Paul F. G. Sims

Pyrimethamine–sulfadoxine resistance in Plasmodium falciparum: what next?

Chemotherapy remains the only practicable tool to control falciparum malaria in sub-Saharan Africa, where >90% of the worlds burden of malaria mortality and morbidity occurs. Resistance is rapidly eroding the efficacy of chloroquine, and the combination pyrimethamine-sulfadoxine is the most commonly chosen alternative. Resistant populations of Plasmodium falciparum were selected extremely rapidly in Southeast Asia and South America. If this happens in sub-Saharan Africa, it will be a public health disaster because no inexpensive alternative is currently available. This article reviews the molecular mechanisms of this resistance and discusses how to extend the therapeutic life of antifolate drugs.

Sulfadoxine resistance in the human malaria parasite Plasmodium falciparum is determined by mutations in dihydropteroate synthetase and an additional factor associated with folate utilization.

Sulfadoxine/pyrimethamine (Fansidar) is widely used in Africa for treating chloroquine‐resistant falciparum malaria. To clarify how parasite resistance to this combination arises, various lines of Plasmodium falciparum were used to investigate the role of naturally occurring mutations in the target enzyme, dihydropteroate synthetase (DHPS), in the parasite response to sulfadoxine inhibition. An improved drug assay was employed to identify a clear correlation between sulfadoxine‐resistance levels and the number of DHPS mutations. Moreover, tight linkage was observed between DHPS mutations and high‐level resistance in the 16 progeny of a genetic cross between sulfadoxine‐sensitive (HB3) and sulfadoxine‐resistant (Dd2) parents. However, we also demonstrate a profound influence of exogenous folate on IC50 values, which, under physiological conditions, may have a major role in determining resistance levels. Importantly, this phenotype does not segregate with dhps genotypes in the cross, but shows complete linkage to the two alleles of the dihydrofolate reductase (dhfr) gene inherited from the parental lines. However, in unrelated lines, this folate effect correlates less well with DHFR sequence, indicating that the gene responsible may be closely linked to dhfr, rather than dhfr itself. These results have major implications for the acquisition of Fansidar resistance by malaria parasites.

Allelic exchange at the endogenous genomic locus in Plasmodium falciparum proves the role of dihydropteroate synthase in sulfadoxine‐resistant malaria

We have exploited the recently developed ability to trans‐ fect the malaria parasite Plasmodium falciparum to investigate the role of polymorphisms in the enzyme dihydropteroate synthase (DHPS), identified in sulfadoxine‐resistant field isolates. By using a truncated form of the dhps gene, specific mutations were introduced into the endogenous gene by allelic replacement such that they were under the control of the endogenous promoter. Using this approach a series of mutant dhps alleles that mirror P.falciparum variants found in field isolates were found to confer different levels of sulfadoxine resistance. This analysis shows that alteration of Ala437 to Gly (A437G) confers on the parasite a 5‐fold increase in sulfadoxine resistance and addition of further mutations increases the level of resistance to 24‐fold above that seen for the transfectant expressing the wild‐type dhps allele. This indicates that resistance to high levels of sulfadoxine in P.falciparum has arisen by an accumulation of mutations and that Gly437 is a key residue, consistent with its occurrence in most dhps alleles from resistant isolates. These studies provide proof that the mechanism of resistance to sulfadoxine in P.falciparum involves mutations in the dhps gene and determines the relative contribution of these mutations to this phenotype.

