Methods and compositions involving sortase B

• US 20060073530A1
• Filed: 08/15/2002
• Published: 04/06/2006
• Est. Priority Date: 08/15/2001
• Status: Abandoned Application

• Current Assignee: Regents of the University of California (University of California), University of Chicago
• Original Assignee: University of Chicago
• Inventors: Schneewind, Olaf, Ton-That, Hung, Su, Kenneth, Mazmanian, Sarkis N.
• Application Number:
  US10/219,783
• Publication Number:
  US 20060073530A1
• Time in Patent Office: Days
• US Class Current: 435/7.320
• CPC Class Codes: A61K 38/4873 Cysteine endopeptidases (3.4.22), e.g. stem bromelain, papain, ficin, cathepsin H
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: G01N 2333/96466 Cysteine endopeptidases (3.4.22)
• CPC Class Codes: G01N 2500/00 Screening for compounds of potential therapeutic value
• CPC Class Codes: G01N 33/56911 Bacteria
• CPC Class Codes: G01N 33/56938 Staphylococcus

Abstract

The present invention provides methods and compositions involving sortase-transamidases, including sortase B, and polypeptides that include a signal sorting sequence of NPQ/KTN/G. Methods of screening for inhibitors of Gram-positive bacteria as well as therapeutic, preventative, and research methods focusing on Gram-positive bacteria are also provided.

Claims

1. A method for screening for a sortase-transamidase inhibitor comprising:

a) incubating at least a first sortase-transamidase and at least a first polypeptide comprising a sorting signal of NPQ/KTN/G, under conditions to allow the first sortase-transamidase to cleave the first polypeptide within the sorting signal;

b) incubating the first sortase-transamidase or the first polypeptide with a candidate inhibitor; and

c) assaying for activity of the first sortase-transamidase, wherein a reduction in activity identifies the compound as a sortase-transamidase inhibitor.

2. The method of claim 1 wherein the sorting signal is at the carboxyl-terminal end of the first polypeptide.

3. The method of claim 2, further comprising incubating the first sortase-transamidase, the first polypeptide, or the candidate compound with a Gram-positive bacterium having a peptidoglycan prior to assaying the activity of the first sortase-transamidase.

4. The method of claim 2, further comprising comparing the activity of the first sortase-transamidase with a sortase-transamidase not incubated with the candidate inhibitor.

5. The method of claim 2, wherein the first sortase-transamidase is srt B.

6. The method of claim 5, wherein the srt B is from Staphylococcus aureus.

7. The method of claim 6, wherein the srt B comprises SEQ ID NO:

4.

8. The method of claim 2, wherein the first sortase-transamidase is substantially purified.

9. The method of claim 1, wherein the first sortase-transamidase is recombinant.

10. The method of claim 1, wherein the first polypeptide is recombinant.

11. The method of claim 2, wherein the first polypeptide comprises 6 contiguous amino acids from SEQ ID NO:

33.

12. The method of claim 1, further comprising:

d) incubating the candidate inhibitor with a second sortase-transamidase and a second polypeptide comprising a sorting signal of LPX₃X₄G, under conditions to allow the second sortase-transamidase to cleave the second polypeptide within the sorting signal; and

e) assaying for activity of the second sortase-transamidase, wherein a reduction in activity identifies the candidate as a sortase-transamidase inhibitor.

13. The method of claim 12 wherein the sorting signal is at the carboxy-terminal end of the second polypeptide.

14. The method of claim 13 wherein the second polypeptide is recombinant.

15. The method of claim 14, wherein the second recombinant polypeptide further comprises a substantially hydrophobic domain of at least 31 amino acids carboxyl to the motif and a charged tail region with at least two positively charged residues carboxyl to the substantially hydrophobic domain, wherein at least one of the positively charged residues is an arginine and the two positively charged residues are located at residues 31-33 from the motif.

16. The method of claim 14, further comprising incubating the second sortase-transamidase, the second recombinant polypeptide, or the candidate compound with a Gram-positive bacterium having a peptidoglycan prior to assaying the activity of the second sortase-transamidase.

17. The method of claim 14, wherein the second recombinant polypeptide comprises at least 5 contiguous amino acids from SEQ ID NO:

7, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, SEQ ID NO;

17, SEQ ID NO;

18, SEQ ID NO;

19, SEQ ID NO;

20, SEQ ID NO;

21, SEQ ID NO;

22, SEQ ID NO;

23, SEQ ID NO;

24, SEQ ID NO;

25, SEQ ID NO;

26, SEQ ID NO;

27, SEQ ID NO;

28, SEQ ID NO;

29, SEQ ID NO;

30, SEQ ID NO;

31 or SEQ ID NO;

32.

