17th International Congress on Toxoplasmosis

A novel component of the cytosolic iron-sulfur biogenesis pathway, TgHCF101, as a potential drug target in Toxoplasma gondii

Cell Biology

Toxoplasma gondii is a unicellular eukaryotic parasite responsible for causing toxoplasmosis in Humans. The disease, asymptomatic in most instances, can be threatening to immunocompromised individuals. The parasite infects about a third of the global population, mainly through undercooked meat consumption, utilizing cysts as a form of persistence. T. gondii, as an obligate intracellular pathogen, scavenges essential nutrients, including iron, crucial to its growth, from its host cell. Both the host and the parasite are using iron; as it is a component of protein cofactors like heme or iron-sulfur (Fe-S) clusters, vital to the parasite. Fe-S clusters represent ancient and indispensable molecular structures formed by specific arrangements of coordinated iron and sulfur atoms. Their biogenesis and specific insertion into target proteins rely on multiprotein complexes fairly conserved across species. Proteins containing Fe-S clusters play crucial roles in cellular functions such as genome maintenance, protein translation, energy conversion and various enzymatic activities implying electron transfer mechanisms. Within T. gondii, three metabolic pathways for Fe-S clusters biogenesis co-exist, located within the mitochondrion, in the plastid of the parasite, or in the cytoplasm. We have characterized the T. gondii homologue of a protein called HCF101, absent from mammals but playing a role in transferring Fe-S clusters to client proteins in the chloroplasts of land plants. Our findings demonstrate that TgHCF101 is essential for parasite viability. However, in contrast to plants, we have shown that it is not associated with any plastid-related function in T. gondii; instead, it is part of the CIA (Cytosolic Iron-Sulfur cluster Assembly) pathway. Even in pan-eukaryotic pathways like the CIA pathway, the involvement of an essential lineage-specific protein like TgHCF101 provides the perspective of discovering parasite-specific features that may be exploited for therapeutic intervention.

Oral presentation

Professor

Exploring redundancies in central carbon metabolism in Toxoplasma parasites

Toxoplasma gondii is an apicomplexan parasite that causes severe disease in immunocompromised individuals, newborns, and livestock. This ubiquitous parasite uses central carbon metabolism pathways to generate energy and macromolecules for its proliferation and survival. In other eukaryotes, the mitochondrial tricarboxylic acid (TCA) cycle plays a key role in energy generation and the provision of biosynthetic intermediates. T. gondii harbours a complete functional mitochondrial TCA cycle, but the enzymes that catalyse the individual metabolic reactions remain understudied, and the overall importance of the TCA cycle is still uncertain. To address this knowledge gap, we investigated the importance and role of the TCA cycle in T. gondii, using a combination of forward and reverse genetics as well as physiological and metabolomic analyses. We found that the loss of some TCA cycle reactions led to severe defects in parasite proliferation and mitochondrial oxygen consumption in vitro, whereas loss of others had minimal impact. Using CRISPR/Cas9 genome-wide screening of TCA cycle mutants we discovered the presence of several functional redundancies. One notable redundancy was in the reactions catalysed by the mitochondrial TCA cycle enzyme malate:quinone oxidoreductase (MQO) and the cytosolic enzyme malate dehydrogenase (MDH), both of which mediate malate oxidation but in different subcellular compartments. Parasites which simultaneously lack both MQO and MDH exhibited a defective TCA cycle, impaired pyrimidine biosynthesis and accumulation of fumarate. We are currently investigating the essential metabolic process(es) that require malate oxidation in the parasite. Overall, our findings enhance the understanding of a key metabolic pathway and the flexible nature of central carbon metabolism in T. gondii.

The regulation of the constant flux of fatty-acids relies on key acyltransferases regulating the nutrient flexibility in Toxoplasma gondii.

