Method for identifying drug-sensitizing antisense DNA fragments and use thereof

Abstract

The invention provides a method for generating and selecting drug-sensitizing antisense DNA fragments. In one embodiment, the method includes identifying a gene of interest using knowledge of bacterial physiology, biochemistry, genetics, genomics, and other means. The method includes PCR amplification of a gene of interest using genomic DNA as a template; fragmentation of the DNA by sonication or other means; selecting DNA fragments no longer than 400 base pairs; ligating the DNA fragments into a suitable expression plasmid with a selectable marker; transforming the plasmids containing the DNA fragments into the organism from which the gene of interest originated; and selecting clones from transformed cells that show a phenotypic difference of the clone grown in the presence of the inducer relative to the phenotype in the absence of inducer.

Description

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows a peptidoglycan biosynthetic pathway in bacteria.

FIG. 2 shows a process for identification of specific antisense fragments.

FIG. 3 shows a validation process for specific sensitized antisense strains

FIGS. 4( a) and (b) show a EC50 Shift Sensitivity of MetRS1 antisense clones to the specific MetRS inhibitor Rx19. (a) Characterization of growth inhibition curve in the presence and absence of xylose. One of the antisense clones (H1) was characterized as to its sensitivity to the MetRS-specific inhibitor Rx19. Upper line: no xylose added. Lower line: +60 mM xylose. The EC50 concentration for each condition was determined. The “EC50 shift” is defined as the ratio of the EC50 in the absence and in the presence of xylose. The EC50 shift in this experiment is calculated to be 711/85.8=8.3. (b) EC50 shift for Rx19 in six different MetRS antisense clones. Clones H1, H2, H6, and E4 showed EC50 shifts for Rx19 greater than 4. Clones A3 and A5 showed EC50 shifts less than 4 even though all six clones had xylose-dependent growth inhibited phenotype and inserts with fragments antisense to the MetRS gene.

FIG. 5 shows an EC50 shift for Rx19 in six different MetRS antisense clones. Clones H1, H2, H6, and E4 showed EC50 shifts for Rx19 greater than 4. Clones A3 and A5 showed EC50 shifts less than 4 even though all six clones had xylose-dependent growth inhibited phenotype and inserts with fragments antisense to the MetRS gene.

FIG. 6 shows an EC50 shifts for MetRS antisense clone H1 on a panel of antibiotics. The sensitivity of MetRS antisense clone to various antibiotics was determined in the absence and presence of xylose. With one exception, the clone showed the same sensitivity to antibiotics in the absence or presence of xylose. For Rx19, the clone was about 8 fold more sensitive in the presence of xylose relative to the sensitivity in the absence of xylose.

FIGS. 7( a) and (b) show the response of murB2 antisense clone to cefotaxime in the presence and absence of a subinhibitory concentration antisense inducer. Upper line: no inducer. Lower line: +40 mM xylose. (a): murB2 antisense clone. (b) metRS antisense clone. The ratio of the IC50+xylose/EC50 (-xylose) gives the ‘IC50 shift’. In (a) the shift is computed to be (2096/6.1)=343. In (b), the metRS antisense clone showed no difference in sensitivity to cefotaxime, indicating that the cefotaxime mechanism of action is unrelated to metRS.

FIGS. 8( a) and (b) show B. anthracis murB-2 antisense strain sensitivity to cephalosporins and other antibiotics. (a) antibiotic panel including cefotaxime, ceftriaxone, cefepime, and cefoxitin, all of which showed greater than 100 fold shift in IC50 in the presence of xylose. (b) antibiotic panel showing greater than 4-fold shift in IC50 for cloxacillin, oxacillin, dicloxacillin, and cefaclor.

FIGS. 9( a)-(c) show an antibiotic dose response in the presence and absence of xylose for S. aureus engineered with murB antisense. (a) Cefoxitin IC50 shift is computed to be 1012/129.1=7.8. (b) Dicloxacillin IC50 shift is computed to be 49.5/8.6=5.8. (c) Antibiotic panel IC50 shift for S. aureus murB antisense strain showing 4-fold or greater sensitivity to cloxacillin, oxacillin, dicloxacillin, pipericillin, cefotaxime, ceftriaxone, cefepime, cefoxitin, and cefozolin.

