ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 2011, p. 2481–2482 0066-4804/11/$12.00 doi:10.1128/AAC.01394-10 Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 55, No. 5
Novel K540N Mutation in Plasmodium falciparum Dihydropteroate Synthetase Confers a Lower Level of Sulfa Drug Resistance than Does a K540E Mutation䌤 Vanshika Lumb and Yagya D. Sharma* Department of Biotechnology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India Received 11 October 2010/Returned for modification 16 December 2010/Accepted 9 February 2011
Sulfadoxine (SDX) and sulfamethoxazole (SMX) each inhibit the Plasmodium falciparum dihydropteroate synthetase (PfDHPS), and certain point mutations in this enzyme yield the drug-resistant parasite. Using a simple Escherichia coli model system, we describe here the effect of the recently reported novel K540N mutation in PfDHPS on the level of SDX/SMX resistance. The survival rate of the transformed E. coli (DHPS-deficient strain) under different SDX or SMX concentrations revealed that the K540N mutation confers a lower level of drug resistance than its contemporary K540E mutation. Further, SMX was more effective than SDX in the E. coli system.
pfpppk-dhps gene was amplified from cDNA by high-fidelity proofreading enzyme Platinum Pfx DNA polymerase (Invitrogen Life Technologies, Carlsbad, CA) using the primers pGEXF (5⬘-CAAGAATTCATACTTTCTGAGG-3⬘) and pGEXR (5⬘-CTCGAGTTACACTTGGTCTATTTTTG-3⬘). The cDNA was denatured at 94°C for 5 min, and PCR amplification was carried out for 35 cycles under the following conditions. The primers were annealed at 48°C for 1 min, with extension for 2 min at 60°C and cycling denaturation at 94°C for 30 s. The final extension was carried out at 60°C for 10 min. The PCR product was subcloned in pGEM-T (Promega Corp., Madison, WI) and then transferred to pGEX 4T-1 (glutathione S-transferase-tagged) (GE Healthcare Life Sciences, Buckinghamshire, NA, England) vector for expression. The resulting pGEX 4T-1–PfPPPK-DHPS was used as a template to modify the gene for improved stability through site-directed mutagenesis using primers InsF (5⬘-GAAATTATAAAATTA TTAAAGAAGAAGAACAAATTCTATAC-3⬘) and InsR (5⬘-CTATAGAATTTGTTCTTCTTCTTTAATAATTTTAT AATTTC-3⬘) as described in reference 9. This PCR product was digested with DpnI and then used to transform the E. coli DH5␣ strain. The amino acid sequence encoded by the PfPPPK-DHPS gene was S436G437K540A581A613 as confirmed by nucleotide sequencing (the mutated amino acid is in boldface). This was used as a template for one-step mutagenesis to create alleles encoding the sequences S436A437K540A581A613, S436A437N540A581A613 and A436G437K540A581A613 by using the primers A437F (5⬘-ATCCTCTGCTCCTTTTGTTATACC-3⬘) and A437R (5⬘-ACTAATTTTTGGATTAGGTATAAC-3⬘) for S436A437K540A581A613, A436F (5⬘-ATCCGCTGGTCCTT TTGTTATACC-3⬘) and A437R for A436G437K540A581A613, and N540F (5⬘-GATAATCTAACAAATTATGATAATC-3⬘) and E540R (5⬘-CATAAACTAGATTATCATAATTTG-3⬘) for S436A437N540A581A613 and the gene tailor mutagenesis kit (Invitrogen Life Technologies, Carlsbad, CA). The PfPPPKDHPS amino acid sequence A436G437K540A581A613 was used as a template to create alleles encoding the A436G437E540A581A613 and A436G437N540A581A613 sequences using primers E540F (5⬘-
Plasmodium falciparum can cause the most severe form of malaria and death in humans. Antifolates are commonly used for P. falciparum malaria treatment. These drugs can specifically inhibit the enzymes involved in the parasite folate biosynthesis pathway. Both sulfadoxine (SDX) and sulfamethoxazole (SMX) target dihydropteroate synthetase (DHPS) by competing with its substrate para-aminobenzoic acid. Five point mutations in the P. falciparum DHPS (PfDHPS) at codons 436, 437, 540, 581, and 613 had been reported to be associated with resistance to sulfonamides (7–9). Different PfDHPS mutant alleles have been reported from different parts of the world, including India. Recently, we have reported a novel K540N mutation in PfDHPS among P. falciparum isolates from Andaman and Nicobar Islands of India (6). This K540N mutation in PfDHPS has now also been reported from other countries (10). Using the Escherichia coli system, we have evaluated here the in vitro effect of this novel PfDHPS mutation on drug resistance levels compared to that of its contemporary K540E mutation. Various cell-based inhibition studies had earlier been used to complement the dhps gene from Mycobacterium, Pneumocystis, and Plasmodium (1, 4, 5). Therefore, we used here the model E. coli system to evaluate the effect of the different mutant Plasmodium falciparum pyrophosphokinase (PfPPPK)-DHPS alleles on the 50% inhibitory concentrations (IC50s) of SDX and SMX. The bacterial strain used for molecular cloning and plasmid amplification was E. coli strain DH5␣ [F⫺ 80dlacZ⌬M15 ⌬(lacZYAargF)U169 endA1 recA1 hsdR17(rk⫺ mk⫹) supE44 thi-1 gyrA relA1]. The DHPS-interrupted E. coli strain C600 [⌬folP::Kmr F⫺ e14⫺ (McrA⫺) thr-1 leuB6 thi-1 ⌬lacY1 glnV44 rfbD1 fhu A21] (3) was used to check the expression of Plasmodium falciparum pyrophosphokinase (pfpppk)-dhps and also for the complementation studies of different constructs (1). The full-length * Corresponding author. Mailing address: Department of Biotechnology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India. Phone: 91-11-26588145. Fax: 91-11-26589286. E-mail:
[email protected]. 䌤 Published ahead of print on 22 February 2011. 2481
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ANTIMICROB. AGENTS CHEMOTHER.
