Graduation Date

Spring 5-6-2023

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Immunology, Pathology & Infectious Disease

First Advisor

Caroline Ng, PhD

Abstract

Malaria remains a significant global health threat, responsible for 691,000 deaths in 2021. Artemisinin-based combination therapies have contributed significantly to decline of malaria disease burden. Upon activation by heme, artemisinins are believed to kill parasites by non-specifically alkylating proteins and causing widespread damage to the parasite. Artemisinin resistance is now prevalent in regions of Southeast Asia and resistance has recently emerged in Africa, where over 90% of all malaria deaths occur. Mutations in Kelch13 (K13) are believed to mediate artemisinin resistance by either reducing parasite hemoglobin digestion, and consequently artemisinin activation, or by increasing parasite stress response mechanisms. We hypothesize that enhanced capacity to restore protein homeostasis (proteostasis) underlies artemisinin resistance in K13 mutant parasites and that proteostasis mechanisms can be targeted to overcome resistance. Here, a novel high content imaging and analysis method is described and utilized 1) to screen putative deubiquitinase inhibitors against artemisinin-resistant parasites and 2) to investigate the role of proteostasis mechanisms in artemisinin resistance. Our screen of putative deubiquitinase inhibitors identified one compound that displayed low micromolar potency against artemisinin sensitive and artemisinin resistant blood stage parasites. By utilizing high content imaging paired with machine learning as well as in vitro selection studies paired with whole genome sequencing, we interrogated the mechanism of action of this compound. To investigate the role of proteostasis mechanisms in artemisinin resistance, we examined the unfolded protein response (UPR) and ubiquitin proteasome system (UPS) in parasites that are isogenic except at K13 and in proteasome mutant parasites. UPR activation was dependent on both parasite stage and K13 genotype, suggesting that K13 plays some role in influencing UPR activation. No difference in proteasome activity was observed between K13 WT and K13 mutant parasites. However, the proteasome appears to be critical for parasite artemisinin response. Regardless of K13 genotype, parasites harboring a mutation in the proteasome display increased sensitivity to artemisinins. These sensitized mutants all showed increased accumulation of K48-linked ubiquitin, a hallmark of proteasome disfunction. Collectively, these data implicate the UPR and UPS as pathways that can be targeted to overcome artemisinin resistance.

Comments

2023 Copyright, the authors

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