Plasmodium falciparum: selection of serine 108 of dihydrofolate reductase during treatment of uncomplicated malaria with co-trimoxazole in Ugandan children.
(1/266)
In vivo testing for resistance of Plasmodium falciparum to co-trimoxazole (trimethoprim/sulfamethoxazole) was performed in Uganda in 41 children with uncomplicated malaria, and blood samples were screened before and after treatment for polymorphisms in the antifolate target genes for dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS). Selection towards a specific genotype at some codons of the DHFR and DHPS genes was observed in samples collected after exposure to co-trimoxazole drug pressure. The alleles 51-isoleucine, 59-arginine, and 108-serine of DHFR were significantly associated with clinical resistance, as was allele 581-alanine of DHPS. Resistance against antifolate combinations probably requires resistance-related polymorphisms in both the DHFR and the DHPS genes. In addition, it appears that the trimethoprim-resistant DHFR genotype differs from that for pyrimethamine at residue 108. (+info)
2.0 A X-ray structure of the ternary complex of 7,8-dihydro-6-hydroxymethylpterinpyrophosphokinase from Escherichia coli with ATP and a substrate analogue.
(2/266)
The X-ray crystal structure of 7,8-dihydro-6-hydroxymethylpterinpyrophosphokinase (PPPK) in a ternary complex with ATP and a pterin analogue has been solved to 2.0 A resolution, giving, for the first time, detailed information of the PPPK/ATP intermolecular interactions and the accompanying conformational change. The first 100 residues of the 158 residue peptide contain a betaalpha betabeta alphabeta motif present in several other proteins including nucleoside diphosphate kinase. Comparative sequence examination of a wide range of prokaryotic and lower eukaryotic species confirms the conservation of the PPPK active site, indicating the value of this de novo folate biosynthesis pathway enzyme as a potential target for the development of novel broad-spectrum anti-infective agents. (+info)
Pyrimethamine-sulfadoxine efficacy and selection for mutations in Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase in Mali.
(3/266)
To assess pyrimethamine-sulfadoxine (PS) efficacy in Mali, and the role of mutations in Plasmodium falciparum dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) in in vivo PS resistance, 190 patients with uncomplicated P. falciparum malaria were treated with PS and monitored for 56 days. Mutation-specific polymerase chain reactions and digestion with restriction endonucleases were used to detect DHFR and DHPS mutations on filter paper blood samples from pretreatment and post-treatment infections. Only one case each of RI and RII level resistance and no cases of RIII resistance or therapeutic failure were observed. Post-PS treatment infections had significantly higher rates of DHFR mutations at codons 108 and 59. No significant selection for DHPS mutations was seen. Pyrimethamine-sulfadoxine is highly efficacious in Mali, and while the low level of resistance precludes assessing the utility of molecular assays for in vivo PS resistance, rapid selection of DHFR mutations supports their role in PS failure. (+info)
Sulfonamide resistance in clinical isolates of Campylobacter jejuni: mutational changes in the chromosomal dihydropteroate synthase.
(4/266)
The characterization of the genetic basis of sulfonamide resistance in Campylobacter jejuni was attempted. The resistance determinant from a sulfonamide-resistant strain of C. jejuni was cloned and was found to show 42% identity with the folP gene (which codes for dihydropteroate synthase, the target of sulfonamides) of the related bacterium Helicobacter pylori. The sequences of the areas surrounding the folP gene in C. jejuni showed similarity to those of the areas surrounding the corresponding gene in H. pylori. The folP gene of C. jejuni, which mediates the resistance, was observed to show particular features when it was compared to other known folP genes. One of these features is the presence of two pairs of direct repeats (15 and 27 bp) within the coding sequence of the gene. Comparison of the C. jejuni folP genes that mediate susceptibility and resistance revealed the occurrence of mutations that changed four amino acid residues. Resistance of C. jejuni to sulfonamides could be associated with one or several of these four mutational substitutions, which all occurred in the five different resistant isolates studied. The codon for one of these changed amino acids was found to be located in the second direct repeat within the coding sequence of the gene. The change made the repeat perfect. The transformation of both the resistance and the susceptibility variants of the gene into an Escherichia coli folP knockout mutant was found to complement the dihydropteroate synthase deficiency, confirming that the characterized sulfonamide resistance determinant codes for the C. jejuni dihydropteroate synthase enzyme. Kinetic measurements established different affinities of sulfonamide for the dihydropteroate synthase enzyme isolated from the resistant and susceptible strains. In conclusion, sulfonamide resistance in C. jejuni was shown to be associated with mutational changes in the chromosomally located gene for dihydropteroate synthase, the target of sulfonamides. (+info)
Low-dose treatment with sulfadoxine-pyrimethamine combinations selects for drug-resistant Plasmodium falciparum strains.
