Clare R. Harding

Iron is required to support essential cellular processes. Due to diverse and dynamic host environments, the obligate intracellular parasite Toxoplasma gondii must adapt to iron-limited conditions. To investigate the adaptations critical to parasite survival under these conditions, we conducted proteomic and metabolomic profiling of Toxoplasma cultured in iron-depleted conditions. We find that iron depletion results in remodeling of the parasite proteome and triggers swift translational repression, prior to decreases in the key translational factor ABCE1. In the context of repressed translation, we also observe a significant rewiring of energy metabolism. Iron-depleted Toxoplasma have altered mitochondrial morphology and a profound reduction in mitochondrial respiration. Untargeted metabolomics revealed changes in central carbon metabolism, with the accumulation of intermediates of glycolysis and the tricarboxylic acid (TCA) cycle. Stable isotope labeling revealed that iron deprivation leads to a fundamental disconnect between these pathways, with reduced incorporation of glucose-derived carbon into cellular macromolecules and disruption of the TCA cycle. Instead, iron-deprived parasites continued to take up glucose and maintain glycolysis for energy generation. Limiting glucose availability, either in culture media or by genetic ablation of glucose uptake, caused a significant increase in sensitivity to iron restriction. Conversely, the limitation of mitochondrially metabolized glutamine improved parasite fitness in iron-depleted conditions. Together, our results establish iron as a key regulator of parasite translation and metabolic flexibility and demonstrate an increased reliance on glycolysis for energy generation and survival under acute iron deprivation. IMPORTANCE: This study determines the effects of iron deprivation on the parasite Toxoplasma gondii . Using proteomics and metabolomics, we reveal iron as a novel regulator of both protein translation and energy metabolism in Toxoplasma, underpinning the importance of this nutrient for essential cellular processes. We find that iron depletion introduces a metabolic bottleneck, whereby parasites become dependent on glucose as their major carbon source. By modulating the parasite’s metabolism by altering carbon source availability, we identify nutrient conditions that improve parasite survival under iron restriction. These data reveal a key role for adaptive plasticity of Toxoplasma central carbon metabolism to drive survival under iron-limited conditions. Understanding the interactions between parasite nutrient availability and metabolism allows us both to map the metabolic flexibility of these parasites and identify potential vulnerabilities.

Copper is a conserved cofactor, required for essential processes including aerobic respiration. Pathogens subvert host copper; however the mechanisms of copper uptake are not well understood. Here, we identify and validate two copper transporters of Toxoplasma gondii and determine the role of copper in parasite metabolism and pathogenesis. We show that Ctr1 is required for copper uptake. Deletion leads to undetectable parasite-associated copper, a significant growth defect and a metabolic shift from mitochondrial respiration with to glycolysis. Absence of Crt1 was fully and rapidly rescued by exogenous copper, which we believe is transported through the lower-affinity transporter Ctr2. Ctr2 is dispensable in rapid growth, however, has a role in parasite persistence in chronic infection, both in vitro and in vivo . Together, these findings reveal for the first time a critical role for copper uptake in shaping metabolic plasticity, and highlights the importance of nutrient availability in regulating apicomplexan metabolism.

Transition metals, such as iron and zinc, are indispensable trace elements for eukaryotic life, acting as co-factors in essential processes ranging from metabolism to DNA replication. These metals can be transported into cells by an evolutionary-conserved family of metal transporters; however, how the ubiquitous mammalian parasite Toxoplasma gondii acquires essential metals has been unknown. Here, we have identified and characterised the first iron and zinc importer in T. gondii . This transporter, named ZFT, localised to the parasite plasma membrane and is essential for the parasite’s life cycle. We find ZFT is regulated by iron availability and overexpression sensitises cells to excess iron and zinc. Using a conditional knockdown system, we find that knockdown of ZFT leads to reduction in mitochondrial respiration and a switch to a more quiescent lifecycle stage. To confirm transport activity, we find that knockdown of ZFT leads to a reduction in parasite-associated zinc and iron, and ZFT expression complements loss of zinc transporter activity in a yeast model. Further, expression of ZFT in Xenopus oocytes demonstrates direct uptake of iron, which is outcompeted in the presence of zinc. Overall, we have identified the first metal uptake transporter in T. gondii and demonstrated the importance of iron and zinc to the parasite. This finding advances our understanding of how this obligate intracellular parasite acquires nutrients from its host.

