Cloning and heterologous expression of Plasmodium ovale dihydrofolate reductase-thymidylate synthase gene

Abstract

Plasmodial bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) is a validated antimalarial drug target. In this study, expression of the putative dhfr-ts of Plasmodium ovale rescued the DHFR chemical knockout and a TS null bacterial strain, demonstrating its DHFR and TS catalytic functions. PoDHFR-TS was expressed in Escherichia coli BL21 (DE3) and affinity purified by Methotrexate Sepharose column. Biochemical and enzyme kinetics characterizations indicated that PoDHFR-TS is similar to other plasmodial enzymes, albeit with lower catalytic activity but better tolerance of acidic pH. Importantly, the PoDHFR from Thai isolate EU266602 remains sensitive to the antimalarials pyrimethamine and cycloguanil, in contrast to P. falciparum and P. vivax isolates where resistance to these drugs is widespread.

Graphical abstract

Fig. S

Schematic representation of generation of full-length podhfr-ts using overlap extension PCR technique and analysis of amplicons. Four PCR reactions were required to generate the complete sequence: (1) pojr-ts (1134 bp) was PCR amplified from P. ovale DNA and ligated into pTZ57R/T; (2) podhfr-jr (863 bp) and pojr-ts (1134 bp) was acquired using pGEMT-podhfr and pTZ57R/T-pojr-ts as template respectively; (3) and (4) podhfr-ts (1920 bp) was obtained by 2-steps PCR overlap extension, in which dhfr-jr and jr-ts contain overlapping sequences that are complementary for extension shown by half arrow heads indicating the direction in which each fragment can act as primers for DNA polymerase. Dashed lines mark newly synthesized sequence or strand. Horizontal arrows represent primers used in PCR reactions: i, ii, iii, iv, and v are 5′Po-link, 3′PoTS, 5′PoDHFR, 3′Po-link, and 5′NhePo, respectively.

Fig. 1

Sequence analyses. (A) Multiple amino acid sequence alignment of PoDHFR-TS variant (EU266602), PoDHFR-TS classic (EU266606), PfDHFR-TS (J03028), PvDHFR-TS (EU478859), PmDHFR-TS (AY846633), and PkDHFR-TS (XM002258192). Numbers in brackets indicate percent homology to PoDHFR-TS variant EU266602. Amino acids A16, N51, C59, S108, and I164 in PfDHFR-TS and F57, S58, T61, S117, I173 in PvDHFR-TS are highlighted in bold. Mutations at these positions have been reported to be associated with antifolate resistance. Residues known to be important for TS activity are shown as underlined bold letters. T225, T258, W303, Y476, and N478 — for which the errors were found in the encoded nucleotides during the cloning process — are denoted in italic with bold letters. (*) amino acid positions with identical residue, (:) amino acid positions with conserved substitution amino acid, (.) amino acid positions with semi-conserved substitution amino acid, and (non-marked) amino acid positions with non-conserved substitution amino acid. (B) The results by LC-MS/MS of tryptic digested fragments of 74 (solid line), 35 (dotted line), and 32 (dash line) kDa proteins matched to DHFR-TS of P. ovale. Highlighted amino acids are N-terminal sequences obtained by Edman N-terminal sequencing of 74, 35, and 32 kDa proteins. Asterisk (*) and hash (#) symbols represent sequences of 35 and 32 kDa protein respectively, identified by Edman N-terminal sequencing. The first three amino acids (bold letters) are extra residues generated from cloning of podhfr-ts into Nhe I site of pET17b.

Fig. 1

Sequence analyses. (A) Multiple amino acid sequence alignment of PoDHFR-TS variant (EU266602), PoDHFR-TS classic (EU266606), PfDHFR-TS (J03028), PvDHFR-TS (EU478859), PmDHFR-TS (AY846633), and PkDHFR-TS (XM002258192). Numbers in brackets indicate percent homology to PoDHFR-TS variant EU266602. Amino acids A16, N51, C59, S108, and I164 in PfDHFR-TS and F57, S58, T61, S117, I173 in PvDHFR-TS are highlighted in bold. Mutations at these positions have been reported to be associated with antifolate resistance. Residues known to be important for TS activity are shown as underlined bold letters. T225, T258, W303, Y476, and N478 — for which the errors were found in the encoded nucleotides during the cloning process — are denoted in italic with bold letters. (*) amino acid positions with identical residue, (:) amino acid positions with conserved substitution amino acid, (.) amino acid positions with semi-conserved substitution amino acid, and (non-marked) amino acid positions with non-conserved substitution amino acid. (B) The results by LC-MS/MS of tryptic digested fragments of 74 (solid line), 35 (dotted line), and 32 (dash line) kDa proteins matched to DHFR-TS of P. ovale. Highlighted amino acids are N-terminal sequences obtained by Edman N-terminal sequencing of 74, 35, and 32 kDa proteins. Asterisk (*) and hash (#) symbols represent sequences of 35 and 32 kDa protein respectively, identified by Edman N-terminal sequencing. The first three amino acids (bold letters) are extra residues generated from cloning of podhfr-ts into Nhe I site of pET17b.

Fig. 2

Bacterial complementation assays to demonstrate DHFR and TS functions of PoDHFR-TS coded sequence. (A) E. coli BL21 (DE3) transformed with either pET17b or pET17bPoDHFR-TS was grown on MM in the absence or presence of 4 μM TMP. (B) E. coli χ 2913recA-DE3 carrying pET17b or pET17bPoDHFR-TS was cultured on MM with and without 50 μg/ml thymidine. MM used in A and B also contained 100 μg/ml ampicillin.

Fig. 3

Coomassie blue-stained SDS-PAGE and X-ray film exposed to [H] FdUMP labeling of recombinant PoDHFR-TS. Lane M, molecular weight markers; (1) uninduced cells carrying pET17b; (2) induced cells carrying pET17b; (3) uninduced cells carrying PoDHFR-TS gene; (4) induced cell carrying PoDHFR-TS gene; (5) insoluble fraction of (4); (6) soluble fraction of (4); (7) purified PoDHFR-TS after MTX-affinity chromatography; (8–10) purified PoDHFR-TS, PvDHFR and TcDHFR-TS; (11–13) autoradiographs of (8–10) following [³H] FdUMP labeling.

Fig. 4

Effect of pH on DHFR (filled circles) and TS (open diamonds) activities of P. ovale. The catalytic constants were determined for reactions performed over pH range of 4–9.5. Each data point is the average value from three experiments.

Fig. 5

Double reciprocal plots of inhibition of DHFR domain of PoDHFR-TS by (A) PYR (0–31.25 nM) and (B) CYC (0–31.25 nM). Assays were performed in the presence of NADPH (100 μM) and H₂folate (2.5–12.5 μM). The final concentrations of inhibitors are indicated on each line.