Major advances in understanding the molecular basis of antimalarial drug resistance have been achieved by using the power of genetics. Since sexual combination occurs in the mosquito vector, gametocytes of cloned parent lines with different genetic backgrounds and drug resistance phenotypes can be mixed, fed to mosquitos, and the resulting haploid progeny in erythrocytes can be cloned and used to identify genetic loci associated with the desired phenotype. Genetic crosses with Plasmodium falciparum have been limited due to limitations of using non-human primates and or costs of mice engrafted with human hepatocytes. Herein we propose a novel approach of using a highly efficient, facile in vitro culture system to produce liver stage P. falciparum for genetic crosses. Backcross breeding is a forward genetics tool that can be used to introduce a specific genetic trait from one line into a second line. Most often this has been used in agriculture to introduce a desired trait (e.g., disease resistance) into an elite breeding line. Herein we propose the first phenotype-assisted backcross with P. falciparum to identify genetic loci that either confer or enhance K13 mediated resistance to artemisinin.
In Aim 1 we will conduct backcross experiments with drug susceptible (Nf54) and an artemisinin-resistant clone of P. falciparum that possesses K13 E252Q mutation and a resistant phenotype in the ring stage survival assay (RSA). Gametocytes of the parent lines will be mixed, fed to mosquitos, and sporozoites used to inoculate 384 well cultures of human hepatocytes. F1 progeny will be collected and subjected to drug selection in a modified RSA to enrich for resistant progeny and then backcrossed with the drug susceptible parent line (Nf54). We will repeat this phenotype-enhanced backcross three more times and clone progeny from backcross generation 4 (BC4) for deep sequencing.
In Aim 2 we will conduct phenotype and genotype analysis of cloned BC4 progeny. Artemisinin resistance phenotypes will be assessed by using a plate-based ring stage survival assay (RSA) in cloned progeny from BC4. QTL analysis will be performed to identify loci associated with artemisinin resistance and the role of K13 E252Q mutation will be assessed. The results from this study could significantly enrich the toolbox for experimental genetics of the human malaria parasite and uncover essential background mutations that either confer or enhance E252Q K13 mutations in artemisinin resistance.
The proposed research is important to public health as malaria remains a key cause of morbidity and mortality for much of the world. The work described here, to conduct genetic crosses to identify the molecular basis of artemisinin resistance, will address one of the key global health concerns hindering the efforts to eradicate malaria. Resistance to artemisinin and artemisinin combination therapies threaten the goobal effort to eliminate malaria.
