacterium selected from the group consisting of Staphylococcus aureus, Enterococcus faecalis, and Bacillus subtilis, a teichoic acid polymerase encoded by DNA having at least about 70% homology with SEQ ID NO;

1, and a teichoic acid polymerase inhibitor to form a mixture;

incubating the mixture under conditions effective, when the teichoic acid polymerase inhibitor is not present, for the teichoic acid polymerase to cause the incorporation of at least one glycerol-3-phosphate moiety into the lipoteichoic acid substrate to form a lipoteichoic acid product having at least one additional glycerol-3-phosphate moiety incorporated therein; and

determining the presence or absence of the lipoteichoic acid product produced when the teichoic acid polymerase inhibitor is present in the mixture relative to the lipoteichoic acid product produced when the teichoic acid polymerase inhibitor is not present in the mixture.

18. The process of claim 17 wherein determining the presence or absence of the lipoteichoic acid product comprises measuring the amount of the lipoteichoic acid product formed.

19. The process of claim 17 wherein determining the presence or absence of the lipoteichoic acid product comprises adding an acid to the mixture after incubation to precipitate the lipoteichoic acid product.

20. The process of claim 17 wherein the CDP-glycerol is CDP[³H]glycerol.

21. The process of claim 20 wherein determining the presence or absence of the lipoteichoic acid product comprises:

adding streptavidin SPA beads comprising a lectin to the mixture after incubation to bind the lipoteichoic acid product; and

measuring the amount of lipoteichoic acid product bound to the SPA beads.

33. The process of claim 17 wherein the teichoic acid polymerase is encoded by DNA represented by SEQ ID NO:

1.

34. The process of claim 17 wherein the amino acid sequence of the teichoic acid polymerase has at least about 70% homology with SEQ ID NO:

2.

35. The process of claim 17 wherein the amino acid sequence of the teichoic acid polymerase comprises an amino acid sequence represented by SEQ ID NO:

2.

22. A process for monitoring enzymatic reactions catalyzed by teichoic acid polymerase, the method comprising:

combining CDP-glycerol, water, a lipoteichoic acid substrate from a bacterium selected from the group consisting of Staphylococcus aureus, Enterococcus faecalis, and Bacillus subtilis, and a teichoic acid polymerase encoded by DNA having at least about 70% homology with SEQ ID NO;

1, to form a mixture;

incubating the mixture under conditions effective for the teichoic acid polymerase to cause the incorporation of at least one glycerol-3-phosphate moiety into the lipoteichoic acid substrate to form a lipoteichoic acid product having at least one additional glycerol-3-phosphate moiety incorporated therein; and

determining the presence or absence of the lipoteichoic acid product.

23. The process of claim 22 wherein determining the presence or absence of the lipoteichoic acid product comprises measuring the amount of the lipoteichoic acid product formed.

24. The process of claim 22 wherein determining the presence or absence of the lipoteichoic acid product comprises adding an acid to the mixture after incubation to precipitate the lipoteichoic acid product.

25. The process of claim 22 wherein the CDP-glycerol is CDP[³H]glycerol.

26. The process of claim 25 wherein determining the presence or absence of the lipoteichoic acid product comprises:

adding streptavidin SPA beads comprising a lectin to the mixture after incubation to bind the lipoteichoic acid product; and

measuring the amount of lipoteichoic acid product bound to the SPA beads.

36. The process of claim 22 wherein the teichoic acid polymerase is encoded by DNA represented by SEQ ID NO:

1.

37. The process of claim 22 wherein the amino acid sequence of the teichoic acid polymerase has at least about 70% homology with SEQ ID NO:

2.

38. The process of claim 22 wherein the amino acid sequence of the teichoic acid polymerase comprises an amino acid sequence represented by SEQ ID NO:

2.

39. A process for determining teichoic acid polymerase activity in a sample, the method comprising:

combining CDP-glycerol, water, a lipoteichoic acid substrate from a bacterium selected from the group consisting of Staphylococcus aureus, Enterococcus faecalis, and Bacillus subtilis, and the sample to form a mixture;

incubating the mixture under conditions effective for a teichoic acid polymerase in the sample to cause the incorporation of at least one glycerol-3-phosphate moiety into the lipoteichoic acid substrate to form a lipoteichoic acid product having at least one additional glycerol-3-phosphate moiety incorporated therein, wherein the teichoic acid polymerase has an amino acid sequence with at least about 70% homology with SEQ ID NO;

2; and

determining the presence or absence of the lipoteichoic acid product.

40. The process of claim 39 wherein determining the presence or absence of the lipoteichoic acid product comprises measuring the amount of the lipoteichoic acid product formed.

41. The process of claim 39 wherein determining the presence or absence of the lipoteichoic acid product comprises adding an acid to the mixture after incubation to precipitate the lipoteichoic acid product.

