Pamukcu, S. | bioRxiv Science

bioRxiv2022

Disruption of the plastid-hosted iron-sulfur cluster biogenesis pathway in Toxoplasma gondii has pleiotropic effects irreversibly impacting parasite viability

Like many other apicomplexan parasites, Toxoplasma gondii contains a plastid harbouring key metabolic pathways, including the SUF pathway that is involved in the biosynthesis of iron-sulfur clusters. These cofactors are key for a variety of proteins involved in important metabolic reactions, potentially including plastidic pathways for the synthesis of isoprenoid and fatty acids. It was shown previously that impairing the NFS2 cysteine desulfurase, involved in the first step of the SUF pathway, leads to an irreversible killing of intracellular parasites. However, the metabolic impact of disrupting the pathway remained unexplored. We have generated another mutant of the pathway, deficient for the SUFC ATPase, and we have investigated in details the phenotypic consequences of TgNFS2 and TgSUFC depletion on parasite homeostasis. Our analysis confirms that Toxoplasma SUF mutants are severely and irreversibly impacted in growth: cell division and membrane homeostasis are particularly affected. Lipidomic analysis suggests a defect in apicoplast-generated fatty acids, along with a simultaneous increase in scavenging of host-derived lipids. However, addition of exogenous lipids did not allow full restauration of growth, suggesting other more important cellular functions were impacted in addition to fatty acid synthesis. For instance, we have shown that the SUF pathway is also key for generating isoprenoid-derived precursors necessary for the proper targeting of GPI-anchored proteins as well as for the parasite gliding motility. Thus, plastid-generated iron-sulfur clusters support the functions of proteins involved in several vital downstream cellular pathways, which implies the SUF machinery may be explored for discovering new potential anti-Toxoplasma targets.

microbiology↗

bioRxiv2021

Differential contribution of two organelles of endosymbiotic origin to iron-sulfur cluster synthesis in Toxoplasma

Iron-sulfur (Fe-S) clusters are one of the most ancient and ubiquitous prosthetic groups, and they are required by a variety of proteins involved in important metabolic processes. Apicomplexan parasites have inherited different plastidic and mitochondrial Fe-S clusters biosynthesis pathways through endosymbiosis. We have investigated the relative contributions of these pathways to the fitness of Toxoplasma gondii, an apicomplexan parasite causing disease in humans, by generating specific mutants. Phenotypic analysis and quantitative proteomics allowed us to highlight notable differences in these mutants. Both Fe-S cluster synthesis pathways are necessary for optimal parasite growth in vitro, but their disruption leads to markedly different fates: impairment of the plastidic pathway leads to a loss of the organelle and to parasite death, while disruption of the mitochondrial pathway trigger differentiation into a stress resistance stage. This highlights that otherwise similar biochemical pathways hosted by different sub-cellular compartments can have very different contributions to the biology of the parasites, which is something to consider when exploring novel strategies for therapeutic intervention. Author summaryToxoplasma gondii is a ubiquitous unicellular parasite that harbours two organelles of endosymbiotic origin: the mitochondrion, and a relict plastid named the apicoplast. Each one of these organelles contains its own machinery for synthesizing iron-sulfur clusters, which are important protein co- factors. In this study, we show that interfering with either the mitochondrial or the plastidic iron- sulfur cluster synthesizing machinery has a profound impact on parasite growth. However, while disrupting the plastidic pathway led to an irreversible loss of the organelle and subsequent death of the parasite, disrupting the mitochondrial pathway led to conversion of the parasites into a stress resistance form. We used a comparative quantitative proteomic analysis of the mutants, combined with experimental validation, to provide mechanistic clues into these different phenotypic outcomes. Although the consequences of disrupting each pathway were manifold, our data highlighted distinct changes at the metabolic level. For instance, the plastidic iron-sulfur cluster synthesis pathway is likely important for maintaining the lipid homeostasis of the parasites, while the mitochondrial pathway is clearly crucial for maintaining their respiratory capacity. Interestingly, we have discovered that other mutants severely impacted for mitochondrial function, in particular the respiratory chain, are able to survive and initiate conversion to the stress resistance form. This illustrates a different capacity for T. gondii to adapt for survival in response to distinct metabolic dysregulations.

microbiology↗