TABLE 1. Effect of various pfdhps mutant alleles on the IC50s of sulfadoxine (SDX) and sulfamethoxazole (SMX) in a model E. coli system Serial no.
PfDHPS mutationa
IC50 value of SDX (M) (mean ⫾ SD)
IC50 value of SMX (M) (mean ⫾ SD)
1 2 3 4 5 6
S436A437K540A581A613 S436G437K540A581A613 A436G437K540A581A613 A436G437E540A581A613 S436A437N540A581A613 A436G437N540A581A613
27.45 ⫾ 3.97 153.55 ⫾ 23.74 250.84 ⫾ 24.74 2,005.23 ⫾ 50.81 38.10 ⫾ 4.58 527.10 ⫾ 34.84
25.30 ⫾ 2.96 102.37 ⫾ 10.16 297.35 ⫾ 19.13 1,363.56 ⫾ 47.47 31.74 ⫾ 3.12 562.10 ⫾ 78.93
a
Mutated amino acids are shown in boldface.
GATGAACTAACAAATTATGATAATC-3⬘)/E540R and N540F/ E540R, respectively. The PCR conditions were the same as those described above except that only 20 instead of 35 cycles of amplification were performed here. The pGEX 4T-1–PfPPPK-DHPS constructs were precultured overnight in LB medium containing 100 g/ml ampicillin. The next day, the culture was diluted with LB medium to an optical density at 600 nm of ⬃0.5. Twenty microliters of this cell suspension was then seeded in 1 ml of Mueller-Hilton medium (Difco, BD Biosciences, San Jose, CA) in 24-well culture plates (Corning Incorporated, Corning, NY) supplemented with various concentrations of SDX (0, 32.26, 64.52, 161.29, 225.81, 322.58, 645.16, 1,612.90, 2,258.06, or 3,225.81 M) or SMX (0, 39.53, 79.05, 197.63, 276.68, 395.25, 790.51, 1,976.28, 2,766.80, or 3,952.57 M) dissolved in dimethyl sulfoxide (DMSO). The culture plates were then grown at 37°C with shaking for 6 h before the growth was measured at A600. The A600 values were plotted against the log of drug concentrations, and 50% cell growth (IC50) was calculated using GraphPad Prism version 4 and sigmoidal curve responses for drug inhibition. One-way analysis of variance (ANOVA) was done to determine the significant differences between the IC50s of different constructs. The IC50s of SDX or SMX for wild-type and mutant PfDHPS alleles are shown in Table 1. These IC50s of SMX were significantly lower (P ⬍ 0.05) than those of SDX for all the PfDHPS alleles except for the allele encoding the A436G437K540A581A613 sequence. Lower IC50s for SMX than SDX have also been reported by using the in vitro P. falciparum culture system (11). The IC50s of SDX or SMX for the wild-type allele encoding the S436A437K540A581A613 sequence were insignificantly lower than the IC50s for the allele encoding the S436A437N540A581A613 sequence. But the IC50s of both drugs for the wild-type allele were significantly lower than those of the other mutant PfDHPS alleles (P ⬍ 0.05). For example, the IC50s of SDX and SMX for the single-mutant allele encoding the S436G437K540A581A613 sequence were 5.59- and 4.04-fold higher, respectively, than that of the wild-type PfDHPS allele. Additional mutations S436A and K540E in PfDHPS significantly increased the drug resistance further (P ⬍ 0.05). Thus, the triple-mutant PfDHPS allele encoding the A436G437E540A581A613 sequence showed the highest IC50 of SDX or SMX. These results were similar to those of earlier reports where additional mutations in this enzyme caused higher levels of SDX resistance (1, 6, 9). However, the novel mutant allele encoding the A436G437N540A581A613 se-