FIG. 10 is a table of antisense fragments.

Claims

1. A method for generating and selecting drug-sensitizing antisense DNA fragments comprising: selecting a gene of interest using means other than genomic surveys of antisense-defined proliferation genes,amplification of the gene of interest using genomic DNA as a template,fragmentation of the DNA by sonication or other means;selecting DNA fragments no longer than 400 base pairs;ligating the DNA fragments into a suitable expression plasmid with a selectable marker;transforming the plasmids containing the DNA fragments into the organism from which the gene of interest originated; andselecting clones from transformed cells that show a discernible phenotypic difference of the clone grown in the presence of the inducer relative to the phenotype in the absence of inducer.

2. The method of claim 1, wherein the discernible phenotype is relative sensitivity to a growth inhibiting compound.

3. The method of claim 1, wherein the discernible phenotype is requirement of a nutrient to the growth medium.

4. The method of claim 1, wherein the discernible phenotype is visible morphology such as shape.

5. The method of claim 1, wherein the discernible phenotype is relative sensitivity to osmotic stress.

6. The method of claim 1 wherein the discernible phenotype is colony size.

7. A method for selecting bacterial strains containing specific drug-sensitizing DNA fragments, comprising: selecting those strains wherein the inserted DNA fragment is oriented antisense to some portion of the gene of interest;selecting those strains from a. wherein the intact mRNA level expressed from the gene of interest is reduced at least 25% in the presence of inducer relative to the absence of inducer; andselecting those strains from b wherein the magnitude of a discernible phenotype is dependent on the concentration of the inducer.

8. The method of claim 7, wherein strains are subjected to a wide range of inducer concentrations to establish a growth response curve to the inducer, such that: at high concentrations of inducer, growth is maximally suppressed;at low or no concentrations of inducer, there is no discernable growth inhibition; orat moderate and empirically determined concentrations, inducer causes 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% growth reduction compared to untreated strains.

9. The method of claim 7, wherein strains are grown in the presence of a wide range of concentrations of growth-inhibiting compound to establish a growth response curve and thereby a concentration that causes a 50% reduction in growth relative to untreated cells (EC50).

10. The method of claim 7, in which the method is conducted both in the presence and in the absence of an empirically determined concentration of inducer that causes between 10% to 50% growth reduction.

11. The method of claim 9, wherein a decrease in growth inhibitor EC50 values of at least four fold is evident in inducer-treated cells relative to cells without inducer treatment.

12. The method of claim 11, wherein the result is an indication that the antibiotic works as a specific inhibitor of the gene of interest.

13. The method of claim 9, wherein no significant change in antibiotic EC50 values is evident in inducer-treated cells relative to cells without inducer treatment.

14. The method of claim 13, wherein the result is an indication that the antibiotic does not work as a specific inhibitor of the gene of interest.