Transition metals, such as iron and zinc, are indispensable trace elements for eukaryotic life, acting as co-factors in essential processes ranging from metabolism to DNA replication. These metals can be transported into cells by an evolutionary-conserved family of metal transporters; however, how the ubiquitous mammalian parasite Toxoplasma gondii acquires essential metals has been unknown. Here, we have identified and characterised the first iron and zinc importer in T. gondii. This transporter, named ZFT, localised to the parasite plasma membrane and is essential for the parasite’s life cycle. We find ZFT is regulated by iron availability and overexpression sensitises cells to excess iron and zinc. Using a conditional knockdown system, we find that knockdown of ZFT leads to reduction in mitochondrial respiration and a switch to a more quiescent lifecycle stage. To confirm transport activity, we find that knockdown of ZFT leads to a reduction is parasite-associated zinc and iron, and ZFT expression complements loss of zinc transporter activity in a yeast model. Further, expression of ZFT in Xenopus oocytes demonstrates direct uptake of iron, which outcompeted in the presence of zinc. Overall, we have identified the first metal uptake transporter in T. gondii and demonstrated the importance of iron and zinc to the parasite. This finding advances our understanding how this obligate intracellular parasite acquires nutrients from its host.

Iron is required to support essential cellular processes. Due to diverse and dynamic host environments, the obligate intracellular parasite Toxoplasma gondii must adapt to iron limited conditions. To investigate the adaptations critical to parasite survival under these conditions, we conducted proteomic and metabolomic profiling of Toxoplasma cultured in iron depleted conditions. We find that iron depletion results in remodelling of the parasite proteome and triggers swift translational repression. This occurs prior to downregulation of the iron-regulated translation factor ABCE1, indicating an upstream, ABCE1-independent mechanism. In the context of repressed translation, we also observe a significant rewiring of energy metabolism. Iron depleted Toxoplasma have altered mitochondrial morphology and a profound reduction in mitochondrial respiration. Untargeted metabolomic analysis revealed tricarboxylic acid cycle (TCA) cycle dysregulation, characterised by accumulation of citrate and fumarate, both substrates of iron-dependent TCA cycle enzymes, and accumulation of glycolytic intermediates. We found iron deprived parasites continue to take up glucose and maintain glycolytic output, comparable to iron replete conditions. Limiting glucose availability either in culture media or by genetic ablation of glucose uptake caused a significant increase in sensitivity to iron restriction. Conversely, limitation of mitochondrially metabolised glutamine improved parasite fitness in iron depleted conditions. Together, our results establish iron as a key regulator of parasite translation and metabolic flexibility and demonstrate carbon source availability as important in Toxoplasma adaptation to iron deprivation. Importance: This study determines the effects of iron deprivation on the parasite Toxoplasma gondii. Using proteomics and metabolomics, we reveal iron as a novel regulator of both translation and energy metabolism in Toxoplasma, underpinning the importance of this nutrient for essential cellular processes. We find that iron depletion introduces a metabolic bottleneck whereby parasites are dependent on glucose as their major carbon source. By modulating the parasite’s metabolism by altering carbon source availability, we identify nutrient conditions that improve parasite survival under iron restriction. These data reveal a key role for adaptive plasticity of Toxoplasma central carbon metabolism to drive survival under iron limited conditions. Understanding the interactions between parasite nutrient availability and metabolism allows us both to map the metabolic flexibility of these parasites, and to identify potential vulnerabilities.

