Leonardo K. Basco

SEQUENCE VARIATIONS IN THE PLASMODIUM VIVAX DIHYDROFOLATE REDUCTASE-THYMIDYLATE SYNTHASE GENE AND THEIR RELATIONSHIP WITH PYRIMETHAMINE RESISTANCE

The gene encoding dihydrofolate reductase-thymidylate synthase of the human malaria parasite, Plasmodium vivax, was isolated by polymerase chain reaction from genomic DNA and cloned. The sequences of the dihydrofolate reductase domain of 30 clinical isolates originating from various geographic areas were compared. Interstrain analysis revealed several genotypic variations, including short tandem repeat arrays which produced length polymorphism between different parasite isolates and point mutations in the putative dihydrofolate reductase active site cavity corresponding to those associated with pyrimethamine resistance in P. falciparum and rodent malaria parasites. Amino acid substitutions Ser-->Asn-117 and Ser-->Arg-58 were associated with decreased level of in vitro pyrimethamine sensitivity. These findings suggest that the P. vivax dihydrofolate reductase domain is characterized by polymorphism that has not been observed in P. falciparum and may explain the resistance of some P. vivax isolates to pyrimethamine. Nucleotide sequence data reported in this paper are available in the EMBL, GenBank and DDJB databases under the accession numbers X98123 (isolate ARI/Pakistan), AJ003050 (isolate CNC/Thailand), AJ003051 (isolate COU/unknown geographic origin), AJ003052 (isolate DUF/French Guiana), AJ003053 (isolate GRO/Madagascar), AJ003054 (isolate HRT/Comoros Islands), AJ003071 (isolate LFT/Cambodia), AJ003072 (isolate LGF/India), AJ003073 (isolate MAN/Comoros Islands), AJ003074 (isolate MAT/Surinam), AJ003075 (isolate PHI/Djibouti), AJ003076 (isolate PIT/Madagascar), AJ003077 (isolate YTZ/Indonesia), AJ222630 (isolate Burma-1), AJ222631 (isolate Burma-151), AJ222632 (isolate Burma-5), AJ222633 (isolate Burma-6), AJ222634 (isolate Burma-98).

Sequence Variations in the Genes Encoding Dihydropteroate Synthase and Dihydrofolate Reductase and Clinical Response to Sulfadoxine-Pyrimethamine in Patients with Acute Uncomplicated Falciparum Malaria

Mutations in dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR) are associated with in vitro resistance to sulfadoxine and pyrimethamine, respectively. The response of 75 patients to sulfadoxine-pyrimethamine was determined, and the genes of the corresponding Plasmodium falciparum isolates were sequenced. Of 12 different unmixed allelic combinations, the triple dhfr mutation Asn-108/Arg-59/Ile-51 was observed in all patients responding with early treatment failure. Some, but not all, patients with an adequate clinical response also harbored isolates with the triple dhfr mutation. Higher initial parasitemia and fever distinguished these 2 patient groups. The dhps genotype apparently had no influence on the clinical outcome. The other dhfr alleles with 1 or 2 mutations and the wild-type allele were found in patients with an adequate clinical response. The triple dhfr mutation is one of the genetic determinants associated with in vivo resistance to sulfadoxine-pyrimethamine.

Analysis of the Plasmodium vivax dihydrofolate reductase–thymidylate synthase gene sequence

A basis for the intrinsic resistance of some Plasmodium vivax isolates to pyrimethamine is suggested following the isolation of the bifunctional gene encoding dihydrofolate reductase-thymidylate synthase (DHFR-TS) of this human malaria parasite. Malaria parasites are dependent on this enzyme for folate biosynthesis. Specific inhibition of the DHFR domain of the enzyme by pyrimethamine blocks pyrimidine biosynthesis, leading to an inhibition of DNA replication. The gene was isolated by the polymerase chain reaction (PCR) from genomic DNA using degenerate oligonucleotides designed to hybridize on the highly conserved regions of the sequence. The nucleotide sequence was completed by screening P. vivax genomic bank. Sequence analysis revealed an open reading frame (ORF) of 1872 nucleotides encoding a deduced protein of 623 amino acids (aa). Alignment with other malarial DHFR-TS genes showed that a 237-residue DHFR domain and a 286-residue TS domain were separated by a 100-aa linker region. Comparison with other malarial species showed low and essentially no isology in the DHFR and junctional domains, respectively, whereas an extensive isology was observed in the TS domain. The characteristic features of the P. vivax DHFR-TS gene sequence include an insertion of a short repetitive tandem array within the DHFR domain that is absent in another human malaria parasite, P. falciparum, and a GC-biased aa composition, giving rise to highly GC-rich DHFR (50.8%), junctional (58.7%), and TS (40.5%) domains, as compared with other malaria parasites. Analysis of the 5 noncoding region revealed the presence of a putative TATA box at 116 nucleotides upstream of the ATG start codon as well as a putative GC box at -636. Comparison of the DHFR sequences from pyrimethamine-sensitive and pyrimethamine-resistant P. vivax isolates revealed two residue changes: Ser Arg-58 and Ser Asn-117. These aa residues correspond to codons 59 and 108 in the P. falciparum DHFR active site in which similar aa substitutions (Cys Arg-59 and Ser Asn-108) are associated with pyrimethamine resistance. These findings may explain the intrinsic resistance of some P. vivax isolates to pyrimethamine.

