PROJECT SUMMARY
The protozoan parasite Plasmodium falciparum causes over 400,000 malaria deaths each year, mostly in young
African children. With no effective vaccine, chemotherapy remains the cornerstone of malaria treatment and
control. Malaria eradication efforts have been hindered by the rise of resistance to first-line antimalarials in
Southeast Asia, including piperaquine (PPQ, used in combination with dihydroartemisinin). Piperaquine
resistance (PPQ-R) and chloroquine resistance are primarily mediated by mutations in the P. falciparum
Chloroquine Resistance Transporter (PfCRT). Mutant PfCRT transports drug away from its site of action in the
asexual blood stage parasite’s digestive vacuole. This acidic organelle degrades endocytosed host hemoglobin
and extrudes globin-derived peptides for parasite protein synthesis. Less understood, however, is how certain
amino acid substitutions confer this efflux mechanism and how they impact native transporter function. To
investigate the effect of these mutations, I propose in Aim 1 to purify contemporary PPQ-R PfCRT isoforms and
perform binding and transport assays with PPQ, other clinically used antimalarials, and positively-charged
peptides as proposed natural substrates. These studies will provide a comprehensive functional characterization
of PPQ-R PfCRT that is currently lacking. It is also important to predict how PPQ-R could spread to or emerge
in other malaria endemic regions. The PfCRT mutations found in SE Asia have yet to be seen on African and
South American backgrounds; however, PPQ is being used in these areas including as first-line treatment or for
uncomplicated malaria. To predict whether the contemporary amino acid substitutions seen in SE Asia could
emerge in other regions to achieve PPQ-R, I propose in Aim 2 to engineer these mutations onto African and
South American PfCRT isoforms in P. falciparum parasites from these regions. Assays will quantify the
susceptibility of these lines to PPQ and other antimalarials, and determine the relative fitness of each line. The
degree of resistance conferred and the fitness cost imposed, along with the regional drug regimen, will be
important in determining which pfcrt alleles predominate and which can emerge and spread in areas of PPQ
use. These aims are predicated on the hypotheses that (1) PPQ-R mutations in PfCRT alter transport of drugs
and natural substrates and that (2) PPQ-R can arise in Africa and South America via the emergence of single
amino acid substitutions observed in SE Asia in mutant PfCRT. These data will also identify whether a gain of
PPQ-R restores parasite susceptibility to chloroquine, as has been seen with most mutations in SE Asia, thereby
creating therapeutic opportunities for new combination therapies. These studies are expected to yield important
new insights into the molecular basis for antimalarial drug resistance, and to leverage that knowledge to predict
the emergence of novel PPQ-R PfCRT isoforms in distinct geographic regions. This will guide the development
of treatment strategies to reduce the global impact and spread of drug-resistant malaria.
