Point mutations in the dihydrofolate reductase-thymidylate synthase gene as the molecular basis for pyrimethamine resistance in Plasmodium falciparum

doi:10.1016/0166-6851(89)90173-4

Molecular and Biochemical Parasitology

Volume 36, Issue 3, October 1989, Pages 253-262

https://doi.org/10.1016/0166-6851(89)90173-4 Get rights and content

Abstract

The dihydrofolate reductase-thymidylate synthase (DHFR-TS) bifunctional complex from pyrimethamine-sensitive (3D7) and drug-resistant (HB3 and 7G8) clones from Plasmodium falciparum was purified to homogeneity. A modified sequence of purification steps with a 10-formylfolate affinity column at its center, allows the isolation of the enzyme complex with a 10-fold higher yield than previously reported, irrespective of the pyrimethamine resistance of the parasites. Titration of the homogeneous DHFR-TS complex with the inhibitor revealed a 500-fold lower affinity of the enzyme from clone 7G8 for the drug than found with the enzyme from clone 3D7. Direct comparison of the homogeneous enzyme preparations on SDS-PAGE revealed no difference in the molecular mass of the DHFR-TS from the 3 clones, nor could a reproducible difference be detected in the peptide patterns obtained after digesting the DHFR-TS complex with various proteases. The amplification of segments from the DHFR-TS coding region of the 3 clones and 7 isolates of P. falciparum by polymerase chain reaction resulted in fragments of the predicted length without any size heterogeneity. The DNA sequence of the DHFR coding region from FCR-3, 3D7, HB3 and 7G8 differs in a total of 4 nucleotides. One point mutation changes amino acid residue 108 from threonine (FCR-3) or serine (3D7) to asparagine (HB3 and 7G8). The presence of asparagine-108 appears to be the molecular basis of pyrimethamine resistance of HB3 and 7G8. The degree of resistance is associated with a point mutation affecting the codon for amino acid 51 in 7G8.

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Cited by (109)

