Studies of an iron transporter of human malaria parasite Plasmodium falciparum

Abstract

Malaria is a serious parasitic disease caused by unicellular protozoa of the genus Plasmodium, which is responsible for high morbidity and mortality rates among those infected. Emerging resistance of Plasmodium falciparum, the most virulent of five Plasmodium parasites that infect humans, to available antimalarial drugs is threatening malaria control efforts and underscores the critical need to identify novel drug targets within the parasite. Given the essential role of iron metabolism in Plasmodium, membrane proteins that function in ferrous iron, Fe²⁺, transport represent attractive drug targets. This research focuses on the P. falciparum vacuolar iron transporter (PfVIT) that plays an important role in Fe²⁺ homeostasis of the parasite by transporting the excess Fe²⁺ into the lumen of endoplasmic reticulum for detoxification via a proton-driven electrochemical gradient. Although important, but perhaps not critical for the viability of asexual blood stages of Plasmodium, given its role as a detoxifier PfVIT could still represent a valid target for drug combination therapy. As such, a keener understanding of both the PfVIT structure and molecular mechanism(s) that regulate the function of the protein are needed.

In this thesis, we present a high-quality three-dimensional structural model of PfVIT homodimer that was constructed using computational structure prediction methods, homology modelling and AlphaFold 2.0. Furthermore, we designed a mutagenic strategy to investigate molecular mechanisms that regulate PfVIT function via phosphorylation and N-terminal-mediated autoinhibition, and the role of the N-terminal tail of PfVIT in subcellular targeting. We constructed a series of N-terminal deletions mutants to precisely map the region of autoinhibition in PfVIT. We also mutated 4 putative phosphorylation sites to residues that mimic either the constitutively modified or constitutively unmodified versions and interrogated all possible combinations of these mutants. In total, we engineered a library of 56 PfVIT mutants and tested the phenotypic effects of these mutants on PfVIT function using the previously described Saccharomyces cerevisiae model of Fe²⁺ transport.

Drawing upon our experimental findings, insights from the structural model, and previous studies on membrane transporters, we propose a model for the regulation of PfVIT function involving both phosphorylation and N-terminal-mediated autoinhibition. Taken together, this extensive structure-function mutagenic study of PfVIT has illuminated multiple regulatory mechanisms that may be exploited to treat human malaria, providing important insights into the key aspects of PfVIT biochemistry. These insights invite a rational, structure-guided design of novel drugs effective against this life-threatening human pathogen.

Thesis is embargoed until 31 July 2026.

Date of Award	Jul 2024
Original language	English
Awarding Institution	Queen's University Belfast
Sponsors	Northern Ireland Department for the Economy
Supervisor	Christopher Law (Supervisor), Edel Hyland (Supervisor) & Geoffrey Gobert (Supervisor)