Transition metals, including iron and zinc, are indispensable metals for almost all life. They play essential roles as cofactors for enzymes involved in a range of biological processes, including DNA replication and metabolism. Toxoplasma gondii is a ubiquitous obligate intracellular apicomplexan parasite which requires iron and zinc for survival. However, in excess, these metals are toxic. Thus, T. gondii has to regulate the uptake of iron and zinc to maintain homeostasis and avoid metal toxicity. Many organisms acquire divalent metals, including iron and zinc, from the environment through an evolutionary conserved family of proteins; Zrt/Irt-like (ZIP) proteins. At the start of my PhD, the mechanisms by which T. gondii acquired these essential metals was not known. The main focus of this work is on the functional characterisation of TGME49₂61720, one of the four ZIPdomain containing proteins predicted to be encoded by the T. gondii genome, which we called ZFT (Zn and Fe transporter). In this thesis, I demonstrate that ZFT localises to the plasma membrane of the parasite and is essential for the replication of T. gondii. I found that the localisation and expression of ZFT is dynamic and dependent on iron and vacuolarstage. I show that overexpression of ZFT leads to a growth defect, and parasites become hypersensitive to excess iron and zinc although protected from iron chelation. I demonstrate that loss of growth upon ZFT knockdown cannot be rescued by either excess iron or zinc, suggesting that ZFT’s function is not redundant in the parasite. Loss of ZFT leads to altered morphology of the apicoplast - although function is maintained - and inhibits mitochondrial respiration, linked to decreased expression of the mitochondrial Fe-S cluster protein SDHB and activity of complex IV of the mitochondrial electron transport chain. ZFT knockdown triggers the partial differentiation of parasites to a more quiescent lifecycle stage. Importantly, I demonstrate that the expression of TgZFT in a yeast model complements the loss of zinc transporter activity, supporting the role of ZFT as a zinc transporter in T. gondii. Finally, I show that knocking down ZFT results in a decrease in parasite-associated iron and zinc, confirming ZFT transporter activity in Toxoplasma gondii. However, as an intracellular pathogen, Toxoplasma acquires iron from its host cell. At the start of my PhD, little was known about the mechanisms by which T. gondii infection impacts host cell iron homeostasis. Thus, the second aim of my PhD project was to start to understand if and how T. gondii manipulates host cell iron homeostasis and trafficking by examining proteins involved in iron homeostasis in the host cell, in the context of infection. Here, I found changes in expression of proteins involved in host iron acquisition (TfR and DMT1) upon infection in both human fibroblasts and murine macrophages, although no changes in iron storage (FTH1) were observed. I show that infected fibroblasts and macrophages take up significantly more transferrin following T. gondii infection. However, whether these changes are host- or parasite-driven is not yet known, and further work is required to elucidate this. In high iron conditions, I show that DMT1 expression levels are maintained in both cell types compared to uninfected/untreated. TfR expression levels are maintained in fibroblasts but are reduced in macrophages, suggesting differences in iron homeostasis between the two cell types. Finally, I demonstrate that the T. gondii zft 3’ UTR confers iron responsiveness in fibroblasts but not in macrophages, further suggesting differences in iron handling between the two cell types. Overall, I have characterised the first iron and zinc uptake transporter in Toxoplasma gondii, identifying the major pathway for the acquisition of these metals by the parasite. Moreover, I demonstrated that infection with T. gondii leads to changes in the expression of iron-related proteins in fibroblasts and macrophages. This work emphasises the importance of both iron and zinc to Toxoplasma gondii and for their ability to survive and persist within the host.
Dana Ossama Nasief Aghabi (Thu,) studied this question.