Metabolism, Biochemistry & Drug Development

To ensure a rapid propagation and pathogenicity within its human host, Toxoplasma gondii require large amount of lipids, through massive scavenging of host resources and de novo fatty acid (FA) synthesis [1, 2]. FAs coming from both sources are remodeled and channeled towards lipid storage, i.e. lipid droplet (LD), avoiding an accumulation of potentially lipotoxic FAs, and being mobilized for parasite division [3]. Incorporation and remodeling of FA in glycerolipids (major membrane lipids) is conducted by a group of protein called acyltransferase. Here, we tried to understand how the vital FA fluxes of the glycerolipid pathway are regulated: we identified two key acyltransferases, TgGPAT (glycerol-3-phosphate acyltransferase) and TgDGAT2 (diacylglycerol acyltransferase). TgGPAT allows bulk membrane biogenesis and parasite formation by generating the key lipid precursor LPA. Metabolic labelling combined to lipidomics reveal major changes on FA substrate (host vs de novo) depending on the nutrient context (rich vs poor). On the other hand, TgDGAT is crucial for LD biogenesis only when the parasite lacks host nutrient. This study reveals that both proteins are essential for glycerolipid metabolism and act in a regulatory manner in different nutrient conditions. The loss of these proteins results in an accumulation of FFAs that cannot be &quot;detoxified&quot; by incorporating them into LDs, leading to parasite death. The nutrient-dependency of these proteins provides insight into the parasite&quot;s metabolic flexibility: they can adapt to different environmental conditions by setting up a specific metabolic program accordingly, rendering proteins non-essential, essential, or vice-versa. Understanding FA remodelling and how these fluxes are regulated in Apicomplexa parasites will be crucial in conceptualizing a novel therapeutic strategy against the parasites. References [1] S. Shunmugam et al., &quot;The flexibility of Apicomplexa parasites in lipid metabolism&quot;, PLoS Pathogens, 2022. doi: 10.1371/journal.ppat.1010313. [2] S. Amiar et al., &quot;Division and Adaptation to Host Environment of Apicomplexan Parasites Depend on Apicoplast Lipid Metabolic Plasticity and Host Organelle Remodeling&quot;, Cell Rep, 2020, doi: 10.1016/j.celrep.2020.02.072. [3] S. Dass et al., &quot;Toxoplasma LIPIN is essential in channeling host lipid fluxes through membrane biogenesis and lipid storage&quot;, Nat Commun, 2021, doi: 10.1038/s41467-021-22956-w.

Genetic screens in physiological contexts nominate metabolic pathways for deep learning-assisted drug discovery

Acquisition of host metabolites is a defining feature of parasitism, yet culture systems are typically replete with excess nutrients that may obscure metabolic relationships between parasites and hosts. We performed two genetic screens to identify metabolic restrictions that occur during infection. First, we conducted a genome-wide screen of Toxoplasma in mice, revealing dozens of metabolic pathways that are required for animal infection. We next assessed which of these pathways could be studied in culture by performing a targeted screen of parasites grown under physiological media conditions. Among the top hits was GTP Cyclohydrolase I (GCH), an enzyme responsible for the first step in the biosynthesis of two metabolites, biopterin and folate. In addition to being synthesized by parasites, these metabolites can be salvaged from the host. We demonstrate that parasites adaptively rely on salvage when host folate levels are artificially elevated by standard culture conditions. As the target of anti-parasitic drugs like pyrimethamine, folate metabolism has long been appreciated as critical for parasite replication. However, anti-folate drugs have traditionally targeted enzymes downstream of the convergence of folate biosynthesis and salvage. Our work demonstrates that targeting biosynthesis alone is sufficient to restrict parasite growth under physiological conditions, nominating new enzymes as putative anti-parasitic targets. As a proof-of-concept, we demonstrate that an inhibitor of mammalian GCH can be repurposed to inhibit parasite growth. We have additionally devised a screening platform to identify compounds that more potently inhibit GCH. Leveraging conservation in bacteria, we created E. coli strains in which bacterial GCH is replaced with parasite or human homologs, enabling rapid screening of large chemical libraries through simple measurements of bacterial growth. An initial screen of 2500 compounds revealed dozens of molecules that selectively inhibit parasite GCH. In ongoing work, we are combining the graph neural network Chemprop and large language models (LLM) to learn the molecular features that enable preferential binding of a compound to parasite GCH, while reducing inhibition of the human homolog. In total, this work demonstrates how examining parasite metabolism in a physiological context can reveal previously overlooked druggable targets, and suggests a new methodology for high-throughput drug screening via bacterial complementation.