Antimalarial effect of cell penetrating peptides derived from the junctional region of Plasmodium falciparum dihydrofolate reductase-thymidylate synthase
2020, Peptides
Citation Excerpt :
There are five species of malaria parasite responsible for human malaria, including Plasmodium falciparum, P. vivax, P. malariae, P. ovale, and P. knowlesi [5–8]. Although malaria is much less prevalent today than in the past two decades, malaria control in many endemic areas is threatened by the emergence and evolution of antimalarial-resistant parasites [9–14]. New antimalarials with novel mechanisms of action are needed to overcome resistant parasites and prevent the re-emergence of malaria [15].
Dihydrofolate reductase-thymidylate synthase of Plasmodium falciparum (PfDHFR-TS) is an important target of antifolate antimalarial drugs. However, drug resistant parasites are widespread in malaria endemic regions. The unique bifunctional property of PfDHFR-TS could be exploited for the design of allosteric inhibitors that interfere with the active dimer conformation. In this study, peptides were derived from the junctional region (JR) of PfDHFR-TS amino acid sequence in the α_j1 helix (JR-helix) and the DHFR domain that is necessary for interaction with α_j1 helix (JR21). Five peptides were synthesized and tested for inhibition of PfDHFR-TS enzyme by Bacterial inhibition assay (BIA) based on the growth of an E. coli DHFR and TS knockout complemented with a recombinant plasmid expressing PfDHFR-TS enzyme. Significant inhibition was observed for JR21 and JR21 conjugated to cell-penetrating octa-arginine peptide (rR8-JR21) with 50 % inhibitory concentration (IC₅₀) of 3.87 and 1.53 μM, respectively. The JR-helix and rR8-JR-helix peptides were inactive. JR21 and rR8-JR21 peptides showed similar growth inhibitory effects on P. falciparum NF54 parasites cultured in vitro. Treatment with rR8-JR21 delayed parasite development, in which an accumulation of ring stage parasites was observed after 12 h of culture. Minimal red blood cell (RBC) hemolysis was observed at the highest dose of peptide tested. The most potent peptide rR8-JR21 not only compromised the development of the P. falciparum, but also inhibited the parasite growth and has low hemolytic effect on human RBCs.
A better resolution for integrating methods for monitoring Plasmodium falciparum resistance to antimalarial drugs
2014, Acta Tropica
Citation Excerpt :
Typically, these are PCR-based, where PCR can be performed as a one-round or nested procedure. Mutation specific (MS)-PCRs through the use of mutation-specific primers were introduced as a sensitive approach to detect resistance-associated SNPs in large-scale studies and as a diagnostic approach to detect SNPs on an individual basis (Zolg et al., 1989; Peterson et al., 1991; Gyang et al., 1992; Plowe et al., 1995; Parzy et al., 1997). Later, PCR followed by RFLP was applied as a simple, rapid and reliable method to discriminate between wild-type and mutant alleles of P. falciparum genes based on the digestion pattern of the PCR product with certain restriction enzymes, eliminating the need for optimal PCR conditions required for MS-PCR approach (Eldin de Pecoulas et al., 1995; Zindrou et al., 1996; Duraisingh et al., 1998).
Effective chemotherapy is the mainstay of malaria control. However, resistance of falciparum malaria to antimalarial drugs compromised the efforts to eliminate the disease and led to the resurgence of malaria epidemics. Three main approaches are used to monitor antimalarial drug efficacy and drug resistance; namely, in vivo trials, in vitro/ex vivo assays and molecular markers of drug resistance. Each approach has its implications of use as well as its advantages and drawbacks. Therefore, there is a need to use an integrated approach that would give the utmost effect to detect resistance as early as its emergence and to track it once spread. Such integration becomes increasingly needed in the era of artemisinin-based combination therapy as a forward action to deter resistance. The existence of regional and global networks for the standardization of methodology, provision of high quality reagents for the assessment of antimalarial drug resistance and dissemination of open-access data would help in approaching an integrated resistance surveillance system on a global scale.
Sulfadoxine-pyrimethamine resistance in Plasmodium falciparum: A zoomed image at the molecular level within a geographic context
2013, Acta Tropica
Citation Excerpt :
Resistance evolution results from stepwise mutations in dhfr gene, starting with S108N that optimally causes both decreased binding affinity for inhibitor drugs and retention of enzyme activity (Sirawaraporn et al., 1997; Yuthavong, 2002). Comparison of the mutation pattern in pyrimethamine-sensitive (3D7) and pyrimethamine-resistant (HB3 and 7G8) P. falciparum clones has confirmed that the presence of S108N/T is responsible for pyrimethamine resistance (Zolg et al., 1989). In Kenyan P. falciparum isolates, S108N significantly affected the in vitro chemosensitivity to pyrimethamine compared to N51I and C59R (Nzila-Mounda et al., 1998).
Antimalarial chemotherapy is one of the main pillars in the prevention and control of malaria. Following widespread resistance of Plasmodium falciparum to chloroquine, sulfadoxine-pyrimethamine came to the scene as an alternative to the cheap and well-tolerated chloroquine. However, widespread resistance to sulfadoxine-pyrimethamine has been documented. In vivo efficacy tests are the gold standard for assessing drug resistance and treatment failure. However, they have many disadvantages, such as influence of host immunity and drug pharmacokinetics. In vitro tests of antimalarial drug efficacy also have many technical difficulties. Molecular markers of resistance have emerged as epidemiologic tools to investigate antimalarial drug resistance even before becoming clinically evident. Mutations in P. falciparum dihydrofolate reductase and dihydrofolate synthase have been extensively studied as molecular markers for resistance to pyrimethamine and sulfadoxine, respectively. This review highlights the resistance of P. falciparum at the molecular level presenting both supporting and opposing studies on the utility of molecular markers.
Red Blood Cell Polymorphism and Susceptibility to Plasmodium vivax
2013, Advances in Parasitology
Citation Excerpt :
