Elucidating the complex genetic and molecular basis of Plasmodium falciparum artemisinin resistance.

Malaria is a disease caused by infection with Plasmodium parasites, of which P. falciparum is the deadliest, and half of the world’s population is at risk of infection. Increased access to artemisinin-based combination therapies (ACTs) and mosquito vector control initiatives has saved an estimated 3.3 million lives since 2000. Despite this progress, malaria remains a major burden to human health, causing approximately 627,000 deaths in 2012. No effective vaccine exists, and artemisinin resistance has emerged in Southeast Asia, making it imperative to understand the mechanism and extent of resistance to prevent a regional crisis from becoming a global disaster. Genomic and transcriptomic analyses of artemisinin-resistant parasites have implicated mutations in the K13 gene, with secondary contributions from genes involved in iron transport and ubiquitin-mediated protein degradation. Using zinc-finger nucleases (ZFNs), it was shown that removing K13 mutations from artemisinin-resistant Cambodian parasites abolishes resistance, while inserting K13 mutations into artemisinin-sensitive parasite lines confers variable degrees of resistance, indicating the influence of a permissive genetic background. The combination of ongoing evaluation of parasites from Southeast Asia and the ability to add or remove candidate mutations from isogenic, well-characterized parasites allows a comprehensive dissection of the resistance mechanism. ZFNs will be used to study the function of K13 and the influence of genetic background on the emergence and spread of artemisinin resistance. Emergence is tied to the mechanism of resistance, whereas spread is tied to transmission. Potential resistance mutations will be added or removed in isogenic parasite lines alone or in combination. To study emergence, both the ring-stage survival assay (RSA0-3h) and a modified [³H]-hypoxanthine incorporation assay will be used to determine artemisinin sensitivity, and K13 function will be explored through changes in the parasite redox metabolome, ubiquitin-mediated proteasomal degradation, identification of interacting partners, and K13 subcellular localization. Based on current data, K13 is hypothesized to function as a negative regulator of oxidative stress responses to iron-mediated redox damage, with resistance-associated mutations reducing its function and thereby increasing antioxidant capability. Transmission will be studied through pairwise fitness comparisons of parasites in both asexual blood stages and sexual mosquito stages. An improved understanding of artemisinin resistance will guide targeted geographical use of non-artemisinin-based second-line or novel therapies to reduce the burden of malaria.

NIH project details

Drug Resistance
Enabling Technologies & Assays
Genetics and Genomics

Jul 2015 — Jun 2018

$168,243

United States