18. The method of claim 17, wherein the second recombinant polypeptide comprises at least 10 contiguous amino acids from SEQ ID NO:

7, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

1, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, SEQ ID NO;

17, SEQ ID NO;

18, SEQ ID NO;

19, SEQ ID NO;

20, SEQ ID NO;

21, SEQ ID NO;

22, SEQ ID NO;

23, SEQ ID NO;

24, SEQ ID NO;

25, SEQ ID NO;

26, SEQ ID NO;

27, SEQ ID NO;

28, SEQ ID NO;

29, SEQ ID NO;

30, SEQ ID NO;

31 or SEQ ID NO;

32.

19. The method of claim 14, wherein the second sortase-transamidase is srt A.

20. The method of claim 19, wherein the srt A is from Staphylococcus aureus.

21. The method of claim 20, wherein the srt A comprises SEQ ID NO:

2.

22. The method of claim 14, wherein the second sortase-transamidase is substantially purified.

23. The method of claim 14, wherein the second sortase-transamidase is recombinant.

24. The method of claim 14, wherein the second sortase-transamidase is obtained from a different species of Gram-positive bacterium than the first sortase transamidase.

25. The method of claim 1, wherein the first sortase-transamidase, the Gram-positive bacterium, or the first polypeptide is attached to a non-reacting structure.

26. The method of claim 1, wherein the first polypeptide further comprises a label.

27. The method of claim 26, wherein the label is radioactive, calorimetric, or enzymatic.

28. The method of claim 1, wherein the activity of the sortase-transamidase is assayed by measuring the amount of cleaved polypeptide.

29. The method of claim 3, wherein the activity of the sortase-transamidase is assayed by measuring the amount of the first polypeptide displayed on the surface of the bacterium.

30. The method of claim 29, wherein the amount of the first recombinant polypeptide displayed on the surface of the bacterium is measured using a proteinaceous compound that specifically binds the first recombinant polypeptide.

31. The method of claim 30, wherein the proteinaceous compound is attached to a label.

32. The method of claim 31, wherein the label is radioactive, calorimetric, or enzymatic.

33. The method of claim 14, further comprising:

d) incubating the candidate inhibitor with a third sortase-transamidase and a third polypeptide comprising a sorting signal, under conditions to allow the third sortase-transamidase to cleave the third polypeptide within the sorting signal; and

e) assaying for activity of the third sortase-transamidase, wherein a reduction in activity identifies the candidate as a sortase-transamidase inhibitor.

34. The method of claim 33 wherein the third polypeptide is recombinant.

35. The method of claim 34, wherein the third sortase-transamidase is obtained from a different species of Gram-positive bacterium than the first and second sortase transamidases.

36. The method of claim 1, further comprising:

d) administering to an animal a Gram-positive bacterium;

b) administering to the animal the candidate inhibitor;

c) assaying the pathogenicity of the bacterium.

37. The method of claim 36, wherein the Gram-positive bacterium is Staphylococcus aureus.

38. The method of claim 36, further comprising comparing the pathogenicity of the bacteria in the animal with a second animal administered a Gram-positive bacterium having a mutation in srt A, srt B, or both.

39. The method of claim 36, wherein the animal is a mouse.

40. The method of claim 36, wherein the pathogenicity of the bacterium is assayed by measuring an amount of bacterium in the animal.

41. The method of claim 36, wherein the pathogenicity of the bacterium is assayed by measuring abscess formation.

42. The method of claim 36, wherein the candidate compound is administered to the animal more than 24 hours after administration of the bacterium to the animal.

43. A method of screening for a sortase-transamidase inhibitor comprising:

a) administering to an animal a Gram-positive bacteriun having a mutation in at least one sortase-transamidase-encoding nucleic acid, wherein the bacterium does not express a functional sortase-transamidase encoded by the nucleic acid;

b) administering to the animal a candidate compound;

c) assaying the pathogenicity of the bacterium.

44. A method for screening for expression of a recombinant polypeptide comprising:

a) incubating a sortase-transamidase, a Gram-positive bacterium having a peptidoglycan, and a polypeptide comprising a sorting signal of NPQ/KTN/G, under conditions to allow the sortase-transamidase to cleave the polypeptide within the sorting signal; and

b) assaying for display of the polypeptide on the surface of the Gram-positive bacterium.

45. The method of claim 44 wherein the sorting signal is at the carboxyl terminal end of the polypeptide.

46. The method of claim 45 wherein the polypeptide is recombinant.

47. The method of claim 46, wherein the sortase-transamidase is srt B.

48. The method of claim 47, wherein the srt B is from Staphylococcus aureus.

49. The method of claim 48, wherein the srt B comprises SEQ ID NO:

4.

