New Scholar Award in Global Infectious Disease
David A. Fidock, Ph.D.
Albert Einstein College of Medicine of Yeshiva University

Genetic Determinants of Chloroquine Resistance in Plasmodium falciparum Malaria.

Malaria is responsible for the deaths of millions of people yearly. Safe, inexpensive treatment and prophylaxis have been impeded by the spread of Plasmodium falciparum strains resistant to the major antimalarial drug chloroquine (CQ). Our investigations into the genetic basis of chloroquine resistance (CQR) have resulted in the identification of the pfcrt (P. falciparum chloroquine resistance transporter) gene, which encodes the transmembrane protein PfCRT that localizes to the digestive vacuole. Data from a genetic cross, analysis of laboratory-adapted field isolates, episomal transformation experiments and analysis of cases of CQ treatment failure implicate a central role for pfcrt point mutations in CQR. Recently, we have also obtained isogenic mutant lines that share the same pfcrt mutations but differ in their degree of resistance. These data support the existence of additional genetic factors that can result in or modulate CQR.

To assess whether mutations in pfcrt are the primary genetic determinant of CQR, we are implementing allelic replacement approaches to exchange the pfcrt allele between CQ-resistant (CQR) and CQ-sensitive (CQS) strains. 3H-hypoxanthine growth inhibition assays will be performed on cloned recombinant parasite lines to assess the phenotypic consequence of modifying the pfcrt sequence. One possible outcome of these experiments would be the demonstration that pfcrt mutations are necessary and sufficient for CQR, on multiple genetic backgrounds. This result will stimulate investigations into the role of individual pfcrt mutations, the physiological consequence of these point mutations and the screening of antimalarial compounds that directly target or otherwise bypass the function of mutant PfCRT and kill CQR parasites. An alternative outcome of these experiments would be the finding that pfcrt mutations confer a CQR phenotype that is partial or depends on the genetic background. In this event, these transformed parasites will serve for subsequent rounds of allelic exchange using a second selectable marker and other candidate determinants including pfmdr1. These data will have important implications for developing diagnostic tools for detecting CQR malaria and also for elaborating mechanistic models of CQR.

To identify additional genetic factors that contribute to CQR, we are investigating isogenic mutant lines that differ in their CQR phenotype and yet harbor the same pfcrt haplotype. These will be investigated by microarray analysis for changes in gene statement that correlate with altered CQ response over the time course of the selection procedures. Transformation studies will then be used to identify which genes can contribute to CQR.

This research is situated in the context of our long-term aim to identify the critical genes involved in P. falciparum resistance to the major family of quinoline antimalarials and understand how resistance is mediated at the molecular, genetic and ultimately pharmacological level. This knowledge will assist in the development of new pharmacological initiatives in the fight against this devastating disease.

Contact Dr. Fidock.