(5/266)
A total of 252 children were enrolled in a drug trial to assess the effect of minimal doses of sulfadoxine (Sdx) and pyrimethamine (Pyr). Parasite samples isolated from these patients were analyzed before and after treatment to investigate the level of drug-resistant strains. The parasite genes encoding dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) were assayed for point mutations that are associated with resistance against drugs. Before treatment, Pyr(r) genotypes of the DHFR gene were found in 42% of all samples, 8% of the patients harbored a mixed parasite population and 50% had a sensitive DHFR genotype. In terms of the DHPS gene, we found mutations in 45% of the parasites. Twenty-four percent had a Ser(436) mutation, and 26% had a Gly(437) mutation. Recrudescent parasites were highly enriched for both Pyr(r) and Sdx(r) strains after treatment (P < 0.001 and P = 0.029, respectively). (+info)
Novel expansions of the gene encoding dihydropteroate synthase in trimethoprim-sulfamethoxazole-resistant Streptococcus pneumoniae.
(6/266)
A study of eight sulfonamide-resistant clinical isolates of Streptococcus pneumoniae revealed chromosomal mutations within the gene encoding dihydropteroate synthase that play a role in conferring resistance to sulfamethoxazole. The presence of the suld mutation, found previously only in a laboratory mutant, was shown to occur in three of the wild-type clinical isolates. The duplication of Ser(61), the other previously defined mutation in the dihydropteroate synthase gene of S. pneumoniae, was observed in only one of the isolates characterized. We report two previously unidentified amino acid alterations, namely, a duplication of Arg(58) and Pro(59) and an insertion of an arginine residue between Gly(60) and Ser(61) in trimethoprim-sulfamethoxazole-resistant strains. The significance of these mutations was confirmed by site-directed mutagenesis and by the transformation of a susceptible strain of S. pneumoniae to sulfamethoxazole resistance. Two resistant isolates did not contain any mutations within the gene encoding dihydropteroate synthase. The results presented suggest the independent generation of resistant mutations among South African clinical isolates. It is also proposed that the mechanism of sulfonamide resistance in S. pneumoniae involves the expansion of a specific region within dihydropteroate synthase, which probably forms part of the sulfonamide binding site. (+info)
Point mutations in dihydrofolate reductase and dihydropteroate synthase genes of Plasmodium falciparum isolates from Venezuela.
(7/266)
The present study was designed to characterize mutations in dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) genes of Plasmodium falciparum in the Bolivar region of Venezuela, where high levels of clinical resistance to sulfadoxine-pyrimethamine (SP, Fansidar; F. Hoffman-La Roche, Basel, Switzerland) has been documented. We used a nested mutation-specific polymerase chain reaction and restriction digestion methods to measure 1) the prevalence of DHFR mutations at 16, 50, 51, 59, 108, and 164 codon positions, and 2) the prevalence of mutations in the 436, 437, 581, and 613 codon sites in DHPS gene. In the case of the DHFR gene, of the 54 parasite isolates analyzed, we detected the presence of Asn-108 and Ile-51 in 96% of the isolates and Arg-50 mutation in 64% of the isolates. Each of these mutations has been associated with high level of resistance to pyrimethamine. Only 2 samples (4%) showed the wild type Ser-108 mutation and none showed Thr-108 and Val-16 mutations that are specific for resistance to cycloguanil. In the case of DHPS gene, we found a mutation at position 437 (Gly) in 100% of the isolates and Gly-581 in 96% of the isolates. The simultaneous presence of mutations Asn-108 and Ile-51 in the DHFR gene and Gly-437 and Gly-581 in the DHPS gene in 96% of the samples tested suggested that a cumulative effect of mutations could be the major mechanism conferring high SP resistance in this area. (+info)
Plasmodium falciparum resistance to sulfadoxine/pyrimethamine in Uganda: correlation with polymorphisms in the dihydrofolate reductase and dihydropteroate synthetase genes.
(8/266)
The efficacy of sulfadoxine/pyrimethamine (S/P) in treatment of uncomplicated falciparum malaria in Africa is increasingly compromised by development of resistance. The occurrence of active site mutations in the Plasmodium falciparum gene sequences coding for dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) is known to confer resistance to pyrimethamine and sulfadoxine. This study investigated the occurrence of these mutations in infected blood samples taken from Ugandan children before treatment with S/P and their relationship to parasite breakthrough by day 7. The results confirm the occurrence of mutations in DHFR and DHPS that were significantly selected under S/P pressure at day 7: a combination of alleles 51-isoleucine and 108-asparagine in DHFR, and 436-serine, 437-alanine, 540-lysine and 581-alanine in DHPS, appears to play a major role in the development of in vivo resistance in P. falciparum strains against S/P. Therefore, earlier results derived from isolates from hyperendemic areas in Tanzania were confirmed by this investigation. (+info)