The first published application of PCR demonstrated how sickle cell anaemia could be diagnosed by amplification of a small segment of the β-globin gene (Saiki et al., 1985). Amplification of malarial parasites from human blood samples was performed in the early 1990s to diagnose species with greater sensitivity than conventional blood smear methods (Barker et al., 1992; Barker et al., 1994) and to identify P. falciparum strains that were carrying mutations associated with drug resistance (Zolg et al., 1989). As powerful high-throughput multiplex assays for diagnosing malarial infections have become routinely available, perspectives on species complexity of malarial infections have changed significantly.
Resistance to Plasmodium vivax blood-stage infection has been widely recognised to result from absence of the Duffy (Fy) blood group from the surface of red blood cells (RBCs) in individuals of African descent. Interestingly, recent studies from different malaria-endemic regions have begun to reveal new perspectives on the association between Duffy gene polymorphism and P. vivax malaria. In Papua New Guinea and the Americas, heterozygous carriers of a Duffy-negative allele are less susceptible to P. vivax infection than Duffy-positive homozygotes. In Brazil, studies show that the Fy^a antigen, compared to Fy^b, is associated with lower binding to the P. vivax Duffy-binding protein and reduced susceptibility to vivax malaria. Additionally, it is interesting that numerous studies have now shown that P. vivax can infect RBCs and cause clinical disease in Duffy-negative people. This suggests that the relationship between P. vivax and the Duffy antigen is more complex than customarily described. Evidence of P. vivax Duffy-independent red cell invasion indicates that the parasite must be evolving alternative red cell invasion pathways.
In this chapter, we review the evidence for P. vivax Duffy-dependent and Duffy-independent red cell invasion. We also consider the influence of further host gene polymorphism associated with malaria endemicity on susceptibility to vivax malaria. The interaction between the parasite and the RBC has significant potential to influence the effectiveness of P. vivax-specific vaccines and drug treatments. Ultimately, the relationships between red cell polymorphisms and P. vivax blood-stage infection will influence our estimates on the population at risk and efforts to eliminate vivax malaria.
Low infectivity of Plasmodium falciparum gametocytes to Anopheles gambiae following treatment with sulfadoxine-pyrimethamine in Mali
2010, International Journal for Parasitology
Sulfadoxine–pyrimethamine (SP) treatment increases the rate of gametocyte carriage and selects SP resistance-conferring mutations in Plasmodium falciparum dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS), raising concerns of increased malaria transmission and spread of drug resistance. In a setting in Mali where SP was highly efficacious, we measured the prevalence of DHFR and DHPS mutations in P. falciparum infections with microscopy-detected gametocytes following SP treatment, and used direct feeding to assess infectivity to Anopheles gambiae sensu lato. Children and young adults presenting with uncomplicated malaria were treated with SP or chloroquine and followed for 28 days. Gametocyte carriage peaked at 67% 1 week after treatment with a single dose of SP. Those post-SP gametocytes carried significantly more DHFR and DHPS mutations than pre-treatment asexual parasites from the same population. Only 0.5% of 1728 mosquitoes fed on SP-treated gametocyte carriers developed oocysts, while 11% of 198 mosquitoes fed on chloroquine-treated gametocyte carriers were positive for oocysts. This study shows that in an area of high SP efficacy, although SP treatment sharply increased gametocyte carriage, the infectiousness of these gametocytes to the vector may be very low. Accurate and robust methods for measuring infectivity are needed to guide malaria control interventions that affect transmission.
Antimalarial drugs resistance
2010, Revue Francophone des Laboratoires
Au cours de leur évolution, les micro-organismes ont su déjouer les pièges qui leur sont tendus par l’environnement et notamment leur hôte (immunité et utilisation de molécules anti-infectieuses). L’émergence et la diffusion de la résistance aux antipaludiques posent un sérieux problème de santé publique. Plasmodium falciparum est maintenant résistant à tous les antipaludiques utilisés même aux derniers commercialisés comme les associations à base d’artémisinine. Les échecs prophylactiques ou thérapeutiques entraînent une ré-émergence du paludisme s’accompagnant d’une augmentation de la transmission, de la morbidité et de la mortalité. La connaissance des mécanismes de résistance permet le développement de nouvelles molécules qui diminueront la résistance, d’identifier les cibles de nouveaux antipaludiques et enfin d’identifier des marqueurs moléculaires pour la surveillance de la résistance aux antipaludiques. La résistance est souvent associée 1) à une altération d’enzymes clé qui sont des cibles d’antipaludiques, 2) à l’altération de l’accumulation de l’antipaludique dans le parasite résultant d’une diminution d’entrée ou d’une augmentation de sortie (efflux) de la molécule, voire aux deux. Des données épidémiologiques, les modes d’action, les mécanismes de résistance et les marqueurs moléculaires de résistance sont présentés pour chaque antipaludique actuellement utilisé.
Throughout the course of evolution, micro-organisms have thwarted traps set by the environment including those designed by man (immunity and use of anti-infectious drugs). The emergence and spread of antimalarial drug resistance poses a severe and increasing public health threat. The Plasmodium falciparum parasite is now resistant to all of the used antimalarial drugs, even to the latest artemisinin-based combination treatments. Failures in prophylaxis or treatments induce the re-emergence of parasite-related morbidity and mortality. Knowledge about resistance mechanisms involved may allow the development of new drugs that minimize or circumvent drug resistance, may allow the identification of new targets for drug development and to identify molecular markers for malaria resistance surveillance. Resistance is often associated with 1) inhibition of alteration of key enzymes that are targets for antimalarial drugs or 2) alteration of drug accumulation into the parasite which results from reduced uptake of the drug, an increased efflux, or a combination of the two processes. Epidemiological data, mode of action, mechanisms of resistance and resistance molecular markers are presented for each antimalarial drugs used at the present time.