Transition metals, such as iron and zinc, are indispensable trace elements for eukaryotic life, acting as co-factors in essential processes ranging from metabolism to DNA replication. These metals can be transported into cells by an evolutionary-conserved family of metal transporters; however, how the ubiquitous mammalian parasite Toxoplasma gondii acquires essential metals has been unknown. Here, we have identified and characterised the first iron and zinc importer in T. gondii . This transporter, named ZFT, localised to the parasite plasma membrane and is essential for the parasite’s life cycle. We find ZFT is regulated by iron availability and overexpression sensitises cells to excess iron and zinc. Using a conditional knockdown system, we find that knockdown of ZFT leads to reduction in mitochondrial respiration and a switch to a more quiescent lifecycle stage. To confirm transport activity, we find that knockdown of ZFT leads to a reduction is parasite-associated zinc and iron, and ZFT expression complements loss of zinc transporter activity in a yeast model. Further, expression of ZFT in Xenopus oocytes demonstrates direct uptake of iron, which outcompeted in the presence of zinc. Overall, we have identified the first metal uptake transporter in T. gondii and demonstrated the importance of iron and zinc to the parasite. This finding advances our understanding how this obligate intracellular parasite acquires nutrients from its host.

Iron is required to support almost all life; however, levels must be carefully regulated to maintain homeostasis. Although the obligate parasite Toxoplasma gondii requires iron, how it responds upon iron limitation has not been investigated. Here, we show that iron depletion triggers significant transcriptional changes in the parasite, including in iron-dependent pathways. We find that a subset of T. gondii transcripts contain stem-loop structures, which have been associated with post-transcriptional iron-mediated regulation in other cellular systems. We validate one of these (found in the 3’ UTR of TGME49_261720) using a reporter cell line. We show that the presence of the stem-loop-containing UTR is sufficient to confer accumulation at the transcript and protein levels under low iron. This response is dose and time-dependent and is specific for iron. The accumulation of transcript is likely driven by an increased reporter mRNA stability under low iron. Interestingly, we find iron-mediated changes in mRNA stability in around 400 genes. To examine the potential mechanism of this stability, we tested aconitase interaction with mRNA in low iron and found 43 enriched transcripts, but no specific interaction with our reporter UTR. However, the endogenous UTR led to maintenance of protein levels and increased survival of the parasite under low iron. Our data demonstrate the existence of iron-mediated post-transcriptional regulation in Toxoplasma for the first time; and suggests iron-mediated regulation may be important to the parasite in low iron environments.

New ferrocenyl-benzimidazole compounds with anti-Toxoplasma gondii activity were synthesized and characterized. The compounds had redox activity and led to the accumulation of reactive oxygen species within parasites.

Advances in sequencing technology have led to a dramatic increase in the number of single-cell transcriptomic datasets available. In the field of parasitology, these datasets typically describe the gene expression patterns of a given parasite species under specific experimental conditions, in specific hosts or tissues, or at different life-cycle stages. However, while this wealth of available data represents a significant resource for further research, the analysis of these datasets often requires significant computational skills, preventing a considerable proportion of the parasitology community from meaningfully incorporating existing single-cell data into their work. Here, we present paraCell, a novel software tool that automates the advanced analysis of published single-cell data without requiring any programming ability. On our free web server, we demonstrated how to visualise data, re-analyse published Plasmodium and Trypanosoma datasets, and present novel Toxoplasma-mouse and Theileria-cow atlases to study the impact of IFN-γ and host genetic susceptibility.

Iron is required to support almost all life; however, levels must be carefully regulated to maintain homeostasis. Although the obligate parasite Toxoplasma gondii requires iron, how it responds when iron becomes limiting has not been investigated. Here, we show that iron depletion triggers significant transcriptional changes in the parasite, including in pathways that require iron. Interestingly, we find that a subset of T. gondii transcripts contain stem-loop structures which have been associated with post-transcriptional iron-mediated regulation in other cellular systems. We validate one of these (found in the 3’ UTR of TGME49_261720) using a reporter cell line. We show that the presence of the stem-loop containing UTR is sufficient to confer accumulation at the transcript and protein levels under low iron. We show that this response is dose and time-dependent and is specific for iron. Using immunoprecipitation, we show that the metabolic enzyme aconitase is capable of binding to mRNA, including fit , and that the presence of the fit UTR leads to stabilisation of the transcript under low iron conditions. These results demonstrate the existence of iron-mediated post-transcriptional regulation in Toxoplasma for the first time, and show that the metabolic enzyme aconitase may have an additional role as an RNA-binding protein.