quence showed significantly lower IC50 values than the mutant allele encoding the A436G437E540A581A613 sequence (P ⬍ 0.05) (Table 1). This result indicates that the novel mutation confers less resistance to SDX or SMX than its contemporary mutation in the pfdhps gene. The mutation K540E causes a loss in the hydrophobic interactions with side chains of lysine and sulfadoxine methoxy groups and thus increases the hydrophilicity and the loop widening to expose the active site (2, 6). In the case of the K540N mutation, binding of SDX is altered because a hydrogen bond could not be formed with the drug due to the short side chain of asparagine compared to that of lysine (6). Earlier, we described the selection of the parasite population with this novel K540N mutation in PfDHPS which was predicted to be due to an unusual drug pressure in the field (6). Although the K540N mutation containing the parasite could be less SDX/SMX resistant than the K540E mutation, its association with two other mutations (triple PfDHPS mutant alleles) among the majority of the isolates is a cause of concern. This work was supported by financial assistance from the Department of Biotechnology (Government of India) and the Indian Council of Medical Research. V.L. received a senior research fellowship from the Council for Scientific and Industrial Research. We acknowledge Gote Swedberg for providing the E. coli C600⌬folP::Kmr strain. Facility of the Bio-Technology Information System (BTIS) of the Biotechnology Department is gratefully acknowledged. We thank R. M. Pandey for statistical analysis and Shalini Narang for preparing the manuscript. REFERENCES 1. Berglez, J., P. Iliades, W. Sirawaraporn, P. Coloe, and I. Macreadie. 2004. Analysis in Escherichia coli of Plasmodium falciparum dihydropteroate synthase (DHPS) alleles implicated in resistance to sulfadoxine. Int. J. Parasitol. 34:95–100. 2. de Beer, T. A., A. I. Louw, and F. Joubert. 2006. Elucidation of sulfadoxine resistance with structural models of the bifunctional Plasmodium falciparum dihydropterin pyrophosphokinase-dihydropteroate synthase. Bioorg. Med. Chem. 14:4433–4443. 3. Fermer, C., and G. Swedberg. 1997. Adaptation to sulfonamide resistance in Neisseria meningitidis may have required compensatory changes to retain enzyme function: kinetic analysis of dihydropteroate synthases from N. meningitidis expressed in a knockout mutant of Escherichia coli. J. Bacteriol. 179:831–837. 4. Iliades, P., S. R. Meshnick, and I. G. Macreadie. 2005. Analysis of Pneumocystis jirovecii DHPS alleles implicated in sulfamethoxazole resistance using an Escherichia coli model system. Microb. Drug Resist. 11:1–8. 5. Iliades, P., S. R. Meshnick, and I. G. Macreadie. 2004. Dihydropteroate synthase mutations in Pneumocystis jirovecii can affect sulfamethoxazole resistance in a Saccharomyces cerevisiae model. Antimicrob. Agents Chemother. 48:2617–2623. 6. Lumb, V., et al. 2009. Emergence of an unusual sulfadoxine-pyrimethamine resistance pattern and a novel K540N mutation in dihydropteroate synthetase in Plasmodium falciparum isolates obtained from Car Nicobar Island, India, after the 2004 tsunami. J. Infect. Dis. 199:1064–1073. 7. Plowe, C. V., et al. 1997. Mutations in Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase and epidemiologic patterns of pyrimethamine-sulfadoxine use and resistance. J. Infect. Dis. 176:1590–1596. 8. Sharma, Y. D. 2005. Genetic alteration in drug resistant markers of Plasmodium falciparum. Indian J. Med. Res. 121:13–22. 9. Triglia, T., J. G. Menting, C. Wilson, and A. F. Cowman. 1997. Mutations in dihydropteroate synthase are responsible for sulfone and sulfonamide resistance in Plasmodium falciparum. Proc. Natl. Acad. Sci. U. S. A. 94:13944– 13949. 10. Vinayak, S., et al. 2010. Origin and evolution of sulfadoxine resistant Plasmodium falciparum. PLoS Pathog. 6:e1000830. 11. Zhang, Y., and S. R. Meshnick. 1991. Inhibition of Plasmodium falciparum dihydropteroate synthetase and growth in vitro by sulfa drugs. Antimicrob. Agents Chemother. 35:267–271.
