State of the ART: Functional genomics of RNA-binding proteins and their role in artemisinin drug resistance in Plasmodium falciparum

PROJECT SUMMARY Plasmodium parasite infections cause an estimated 229 million malaria cases and 409,000 deaths per year worldwide, posing a significant global public health burden. Artemisinin (ART) is the most effective first-line drug in combination therapies to treat Plasmodium falciparum (Pf) malaria, the deadliest malaria-causing parasite, but a major problem plaguing disease elimination efforts is the emergence and spread of partial ART resistance, characterized by delayed parasite clearance following ART treatments. In Pf, this has led to reduced efficacy as well as an increased risk of resistance to partner drugs. The threat of multi-drug resistant parasites therefore looms large, prompting a critical need to understand the molecular mechanisms of ART resistance, a complex phenomenon whose associated molecular markers remain poorly described. Studies on Pf ART resistance have largely focused on mutations in the Kelch 13 protein (K13) propeller domain, which disrupt digestion of hemoglobin required for ART activation. This disruption gives the parasite a fitness advantage when faced with ART, and can partially account for resistance. But while ART is dependent on hemoglobin digestion, its activity is dependent on other, yet-to-be elucidated mechanisms. In the present work, ART sensitivity in a Pf field isolate harboring a K13 mutation was modulated using an in vitro evolution strategy. Single-cell RNA-sequencing was then performed to elucidate the transcriptional signatures of ART-resistant and -sensitive ring forms. The most striking distinction between ART-resistant and -sensitive rings is the greater expression of genes encoding for RNA-binding proteins (RBPs) in the former group. RBPs are post-transcriptional regulators of gene expression that coordinate growth and life-stage transitions in Plasmodium parasites, and are also known to mediate stress responses, including to drug exposure. However, despite evidence of altered Pf stress response pathways implicated in ART resistance, the role of RBPs remains unknown. The proposed project will use state of the art techniques and phenotypic assays to assess the extent to which post-transcriptional gene regulation via RBPs governs ART resistance. In aim 1, I will use long-read, short-read, and Chromosome Conformation Capture sequencing to determine the genetic signatures underlying the observed cellular and transcriptomic phenotypes in ART-resistant and -sensitive parasites; in aim 2, I will use inducible knockdown studies to assess the extent to which two candidate RBPs mediate ART resistance; and in aim 3, I will use high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (eCLIP-seq) to identify the RNA interactors of the candidate RBPs. The outcomes of these studies will expand our understanding of ART resistance mechanisms and guide new therapeutic strategies to tackle ART resistance.