15. The method of claim 1, wherein the drug is an antibiotic acting through inhibition of DNA biosynthesis.

16. The method of claim 1, wherein the drug is an antibiotic acting through inhibition of RNA biosynthesis.

17. The method of claim 1, wherein the drug is an antibiotic acting through inhibition of protein biosynthesis.

18. The method of claim 1, wherein the drug is an antibiotic acting through inhibition of fatty acid biosynthesis.

19. The method of claim 1, wherein the drug is an antibiotic acting through inhibition of cell wall biosynthesis.

20. The method of claim 1, wherein the drug is an antibiotic acting through inhibition of amino acid biosynthesis.

21. The method of claim 1, wherein the drug is an antibiotic acting through inhibition of nucleotide biosynthesis.

22. The method of claim 1, wherein the drug is an antibiotic acting through inhibition of vitamin biosynthesis.

23. The method of claim 1, wherein the drug is an antibiotic acting through inhibition of isoprenoid biosynthesis.

24. The method of claim 1, wherein the drug is an antibiotic acting through inhibition of co-factor biosynthesis

25. The method of claim 1, wherein the drug is an antibiotic acting through inhibition of MurB.

26. The method of claim 1, wherein the drug is an antibiotic acting through inhibition of UppS.

27. The method of claim 1, wherein the drug is an antibiotic acting through inhibition of DHFR.

28. The method of claim 1, wherein the drug is a beta-lactam antibiotic.

29. The method of claim 1, wherein the drug is a diaminopyrimidine antibiotic.

30. The method of claim 1, wherein the drug is macrolide antibiotic.

31. The method of claim 1, wherein the drug is daptomycin.

32. The method of claim 1, wherein the drug is a fluoroquinolone antibiotic including ciprofloxacin.

33. The method of claim 1, wherein the drug is a tetracycline.

34. The method of claim 1, wherein the drug is an oxazolidinone.

35. The method of claim 1, wherein the drug is fosfomycin.

36. The method of claim 1, wherein the drug is fosmidomycin.

37. The method of claim 1, wherein the drug is mupirocin.

38. The method of claim 1, wherein the drug is a lipopeptide.

39. The method of claim 1, wherein the drug is a glycopeptide.

40. The method of claim 1, wherein the drug is vancomycin.

41. The method of claim 1, wherein the drug is trimethoprim.

42. The method of claim 1, wherein said antisense-oriented fragment is a fragment of the Bacillus anthracis gene murB-2 and is comprised of the sequence specified for SEQ ID: Ba-murB2-C1.

43. The method of claim 1, wherein said antisense-oriented fragment is a fragment of the Bacillus anthracis gene murB-2 and is comprised of the sequence specified for SEQ ID: Ba-murB2-H1.

44. The method of claim 1, wherein said antisense-oriented fragment is a fragment of the Bacillus anthracis gene murB-2 and is comprised of the sequence specified for SEQ ID: Ba-murB2-D1.

45. The method of claim 1, wherein said antisense-oriented fragment is a fragment of the Bacillus anthracis gene murB-2 and is comprised of the sequence specified for SEQ ID: Ba-murB2-D2.

46. The method of claim 1, wherein said antisense-oriented fragment is a fragment of the Bacillus anthracis gene metS and is comprised of the sequence specified for SEQ ID: Ba-metRS-H1.

47. The method of claim 1, wherein said antisense-oriented fragment is a fragment of the Bacillus anthracis gene metS and is comprised of the sequence specified for SEQ ID: Ba-metRS-H2.

48. The method of claim 1, wherein said antisense-oriented fragment is a fragment of the Bacillus anthracis gene metS and is comprised of the sequence specified for SEQ ID: Ba-metRS-H6.

49. The method of claim 1, wherein said antisense-oriented fragment is a fragment of the Bacillus anthracis gene metS and is comprised of the sequence specified for SEQ ID: Ba-metRS-E4.

50. The method of claim 1, wherein said antisense-oriented fragment is a fragment of the Bacillus anthracis gene uppS and is comprised of the sequence specified for SEQ ID: Ba-uppS-UG9.

51. The method of claim 1, wherein said antisense-oriented fragment is a fragment of the Bacillus anthracis gene uppS and is comprised of the sequence specified for SEQ ID: Ba-uppS-UA3.

52. The method of claim 1, wherein said antisense-oriented fragment is a fragment of the Bacillus anthracis gene dfrA and is comprised of the sequence specified for SEQ ID: Ba-dfrA-2G1.

53. The method of claim 1, wherein said antisense-oriented fragment is a fragment of the Bacillus anthracis gene dfrA and is comprised of the sequence specified for SEQ ID: Ba-dfrA-2G6.

54. The method of claim 1, wherein said antisense-oriented fragment is a fragment of the Staphylococcus aureus gene murB and is comprised of the sequence specified for SEQ ID: Sa-murB-E9.

55. The method of claim 1, wherein said antisense-oriented fragment is a fragment of the Staphylococcus aureus gene murB and is comprised of the sequence specified for SEQ ID: Sa-murB-F7.

56. The method of claim 1, wherein said antisense-oriented fragment is a fragment of the Staphylococcus aureus gene murB and is comprised of the sequence specified for SEQ ID: Sa-murB-B9.