Kinetic properties of dihydrofolate reductase from wild-type and mutant Plasmodium vivax expressed in Escherichia coli

Antifolate drugs inhibit malarial dihydrofolate reductase (DHFR). In Plasmodium falciparum, antifolate resistance has been associated with point mutations in the gene encoding DHFR. Recently, mutations at homologous positions have been observed in the P. vivax gene. Since P. vivax cannot be propagated in a continuous in vitro culture for drug sensitivity assays, the kinetic properties of DHFR were studied by expression of the DHFR domain in Escherichia coli. Induced expression yielded a protein product that precipitated as an inclusion body in E. coli. The soluble, active DHFR recovered after denaturation and renaturation was purified to homogeneity by affinity chromatography. Kinetic properties of the recombinant P. vivax DHFR showed that the wild-type DHFR (Ser-58 and Ser-117) and double mutant DHFR (Arg-58 and Asn-117) have similar K(m) values for dihydrofolate and NADPH. Antifolate drugs (pyrimethamine, cycloguanil, trimethoprim, and methotrexate), but not proguanil (parent compound of cycloguanil) inhibit DHFR activity, as expected. The kinetics of enzyme inhibition indicated that point mutations (Ser58Arg and Ser117Asn) are associated with lower affinity between the mutant enzyme and pyrimethamine and cycloguanil, which may be the origin of antifolate resistance.

Chloroquine Resistance in Plasmodium falciparum and Polymorphism of the CG2 Gene

A distinct genotype (designated Dd2-type profile) consisting of 12 point mutations and 3 repetitive regions of the CG2 gene, a candidate gene for chloroquine resistance, has been associated with in vitro resistance in laboratory-adapted strains of Plasmodium falciparum. The DNA sequence of clinical isolates, characterized by in vitro and in vivo tests, was analyzed to evaluate whether the genotype corresponds to the phenotype in naturally occurring parasites. Eight of 11 chloroquine-resistant isolates had the Dd2 genotype. One resistant isolate (by in vitro assay) with a sensitive CG2 genotype was sensitive in vivo. Two resistant isolates and 6 sensitive isolates were multiple infections with mixed alleles. No typical CG2 genotype was found corresponding to the chloroquine-sensitive isolates. These results suggest a strong association between the drug-resistant and CG2 genotypes and support the hypothesis that the CG2 gene may be implicated in chloroquine resistance.

HETEROGENEITY IN THE CIRCUMSPOROZOITE PROTEIN GENE OF PLASMODIUM MALARIAE ISOLATES FROM SUB-SAHARAN AFRICA

Polymorphism of the circumsporozoite protein (CSP) of Plasmodium malariae was characterized by comparing gene sequences of twelve field isolates obtained in Yaoundé, Cameroon, Central Africa, and one clinical isolate originating from Côte dIvoire, West Africa. The length of the CSP gene ranged from 1266 to 1302 bp. The size polymorphism was due to variation in the number of tandem repeat units. All P. malariae isolates displayed a highly conserved 5 non-repeat region. Seven non-synonymous and two synonymous nucleotide variations were observed in the 3 non-repeat region. In the deduced amino acid sequence the repetitive sequences consisted of a varying number of major (Asn Ala Ala Gly (NAAG); range between 42 and 46 units) and minor (Asn Asp Ala Gly (NDAG) or Asn Asp Gln Gly (NDEG); n = six or seven units) tetrapeptide units. None of the isolates had an identical sequence at nucleotide level. These findings suggest that polymorphism in CSP is essentially limited to the tandem repeat domain.