Resistance to antifolates in Plasmodium falciparum monitored by sequence analysis of dihydropteroate synthetase and dihydrofolate reductase alleles in a large number of field samples of diverse origins

Resistance of Plasmodium falciparum to antifolate chemotherapy is a significant problem where combinations such as Fansidar (pyrimethamine-sulfadoxine; PYR-SDX) are used in the treatment of chloroquine-resistant malaria. Antifolate resistance has been associated with variant sequences of dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS), the targets of PYR and SDX respectively. However, while the nature and distribution of mutations in the dhfr gene are well established, this is not yet the case for dhps. We have thus examined by DNA sequence analysis 141 field samples from several geographical regions with differing Fansidar usage (West and East Africa, the Middle East and Viet Nam) to establish a database of the frequency and repertoire of dhps mutations, which were found in 60% of the samples. We have also simultaneously determined from all samples their dhfr sequences, to better understand the relationship of both types of mutation to Fansidar resistance. Whilst the distribution of mutations was quite different across the regions surveyed, it broadly mirrored our understanding of relative Fansidar usage. In samples taken from individual patients before and after drug treatment, we found an association between the more highly mutated forms of dhps and/or dhfr and parasites that were not cleared by antifolate therapy. We also report a novel mutation in a Pakistani sample at position 16 of DHFR (A16S) that is combined with the familiar C59R mutation, but is wild-type at position 108. This is the first observation in a field sample of a mutant dhfr allele where the 108 codon is unchanged.

Prevalence of Toxoplasma gondii in commercial meat products as monitored by polymerase chain reaction – food for thought?

DNA was extracted from 71 meat samples obtained from UK retail outlets. All of these DNA preparations gave the expected polymerase chain reaction products when amplified with primers specific for the species from which the meat originated. A second polymerase chain reaction analysis, using primers specific for the Toxoplasma gondii SAG2 locus, revealed the presence of this parasite in 27 of the meat samples. Restriction analysis and DNA sequencing showed that 21 of the contaminated meats contained parasites genotyped as type I at the SAG2 locus, whilst six of the samples contained parasites of both types I and II. Toxoplasma- positive samples were subjected to further polymerase chain reaction analysis to determine whether any carried an allele of the dihydropteroate synthase gene that has recently been shown to be causally associated with sulfonamide resistance in T. gondii. In all cases, this analysis confirmed that parasites were present in the samples and, additionally, revealed that none of them carried the drug-resistant form of dihydropteroate synthase. These results suggest that a significant proportion of meats commercially available in the UK are contaminated with T. gondii. Although none of the parasites detected in this study carried the sulfonamide-resistance mutation, a simplified procedure for monitoring this situation merits development.

Quantitative proteomics of the human malaria parasite Plasmodium falciparum and its application to studies of development and inhibition

The ability to measure accurately comparative levels of protein expression after drug challenge, metabolic stress, developmental programming or other perturbation represents one of the most important goals in post‐genomics malaria research. We describe here a simple and robust quantitative methodology that is ideally suited to in vitro experiments designed to study changes in the proteome of the most important of the human parasites, the lethal species Plasmodium falciparum. The metabolic labelling technique we have developed uses parasite uptake of heavy isotope‐containing isoleucine during normal growth followed by two‐dimensional separation of individual proteins and mass spectrometry. The method is applicable to essentially each of the ≈ 5300 proteins of P. falciparum predicted from the completed genome sequence, permitting facile identification and accurate comparative quantification of labelled peptides from any of these proteins synthesized by in vitro cultures subjected to different stimuli. We demonstrate its application to the study of cell cycle changes, where we observe divergent patterns of protein and reported transcript levels indicative of modulation at the translational level. Our data also provide evidence for significant levels of post‐translational modification in the parasite, and we measure differences among variants of phosphoethanolamine N‐methyltransferase and actin‐I across the cell cycle. We have also monitored parasite responses to equipotent doses of the clinical antimalarial inhibitors pyrimethamine and tetracycline and observed differential effects for a number of proteins unrelated to likely targets of these drugs.

