Escherichia coli cell line with thyA knockout, folA knockout, and one or both of folP knockout, and folK knockout

Abstract

In this invention, cell lines are created for enzyme inhibitory testing of inhibitors against Plasmodium falciparum DHFR-TS and HPPK-DHPS. Provided the complementing DHFR-TS and HPPK-DHPS have sufficient activities to support growth of the surrogates in un-supplemented medium, the same surrogates could be used for screening inhibitors of targets against other parasite and pathogen species e.g. Plasmodium vivax, Trypanosoma brucei, Trypanosoma cruzi, Toxoplasma gondii or Mycobacterium tuberculosis. The cell lines in this invention are Escherichia coli strain whose thyA, folA, folK, and folP genes were disrupted using genetic knockout coupled with elimination of antibiotic resistance markers. The thyA KO, folP KO, folK KO, thyAfolA KO, folKfolP KO, thyAfolAfolP KO, thyAfolAfolK KO and thyAfolAfolKfolP KO E. coli cell lines are easy and convenient for testing single and combination drugs as plasmids bearing complementing parasite genes can be introduced simply by transformation using standard antibiotic selection.

Claims

1. An Escherichia coli (E. coli) cell line in which its thymidylate synthase (thyA), dihydrofolate reductase (folA), and one or both of its dihydropteroate synthase (folP) and 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase (folK) genes have been disrupted by genetic knockout, and wherein antibiotic resistance genes used to create the genetic knockout have been functionally eliminated.

2. The cell line of claim 1, wherein the cell line is E. coli type BL21(DE3).

3. The cell line of claim 1, wherein cells of the cell line comprise one or both of a parasite dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene for expression of a parasite DHFR-TS enzyme, and a parasite 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase-dihydropteroate synthase (HPPK-DHPS) gene for expression of a parasite HPPK-DHPS enzyme.

4. The cell line of claim 3, wherein one or both of the parasite DHFR-TS gene and the parasite HPPK-DHPS gene are of a parasite selected from the group consisting of Plasmodium falciparum, Plasmodium vivax, Trypanosoma brucei, Trypanosoma cruzi, Toxoplasma gondii, and Mycobacterium tuberculosis.

5. The cell line of claim 3, wherein one or both of the parasite DHFR-TS gene and the parasite HPPK-DHPS gene are present on plasmids.

6. The cell line of claim 5 wherein the cells comprise the parasite DHFR-TS gene.

7. The cell line of claim 5, wherein the cells comprise the parasite HPPK-DHPS gene.

8. The cell line of claim 5, wherein the cells comprise the parasite DHFR-TS gene and the parasite HPPK-DHPS gene.

9. The cell line of claim 8, wherein the heterologous expression of the parasite DHFR-TS enzyme and the parasite HPPK-DHPS enzyme permits the cells to grow on minimal media without thymidine and folinic acid supplementation.

10. The cell line of claim 1, wherein cells of the cell line are sensitive to one or both of ampicillin and chloramphenicol.

11. The cell line of claim 1, wherein the thyA, folA, folP and folK genes have been disrupted by genetic knockout (E. coli thyAfolAfolPfolK KO).

12. The cell line of claim 1, wherein the thyA, folA and folP genes have been disrupted by genetic knockout (E. coli thyAfolAfolP KO).

13. The cell line of claim 1, wherein the thyA, folA and folK genes have been disrupted by genetic knockout (E. coli thyAfolAfolK KO).

14. A method of identifying an inhibitor of a parasite DHFR-TS enzyme or a parasite HPPK-DHPS enzyme, comprising: a) growing the E. coli cell line of claim 8 in the presence and absence of a candidate drug in media; and b) identifying the candidate drug as an inhibitor of the parasite DHFR-TS enzyme or the parasite HPPK-DHPS enzyme when the cell line grows more quickly in the absence as compared to the presence of the candidate drug.

15. The method of claim 14, wherein the media does not comprise a thymidine supplement and a folinic acid supplement.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1A: Invention step for creating a knockout gene (thyA KO or folA KO or folP KO or folK KO) in E. coli by a double crossover strategy. FIG. 1B: PCR results for verification of gene knockouts in E. coli.

