Mutagenesis of dihydrofolate reductase from Plasmodium falciparum: analysis in Saccharomyces cerevisiae of triple mutant alleles resistant to pyrimethamine or WR99210

Ferlan, Jill T; Mookherjee, Somnath; Okezie, Ihuoma Nicole; Fulgence, Lucy; Sibley, Carol Hopkins

doi:10.1016/s0166-6851(01)00207-9

Cited by 35 publications

(28 citation statements)

References 66 publications

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“…2 and 3). This correlates well with in vitro data that demonstrates a higher level of resistance to pyrimethamine than to cycloguanil and essentially no effect on WR99210 in the presence of the S108N mutation alone or in combination with N51I (S108N/N51I) (Ferlan et al, 2001).…”

Section: H Relationship Of Point Mutations To Dihydrofolate Reductassupporting

confidence: 89%

Mechanisms of Resistance of Malaria Parasites to Antifolates

2005

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Section: H Relationship Of Point Mutations To Dihydrofolate Reductassupporting

confidence: 89%

Mechanisms of Resistance of Malaria Parasites to Antifolates

2005

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“…We have established an expression system for the P. vivax dhfr gene in yeast cells that lack endogenous DHFR activity. For alleles of P. falciparum, the in vitro sensitivity of these engineered yeast lines to antifolate drugs reflects the sensitivity of the parasite from which the dhfr allele was isolated (17,22,66). To define the range of pyrimethamine sensitivity represented in these P. vivax alleles, we created yeast lines dependent upon each of 7 alleles: the wild type and two double-, two triple-, and two quadruple-mutant alleles.…”

Section: P Vivax Dhfr Alleles Identified In Indonesia and Pngmentioning

confidence: 99%

Novel Plasmodium vivax dhfr Alleles from the Indonesian Archipelago and Papua New Guinea: Association with Pyrimethamine Resistance Determined by a Saccharomyces cerevisiae Expression System

Hastings

Maguire

Bangs

et al. 2005

Antimicrob Agents Chemother

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In plasmodia, the dihydrofolate reductase (DHFR) enzyme is the target of the pyrimethamine component of sulfadoxine-pyrimethamine (S/P). Plasmodium vivax infections are not treated intentionally with antifolates. However, outside Africa, coinfections with Plasmodium falciparum and P. vivax are common, and P. vivax infections are often exposed to S/P. Cloning of the P. vivax dhfr gene has allowed molecular comparisons of dhfr alleles from different regions. Examination of the dhfr locus from a few locations has identified a very diverse set of alleles and showed that mutant alleles of the vivax dhfr gene are prevalent in Southeast Asia where S/P has been used extensively. We have surveyed patient isolates from six locations in Indonesia and two locations in Papua New Guinea. We sequenced P. vivax dhfr alleles from 114 patient samples and identified 24 different alleles that differed from the wild type by synonymous and nonsynonymous point mutations, insertions, or deletions. Most importantly, five alleles that carried four or more nonsynonymous mutations were identified. Only one of these highly mutant alleles had been previously observed, and all carried the 57L and 117T mutations. P. vivax cannot be cultured continuously, so we used a yeast assay system to determine in vitro sensitivity to pyrimethamine for a subset of the alleles. Alleles with four nonsynonymous mutations conferred very high levels of resistance to pyrimethamine. This study expands significantly the total number of novel dhfr alleles now identified from P. vivax and provides a foundation for understanding how antifolate resistance arises and spreads in natural P. vivax populations.Plasmodium vivax causes a severe and debilitating febrile illness. This mosquito-borne parasite infects an estimated 80 million people each year and is a prevalent cause of malaria outside sub-Saharan Africa (39). Unfortunately, P. vivax cannot be maintained in continuous culture, so despite its prevalence, the study of vivax has lagged markedly behind that of Plasmodium falciparum. This difference is particularly true with respect to the genetics and epidemiology of drug resistance in P. vivax. Chloroquine has been the first-line antimalarial treatment in most areas of endemicity, but resistance to this drug has virtually eliminated its usefulness against P. falciparum (61, 63, 65) and chloroquine resistance has now been observed with P. vivax as well (1,21,34,45,52,55,58,59). Fansidar, a fixed combination of sulfadoxine and pyrimethamine (S/P), is most often the alternative chosen when chloroquine-resistant P. falciparum parasites render chloroquine ineffective (4). However, S/P has not been recommended for primary therapy of vivax malaria due to the poor clinical efficacy reported when the drug was introduced in the 1950s (13,14,26,49,64,68).In both P. falciparum and P. vivax, the dihydrofolate reductase (DHFR, E.C. 1.5.1.3) enzyme is the therapeutic target of the pyrimethamine component of S/P (12,18,23,26,27,43,44,60). A combination of in vitro analysis of cultu...

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“…Although pyrimethamine and other antifolates are currently the first-line treatment for malaria in many parts of Africa (12)(13)(14), high-level resistance has been reported elsewhere (4,13,15). The genetic basis of pyrimethamine resistance in Plasmodium falciparum is associated with a small number of amino acid replacements in the parasite DHFR (3,4,(16)(17)(18). Each of these replacements has been observed in clinical isolates of resistant strains (14,(19)(20)(21).…”

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confidence: 99%

Stepwise acquisition of pyrimethamine resistance in the malaria parasite

Lozovsky

Chookajorn

Brown

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

266

328

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The spread of high-level pyrimethamine resistance in Africa threatens to curtail the therapeutic lifetime of antifolate antimalarials. We studied the possible evolutionary pathways in the evolution of pyrimethamine resistance using an approach in which all possible mutational intermediates were created by site-directed mutagenesis and assayed for their level of drug resistance. The coding sequence for dihydrofolate reductase (DHFR) from the malaria parasite Plasmodium falciparum was mutagenized, and tests were carried out in Escherichia coli under conditions in which the endogenous bacterial enzyme was selectively inhibited. We studied 4 key amino acid replacements implicated in pyrimethamine resistance: N51I, C59R, S108N, and I164L. Using empirical estimates of the mutational spectrum in P. falciparum and probabilities of fixation based on the relative levels of resistance, we found that the predicted favored pathways of drug resistance are consistent with those reported in previous kinetic studies, as well as DHFR polymorphisms observed in natural populations. We found that 3 pathways account for nearly 90% of the simulated realizations of the evolution of pyrimethamine resistance. The most frequent pathway (S108N and then C59R, N51I, and I164L) accounts for more than half of the simulated realizations. Our results also suggest an explanation for why I164L is detected in Southeast Asia and South America, but not at significant frequencies in Africa.adaptive landscape ͉ drug resistance ͉ evolution

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Mutagenesis of dihydrofolate reductase from Plasmodium falciparum: analysis in Saccharomyces cerevisiae of triple mutant alleles resistant to pyrimethamine or WR99210

Cited by 35 publications

References 66 publications

Mechanisms of Resistance of Malaria Parasites to Antifolates

Mechanisms of Resistance of Malaria Parasites to Antifolates

Novel Plasmodium vivax dhfr Alleles from the Indonesian Archipelago and Papua New Guinea: Association with Pyrimethamine Resistance Determined by a Saccharomyces cerevisiae Expression System

Stepwise acquisition of pyrimethamine resistance in the malaria parasite

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