The identification, molecular cloning and characterisation of a gene from Phanerochaete chrysosporium that shows strong homology to the exo-cellobiohydrolase I gene from Trichoderma reesei

We have identified a genomic DNA fragment from the lignin-degrading fungus Phanerochaete chrysosporium (P.c.) that hybridizes to a DNA probe encoding part of the exo-cellobiohydrolase I (CBHI) gene of Trichoderma reesei (T.r.). This fragment has been subcloned and its nucleotide sequence determined. We demonstrate that it could encode a 516 residue protein that shows strong homology with the known protein sequence of CBHI from T.r. Comparison of the two nucleotide sequences identifies two regions within the P.c. sequence that are not represented in T.r. Further inspection of these regions reveals sequences closely related to the conserved elements of filamentous fungal introns. We conclude that the P.c. genomic sequence contains the same number of introns (2) as found in T.r. but that these are located at different relative positions within the two genes. The transcript from the P.c. sequence is induced in the presence of cellulose but not by glucose and we therefore conclude that this sequence represents the first cellulase gene to have been described from this organism.

Characterisation of the dihydrofolate reductase-thymidylate synthetase gene from human malaria parasites highly resistant to pyrimethamine.

To investigate the genetic basis of drug resistance in human malaria parasites, we have sequenced the entire dihydrofolate reductase thymidylate synthetase DHFR-TS bifunctional gene from the highly pyrimethamine-resistant K1 isolate of Plasmodium falciparum. The protein is predicted to consist of 607 amino acids (aa), (71,685 Da), with an N-terminal methionine encoded by the second start codon of the open reading frame. Compared to the sequence from drug-sensitive parasites, there are two nucleotide changes in the coding region which bring about a substitution of Arg for Cys at aa position 59 and Asn for Thr at aa position 108. Both changes occur in regions of the DHFR domain involved in inhibitor and cofactor binding and are hence strongly implicated in drug resistance. The gene is present as a single copy in both K1 and drug-sensitive FCR3 isolates, and is assigned to chromosome 4. Codon usage follows the pattern observed in that of malarial surface antigen genes, with the exception fo codons corresponding to Val and Pro. The Asn and Lys contents of the predicted protein are exceptionally high, these residues being particularly concentrated in the DHFR and junction domains.

Global absolute quantification of a proteome: Challenges in the deployment of a QconCAT strategy

In this paper, we discuss the challenge of large‐scale quantification of a proteome, referring to our programme that aims to define the absolute quantity, in copies per cell, of at least 4000 proteins in the yeast Saccharomyces cerevisiae. We have based our strategy on the well‐established method of stable isotope dilution, generating isotopically labelled peptides using QconCAT technology, in which artificial genes, encoding concatenations of tryptic fragments as surrogate quantification standards, are designed, synthesised de novo and expressed in bacteria using stable isotopically enriched media. A known quantity of QconCAT is then co‐digested with analyte proteins and the heavy:light isotopologues are analysed by mass spectrometry to yield absolute quantification. This workflow brings issues of optimal selection of quantotypic peptides, their assembly into QconCATs, expression, purification and deployment.

Utilization of exogenous folate in the human malaria parasite Plasmodium falciparum and its critical role in antifolate drug synergy

The antifolate combination pyrimethamine/sulphadoxine (PYR/SDX; Fansidar) is frequently used to combat chloroquine‐resistant malaria. Its success depends upon pronounced synergy between the two components, which target dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) in the folate pathway. This synergy permits clearance of parasites resistant to either drug alone, but its molecular basis is still unexplained. Plasmodium falciparum can use exogenous folate, which is normally present in vivo, bypassing SDX inhibition of DHPS and, apparently, precluding synergy under these conditions. However, we have measured parasite inhibition by SDX/PYR combinations in assays in which folate levels are strictly controlled. In parasites that use exogenous folate efficiently, SDX inhibition can be restored by levels of PYR significantly lower than those required to inhibit DHFR. Isobolograms show that the degree of synergy between PYR and SDX is highly dependent upon prevailing folate concentrations and are indicative of PYR acting to block folate uptake and/or utilization. No significant synergy was observed at physiological drug levels when PYR/SDX acted on purified DHFR, whether wild type or mutant. We conclude that the primary basis for antifolate synergy in these organisms arises from PYR targeting a site (or sites) in addition to DHFR, which restores DHPS as a relevant target for SDX.