Analysis of Plasmodium falciparum chloroquine resistance transporters in Saccharomyces cerevisiae.

摘要

The evolution of drug resistance in malaria parasites continues to hamper global eradication campaigns and wreak havoc in endemic countries. Mutations in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) have been identified as the determinant for chloroquine (CQ) resistance (CQR) in malaria parasites. PfCRT is an integral membrane protein that localizes to the digestive vacuole (DV) during the intraerythrocytic stages of the parasite's life cycle and controls an essential, yet unknown, endogenous function. Mutant proteins modify the biochemical process that the parasite exploits in order to evade toxic by-products released during obligate hemoglobin digestion. The purpose of this study was to further elucidate key features of drug resistance in malaria parasites using yeast as model system.;To further characterize the structure and function of PfCRT, I have employed a galactose-inducible heterologous system in S. cerevisiae wherein expression of CRT proteins in the plasma membrane (PM) of growing yeast leads to CQ hypersensitivity phenotype. I have developed several drug sensitivity assays to measure PfCRT-mediated CQ transport function associated with chloroquine sensitive (CQS) and CQR isoforms, including the PfCRT orthologue PvCRT. My results suggest that both mutant CQR- and CQS- associated isoforms are capable of PfCRT-mediated CQ transport and that CQR associated isoforms are stimulated by membrane potential. Improvements to the yeast system have allowed me to reliably distinguish between 15 different naturally occurring CQR isoforms from around the globe. My interpretation of these results, that mutations in PfCRT are not enough to fully recapitulate CQR in the parasite, suggest that additional mutations are required to fully modulate CQR phenomena.;I further exploit the yeast system to elucidate mechanisms underlying the toxicity and tolerance of CQ to yeast. My results suggest that vacuolar membrane potential and mitochondrial function play a key role in mediating cytocidal CQ resistance in yeast. My results from attempts to identify the natural physiologic substrate of PfCRT suggest that PfCRT may play a role in ion homeostasis.;This study demonstrates the use of a model eukaryotic heterologous system to elucidate key features of a resistance protein from malaria parasites and highlights the potential of using this system to characterize its endogenous substrate.