Investigations into the genetic and biochemical basis of chloroquine resistance in malaria parasites
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Chloroquine resistance (CQR) in the human malaria parasite Plasmodium falciparum has been associated with multiple mutations in the transmembrane protein P. falciparum Chloroquine Resistance Transporter (PfCRT). However, the precise role of the individual PfCRT polymorphisms had previously not been genetically defined. All CQ-resistant parasites express a K76T polymorphism in PfCRT, which has been strongly correlated with in vitro CQR as well as in vivo CQ treatment failure. Nevertheless mutations in additional genes have been postulated to also contribute to resistance. Using allelic exchange, we here show that CQR is critically determined by the single PfCRT polymorphism K76T. Replacement of the mutant threonine with wild-type lysine in PfCRT (generating back-mutant parasite lines) resulted in complete loss of CQR in geographically distinct CQ-resistant lines. Verapamil (VP) chemosensitization of CQR, a characteristic feature of all CQ-resistant lines, was also lost in these lines. Biochemical analyses revealed an increased apparent binding affinity of CQ for its target hematin, which resulted in a significantly increased binding of CQ to hematin in the back-mutant lines. The PfCRT polymorphisms preceding residue 76 were found to dictate the degree of VP chemosensitization of CQR, which differs between CQ-resistant lines. We also discovered that the K76T mutation is tailor-made to confer resistance to drugs that structurally closely resemble CQ, as subtle alterations in the CQ side-chain length can overcome this resistance. Thus, our findings establish the PfCRT K76T mutation as the critical component of CQR and suggest that CQ accessibility and binding to hematin are determined by drug-protein interactions involving this mutant residue. These data validate the use of K76T as a molecular marker of CQR in field-based studies assessing the prevalence of CQR in endemic regions. Also, we have generated transgenic P. berghei expressing both mutant and wild-type pfcrt alleles in the place of its ortholog pfcrt. These recombinant rodent malaria parasites are being used to test whether mutant pfcrt can confer in vivo CQ treatment failure.