: The emergence and spread of chloroquine resistant (CQR) strains of Plasmodium falciparum, first reported in Asia and South America, drastically compromises efforts to treat and control malaria. Chloroquine (CQ) interferes with the process of heme polymerization in the parasite digestive vacuole (DV) and chloroquine resistance (CQR) is associated with reduced accumulation of this drug. Our long-term objective is to understand CQR at the genetic and physiological level. Recent work has led to the identification of the pfcrt (P. falciparum chloroquine resistance transporter) gene, which encodes the DV transmembrane protein PfCRT. Mutations in pfcrt segregate with the verapamil (VP)-reversible CQR phenotype in a genetic cross and show a strong association with CQR in laboratory-adapted field isolates. These data and CQ selection experiments implicate a central role for pfcrt mutations in CQR. This now raises the critical question of whether CQR results from mutations in this gene alone or whether multiple genes are necessary for this phenotype. Significant insight into the CQR mechanism can also now be gained by defining the physiological function(s) of PfCRT. The first specific aim of this application is to assess the contribution of individual pfcrt mutations to CQR. Chloroquine-sensitive (CQS) and chloroquine-resistant (CQR) P. falciparum lines will be transformed with episomal transgene constructs that express pfcrt sequences from CQR and CQS parasites and transformed lines will be assayed for changes in parasite response to CQ+/-VP. These assays provide a rapid screen to identify key pfcrt mutations involved in CQR. The second specific aim will determine by allelic exchange whether pfcrt mutations are necessary and sufficient to confer P. falciparum CQR in both New and Old World strains. The third specific aim will determine the physiological function of PfCRT and how this relates mechanistically to CQR. Single-cell pH measurements on pfcrf-modified lines will be performed to test the model that pfcrt mutations can cause CQR by acidification of the DV pH. pfcrt sequences will be expressed in Xenopus laevis oocytes to assess for transport of CQ and candidate physiological substrates. These studies will yield data that should stimulate new strategies to counter the spread of CQR malaria and contribute significantly to understanding the molecular and physiological basis of CQR.
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