(2) FIG. 2A-B: Complementation assay of dihydrofolate reductase-thymidylate synthase and 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase-dihydropteroate synthase enzyme in invention. FIG. 2A: Assay of a quadruple mutant PEcTFPK without any plasmid (spot 1) and with plasmids containing Pfdhfr-ts (WT) and Pfhppk-dhps (WT) (spots 2-5). FIG. 2B: Assay of a quadruple mutant PEcTFPK with plasmids containing variants of Pfdhfr-ts and Pfhppk-dhps. Spot 1: PEcTFPK (WT/WT), A quadruple thyAfolAfolPfolK KO E. coli BL21(DE3) with plasmids for expressing Pfdhfr-ts (WT) and Pfhppk-dhps (WT) Spot 2: PEcTFPK (WT/K1), A quadruple thyAfolAfolPfolK KO E. coli BL21(DE3) with plasmids for expressing Pfdhfr-ts (WT) and Pfhppk-dhps (A437G+A581G, K1) Spot 3: PEcTFPK (WT/V1S), A quadruple thyAfolAfolPfolK KO E. coli BL21(DE3) with plasmids for expressing Pfdhfr-ts (WT) and Pfhppk-dhps (S436F+A437G+A613T, V1S) Spot 4: PEcTFPK (K1/WT), A quadruple thyAfolAfolPfolK KO E. coli BL21(DE3) with plasmids for expressing Pfdhfr-ts (C59R+S108N, K1) and Pfhppk-dhps (WT) Spot 5: PEcTFPK (K1/K1), A quadruple thyAfolAfolPfolK KO E. coli BL21(DE3) with plasmids for expressing Pfdhfr-ts (C59R+S108N, K1) and Pfhppk-dhps (A437G+A581G, K1) Spot 6: PEcTFPK (K1/V1S), A quadruple thyAfolAfolPfolK KO E. coli BL21(DE3) with plasmids for expressing Pfdhfr-ts (C59R+S108N, K1) and Pfhppk-dhps (S436F+A437G+A613T, V1S) Spot 7: PEcTFPK (V1S/WT), A quadruple thyAfolAfolPfolK KO E. coli BL21(DE3) with plasmids for expressing Pfdhfr-ts (N51I+C59R+S108N+I164L, V1S) and Pfhppk-dhps (WT) Spot 8: PEcTFPK (V1S/K1), A quadruple thyAfolAfolPfolK KO E. coli BL21(DE3) with plasmids for expressing Pfdhfr-ts (N51I+C59R+S108N+I164L, V1S) and Pfhppk-dhps (A437G+A581G, K1) Spot 9: PEcTFPK (V1S/V1S), A quadruple thyAfolAfolPfolK KO E. coli BL21(DE3) with plasmids for expressing Pfdhfr-ts (N51I+C59R+S108N+I164L, V1S) and Pfhppk-dhps (S436F+A437G+A613T, V1S)

(3) FIG. 3A-B: Isobologram of pyrimethamine and sulfathiazole against the invented host carrying plasmids for expressing Pfdhfr-ts (WT) and Pfhppk-dhps (WT). Synergistic effect was observed from both invented host, a triple mutant, PEcATFP (FIG. 3A) and a quadruple mutant, PEcTFPK (FIG. 3B).

