Sequence Variation of the Hydroxymethyldihydropterin Pyrophosphokinase: Dihydropteroate Synthase Gene in Lines of the Human Malaria Parasite, Plasmodium falciparum, with Differing Resistance to Sulfadoxine

Abstract

Dihydropteroate synthase (H2Pte synthase) is the target of the sulfur‐based antimalarial drugs, which are frequently used in synergistic combination with inhibitors of dihydrofolate reductase (H2folate reductase) to combat chloroquine‐resistant malaria. We have isolated the H2Pte synthase coding sequence of the most pathogenic human parasite Plasmodium falciparum. It forms part of a longer coding sequence, located on chromosome 8, that also specifies 6‐hydroxymethyl‐7,8‐dihydropterin pyrophosphokinase (CH2OH‐H2pterinPP kinase) at its 5′ proximal end. This domain is unusually large, with two long insertions relative to other CH2OH‐H2pterinPP kinase molecules. To investigate a possible genetic basis for clinical resistance to sulfa drugs, we sequenced the complete H2Pte synthase domains from eleven isolates of P. falciparum with diverse geographical origins and levels of sulfadoxine resistance. Overall, point mutations in five positions were observed, affecting four codons. Parasite lines exhibiting high‐level resistance were found to carry either a double mutation, altering both Ser436 and Ala613, or a single mutation affecting Ala581. The mutations at positions 436 and 581 have the same location relative to each of two degenerate repeated amino acid motifs that are conserved across all other known H2Pte synthase molecules. The amino acid alteration at residue 613 is identically positioned relative to a different conserved motif. The fourth amino acid residue (437) affected by mutation, though adjacent to the apparently crucial residue 436, shows no obvious correlation with resistance. Although these mutations have no exact counterparts in any other organism, that at position 581 falls within a region of three amino acids where H2Pte synthase is modified in various ways in a number of sulfonamide‐resistant pathogenic bacteria. Copy‐number analysis indicated that there was no amplification of the H2Pte synthase domain in resistant parasite lines of P. falciparum, compared to sensitive lines.

Epidemiology and clinical relevanceofPneumocystis jiroveciiFrenkel, 1976 dihydropteroate synthase gene mutations

“…A potential association of point mutations at P. jirovecii DHPS codons 55 and 57 with sulpha or sulphone resistance has already been reported by several authors. These sequence variations are located at one of the active sites of the enzyme and are similar to point mutations that lead to sulpha drugs resistance in other organisms (Lopez et al, 1987;Brooks et al, 1994;Kazanjian et al, 1998;Triglia et al, 1998;Vedantam et al, 1998;Helweg-Larsen et al, 1999;Armstrong et al, 2000;Huang et al, 2004). For this reason, mutations at codons 55 and 57 are the most studied sequence variations in the P. jirovecii DHPS locus.…”

“…Mutations at or very near these positions may be implicated in the re-arrangement of protein structure affecting substrate and sulpha biding to DHPS enzyme. Identical mutations have been shown to confer resistance to sulpha drugs in other microorganisms such as P. falcipasrum, E. coli, and S. pneumoniae (Lopez et al, 1987;Brooks et al, 1994;Triglia et al, 1998;Vedantam et al, 1998;Ma et al, 1999;Armstrong et al, 2000). The potential effect of sequence variations, other than the mutations at codons 55 and 57, should be investigated in further studies.…”

“…However, in the rat model, the effect of TMP-SMZ is almost entirely attributed to sulfamethoxazole (Walzer et al, 1992). The molecular mechanisms of antifolate resistance, particularly in the DHPS locus, have been studied extensively in numerous bacteria and some protozoa, like Plasmodium falciparum, whereas DHPS point mutations are associated with sulpha resistance (Brooks et al, 1994;Triglia et al, 1998). Recently, mutant sequences of P. jirovecii DHPS gene have been detected.…”

Adecuación de una prueba radiométrica para la detección de resistencia múltiple de Plasmodium falciparum a medicamentos

“…Finalmente cabe anotar; primero, que la resistencia"in vitro" a pirimeiamina y sulfadoxina es producida por mutaciones puntuales que generan alteraciones de aminoácidos en las enzimas Dihidrofolato reductasa (DHFR) y Dihidropteroato sintetasa (DHPS) respectivamente (29)(30)(31)(32)(33); y segundo, que en l a actualidad los ensayos de restricción enzimática…”

Resistência à sulfadoxina-pirimetamina em Maputo, Moçambique: presença de mutações nos genes dhfr e dhps do Plasmodium falciparum

“…Maputo é a capital de Moçambique, um País onde a malária é endêmica todo ano, atingindo o seu ponto mais alto após a época chuvosa (dezembro a abril). Com uma intensidade de transmissão variável, que depende da precipitação, altitude e temperaturas, o Plasmodium falciparum é o parasita mais prevalente, sendo responsável por cerca de 9�% de todas infecções 3 . Os primeiros registros de resistência à cloroquina datam de 1983 4 e, nos últimos anos, tem sido realizadas várias investigações relacionadas com resistência e este e outros antimaláricos na região 5 6 7 .…”