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Molecular and Biochemical Parasitology

Abstract

References (27)

A bifunctional thymidylate synthetase-dihydrofolate reductase in protozoa

Mol. Biochem. Parasitol.

The establishment of genomic DNA libraries for the human malaria parasite Plasmodium falciparum and identification of individual clones by hybridisation

Mol. Biochem. Parasitol.

A convenient procedure for purification of thymidylate synthase from 11210 cells

Anal. Biochem.

Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors

Gene

A rapid boiling method for the preparation of bacterial plasmids

Anal. Biochem.

Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis

J. Biol. Chem.

Plasmodium falciparum: induction, selection, and characterization of pyrimethamine-resistant mutants

Exp. Parasitol.

Pyrimethamine resistant Plasmodium falciparum: overproduction of dihydrofolate reductase by a gene duplication

Mol. Biochem. Parasitol.

Altered dihydrofolate reductase in pyrimethamine-resistant Plasmodium falciparum

Mol. Biochem. Parasitol.

Folates in pyrimidine nucleotide biosynthesis

Dihydrofolate reductase

Biochemistry of Plasmodium (Malarial Parasites)

Microbiol. Rev.

Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction

Cited by (109)

Antimalarial effect of cell penetrating peptides derived from the junctional region of Plasmodium falciparum dihydrofolate reductase-thymidylate synthase

A better resolution for integrating methods for monitoring Plasmodium falciparum resistance to antimalarial drugs

Sulfadoxine-pyrimethamine resistance in Plasmodium falciparum: A zoomed image at the molecular level within a geographic context

Red Blood Cell Polymorphism and Susceptibility to Plasmodium vivax

Low infectivity of Plasmodium falciparum gametocytes to Anopheles gambiae following treatment with sulfadoxine-pyrimethamine in Mali

Antimalarial drugs resistance