(4) Table 1 Growth rate of quadruple mutant PEcTFPK expressing different Pfdhfr-ts and Pfhppk-dhps variants in comparison with E. coli BL21(DE3) in minimal media with L-arabinose. PEcTFPK (WT/WT): A quadruple thyAfolAfolPfolK KO E. coli BL21(DE3) with plasmids for expressing Pfdhfr-ts (WT) and Pfhppk-dhps (WT) PEcTFPK (WT/K1): A quadruple thyAfolAfolPfolK KO E. coli BL21(DE3) with plasmids for expressing Pfdhfr-ts (WT) and Pfhppk-dhps (A437G+A581G, K1) PEcTFPK (WT/V1S): A quadruple thyAfolAfolPfolK KO E. coli BL21(DE3) with plasmids for expressing Pfdhfr-ts (WT) and Pfhppk-dhps (S436F+A437G+A613T, V1S) PEcTFPK (K1/WT): A quadruple thyAfolAfolPfolK KO E. coli BL21(DE3) with plasmids for expressing Pfdhfr-ts (C59R+S108N, K1) and Pfhppk-dhps (WT) PEcTFPK (K1/K1): A quadruple thyAfolAfolPfolK KO E. coli BL21(DE3) with plasmids for expressing Pfdhfr-ts (C59R+S108N, K1) and Pfhppk-dhps (A437G+A581G, K1) PEcTFPK (K1/V1S): A quadruple thyAfolAfolPfolK KO E. coli BL21(DE3) with plasmids for expressing Pfdhfr-ts (C59R+S108N, K1) and Pfhppk-dhps (S436F+A437G+A613T, V1S) PEcTFPK (V1S/WT): A quadruple thyAfolAfolPfolK KO E. coli BL21(DE3) with plasmids for expressing Pfdhfr-ts (N51I+C59R+S108N+I164L, V1S) and Pfhppk-dhps (WT) PEcTFPK (V1S/K1): A quadruple thyAfolAfolPfolK KO E. coli BL21(DE3) with plasmids for expressing Pfdhfr-ts (N51I+C59R+S108N+I164L, V1S) and Pfhppk-dhps (A437G+A581G, K1) PEcTFPK (V1S/V1S): A quadruple thyAfolAfolPfolK KO E. coli BL21(DE3) with plasmids for expressing Pfdhfr-ts (N51I+C59R+S108N+I164L, V1S) and Pfhppk-dhps (S436F+A437G+A613T, V1S)

(5) TABLE-US-00001 Fitness of dhps Fitness of dhfr mutant (under mutant (under same same dhfr dhps background hppk- % growth of background shown shown in the Cell dhfr-ts* dhps** cell*** in the parenthesis).sup.$ parenthesis).sup.# BL21(DE3) thyA + folK + 100 0.00 folA folP PEcTFPK(WT/WT) WT WT 74.94 0.90 1.00 (WT-dhfr) 1.00 (WT-dhps) PEcTFPK(WT/K1) WT K1 89.23 1.15 1.19 (WT-dhfr) 1.00 (K1-dhps) PEcTFPK(WT/V1S) WT V1S 59.60 0.85 0.79 (WT-dhfr) 1.00 (V1S-dhps) PEcTFPK(K1/WT) K1 WT 71.57 1.73 1.00 (K1-dhfr) 0.95 (WT-dhps) PEcTFPK(K1/K1) K1 K1 77.32 2.08 1.08 (K1-dhfr) 0.87 (K1-dhps) PEcTFPK(K1/V1S) K1 V1S 77.00 1.07 1.08 (K1-dhfr) 1.29 (V1S-dhps) PEcTFPK(V1S/WT) V1S WT 67.85 1.35 1.00 (V1S-dhfr) 0.90 (WT-dhps) PEcTFPK(V1S/K1) V1S K1 63.47 0.74 0.93 (V1S-dhfr) 0.71 (K1-dhps) PEcTFPK(V1S/V1S) V1S V1S 78.05 0.74 1.15 (V1S-dhfr) 1.31 (V1S-dhps) *mutation at C59R + S108N (K1) and N51I + C59R + S108N + I164L (V1S) of PfDHFR **mutation at A437G + A581G (K1) and S436F + A437G + A613T (V1S) of PfDHPS ***values are mean growth relative to untreated control (100%) standard deviation .sup.$ratio of growth rate of PEcTFPK expressing dhps mutant and that expressing WT dhps under the same dhfr type .sup.#ratio of growth rate of PEcTFPK expressing dhfr mutant and that expressing WT dhfr under the same dhps type

BEST MODE FOR CARRYING OUT THE INVENTION

(6) As described above in disclosure of invention section

INDUSTRIAL APPLICABILITY

(7) As described above in disclosure of invention section

REFERENCES

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