42. The process of claim 39 wherein the CDP-glycerol is CDP[³H]glycerol and wherein determining the presence or absence of the lipoteichoic acid product comprises:

adding streptavidin SPA beads comprising a lectin to the mixture after incubation to bind the lipoteichoic acid product; and

measuring the amount of lipoteichoic acid product bound to the SPA beads.

43. A process for determining the presence or absence of lipoteichoic acid from a bacterium selected from the group consisting of Staphylococcus aureus, Enterococcus faecalis, and Bacillus subtilis in a sample, the method comprising:

combining CDP-glycerol, water, a teichoic acid polymerase having an amino acid sequence with at least about 70% homology with SEQ ID NO;

2, and the sample to form a mixture;

incubating the mixture under conditions effective for the teichoic acid polymerase to cause the incorporation of at least one glycerol-3-phosphate moiety into a lipoteichoic acid present in the sample to form a lipoteichoic acid product having at least one additional glycerol-3-phosphate moiety incorporated therein; and

determining the presence or absence of the lipoteichoic acid product.

44. The process of claim 43 wherein determining the presence or absence of the lipoteichoic acid product comprises measuring the amount of the lipoteichoic acid product formed.

45. The process of claim 43 wherein determining the presence or absence of the lipoteichoic acid product comprises adding an acid to the mixture after incubation to precipitate the lipoteichoic acid product.

46. The process of claim 43 wherein the CDP-glycerol is CDP[³H]glycerol and wherein determining the presence or absence of the lipoteichoic acid product comprises:

adding streptavidin SPA beads comprising a lectin to the mixture after incubation to bind the lipoteichoic acid product; and

measuring the amount of lipoteichoic acid product bound to the SPA beads.

47. A process for screening teichoic acid polymerase inhibitors, the method comprising:

combining CDP-glycerol, water, a lipoteichoic acid substrate from a bacterium selected from the group consisting of Staphylococcus aureus, Enterococcus faecalis, and Bacillus subtilis, a teichoic acid polymerase having an amino acid sequence with at least about 70% homology with SEQ ID NO;

2, and a teichoic acid polymerase inhibitor to form a mixture;

incubating the mixture under conditions effective, when the teichoic acid polymerase inhibitor is not present, for the teichoic acid polymerase to cause the incorporation of at least one glycerol-3-phosphate moiety into the lipoteichoic acid substrate to form a lipoteichoic acid product having at least one additional glycerol-3-phosphate moiety incorporated therein; and

determining the presence or absence of the lipoteichoic acid product produced when the teichoic acid polymerase inhibitor is present in the mixture relative to the lipoteichoic acid product produced when the teichoic acid polymerase inhibitor is not present in the mixture.

48. The process of claim 47 wherein determining the presence or absence of the lipoteichoic acid product comprises measuring the amount of the lipoteichoic acid product formed.

49. The process of claim 47 wherein determining the presence or absence of the lipoteichoic acid product comprises adding an acid to the mixture after incubation to precipitate the lipoteichoic acid product.

50. The process of claim 47 wherein the CDP-glycerol is CDP[³H]glycerol and wherein determining the presence or absence of the lipoteichoic acid product comprises:

adding streptavidin SPA beads comprising a lectin to the mixture after incubation to bind the lipoteichoic acid product; and

measuring the amount of lipoteichoic acid product bound to the SPA beads.

51. A process for monitoring enzymatic reactions catalyzed by teichoic acid polymerase, the method comprising:

combining CDP-glycerol, water, a lipoteichoic acid substrate from a bacterium selected from the group consisting of Staphylococcus aureus, Enterococcus faecalis, and Bacillus subtilis, and a teichoic acid polymerase having an amino acid sequence with at least about 70% homology with SEQ ID NO;

2 to form a mixture;

incubating the mixture under conditions effective for the teichoic acid polymerase to cause the incorporation of at least one glycerol-3-phosphate moiety into the lipoteichoic acid substrate to form a lipoteichoic acid product having at least one additional glycerol-3-phosphate moiety incorporated therein; and

determining the presence or absence of the lipoteichoic acid product.

52. The process of claim 51 wherein determining the presence or absence of the lipoteichoic acid product comprises measuring the amount of the lipoteichoic acid product formed.

53. The process of claim 51 wherein determining the presence or absence of the lipoteichoic acid product comprises adding an acid to the mixture after incubation to precipitate the lipoteichoic acid product.

54. The process of claim 51 wherein the CDP-glycerol is CDP[³H]glycerol and wherein determining the presence or absence of the lipoteichoic acid product comprises:

adding streptavidin SPA beads comprising a lectin to the mixture after incubation to bind the lipoteichoic acid product; and

measuring the amount of lipoteichoic acid product bound to the SPA beads.