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Novel polymorphisms in plasmodium falciparum ABC transporter genes are associated with major ACT antimalarial drug resistance

Publication . Veiga, Maria Isabel; Pousão-Ferreira, Pedro; Jornhagen, Louise; Malmberg, Maja; Kone, Aminatou; Schmidt, Berit Aydin; Petzold, Max; Bjorkman, Anders; Nosten, Francois; Gil, José Pedro

Chemotherapy is a critical component of malaria control. However, the most deadly malaria pathogen, Plasmodium falciparum, has repeatedly mounted resistance against a series of antimalarial drugs used in the last decades. Southeast Asia is an epicenter of emerging antimalarial drug resistance, including recent resistance to the artemisinins, the core component of all recommended antimalarial combination therapies. Alterations in the parasitic membrane proteins Pgh-1, PfCRT and PfMRP1 are believed to be major contributors to resistance through decreasing intracellular drug accumulation. The pfcrt, pfmdr1 and pfmrp1 genes were sequenced from a set of P. falciparum field isolates from the Thai-Myanmar border. In vitro drug susceptibility to artemisinin, dihydroartemisinin, mefloquine and lumefantrine were assessed. Positive correlations were seen between the in vitro susceptibility responses to artemisinin and dihydroartemisinin and the responses to the arylamino-alcohol quinolines lumefantrine and mefloquine. The previously unstudied pfmdr1 F1226Y and pfmrp1 F1390I SNPs were associated significantly with artemisinin, mefloquine and lumefantrine in vitro susceptibility. A variation in pfmdr1 gene copy number was also associated with parasite drug susceptibility of artemisinin, mefloquine and lumefantrine. Our work unveils new candidate markers of P. falciparum multidrug resistance in vitro, while contributing to the understanding of subjacent genetic complexity, essential for future evidence-based drug policy decisions.

2011-05Journal article

Open Access

PfMDR1: mechanisms of transport modulation by functional polymorphisms

Publication . Ferreira, Pedro; Holmgren, Gabrielle; Veiga, Maria Isabel; Uhlen, Per; Kaneko, Akira; Gil, José Pedro

ATP-Binding Cassette (ABC) transporters are efflux pumps frequently associated with multidrug resistance in many biological systems, including malaria. Antimalarial drug-resistance involves an ABC transporter, PfMDR1, a homologue of P-glycoprotein in humans. Twenty years of research have shown that several single nucleotide polymorphisms in pfmdr1 modulate in vivo and/or in vitro drug susceptibility. The underlying physiological mechanism of the effect of these mutations remains unclear. Here we develop structural models for PfMDR1 in different predicted conformations, enabling the study of transporter motion. Such analysis of functional polymorphisms allows determination of their potential role in transport and resistance. The bacterial MsbA ABC pump is a PfMDR1 homologue. MsbA crystals in different conformations were used to create PfMDR1 models with Modeller software. Sequences were aligned with ClustalW and analysed by Ali2D revealing a high level of secondary structure conservation. To validate a potential drug binding pocket we performed antimalarial docking simulations. Using aminoquinoline as probe drugs in PfMDR1 mutated parasites we evaluated the physiology underlying the mechanisms of resistance mediated by PfMDR1 polymorphisms. We focused on the analysis of well known functional polymorphisms in PfMDR1 amino acid residues 86, 184, 1034, 1042 and 1246. Our structural analysis suggested the existence of two different biophysical mechanisms of PfMDR1 drug resistance modulation. Polymorphisms in residues 86/184/1246 act by internal allosteric modulation and residues 1034 and 1042 interact directly in a drug pocket. Parasites containing mutated PfMDR1 variants had a significant altered aminoquinoline susceptibility that appears to be dependent on the aminoquinoline lipophobicity characteristics as well as vacuolar efflux by PfCRT. We previously described the in vivo selection of PfMDR1 polymorphisms under antimalarial drug pressure. Now, together with recent PfMDR1 functional reports, we contribute to the understanding of the specific structural role of these polymorphisms in parasite antimalarial drug response.

2011-09Journal article

Open Access

Polymorphism in PfMRP1 (Plasmodium falciparum multidrug resistance protein 1) Amino Acid 1466 associated with resistance to Sulfadoxine-Pyrimethamine treatment

Publication . Dahlstrom, Sabina; Veiga, M. Isabel; Martensson, Andreas; Bjorkman, Anders; Gil, J. P.

Sulfadoxine-pyrimethamine (SP) remains widely recommended for intermittent preventive treatment against Plasmodium falciparum malaria for pregnant women and infants in Africa. Resistance to SP is increasing and associated primarily with mutations in the P. falciparum dhfr (Pfdhfr) and Pfdhps genes. This study aimed to explore the hypothetical association of genetic alterations in the P. falciparum multidrug resistance protein gene (Pfmrp1) with the in vivo response to SP by detecting the selection of single nucleotide polymorphisms (SNPs) following standard single-dose treatment administered to children with acute uncomplicated P. falciparum malaria in Tanzania. We detected significant selection of parasites carrying the Pfmrp1 1466K allele in samples from children with recrudescent infections, with 12 (100%) of 12 such samples being positive for this allele, compared to 52 (67.5%) of 77 baseline samples (P=0.017), in parallel with the selection of the Pfdhfr Pfdhps quintuple mutant haplotype in cases of recrudescence (P=0.001). There was no association between the 1466K SNP and the Pfdhfr Pfdhps quintuple mutation, indicating independent selections. Our data point for the first time to a role for a P. falciparum multidrug resistance protein homologue in the antimalarial activity of SP. Moreover, they add to the growing evidence of the potential importance of Pfmrp1 in antimalarial drug resistance.

2009-06Journal article

Open Access

Prevalence of resistance associated polymorphisms in Plasmodium falciparum field isolates from southern Pakistan

Publication . Ghanchi, Najia Karim; Ursing, Johan; Beg, Mohammad A.; Veiga, Maria I.; Jafri, Sana; Martensson, Andreas

Background: Scarce data are available on Plasmodium falciparum anti-malarial drug resistance in Pakistan. The aim of this study was, therefore, to determine the prevalence of P. falciparum resistance associated polymorphisms in field isolates from southern Pakistan. Methods: Blood samples from 244 patients with blood-slide confirmed P. falciparum mono-infections were collected between 2005-2007. Single nucleotide polymorphisms in the P. falciparum chloroquine resistance transporter (pfcrt K76T), multi drug resistance (pfmdr1 N86Y), dihydrofolate reductase (pfdhfr A16V, N51I, C59R, S108N, I164L) and dihydropteroate synthetase (pfdhps A436S, G437A and E540K) genes and pfmdr1 gene copy numbers were determined using PCR based methods. Results: The prevalence of pfcrt 76T and pfmdr1 86Y was 93% and 57%, respectively. The prevalence of pfdhfr double mutations 59R + 108N/51R + 108N was 92%. The pfdhfr triple mutation (51I, 59R, 108N) occurred in 3% of samples. The pfdhfr (51I, 59R, 108N) and pfdhps (437G, 540E) quintuple mutation was found in one isolate. Pfdhps 437G was observed in 51% and 540E in 1% of the isolates. One isolate had two pfmdr1 copies and carried the pfmdr1 86Y and pfcrt 76T alleles. Conclusions: The results indicate high prevalence of in vivo resistance to chloroquine, whereas high grade resistance to sulphadoxine-pyrimethamine does not appear to be widespread among P. falciparum in southern Pakistan.

2011-01Journal article

Open Access