RESUMO
Identifying the source of resurgent parasites is paramount to a strategic, successful intervention for malaria elimination. Although the malaria incidence in Panama is low, a recent outbreak resulted in a 6-fold increase in reported cases. We hypothesized that parasites sampled from this epidemic might be related and exhibit a clonal population structure. We tested the genetic relatedness of parasites, using informative single-nucleotide polymorphisms and drug resistance loci. We found that parasites were clustered into 3 clonal subpopulations and were related to parasites from Colombia. Two clusters of Panamanian parasites shared identical drug resistance haplotypes, and all clusters shared a chloroquine-resistance genotype matching the pfcrt haplotype of Colombian origin. Our findings suggest these resurgent parasite populations are highly clonal and that the high clonality likely resulted from epidemic expansion of imported or vestigial cases. Malaria outbreak investigations that use genetic tools can illuminate potential sources of epidemic malaria and guide strategies to prevent further resurgence in areas where malaria has been eliminated.
Assuntos
Antimaláricos/farmacologia , Cloroquina/farmacologia , Surtos de Doenças , Resistência a Medicamentos/genética , Malária Falciparum/epidemiologia , Plasmodium falciparum/isolamento & purificação , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Análise por Conglomerados , Colômbia , Código de Barras de DNA Taxonômico , Feminino , Loci Gênicos/genética , Haplótipos , Humanos , Malária Falciparum/parasitologia , Masculino , Pessoa de Meia-Idade , Panamá/epidemiologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Polimorfismo de Nucleotídeo Único , Proteínas de Protozoários/genética , Adulto JovemRESUMO
The function of Plasmodium falciparum chloroquine resistance transporter (PfCRT) can be quantified using a Saccharomyces cerevisiae model system [Baro, N. K., Pooput, C., and Roepe, P. D. (2011) Biochemistry 50, 6701-6710]. We further optimized this system to distinguish PfCRT isoforms found in P. falciparum strains and isolates from across the globe. We created and expressed 13 naturally occurring pfcrt alleles associated with a range of chloroquine resistant (CQR) phenotypes. Using galactose induction of PfCRT, we quantified PfCRT and chloroquine (CQ)-dependent yeast growth inhibition and [3H]CQ transport specifically due to a given PfCRT isoform. Surprisingly, we found poor correlation between these parameters and the CQ IC50 observed in strains of malaria harboring the same isoforms. This suggested that an increased level of CQ transport due to PfCRT mutation is necessary, but not sufficient, for the range of CQ IC50 values observed in globally distributed CQR P. falciparum isolates.
Assuntos
Cloroquina/farmacologia , Proteínas de Membrana Transportadoras/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Alelos , Sequência de Bases , Química Farmacêutica , Cloroquina/química , Desenho de Fármacos , Galactose/química , Haplótipos , Concentração Inibidora 50 , Proteínas de Membrana Transportadoras/genética , Dados de Sequência Molecular , Mutação , Fenótipo , Plasmídeos/metabolismo , Plasmodium falciparum/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas de Protozoários/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Previous work from our laboratory optimized MeOH-inducible expression of the P. falciparum malarial parasite transporter PfCRT in P. pastoris yeast. These strains are useful for many experiments but do not allow for inducible protein expression under ambient growth conditions. We have therefore optimized galactose-inducible expression of PfCRT in S. cerevisiae yeast. We find that expression of PfCRT confers CQ hypersensitivity to growing yeast and that this is due to plasma membrane localization of the transporter. We use quantitative analyses of growth rates to compare hypersensitivity for yeast expressing various PfCRT isoforms. We also report successful high level inducible expression of the P. vivax orthologue, PvCRT, and compare CQ hypersensitivity for PvCRT vs PfCRT expressing yeast. We test the hypothesis that hypersensitivity is due to increased transport of CQ into yeast expressing the transporters via direct (3)H-CQ transport experiments and analyze the effect that membrane potential has on transport. The data suggest important new tools for rapid functional screening of PfCRT and PvCRT isoforms and provide further evidence for a model wherein membrane potential promotes charged CQ transport by PfCRT. Data also support our previous conclusion that wild type PfCRT is capable of CQ transport and provide a basis for understanding the lack of correspondence between PvCRT mutations and resistance to CQ in the important malarial parasite P. vivax.