Studies of vacuolar iron transporter homologue from Plasmodium falciparum in a yeast model system

Abstract

Malaria is a serious and often a fatal disease in humans, caused by a parasite commonly transmitted by the female Anopheles mosquito. Plasmodium falciparum is by far the deadliest of all malaria parasites. According to the most current World Malaria Report, there were 247 million cases of malaria in 2021, up from 245 million in 2020. With the lack of a roll-out malarial vaccination programme and emerging resistance against the drugs commonly used to control infection, there is a pressing need to gain a deeper understanding of the biochemistry and physiology of the malaria parasite. Plasmodium falciparum pathogenicity derives in part from an ability to influence the physiology of its human host during developmental stages, specifically within human blood. Plasmodium falciparum feeds on haemoglobin, develops and divides within erythrocytes, including modifying host cells so they can attach to blood vessel walls. This knowledge can be exploited to develop effective strategies for controlling the disease. One specific area that necessitates further investigation is the role and mechanism of Plasmodium membrane transporter proteins. The malaria parasite undergoes a complex life cycle during which it must regulate the uptake and efflux of nutrients, ions, and other metabolites across its membrane systems. Iron metabolism is the key to the survival of plasmodial cells, especially during the intraerythrocytic stage. Iron is an essential cofactor in several vital enzymatic reactions in critical cellular processes such as energy production, respiration, and DNA synthesis. Intracellular levels of labile divalent iron need to be tightly regulated by the parasite to ensure an adequate iron supply for all essential biological processes while managing the excess iron before it becomes toxic due to generating oxygen-derived radicals and other damaging species via the Fenton reaction. Integral membrane proteins responsible for ferrous iron transport via various membranes play a crucial role in iron homeostasis. One group of these proteins are members of the poorly understood Vacuolar Iron Transporter (VIT) family that have been characterised in the plant called VIT1 and in yeast called CCC1, Ca2+ sensitive cross-complementer 1. These proteins are generally responsible for importing excess iron into the vacuole to protect from cellular toxicity and storing it during iron deficiency. Slavic et al. (2016) conducted an in vivo study that presents compelling evidence suggesting that interfering with Plasmodium falciparum PfVIT (Plasmodium falciparum Vacuolar Iron Transporter) function could be a promising approach for treating malaria infection. This thesis presents an extensive molecular investigation of PfVIT, building upon prior research, to unravel its intricate structure and function. The study thoroughly explores its molecular features, structure-function relationships, and computational insights. A meticulous and accurate molecular model of PfVIT's structure has been successfully generated, laying the foundation for deeper analyses. The first expression of PfVIT in Saccharomyces cerevisiae using a heterologous overexpression framework was a big step forward because it helped proteins fold correctly. This innovative approach provides unprecedented opportunities to scrutinize PfVIT's functionality and distinctive metal ion recognition capabilities. Notably, molecular dynamics simulations have formulated a dynamic model of PfVIT's translocation activity, enhancing our grasp of its behaviour. Intriguingly, the overexpression of PfVIT in Saccharomyces cerevisiae serves as a gateway to unearthing crucial insights into its functional attributes and the underlying mechanisms of metal ion recognition. Mutagenesis studies have shown that specific residues, like the methionine at position 161, are essential for the transporter activity of PfVIT. Significantly, an exploration into the conserved residue D54 has highlighted its involvement in nickel transport. These findings have profound implications for comprehending VIT proteins and their therapeutic potential for combating malaria. Experimental evidence underscores the indispensable nature of PfVIT's MBD for effective cation transport, as any disruption to this domain results in functional impairment. This invaluable insight positions the research outcomes as a springboard for fellow researchers, guiding the development of targeted inhibitors against PfVIT, thus advancing malaria treatment strategies.