Genetic deletion of an essential lipid storage enzyme attenuates the virulence of Toxoplasma gondii

The ability of Toxoplasma gondii to infect a wide range of hosts is greatly influenced by its capability to salvage essential nutrients from the host cell and its environment. Toxoplasma relies on lipids for its critical functions, acquiring fatty acids from the host cell, in addition to de novo synthesis, to support its growth and ensure survival. Fatty acids are important components for Toxoplasma's membrane biogenesis, energy generation, and other metabolic processes. However, excessive free fatty acids (FFA) are toxic to the parasite. To evade lipotoxicity, Toxoplasma expresses lipid-esterifying enzymes to divert surplus FFA into major storage neutral lipids, such as triacylglycerol (TAG) and cholesteryl ester (CE), stored within intracellular lipid droplets (LD). One such enzyme, the acyl-CoA:diacylglycerol acyltransferase (TgDGAT1) catalyzes the final step in the acyl-CoA-dependent synthesis of TAG. TgDGAT1, a member of the membrane-bound O-acyltransferase (MBOAT) superfamily, shares structural and functional features with enzymes in the DGAT1 family. Interestingly, the DGAT1 inhibitor T863 arrests the growth and multiplication of Toxoplasma in cultured mammalian cells, suggesting an important role for this enzyme in storing energy-rich FAs as TAGs and protecting the parasite from FFA-induced toxicity. However, the direct effect of loss of DGAT1 enzyme activity in the parasite remains unexplored. In this study, we generated a genetically modified Toxoplasma type I strain devoid of the TgDGAT1 gene, rendering it incapable of storing FFAs as TAGs within LDs. Of interest, we note that RH ∆DGAT1 parasites can grow and multiply in vitro under lipid-depleted conditions, showing a moderate growth defect. However, in standard growth media, the mutant's inability to store fatty acids severely impairs its ability to thrive within host mammalian cells. Additionally, EM studies reveal gross morphological abnormalities, with skinny parasites disorganized in the parasitophorous vacuole. We further show that the RH ∆DGAT1 strain is avirulent in mice, even at higher infection doses. Active immunization with these genetically attenuated whole-cell tachyzoites boosts the host immune response, providing protective immunity against subsequent acute lethal challenges by wild-type parasites. Overall, our findings suggest that unraveling the mechanisms of fatty acid storage in Toxoplasma is crucial for developing strategies to disrupt the parasite's survival mechanisms.

A drug repositioning strategy identifies a compound inhibiting GSK3 activity in Toxoplasma gondii

The phylum Apicomplexa encompasses many of the world&quot;s pre-eminent protozoan pathogens, including Toxoplasma gondii causing toxoplasmosis, a highly prevalent foodborne parasitic disease. Despite its widespread impact, existing therapies exhibit limited effectiveness, highlighting the urgent need for potent drugs targeting both T. gondii and related pathogens within the phylum. Here, compound 209 (C209) was identified in a phenotypic screen using a repurposed drug library aiming to uncover novel antiparasitic compounds and their corresponding targets. Previously assessed in clinical trials for cancer therapy, C209 demonstrated significant parasiticidal activity by disrupting the intracellular development of tachyzoites in the nanomolar range. Using a forward genetic approach, we identified the essential cell cycle-associated kinase TgGSK3 as the primary target of C209, identifying three single nucleotides substitutions conferring resistance to the drug. Through high-resolution crystallization and structure determination of the GSK3/C209 complex, we elucidated the compound's unique binding mode which displays a remarkable fold of its own and we could rationalize the interfering effect of the three amino acid mutations within the compound binding cavity. Biochemical analysis confirmed this inhibition, revealing a 2- to 18-fold loss of compound binding affinity in mutated proteins and a 2- to 5-fold decrease in kinase activity compared to the native protein. The conservation of GSK3 in Cryptosporidium has triggered interest in its potential as a new target offering an avenue for the development of broader anti-parasitic drugs.

Session V: Metabolism, Biochemistry & Drug Development

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