Sloan, M. A. | bioRxiv Science

bioRxiv2025

Global translational and metabolic remodelling during iron deprivation in Toxoplasma gondii

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. ImportanceThis 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 parasites 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.

microbiology↗

bioRxiv2024

The Toxoplasma gondii mitochondrial transporter ABCB7 is essential for cytosolic iron-sulfur cluster biogenesis and protein translation

Iron-sulfur (Fe-S) clusters are ubiquitous inorganic cofactors required for numerous essential cellular pathways. Since they cannot be scavenged from the environment, Fe-S clusters are synthesised de novo in cellular compartments such as the apicoplast, mitochondrion and cytosol. The cytosolic Fe-S cluster biosynthesis pathway relies on transport of an intermediate from the mitochondrial pathway. An ATP binding cassette (ABC) transporter called ABCB7 is responsible for this role in numerous commonly studied organisms, but its role in the medically important apicomplexan parasites has not yet been studied. Here we identify and characterise the Toxoplasma gondii ABCB7 homolog. Genetic depletion shows that it is essential for parasite growth, and that disruption triggers partial stage conversion. Characterisation of the knock-down line highlights a defect in cytosolic Fe-S cluster biogenesis leading to defects in protein translation and other pathways including DNA and RNA replication and metabolism. Our work provides support for a broad conservation of the connection between mitochondrial and cytosolic in Fe-S cluster biosynthesis and reveal its importance for parasite survival.

microbiology↗

bioRxiv2023

Keeping FIT: Iron-mediated post-transcriptional regulation in Toxoplasma gondii

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.

microbiology↗

bioRxiv2020

Tools for the genetic manipulation of Herpetomonas muscarum

Trypanosomatid parasites are causative agents of important human and animal diseases such as sleeping sickness and leishmaniasis. Most trypanosomatids are transmitted to their mammalian hosts by insects, often belonging to Diptera (or true flies). With resistance to both vector-targeted pesticides and trypanocidal drugs being reported, there is a need for novel transmission blocking strategies to be developed. Studies using the blood-feeding vectors themselves are not broadly accessible, as such, new model systems are being developed to unpick insect-trypanosmatids interactions. One such case is the interactions between the model dipteran Drosophila melanogaster and its natural trypanosomatid Herpetomonas muscarum. Our previous work has found that much of the transcriptomic changes triggered in H. muscarum after ingestion by Drosophila reflect what is known for disease-causing trypanosomatids. Here we describe a set of tools to genetically manipulate the parasite and therefore create a truly tractable insect-parasite interaction system from both sides of this association. These include transgenic fluorescently tagged parasites to follow infection dynamics in the fly gut as well as iterations of plasmids that can be used for generating knock-in and knock-out strains. The tools presented in this short report will facilitate further characterisation of trypanosomatid establishment in a model dipteran.

microbiology↗

bioRxiv2019

The Phlebotomus papatasi transcriptomic response to trypanosomatid-contaminated blood is robust but non-specific

Leishmaniasis, caused by parasites of the genus Leishmania, is a disease that effects up to 8 million people worldwide. Parasites are transmitted to human and animal hosts through the bite of an infected sand fly. Novel strategies for disease control, require a better understanding of the key step for transmission namely, the establishment of infection inside the fly. In this work we wanted to identify fly transcriptomic signatures associated with infection success or failure. We used next generation sequencing to describe the transcriptome of the sand fly Phlebotomus papatasi when fed with blood alone or with blood containing one of three trypanosomatids: Leishmania major, Leishmania donovani and Herpetomonas muscarum: a parasite not transmitted to humans. Of these, only L. major was able to successfully establish an infection in P. papatasi. However, the transcriptional signatures observed were not specific to success or failure of infection but a generalised response to the blood meal. This implies that sand flies perceive Leishmania as just a feature of their microbiome landscape and that any strategy to tackle transmission should focus on the response towards the blood meal rather than parasite establishment. Authors summaryLeishmania are parasites that cause leishmaniasis, a group of serious diseases that affect millions of people, mainly across the subtropics and tropics. They are transmitted to humans by phlebotomine sand flies. However, despite establishment in the insects midgut being key to transmission, early infection events inside the insect are still unclear. Here, we study the gene expression response of the insect vector to a Leishmania parasite that is able to establish infection (L. major) one that is unable to do so (L. donovani) as well as one that is not a natural parasite of sand flies (Herpetomonas muscarum). We found that responses following any of the infected blood meals was very similar to uninfected blood meal. However, changes post-blood meal from day 1 to day 9 were dramatic. As a blood feeding insect can accumulate three times its weight in one blood meal, this seems to be the most important physiological change rather than the presence of the parasite. The latter might be just one in a number of microbes the insect encounters. This result will generate new thinking around the concept of stopping transmission by controlling the parasite inside the insect.

immunology↗

bioRxiv2019

Transcriptional and genomic parallels between the monoxenous parasite Herpetomonas muscarum and Leishmania

Trypanosomatid parasites are causative agents of important human and animal diseases such as sleeping sickness and leishmaniasis. Most trypanosomatids are transmitted to their mammalian hosts by insects, often belonging to Diptera (or true flies). These are called dixenous trypanosomatids since they infect two different hosts, in contrast to those that infect just insects (monoxenous). However, it is still unclear whether dixenous and monoxenous trypanosomatids interact similarly with their insect host, as fly-monoxenous trypanosomatid interaction systems are rarely reported and under-studied - despite being common in nature. Here we present the genome of monoxenous trypanosomatid Herpetomonas muscarum and discuss its transcriptome during in vitro culture and during infection of its natural insect host Drosophila melanogaster. The H. muscarum genome is broadly syntenic with that of human parasite Leishmania major. We also found strong similarities between the H. muscarum transcriptome during fruit fly infection, and those of Leishmania during sand fly infections. Overall this suggests Drosophila-Herpetomonas is a suitable model for less accessible insect-trypanosomatid host-parasite systems such as sand fly-Leishmania.\n\nAuthor SummaryTrypanosomes and Leishmania are parasites that cause serious Neglected Tropical Diseases (NTDs) in the worlds poorest people. Both of these are dixenous trypanosomatids, transmitted to humans and other mammals by biting flies. They are called dixenous as they can establish infections in two different types of hosts - insect vectors and mammals. In contrast, monoxenous trypanosomatids usually only infect insects. Despite establishment in the insects midgut being key to transmission of NTDs, events during early establishment inside the insect are still unclear in both dixenous and monoxenous parasites. Here, we study the interaction between a model insect - the fruit fly Drosophila melanogaster - and its natural monoxenous trypanosomatid parasite Herpetomonas muscarum. We show that both the genome of this parasite, and gene regulation at early stages of infection have strong parallels with Leishmania. This work has begun to identify evolutionarily conserved aspects of the process by which trypanosomatids establish in insects, thus potentially highlighting key checkpoints necessary for transmission of dixenous parasites. In turn, this might inform new strategies to control trypanosomatid NTDs.

microbiology↗