Omics Profiling and Spatio-Temporal Consequences of Mutations of PfKelch13 in Artemisinin Drug Resistance

Artemisinin Combination Therapies (ACTs) are frontline, fast-acting drugs that have been key in helping reduce the burden of malaria cases and deaths. However, malaria parasites are developing resistance to artemisinins, threatening global malaria control and elimination. Through the use of whole-genome sequencing (WGS) of artemisinin resistant parasites, mutations in pfkelch13 (K13) were shown to be associated with resistance both in vitro and in vivo. From its sequence and predicted molecular structure, K13 was predicted to be a substrate adapter involved in target identification, binding, ubiquitination and subsequent degradation of select substrates, keeping their levels low to maintain proper cellular homeostasis. Identification of a molecular marker of resistance led to the identification of underlying mechanisms. Two major K13 effector mechanisms have been proposed to overcome artemisinin-induced proteopathy and death. They are, firstly, proteostatic dysregulation of parasite phosphatidylinositol-3-kinase (PI3K) resulting in elevation of parasite PI3P and second, the upregulation of parasite oxidative stress and protein damage pathways via the unfolded protein response (UPR). However, how these two mechanisms were related (if they were at all) and the dynamics of PI3P, K13 and proteostasis systems that included the UPR had not yet been delineated.

Our work used bioinformatic and computational methods along with cryoimmunoelectron microscopy to show that K13 concentrates at PI3P vesicles / tubules in the parasite endoplasmic reticulum (ER) in P. falciparum infected erythrocytes. We also showed that K13 copurified with the parasite virulence adhesin PfEMP1. Isolation and subsequent analysis of the PfEMP1-K13 immunoproteome showed that this proteome is comprehensively enriched in multiple systems of proteostasis including protein export, quality control, protein folding in the ER and cytoplasm and the UPR. These findings were validated by showing that the major resistance marker K13C580Y quantitatively increased PI3P tubules and vesicles, exporting them throughout the parasite and the host red cell. The amplification of these vesicles containing signatures of proteostatic control that disseminate throughout the parasite might be involved in neutralizing the toxic proteopathy artemisinins cause in the parasite.

While these findings were focused on the major resistance mutation in South East Asia (SEA), K13C580Y, we expanded our studies to encompass all known K13 polymorphisms identified in Asia and Africa (over 200 polymorphisms). Since different polymorphisms lead to different levels of resistant (if at all), we developed an in silico tool to separate resistance causing mutations from sensitive ones. We can use this tool to distinguish between sensitive polymorphisms and resistance mutations in malaria endemic regions.

Overall, through our computational analyses presented here, we provide several insights into the role and function of the artemisinin resistance marker PfK13. We show that K13 is a marker for parasite PI3P, concentrating in vesicles in the ER. Expansion of these vesicles (containing pathways of protein proteostasis and the UPR) might confer resistance by disseminating resistance intermediates to mitigate the promiscuous artemisinin-induced proteopathy throughout the infected erythrocyte. We also develop an in silico tool to show how different polymorphisms along K13 might be involved in this mechanism of resistance.