50. The method of claim 46, wherein the sortase-transamidase is substantially purified.

51. The method of claim 46, wherein the sortase-transamidase is recombinant.

52. The method of claim 46, wherein the polypeptide comprises 6 amino acids of SEQ ID NO:

33.

53. The method of claim 46, wherein the polypeptide is assayed using a proteinaceous compound that specifically binds the polypeptide.

54. The method of claim 53, wherein the proteinaceous compound is labeled.

55. The method of claim 54, wherein the proteinaceous compound is labeled with an enzymatic marker, a colorimetric marker, or a radiolabel.

56. The method of claim 53, wherein the proteinaceous compound is an antibody.

57. The method of claim 53, wherein the proteinaceous compound is a ligand for the polypeptide.

58. The method of claim 53, wherein the proteinaceous compound is a substrate for the polypeptide.

59. A method of diagnosing or treating a Gram-positive bacteria infection in an organism comprising:

a) linking an antibiotic or a detection reagent to a polypeptide comprising a sorting signal of NPQ/KTN/G; and

b) introducing the linked polypeptide to the organism, wherein the linked polypeptide becomes covalently cross-linked to the bacterium.

60. The method of claim 59 wherein the sorting signal is at the carboxyl terminal end of the polypeptide.

61. The method of claim 60, wherein the antibiotic or detection reagent is conjugated to the polypeptide.

62. A conjugate comprising an antibiotic or detection reagent conjugated to a polypeptide comprising a sorting signal of NPQ/KTN/G at its carboxyl-terminal end.

63. A recombinant cleaved polypeptide displayed on the surface of a Gram-positive bacterium comprising a sortase-transamidase by covalent linkage, wherein the polypeptide comprised an amino acid sequence of NPQ/KTN/G prior to being cleaved by the sortase-transamidase.

64. The recombinant cleaved polypeptide of claim 63, wherein the bacterium is Staphylococcus aureus.

65. A vaccine comprising a Gram-positive bacterium displaying a recombinant cleaved polypeptide on its surface, wherein the polypeptide is covalently attached to the bacterium and comprises a sorting sequence comprising an amino acid sequence of NPQ/KTN/G or LPX₃X₄G prior to being cleaved by a sortase-transamidase in the bacterium.

66. The vaccine of claim 65, wherein the polypeptide comprises a sorting sequence comprising an amino acid sequence of NPQ/KTN/G prior to being cleaved by a sortase-transamidase in the bacterium.

67. The vaccine of claim 66, wherein the polypeptide comprises 6 contiguous amino acids from SEQ ID NO:

7, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, SEQ ID NO;

17, SEQ ID NO;

18, SEQ ID NO;

19, SEQ ID NO;

20, SEQ ID NO;

21, SEQ ID NO;

22, SEQ ID NO;

23, SEQ ID NO;

24, SEQ ID NO;

25, SEQ ID NO;

26, SEQ ID NO;

27, SEQ ID NO;

28, SEQ ID NO;

29, SEQ ID NO;

30, SEQ ID NO;

31, SEQ ID NO;

32, or SEQ ID NO;

33.

68. The vaccine of claim 65, wherein the Gram-positive bacterium is Staphylococcus aureus.

69. The method of claim 65, wherein the Gram-positive bacterium comprises a genetic mutation, wherein the mutation reduces the pathogenicity of the bacterium compared to a bacterium lacking the mutation.

70. A method for inducing an immune response in an animal comprising administering to the animal the vaccine of claim 65 in an amount effective to induce an immune response against the antigen.

71. A method of making a vaccine comprising incubating a sortase-transamidase, a Gram-positive bacterium having a peptidoglycan, and a recombinant polypeptide comprising a sorting signal of NPQ/KTN/G at its carboxyl-terminal end and an antigen, under conditions to allow the sortase-transamidase to cleave the polypeptide within the sorting signal.

72. The method of claim 71, wherein the antigen is a tumor-associated antigen or a virus antigen or a bacterial antigen.

73. The method of claim 71, wherein the Gram-positive bacterium comprises a genetic mutation, wherein the mutation reduces the pathogenicity of the bacterium compared to a bacterium lacking the mutation.

74. The method of claim 71, wherein the polypeptide comprises 6 contiguous amino acids of SEQ ID NO:

33.

75. A method for identifying a sorting signal comprising:

a) searching a protein database for homologous amino acid sequences in Gram-positive bacterium using a query lacking the sequence of LPX₃X₄G and NPQTN;

b) identifying at least a first protein sequence that has at least 10% identity or 20% homology to a second protein sequence;

c) obtaining a polypeptide comprising an identified protein sequence; and

d) incubating the polypeptide with a sortase-transamidase under conditions to allow the polypeptide to be cleaved.